Abstract
Depression, a major global health issue, is closely associated with
imbalances in gut microbiota and altered intestinal functions. This
study investigates the antidepressant potential of a composite of
Geniposide (GP) and Nanocrystalline Cellulose (NCC), focusing on its
effects on the gut-brain axis. Utilizing network pharmacology, GP was
identified as a key compound targeting the BCL2 gene in depression
management. Experimental approaches, including a chronic unpredictable
mild stress (CUMS) model in mice, cellular assays, and fecal microbiota
transplantation (FMT), were used to evaluate the composite’s
effectiveness. Results indicate that GP activates the adenosine
monophosphate-activated protein kinase (AMPK) pathway by upregulating
BCL2, enhancing intestinal barrier integrity, and balancing gut flora.
These mechanisms contribute to its positive effects on hippocampal
function and depressive-like behaviors in mice, suggesting that the
GP-NCC composite could be a promising avenue for developing depression
therapies that target gut health.
graphic file with name 42003_2025_7934_Figa_HTML.jpg
Subject terms: Biotechnology, Diseases
__________________________________________________________________
NCC-GP complex enhances gut-brain axis modulation to treat depression.
It strengthens the intestinal barrier, balances gut microbiota, and
activates the BCL2-AMPK pathway, offering a novel therapeutic approach.
Introduction
Depression is a prevalent mental disorder that significantly impacts
the quality of life and social functioning of individuals^[34]1–[35]3.
With the rise in societal stress and lifestyle changes, the incidence
of depression is increasing and has emerged as a crucial issue in
global health^[36]4. Research indicates that the development of
depression is closely associated with various factors, including
genetics, environment, and lifestyle, necessitating comprehensive
regulation and treatment^[37]5–[38]7.
Geniposide (GP), a vital active ingredient of Gardenia jasminoides J.
Ellis traditional medicine, exhibits diverse biological activities and
has been found to have a positive impact on mood regulation, cognitive
enhancement, and anti-inflammatory effects^[39]8,[40]9. Simultaneously,
nanocrystalline cellulose (NCC), known for its excellent
biocompatibility and drug delivery performance, is increasingly being
utilized in the pharmaceutical field for drug applications^[41]10.
Leveraging the advantages of GP and NCC, exploring a novel therapeutic
strategy for depression by combining the two holds significant research
significance.
Recent studies have revealed a close association between intestinal
flora, intestinal barrier function, and the onset and progression of
Depression^[42]11–[43]13. The gut, being one of the body’s largest
immune organs, plays a vital role in maintaining the balance of
intestinal flora, intestinal barrier function, and
neurotransmitters^[44]14–[45]16. Imbalanced intestinal flora and
compromised intestinal barrier function may lead to inflammatory
responses and neurological abnormalities, subsequently affecting mood
and behavior^[46]17–[47]19.
The rise of network pharmacology has brought new perspectives and
methodologies to traditional medicine research^[48]20,[49]21. In this
study, through network pharmacology screening, GP was identified as the
primary active component for treating Depression in Gardenia
jasminoides J. Ellis. Furthermore, the regulatory role of GP’s target,
BCL2, is crucial in depression treatment^[50]22. BCL2’s modulation
plays a significant role in processes such as cell apoptosis and
inflammatory responses, thus warranting further investigation into its
role in Depression^[51]23,[52]24.
A comprehensive research approach was undertaken. Initially, utilizing
network pharmacology, the main active component and key targets of GP
in treating depression were identified, followed by the construction of
a mouse chronic unpredictable mild stress (CUMS) model. Sequencing data
and proteomic data of mouse intestinal tissue pre-and post-GP treatment
were obtained using high-throughput sequencing and proteomics
techniques, enabling the identification of key factors related to
CUMS^[53]25.
The aim of this study is to elucidate the molecular mechanisms of the
GP and NCC composite in alleviating depression-like behaviors by
improving intestinal barrier function and restoring microbial balance
to exert a positive effect on depression. Through experimentation, it
was demonstrated that NCC-GP can balance intestinal flora, repair the
intestinal barrier, and affect the brain’s neurological system via the
gut-brain axis, thereby holding potential value and scientific
significance for treating depression. This research provides valuable
insights into new approaches and strategies in depression therapy,
offering a scientific basis for clinical applications.
Results
Analysis of the potential mechanisms of GP in the treatment of depression
based on network pharmacology
Depression, also known as depressive disorder, is characterized by
persistent and prominent mood disturbances and cognitive impairments.
Patients often exhibit suicidal tendencies^[54]26–[55]28. According to
the World Health Organization, the global prevalence of depression is
expected to reach 15%-20%^[56]29,[57]30, with high rates of disability
and mortality. It is one of the leading causes of Disability-Adjusted
Life Years (YLD) loss^[58]31. Gardenia jasminoides J. Ellis, a shrub in
the Rubiaceae family, has been traditionally used in Chinese medicine
for jaundice hepatitis, sprains, hypertension, diabetes, and other
conditions^[59]32–[60]35. Recent studies have shown that Gardenia
jasminoides J. Ellis and its bioactive components exhibit significant
anti-tumor activity, can lower blood sugar and lipids, protect the
liver and gallbladder, gastric mucosa, and improve depression
symptoms^[61]36–[62]38.
In order to investigate the potential application of Gardenia
jasminoides J. Ellis and its bioactive components in depression, we
conducted an in-depth analysis of its bioactive components and targets
based on network pharmacology. The workflow of this analysis is
depicted in Fig. [63]1A. Using OB > 10% and DL > 0.4 as selection
criteria, we identified 19 effective bioactive components from Gardenia
jasminoides J. Ellis in the traditional Chinese medicine systems
pharmacology database and analysis platform (TCMSP) database
(Table [64]S1). By mapping these effective bioactive components to
their corresponding 867 target proteins in the Uniprot database, we
obtained a total of 251 target genes. Subsequently, using the GeneCards
database with “depression” as the key term, we screened
depression-related genes with a Relevance score > 5 threshold. The
intersection of depression-related genes and the target genes of
effective bioactive components was considered the potential therapeutic
target of the bioactive components (Fig. [65]1B).
Fig. 1. Potential target screening and functional enrichment analysis of
Gardenia jasminoides J. Ellis components for treating depression.
[66]Fig. 1
[67]Open in a new tab
A Illustrative Overview of the Bioinformatics Analysis Process; B Venn
Diagram Showing the Intersection Genes between Depression-Related Genes
and Gardenia jasminoides J. Ellis Active Compound Target Genes; C
Network Illustrating the Drug-Active Component-Target-Depression
Relationship of Gardenia jasminoides J. Ellis, where green and red
colors denote Gardenia jasminoides J. Ellis and Depression,
respectively, yellow signifies 52 active compounds with target genes,
and blue represents 47 intersecting target genes; D Hierarchical Tree
Map displaying the enrichment of target genes in different GO
categories (Biological Process, Cellular Component, Molecular
Function); E Network Diagram of KEGG Pathway Enrichment Analysis; F
Protein Interaction Node Statistics Chart of the 47 intersecting target
genes; G PPI Network Graph based on the Degree value of the 47
intersecting target genes, wherein larger nodes with redder color
indicate higher importance in the network, and thicker, redder lines
denote stronger interaction relationships.
Subsequently, we selected 47 intersecting target genes as potential
targets and integrated them with the 52 active compounds of Gardenia
jasminoides J. Ellis into Cytoscape, resulting in the construction of a
network model representing the Chinese herbal medicine-active
compounds-target genes-Depression (Fig. [68]1C). Through this network,
crucial active compounds and their target genes can be identified. To
elucidate the roles of these potential therapeutic targets in the
treatment of depression, functional enrichment analysis was conducted
on these genes. The Gene Ontology (GO) analysis revealed that the BP of
the potential targets was significantly enriched in cellular responses
to oxidative stress, regulation of cell development, and positive
regulation of monooxygenase activity, among others. The CCs were mainly
enriched in transport vesicles, endoplasmic reticulum lumens, and
phagocytic cups. Likewise, the MFs were primarily enriched in cytokine
activity, cytokine receptor binding, and signaling receptor activator
activity (Fig. [69]1D, Fig. [70]S1). Furthermore, based on the Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis,
these genes were found to mainly participate in pathways such as the
AMPK signaling pathway, Neurotrophin signaling pathway, and
Dopaminergic synapse, which are potentially related to the occurrence
and development of Depression (Fig. [71]1E).
To facilitate the subsequent selection of key therapeutic targets, the
aforementioned potential targets were subjected to protein-protein
interaction (PPI) analysis using the String database. A PPI network
graph was then redrawn based on the Degree value, revealing clear
interaction relationships among the targets (Fig. [72]1F). A
statistical graph based on the number of adjacent nodes showed that 11
targets had a Number of adjacent nodes > 30, namely IL6, AKT1, IL1B,
INS, TNF, BCL2, FOS, BDNF, IL10, CXCL8, and IFNG. These genes were
proposed as potential candidates for further exploration in the
treatment of depression (Fig. [73]1G).
Moreover, further analysis demonstrated that the 11 therapeutic targets
corresponded to 13 active compounds (Table [74]S2). Among these 11
targets identified, only BCL2 was found to be a target of GP, prompting
us to choose BCL2 for further investigation, with different pathways
enriched by these compounds’ target genes. For example, the target
genes of caffeic acid, chexanal, farnesol, and methyl palmitate were
mainly associated with cell apoptosis and inflammation. On the other
hand, the target genes of kaempferol, lauric acid, methyl palmitate,
oleic acid, paeonol, quercetin, rutin, and ursolic acid were primarily
involved in intracellular receptor signaling, receptor activity
regulation, and hormone-mediated signal transduction pathways. The
target genes of β-sitosterol and GP were associated with multiple
pathways like signal transduction, substance metabolism, cell cycle,
and regulation of cell structure. Given that GP is involved in various
neuro-signaling pathways, as well as depression-related apoptosis and
inflammation pathways within the PPI network, we speculate that GP may
serve as the main active component for treating depression. Research
has shown that GP significantly improves some depressive-like behaviors
in mice and rats, suppresses neuroinflammation, and enhances cognitive
dysfunction and depressive/anxiety symptoms. Previous studies by the
research group have also corroborated these findings^[75]39,[76]40.
Thus, it is speculated that GP may serve as the main effective
component for treating depression, with BCL2. BCL2 was prioritized due
to its robust regulatory effects on apoptosis and intestinal barrier
function, both of which are critical in the pathophysiology of
depression.
The crucial role of the gut-brain axis in depression treatment: evidence of
the therapeutic effect of GP
The interaction between the digestive system and brain function has
emerged as a vital area of focus in psychiatric research in recent
years. These multifaceted interactions occur within the
microbiota-gut-brain axis. The microbial community throughout the
entire gastrointestinal tract, known as intestinal flora, closely
correlates with anxiety and depressive disorders^[77]41,[78]42.
Emerging scientific evidence indicates that the intestinal flora plays
a significant role in regulating the central nervous system
(CNS)^[79]43. During negative emotional states such as stress, anxiety,
or depression, there is an increase in pro-inflammatory markers
expression and alterations in the intestinal flora and gut
permeability, leading to dysregulation of the gut-brain axis, which
subsequently causes dysfunction in the CNS^[80]44.
To validate the therapeutic effect of GP on Depression, we initially
established a CUMS animal model, with the CUMS procedures conducted
prior to behavioral tests such as the FST and SPT. Subsequently, we
assessed the success of modeling through the tail suspension test
(TST), sucrose preference test (SPT), forced swim test (FST), Morris
water maze (MWM), and histopathological staining of the hippocampal
tissues in mice (Fig. [81]2A). The results consistently demonstrated
that, compared to the control group, the CUMS-induced mice exhibited
significantly reduced sucrose preference and memory but increased tail
suspension and forced swim times, indicative of pronounced
depressive-like behavior. The hippocampal staining results showed
significant changes in the hippocampal structure of the depression mice
induced by CUMS, with a notable increase in apoptotic neurons and a
significantly lower proportion of neuron cells compared to the control
group (Fig. [82]S2A-G).
Fig. 2. Validation of the therapeutic effect of GP on Depression.
[83]Fig. 2
[84]Open in a new tab
A Overview of Mouse Depressive-Like Behavior Detection (Created by
BioRender); B–E Evaluation of depressive-like behavior in mice by TST,
SPT, FST, and MWM experiments; F H&E staining results of the
hippocampal tissues of mice in various groups, Scale bar=250 / 25 μm
for the leftmost column, and Scale bar=25 μm for the remaining two
columns; G Assessment of neuron cell counts in the hippocampal tissues
of mice across different groups by Nissl staining, Scale bar=250 / 25
μm; H TUNEL staining to detect neuron cell apoptosis in the hippocampal
tissues of mice in different groups, Scale bar=250/ 25 μm; One-way
ANOVA was used for comparisons among different groups; I
Immunohistochemical analysis of GFAP and BDNF expression in hippocampal
tissues, Scale bar=50μm; J Weight changes in mice from each group; K
ELISA analysis of inflammatory factor content in colonic tissues of
mice in each group; L Immunohistochemical evaluation of E-cadherin
content in colonic tissues of mice in various groups, Scale bar=100 μm;
M H&E staining results of colonic tissues of mice in different groups,
Scale bar=100 μm; N–Q Assessment of depressive-like behavior in mice by
TST, SPT, FST, and MWM experiments. For the comparison of two sets of
data, the independent samples t-test was utilized, whereas for the
comparison of data among multiple groups, ANOVA was employed. To
compare data across different time points within each group, two-way
ANOVA was conducted. The line between groups represents the specific
p-value, with p < 0.05 indicating a statistically significant
difference between groups, with 6 mice in each group.
In the colonic epithelial tissue, E-cadherin plays a critical role in
maintaining cellular barrier function and structural integrity^[85]45.
Subsequent examinations of the mice revealed changes in body weight and
colonic tissue induced by CUMS; the results showed that CUMS-induced
mice experienced a significant decrease in body weight compared to the
control group. In the colonic tissue, levels of TNF-α, IL-1β, and IL-6
were markedly elevated by enzyme-linked immunosorbent assay (ELISA)
(Fig. [86]S2I). The H&E staining results showed that the colonic tissue
of CUMS mice exhibited inflammatory cell infiltration in the lamina
propria, goblet cell loss, mucosal hyperplasia, crypt abscesses, and
crypt ulcers. Immunohistochemistry results revealed a substantial
decrease in E-cadherin content in the colonic epithelial tissue of mice
(Fig. [87]S2H–K). Furthermore, follow-up FMT experiments confirmed that
alleviation of depressive behavior in CUMS-induced mice occurred when
they were orally administered fecal pellets from the control group,
underscoring the pivotal role of intestinal flora in the onset and
progression of Depression (Fig. [88]S2L–O).
Subsequently, we treated the successfully CUMS modeled mice with
100 mg/kg of GP. The selective serotonin reuptake inhibitor (SSRI)
fluoxetine was used as a positive control for comparison and
evaluation. Behavioral tests revealed a significant therapeutic effect
of GP on the mice compared to the DMSO group. The therapeutic effects
of the SSRI fluoxetine were comparable to those of GP, with no
significant differences (Fig. [89]2B–E). H&E staining results
demonstrated that fluoxetine, as a positive control, exhibited
significant therapeutic effects compared to the negative control.
Additionally, treatment with GP significantly alleviated hippocampal
lesions in mice. Nissl staining and TUNEL staining results showed a
significant increase in the proportion of neuronal cells in the
hippocampal tissue of mice post-GP or fluoxetine treatment, accompanied
by a marked reduction in apoptotic cell numbers (Fig. [90]2F–H). In the
CUMS model, GFAP and BDNF immunoexpression were significantly reduced.
GP or fluoxetine treatment markedly increased the expression of these
key neuroproteins (Fig. [91]2I). Furthermore, GP or fluoxetine
treatment resulted in a significant increase in mouse body weight and a
significant alleviation of inflammation in colonic tissue, with a
notable elevation in E-cadherin content (Fig. [92]2J–L). Moreover, FMT
experiments showed that administering fecal pellets from GP-treated
mice to the DMSO-treated mice relieved depressive behavior in both
groups (Fig. [93]2N–Q).
Overall, these findings collectively suggest that GP can treat
depression by repairing the intestinal barrier, balancing intestinal
flora, and further impacting the hippocampal tissue in the brain. We
speculate that GP alleviates depressive symptoms by modulating GFAP and
BDNF expression in the hippocampus.
Mechanistic analysis of the therapeutic effect of GP on depression: BCL2 as a
key downstream target
Studies have shown that BCL2 may play a potential role in improving
intestinal barrier dysfunction^[94]46. Recent research indicates a
close relationship between the gut-brain axis and neurological
disorders such as depression^[95]47,[96]48. To analyze GP’s target in
improving intestinal dysfunction associated with depression, tandem
mass tag (TMT)-labeled quantitative proteomics was used to analyze
colonic tissues from six mice treated with DMSO or 100 mg/kg GP
(GP100). A total of 10,971 proteins were identified, yielding a set of
108 differentially expressed proteins (DEPs), including 38 upregulated
and 70 downregulated proteins (Fig. [97]3A–C). High-throughput
sequencing of colonic tissues from the DMSO and GP100 groups identified
306 differentially expressed genes (Fig. [98]3D, E). LASSO regression
and multivariable Cox analysis identified gene set B (Fig. [99]3F, G),
while a random forest algorithm was used to evaluate gene importance,
yielding gene set C (Fig. [100]3H). Depression-related genes were
extracted using SVM-RFE to obtain gene set D (Fig. [101]3I). The
intersection of these sets revealed a key factor: BCL2 (Fig. [102]3J).
Proteomics and transcriptomics confirmed significant upregulation of
BCL2 expression after GP treatment, validated by reverse transcription
quantitative polymerase chain reaction (RT-qPCR). When drug molecules
interact with targets, they require active conformations with geometric
and energy matching. Therefore, we employed molecular docking
techniques to predict the binding of GP to the active pocket of BCL2.
The detailed analysis process is illustrated in Fig. [103]3K.
Initially, the 2D and 3D molecular structures of GP were obtained from
the PubChem database (Fig. [104]S3). Subsequently, molecular docking
analysis of BCL2 and GP was conducted using software such as
AutoDockTools 1.5.6 and Vina 1.5.6. The 3D model displayed the binding
mode of the target protein receptor with the compound GP and its
interactions with surrounding amino acid residues (Fig. [105]3L).
Molecular docking demonstrated the binding affinity between GP and
BCL2, with BCL2 upregulation confirmed by transcriptomics, proteomics,
and RT-qPCR. Results from proteomics and transcriptomics studies
demonstrated a significant upregulation of BCL2 expression levels
following GP treatment, which was further validated using RT-qPCR in
the colon tissues of mice treated with GP (Fig. [106]3M).
Fig. 3. Machine learning algorithm and proteomics combined identification of
key targets for GP treatment in depression.
[107]Fig. 3
[108]Open in a new tab
A Proportion of differentially expressed proteins (DEPs) between
colonic tissues of three DMSO group mice and three GP100 group mice. B
Comparison of the number of upregulated and downregulated DEPs, with
red representing upregulated proteins and blue representing
downregulated proteins, N = 6. C Heatmap showing the differential
expression of the top 20 DEPs in the proteomics data, with DMSO (N = 3)
and GP100 (N = 3) representing colonic tissue samples from three mice
in each group. D Volcano plot of differentially expressed mRNA between
colonic tissues of three DMSO group mice and three GP100 group mice in
high-throughput sequencing data. E Heatmap showing the differential
expression of selected DEGs in the high-throughput sequencing data,
with DMSO (N = 3) and GP100 (N = 3) representing colonic tissue samples
from three mice in each group. F–G Lasso coefficient screening plot. H
Random forest algorithm result plot. I SVM-RFE algorithm result plot. J
Venn diagram showing the overlap of Depression-related mRNA and DEPs
identified by three machine learning algorithms: lasso regression,
random forest, and SVM-RFE. K Schematic workflow of bioinformatics
analysis in Fig. 3 (Created by BioRender). L Molecular docking result
between GP molecule and target protein BCL2. M RT-qPCR analysis of BCL2
expression changes in colonic tissues of mice after GP treatment,
analyzed by independent t-test (N = 6 mice per group). In the volcano
plot, blue dots represent significantly downregulated mRNA in the GP100
group, red dots represent significantly upregulated mRNA in the GP100
group, and gray dots represent mRNA with no significant difference. The
line between groups represents the specific p-value, with p < 0.05
indicating a statistically significant difference between groups.
In conclusion, the aforementioned results indicate that BCL2 is a
downstream target of GP in the treatment of depression.
GP promotes intestinal barrier integrity by regulating BCL2 expression in
colonic epithelial cells
To validate whether BCL2 is a target of GP, we established an in vitro
cell model of colonic epithelial cells induced by LPS in mice
(Fig. [109]4A). ELISA and in vitro barrier integrity experiments showed
a significant upregulation of inflammatory factors and a decrease in
intestinal barrier integrity in colonic epithelial cells, confirming
the successful model construction (Fig. [110]4B, C). RT-qPCR results
revealed a significant downregulation of BCL2 expression in colonic
epithelial cells after LPS induction, which was significantly
upregulated upon treatment with GP (Fig. [111]S4A, B). Subsequently, we
used lentivirus to knock down or overexpress BCL2, verifying lentiviral
transfection efficiency using RT-qPCR and Western blot techniques. The
results demonstrated a significant increase in BCL2 expression after
overexpressing lentivirus, with the most significant knockdown
efficiency observed in sh-BCL2-1, which was consequently chosen for
further experiments (Fig. [112]S4C–F).
Fig. 4. GP-mediated BCL2 expression alleviates depression.
[113]Fig. 4
[114]Open in a new tab
A Schematic diagram of in vitro cell model; B Determination of
inflammatory factor levels in cells of each group using ELISA; C
Results of in vitro intestinal barrier integrity in different cell
groups. The left graph shows TEER values representing cell barrier
integrity, and the right graph shows FITC-Dex values representing cell
barrier permeability; D Schematic diagram of cell experiment process
(Created by BioRender); E Measurement of cell viability in each group
using MTT assay; F Evaluation of cell proliferation capacity in each
group using EdU assay (Scale bar=50 μm); G Re-assessment of
inflammatory factor levels in cells of each group using ELISA; H
Detection of E-cadherin expression in cells of each group using Western
blot; I Same as (C), depicting the results of in vitro intestinal
barrier integrity in different cell groups; J Assessment of cell
viability in each group using MTT assay; K Evaluation of cell
proliferation capacity in each group using EdU assay (Scale bar=50 μm);
L Reinforced assessment of inflammatory factor levels in cells of each
group using ELISA; M Re-examination of E-cadherin expression in cells
of each group using Western blot; N Reiteration of in vitro intestinal
barrier integrity results in different cell groups, with TEER values
indicating cell barrier integrity on the left and FITC-Dex values
representing cell barrier permeability on the right. Comparisons
between two groups were analyzed using t-tests, while one-way ANOVA was
used for comparisons among different groups. The line between groups
represents the specific p-value, with p < 0.05 indicating a
statistically significant difference between groups. Each cell
experiment was replicated three times.
Next, we overexpressed BCL2 with DMSO treatment and knocked down BCL2
with GP treatment. Cell viability was measured using the MTT assay,
cell proliferation capacity was assessed with EdU incorporation,
changes in inflammatory factor levels were evaluated by ELISA,
E-cadherin expression was analyzed by Western blot, and the integrity
of the intestinal barrier was assessed through in vitro barrier
integrity experiments (permeability assessment) (Fig. [115]4D). The
results showed that in the DMSO group, overexpression of BCL2 led to
increased cell viability, enhanced cell proliferation, reduced
inflammatory factor levels, significant upregulation of E-cadherin
expression, and improved integrity of the intestinal barrier. The
results showed that there was no significant difference in the
therapeutic effects between the GP-treated group and the BCL2
overexpression group. When GP was combined with the BCL2 overexpression
group, GP significantly enhanced the therapeutic effects compared to
the BCL2 overexpression group alone, further supporting the role of
BCL2 in the mechanism of GP action (Fig. [116]4E–I). Conversely, in the
GP treatment group, cell viability decreased, proliferation capacity
diminished, inflammatory factor levels increased, E-cadherin expression
significantly decreased, and the in vitro intestinal barrier integrity
declined after knocking down BCL2 (Fig. [117]4J–N).
These results convincingly demonstrate that GP can enhance the
integrity of the intestinal barrier by upregulating BCL2 expression in
colonic epithelial cells.
GP alleviates depression by activating the BCL2-mediated AMPK pathway
Numerous studies have shown that activation of the AMPK signaling
pathway is beneficial in promoting the balance of intestinal
flora^[118]49,[119]50. Dorsomorphin, a selective ATP-competitive AMPK
inhibitor, has been proven to suppress the phosphorylation levels of
AMPK^[120]51,[121]52. The results in Fig. [122]1E suggest a potential
association between GP and the treatment of depression through the AMPK
pathway, while predictive results from the GeneMANIA database indicate
interactions between BCL2 and AMPK (including PRKAA1 and PRKAA2)
(Fig. [123]5A). Therefore, we hypothesize that GP might activate the
AMPK pathway by mediating the expression of BCL2, promoting the balance
of intestinal flora and thus alleviating depression.
Fig. 5. GP mediates BCL2 expression to activate the AMPK pathway, alleviating
depression.
[124]Fig. 5
[125]Open in a new tab
A Predicted results from the GeneMANIA database; B Evaluation of AMPK
phosphorylation levels in cells of each group using Western blot; C
Detection of BCL2 expression levels and AMPK phosphorylation levels in
cells of each group through Western blot; D Assessment of cell
viability in each group with MTT assay; E Examination of cell
proliferation capacity in each group using EdU experiment (Scale bar=50
μm); F Measurement of inflammatory factor levels in cells of each group
with ELISA; G Assessment of E-cadherin expression in cells of each
group, analyzed by Western blot; H Measurement of in vitro intestinal
barrier integrity in cells of each group, where TEER values in the left
graph represent cell barrier integrity, and FITC-Dex values in the
right graph represent cell barrier permeability; I Presentation of H&E
staining results of mouse hippocampal tissues in each group, Scale
bar=250 / 25 μm; J Examination of the number of neuronal cells in mouse
hippocampal tissues of each group through Nissl staining, Scale bar=250
/ 25 μm; K Detection of neuronal cell apoptosis status in mouse
hippocampal tissues of each group using TUNEL staining, Scale bar=250 /
25 μm; L Recording of the body weight changes in mice of each group; M
Demonstration of H&E staining results of mouse colon tissues in each
group, with Scale bar=100 μm; N Determination of in vivo barrier
integrity experiment results; (O-R) Evaluation of depressive-like
behaviors in mice of each group using TST, SPT, FST, and MWM
experiments. One-way ANOVA was used for comparisons among different
groups. The line between groups represents the specific p-value, with
p < 0.05 indicating a statistically significant difference between
groups. Cell experiments were repeated three times, with 6 mice in each
group.
To validate our hypothesis, we initially examined the phosphorylation
levels of AMPK upon knocking down or overexpressing BCL2, revealing a
decrease in AMPK phosphorylation levels with BCL2 knockdown and an
increase with BCL2 overexpression (Fig. [126]5B). Subsequently, while
overexpressing BCL2, we inhibited the phosphorylation levels of AMPK
using Dorsomorphin (Dor). The results showed that compared to the
oe-NC + DMSO group, the cells in the oe-BCL2 + DMSO group exhibited
significantly enhanced phosphorylation levels of AMPK, increased
expression of BCL2, enhanced cell viability and proliferation
capabilities, reduced cellular inflammation, upregulated E-cadherin
expression, and improved intestinal barrier integrity. Conversely,
compared to the oe-BCL2 + DMSO group, the cells in the oe-BCL2 + Dor
group showed a significant decrease in AMPK phosphorylation levels, no
significant change in BCL2 expression, weakened cell viability and
proliferation capabilities, exacerbated cellular inflammation,
downregulated E-cadherin expression, and decreased intestinal barrier
integrity (Fig. [127]5C–H).
Subsequently, we verified this mechanism in vivo. Western blot analysis
revealed a significant increase in AMPK phosphorylation levels and BCL2
expression in mouse colon tissues of the oe-BCL2 + DMSO group compared
to the oe-NC + DMSO group (Fig. [128]S5A). Further examinations on the
impact on mouse depression revealed that compared to the oe-NC + DMSO
group, the inflammation infiltration in mouse colon tissues of the
oe-BCL2 + DMSO group was alleviated, intestinal barrier integrity was
strengthened, hippocampal tissue lesions were mitigated, the proportion
of neuronal cells significantly increased, depression-like behaviors in
mice were alleviated, accompanied by a slight increase in body weight.
In contrast, the oe-BCL2 + Dor group showed a worsening of colorectal
tissue inflammation, a significant decrease in intestinal barrier
integrity, exacerbation of hippocampal tissue lesions, reduction in
neuronal cells, worsening of depression in mice, and decrease in body
weight (Figs. [129]5I–N and Fig. [130]S5B–F). Furthermore, results from
FMT experiments confirmed that administration of fecal pellets from
oe-BCL2 + DMSO group mice to the oe-NC + DMSO/oe-BCL2 + Dor group mice
alleviated depression behaviors, highlighting the essential role of
intestinal flora in the development of Depression (Fig. [131]5O–R).
In conclusion, the results demonstrate that GP can alleviate depression
by upregulating the expression of BCL2 in colonic epithelial tissues to
activate the AMPK pathway.
GP complexed with NCC: enhancing oral drug release and intestinal absorption
Table [132]S1 results revealed a mere 14.64% oral bioavailability of
GP. Previous studies have highlighted that NCC, originating from plant
fibers, boasts excellent biocompatibility and high biosafety, enabling
the promotion of intestinal barrier repair and the balance of
intestinal flora^[133]53. Additionally, NCC presents a substantial
surface area and negative charge, facilitating the binding of a
plethora of drugs, thus unleashing the potential for high drug loading
and optimal dosage control^[134]54. Building upon these investigations,
we successfully synthesized the GP complex with NCC (Fig. [135]6A).
Fig. 6. Synthesis and characterization of NCC-GP.
[136]Fig. 6
[137]Open in a new tab
A Schematic representation of the preparation and physical
characterization process of the NCC-GP composite (Created by
BioRender); B Scanning electron microscope images of the NCC-GP
composite (Scale bar=500 nm); C Determination of the binding efficiency
of NCC with GP using UV-Vis spectrophotometry; D Assessment of the Zeta
potential of NCC-GP solution by dynamic light scattering (DLS); E
Evaluation of the drug release rate of NCC-GP composite using UV-Vis
spectrophotometry; F Investigation of intracellular uptake of
fluorescently labeled NCC-GP after cellular internalization (Scale
bar=50 μm). Cell experiments were repeated thrice.
In the preparation process, we initially examined the morphology of NCC
using scanning electron microscopy, revealing individual NCC fibers to
be approximately 10 nm wide and 500 nm long (Fig. [138]6B). Following
the preparation of NCC-GP, the drug binding rate was calculated using a
UV-visible spectrophotometer. The results indicated a significant
increase in the quantity of GP bound to NCC with an escalation in the
drug mass added to the NCC suspension. When PBS was utilized as the
dispersing medium, over 100 μg of GP was shown to bind to NCC, with a
binding efficiency exceeding 50% (Fig. [139]6C). Additionally, no
significant alteration in zeta potential was observed after GP was
bound to NCC (Fig. [140]6D).
Subsequently, we assessed the drug release efficacy of NCC-GP. The
findings demonstrated a rapid release of GP from NCC, with
approximately 60% of the bound GP being released within 4 hours. By the
end of day 1, drug release had stabilized, surpassing an 80% release
rate of GP (Fig. [141]6E). Furthermore, visualization of the uptake of
fluorescently labeled NCC by murine colonic epithelial cells indicated
substantial uptake of both NCC and NCC-GP, with no apparent cellular
breakdown within 24 hours, suggesting the absence of significant
cytotoxicity with NCC and NCC-GP (Fig. [142]6F).
In conclusion, we have successfully developed a GP complexed with NCC,
exhibiting a rapid and cost-effective drug release rate, along with
efficient uptake by colonic epithelial cells.
NCC-GP enhanced GP’s restorative effect on the intestinal barrier
In this study, the toxicity and therapeutic effects of NCC and NCC-GP
on colonic epithelial cells were further investigated through in vitro
cell experiments. Initially, Western blot analysis was employed to
assess the impact of NCC-GP on the expression of BCL2 and the
phosphorylation levels of AMPK. The results demonstrated that compared
to the NCC/DMSO group, NCC-GP exhibited similar effects to GP,
significantly upregulating the expression of BCL2 and the
phosphorylation levels of AMPK (Fig. [143]7A). Subsequently, cells from
the Normal group were treated with NCC or NCC-GP, and the toxicity and
therapeutic effects of NCC-GP on the cells were evaluated using the MTT
assay, EdU labeling, and TUNEL staining, with DMSO serving as the
control group. The findings revealed no significant changes in the
viability, proliferation capacity, or apoptosis status of cells in the
NCC + Nor/NCC-GP + Nor groups compared to the DMSO + Nor group,
indicating that NCC and NCC-GP did not exhibit significant toxicity to
the cells (Fig. [144]7B–D). Moreover, compared to the DMSO group, the
viability and proliferation capacity of cells in the NCC group, GP
group, and NCC-GP group gradually increased while the apoptotic ability
decreased. Among these groups, cells in the NCC-GP group demonstrated
the highest viability and proliferation capacity and the lowest
apoptosis rate (Fig. [145]7E–G).
Fig. 7. Effects of NCC-GP on colonic epithelial cells.
[146]Fig. 7
[147]Open in a new tab
A Evaluation of BCL2 expression levels and AMPK phosphorylation levels
in cells of each group using Western blot; B Assessment of cell
viability in each group with MTT assay; C Measurement of cell
proliferation capacity in each group through EdU experiment (Scale
bar=50 μm); D Evaluation of cell apoptosis status in each group using
TUNEL staining (Scale bar=50 μm); E Reassessing cell viability in each
group with MTT assay; F Analysis of cell proliferation capacity in each
group through EdU experiment (Scale bar=50 μm); G Determination of cell
apoptosis status in each group using TUNEL staining (Scale bar=50 μm);
H Measurement of inflammatory factor levels in cells of each group with
ELISA; I Detection of E-cadherin expression in cells of each group
through Western blot; J Assessment of in vitro intestinal barrier
integrity in cells of each group, where TEER values in the left graph
represent cell barrier integrity, and FITC-Dex values in the right
graph represent cell barrier permeability. Comparisons between two
groups were analyzed using t-tests, while one-way ANOVA was used for
comparisons among different groups. The line between groups represents
the specific p-value, with p < 0.05 indicating a statistically
significant difference between groups. Each cell experiment was
repeated three times.
Subsequent analysis involved measuring the levels of inflammatory
factors within the cells using ELISA. The results illustrated a gradual
reduction in the inflammatory factor levels in cells from the NCC
group, GP group, and NCC-GP group compared to the DMSO group, with the
NCC-GP group showing the most significant alleviating effect on
cellular inflammation (Fig. [148]7H). Further Western blot and
immunohistochemistry experiments yielded consistent outcomes,
indicating that the expression of E-cadherin in cells from the NCC
group, GP group, and NCC-GP group significantly increased compared to
the DMSO group. This led to an enhancement in the integrity of the in
vitro intestinal barrier, with NCC-GP demonstrating the most
significant effect (Fig. [149]7I, J).
In summary, the results suggest that NCC-GP effectively enhances GP’s
reparative action on the intestinal barrier.
Intestinal barrier repair and depression treatment: mechanisms and efficacy
of NCC-GP
In the final analysis, it was verified in vivo that NCC-GP mediates the
alleviation of depression through enhancement of the intestinal barrier
in the gut-brain axis. Western blot analysis revealed significantly
increased levels of BCL2 expression and AMPK phosphorylation in the
colon tissues of mice treated with NCC-GP, consistent with the results
of in vitro cell experiments (Fig. [150]8A). Subsequently, behavioral
tests were conducted to assess the degree of depression in mice,
showing a noticeable reduction in depressive behaviors in the NCC
group, GP100 group, and NCC-GP group compared to the DMSO group, with
the efficacy gradually increasing and being most pronounced in the
NCC-GP group (Fig. [151]8B–E). H&E staining results indicated
alleviation of lesions in the hippocampal tissues of mice in the NCC
group, GP100 group, and NCC-GP group, with the therapeutic effect
progressively increasing. Nissl and TUNEL staining further demonstrated
a gradual increase in the proportion of neuronal cells and a reduction
in apoptosis, with the most significant effect observed in the NCC-GP
group (Fig. [152]8F–H). Additionally, compared to the DMSO group, mice
in the NCC group, GP100 group, and NCC-GP group exhibited gradual
weight gain, reduced intestinal inflammation, increased E-cadherin
levels, and improved integrity of the intestinal barrier
(Fig. [153]8I–L).
Fig. 8. In vivo validation of NCC-GP alleviating depression via the gut-brain
axis.
[154]Fig. 8
[155]Open in a new tab
A Expression levels of BCL2 and AMPK in each group of cells were
detected using Western blot; B–E Depressive-like behaviors of mice in
each group were assessed through TST, SPT, FST, and MWM experiments; F
display of H&E staining results of hippocampal tissues in each group of
mice, Scale bar = 250 μm / 25 μm; G Neuronal cell count in the
hippocampal tissues of each group of mice was evaluated through Nissl
staining, Scale bar = 250 / 25 μm; H Apoptosis of neuronal cells in the
hippocampal tissues of each group of mice was examined using TUNEL
staining, Scale bar = 250 / 25 μm; I Monitoring of weight changes in
each group of mice with analyzed using two-way ANOVA; J Presentation of
H&E staining results of colonic tissues in each group of mice, Scale
bar = 100 μm; K Content of E-cadherin in colonic tissues of each group
of mice was detected through Western blot; L Display of in vivo barrier
integrity experiment results; M–P Assessment of depressive-like
behaviors in each group of mice through TST, SPT, FST, and MWM
experiments; Q H&E staining results of hippocampal tissues in each
group of mice, Scale bar = 250/25 μm; R Neuronal cell count in the
hippocampal tissues of each group of mice was evaluated via Nissl
staining, Scale bar = 250 / 25 μm; S Evaluation of neuronal cell
apoptosis in the hippocampal tissues of each group of mice using TUNEL
staining, Scale bar = 250 / 25 μm. Comparisons between two groups were
analyzed using t-tests, while one-way ANOVA was used for comparisons
among different groups. The line between groups represents the specific
p-value, with p < 0.05 indicating a statistically significant
difference between groups, with 6 mice per group.
Furthermore, results from FMT experiments indicated that administering
fecal pellets from NCC mice or NCC-GP mice via gavage to DMSO group
mice led to relief in depressive behaviors, with the NCC-GP group
showing the best therapeutic efficacy (Fig. [156]8M–P). H&E staining
illustrated a notable alleviation of lesions in the hippocampal tissues
of mice in the DMSO + FMT-NCC group and DMSO + FMT-NP group, while
Nissl and TUNEL staining revealed a significant increase in the
proportion of neuronal cells and a marked decrease in apoptosis in
these groups (Fig. [157]8Q–S).
Finally, an assessment of the in vivo toxicity of NCC-GP in mice was
conducted, with major organs subjected to H&E staining post-treatment,
revealing no significant pathological changes (Fig. [158]S6),
indicating the excellent biocompatibility of NCC-GP. These findings
collectively demonstrate that NCC-GP can ameliorate depression by
alleviating intestinal inflammation, repairing the intestinal barrier,
balancing the intestinal flora, and further influencing the hippocampal
tissues in the brain through the gut-brain axis.
Discussion
In previous studies, GP, as the main component of the traditional
Chinese medicine Gardenia jasminoides J. Ellis, has been a subject of
considerable attention due to its confirmed antidepressant
effect^[159]55,[160]56. However, to the best of our knowledge, this
study investigates a previously unreported mechanism of the GP-NCC
combination in regulating intestinal barrier function and balancing
intestinal flora. By employing various research tools, including
network pharmacology screening, proteomic techniques, and machine
learning algorithms, this study delves deeper into unveiling the
molecular mechanism of GP in combination with NCC on depression-like
behaviors, offering a fresh perspective for depression treatment
research.
Through the construction of a CUMS model and in vitro experimental
validation, we discovered that GP improves intestinal barrier function
and alleviates depression-like behaviors in mice by upregulating the
expression of BCL2 to activate the AMPK pathway. This finding aligns
with previous research on the regulatory role of GP in cellular
survival environments. However, our study further elucidates its key
factors correlated with CUMS, providing a new angle for mechanistic
exploration^[161]57,[162]58.
In vitro cell experiments revealed that GP modulates the vitality,
proliferative capacity, and apoptosis of colonic epithelial cells by
regulating BCL2 expression, thereby enhancing the integrity of the
intestinal barrier. This discovery supports previous research findings,
validating the significant role of GP in regulating cellular functions
and inflammatory responses. Concurrently, the compound of GP and NCC,
NCC-GP, demonstrates remarkable effects in intestinal barrier repair,
offering new avenues and possibilities for depression
treatment^[163]22,[164]59,[165]60.
By preparing the composite of NCC and GP, NCC-GP, we confirmed its
reparative effect on the intestinal barrier. This innovative
preparation method opens new pathways for the application of
traditional Chinese medicine ingredients in depression therapy while
also unveiling fresh possibilities for the potential use of
nanotechnology in treating neurological and psychiatric
disorders^[166]61.
Animal experiments further substantiated the effectiveness of NCC-GP in
alleviating depression-like behaviors, reinforcing the compound’s
therapeutic effect on depression through the restoration of the
intestinal barrier and microbiota balance^[167]62. Compared to previous
research outcomes, our study offers a more comprehensive and in-depth
experimental validation, providing more reliable evidence for the
application of this compound in depression treatment.
Based on the aforementioned results, we can preliminarily conclude the
following: GP, by upregulating the expression of BCL2 in colonic
epithelial tissues, activates the AMPK pathway to alleviate depression.
The complex formed by NCC binding with GP can further impact the
brain’s hippocampal tissue through the gut-brain axis by alleviating
intestinal inflammation, repairing the intestinal barrier, and
balancing the intestinal flora, ultimately treating Depression. This
study introduces a novel therapeutic strategy to address the low oral
bioavailability issue of GP, which conventional treatments struggle to
overcome. Our research lays a theoretical foundation for understanding
the development of depression and discovering new therapeutic targets.
Nevertheless, the study has certain limitations. While positive results
were observed in in vitro cell experiments and animal models, the
successful translation of these findings into clinical applications
requires further validation. Additionally, the long-term safety and
potential side effects remain unclear, prompting our future endeavors
to delve deeper into these areas.
The study’s scientific and clinical significance lies in the in-depth
exploration of the active component GP from the traditional Chinese
medicine Gardenia jasminoides J. Ellis and its binding with NCC to
treat depression, elucidating the molecular mechanisms involved.
Through network pharmacology screening and experimental validation, GP
was identified as the primary active component for treating depression,
revealing its mechanism of action in treating depression by modulating
key pathways such as BCL2 and AMPK, thus providing new pharmacological
evidence for traditional Chinese medicine in depression treatment.
Furthermore, utilizing high-throughput sequencing and proteomic
technologies, the study unveiled the impact of GP on balancing
intestinal flora and improving intestinal barrier function in mice
exhibiting depressive behaviors, offering crucial insights into the
relationship between depression and the gut-brain axis.
In terms of clinical applications, our study offers a theoretical basis
for developing new drugs to treat depression. Moreover, the
cost-effective preparation method of the complex formed by NCC and GP
exhibits a favorable drug release profile, making it a promising
candidate for a novel depression treatment. Additionally, the new
strategy for treating depression through balancing intestinal flora,
repairing the intestinal barrier, and regulating brain neural tissues
via the gut-brain axis presents fresh perspectives for the treatment of
mental illnesses.
Nonetheless, the study has some limitations. Firstly, the research was
conducted on mouse models, and further confirmation of its efficacy and
safety in humans is needed. The study results indicated that the
absolute bioavailability of GP followed the order: i.m. (72.69%) > i.n.
(49.54%) > i.g. (9.74%). It was also found that GP can permeate the
skin both in vitro and in vivo, allowing for rapid distribution into
subcutaneous tissues and the bloodstream in mice after administration
with ShanYi aerosol^[168]63,[169]64. We acknowledge that
pharmacokinetic and pharmacodynamic data on NCC are currently limited,
and future studies will explore the pharmacokinetics and pharmacology
of GP treatment in greater depth. We also plan to include germ-free
mice or antibiotic-treated groups in subsequent research to further
confirm whether GP exerts its antidepressant effects through modulation
of the gut microbiota. By introducing such controls, we aim to delve
deeper into the mechanism of GP in the CUMS mouse model. Secondly,
despite unveiling the molecular mechanisms of GP in treating depression
through various technological approaches, further investigation into
its effects and applicability across different types of depression
patients is required to better guide clinical applications. Based on
previous CUMS research selections^[170]65,[171]66, male mice were
chosen in this study for several scientific considerations: The
endocrine cycles of female mice significantly impact the
reproducibility of behavioral experiments, especially in depression
models, where fluctuations in female hormones can interfere with
experimental outcomes, increasing the variability and complexity of
data interpretation. In contrast, male mice have more stable endocrine
profiles, facilitating clearer and more consistent results.
Standardized research protocols often prioritize male mice to minimize
sex-related variables when investigating new biological mechanisms, as
this is a common strategy in experimental model design. This choice not
only helps to clarify the relationship between experimental
interventions and improvements in depressive symptoms but also provides
a comparative basis for future studies in female models. Building on
this study, we will explore whether the mechanisms of GP-mediated BCL2
upregulation and AMPK pathway activation in alleviating depression are
equally effective in female mice and will address potential sex
differences.
Looking ahead, future exploration can focus on uncovering the potential
mechanisms and clinical applications of the complex formed by GP and
NCC in depression treatment. By integrating the concept of personalized
medicine and conducting clinical trials tailored to different types of
depression, patients can lead to individualized precision therapies.
Preclinical studies have shown that subdiaphragmatic vagotomy (SDV) can
block depressive-like behaviors in mice following fecal microbiota
transplantation (FMT) from mice exhibiting depressive-like
behaviors^[172]67. We will further investigate whether SDV affects the
antidepressant effects of the GP-NCC complex in the CUMS model, which
promises to be an intriguing area of exploration. Furthermore,
investigating the combined use of GP with other drugs or treatment
modalities may unveil additional treatment strategies, providing more
choices and hope for patients with depression.
Materials and methods
Gardenia bioactive components
Chemical components of Gardenia jasminoides J. Ellis were retrieved
using the TCMSP. Active components with oral bioavailability (OB) > 10%
and drug-likeness (DL) > 0.4 were selected as effective, along with
their respective targets. The identified targets were converted into
corresponding genes using the Uniprot database
([173]http://www.uniprot.org/)^[174]68.
Analysis of active ingredient targets
Genes related to depression were searched in the GeneCards database
([175]https://www.genecards.org/) using “depression” as the keyword,
with a relevance score > 5 as the threshold for selecting
depression-related genes. An intersection analysis was conducted using
the XenoTox academic online analysis platform between genes related to
depression and the target genes of the aforementioned active components
to create a Venn diagram, obtaining the intersection genes as potential
therapeutic targets. The selected potential therapeutic targets and
active components were imported into Cytoscape software (version
3.10.0) to construct a network named “Chinese Medicine - Active
Components - Targets - Depression.” Subsequently, core components and
targets were identified based on this network, and further analysis of
the underlying mechanisms was conducted^[176]69.
PPI
The potential therapeutic targets identified through the aforementioned
screening were mapped onto the String database platform
([177]https://string-db.org/), with Homo sapiens selected as the
research species for PPI analysis. A medium confidence level
interaction score (0.400) was set, unconnected nodes were hidden, and
other parameters were kept at default settings to obtain a PPI network
of potential therapeutic targets for depression treatment, with the
protein interaction data downloaded. The connectivity node count
statistics graph for each protein was generated using R software.
Subsequently, the PPI network graph was redrawn using Cytoscape
software based on the Degree values and the proteins’ importance in the
network as determined in the PPI analysis^[178]70.
Functional enrichment analysis
GO and KEGG functional enrichment analyses were conducted on the
potential therapeutic targets of active compounds to differentiate the
enriched pathways of different target genes of active compounds. The
“ClusterProfiler” package in R software was utilized for GO and KEGG
enrichment analyses. The GO enrichment analysis included three
categories: biological process (BP), cellular component (CC), and
molecular function (MF), with a significance threshold of P < 0.05 for
selection and visualization of the enriched pathways and genes. KEGG
enrichment analysis was performed with a significant enrichment
selection criteria of P < 0.05, followed by visualization of the
enriched pathways and genes. Based on the differential enrichment
pathways of the targets, key active compounds and their targets were
selected in conjunction with relevant literature to speculate on the
potential mechanisms of action^[179]71.
Molecular simulation docking
GP’s 2D and 3D chemical structures were obtained from the PubChem
database ([180]https://pubchem.ncbi.nlm.nih.gov/) with Compound CID:
187808 and saved in “SDF” format. The crystal structure of the
candidate target protein BCL2 (PDB ID: 1G5M) for GP was downloaded from
the Protein Data Bank (PDB) database ([181]https://www.rcsb.org). The
compound GP’s structure was converted to a three-dimensional structure
using Chem3DUltra 14.0 software and subjected to energy minimization
using the MM2 algorithm. Subsequently, the target protein receptor
underwent dehydration and organic compound removal using PyMOL
software. AutoDockTools 1.5.6 was employed for hydrogenation and charge
computation of the target protein receptor molecules. The compound and
target protein receptor were converted into “pdbqt” files, and
appropriate box center and grid parameters were set. Finally, Vina
1.5.6 was utilized to assess molecular docking and calculate the
binding free energy. By integrating PPI network analysis, pharmacophore
modeling, and molecular docking analysis, drug-target interactions were
identified^[182]72.
Cell culture
Mouse colonic epithelial cells (CP-M159, Procell, China) were cultured
in a specialized medium (CM-M159, Procell) containing 10% FBS
(12484028, Gibco, USA) and 1% penicillin/streptomycin (15140148, Gibco,
USA). To establish an in vitro model of colonic epithelial cells,
100 μg/mL of LPS (L5293, Sigma-Aldrich, USA) was added to the culture
medium and the cells were treated for 24 hours. For studying the action
of AMPK, 10 μM Dorsomorphin (AMPK inhibitor; HY-13418A, MedChemExpress,
USA) was added to the cell culture medium, followed by subsequent
experiments after 18 hours^[183]51.
The cell groups were designated as follows: Control/Nor group (normal
mouse colonic epithelial cells), LPS group (mouse colonic epithelial
cells treated with LPS), DMSO group (mouse colonic epithelial cells
cultured with 10 μM DMSO in the medium), Dor group (mouse colonic
epithelial cells cultured with 10 μM Dorsomorphin in the medium).
293 T cell line was obtained from ATCC (CRL-3216) and cultured in DMEM
medium (11965092, Gibco, USA) containing 10% FBS, 10 μg/mL
streptomycin, and 100 U/mL penicillin. All cells were cultured at 37 °C
with 5% CO[2] in a humidified CO[2] incubator. Passaging was performed
when the cells reached 80%-90% confluence^[184]73.
Construction of virus vector and cell transfection
Mouse cDNA sequences were analyzed for potential short hairpin RNA
(shRNA) target sequences based on GeneBank. Three sequences targeting
BCL2 were initially designed, with one sequence lacking interfering
sequences serving as the negative control (sh-NC). The primer sequences
are listed in Table [185]S3, and the oligonucleotides were synthesized
by GenePharma® (Shanghai, China). A lentiviral packaging system was
constructed using pLKO.1 (lentivirus gene silencing vector). The
packaged virus and target vector were co-transfected into 293 T cells
using Lipofectamine 2000 (11668500, Invitrogen, USA) with a cell
confluence of 80-90%. After 48 hours of cell culture, the supernatant
was collected, and virus particles were present in the filtered
supernatant after centrifugation. The virus was harvested during the
logarithmic growth phase for virus titer detection.
Lentivirus for BCL2 overexpression was constructed and packaged by
Genechem (Shanghai, China), with the overexpression vector being
LV-PDGFRA. The packaged virus and target vector were co-transfected
into 293 T cells using Lipofectamine 2000 (11668500, Invitrogen, USA)
with a cell confluence of 80-90%. After 48 hours of cell culture, the
supernatant was collected, and virus particles were present in the
filtered supernatant after centrifugation. The virus was harvested
during the logarithmic growth phase for virus titer detection.
Additionally, for the convenience of subsequent bioluminescence imaging
experiments, the silence or overexpression vectors carried the
GFP/mCherry genes.
When the cells reached the logarithmic growth phase, they were digested
with trypsin, suspended, and seeded at a concentration of 5 × 10^4
cells/mL in 6-well plates, with 2 mL per well. Prior to establishing
the in vitro cell model, lentivirus for BCL2 overexpression or
knockdown (MOI = 10, virus titer of 1 × 10^8 TU/mL) was added
separately to the cell culture medium for 48 hours, followed by
screening stable cell lines with 2 µg/mL puromycin (UC0E03,
Sigma-Aldrich, USA) for 2 weeks^[186]74–[187]77.
The cell transfection groups were as follows: sh-NC group (mouse
colonic epithelial cells without BCL2 knockdown), sh-BCL2 group (mouse
colonic epithelial cells with BCL2 knockdown), oe-NC group (mouse
colonic epithelial cells without BCL2 overexpression), and oe-BCL2
group (mouse colonic epithelial cells with BCL2 overexpression).
Construction and grouping of the CUMS model in mice
Male C57BL/6 J mice weighing 20-25 grams and aged 8-10 weeks were
purchased from Hunan Siley Jingda Experimental Animal Co., Ltd., in
Hunan, China. The mice were housed individually in cages in an
SPF-grade animal facility maintained at a humidity of 60%-65% and a
temperature of 25 ± 2 °C, with a 12-hour light-dark cycle and ad
libitum access to food and water. After one week of acclimation, the
mice were observed for their health status before the start of the
experiment. The experimental procedures and animal usage were approved
by the Animal Ethics Committee^[188]78.
The mice were divided into a control group and a CUMS group. In the
CUMS group, mice were subjected to the following mild stressors
(Fig. [189]S7) individually: (i) 24-hour fasting; (ii) 24-hour water
deprivation; (iii) 1-hour empty bottle exposure; (iv) 7-hour cage tilt
(45 °); (v) overnight illumination; (vi) 24-hour living in dirty cages
(bedding with 100 g sawdust and 200 mL water); (vii) 30 minutes of
forced swimming at 8 °C; (viii) 2 hours of restraint; (ix) 24-hour
exposure to a novel object (such as a piece of plastic). These stress
stimuli were randomly applied over a span of 6
weeks^[190]79,[191]80.After successful modeling with CUMS, behavioral
tests such as the FST and SPT were conducted.
The grouping information of the mice in the modeling experiment is as
follows (6 mice per group): Control group (non-modeled control mice);
CUMS group (mice modeled with CUMS); DMSO group (mice modeled and
treated with DMSO); GP100 group (mice modeled and treated with
100 mg/kg GP); SSRI group (mice modeled treated with fluoxetine at a
dosage of 20 mg/kg)；CUMS + FMT group (CUMS modeled mice subjected to
FMT with 200 µL of fecal pellets from control mice); DMSO + FMT-GP
group (DMSO-treated mice subjected to FMT with 200 µL of fecal pellets
from GP100 group); oe-NC + DMSO group (mice injected with lentivirus
constructed with oe-NC vector and intraperitoneally injected with
10 mg/kg of DMSO); oe-BCL2 + DMSO group (mice injected with lentivirus
constructed with oe-BCL2 vector and intraperitoneally injected with
10 mg/kg of DMSO); oe-BCL2 + Dor group (mice injected with lentivirus
constructed with oe-BCL2 vector and intraperitoneally injected with
10 mg/kg of Dorsomorphin); oe-NC + DMSO + FMT-BCL2 group (mice from
oe-NC + DMSO group subjected to FMT with 200 µL of fecal pellets from
oe-BCL2 + DMSO group); oe-BCL2 + Dor + FMT-BCL2 group (mice from
oe-BCL2 + Dor group subjected to FMT with 200 µL of fecal pellets from
oe-BCL2 + DMSO group); NCC group (mice treated with 100 mg/kg NCC by
gastric lavage); NCC-GP group (mice treated with 100 mg/kg NCC-GP by
gastric lavage); DMSO + FMT-NCC group (DMSO-treated mice subjected to
FMT with 200 µL of fecal pellets from NCC group); DMSO + FMT-NP group
(DMSO-treated mice subjected to FMT with 200 µL of fecal pellets from
NCC-GP group). GP (HY-N0009, MedChemExpress, USA) was dissolved in DMSO
(D2650, Sigma-Aldrich, USA) to prepare a 10 μM stock solution. The
dosage of GP was based on previous studies demonstrating its
antidepressant effects in mouse models^[192]81. The fluoxetine dosage
followed established research, with fluoxetine hydrochloride obtained
from Dar Al Dawa Pharmaceuticals (Jordan). Fluoxetine hydrochloride was
dissolved in 0.03% sodium carboxymethylcellulose and administered daily
by gastric gavage at a dose of 20 mg/kg^[193]82. Sixty minutes before
CUMS induction, mice were orally gavaged with a dose of 10 mL/kg once
daily, Lentiviruses overexpressing BCL2 were constructed and packaged
by Genechem (Shanghai, China), with LV-PDGFRA as the viral gene
overexpression vector. Packaging plasmids (1 µg) and target plasmid
(1 µg) were co-transfected into 293 T cells in a 6-well plate (80-90%
cell confluence) using Lipofectamine 2000 (11668500, Invitrogen, USA)
at a ratio of 1:2. After 48 hours of culture, the supernatant
containing viral particles was collected and centrifuged. The viral
titer was subsequently determined. Mice in the lentivirus-transfected
groups were injected with 1 × 10^8 TU/mL lentivirus into the colon
tissue to establish overexpression or knockdown models, with two
injections per week.
Behavioral tests were conducted six weeks after Chronic Unpredictable
Mild Stress (CUMS) induction to confirm the successful establishment of
the CUMS mouse model. Mice were deeply anesthetized with an appropriate
concentration of isoflurane gas administered through a breathing mask.
Blood samples were collected via retro-orbital puncture following
cervical dislocation, and serum was separated by centrifugation at
4 °C, 2500 rpm for 10 minutes, then stored at -80 °C. Subsequently, the
brain and hippocampal tissues were collected, colonic tissues were
dissected, frozen in liquid nitrogen, and stored at
-80 °C^[194]52,[195]79.
Tail suspension test (TST)
Each mouse had its tail taped to a position 60 centimeters above the
ground and was filmed for 6 minutes. To prevent climbing behavior
during the test, a transparent cylindrical obstacle with a diameter of
1.5 cm and a length of 4 cm (XinRuan Technology) was placed on the tail
of each mouse. An observer blinded to the mouse groups recorded the
total immobility time of each mouse during the entire 6 minutes
(passively suspended without any movement)^[196]80,[197]83.
Sucrose preference test (SPT)
Initially, animals were trained to drink a 1% sucrose solution from two
different bottles (conducted 48 hours before the formal experiment).
After 24 hours, the animals were given free access to a 1% sucrose
solution and water from the two different bottles. To prevent side
preference, the positions of the two bottles were switched. The
consumption of sucrose from the two bottles was measured in the
following 24 hours, and the sucrose preference (%) was calculated using
the formula: Sucrose Consumption / (Sucrose Consumption + Water
Consumption) × 100%^[198]80,[199]84.
FST
Mice were placed in a cylindrical acrylic tank filled with water at a
temperature of 23 ± 1 °C and a depth of 13 cm (diameter 10 cm, height
20 cm). The mice were forced to swim for 6 minutes, and the immobility
time during the last 5 minutes was recorded using the Visutrack motion
tracking system from Shanghai Xinhua Software Information Technology
Co., Ltd.^[200]80.
MWM
The MWM utilized in this study consists of a circular water tank with a
diameter of 120 cm, filled with water maintained at a temperature of
22 ± 2 °C. The platform within the maze is utilized as a spatial
reference point and is surrounded by walls bearing distinct shapes. A
camera mounted above the maze records the swimming paths of the mice as
they navigate through the water maze. During the acquisition phase
trials, the platform is submerged 1-2 cm below the water’s surface,
with mice placed in one of the cardinal directions (north, south, east,
or west) facing the walls of the tank. Mice are given 60 seconds to
locate the platform, and if unsuccessful, they are guided to it and
allowed to remain there for 10 seconds. Four trials are conducted each
day, separated by at least one hour, with the escape latency for
spatial memory acquisition recorded in each trial. On the 7th day, the
platform is removed for a probe test (Fig. [201]S8), where the time
spent in each quadrant, the number of crossings in the target
(platform) area, average speed, and total distance covered are
measured^[202]80.
Histopathological staining techniques
Hematoxylin and eosin (H&E) staining: Mouse hippocampal or colon tissue
samples were fixed, sectioned, dewaxed in xylene, rehydrated in 100%,
95%, and 70% ethanol, and then embedded or rinsed in water. The
prepared sections were stained with hematoxylin staining solution
(H8070, Solarbio, Beijing) for 5-10 minutes at room temperature,
followed by rinsing with distilled water, dehydration in 95% ethanol,
and immersion in eosin staining solution (G1100, Solarbio, Beijing) for
5-10 minutes. The sections were dehydrated, cleared, and
coverslipped^[203]85.
Nissl staining: Intact hippocampal tissue sections were air-dried at
room temperature, fixed for 30 seconds, placed in cresyl violet
staining solution (C0117, Beyotime Biotechnology Co., Ltd, Shanghai)
for 1 hour in the dark, sequentially differentiated in 70%, 80%, and
95% ethanol (10 seconds each), followed by staining differentiation
solution (ethanol: chloroform: diethyl ether = 1:1:1) until clear
differentiation was observed. The sections were immersed twice in
ethanol (5 minutes each), permeated twice in dichloromethane (4 minutes
each), and mounted in Canada balsam. Neuronal counts were conducted
using a fluorescence microscope (BX63, Olympus).
TUNEL staining: Frozen tissue sections were immersed in DMSO and
incubated in 200 mL citrate buffer (0.1 mL, pH 6.0), heated in a
microwave at 90-95 °C for 1 minute at 680 W (80% power), cooled by
adding 80 mL double-distilled water (20-25 °C). Subsequently, the
sections were incubated in 20% normal bovine serum at room temperature
for 30 minutes, followed by a 90-minute incubation with 50 μL TUNEL
reaction mixture (negative control without TUNEL reaction mixture). The
sections were then incubated at room temperature for 10 minutes with 3%
H[2]O[2], followed by a 90-minute incubation at 37 °C, incubated for
30 minutes at 37 °C with peroxidase (POD) (50 μL per section),
visualized using diaminobenzidine (DAB)/H[2]O[2], counterstained with
hematoxylin, dehydrated, cleared, and mounted in neutral balsam. Cell
observations were made using a fluorescence microscope. The number of
TUNEL-positive cells with dark brown nuclear staining was counted, and
the apoptotic cell count was determined^[204]86.
High-throughput sequencing and analysis
Three samples of colonic epithelial tissue were randomly selected from
mice in the DMSO treatment group and three from the 100 mg/kg GP
treatment group. Total RNA was extracted using the Total RNA Isolation
Kit (12183555, Invitrogen, USA). The total RNA OD value was measured
quantitatively using a UV spectrophotometer (BioSpectrometer basic,
Eppendorf, USA), and RNA integrity was assessed via agarose gel
electrophoresis. High-quality total RNA was reverse-transcribed to cDNA
to construct an RNA library, and sequencing was performed on Illumina’s
NextSeq 500. The raw image data obtained from sequencing were converted
to raw reads through base calling. For quality control, sequencing
adapters were removed and low-quality sequences were filtered out using
cutadapt, leaving “clean reads.” These reads were aligned to the mouse
reference genome, followed by gene expression quantification using the
R package to obtain a gene expression matrix^[205]87.
The “limma” package in R was used to identify differentially expressed
genes in the high-throughput sequencing data, using |log2FC | > 2 &
P.value < 0.05 as the selection criteria. Volcano and heatmaps were
created using the ggplot2 and pheatmap packages,
respectively^[206]88–[207]90.
Protein sample preparation and analysis in proteomics
Colonic epithelial tissue samples from three mice in the DMSO-treated
group and three from the 100 mg/kg GP-treated group were ground in a
mortar with liquid nitrogen and transferred to a 5 cm³ centrifuge tube.
Subsequently, ultrasonication was performed using an ultrasonic cell
disruptor (SCIENTZ-IID, Scientz, Ningbo) under ice-cold conditions. The
processing medium consisted of phenol extraction buffer containing
10 mM DTT (R0861, Solarbio, Beijing), a 1% proteinase inhibitor
cocktail (P6731, Solarbio, Beijing), and 2 mM EDTA (E1170, Solarbio,
Beijing). This step was repeated 8 times. Afterward, an equal volume of
Tris-saturated phenol (HC1380, BIOFOUNT, Beijing) at pH=8.0 was added,
followed by vortex mixing for 4 minutes. The mixture was then
centrifuged at 5000 g for 10 minutes at 4 °C, and the upper layer of
phenol was transferred to a new centrifuge tube. Ammonium sulfate
(0.1 M, 101217, Sigma-Aldrich, USA) saturated methanol (106035,
Sigma-Aldrich, USA) was added to the phenol solution in a volumetric
ratio of 1:5 and left overnight to precipitate the proteins. After
centrifugation at 4 °C for 10 minutes, the supernatant was removed. The
remaining precipitate was washed once with ice-cold methanol and three
times with ice-cold acetone. The washed proteins were then re-dissolved
in 8 M urea (U8020, Solarbio, Beijing), and the protein concentration
was determined using a BCA assay kit (P0012, Beyotime Biotechnology
Co., Ltd, Shanghai)^[208]91. The procedures were carried out according
to the manufacturer’s instructions.
Protein digestion and mass spectrometry analysis workflow
Each sample received 50 µg for enzymatic digestion. Initially, the
protein solution was mixed with DTT to a concentration of 5 mM and left
at 56 °C for 30 minutes. Subsequently, acrylamide was added to reach a
concentration of 11 mM and left at room temperature for 15 minutes.
Finally, the urea concentration in the samples was diluted below 2 M,
followed by the addition of trypsin (25200056, Thermo Fisher, USA) at a
ratio of 1:50 (w/w) for overnight digestion at 37 °C. Subsequently,
trypsin was added again at a ratio of 1:100 (trypsin: protein) for
another 4 hours of digestion.
After digestion, peptide desalting was performed using the HyperSep™
C18 purification column (60108-302, Thermo Fisher, USA), followed by
vacuum drying. The peptides were reconstituted in 0.5 M TEAB (90114,
Thermo Fisher, USA) and processed according to the manufacturer’s
instructions of the TMT reagent kit (90064CH, Thermo Fisher, USA). In
brief, a unit of TMT reagent was thawed and dissolved in acetonitrile
(113212, Sigma-Aldrich, USA). The peptide mixture was then incubated at
room temperature for 2 hours, desalted and dried using a vacuum
centrifuge. Equal amounts of labeled peptides from each group were
mixed and subjected to hierarchical separation using the Pierce™
High-pH Reversed-Phase Peptide Isolation Kit (84868, Thermo Fisher,
USA). Subsequently, the samples were collected and combined into 15
fractions, and the dried peptides in each fraction were dissolved in
0.1% formic acid (159002, Sigma-Aldrich, USA).
Each sample was prepared by taking 2 µg of peptide and separated using
the nano-UPLC liquid chromatography system Easy nLC 1200 (Thermo
Fisher, USA). The samples were first pre-treated using a Trap C18
column (100 µm × 20 mm, 5 µm), followed by gradient separation using a
C18 analytical column (75 µm × 150 mm, 3 µm) at a flow rate of 300
nL/min. The mobile phase A consisted of a 0.1% formic acid aqueous
solution, while mobile phase B consisted of a 0.1% formic acid aqueous
solution with 95% acetonitrile. The gradient elution process included:
0-2 minutes, 2%-8% B; 2-71 minutes, 8%-28% B; 71-79 minutes, 28%-40% B;
79-81 minutes, 40%-100% B; 81-90 minutes, 100% B. The liquid
chromatography-separated peptides were analyzed by a Q-Exactive HF-X
mass spectrometer (Thermo Fisher, USA). The analysis duration was
60 minutes, with an electrospray voltage set at 2.1 kV, operating in
positive ion mode, with a precursor ion scan range of 350-1200 m/z and
a primary mass resolution of 60,000 at m/z 200. The AGC target was set
at 3e6, and the maximum injection time for the primary scan was 30 ms.
The secondary mass resolution was 15,000 @ m/z 200 with an AGC target
of 1e6 and a maximum injection time of 25 ms. The MS2 activation type
was set as HCD, with an isolation window of 20 Th and a normalized
collision energy of 32^[209]91,[210]92.
Database retrieval and data processing
The LC-MS/MS data obtained were processed using MaxQuant software
(v1.5.2.8), which involved peptide identification and protein
quantification. The UniProt 14.1 database (2009)
([211]https://www.uniprot.org/) was utilized for tandem mass
spectrometry retrieval in conjunction with a decoy database. Trypsin/P
was designated as the cleaving enzyme, allowing for up to 2 missed
cleavages. The initial search was set at 20 ppm, while the main search
was set at 5 ppm with a fragment ion mass tolerance of 0.02 Da.
Criteria for peptide false discovery rate (FDR) ≤ 0.01, protein
FDR ≤ 0.01, and peptide score distribution were established for
database qualification. Differential expression proteins (DEPs) between
colonic tissue samples were screened using the “limma” package in R
software (with thresholds set at |log2FC | > 2 and
P.value < 0.05)^[212]91,[213]93.
PPI network analysis
DEPs identified in colonic tissue samples were utilized to construct
PPI networks by importing them into the STRING database
([214]https://cn.string-db.org/) and further analyzed using Cytoscape
v3.10.0 software to generate the protein interaction network
diagram^[215]94.
MTT assay for assessing colonic epithelial cell viability
The MTT assay involved seeding the cells onto a 96-well cell culture
plate at a density of 3-5 × 10^4 cells/mL and then culturing them for
48 hours. Subsequently, MTT solution (10 mg/mL, ST316, Beyotime
Biotechnology Co., Ltd, Shanghai, China) was added to the cell
suspension and incubated for 4 hours, followed by the addition of
dimethyl sulfoxide (DMSO) with shaking for 10 minutes. Absorbance at
490 nm was measured using a spectrophotometer (Laspec, China). Cell
viability was calculated as (ΔA_sample - ΔA_blank)/(ΔA_control -
ΔA_blank), where ΔA_sample represents the absorbance difference of the
sample, ΔA_blank is the absorbance difference of the blank, and
ΔA_control is the absorbance difference of the control group^[216]95.
EdU experiment
Colonic epithelial cells were seeded in a 24-well plate and incubated
in a culture medium containing EdU ([217]C10337, Invitrogen, USA) at a
concentration of 10 µmol/L for 2 hours in a CO[2] incubator.
Subsequently, the culture medium was removed, and the cells were fixed
with a DMSO solution containing 4% paraformaldehyde at room temperature
for 15 minutes. The cells were then washed twice with DMSO containing
3% BSA, permeabilized at room temperature with DMSO containing 0.5%
Triton X-100 for 20 minutes, followed by another two washes with DMSO
containing 3% BSA. 100 µL of staining solution was added to each well
and incubated at room temperature in the dark for 30 minutes. DAPI
nuclear staining was performed for 5 minutes, and the slides were then
observed using a fluorescence microscope (BX63, Olympus, Japan) to
randomly examine 6-10 fields of view and record the number of positive
cells in each field. The EdU labeling rate (%) was calculated as the
number of positive cells divided by the sum of positive and negative
cells multiplied by 100%^[218]96. Each experiment was repeated three
times.
TUNEL staining
Colonic epithelial cells from each group were fixed with 4%
paraformaldehyde at room temperature for 15 minutes, followed by
permeabilization with 0.25% Triton X-100 for 20 minutes. Samples were
then blocked with 5% bovine serum albumin (BSA, 36101ES25, Yeasen
Biotechnology (Shanghai) Co., Ltd., China) and stained with TUNEL
(C1086, Beyotime Biotechnology Co., Ltd, Shanghai, China) reagent.
Subsequently, the sections were counterstained in the dark with a DAPI
staining solution (C1002, Beyotime Biotechnology Co., Ltd, Shanghai,
China). Apoptotic cells were visualized under a confocal microscope
(LSM 880, Carl Zeiss AG, Germany). TUNEL-positive cells (green
fluorescence) indicated apoptotic cells, while DAPI labeled the cell
nuclei, with blue fluorescence representing the total cell count. Five
different fields were selected from each group to calculate the
percentage of apoptotic cells, which is calculated as the number of
apoptotic cells divided by the total cell count multiplied by
100%^[219]97.
Substance permeability assessment (SPE)
To establish a single-cell barrier model, 0.3 × 10^5 murine colonic
epithelial cells were suspended on polycarbonate membranes of transwell
chambers (3413, Corning, USA) and cultured at 37 °C with 5% CO[2] for
21 days. The transepithelial electrical resistance (TEER) of the
monolayer was measured using the Millicell-ERS resistance system
(Millipore, Billerica, MA, USA), and after 21 days of cultivation, the
average TEER value exceeded 400 Ω·cm^2 (Fig. [220]S9). Induction of
barrier dysfunction in the single-cell barrier was achieved by treating
with 100 μg/mL lipopolysaccharide (LPS) for 48 hours, followed by
replacing the culture medium in the upper and lower chambers with a
serum-free medium. Fluorescein isothiocyanate (FITC)-labeled dextran
(70 kDa, 2 mg/mL; FD70, Sigma-Aldrich, USA) was added to the upper
chamber, and the concentration of albumin-FITC in the lower chamber was
measured at 495 nm wavelength using an ELISA^[221]98.
Preparation of GP and NCC composite (NCC-GP)
GP was dissolved in 10 mM phosphate buffer (AM9624, Invitrogen, USA) at
pH 7.4. A 1.5 mL GP solution was added to a 2 mL microcentrifuge tube
containing a 4 mg/mL suspension of NCC (C909405, MACKLIN, China), and
the mixture was stirred at 37 °C with a stirring speed of 8 revolutions
per minute for 30 minutes. The suspension, containing phosphate buffer,
formed a flocculated NCC/GP suspension, which was centrifuged at
18000 g for 10 minutes to precipitate the GP and NCC composite. The
concentration of unbound GP ([drug[unbound]]) in the supernatant was
determined using a Varian 50 Bio UV Vis spectrophotometer (Varian,
Inc., Canada) at wavelengths of 482 nm and 364 nm. The concentration of
GP bound to NCC ([drug[bound]]) was calculated using the formula:
[MATH: [drugbound]=[drugadded]−[drugunbound]
:MATH]
^[222]54.
Characterization of NCC physicochemical properties
The zeta potential of NCC in phosphate buffer was measured in an
Eppendorf UVette centrifuge tube using Zeta Plus (Brookhaven
Instruments Corp., USA) at a scattering angle of 90 °. Three
measurements were conducted. The structure and morphology of NCC were
examined using scanning electron microscopy (JEOL 6700, JEOL, Japan).
The method for scanning electron microscopy examination involved
dropping the NCC suspension onto a clean silicon wafer to form a small
droplet (approximately 5 μL), followed by overnight drying for
preparation^[223]99.
Drug release study
For the release study, the NCC samples loaded with the drug were
resuspended in PBS and incubated at 37 °C with a speed of 8 rpm. At
specified time points, the suspension was centrifuged at 18,000 g for
10 minutes to remove the supernatant, and drug quantification was
performed by measuring GP using UV-Vis spectroscopy or high-performance
liquid chromatography (HPLC). The amount of unbound drug in the
supernatant was determined by HPLC using a Waters HPLC system equipped
with Millennium software and UV-visible detection (Waters Ltd, Canada).
A Novapak C18 column with an injection volume of 20 μL, a flow rate of
1 mL/min, and a mobile phase consisting of acetonitrile and formic acid
water (containing 0.05% formic acid) was used for separation. Detection
was performed at 273 nm. At each sampling point, fresh PBS was added to
the tube, and the NCC/GP nanocomplex was resuspended^[224]54,[225]61.
Study on cell binding and uptake
The test cells were seeded at a concentration of 10,000 cells/well in a
96-well cell culture plate and allowed to equilibrate overnight. A
suspension of 2 mg NCC-GP in a 5 mm NaCl solution was prepared and
incubated at 37 °C for 30 minutes. The suspension was centrifuged at
18000 g for 30 seconds to remove the supernatant. The pellet was
resuspended in a 5 mm NaCl solution containing 100 μg of fluorescein
(HY-D0251, MedChemExpress, USA) per milliliter and then further
incubated at 37 °C for 30 minutes. After another centrifugation at
18000 g for 30 seconds and removal of the supernatant, the pellet was
resuspended in DMSO and diluted to obtain a 10 mg/mL sample.
Subsequently, 200 μL of NCC-GP suspension was added to each well of the
cell culture plate, and the cells were allowed to incubate for
24 hours. The culture medium was removed, and the cells were washed
three times with PBS, followed by lysis with 2% Triton X-100. The
fluorescence intensity of the bound NCC-GP/fluorescein on the cells was
measured using a fluorescence spectrophotometer with excitation and
emission wavelengths of 480 and 520 nm, respectively. These
fluorescence intensity values were converted to the mass of bound
NCC-GP/fluorescein on the cells using a standard curve previously
constructed. The BCA total protein analysis kit (P0012, Beyotime
Biotechnology Co., Ltd., Shanghai, China) was used to ensure an equal
number of cells in each well.
For the observation of cell uptake of NCC, the test cells were cultured
following the binding study protocol. Disinfected glass coverslips with
a diameter of 1 cm were placed at the bottom of a 6-well tissue culture
plate, and 50,000 cells were added to each well and incubated overnight
under culture conditions. The cells were then incubated at 37 °C with
a suspension of 1.25 mg/mL NCC/cetyltrimethylammonium bromide
(CTAB)/fluorescein for varying durations. The coverslips were removed,
washed three times in PBS, treated with DAPI dye for 30 minutes, washed
again, and then treated with a 3.7% formaldehyde solution in deionized
water for 30 minutes. The coverslips with the cells facing down were
mounted on glass microscope slides and sealed with transparent nail
polish. Visualization of the fluorescein and DAPI-stained cell nuclei
was performed using an FV-1000 laser confocal scanning microscope
(Olympus, Japan)^[226]54.
The subsequent cell experimental groups were as follows: (1) DMSO + Nor
group: untreated normal colonic epithelial cells cultured with 200 μL
of DMSO solution; (2) NCC + Nor group: untreated normal colonic
epithelial cells cultured with 200 μL of 10 mg/mL NCC solution; (3)
NCC-GP + Nor group: untreated normal colonic epithelial cells cultured
with 200 μL of 10 mg/mL NCC-GP solution; (4) DMSO group: LPS-stimulated
colonic epithelial cells cultured with 200 μL of DMSO solution; (5) GP
group: LPS-stimulated colonic epithelial cells cultured with 200 μL of
10 mg/mL GP solution; (6) NCC group: LPS-stimulated colonic epithelial
cells cultured with 200 μL of 10 mg/mL NCC solution; (7) NCC-GP group:
LPS-stimulated colonic epithelial cells cultured with 200 μL of
10 mg/mL NCC-GP solution.
ELISA
Colonic epithelial cells and murine colonic tissue levels of
inflammatory cytokines were determined using ELISA kits purchased from
Invitrogen (USA), as indicated in Table [227]S4^[228]100,[229]101.
RT-qPCR
Total RNA was extracted from tissues or cells using Trizol reagent
(15596026, Invitrogen, USA). Subsequently, the concentration and purity
of the total RNA were assessed at 260/280 nm using NanoDrop LITE
(ND-LITE-PR, Thermo Scientific™, Germany). Next, cDNA was synthesized
from the extracted total RNA using the PrimeScript RT reagent Kit with
gDNA Eraser (RR047Q, TaKaRa, Japan). The expression levels of various
genes were then determined by RT-qPCR using SYBR Green PCR Master Mix
(4364344, Applied Biosystems, USA) and the ABI PRISM 7500 sequence
detection system (Applied Biosystems). The primers for the genes were
synthesized by TaKaRa (Table [230]S5), with GAPDH serving as the
reference gene. Finally, the relative expression levels of the genes
were analyzed using the 2^-ΔΔCt method, where △△Ct = (average Ct value
of the target gene in the experimental group - average Ct value of the
reference gene in the experimental group) - (average Ct value of the
target gene in the control group - average Ct value of the reference
gene in the control group)^[231]102–[232]104. All RT-qPCR analyses were
performed in triplicate.
Western blot
To begin, tissues or cells were collected and lysed using an enhanced
RIPA lysis buffer containing protease inhibitors (P0013B, Beyotime
Biotechnology Co., Ltd, Shanghai, China). The protein concentration was
then determined using a BCA protein quantification kit (P0012, Beyotime
Biotechnology Co., Ltd, Shanghai, China). Subsequently, proteins were
separated by 10% SDS-PAGE and transferred onto a PVDF membrane (FFP39,
Beyotime Biotechnology Co., Ltd). The membrane was blocked with 5% BSA
(ST023, Beyotime Biotechnology Co., Ltd) at room temperature for
2 hours to prevent nonspecific binding, followed by the addition of
diluted primary antibodies and incubation at room temperature for
1 hour. The primary antibodies used were rabbit anti-human (details in
Table [233]S6). After washing the membrane, an HRP-conjugated goat
anti-rabbit secondary antibody (ab6721, 1:2000, Abcam, UK) was added
and incubated at room temperature for 1 hour. Pierce™ ECL Western
Blotting Substrate (32209, Thermo Scientific™, Germany) A and B
solutions were mixed in equal amounts in a dark room, added to the
membrane, and exposed in a gel imaging system. Images were captured
using a Bio-Rad imaging system (BIO-RAD, USA) and the bands in the
Western blot images were quantified for grayscale using Image J
analysis software, with GAPDH as an internal control^[234]103. Each
experiment was repeated 3 times.
Assessment of intestinal barrier integrity
The test is based on examining the integrity of the intestinal barrier
using 4 kD fluorescein isothiocyanate-dextran (FD4, Sigma-Aldrich).
After 4 hours of fasting, mice were orally administered FITC-dextran
(0.6 g per kg body weight). One hour later, 200 μL of blood was
collected into Microvette tubes (Sarstedt, France, Marnay). The tubes
were then centrifuged at 10,000 g for 5 minutes at room temperature to
extract serum. The collected serum was diluted with an equal volume of
DMSO and analyzed for FITC concentration using an excitation wavelength
of 485 nm and an emission wavelength of 535 nm. All analyses were
conducted under blinded experimental conditions^[235]45.
FMT
Recipient mice were provided with a synthetic antibiotic combination in
their drinking water for six consecutive days, including vancomycin
(0.5 g/L; V2002), ampicillin (1 g/L; 171254), streptomycin (5 g/L;
85886), neomycin (1 g/L; N6386), and metronidazole (0.5 g/L; M1547),
all sourced from Sigma-Aldrich. After 24 hours, the animals were orally
administered the microbial inoculum twice, with a three-day interval
between administrations. The inoculated animals were housed in separate
sterile isolators. Fresh fecal pellets were collected directly from the
donor mouse’s rectum, homogenized in 1 mL of sterile phosphate-buffered
saline (DMSO) containing 100 mg (approximately 5-6 fecal pellets). The
homogenate was then filtered through a 20 μm nylon filter to remove
large particles and fibrous materials. The solution was collected and
administered to recipient mice orally in a volume of 200 µL to minimize
microbial content changes within 15 minutes. Prior to any analysis, the
recipient animals were housed in isolators for eight weeks. The choice
of an eight-week delay between FMT and analysis was made because the
C57BL/6 J mouse strain is considered to have relatively strong stress
resistance and requires at least eight weeks to exhibit depression-like
behavior (Fig. [236]S10)^[237]79.
Immunohistochemistry (IHC) staining
For a list of antibodies, please refer to Table [238]S7. Mouse tissues
were fixed with 4% paraformaldehyde overnight and then processed for
paraffin embedding with a slice thickness of 4 μm, followed by
deparaffinization in xylene. The tissue sections were rehydrated in a
series of graded ethanol washes (absolute ethanol, 95% ethanol, and 75%
ethanol for 3 minutes each). Antigen retrieval was achieved by boiling
the sections in 0.01 M citrate buffer for 15-20 minutes, followed by
quenching endogenous peroxidase activity at room temperature for
30 minutes with 3% H[2]O[2]. The sections were then blocked with goat
serum blocking solution at room temperature for 20 minutes, excess
liquid was removed, and primary antibodies were applied and left to
incubate at room temperature for 1 hour. Subsequently, the sections
were washed with DMSO and incubated with a secondary IgG goat
anti-mouse antibody at 37 °C for 20 minutes, followed by washing with
DMSO and incubation with SP (streptavidin-peroxidase) at 37 °C for
30 minutes. After washing with DMSO, the sections were developed using
DAB (P0202, Beyotime Biotechnology) for 5-10 minutes, followed by a
10-minute water rinse to stop the reaction. Counterstaining was
performed with hematoxylin (C0107, Beyotime Biotechnology) for
2 minutes, differentiation with acid alcohol, a 10-minute water rinse,
dehydration in graded alcohols (xylene clearing), and mounting with 2-3
drops of neutral resin. Observations and counting were done under a
bright-field microscope: 5 high-power fields per section were randomly
selected, with 100 cells counted per field, to determine the
Ki67-positive cell percentage^[239]105.
Statistics and reproducibility
Our study utilized the R statistical programming language, version
4.2.1, with code compilation facilitated through the integrated
development environment RStudio (version 2022.12.0-353). For file
processing, Perl programming language version 5.30.0 was employed.
Additionally, data analysis involved the utilization of GraphPad Prism
software (version 8.0).
Descriptive statistics were presented using mean ± standard deviation
for continuous variables. Group comparisons were conducted using an
independent samples t-test^[240]106. Analysis of variance (ANOVA) was
employed for comparing data among different groups, while two-way ANOVA
was utilized for assessing data variations across different time
points. Post-hoc testing was performed using Bonferroni adjustment. The
threshold for statistical significance was set at P < 0.05^[241]107.
For animal experiments, each group consisted of at least six mice
(n = 6 per group), determined based on power analysis to ensure
sufficient statistical significance. Behavioral tests were conducted in
biological triplicates, considering each mouse as an independent
biological replicate. In vitro experiments, including cellular assays,
Western blot, RT-qPCR, and ELISA, were performed in technical
triplicates and repeated in at least three independent biological
replicates (n ≥ 3). Details of sample sizes and statistical tests
applied for specific experiments are provided in the figure legends and
Methods section.
To ensure reproducibility, all experiments were performed independently
at least three times, and results were verified across different
experimental batches. Blinding was applied where applicable,
particularly in behavioral assessments and histological analyses, to
minimize bias. Data consistency was evaluated across independent
experiments, ensuring robust and reproducible findings.
Reporting summary
Further information on research design is available in the [242]Nature
Portfolio Reporting Summary linked to this article.
Supplementary information
[243]Supplementary Information^ (3.4MB, pdf)
[244]Reporting summary^ (1.6MB, pdf)
Acknowledgements