ABSTRACT
Non‐alcoholic steatohepatitis (NASH) poses a serious threat to human
health. Alisol B 23‐Acetate (AB23A) has shown beneficial effects on
NASH, but its mechanism of action remains unclear. We conducted
in vitro experiments by inducing L02 cell damage with free fatty acids
(FFA) and administering various concentrations of AB23A. We found that
AB23A intervention reduced triglyceride (TG) levels in FFA‐induced L02
cells and improved cellular steatosis. Transcriptomic analysis revealed
that AB23A intervention significantly downregulated glucose‐regulated
protein 94 (Grp94), indicating that AB23A primarily regulates the
protein processing pathway in the endoplasmic reticulum. Within this
pathway, AB23A intervention also significantly downregulated
endoplasmic reticulum stress (ERS)‐related genes (PERK, eIF2α, ATF4)
and ER‐associated degradation (ERAD)‐related genes (FBXO2, DERL,
HSP90AA1). When we silenced GRP94, the regulatory effects of AB23A on
TG levels, cellular steatosis, ERS‐related proteins (p‐PERK/PERK,
p‐eIF2α/eIF2α, ATF4), and ERAD‐related proteins (FBXO2, DERL, HSP90α)
disappeared. In vivo, AB23A intervention promoted recovery of the liver
index in NASH mice, reduced hepatic inflammatory infiltration and lipid
deposition, improved serum alanine aminotransferase (ALT) and aspartate
aminotransferase (AST) activities, and reduced liver TG levels. RT‐qPCR
and Western blot results demonstrated that AB23A intervention
dose‐dependently downregulated the gene and protein expression of GRP94
and ERS‐ and ERAD‐related factors. There was no significant difference
between the effects of high‐dose AB23A intervention and PPC
intervention. This study demonstrated, through both in vitro and
in vivo experiments, that AB23A improves hepatic steatosis. This effect
may be related to the downregulation of GRP94, which suppresses ERS and
ERAD, thereby restoring ER homeostasis.
Keywords: Alisol B 23‐acetate, endoplasmic reticulum stress,
ER‐associated degradation, GRP94, non‐alcoholic steatohepatitis,
transcriptomics
__________________________________________________________________
Alisol B 23‐Acetate (AB23A) alleviated hepatic injury and lipid
deposition in MCD diet‐induced non‐alcoholic steatohepatitis (NASH)
mice. Alisol B 23‐Acetate reduced liver cell damage by restoring
endoplasmic reticulum homeostasis. AB23A down‐regulated GRP94,
suppressing ER stress and ER‐associated degradation (ERAD).
graphic file with name FSN3-13-e70086-g006.jpg
1. Introduction
There is an increasing prevalence of worldwide obesity and metabolic
syndrome. Non‐alcoholic fatty liver disease (NAFLD) is one of the most
common chronic liver diseases globally, with its incidence now over 25%
(Wei et al. [48]2024). NAFLD encompasses multiple stages, ranging from
simple fatty liver to non‐alcoholic steatohepatitis (NASH) and can
progress to advanced liver fibrosis and hepatocellular carcinoma. NASH,
a more severe form of NAFLD, serves as a critical intermediate stage in
the progression from NAFLD to liver fibrosis and liver cancer.
Approximately 20% of NAFLD patients will develop NASH, and alarmingly,
about 20% of NASH patients will progress to liver cirrhosis (Povsic
et al. [49]2019; Sheka et al. [50]^2020). Given the serious threat NASH
poses to human health, in‐depth research on the mechanisms underlying
NASH development is crucial, especially for the search for effective
intervention drugs.
Hepatocytes are rich in endoplasmic reticulum (ER), an essential
organelle involved in the synthesis and processing of numerous proteins
and lipids. During the development of NASH, abnormal lipid accumulation
can disrupt ER function in hepatocytes, leading to the buildup of
unfolded or misfolded proteins in the ER. This triggers the cell's
defense mechanism, causing ER stress (ERS) and activating the unfolded
protein response (UPR). As a result, protein synthesis is blocked, and
ER‐associated degradation (ERAD) is induced (Ajoolabady
et al. [51]2023). A randomized controlled trial revealed that severe ER
stress exists in the livers of NASH patients, and promoting the
restoration of ER homeostasis can effectively improve liver function
(Okada et al. [52]2018). Consequently, restoring ER homeostasis has
become a crucial target for controlling NASH progression.
Glucose‐regulated protein 94 (Grp94) is a principal molecular chaperone
protein in the ER and a member of the HSP90 family, which is involved
in the folding and quality control of various proteins (Xu
et al. [53]2024). It plays a significant role in the development of
NASH, with studies indicating its activation in NASH mouse models
(Lebeaupin et al. [54]2018; Li et al. [55]^2020). Notably, The
regulation of GRP94 is crucial for maintaining the homeostasis of the
ER (Marzec et al. [56]2012).
Natural products hold great potential in restoring ER homeostasis.
Studies have shown that artemisinin (Yin et al. [57]2021), curcumin
(Zhou et al. [58]2021), and ginsenoside Rb1 (Shaukat et al. [59]2021)
can inhibit ERS‐related proteins, particularly protein kinase‐like ER
kinase (PERK) phosphorylation, to alleviate ERS. Alisol B 23‐Acetate
(AB23A), an important active ingredient in the traditional Chinese
medicine Alisma orientale (Sam.) Juzep., has demonstrated beneficial
effects on NASH (Li et al. [60]2024). Furthermore, Zexie decoction, a
traditional Chinese medicine formula with Alisma orientale (Sam.)
Juzep. as its main ingredient, has been widely used in treating NAFLD
and effectively protecting liver function (Li et al. [61]2024).
However, the precise mechanism by which AB23A exerts its therapeutic
effects on NASH remains to be explored.
In this study, we first conducted in vitro experiments using
FFA‐induced L02 cells and administered different concentrations of
AB23A to evaluate its effect on improving cellular steatosis. Based on
these findings, we hypothesized that AB23A improves NASH by restoring
ER homeostasis. To validate this hypothesis, we evaluated the effects
of AB23A on ERS‐ and ERAD‐related factors after silencing GRP94.
Furthermore, we conducted in vivo experiments to verify the anti‐NASH
effects of AB23A.
2. Materials and Methods
2.1. Materials
The comprehensive information regarding reagents, kits, antibodies, and
other materials utilized in this study is provided in Appendix [62]S1.
2.2. In Vitro Experiments
2.2.1. Cell Culture
L02 cells were provided by iCell Bioscience Inc. (Shanghai, China).
Subsequently, we cultured L02 cells in RPMI‐1640 medium supplemented
with 10% fetal bovine serum, 100 μg/mL penicillin, and 100 μg/mL
streptomycin. The cells were maintained in a constant temperature
incubator with 5% CO[2] at 37°C. Passaging was performed when the cell
density reached 60%–70%.
2.2.2. Cell Transfection
Following cell plating, allow the cells to grow to a confluency rate of
30%–40%. Subsequently, replace the medium with a fresh medium
containing Lipo2000 transfection reagent, siGRP94 plasmid, and the
empty vector (siNC) for a 6‐h incubation period. The sequence of
siNC:siNC: Sense sequence: UUCUCCGAACGUGUCACGUTT, Antisense sequence:
ACGUAGCACGUUCGGAGAATT. The sequence of siGRP94: siGRP94‐1: Sense
sequence: GAAGAAGCAUCUGAUUACC, Antisense sequence: GGUAAUCAGAUGCUUCUUC;
siGRP94‐2: Sense sequence: GGUCAGAGCUGACGAUGAAGU, Antisense sequence:
UUCAUCGUCAGCUCUGACCGA. Afterward, replace the medium with RPMI‐1640
medium supplemented with 10% fetal bovine serum and
penicillin–streptomycin, and continue culturing for 24 h.
2.2.3. MTT Assay
Following the plating of L02 cells, we treated them with varying
concentrations of AB23A (0, 20, 40, 80, and 160 μM) for 24 h.
Subsequently, 10 μL of MTT solution (5 mg/mL) was added to each well.
After a 4‐h incubation period, the medium was discarded, and 100 μL of
DMSO was added. The absorbance of the samples was measured at 490 nm,
and the relative cell viability was calculated based on the absorbance
values.
2.2.4. Establishment of NASH Cell Model
After the cells were plated, they were allowed to grow until cell
confluence reached 80%. Subsequently, the cells were stimulated with
1 mM FFA (palmitic acid: oleic acid at a ratio of 1:2) for 24 h to
establish a NASH cell model. The model was evaluated by measuring the
TG level in the cells and performing Oil Red O staining on the cells.
2.2.5. Transcriptomic Analysis
After plating, the cells were cultured in an incubator for 24 h and
subsequently divided into Control, FFA, and FFA + 80 μM AB23A groups.
These groups underwent further culturing for 24 h. Following this, all
media were removed, and the cells were washed twice with cold PBS.
Total RNA was isolated using TRIzol, and the purity, concentration, and
integrity of the RNA samples were assessed to ensure the utilization of
qualified samples for transcriptome sequencing. After passing quality
control, library construction was performed, and sequencing was
conducted on the Illumina platform. DESeq2 software was employed to
analyze the differentially expressed genes (DEGs) between the control,
FFA, and FFA + 80 μM AB23A groups. Genes with |Log[2](FoldChange)| ≥ 1
and p [adj] ≤ 0.05 were identified as DEGs, and KEGG analysis was
conducted on these DEGs.
2.3. In Vivo Experiment
2.3.1. Animals
We obtained sixty healthy male C57BL/6 mice, aged 6–8 weeks, with a
body weight of approximately 22 g from Si Pei Fu (Beijing)
Biotechnology Co. Ltd. (Production License No.: SYXK (Beijing)
2019–0030). The mice were housed under standard conditions, including a
12‐h light–dark cycle, an ambient temperature of approximately 25°C,
and an ambient humidity of approximately 50%. Food and water were
provided ad libitum. Approval for all animal experiments was obtained
from the Ethical Review Committee of Animal Experiments at Yunnan
University of Chinese Medicine (Approval No.: R‐062024G256).
2.3.2. Modeling, Grouping, and Administration
After a week of adaptive feeding, we randomly assigned the 60 C57BL/6
mice into six groups: Control group, NASH group, Polyene
Phosphatidylcholine (PPC) intervention group, and three AB23A
intervention groups with different doses, namely Low‐dose AB23A
(L‐AB23A), Medium‐dose AB23A (M‐AB23A), and High‐dose AB23A (H‐AB23A).
With the exception of the Control group, all other five groups received
a methionine and choline deficiency (MCD) diet, as established in
previous studies (Li et al. [63]2024). Additionally, the PPC
intervention group received 88 mg/kg/d of PPC via oral administration,
while the low, medium, and high‐dose AB23A intervention groups received
15, 30, and 60 mg/kg/d of AB23A via oral administration, respectively.
The dosage settings for PPC and AB23A were determined based on prior
research (Li et al. [64]2024). The Control and NASH groups were
administered an equal volume of saline daily via gastric gavage as a
parallel control. The administration of drugs continued for 6 weeks,
during which we recorded the mice's body weights weekly. After 6 weeks,
we euthanized the mice and collected blood. The serum was separated by
centrifugation and stored frozen. We promptly removed and weighed the
liver, calculating the liver index. The liver index was calculated
using the following formula: liver index (%) = liver weight (g)/body
weight (g) × 100. Subsequently, the left lateral lobe from identical
locations was fixed for pathological sectioning to observe pathological
damage, while the remaining liver lobes were frozen for RT‐qPCR and
Western blot analysis.
2.4. Biochemical Detection
We employed reagent kits to measure the alanine aminotransferase (ALT)
and aspartate aminotransferase (AST) activities in the serum of mice
from each group. Furthermore, we collected liver tissues and cells
cultured in vitro, normalizing the total protein using the BCA method.
The triglyceride (TG) levels in both liver tissues and cultured cells
were detected using reagent kits.
2.5. Pathological Staining
To assess pathological changes and inflammation in liver tissue, we
prepared paraffin sections of liver tissue, stained them with
hematoxylin and eosin (HE), and observed them under an optical
microscope. For observing the level of lipid deposition in hepatocytes,
we prepared frozen sections of liver tissue, stained them with Oil Red
O, and examined them under an optical microscope. Quantification of Oil
Red O‐positive areas was conducted using ImageJ software.
2.6. RT‐qPCR
We extracted total RNA from liver tissue using a total RNA extraction
kit and measured the RNA concentration. Subsequently, we obtained cDNA
using a reverse transcription kit. We utilized qPCR to determine the
target gene mRNA expression levels. The
[MATH:
2−ΔΔC
T
:MATH]
method relative to β‐actin was used to compute the relative expression
levels of each target mRNA. Primer sequences are provided in
Appendix [65]S1.
2.7. Western Blot
We extracted total proteins from liver tissue samples or cells cultured
in vitro and determined their concentrations using a BCA kit.
Subsequently, we separated the total liver protein using SDS‐PAGE and
transferred it to a PVDF membrane. To minimize non‐specific binding, we
blocked the PVDF membrane with a 5% skim milk powder solution for 1 h.
Following this, we incubated the membrane with target protein
antibodies at 4°C for over 12 h to ensure adequate binding between the
antibodies and target proteins. After multiple washes, the membrane was
incubated with HRP‐labeled secondary antibodies for 1 h at room
temperature. Upon completion of the incubation, we washed the membrane
multiple times to eliminate excess reagents and unbound antibodies.
Finally, we utilized ECL imaging technology to visualize the protein
bands on the membrane, and ImageJ software facilitated precise
quantitative analysis of the results.
2.8. Statistic Analysis
We conducted statistical analysis using the SPSS Pro online data
analysis platform. All data were presented as mean ± standard deviation
(SD). We applied the Shapiro–Wilk test to evaluate the normality of the
data distribution. To assess the significance of differences between
groups, the Student's unpaired t‐test was utilized, as well as one‐way
or two‐way ANOVA, with Tukey's and Bonferroni's post hoc tests for
multiple comparisons. If the data did not follow a normal distribution,
the rank‐sum test was employed for analysis. For statistical
significance, we set a threshold of a p‐value of less than 0.05
(p < 0.05).
3. Results
3.1. AB23A Intervention Improved FFA‐Induced Steatosis of L02 Cells
We initiated our investigation by conducting an MTT assay to assess the
cytotoxicity of AB23A on L02 cells. The outcomes revealed that AB23A
concentrations below 80 μM had no significant impact on the viability
of L02 cells (Figure [66]1A). Consequently, we opted for 20, 40, and
80 μM AB23A for subsequent analysis. We categorized the cells into
several groups: Control, Control + 80 μM AB23A, FFA, FFA + 20 μM AB23A,
FFA + 40 μM AB23A, and FFA + 80 μM AB23A. By evaluating the TG levels
of cells in each group and observing the level of lipid deposition in
cells through Oil Red O staining, we assessed the impact of AB23A on
hepatocyte steatosis in vitro. The findings illustrated that
FFA‐induced L02 cells exhibited elevated TG levels and severe
steatosis. AB23A intervention mitigated TG levels and ameliorated cell
steatosis, with 80 μM AB23A demonstrating the most pronounced effect
(Figure [67]1B–D). Thus, we selected the 80 μM AB23A intervention group
for further analysis.
FIGURE 1.
FIGURE 1
[68]Open in a new tab
AB23A intervention improves FFA‐induced steatosis of L02 cells. MTT
results showing that AB23A at concentrations below 80 μM has no
significant effect on the viability of L02 cells (A). (B–D) Effect of
AB23A on hepatocyte steatosis in vitro assessed by the TG levels in
cells from each group and the level of lipid deposition in cells via
Oil Red O staining. (B) AB23A intervention reduces the TG levels in
FFA‐induced L02 cells. (C, D) Oil Red O staining results showing that
AB23A intervention improves FFA‐induced steatosis in L02 cells. Cell
groups: Control, Control+80 μM AB23A, FFA, FFA + 20 μM AB23A,
FFA + 40 μM AB23A, and FFA + 80 μM AB23A. Data are presented as the
mean ± SD. In (A) n = 6. **p < 0.01 compared to the 0 μM group. In
(B–D) n = 3. ^# p < 0.05, ^## p < 0.01 compared to the Control group;
*p < 0.05, **p < 0.01 compared to the FFA group. One‐way or two‐way
ANOVA followed by post hoc analysis with Tukey's test for comparison
between more groups.
3.2. AB23A Intervention Down‐Regulated GRP94 Expression
We organized the cells into three groups: Control group, FFA group, and
FFA + 80 μM AB23A group, and conducted transcriptome analysis. DEGs
were identified between the FFA group vs. Control group and the
FFA + 80 μM AB23A group vs. FFA group, based on the criteria of
|Log[2](FoldChange)| ≥ 1 and p [adj] ≤ 0.05. Detailed information
regarding the DEGs is provided in Appendix [69]S1. Our findings
revealed that compared to the Control group, GRP94 exhibited
upregulation in the FFA group, whereas after AB23A intervention, the
downregulation of GRP94 was most pronounced (Figure [70]2A). Western
blot results demonstrated that relative to the Control group, the
protein expression of GRP94 increased in the FFA group, whereas after
AB23A intervention, a significant reduction in GRP94 protein expression
was observed (Figure [71]2B). Subsequently, KEGG pathway enrichment
analysis was performed, revealing enrichment of GRP94 in the “Protein
processing in endoplasmic reticulum” pathway. Consequently, our
subsequent analysis focused on the effects of AB23A on this pathway
(Figure [72]2C).
FIGURE 2.
FIGURE 2
[73]Open in a new tab
AB23A intervention down‐regulates GRP94 expression. Cells were divided
into Control, FFA, and FFA + 80 μM AB23A groups and transcriptomic
analysis was performed. Using the criteria of |Log[2](FoldChange)| ≥ 1
and padj ≤ 0.05, DEGs were screened between the FFA group vs. Control
group and the FFA + 80 μM AB23A group vs. FFA group. (A, B) DEGs
visualized using volcano plots. We found that AB23A intervention
significantly downregulates GRP94 expression (A), which was also
confirmed by western blot. (C–E) KEGG pathway enrichment analysis
revealing that GRP94 was enriched in the “Protein processing in
endoplasmic reticulum” pathway (C). In this pathway, ERS‐related genes
(PERK, eIF2α, ATF4) and ERAD‐related genes (FBXO2, DERL, HSP90AA1) are
highlighted in the heatmap (D), and the relationship between GRP94,
ERS, and ERAD is simplified and presented (E). Data are presented as
the mean ± SD. n = 3 per group. ^## p < 0.01 compared to the Control
group; **p < 0.01 compared to the FFA group. One‐way or two‐way ANOVA
followed by post hoc analysis with Bonferroni test for comparison
between more groups.
Upon visualization of the heatmap of genes associated with the “Protein
processing in endoplasmic reticulum” pathway, we observed that, in
addition to significantly downregulating GRP94 expression, AB23A also
markedly downregulated ERS‐related genes (PERK, eIF2α, ATF4) and
ERAD‐related genes (FBXO2, DERL, HSP90AA1) (Figure [74]2D). Previous
study (Li et al. [75]2024) have highlighted GRP94 as a pivotal protein
promoting ERS and ERAD, and down‐regulating GRP94 can ameliorate ERS
and ERAD. We plotted the relationship between GRP94, ERS and ERAD
(Figure [76]2E). Building upon this, we speculate that AB23A may
improve FFA‐induced L02 cell steatosis by down‐regulating GRP94,
thereby improving ERS and ERAD processes.
3.3. AB23A Improves ERS and ERAD by Down‐Regulating GRP94
To validate the aforementioned speculation, we proceeded to assess the
effects of AB23A on FFA‐induced L02 cell steatosis and the expression
of ERS and ERAD‐related proteins following the silencing of the GRP94
gene. We selected two different GRP94 siRNAs to reduce the potential
risk of off‐target effects. The results showed that both siRNAs
effectively suppressed the expression of GRP94 (Figure [77]3A).
Subsequently, we allocated the cells into the following groups:
Control+siNC, FFA + siNC, FFA + 80 μM AB23A + siNC, FFA + siGRP94‐1,
FFA + siGRP94‐1 + 80 μM AB23A, FFA + siGRP94‐2, and
FFA + siGRP94‐2 + 80 μM AB23A. The results demonstrated that relative
to the Control+siNC group, both the TG level and lipid deposition level
in the FFA + siNC group exhibited a significant increase, which was
subsequently reversed by AB23A intervention. However, silencing GRP94
annulled the ameliorative effect of AB23A on FFA‐induced L02 cell
steatosis (Figure [78]3B–D). Western blot results unveiled that in
comparison to the Control+siNC group, the protein expression of GRP94,
p‐PERK/PERK, p‐eIF2α/eIF2α, ATF4, FBXO2, DERL, and HSP90α was
upregulated in the FFA + siNC group, with AB23A intervention resulting
in the downregulation of these proteins. Upon silencing GRP94, the
expression of GRP94 protein in all groups nearly vanished, and the
regulatory effects of AB23A on ERS‐related proteins (p‐PERK/PERK,
p‐eIF2α/eIF2α, ATF4) and ERAD‐related proteins (FBXO2, DERL, HSP90α)
were nullified (Figure [79]4A–C). This suggests that AB23A improves
FFA‐induced L02 cell steatosis by
[MATH: downregulatingGRP94 :MATH]
and thereby enhancing ERS and ERAD.
FIGURE 3.
FIGURE 3
[80]Open in a new tab
Silencing GRP94 abolishes the improvement effect of AB23A intervention
on FFA‐induced steatosis in L02 cells. The effect of AB23A intervention
on FFA‐induced steatosis in L02 cells was evaluated by silencing the
GRP94 gene. Western blot results demonstrated that both siRNAs
significantly suppressed the expression of GRP94 (A, B). **p < 0.01
compared to the siNC group. The results show that after silencing
GRP94, the effect of AB23A intervention on reducing the TG levels (C)
and improving steatosis (D, E) in FFA‐induced L02 cells disappears.
Cell groups: Control+siNC, FFA + siNC, FFA + 80 μM AB23A + siNC,
FFA + siGRP94‐1, FFA + siGRP94‐1 + 80 μM AB23A, FFA + siGRP94‐2, and
FFA + siGRP94‐2 + 80 μM AB23A. Data are presented as the mean ± SD.
n = 3 per group. ^## p < 0.01 compared to the Control+siNC group;
*p < 0.05, **p < 0.01 compared to the FFA + siNC group. One‐way or
two‐way ANOVA followed by post hoc analysis with Tukey's test for
comparison between more groups.
FIGURE 4.
FIGURE 4
[81]Open in a new tab
AB23A intervention improves ERS and ERAD via down‐regulated of GRP94.
The effect of AB23A intervention on the expression of ERS and
ERAD‐related proteins was evaluated by silencing the GRP94 gene.
Western blot results demonstrated that both siRNAs significantly
suppressed the expression of GRP94 (A) the effect of AB23A on
downregulating the expression of ERS‐related proteins (p‐PERK/PERK,
p‐eIF2α/eIF2α, ATF4) (B) and ERAD‐related proteins (FBXO2, DERL,
HSP90α) (C) abolishes. Data are presented as the mean ± SD. n = 3 per
group. ^# p < 0.05, ^## p < 0.01 compared to the Control+siNC group;
*p < 0.05, **p < 0.01 compared to the FFA + siNC group. One‐way or
two‐way ANOVA followed by post hoc analysis with Tukey's test for
comparison between more groups.
3.4. AB23A Can Improve Steatosis in Hepatocytes of NASH Mice and Restore ER
Homeostasis
Building upon the in vitro experiments, we further substantiated the
therapeutic efficacy of AB23A on NASH mice and its regulatory role in
ER homeostasis through in vivo experiments. Firstly, we found that
H‐AB23A can significantly alleviate MCD‐induced weight loss
(Figure [82]S1A). Furthermore, AB23A did not affect food intake
(Figure [83]S1B). Compared to the control group, the liver index of
NASH mice exhibited a significant reduction. However, post AB23A
treatment, the liver index of the mice exhibited some level of
recovery, with the H‐AB23A group manifesting the most notable
improvement (Figure [84]5A). HE staining and Oil Red O staining
unveiled that while hepatic cords in the Control group were orderly
arranged with a normal structure, the liver of NASH mice displayed
severe fatty changes characterized by disorganized hepatic cord
arrangement and substantial inflammatory cell infiltrations. Following
AB23A intervention, these symptoms demonstrated varying degrees of
improvement (Figure [85]5B–D). Furthermore, subsequent to AB23A
treatment, the activities of ALT and AST in serum along with the liver
TG level exhibited varying degrees of improvement relative to the NASH
group, indicative of AB23A's ability to effectively restore liver
function and ameliorate hepatic steatosis in NASH mice
(Figure [86]5E–G).
FIGURE 5.
FIGURE 5
[87]Open in a new tab
AB23A can improve hepatocyte steatosis in NASH mice. NASH mouse model
was established through a MCD diet. Different doses of AB23A
intervention were administered to evaluate the therapeutic effect of
AB23A on NASH mice. (A) AB23A intervention promotes the recovery of
liver index in NASH mice. (B–D) AB23A intervention reduces inflammatory
infiltration in the liver of NASH mice (B) and improves lipid
deposition in the liver (C, D). (E‐G) AB23A intervention improves serum
ALT (E), AST activity (F), and liver TG levels (G). Data are presented
as the mean ± SD. n = 10 per group. ^## p < 0.01 compared to the
Control group; *p < 0.05, **p < 0.01 compared to the NASH group.
One‐way or two‐way ANOVA followed by post hoc analysis with Tukey's
test for comparison between more groups.
Additionally, we evaluated the effect of AB23A on ER homeostasis
through RT‐qPCR and Western blot analyses. The outcomes revealed that
compared to the Control group, the expression of GRP94, p‐PERK/PERK,
p‐eIF2α/eIF2α, ATF4, FBXO2, DERL, and HSP90α genes and proteins was
elevated in NASH mice, while AB23A intervention led to the
downregulation of these genes and proteins in a dose‐dependent manner.
Furthermore, we employed PPC as a positive control for intervention,
and the results indicated no significant differences in the
aforementioned indicators between the H‐AB23A group and the PPC group
(Figure [88]6A–F).
FIGURE 6.
FIGURE 6
[89]Open in a new tab
AB23A intervention can restore ER homeostasis in NASH mice hepatocytes.
The effect of AB23A on ER homeostasis was investigated in hepatocytes
of NASH mice using RT‐qPCR and Western blot. The results show that
AB23A intervention downregulates the gene and protein expression of
GRP94 (A, B), as well as the expression of ERS‐related genes and
proteins (C, D) and ERAD‐related genes and proteins (E, F). Data are
presented as the mean ± SD. n = 3 per group. ^## p < 0.01 compared to
the Control group; *p < 0.05, **p < 0.01 compared to the NASH group.
One‐way or two‐way ANOVA followed by post hoc analysis with Bonferroni
test for comparison between more groups.
4. Discussion
Numerous natural products, including betaine (Chen et al. [90]2021) and
resveratrol (Binmowyna et al. [91]2024), have demonstrated efficacy in
ameliorating hepatic steatosis in NASH animal models within laboratory
settings. This study aims to assess the therapeutic potential of AB23A,
the principal active compound derived from Alisma orientale (Sam.)
Juzep., in NASH treatment through both in vitro and in vivo
experimentation. In the in vitro phase, L02 cells subjected to FFA
stimulation exhibited pronounced lipid accumulation, accompanied by an
increase in cellular TG levels. In the subsequent in vivo phase,
NASH‐afflicted mice, following a 6‐week MCD diet regimen, displayed
marked hepatic steatosis, evidenced by a significant decrease in liver
index and a notable rise in TG levels. These observations confirm the
successful establishment of the NASH model in both experimental
settings. Upon AB23A intervention, reductions in hepatic lipid
deposition were evident in both in vitro and in vivo experiments, along
with improvements in key indicators such as ALT, AST, and TG levels.
These preliminary results underscore the potential therapeutic efficacy
of AB23A in alleviating hepatic steatosis and addressing NASH.
Furthermore, transcriptome analysis following AB23A intervention
revealed a significant downregulation in the expression of GRP94.
Additionally, within the KEGG‐enriched pathways scrutinized, AB23A
intervention notably modulated “Protein processing in endoplasmic
reticulum,” primarily linked to improving ERS and ERAD function,
crucial for maintaining ER homeostasis wherein GRP94 assumes
significance (Ajoolabady et al. [92]2023; Krshnan et al. [93]^2022).
The ER serves as a pivotal organelle for lipid and sterol synthesis,
processing, and metabolism, thereby playing a critical role in hepatic
lipid homeostasis. In the progression of NASH, excessive lipid
accumulation within hepatocytes precipitates ERS and ERAD
dysregulation, exacerbating lipid metabolic disturbances and fostering
NASH advancement (Dreher and Hoppe [94]2018; Lebeaupin
et al. [95]^2018; Rennert et al. [96]^2020). Notably, interventions
with ERS inducers have demonstrated a propensity to escalate
hepatocytic lipid accumulation, perturbing ER equilibrium (Parafati
et al. [97]2018). Conversely, inhibitory measures targeting GRP94 have
exhibited efficacy in restoring ER homeostasis and ameliorating
pathological sequelae across diverse ailments, including cancer and
immune‐mediated inflammation (Pugh et al. [98]2022). The potential
direct impact of AB23A on GRP94 awaits further validation through
experiments such as cellular thermal shift assay (CETSA). Moreover, the
function and activity of GRP94 may be influenced by more intricate
mechanisms. For instance,
3‐(1,5‐diphenyl‐4,5‐dihydro‐1H‐pyrazol‐3‐yl)‐7‐hydroxy‐2H‐chromen‐2‐one
(HCP1), a small molecule compound, can specifically bind to the third
site of GRP94, thereby directly inhibiting its activity and affecting
its role in protein folding and quality control within the ER (Wei
et al. [99]2019). Additionally, Protein kinase CK2 (CK2), a
multifunctional protein kinase composed of two α and two β subunits,
has its CK2α subunit primarily responsible for the phosphorylation of
GRP94 at Ser306 and Thr786 in the ER (Kim et al. [100]2024).
Furthermore, GRP94 can be acetylated and binds to histone deacetylase 6
(HDAC6), a known activator of HSP90 proteins. Inhibition of HDAC6 leads
to a decrease in PC2 levels, suggesting a synergistic role of HDAC6 and
GRP94 in regulating PC2 levels (Yao, Outeda, et al. [101]^2021; Yao,
Ren, et al. [102]^2021). Therefore, the upregulation of GRP94 may be
associated with factors such as HCP1, CK2α, and HDAC6. These molecules
could mediate AB23A's indirect effect on GRP94.
Moreover, our investigation revealed that AB23A intervention
downregulated the expression of ERS‐associated proteins (PERK, eIF2α,
ATF4). PERK, a type I transmembrane protein situated on the endoplasmic
reticulum membrane, upon sensing unfolded protein accumulation,
undergoes oligomerization and autophosphorylation, culminating in eIF2α
phosphorylation and instigating the UPR to reinstate ER homeostasis
(Fan and Jordan [103]2022). However, excessive ERS can precipitate a
breakdown in the PERK‐eIF2α compensatory mechanism, with phosphorylated
eIF2α activating ATF4, ultimately provoking apoptosis (Yao, Ren,
et al. [104]^2021). Studies corroborate that inhibitory measures
targeting the PERK‐mediated signaling cascade can mitigate cell damage
stemming from ERS (Rozpedek‐Kaminska et al. [105]^2020). Notably, AB23A
intervention downregulates PERK expression and its downstream
effectors, suggesting a potential avenue through which AB23A may
mitigate hepatic cell injury.
In addition, we observed that AB23A intervention downregulated the
expression of ERAD‐related proteins (FBXO2, DERL, HSP90α). FBXO2, DERL,
and HSP90α constitute integral components of ERAD. FBXO2, a ubiquitin
ligase substrate adaptor protein predominantly localized in the
cytoplasm, functions as a sensor for misfolded proteins, facilitating
their identification and subsequent degradation (Yuan
et al. [106]2017). Research indicates upregulated FBXO2 expression in
liver tissue from NAFLD patients, with FBXO2 overexpression linked to
lipid accumulation in HepG2 cells (Liu et al. [107]2023). DERL, a
member of the Derlin family situated on the endoplasmic reticulum
membrane, likewise participates in the degradation of misfolded
proteins through ERAD. Studies underscore the pivotal role of Derlin
family members in modulating NASH progression by regulating ERAD
activation and ERS (Wang et al. [108]2024). HSP90α, a stress‐responsive
subtype of the HSP90 family encoded by HSP90AA1, exhibits marked
upregulation under cellular stress conditions, fostering cancer
metastasis and inflammatory cascades (Zuehlke et al. [109]2015).
AB23A's suppressive impact on FBXO2, DERL, and HSP90α underscores its
potential in NASH treatment via anti‐ERAD mechanisms. Notably, GRP94, a
central protein orchestrating both ERS and ERAD processes, emerges as a
crucial regulator. Inhibition of GRP94 holds promise in ameliorating
both pathways (Marzec et al. [110]2012). Notably, upon silencing GRP94,
the ameliorative effects of AB23A intervention on ERS and ERAD
diminished, implicating AB23A in restoring ER homeostasis by
[MATH: downregulatingGRP94 :MATH]
, subsequently attenuating ERS and ERAD processes.
Based on our previous study (Li et al. [111]2024), AB23A's mechanism in
treating NASH likely involves regulating alanine, aspartate, and
glutamate metabolism, D‐glutamine and D‐glutamate pathways, and
arginine biosynthesis. Interestingly, glutamine has been proved to
attenuates endoplasmic reticulum stress and apoptosis in TNBS‐induced
colitis (Crespo et al. [112]2012). Therefore, it remains to be
investigated whether AB23A's ability to inhibit GRP94 could lead to the
upregulation of glutamine, which may further suppress ER stress and
mitigate NASH progression. Moreover, Pharmacokinetic studies are
crucial for optimizing dosing regimens, understanding the drug's
bioavailability, and ensuring its therapeutic potential is achieved
without adverse effects. We plan to assess the pharmacokinetic
properties of AB23A through in vivo experiments in future studies,
which will provide valuable insights for its application.
In conclusion, our study, conducted through in vitro and in vivo
experiments, underscores the potential of AB23A in ameliorating hepatic
steatosis, likely via GRP94 inhibition, consequently impeding ERS and
ERAD processes while restoring ER equilibrium (Figure [113]7).
Nevertheless, there remains scope for refinement in our experimental
approaches.
FIGURE 7.
FIGURE 7
[114]Open in a new tab
AB23A down‐regulated GRP94, thereby suppressing ERS and ERAD, restoring
ER homeostasis, and improving hepatocyte steatosis.
Author Contributions
Fei Qu: conceptualization (equal), data curation (equal), writing –
original draft (equal). Yuming Wang: data curation (equal), formal
analysis (equal), writing – original draft (equal). Yanping Zhang:
investigation (equal), project administration (equal). Feng Chen:
methodology (equal), software (equal). Yuanliang Ai: investigation
(equal), methodology (equal). Weibo Wen: conceptualization (equal),
supervision (equal), validation (equal). Jiabao Liao: resources
(equal), software (equal). Hanzhou Li: investigation (equal),
methodology (equal). Huan Pei: validation (equal), visualization
(equal). Mingxi Lu: methodology (equal). Ling Yang: supervision
(equal), validation (equal). Ning Wang: resources (equal), software
(equal). Huantian Cui: resources (equal), visualization (equal),
writing – review and editing (equal).
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Appendix S1.
[115]FSN3-13-e70086-s001.docx^ (289.9KB, docx)
Funding: This work was supported by Zhejiang TCM science and technology
plan (2024ZL1072); the Science and Technology Department of Zhejiang
Province, explore public welfare (LTGY24H270006) and the Joint Special
Project for Basic Research of Traditional Chinese Medicine in Yunnan
Province (202401AZ070001‐038).
Fei Qu, Yuming Wang and Yanping Zhang contributed equally to this work.
Contributor Information
Ling Yang, Email: 27489722@qq.com.
Ning Wang, wnworkemail@163.com.
Huantian Cui, Email: 1762316411@qq.com.
Data Availability Statement
Data will be made available upon request.
References