Abstract
Introduction
Lithospermum erythrorhizon extract (LEE), rich in shikonin and its
derivatives, has been traditionally valued for anti-inflammatory and
wound-healing properties.
Objective
This study aimed to investigate the immunoprotective effects and
underlying mechanisms of LEE in a rat model of dexamethasone-induced
immunosuppression.
Methods
One hundred SPF Sprague–Dawley rats were randomized into control,
model, and LEE treatment groups (10, 20, 40 mg/kg). Immunosuppression
was induced with dexamethasone (7.5 mg/kg, i.p.) for 7 days, followed
by oral LEE for 21 days. Body weight, food consumption, hematology, and
serum biochemistry were assessed. Immunomodulatory effects were
evaluated via cytokine profiles, immunoglobulin and complement levels,
lymphocyte subtypes and proliferation, immune organ indices, and
histopathology. Potential targets and pathways were predicted by
network pharmacology and validated by RT-qPCR.
Results
LEE significantly improved body weight, white blood cell counts (WBC),
lymphocyte (LYMPH%), and CD4^+/CD8^+ ratio. It downregulated
pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-1β,
interleukin-6) (TNF-α, IL-1β, IL-6) and upregulated anti-inflammatory
cytokine interleukin-4 (IL-4), while restoring immunoglobulin G, M and
A (IgG, IgM, IgA) and complement 3 and 4 (C3, C4) levels. LEE also
enhanced ConA- and LPS-induced lymphocyte proliferation, and alleviated
spleen and thymus atrophy, as evidenced by increased organ indices and
improved histopathology. Network pharmacology highlighted MAPK
signaling, particularly the p38 and JNK- as central pathways, which was
supported by RT-qPCR showing upregulation of Akt1, Mapk3, Mapk14,
Pik3ca, and Mapk1.
Conclusion
LEE effectively ameliorates dexamethasone-induced immunosuppression by
restoring immune cell activity, regulating cytokine balance, and
preserving immune organ structure, primarily via MAPK pathway
regulation. This study provides a scientific basis for the development
of LEE as a natural immunomodulatory agent in managing
immunosuppression in mammals.
Keywords: Lithospermum erythrorhizon extract, immunosuppression,
inflammation, MAPK signaling, network pharmacology
1. Introduction
Immunosuppression, characterized by a compromised immune system that
diminishes its ability to defend against pathogens and malignancies,
represents a formidable challenge in healthcare. This condition can
result from diverse causes, including pharmacological interventions,
chronic diseases, and specific medical procedures. The impaired immune
response significantly increases susceptibility to infections, delays
wound healing, and elevates the risk of cancer progression and
transplant rejection, all of which complicate clinical outcomes and
hinder recovery ([32]1–3). Notably, one critical yet often
underappreciated factor contributing to immunosuppression is chronic
inflammation, which disrupts immune homeostasis by inducing cytokine
imbalances and promoting immune cell exhaustion ([33]4). Efforts to
mitigate immunosuppression require a delicate balance—refining
therapeutic approaches to preserve immune functionality while
minimizing adverse effects. Central to this endeavor is understanding
the interplay between immunosuppression and inflammation, as chronic
inflammatory responses can exacerbate immune dysfunction and drive
disease progression. This intersection highlights the pressing need for
innovative strategies, including the exploration of natural products
with immunomodulatory and anti-inflammatory potential, to restore
immune homeostasis and enhance therapeutic outcomes.
Inflammation, a pivotal component of the body’s innate defense system,
serves as a double-edged sword in immunological balance. While acute
inflammation is essential for combating infections and initiating
tissue repair, chronic or dysregulated inflammation can paradoxically
drive immunosuppression. Chronic inflammation disrupts cytokine
homeostasis, as evidenced by elevated levels of pro-inflammatory
mediators such as tumor necrosis factor-α (TNF-α) and interleukin-1β
(IL-1β), which not only amplify the inflammatory cascade but also
suppress T cell activity and impair immune coordination ([34]5, [35]6).
Moreover, chronic inflammatory states are associated with diminished
immunoglobulin synthesis, resulting in reduced antibody production and
a compromised humoral immune response. Pathological remodeling of
primary lymphoid organs, including the spleen and thymus, further
exacerbates immune dysfunction by impairing the maturation and
functionality of immune cells ([36]7). Breaking this
inflammatory-immunosuppressive loop remains a critical goal in the
development of next-generation immunotherapeutics. At the molecular
level, chronic inflammation is closely linked to the dysregulation of
key signaling pathways, particularly the nuclear factor κB (NF-κB) and
mitogen-activated protein kinase (MAPK) pathways. Persistent activation
of the NF-κB pathway leads to sustained expression of pro-inflammatory
cytokines, chemokines, and adhesion molecules, thereby maintaining a
chronic inflammatory state that exhausts immune resources ([37]8).
Simultaneously, activation of the MAPK signaling cascade—especially
through the p38 (MAPK) and JNK (c-Jun N-terminal Kinase)
branches—contributes to the upregulation of IL-1β and TNF-α, which in
turn suppress T-cell differentiation and inhibit antigen-presenting
functions ([38]9). These molecular events serve as critical mediators
connecting unresolved inflammation to functional immunosuppression.
This intricate interplay between inflammation and immunosuppression
underscores the critical need for interventions capable of breaking
this vicious cycle by modulating the signaling networks at the
crossroads of immune activation and suppression.
Natural medicines have long been recognized for their potential to
enhance anti-inflammation and immune enhancement function. Among these,
Lithospermum erythrorhizon, a traditional Chinese medicinal herb rich
in bioactive compounds such as shikonin, β,β-dimethylacrylshikonin and
other naphthoquinones, has long been recognized for its
anti-inflammatory effects. Research has shown that shikonin effectively
suppresses pro-inflammatory cytokines, thereby reducing chronic
inflammation and alleviating related conditions ([39]10). Chemically,
over 80 distinct compounds have been identified from Lithospermum
erythrorhizon, primarily includes shikonin, acetylshikonin,
isobutyrylshikonin, and β,β-dimethylacrylshikonin, which are
responsible for the characteristic red pigment of the root ([40]Figure
1) ([41]11). These compounds have shown significant potential in
modulating immune responses and exerting anti-inflammatory effects.
Among them, shikonin has been widely studied for its immunomodulatory
activity, including its ability to enhance macrophage phagocytosis,
stimulate dendritic cell maturation, and promote antigen presentation
([42]12). Acetylshikonin has demonstrated the ability to inhibit
pro-inflammatory cytokine production, thus aiding in the regulation of
immune homeostasis and the prevention of excessive immune activation
([43]13). Furthermore, Li et al. indicates that shikonin derivatives
can influence T-cell differentiation and boost the activity of natural
killer (NK) cells, contributing to the body’s defense against pathogens
and tumors ([44]14). In addition, some studies have shown that shikonin
and its analogs can modulate the expression of key cytokines such as
interleukin-2 (IL-2), interferon-γ (IFN-γ), and TNF-α, which are
essential for orchestrating immune responses ([45]15, [46]16). These
findings highlight the potential of Lithospermum erythrorhizon extract
(LEE) as a natural source of immunoregulatory agents, offering
promising prospects for enhancing immune health and developing novel
therapeutic strategies. By exploring the therapeutic synergy of natural
compounds, we aim to uncover innovative strategies to address the
intertwined challenges of inflammation and immune dysregulation.
Despite growing interest in natural immunomodulators, the molecular
targets and pathways through which LEE exerts its immunoprotective
effects remain insufficiently elucidated. To bridge this gap, network
pharmacology has emerged as a powerful systems-level approach that
integrates compound-target prediction, protein–protein interaction
(PPI) analysis, and pathway enrichment, allowing the identification of
key regulatory networks involved in disease modulation. In this study,
network pharmacology was applied to systematically screen and analyze
the potential targets of LEE in the context of immunosuppression. Among
the core targets identified were protein kinase Bα (Akt1),
mitogen-activated protein kinase 3 (Mapk3), mitogen-activated protein
kinase 1 (Mapk1), mitogen-activated protein kinase 14 (Mapk14), and
Pik3ca, which are central components of the MAPK and PI3K-Akt signaling
pathways—both of which are critical for immune cell survival,
differentiation, and inflammatory regulation.
Figure 1.
[47]Chemical structures labeled A to H, showing various organic
compounds with hydroxyl and carbonyl functional groups. Each structure
varies in its arrangement, with aromatic rings and different side
chains.
[48]Open in a new tab
Chemical structures of the major compounds identified in LEE and their
derivatives. (A) Shikonin; (B) Acetylshikonin; (C)
β,β-Dimethylacrylshikonin; (D) β-Hydroxyisovalerylshikonin; (E)
DL-Acetylshikonin; (F) (2-Methylbutyryl) shikonin; (G) Lithospermidin
B; (H) Deoxyshikonofuran.
Therefore, this study aims to comprehensively investigate the
immunoprotective effects of LEE in a rat model of dexamethasone-induced
immunosuppression, by integrating traditional pharmacodynamic
evaluation with network pharmacology and gene expression validation,
thus providing mechanistic insight into its multi-target
immunomodulatory actions and supporting its potential clinical
application as a natural immunopotentiator.
2. Materials and methods
2.1. Animals
SPF SD rats, weighing 80 ~ 100 g, were purchased from Vitalriver Co.,
Ltd., (animal license number SCXK 2021–0006). The rats were housed at
the Center for Drug Safety Evaluation and Research at Zhejiang
University, under controlled environmental conditions: temperature
maintained at 20 ~ 26 °C, relative humidity at 40 ~ 70%, and a 12-h
light cycle (from 8:00 to 20:00). They were provided ad libitum access
to food and water and underwent a 7-day acclimatization period. All
animal procedures were conducted in accordance with the guidelines
approved by the Institutional Animal Care and Use Committee of the
Center for Drug Safety Evaluation and Research, Zhejiang University
(IACUC No. 23-s087).
2.2. Chemicals
Lithospermum erythrorhizon root was purchased from Shaanxi Honghao
Bio-Tech Co., Ltd. (Shanxi, China). Shikonin (565850),
β,β-Dimethylacrylshikonin (SML3463), acetylshikonin (TA9H93CFC329),
pentobarbital sodium (20121030) and 3-(4, 5-dimethyl-2-thiazolyl)-2,
5-diphenyl-2-H-tetrazolium bromide (MTT, M2128) were purchased from
Sigma-Aldrich Inc. (St. Louis, MO, United States). Isobutyrylshikonin
([49]B11182) was purchased from Shanghai Shifeng Bio-Technology Co.,
Ltd. Anhydrous ethanol, methanol, acetonitrile, cyclohexane, petroleum
ether, ethyl acetate, and acetone were purchased from Sinopharm
Chemical Reagent Co., Ltd. (Beijing, China). Sodium chloride injection
was purchased from Hangzhou Minsheng Pharmaceutical (Hangzhou, China).
Phosphate-buffered saline (PBS) (10010023), RPMI-1640 medium (7200047),
TNF-α (400-14) and interleukin-1β (IL-1β, 400-01B) ELISA kits were the
products of Gibco (Thermo Fisher Scientific, Waltham, United States).
Interleukin-4 (IL-4, SEKR-0004), interleukin-6 (IL-6, SEKR-0005),
immunoglobulin A (IgA, SEKR-0018), immunoglobulin G (IgG, SEKR-0020),
immunoglobulin M (IgM, SEKR-0021) ELISA kits were the products of
Solarbio Co. Ltd. (Beijing, China). Anti-CD4 antibody (OX-35) and
Anti-CD8 antibody (341) were the products of Abcam (Cambridge, United
Kingdom). Alanine aminotransferase (ALT, 336794), aspartate
aminotransferase (AST, 348556), albumin (Alb, 322,975), creatinine (Cr,
340,775), glucose (Glu, 330,126), total cholesterol (TC, 347559), total
protein (TP, 335972), urea nitrogen (BUN, 354868) and triglyceride (TG,
241813) were purchased from Roche Diagnostics GmbH (North America,
United States). Complement 3 (C3, CSB-E08666r) and Complement 4 (C4,
CSB-E08706r) ELISA kits were the products of Cusabio Technology LLC.
(Houston, United States). RNAiso™ Plus kit (9108) and PrimeScript™ RT
reagent kit (6210A) were the products of Takara (Dalian, China).
Diluent PK-30 (G7444), Leukolysin FFD-200A (R7078), Dye solution
FS-800A (A7114), Basophilic hemolysin FBA-200A (R7070) and Hemolysin
SLS-220A (A7016) were purchased from Jinan Xisen Meikang Medical
Electronics Co., Ltd (Jinan, China).
2.3. Instruments
High performance liquid chromatography (HPLC, LC-20A, Shimadzu);
Electronic balance (PL2001-L, Mettler Toledo); Electronic Analysis
Balance (MS105DU, Mettler Toledo); Electronic Analysis Balance (AL104,
Mettler Toledo); Clean Bench (JB-VD-650 U, Suzhou Jiabao Purification
Engineering Equipment); pH Meter (PB-21, Sartorius); Water Purification
Equipment (Purelab OptionS7, ELAG); Hematology analyzer (XT-2000i,
Sysmex); Serum chemistry analyzer (Cobas C311, Roche); Flow cytometer
(FACSCalibur) (BD Biosciences, United States); Microplate reader
(Multiskan FC, Thermo Fisher Scientific); PCR (CFX96 Touch, Bio-Rad);
RNA/DNA quantification analyzer (NanoDrop^® ND-1000, Thermo Fisher
Scientific); Microscope (DM4000, Leica); Biological Microscope (E5,
Ningbo Shunyu).
2.4. Methods
2.4.1. LEE preparation
The Lithospermum erythrorhizon extract (LEE) was prepared by a
combination of optimized ultrasonic-assisted extraction and
high-pressure extraction methods, with slight modifications based on
the protocol reported by Kim et al. ([50]17). Briefly, dried roots of
L. erythrorhizon were ground into coarse powder and extracted using 70%
ethanol at a solid-to-liquid ratio of 1:20 (w/v). The extraction
process involved ultrasonic treatment at 40 kHz for 30 min followed by
high-pressure extraction at 100 bar for 20 min. The combined extract
was filtered, concentrated under reduced pressure, and then lyophilized
to obtain a dry powder. The yield of the final extract was
approximately 43.26% (w/w) based on the dry weight of raw materials.
HPLC analysis indicated that the extract contained 5.62% (w/w) of
shikonin and its derivatives as the major active components.
2.4.2. Experimental design
A stratified randomization approach was used to allocate 100 selected
animals into five groups ([51]Table 1): control, model, low dose
(10 mg/kg), medium dose (20 mg/kg), and high dose (40 mg/kg), with 20
rats per group. The dosages of LEE were determined based on preliminary
dose-ranging studies (data not shown), while immunosuppression was
induced by intraperitoneal injection of the dexamethasone (7.5 mg/kg,
i.p., once daily for 7 consecutive days), according to previously
research ([52]18). Animals in the dose groups received daily oral
administration of LEE for 21 consecutive days following dexamethasone
treatment, the first day of dexamethasone administration was designated
as Day 1. Body weight and food consumption were recorded weekly. At the
end of the LEE administration period, blood samples were collected for
hematology and serum chemistry analysis. Additionally, spleen (n = 10)
was harvested for the evaluation of cytokine, immunoglobulin,
complement level and messenger ribonucleic acid (mRNA) expression.
Lymphocytes were isolated and analyzed for subtype classification and
proliferation capacity. Organ-body weight ratios and histopathology
examinations (n = 10) were also performed. To further elucidate the
potential mechanisms of LEE, network pharmacology analysis was
conducted to predict candidate targets, which were subsequently
validated by real-time quantitative PCR (RT-qPCR) in spleen tissues.
Details of the experimental grouping and study design are provided in
[53]Table 1 and [54]Figure 2.
Table 1.
Grouping.
SN Group N Dexamethasone (mg/kg) LEE (mg/kg)
1 Control 20 0 0
2 Model 20 7.5 0
3 Low dose 20 7.5 10
4 Medium dose 20 7.5 20
5 High dose 20 7.5 40
[55]Open in a new tab
Figure 2.
[56]Timeline diagram depicting a research experiment involving mice.
Days 1, 8, 15, 22, and 29 mark checkpoints for measuring body weights
and food consumption. Mice receive Dexamethasone injections daily for
seven days (7.5 mg/kg, intraperitoneally) followed by daily oral
administration of LEE for 21 days. Necropsy is performed on day 29 with
tissue and liquid collected. Tissue analysis includes organ-body weight
ratio, histopathology, cytokine levels, immunoglobulin, complement,
mRNA, and network pharmacology. Blood collection is used for hematology
and serum chemistry. Lymphocytes are isolated for subtype proliferation
studies.
[57]Open in a new tab
Schematic diagram of the experimental design and timeline. A total of
100 rats were randomly assigned into five groups (n = 20 per group):
control, model, low dose (10 mg/kg), medium dose (20 mg/kg), and high
dose (40 mg/kg) of LEE. Immunosuppression was induced via daily
intraperitoneal injection of dexamethasone (7.5 mg/kg) for 7
consecutive days (Days 1–7). LEE was administered orally once daily for
21 days (Days 8–28). Body weight and food consumption were monitored
weekly. At the end of the treatment period, blood samples were
collected for hematology and serum chemistry analysis. Additionally,
spleen was harvested for the evaluation of cytokine, immunoglobulin,
complement level and mRNA expression. Lymphocytes were isolated and
analyzed for subtype classification and proliferation capacity.
Organ-body weight ratios and histopathology examinations were also
performed. To further elucidate the potential mechanisms of LEE,
network pharmacology analysis was conducted to predict candidate
targets, which were subsequently validated by RT-qPCR in spleen
tissues.
2.4.3. Body weights
Animals were weighed prior to the first dexamethasone administration,
and subsequently on Day 8, 15, 22, and 29 of the dosing periods.
2.4.4. Food consumption
Food consumption was measured weekly by subtracting the remaining feed
from the total feed added over 7-day intervals. Average daily food
consumption per rat was calculated using the following formula:
[MATH: Food
Consumption=(Food Addition−Food
Remaining)∕DaysAnimal NumbersperCage :MATH]
2.4.5. Hematology examination
At the end of the administration period, blood samples were collected
from the anesthetized rats via the abdominal aorta using vacuum blood
collection needles. The samples were immediately transferred into EDTA
dipotassium salt (EDTA-K2) anticoagulant tubes. Hematology parameters
were analyzed as outlined in [58]Supplementary Table 1 ([59]19).
2.4.6. Serum chemistry examination
Following the final dosing, blood was collected from the abdominal
aorta of anesthetized rats using vacuum blood collection needles and
transferred into tubes containing coagulation accelerators. After
clotting and centrifugation, serum chemistry parameters were measured
as detailed in [60]Supplementary Table 2 ([61]20).
2.4.7. Cytokine measurement
On day 29 (following the final administration), the spleen homogenates
were prepared and centrifuged to collect the supernatant (1:5 dilution)
for cytokine analysis. Levels of TNF-α, IL-1β, IL-4, and IL-6 were
quantified using ELISA kits and expressed in pg./mL, following the
manufacturer’s instructions ([62]21).
2.4.8. Immunoglobulin and complement measurement
On day 29, the spleen was harvested to access the immunoglobulin and
complement levels. IgG, IgM, IgA, C3, and C4 levels were determined
using ELISA kits according to the manufacturer’s instructions ([63]22).
2.4.9. Real-time quantitative PCR expression
Total RNA was extracted from spleen tissues using the RNAiso™ Plus kit
according to the manufacturer’s instructions. RNA purity and integrity
were confirmed by evaluating the 260/280 nm absorbance ratio (ranging
from 1.8 to 2.0), and RNA concentration was determined using a
NanoPhotometer spectrophotometer. cDNA synthesis was then performed
using the PrimeScript™ RT reagent kit, following the manufacturer’s
protocol. Primers used for RT-qPCR were designed with Primer 5 software
and synthesized by Takara (Takara, Dalian, China) and are listed in
[64]Supplementary Table 3. β-Actin was used as the internal control to
normalize gene expression. The mRNA expression levels of Interferon-γ
(IFN-γ), IL-6, IL-1β, vascular cell adhesion molecule-1 (VCAM-1),
intercellular adhesion molecule 1 (ICAM-1) and NF-κB were detected by
performing RT-qPCR reactions on Bio-Rad T100 (Bio-Rad Laboratories,
Inc., United States) ([65]23). The comparative Ct value method was used
to quantify mRNA expression relative to β-actin expression using the
2^−ΔΔCT method, and results were expressed as mean ± standard deviation
(SD).
2.4.10. Lymphocyte subtype analysis
On day 29 (after the administration period), spleens were collected and
processed for cell suspensions. Cell viability was assessed via trypan
blue exclusion, and the cell suspension was adjusted to a concentration
of 5.0 × 10^6 cells/mL. For flow cytometry analysis, cells were stained
with CD4-FITC and CD8-PE antibodies, incubated in the dark at room
temperature for 30 min, centrifuged at 1,200 g for 5 min, and
resuspended in 0.3 mL PBS. The CD4^+, CD8^+, and CD4^+/CD8^+ ratios
were analyzed using FlowJo V10 software ([66]24).
2.4.11. Lymphocyte proliferation assay
Lymphocytes were prepared as described in section 2.4.10. Proliferative
capacity was assessed using the MTT assay ([67]25). Briefly, cells were
stimulated with Concanavalin A (ConA, 5 μg/mL) and Lipopolysaccharide
(LPS, 8 μg/mL), and optical density (OD) was measured at 570 nm after
the addition of MTT and solubilization in DMSO (with 0.04 N HCl). The
stimulation index (SI) was calculated as follows: SI = OD value of
mitogen-stimulated cells (ConA-5 μg/mL, LPS-8 μg/mL) divided by OD
value of non- mitogen-stimulated cells.
2.4.12. Organ weights
During necropsy, the absolute weights of the spleen and thymus were
measured. The “organ-body weight ratio” was calculated using the
following formula.
[MATH: Organ−Body Weight Ratio=Absolute Weight of Organ(g)Body Weights(kg)×1000<
/mn>×100% :MATH]
2.4.13. Histopathology examination
Spleens and thymuses from all groups were fixed in buffered formalin,
sectioned, and stained with hematoxylin and eosin (HE method) for
histopathology evaluation.
2.4.14. Computational system pharmacology analysis
2.4.14.1. Identification and screening of active components
Active compounds of LEE were initially identified through the
Traditional Chinese Medicine Systems Pharmacology (TCMSP)
database,[68]^1 using oral bioavailability (OB ≥ 30%) and drug-likeness
(DL ≥ 0.18) as screening criteria. To ensure relevance to the test
extract, these compounds were further cross-validated with the main
constituents identified by HPLC analysis performed in the current
study. This dual-approach ensured both database-driven and
experimentally-confirmed reliability of selected bioactive molecules
([69]26).
2.4.14.2. Target prediction
The molecular targets of the screened active compounds were predicted
using SwissTargetPrediction[70]^2 (based on chemical similarity and
known bioactivity information in humans) and Similarity Ensemble
Approach (SEA)[71]^3 (which uses ligand-based chemical similarity to
infer biological targets). Predicted targets were standardized using
UniProt database annotations[72]^4 and limited to Homo sapiens proteins
for consistency ([73]26).
2.4.14.3. Screening of potential compound targets associated with
immunosuppression
To determine the potential therapeutic relevance of the predicted
targets, immunosuppression-related genes were collected from several
publicly available databases, including GeneCards, OMIM, and DisGeNET,
using “immunosuppression” and related keywords. Compound-related
targets were intersected with disease-related genes to identify common
targets using Venny 2.1.0.[74]^5 The overlapping genes were then
imported into Cytoscape (v3.9.1) to construct a compound–target–disease
network. Network topology was analyzed to identify key nodes with high
degree values representing potential core targets ([75]26).
2.4.14.4. Protein–protein interaction network analysis
The intersected targets were submitted to the STRING database
(v11.5)[76]^6 to construct a protein–protein interaction (PPI) network
with a minimum required interaction score of 0.7 (high confidence). The
resulting PPI network was imported into Cytoscape, and topological
analysis was performed using the CytoHubba plugin to determine core
targets based on multiple centrality parameters, including degree,
betweenness centrality, and closeness centrality. The top-ranked hub
genes were selected for further functional and experimental validation
([77]26).
2.4.14.5. Gene ontology and pathway analysis
To explore the biological significance of the overlapping targets, Gene
Ontology (GO) functional enrichment (covering biological processes,
molecular functions, and cellular components) and Kyoto Encyclopedia of
Genes and Genomes (KEGG) pathway enrichment analyses were conducted
using the Metascape platform.[78]^7 Enrichment terms with p < 0.05 and
enrichment factors > 1.5 were considered statistically significant. The
results were visualized using bar plots and bubble charts to highlight
key biological processes and signaling pathways, particularly those
involved in immune regulation, inflammation, and cellular signaling
cascades ([79]26).
2.4.15. Validation of mechanism by RT-qPCR
To experimentally validate the core regulatory targets identified from
the network pharmacology analysis, RT-qPCR was performed. Total RNA was
extracted from spleen tissues using the RNAiso™ Plus kit, and cDNA
synthesis was conducted using the PrimeScript™ RT reagent kit according
to the manufacturer’s protocol, as described in Section 2.4.9. The mRNA
expression levels of Akt1, Mapk3, Mapk1, Mapk14, and
Pik3ca—representing key nodes in the MAPK and PI3K-Akt signaling
pathways—were measured and normalized to the housekeeping gene GAPDH.
The relative expression levels were calculated using the 2^-ΔΔCT
method. All reactions were performed in triplicate. Data were expressed
as mean ± standard deviation (SD) from at least three independent
biological replicates. The primer sequences used in this study are
listed in [80]Supplementary Table 3 ([81]27).
2.4.16. Statistics and analysis
Statistical analysis was performed using SPSS Statistics for Mac
(Version 20, IBM, United States) with one-way analysis of variance
(ANOVA), followed by Tukey’s post-hoc test for multiple comparisons
between groups. All tests were two-sided, and statistical significance
was defined as p < 0.05, p < 0.01 or p < 0.001. Results were expressed
as means ± standard deviation (SD). GraphPad InStat software (Version
7, GraphPad, United States) was used to assist with statistical
visualization.
3. Results
3.1. LEE preparation
The optimized extraction procedure involved three successive rounds of
reflux extraction with 70% ethanol (v/v), each lasting 2 h, using a
solid-to-liquid ratio of 1:20 (w/v). After pooling the extracts, the
combined solution was filtered and concentrated under reduced pressure,
followed by vacuum drying to obtain a purple-red powder. The final
extract yield was calculated based on the dry weight of the raw
material. Quantitative analysis using validated HPLC methods revealed
that the major active constituents in the extract were shikonin (3.28%,
w/w), acetylshikonin (1.09%, w/w), isobutyrylshikonin (0.37%, w/w), and
β,β-dimethylacrylshikonin (0.88%, w/w), as summarized in [82]Table 2.
These naphthoquinone derivatives are known for their immunomodulatory
and anti-inflammatory properties and serve as key pharmacologically
active markers of Lithospermum erythrorhizon.
Table 2.
HPLC analysis of active ingredients in LEE (paste 5 mg/mL).
Items Percentage (%)
Shikonin 3.28
Acetylshikonin 1.09
β,β-Dimethylacrylshikonin 0.88
Isobutyrylshikonin 0.37
[83]Open in a new tab
3.2. Body weights
There were no significant differences in body weight among the groups
prior to modeling. By Day 8 (after 1 week of administration), the model
group exhibited a significant reduction in body weight compared to the
control group (p < 0.01), whereas the medium and high dose groups
showed significantly higher body weights than the model group
(p < 0.05). On Day 15, body weights in the medium and high dose groups
remained significantly higher than those in the model group (p < 0.05).
By Day 22, all treatment groups exhibited significantly increased body
weights compared to the model group (p < 0.01), with no significant
differences observed between the medium or high dose groups and the
control group (p > 0.05). On Day 29, the medium and high dose groups
continued to show significantly higher body weights than the model
group (p < 0.001), and their weights were comparable to those of the
control group. These results suggest that LEE effectively mitigated
dexamethasone-induced weight loss ([84]Supplementary Table 4).
3.3. Food consumption
No significant differences in food consumption were observed among the
treatment groups compared to the control group throughout the study
period. Detailed results are presented in [85]Supplementary Table 5.
3.4. Hematology examination
Compared with the control group, the model group exhibited a
significant reduction in white blood cell (WBC) count (p < 0.001). Both
the medium and high dose treatment groups showed significantly
increased WBC counts compared to the model group (p < 0.001), with the
high dose group nearly restoring WBC levels to those of the control
group. In addition, dexamethasone administration significantly reduced
the percentage of lymphocytes (LYMPH%) (p < 0.001). After treatment
with LEE, LYMPH% significantly increased (p < 0.001), showing no
significant difference from the control group (p > 0.05). These
findings are summarized in [86]Table 3.
Table 3.
Hematology examination (mean ± SD, n = 20).
SN Group Parameters
WBC (10^9/L) NEUT% (%) LYMPH% (%) MONO% (%) EO% (%)
1 Control 9.1 ± 1.5^a 12.3 ± 2.8 88.2 ± 3.3^a 2.2 ± 0.8 0.6 ± 0.2
2 Model 5.9 ± 1.9^c 9.4 ± 3.8 67.4 ± 4.7^b 3.0 ± 0.3 0.7 ± 0.4
3 Low dose 6.9 ± 1.0^bc 10.1 ± 3.2 84.2 ± 3.4^ab 2.6 ± 0.6 0.7 ± 0.3
4 Medium dose 8.0 ± 1.7^b 11.4 ± 2.4 85.2 ± 3.0^ab 2.9 ± 1.0 0.5 ± 0.2
5 High dose 8.7 ± 1.4^ab 12.3 ± 3.2 84.2 ± 3.1^ab 2.7 ± 0.7 0.7 ± 0.3
BASO% (%) RBC (10^12/L) Hb (g/dL) Hct (%) MCV (fL)
1 Control 0.0 ± 0.0 7.3 ± 0.2 14.3 ± 0.6 40.8 ± 1.2 57.6 ± 1.0
2 Model 0.0 ± 0.0 6.7 ± 0.3 13.8 ± 0.5 40.9 ± 1.1 56.3 ± 1.4
3 Low dose 0.0 ± 0.0 6.9 ± 0.5 14.0 ± 0.4 41.0 ± 1.7 58.8 ± 2.6
4 Medium dose 0.0 ± 0.0 7.1 ± 0.2 14.1 ± 0.5 40.1 ± 1.5 57.1 ± 1.3
5 High dose 0.0 ± 0.0 6.8 ± 0.4 13.7 ± 0.5 41.4 ± 1.4 59.4 ± 2.3
MCH (pg) MCHC (g/dL) PLT (10^9/L)
1 Control 20.9 ± 0.3 34.7 ± 0.6 1,017 ± 125
2 Model 18.7 ± 0.6 29.3 ± 0.5 877 ± 280
3 Low dose 20.5 ± 1.0 34.0 ± 0.2 933 ± 241
4 Medium dose 19.1 ± 0.4 34.4 ± 0.3 1,033 ± 188
5 High dose 20.0 ± 0.4 33.8 ± 0.7 961 ± 172
[87]Open in a new tab
Different letters are significantly different (P < 0.05, P < 0.01, or P
< 0.001).
3.5. Serum chemistry examination
No significant differences were observed in serum chemistry parameters
among the experimental groups compared to the control group. Detailed
data are presented in [88]Table 4.
Table 4.
Serum chemistry examination (mean ± SD, n = 20).
SN Group Parameters
TP (g/L) Alb (g/L) ALT (U/L) AST (U/L) BUN (mmol/L)
1 Control 55.7 ± 2.5 38.5 ± 2.3 32.2 ± 6.7 136.4 ± 19.2 4.7 ± 0.5
2 Model 55.1 ± 3.9 39.2 ± 2.6 31.4 ± 5.8 120.3 ± 16.9 4.5 ± 1.0
3 Low dose 54.9 ± 4.7 41.9 ± 3.2 34.2 ± 7.3 115.3 ± 26.6 4.8 ± 0.6
4 Medium dose 53.0 ± 3.3 38.7 ± 2.4 31.5 ± 4.5 120.3 ± 20.5 4.4 ± 0.7
5 High dose 51.9 ± 1.2 40.3 ± 1.1 33.6 ± 4.4 119.1 ± 22.7 4.3 ± 0.6
Cr (μmol/L) Glu (mmol/L) TG (mmol/L) TC (mmol/L)
1 Control 29.0 ± 6.2 6.6 ± 0.5 0.6 ± 0.3 1.3 ± 0.2
2 Model 28.7 ± 5.4 6.0 ± 1.3 0.4 ± 0.3 1.7 ± 0.3
3 Low dose 27.4 ± 7.5 6.8 ± 0.6 0.5 ± 0.2 1.8 ± 0.1
4 Medium dose 26.3 ± 2.9 6.7 ± 0.8 0.6 ± 0.2 1.3 ± 0.3
5 High dose 25.5 ± 3.8 5.6 ± 0.7 0.7 ± 0.4 1.6 ± 0.4
[89]Open in a new tab
3.6. Cytokine measurement
Following dexamethasone administration, the model group exhibited
significantly elevated levels of pro-inflammatory cytokines TNF-α,
IL-1β, and IL-6, along with a marked reduction in the anti-inflammatory
cytokine IL-4 compared to the control group (p < 0.001). Treatment with
LEE significantly reversed these changes in a dose-dependent manner. In
both the medium and high dose groups, TNF-α and IL-6 levels were
restored to levels comparable to those of the control group (p > 0.05),
while IL-1β levels were significantly reduced and IL-4 levels
significantly increased (p < 0.01 or p < 0.001). These findings suggest
that LEE can effectively regulate cytokine secretion and mitigate
immunosuppression. Detailed results are presented in [90]Figures
3A–[91]D.
Figure 3.
[92]Bar graphs labeled A to I compare the levels of various cytokines
and immunoglobulins (TNF-α, IL-1β, IL-4, IL-6, IgG, IgM, IgA, C3, and
C4) across different groups: Control, Model, Low Dose, Medium Dose, and
High Dose. Different letters above bars in each graph denote
statistically significant differences. Each graph displays values in
picograms or nanograms per milliliter, indicating dose-dependent
effects on immune markers.
[93]Open in a new tab
Effect of LEE on cytokine, immunoglobulin and complement levels in
dexamethasone-induced immunosuppressed rats. Rats were
intraperitoneally administrated dexamethasone (7.5 mg/kg, i.p., once
daily for 7 consecutive days) to induce immunosuppression.
Subsequently, animals in the treatment groups received daily oral doses
of LEE for 21 consecutive days (low dose, 10 mg/kg; medium dose,
20 mg/kg; high dose, 40 mg/kg) The spleen was collected to assess the
levels of cytokine (TNF-a, IL-1b, IL-4, and IL-6) (A–D),
immunoglobulins (IgG, IgM, and IgA) (E–G) and complement proteins (C3
and C4) (H,I) as described in the methods section. LEE significantly
downregulated pro-inflammatory cytokines while restoring
anti-inflammatory cytokine IL-4 and humoral immune factors
(immunoglobulins and complements), indicating improved immune balance.
Data are expressed as mean ± SD (n = 10). *,**,*** indicated
statistically significant difference compared with the control group,
and #,##,### indicated statistically significant difference compared
with the model group (p < 0.05, p < 0.01, or p < 0.001).
3.7. Immunoglobulin and complement measurement
Dexamethasone administration significantly reduced the levels of IgG,
IgM, and IgA in the spleen compared to the control group (p < 0.001),
indicating a marked immunosuppressive effect. Treatment with LEE
notably reversed this trend. In particular, the medium and high dose
groups showed significantly increased IgG levels compared to the model
group (p < 0.01). In terms of complement proteins, C3 levels were
significantly decreased following dexamethasone treatment (p < 0.001),
whereas C4 levels remained unchanged (p > 0.05). After treatment with
LEE, C3 levels increased, though not significantly compared to the
control group (p > 0.05). However, C4 levels were significantly
elevated in both the medium and high dose groups compared to the
control group (p < 0.01), suggesting a possible regulatory role of the
extract on complement pathways. These findings indicate that LEE can
partially restore immunoglobulin levels and modulate complement
activity, thereby contributing to the improvement of immune function
under immunosuppressed conditions. The detailed results are presented
in [94]Figures 3E–[95]I.
3.8. Real-time quantitative PCR expression
Compared to the control group, dexamethasone-induced immunosuppression
significantly reduced the mRNA expression of IFN-γ in the spleen, while
the mRNA levels of IL-6, IL-1β and NF-κB were significantly upregulated
(p < 0.01 or p < 0.001). Following treatment with LEE, the medium and
high dose groups exhibited a significant increase in IFN-γ expression
and decreases in IL-6, IL-1β, and NF-κB expression (p < 0.05 or
p < 0.01), indicating that the extract effectively regulated
immune-related gene transcription. These findings are illustrated in
[96]Figure 4.
Figure 4.
[97]Six bar charts labeled A to F show the effects of different doses
in five categories: Control, Model, Low Dose, Medium Dose, and High
Dose. Measurements are presented for IFN-γ, IL-6, IL-1β, VCAM-1,
ICAM-1, and NF-κB. Each chart uses distinct patterns for the bars, with
labeled statistical differences (a, b, c) indicated above some bars.
[98]Open in a new tab
Effect of LEE on relative mRNA expressions levels in
dexamethasone-induced immunosuppressed rats. Rats were
intraperitoneally administrated dexamethasone (7.5 mg/kg, i.p., once
daily for 7 consecutive days), followed by daily oral administration of
LEE at different doses for 21 consecutive days (low dose, 10 mg/kg;
medium dose, 20 mg/kg; high dose, 40 mg/kg). LEE treatment
downregulated pro-inflammatory and adhesion-related genes (IL-6, IL-1β,
VCAM-1, ICAM-1, NF-kB), while restoring IFN-γ expression, suggesting
attenuation of inflammatory signaling and improved immune regulation.
The spleen was harvested for quantitative PCR analysis of IFN-γ, IL-6,
IL-1β, VCAM-1, ICAM-1, and NF-KB mRNA levels (A–F). Data are expressed
as mean ± SD (n = 10). *,**,*** indicated statistically significant
difference compared with the control group, and #,##,### indicated
statistically significant difference compared with the model group
(p < 0.05, p < 0.01, or p < 0.001).
3.9. Lymphocyte subtype analysis
As shown in [99]Figures 5A–[100]F, dexamethasone administration
significantly reduced the CD4^+/CD8^+ T lymphocyte ratio compared to
the control group (p < 0.001), indicating an imbalance in T cell
subpopulations due to immunosuppression. Treatment with LEE mitigated
this effect in a dose-dependent manner. Notably, the high dose group
restored the CD4^+/CD8^+ ratio to a level comparable to that of the
control group (p > 0.05), suggesting that LEE can effectively regulate
T lymphocyte homeostasis under immunosuppressive conditions.
Figure 5.
[101]Flow cytometry plots (A-E) display CD4 and CD8 cell populations
under different conditions: Control, Model, Low Dose, Medium Dose, and
High Dose. Bar graphs (F-G) show CD4+/CD8+ ratios and stimulation
indices (SI) for ConA and LPS, comparing Control, Model, Low, Medium,
and High Doses. Different doses affect cell ratios and responses, with
statistical significance indicated by different letters.
[102]Open in a new tab
Effect of LEE on lymphocyte subtype ratio and lymphocyte proliferation
stimulation index (SI) in dexamethasone-induced immunosuppressed rats.
Rats were intraperitoneally administrated dexamethasone (7.5 mg/kg,
i.p., once daily for 7 consecutive days) to induce immunosuppression,
followed by daily oral administration of LEE at various doses for 21
consecutive days (low dose, 10 mg/kg; medium dose, 20 mg/kg; high dose,
40 mg/kg). At the end of the treatment period, spleens were harvested,
and splenic lymphocytes were prepared. The CD4^+/CD8^+ T cell ratio was
analyzed by flow cytometry (A–E), as described in the methods section.
For lymphocyte proliferation analysis, isolated splenic lymphocytes
were cultured in vitro and stimulated with either ConA (T cell
proliferation) or LPS (B cell proliferation). Proliferative responses
were evaluated using the MTT assay, and the stimulation index (SI) was
calculated accordingly. LEE significantly increased the CD4^+/CD8^+
ratio and enhanced both T and B cell proliferation, indicating
restoration of cellular immune competence. Data are presented as mean ±
standard deviation (SD) (n = 10). *,**,*** indicated statistically
significant difference compared with the control group, and #,##,###
indicated statistically significant difference compared with the model
group (F, CD4^+/CD8^+ ratios) (G, SI) (p < 0.05, p < 0.01, or
p < 0.001).
3.10. Lymphocyte proliferation assay
As shown in [103]Figure 5G, dexamethasone treatment significantly
suppressed lymphocyte proliferation, as indicated by a decreased SI in
response to both ConA and LPS. LEE treatment effectively alleviated
this suppression. Specifically, the high dose group significantly
restored the ConA-induced SI compared to the model group (p < 0.05),
although no significant difference was observed compared to the control
group (p > 0.05). For LPS-induced SI, both the medium and high dose
groups showed significant improvement compared to the model group, and
the values remained statistically indistinguishable from those of the
control group (p > 0.05). These results suggest that LEE enhances both
T cell (ConA-induced) and B cell (LPS-induced) proliferative responses
under immunosuppressive conditions.
3.11. Organ weights
As shown in [104]Figure 6A, dexamethasone administration significantly
reduced both spleen and thymus indices compared to the control group
(p < 0.001), reflecting immunosuppression-induced atrophy of immune
organs. Oral administration of LEE effectively ameliorated these
reductions. Notably, the medium and high dose LEE groups exhibited
significantly higher spleen and thymus indices than the model group
(p < 0.05 or p < 0.01), with no statistically significant differences
from the control group (p > 0.05). These findings indicate that LEE can
restore immune organ indices and potentially reverse
dexamethasone-induced immune organ atrophy.
Figure 6.
[105]Graph A shows bar charts comparing spleen and thymus indices
across different treatments: Control, Model, Low Dose, Medium Dose, and
High Dose. Images B and C display histological sections of the spleen
and thymus, respectively, under the same treatment conditions, with
varying levels of cellular density and structure.
[106]Open in a new tab
Effect of LEE on immune organ indices and histopathology changes in
dexamethasone-induced immunosuppressed rats. Rats were
intraperitoneally administrated dexamethasone (7.5 mg/kg, i.p., once
daily for 7 consecutive days) to induce immunosuppression. Following
this, animals in the LEE-treated groups received daily oral
administration of LEE at various doses for 21 consecutive days (low
dose, 10 mg/kg; medium dose, 20 mg/kg; high dose, 40 mg/kg). At the end
of the treatment, the spleen and thymus were excised, and organ indices
(organ-body weight ratio) were calculated as described in the methods
section (A). For histopathological evaluation, the collected organs
were fixed, sectioned, and stained with hematoxylin and eosin (HE)
according to standard protocols. Representative histological images of
spleen and thymus tissues are presented (B,C) showed that LEE mitigated
dexamethasone-induced atrophy and structural damage, with clearer
tissue architecture compared to the model group. Scale bar = 50 μm.
Quantitative data are expressed as mean ± standard deviation (SD)
(n = 10). *,**,*** indicated statistically significant difference
compared with the control group, and #,##,### indicated statistically
significant difference compared with the model group (p < 0.05,
p < 0.01, or p < 0.001).
3.12. Histopathology examination
As shown in [107]Figures 6B,[108]C, dexamethasone-induced
immunosuppression caused marked histopathological alterations in the
spleen and thymus, including reduced cortical-medullary
differentiation, lymphocyte depletion, and tissue atrophy. These
pathological changes are consistent with structural damage and
functional decline of immune organs. In contrast, treatment with LEE
notably alleviated these histological lesions. The medium and high dose
groups exhibited relatively preserved tissue architecture, with
improved lymphocyte density and restoration of thymic and splenic
microstructures. These findings further support the protective effect
of LEE against immunosuppression-induced organ damage.
3.13. Computational system pharmacology analysis
3.13.1. Potential target genes and the PPI network analysis of LEE in
immunosuppression protection
A total of 85 overlapping genes were identified by intersecting
differentially expressed genes associated with immunosuppression and
predicted targets of active compounds in LEE ([109]Figure 7A). A
protein–protein interaction (PPI) network was constructed using the
STRING database, comprising 85 nodes and 276 edges ([110]Figure 7B).
This network was further visualized and analyzed using Cytoscape
software ([111]Figure 7C). Topological analysis of the PPI network
indicated that Akt1, Mapk3, Pik3ca, Mapk14, and Mapk1 were core nodes
with high connectivity (degree > 10), suggesting they may serve as key
targets through which LEE exerts its immunomodulatory effects (see
[112]Supplementary Table 6).
Figure 7.
[113]Three-panel image showing: A) Venn diagram with two overlapping
circles, blue (1414, 86.7%) and yellow (131, 8%), sharing 85 (5.2%). B)
Network diagram with multicolored nodes and connecting lines. C)
Circular network diagram with blue nodes labeled, connected by gray
lines.
[114]Open in a new tab
Potential target genes of LEE in immunosuppression. (A) The STRING
protein–protein interaction (PPI) network of 85 potential target. (B)
Venn diagram showing the intersection of differentially expressed genes
in immunosuppression and predicted targets of LEE. (C) Visualized PPI
network constructed using Cytoscape, displaying topological features of
the target genes. These analyses suggested that LEE exerted
immunomodulatory effects through a core set of overlapping genes,
providing mechanistic insight into its potential molecular targets.
3.13.2. Potential action mechanisms of LEE in immunosuppression protection
The 85 potential target genes were subjected to GO and KEGG enrichment
analysis using the Metascape platform. GO enrichment results suggested
that LEE may exert its therapeutic effects against inflammation-induced
immunosuppression by modulating key biological processes such as
phosphorylation, protein binding, gene expression, apoptosis, and cell
proliferation. These effects are associated with cellular components
including the cytoplasm, cytosol, nucleus, protein-containing
complexes, and the plasma membrane ([115]Figure 8A). KEGG pathway
analysis revealed that the MAPK signaling pathway showed the highest
level of enrichment among the those potentially implicated in the
immunoregulatory effects of LEE ([116]Figure 8B). This pathway, along
with others such as PI3K-Akt, is well known for its involvement in
modulating immune activation, promoting cell survival, and maintaining
inflammatory homeostasis. To validate the relevance of these pathways,
five representative hub genes were selected for RT-qPCR verification
(Akt1, Mapk3, Pik3ca, Mapk14, and Mapk1), all of which are central to
the aforementioned signaling cascades. The RT-qPCR results showed that
LEE treatment significantly upregulated the mRNA expression levels of
these genes in rat spleen tissue (p < 0.05, p < 0.01, or p < 0.001),
providing experimental confirmation of the bioinformatics predictions
and supporting the immunorestorative role of LEE under
dexamethasone-induced immunosuppression ([117]Figures 8C–[118]G).
Figure 8.
[119]Panel A shows enrichment bar graphs for biological processes,
cellular components, and molecular functions. Panel B displays a dot
plot of enriched pathways related to cancer. Panels C to G present bar
charts comparing different treatments—Control, Model, Low Dose, Medium
Dose, and High Dose—on the expression levels of proteins Akt1, Mapk3,
Pik3ca, Mapk14, and Mapk1. Error bars and statistical significance
markers are included.
[120]Open in a new tab
Potential action mechanisms of LEE and validation of potential target
genes of LEE in immunosuppression. (A) GO enrichment analysis of
biological processes, molecular functions, and cellular components
associated with LEE target genes. (B) KEGG pathway enrichment analysis
highlighting the MAPK signaling pathway as the most relevant mechanism.
(C–G) RT-qPCR validation of mRNA expression levels of five key target
genes (Akt1, Mapk3, Pik3ca, Mapk14, and Mapkl) in spleen tissues of
rats treated with LEE. These results indicated that LEE regulated
immune responses mainly through the MAPK signaling axis and modulated
expression of critical genes involved in inflammatory and immune
pathways, thereby supporting the network pharmacology predictions. Data
are expressed as mean ± SD (n = 10). *,**,*** indicated statistically
significant difference compared with the control group, and #,##,###
indicated statistically significant difference compared with the model
group (p < 0.05, p < 0.01, or p < 0.001).
4. Discussion
The present study demonstrated that Lithospermum erythrorhizon extract
(LEE) significantly mitigated dexamethasone-induced immunosuppression
in rats by modulating multiple immune parameters, restoring immune
organ indices, regulating cytokine profiles, and altering the
expression of key immune-related genes. These findings highlight the
potential of LEE as a promising immunopotentiator with
anti-inflammatory and immunoregulatory properties.
Immunosuppression poses significant health risks and can lead to more
severe diseases. The body is usually exposed to many harmful factors
such as inflammation, chronic diseases, chemotherapy and radiation
therapy, autoimmune disorders or aging which may aggravate
immunosuppression. These changes underscore the severe consequences of
immunosuppression on overall health. Heikki et al. has documented these
effects, highlighting the correlation between immunosuppression, weight
loss, and hematological alterations ([121]28). Studies have shown that
dexamethasone promoted the secretion of pro-inflammatory cytokines
while inhibiting the production of immunoglobulins and complement,
ultimately impairing the function of immune organs ([122]29–32). Given
the established link between chronic inflammation and immunosuppressive
states, we selected dexamethasone as a model compound to mimic chronic
inflammation-induced immunosuppression, allowing for the evaluation of
potential therapeutic interventions. In the present study,
dexamethasone could decrease the body weights ([123]Supplementary Table
4), hematology parameters of WBC and LYMPH% ([124]Table 3), IL-4
([125]Figure 3C), IgG, IgM, IgA, C3, and C4 ([126]Figures 3E–[127]I),
CD4+/CD8 + ratio ([128]Figure 5F), lymphocyte proliferation SI
([129]Figure 5G), the spleen and thymus-body ratios ([130]Figure 6A),
increase the TNF-α, IL-1β, and IL-6 ([131]Figures 3A,[132]B,[133]D),
and cause the change of immune gene mRNA expression ([134]Figure 4),
resulting in significantly spleen and thymus atrophy ([135]Figures
6A,[136]B).
Immunosuppression is often accompanied by a deterioration in general
health status, typically manifesting as body weight loss and reduced
appetite due to metabolic imbalances and inflammatory stress ([137]33).
In this study, rats treated with dexamethasone exhibited a significant
reduction in body weight and food consumption, consistent with
previously reported models of glucocorticoid-induced immunosuppression
([138]34). Oral administration of LEE significantly mitigated these
effects, suggesting an overall improvement in metabolic and systemic
health ([139]Supplementary Tables 4, 5). The observed improvements
reflect LEE’s capacity to counteract inflammation-induced catabolic
effects and support physiological homeostasis. Hematological indicators
such as WBC and LYMPH% are key parameters in evaluating immune
competence. Dexamethasone treatment significantly suppressed these
indices, reflecting leukopenia and lymphopenia, both of which are
hallmarks of immunosuppression ([140]35). Our results showed that LEE
administration significantly reversed these declines, indicating
enhanced hematopoietic and immunoregulatory function ([141]Table 3).
Additionally, serum biochemical indicators remained largely stable
across all LEE-treated groups ([142]Table 4), suggesting that LEE does
not induce hepatic or renal toxicity at the administered doses, which
is consistent with its traditional use in herbal medicine ([143]36).
Although certain naphthoquinone derivatives from Lithospermum
erythrorhizon, particularly shikonin, have been reported to exhibit
redox-active properties that could theoretically provoke oxidative
stress or off-target cytotoxicity at high concentrations ([144]37),
such effects were not evident in this study. Neither weight loss nor
biochemical signs of oxidative injury were observed. These findings
suggest that, under the dosing regimen used here, LEE exhibits a
favorable safety profile without evidence of pro-oxidative liabilities.
Nonetheless, further mechanistic and toxicokinetic studies would be
warranted to fully characterize long-term safety and potential
tissue-specific effects of shikonin-containing extracts.
Chronic inflammation disrupts cytokine equilibrium and impairs both
cellular and humoral immunity ([145]7). Consistent with this,
dexamethasone elevated pro-inflammatory cytokines (TNF-α, IL-1β, IL-6)
while reducing IL-4. LEE treatment effectively downregulated the
expression of pro-inflammatory cytokines while restoring
anti-inflammatory cytokines ([146]Figures 3A–[147]D), suggesting a
rebalancing of Th1/Th2 responses. Moreover, LEE significantly elevated
serum levels of immunoglobulins (IgG, IgM, IgA) and complements (C3,
C4) ([148]Figures 3E–[149]I), indicating restoration of humoral immune
competence. These findings align with previous reports on shikonin’s
immunomodulatory effects ([150]38, [151]39).
RT-qPCR analysis further supported the immunoregulatory role of LEE by
showing restored mRNA expression of IFN-γ and suppressed expression of
IL-6, IL-1β, NF-κB, VCAM-1, and ICAM-1 ([152]Figure 4). NF-κB, a master
regulator of inflammation, plays a pivotal role in immune dysfunction
under chronic stress ([153]40). The suppression of NF-κB and its
downstream adhesion molecules by LEE suggests a molecular mechanism
through which it may inhibit leukocyte migration and cytokine storms,
thereby alleviating systemic inflammation. Immunosuppression typically
alters lymphocyte subtypes, especially the CD4+/CD8 + ratio, leading to
impaired adaptive immunity ([154]41). Our results showed that LEE
restored the CD4+/CD8 + balance and enhanced ConA- and LPS-induced
lymphocyte proliferation ([155]Figure 5). These outcomes confirm that
LEE supports both helper T cell function and B cell activation. This is
consistent with studies reporting that shikonin derivatives can promote
dendritic cell maturation and antigen presentation ([156]42, [157]43).
The spleen and thymus are primary lymphoid organs essential for immune
cell development and homeostasis ([158]44). In this study,
dexamethasone administration induced notable atrophy of these organs,
as reflected by the significant decline in organ-body weight ratios and
histopathological evidence of structural disintegration. HE revealed
reduced white pulp density in the spleen and cortical thinning in the
thymus, both indicative of immunosuppressive damage ([159]Figures
6B,[160]C). Remarkably, LEE treatment reversed these pathological
changes in a dose-dependent manner, significantly restoring spleen and
thymus indices and preserving tissue architecture ([161]Figure 6A).
These findings suggest that LEE confers protective effects on central
immune organs, thereby enhancing host immune competence under
conditions of pharmacologically induced immunosuppression.
To further explore the mechanisms underlying LEE’s immunoregulatory and
anti-inflammatory effects, we employed a network pharmacology approach.
From the intersection of predicted LEE-related targets and
immunosuppression-associated genes, 85 overlapping targets were
identified. Protein–protein interaction (PPI) analysis revealed several
key hub genes, including Akt1, Mapk3, Pik3ca, Mapk14, and Mapk1—all of
which are central regulators in immune and inflammatory signaling
pathways ([162]Figure 7; [163]Supplementary Table 6). KEGG pathway
enrichment analysis identified the MAPK signaling cascade as the most
significantly involved pathway ([164]Figure 8), implicating its role in
the modulation of cytokine production and immune cell activity
([165]45). To validate these findings, RT-qPCR analysis demonstrated
that LEE treatment significantly upregulated the mRNA expression of
these five hub genes in spleen tissue ([166]Figure 8), confirming their
involvement in the observed immunomodulatory and anti-inflammatory
effects. Notably, the MAPK pathway, particularly the p38 and JNK
branches, has been well-documented to regulate pro-inflammatory
cytokine expression (e.g., TNF-α, IL-1β) and T cell differentiation
([167]46). These results collectively suggest that LEE mitigates
immunosuppression induced by inflammatory, at least in part, by
modulating MAPK-dependent inflammatory and immune signaling pathways.
While several natural compounds—such as curcumin, resveratrol—have been
investigated for their immunomodulatory potential ([168]47, [169]48),
LEE appears to exert a more selective mechanisms and broader spectrum
of regulatory activity both inflammatory and structural components of
the immune microenvironment. Unlike many conventional
immunopotentiators, which may enhance immune activation at the expense
of inflammatory control, LEE appears to rebalance immune homeostasis by
concurrently restoring adaptive immunity and downregulating excessive
inflammatory signals. These properties, combined with its favorable
safety profile, position LEE as a distinctive botanical candidate with
dual immunorestorative and anti-inflammatory capabilities, meriting
further investigation alongside established immunomodulatory agents.
Taken together, our findings demonstrate that LEE exerts multifaceted
immunoprotective effects by preserving immune organ structure,
enhancing key immune parameters, and regulating molecular pathways
associated with inflammation and immune activation. Such balanced
regulation is particularly relevant in veterinary contexts where
overactivation of the immune system may predispose to autoimmune
complications or chronic inflammatory diseases. Thus, LEE may represent
a promising candidate for integrative veterinary approaches aimed at
improving animal health, reducing reliance on antibiotics, and
enhancing resilience against infections. These results support the
therapeutic potential of LEE as a natural immunopotentiator for the
management of inflammation-related immune dysregulation. Given its dual
regulatory activity and favorable safety profile, LEE holds promise as
a translational candidate for future clinical development targeting
immune dysfunction and chronic inflammatory conditions.
While this study demonstrated that LEE mitigates dexamethasone-induced
immunosuppression in rats, further work is warranted. Validation in
diverse preclinical models, including large animals, is needed to
confirm reproducibility and assess species-specific responses.
Pharmacokinetic and toxicokinetic studies should define optimal dosing
and long-term safety. Ultimately, translational studies—such as pilot
human trials or controlled veterinary trials—will be essential to
establish the therapeutic value of LEE in managing immunosuppression
and chronic inflammation.
5. Conclusion
In conclusion, this study demonstrates that LEE exerts potent
immunomodulatory effects in a rat model of dexamethasone-induced
immunosuppression. LEE significantly improved body weight,
hematological parameters, cytokine balance, immunoglobulin and
complement levels, lymphocyte proliferation, and immune organ indices,
while alleviating thymus and spleen atrophy. Mechanistically, LEE
modulated the expression of key immune-related genes such as IFN-γ,
IL-1β, and NF-κB, and was found to target multiple signaling pathways
through network pharmacology analysis, particularly the MAPK pathway.
RT-qPCR validation further confirmed the upregulation of central nodes
including Akt1, Mapk3, Mapk14, and Pik3ca, suggesting that LEE may
restore immune homeostasis via the p38/MAPK signaling axis. These
findings provide pharmacological evidence supporting the potential
application of LEE as a natural immunopotentiator for preventing or
alleviating inflammation-associated immunosuppression. Future studies
are warranted to explore its clinical relevance and to further
elucidate its molecular targets in disease contexts.
Acknowledgments