Network pharmacology and in vivo study: Unraveling the therapeutic mechanisms of Panax ginseng in potentially treating ulcerative colitis

Abstract

BACKGROUND

Ulcerative colitis (UC), a chronic and challenging condition, necessitates the development of more effective treatments owing to the unsatisfactory efficacy and side effects associated with current medications. Traditional Chinese medicine (TCM), known for its multi-stage and multi-targeted approach, has a long history in treating gastrointestinal diseases and offering a promising alternative UC treatment. Panax ginseng (P. ginseng), a commonly used remedy for UC in TCM, exemplifies this potential, although the specific components and mechanisms through which its therapeutic effects are exerted remain to be fully elucidated, highlighting the need for further research to unlock its full potential as a treatment option.

AIM

To investigate the key constituents and biological pathways through which P. ginseng exerts therapeutic effects on UC.

METHODS

Network pharmacology investigated the UC-alleviating mechanism of P. ginseng, including “active ingredient-target-disease” network analysis, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. Panaxadiol (PD; active ingredient of P. ginseng) was tested in a mouse model of 3% dextran sulfate sodium-induced UC, with assessments of body weight, Disease Activity Index scores, and colon length. Colitis and intestinal barrier integrity were analyzed via hematoxylin-eosin and Alcian blue and periodic acid-Schiff staining, immunohistochemistry, real time-quantitative PCR, and western blotting.

RESULTS

By integrating and analyzing the targets of P. ginseng and UC, 15 critical hub genes were discovered. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed the mechanisms involved to be linked to MAPK and PI3K-Akt signaling. Among the 10 main active ingredients identified as potentially effective, PD was most abundant and was validated in vivo to mitigate weight loss, reduce Disease Activity Index scores, and prevent colon shortening. PD also reduced inflammation and suppressed expression of pro-inflammatory cytokines and mediators. In addition, PD increased expression of mucin and tight junction proteins. Ultimately, PD counteracted effects of dextran sulfate sodium by inhibiting phosphorylation of NF-кB and MAPK, while increasing phosphorylation of AMPK and expression of NRF2 and NQO1.

CONCLUSION

PD alleviates colitis and aids intestinal barrier repair, partly via modulation of the MAPK/NF-кB and AMPK/NRF2/NQO1 pathways. These findings also suggest new research methods for treatment of UC with TCM.

Key Words: Ulcerative colitis; Panax ginseng; Network pharmacology; Panaxadiol; MAPK; NF-κB; AMPK; NRF2

Core Tip: Panax ginseng (P. ginseng) is widely used to treat ulcerative colitis (UC) and known for its therapeutic efficacy. However, the specific components and mechanisms underlying its action require further clarification. Using network pharmacology analysis, we identified the main components of P. ginseng in treating UC, with panaxadiol (PD) emerging as the most abundant and potentially critical component. Next, we validated the role of PD in a mouse model of dextran sulfate sodium-induced colitis. The results demonstrated that PD effectively alleviates colitis and helps repair the intestinal barrier, partly via modulation of the MAPK/NF-кB and AMPK/NRF2/NQO1 signaling pathways.

INTRODUCTION

Ulcerative colitis (UC) is an inflammatory bowel disease that affects the rectum and colon to varying degrees, posing a lifelong challenge that can lead to significant complications and impaired quality of life[1]. With an estimated global prevalence of five million cases in 2023 and an increasing incidence rate worldwide, the pathogenesis of UC remains only partially understood, although it is believed to involve a combination of immune response, gut microbiota, genetic predisposition, and environmental factors[2]. Currently, UC is primarily treated with aminosalicylic acid, corticosteroids, thiopurines, biologics (anti-integrins and anticytokines), and small molecules (sphingosine-1-phosphate receptor modulators and Janus kinase inhibitors). However, these treatments pose challenges such as drug unresponsiveness, drug dependence, adverse effects like fever, infection, and diarrhea, and high recurrence rates[3,4]. Consequently, there is an urgent need to explore more effective and satisfactory treatment options. Traditional Chinese medicine (TCM), known for its multi-target and multi-pathway approach, holds promise as a potential breakthrough in the treatment of UC[5].

Panax ginseng (P. ginseng), one of the most well-known TCM, has a long history as an herbal medicine for treating various diseases. Research of P. ginseng and its extracts has provided evidence of strong immune regulation and anti-inflammatory abilities in the intestinal system; these include regulating immune balance, regulating the expression of inflammatory mediators and cytokines, restoring gut microbiota and metabolic imbalances, promoting healing of intestinal mucosa, preventing colitis-related colorectal cancer, reducing antibiotic induced diarrhea, and alleviating symptoms of irritable bowel syndrome[6].

Indeed, P. ginseng is a crucial component in many effective prescriptions for UC treatment, such as Shenling Baizhu powder, Sijunzi decoction, and Lizhong decoction. P. ginseng exhibits significant pharmacological activities, including anti-inflammatory, antioxidant, anticancer, and immunoregulatory properties[7-9]. Numerous studies have shown that prescriptions containing P. ginseng, as well as ginseng extracts or individual ginseng components, can regulate intestinal flora, alleviate intestinal inflammation, and repair the intestinal mucosa in cases of UC[10-12]. However, there is still a lack of systematic research on the main components and specific mechanisms by which P. ginseng exerts its therapeutic effects in UC.

Network pharmacology is a comprehensive discipline that combines systems biology and network informatics to guide drug discovery and development. It is a systematic and effective method for studying the complex relationship between TCM and diseases[13]. In this study, network pharmacology was employed for the first time to explore the targets, signaling pathways, and main active ingredients of P. ginseng in the treatment of UC. The study also validated the therapeutic effects of panaxadiol (PD), one of the primary active components of P. ginseng. In addition, the findings provide a reliable reference for future experimental research and new drug development.

MATERIALS AND METHODS

Screening of active ingredients of P. ginseng and prediction of relative targets

Using “Panax ginseng CAM” as the keyword, the compound was searched in the TCM Systems Pharmacology Database and Analysis Platform (TCMSP) (https://tcmspw.com/tcmsp.php). The active ingredients of P. ginseng were screened based on the criteria of oral bioavailability ≥ 30% and drug-likeness ≥ 0.18. The gene targets corresponding to each active ingredient were predicted using the SwissTargetPrediction database (http://swisstargetprediction.ch/).

Acquisition of disease targets of UC

The keyword “ulcerative colitis” was used to retrieve disease-related targets from GeneCards (https://www.genecards.org/), DisGeNET (https://www.disgenet.org), and Online Mendelian Inheritance in Man (OMIM) (https://omim.org/) databases. The targets identified from each database were then merged, with duplicates removed, to obtain the common targets associated with UC. Venn diagram was constructed using the OmicShare online tool (https://omicshare.com) to visually present the intersection.

Construction and analyses of the protein-protein interaction network

The potential targets of the active ingredients of P. ginseng and the disease targets of UC were intersected to identify common genes for further research. These intersection genes were then imported into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (https://string-db.org) to generate a protein-protein interaction (PPI) network. The resulting gene network was subsequently imported into Cytoscape 3.7.2 (https://cytoscape.org) to further analyze the interaction network. Using the Centiscape 2.2 plugin, three key parameters-betweenness centrality (termed BC), closeness centrality (termed CC), and degree centrality (termed DC) - were calculated to assess the attributes of the nodes within the network. Nodes with higher quantitative values for all three parameters were considered more important within the network. Target nodes that exceeded the median values for these parameters were selected to construct a new PPI network. The core PPI network was ultimately obtained through repeated refinement.

Enrichment analyses of gene ontology and pathway

The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and Gene Ontology (GO) function analysis, including biological process, cellular component, and molecular function categories, were performed on the intersection genes using the DAVID database (https://david.ncifcrf.gov/). Functional terms and pathways with a P value < 0.05 were considered significant and retained for further analysis. The results were sorted by gene ratio in descending order, and the top 10 results were selected for further analysis. The results of the GO function analysis were visualized using histograms and bubble charts. Additionally, the identified pathways and corresponding genes were imported into Cytoscape 3.7.2 for visualization as relevant pathway maps.

Identifying the main active ingredients of P. ginseng for treating UC

The active ingredients of P. ginseng and their corresponding targets were imported into Cytoscape 3.7.2 to construct a drug-ingredient-target network. Non-intersecting genes were removed, resulting in the creation of a refined network. The CytoNCA plugin was then used to analyze this network, with three main parameters, namely betweenness, closeness, and degree, being calculated. The main active ingredients of P. ginseng in the treatment of UC were identified based on the median value of the degree parameter.

Establishment of the UC animal model and treatments

Eight-week-old female C57BL/6 mice were obtained from the Laboratory Animal Center of Shanxi Provincial People’s Hospital. All mice were housed in a specified pathogen-free environment at 22-23 °C with a 12-hour light/dark cycle and had free access to water and food. A total of 32 mice were randomly divided into four groups, with eight mice per group, as follows: The control group; the PD group; the dextran sulfate sodium (DSS) group; and the DSS + PD group. The control group and PD group were provided normal drinking water, whereas the DSS group and DSS + PD group received 3% DSS (w/v, 36-50 kDa; Yeasen Biotech Co., Ltd., Shanghai, China) in their drinking water from day 4 to day 10. The control group and DSS group received olive oil (10 µL/g; Yuanye Bio Sci & Tech Co., Ltd, Shanghai, China) once daily by oral gavage. Meanwhile, the PD group and DSS + PD group received PD (30 mg/kg weight. purity ≥ 98%; Solarbio, Beijing, China) dissolved in olive oil (10 µL/g) once daily by oral gavage throughout the experimental period. The PD dose was determined by reference to the previous literature[14]. All animal procedures were approved by the Shanxi Provincial People’s Hospital Institutional Animal Ethics Committee [(2024) No. 506].

Macroscopic grading and histological analysis of the animal model

The Disease Activity Index (DAI), which is based on weight loss, stool consistency, and the degree of intestinal bleeding, was used to evaluate the macroscopic severity of DSS-induced colitis[15]. The body weight, stool consistency, and presence of blood in the stool of each mouse were recorded throughout the progression of the DSS-induced UC in the mouse model. At the end of the experiment, the mice were sacrificed by cervical dislocation, and their colon tissues were promptly collected and measured for length.

The colonic tissues were fixed in 4% paraformaldehyde (Servicebio, Hubei, China), embedded in paraffin, sectioned into 3 μM slices, and then stained with hematoxylin-eosin (HE) (Servicebio) and Alcian blue and periodic acid-Schiff (commonly referred to as AB/PAS) stain (Solarbio) for observation under an optical microscope (CKX53; Olympus, Tokyo, Japan). Histopathological scores were calculated according to the criteria outlined in Table 1[16].

Table 1 Scoring criteria for inflammation-associated histological changes.

Score	Inflammation	Crypt damage	Ulceration	Edema
0	No infiltrate	None	None	Absent
1	Occasional cells limited to submucosa	Some crypt damage, spaces between crypts	Small, focal ulcers	Present
2	Significant presence of inflammatory cells in submucosa limited to focal areas	Larger spaces between crypts, loss of goblet cells, some shortening of crypts	Frequent small ulcers	-
3	Infiltrate is present in both submucosa and lamina propria, limited to focal areas	Large areas without crypts, surrounded by normal crypts	Large areas lacking surface epithelium	-
4	A large amount of infiltrate in the submucosa, lamina propria, and surrounding blood vessels, covering large areas of mucosa	No crypts	-	-
5	Transmural inflammation (mucosa to muscularis)	-	-	-

Immunohistochemistry

After dewaxing the paraffin-embedded colon sections, antigen retrieval was performed using citric acid (Solarbio). The sections were then washed with 3% hydrogen peroxide (Solarbio) at room temperature for 25 minutes and incubated with 3% bovine serum albumin (Solarbio) for 30 minutes. Subsequently, the sections were incubated with the primary antibody (Servicebio) at 4 °C overnight, followed by incubation with the corresponding secondary antibody. Freshly prepared diaminobenzidine (Solarbio) colorimetric solution was then dropped onto the sections for color development. The development time was monitored under a microscope and stopped by rinsing with tap water. The cell nuclei were stained with hematoxylin (Solarbio), followed by dehydration, and the sections were sealed with synthetic resin. Finally, the slides were observed under a microscope, where a dark yellow color indicated a positive result.

Western blot analysis

RIPA lysis buffer containing 1% protein phosphatase inhibitors and 1% phenylmethyl sulfonyl fluoride (all, Solarbio) was added to the colon tissue homogenate. After sufficient lysis and centrifugation at 4 °C for 15 minutes (5430R; Eppendorf, Hamburg, Germany), the supernatant was collected. Protein concentration was measured using the bicinchoninic acid kit (Thermo Fisher Scientific, Waltham, MA, United States). Equal amounts of protein extract (40 μg) were loaded into the wells of a 10% sodium dodecyl sulfate-polyacrylamide gel for electrophoretic separation. The isolated proteins were then transferred onto a polyvinylidene fluoride membrane (Immobilion-PSQ; Millipore, Burlington, MA, United States). The membrane was blocked with 5% skimmed milk (Solarbio) at room temperature for 2 hours. After washing twice with Tris-buffered saline containing Tween (TBST) solution, the membranes were incubated overnight at 4 °C with primary antibodies, including p-P38, P38, p-ERK1/2, ERK1/2, p-JNK1/2, JNK1/2, NF-кB p-P65, P65, phospho-AMPKα (Thr172), AMPK, NRF2, NQO-1, and GAPDH (all from Proteintech, Wuhan, China, except for phospho-AMPKα from Cell Signaling Technology, Danvers, MA, United States), each diluted 1:1000. After washing three times with TBST, the membranes were incubated with enzyme-linked anti-mouse or anti-rabbit secondary antibody (Proteintech), diluted 1:2000 at room temperature for 2 hours, followed by three additional washes with TBST buffer. Finally, the ChemiDoc XRS+ Image Lab System (Bio-Rad, Hercules, CA, United States) was used to detect the protein bands after reacting with a developer (Boster Bio, Pleasanton, CA, United States). The intensity of the bands was quantified using ImageJ software and compared to GAPDH to determine the relative expression levels of the proteins.

Real time-quantitative PCR

Total RNA was extracted from colon tissue homogenates using RNAiso Plus (TaKaRa, Shiga, Japan), and the RNA concentrations were determined. A reverse transcription kit (TaKaRa) was used to reverse transcribe RNA into cDNA at equal concentrations. The Real time-quantitative (RT-q) PCR test sample consisted of cDNA, primers (primer sequences shown in Table 2), and 2 × M5 HiPer SYBRGreen qPCR SuperMix (Mei5 Biotechnology Co. Ltd., Beijing, China). RT-qPCR detection was performed using the CFX96 Real-Time PCR Detection System (CFX Connect; Bio-Rad). Each sample was tested in triplicate, and the relative expression levels of mRNA were calculated using the 2^-ΔΔCT method, with β-actin serving as the reference gene.

Table 2 Primers for real time-quantitative PCR.

Gene	Forward	Reverse
TNF-α	5′-CCCCAAAGGGATGAGAAGTTC-3′	5′-CCTCCACTTGGTGGTTTGCT-3′
IL-1β	5′-GTTCCCATTAGACAACTGCACTACAG-3′	5′-GTCGTTGCTTGGTTCTCCTTGTA-3′
IL-6	5′-CCAGAAACCGCTATGAAGTTCC-3′	5′-GTTGGGAGTGGTATCCTCTGTGA-3′
COX2	5′-CAGTTTATGTTGTCTGTCCAGAGTTTC-3′	5′-CCAGCACTTCACCCATCAGTT-3
iNOS	5′-GAACTGTAGCACAGCACAGGAAAT-3′	5′-CGTACCGGATGAGCTGTGAAT-3′
β-Actin	5′-GTCAGGTCATCACTATCGGCAAT-3′	5′-AGAGGTCTTTACGGATGTCAACGT-3′

Statistical analysis

In this study, all data are presented as mean ± standard error of the mean. Statistical analyses were conducted using GraphPad Prism software version 8.0 (La Jolla, CA, United States). The significance of differences between groups was assessed using one-way ANOVA, followed by Tukey’s multiple range test. It is important to note that due to the small sample size, the statistical method of ANOVA has certain limitations; however, considering that the mean can better represent the data than the median we chose this method as it is commonly used in the literature. A P value of < 0.05 indicated a statistically significant difference. For each experiment, three or more biological replicates were performed.

RESULTS

Screening of drug-disease targets

In total, 20 active ingredients of P. ginseng were identified through the TCMSP database and subsequent Absorption, Distribution, Metabolism and Excretion (known as ADME) screening (criteria: Oral bioavailability ≥ 30, drug-likeness ≥ 0.18). Using the SwissTargetPrediction database, potential targets for these active ingredients were predicted. After removing duplicates, 632 unique targets were identified for P. ginseng.

DisGeNET, GeneCards, and OMIM databases were searched to obtain the known target genes related to UC. After merging the data from each database and removing duplicates, a total of 2157 disease targets were identified for UC. By intersecting the potential targets of P. ginseng with these UC-related targets, 223 common targets were identified (Figure 1A).

Figure 1 Protein-protein interaction network and topological analysis. A: Venn diagram of the active ingredient of Panax ginseng (P. ginseng) and potential targets of ulcerative colitis (UC); B: Protein-protein interaction network acquired from the Search Tool for the Retrieval of Interacting Genes/Proteins database platform; C: Screening of hub genes for P. ginseng for the treatment of UC.

PPI network and topological analysis

By inputting the 223 intersection targets of P. ginseng for treating UC into the STRING database platform, a PPI network was constructed (Figure 1B). To further visualize protein interactions, the PPI network was imported into Cytoscape 3.7.2, resulting in a new PPI network consisting of 221 nodes and 3853 connecting edges. In this network, the thickness of the lines indicates the strength of the connections between genes. We then used the Centiscape2.2 plugin to identify core targets. The selection criteria were based on the following corresponding median values: BC > 218.371040723978; CC > 0.00232353904184756; and DC > 34.8687782805429. This resulted in a new PPI network with 40 nodes and 568 edges. By refining the selection criteria to BC > 10.600000000000009, CC > 0.020623666641088337, and DC > 28.4, we extracted a final PPI network with 15 nodes and 105 edges. Ultimately, we identified 15 hub genes, namely IL6, AKT1, TNF, PPARG, EGFR, STAT3, CASP3, BCL2, NF-кB1, PTGS2, ESR1, MMP9, ERBB2, SRC, and TLR4 (Figure 1C).

Enrichment analysis of GO and KEGG pathways

The 223 intersection genes were inputted into the DAVID database for GO biological processes and KEGG pathway enrichment analysis. The top 10 results were visualized as histograms (Figure 2A) and bubble charts (Figure 2B). The targets identified in the biological processes are closely associated with signal transduction, phosphorylation, protein phosphorylation, negative regulation of the apoptotic process, positive regulation of transcription from RNA polymerase II promoter, positive regulation of cell proliferation, positive regulation of transcription (DNA-templated), inflammatory response, negative regulation of transcription from RNA polymerase II promoter, and response to xenobiotic stimulus. Regarding cellular components, P. ginseng mainly affects the plasma membrane, cytoplasm, cytosol, membrane, nucleus, nucleoplasm, extracellular region, extracellular exosome, extracellular space, and cell surface. At the molecular level, the function of P. ginseng is mainly related to protein binding, identical protein binding, ATP binding, protein kinase activity, protein homodimerization activity, protein serine/threonine kinase activity, zinc ion binding, protein kinase binding, enzyme binding, and protein tyrosine kinase activity.

Figure 2 Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis. A: Histogram of Gene Ontology (GO) enrichment analysis, displaying categories for biological process, cellular component, and molecular function; B: Bubble chart of GO enrichment analysis, where the X-axis represents the enrichment value. The color intensity of the dots indicates the P value (with redder dots indicating lower P values), and the size of the dots reflects the number of counts; C: Network analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Purple nodes represent the top 10 KEGG pathways, and the yellow nodes represent the intersection genes between Panax ginseng and ulcerative colitis. BP: Biological process; CC: Cellular component; MF: Molecular function.

A total of 169 enrichment results were obtained by KEGG pathway analysis. The top 10 pathways (Figure 2C) were selected based on gene ratio and a significance threshold of P < 0.05. These pathways mainly included signaling pathways such as Pathways in cancer, PI3K-Akt signaling pathway, Hepatitis B, Kaposi sarcoma-associated herpesvirus infection, Proteoglycans in cancer, MAPK signaling pathway, human T-cell leukemia virus infection, human cytomegalovirus infection, Ras signaling pathway, and Chemical carcinogenesis - receptor activation. These findings suggest that the active ingredients of P. ginseng may treat UC by primarily targeting these pathways.

Identifying the main active ingredients

Cytoscape 3.7.2 was used to construct a network relationship among the 223 intersection genes and active ingredients of P. ginseng (Figure 3). The resulting network included 239 nodes and 597 connecting edges. Owing to the large number of genes, those with degree values ≤ 2 were removed, resulting in a refined network including 103 nodes and 417 connecting edges. The degree values of the active ingredients in the network were then calculated (Table 3). Based on degree values greater than the median, 10 main active ingredients of P. ginseng for UC treatment were identified; these are Gomisin B, Ginsenoside-Rh4_qt, Deoxyharringtonine, Ginsenoside Rg5_qt, PD, Girinimbin, Celabenzine, Alexandrin_qt, Aposiopolamine, and Suchilactone.

Figure 3 Screening of the main active ingredients of Panax ginseng for the treatment of ulcerative colitis. Yellow nodes represent Panax ginseng, purple nodes represent its active ingredients, and cyan nodes represent intersection genes with a degree value > 2.

Table 3 Parameters of active ingredients of Panax ginseng.

Active ingredient	Degree	Betweenness	Closeness
Gomisin B	35.0	1180.6964	0.46575344
Ginsenoside-Rh4_qt	32.0	1050.481	0.4573991
Deoxyharringtonine	31.0	1032.064	0.45333335
Ginsenoside Rg5_qt	30.0	872.32416	0.4493392
Panaxadiol	29.0	995.441	0.44541484
Girinimbin	28.0	1012.3926	0.44155845
Celabenzine	26.0	677.8078	0.43404254
Alexandrin_qt	25.0	728.7032	0.43037975
Aposiopolamine	24.0	684.26025	0.42677826
Suchilactone	24.0	540.3291	0.42323652
Arachidonate	23.0	651.64233	0.42323652
Kaempferol	19.0	426.3942	0.40963855
Beta-sitosterol	18.0	342.33905	0.4063745
Stigmasterol	18.0	352.47723	0.4063745
Inermin	17.0	323.1763	0.40316206
Fumarine	15.0	223.88904	0.39688715
Frutinone A	11.0	144.62103	0.38490567
Ginsenoside rh2	6.0	32.973564	0.3709091
Dianthramine	4.0	21.07494	0.3655914
Diop	2.0	2.9129128	0.36042404

Among these, PD is one of the three major ginsenosides, accounting for 8.75% of the total ginsenoside hydrolysates. PD exhibits numerous biological activities, including anticancer[17-19], immune-regulatory[20], neuroprotective[21], and radioprotective effects[22], with particularly strong anti-inflammatory and antioxidant properties demonstrated in many studies. It has been reported that PD significantly inhibits IL-1β secretion by suppressing the activation of non-canonical caspase-8 inflammasome and MAPKs in macrophages[14]. In addition, Mi et al[23] confirmed that PD markedly reduces inflammation and oxidative stress in both mice with non-alcoholic fatty liver disease and in vitro models. However, the underlying bioactive interactions between PD and UC remain unclear. Therefore, we further investigated and validated the therapeutic effects of PD on UC in a mouse model.

PD alleviated weight loss, reduced DAI scores, and prevented colon shortening in DSS-induced mice

Experimental colitis induced by DSS closely mimics the symptoms and histopathological features of UC, making it a widely accepted animal model for UC research. Key indicators used to assess the severity of DSS-induced colitis include weight change, DAI, colon length, and intestinal injury. In this study, mice in the DSS group exhibited significant weight loss starting from the 5^th day of DSS administration, whereas those in the DSS + PD group showed a milder weight loss trend (Figure 4A). The DAI, which reflects the overall disease severity, where higher scores indicate greater severity, was lower in the DSS + PD group compared to the DSS group (Figure 4B). Additionally, colon shortening, another indicator of colitis severity, was significantly more pronounced in the DSS group but less severe in the DSS + PD group (Figure 4C).

Figure 4 Panaxadiol alleviated weight loss, reduced the Disease Activity Index score, and prevented colon shortening in mice with dextran sulfate sodium-induced colitis. A: Body weight changes; B: Disease Activity Index score; C: Colon length. Data are expressed as mean ± standard error of the mean (n = 8). ^aP < 0.05; DAI: Disease Activity Index; DSS: Dextran sulfate sodium; PD: Panaxadiol.

PD relieved intestinal inflammation in DSS-induced mice

We performed HE staining on colon sections to evaluate the alleviating effect of PD in DSS-induced colitis. The DSS group exhibited significant infiltration of inflammatory cells, glandular destruction, crypt loss, and formation of crypt abscesses, whereas these pathological damages were alleviated in the DSS + PD group (Figure 5A), as indicated by the histological scores (Figure 5B). RT-qPCR analysis revealed that DSS treatment increased the expression of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β, which were reduced by pretreatment with PD (Figure 5C). Additionally, DSS increased the expression of iNOS and COX-2, whereas PD treatment reversed this trend (Figure 5D). These data indicate that PD effectively reduces intestinal inflammation in mice with DSS-induced colitis.

Figure 5 Panaxadiol attenuated intestinal inflammation in mice with dextran sulfate sodium-induced colitis. A: Representative images of hematoxylin-eosin staining (× 40 and × 200 magnification); B: Histopathological score; C and D: Panaxadiol treatment reduces pro-inflammatory cytokines (C) and pro-inflammatory mediators (D) in mice with dextran sulfate sodium-induced colitis. Data are expressed as mean ± standard error of the mean (n = 6). ^aP < 0.05; ^bP < 0.01; DSS: Dextran sulfate sodium; PD: Panaxadiol.

PD maintained the integrity of the intestinal epithelial barrier in mice with DSS-induced colitis

In this study, we investigated the function of the intestinal epithelial barrier, focusing on the mucus barrier and tight junction proteins. Using AB/PAS staining, we found that DSS significantly reduced mucin expression compared to the control group, whereas treatment with PD mitigated this decrease (Figure 6A). Immunohistochemistry analysis revealed that DSS lowered the expression of tight junction proteins (claudin-1, occludin, and ZO-1) in colon tissue, but PD treatment increased their expression (Figure 6B-D). Overall, our results demonstrate that while DSS disrupted the intestinal epithelial barrier, PD treatment enhanced the expression of mucin and tight junction proteins, thereby maintaining the integrity of the intestinal epithelial barrier in mice with DSS-induced colitis.

Figure 6 Panaxadiol maintained the integrity of the intestinal epithelial barrier in mice with dextran sulfate sodium-induced colitis. A: Alcian blue and periodic acid-Schiff staining (× 40); B-D: Immunohistochemistry analyses of claudin-1, occludin, and ZO-1 (× 100 magnification). Data are expressed as mean ± standard error of the mean (n = 8). ^aP < 0.05; ^bP < 0.01; DSS: Dextran sulfate sodium; PD: Panaxadiol.

PD regulated the activation of MAPK/NF-кB and AMPK/NRF2/NQO1 signaling in mice with DSS-induced colitis

NF-κB and MAPKs (including p38, ERK1/2, and JNK1/2) are crucial regulators of pro-inflammatory mediators and cellular responses to cytokines, with known interactions between their signaling pathways[24]. To investigate the effect of PD on these pathways, western blot analysis was conducted on colon tissue homogenates. The results revealed that DSS treatment significantly increased the phosphorylation levels of NF-κB p65, p38, ERK1/2, and JNK1/2 compared to the control group. However, PD treatment effectively attenuated these elevated phosphorylation levels (Figure 7A), suggesting its potential role in modulating inflammatory responses through these signaling pathways.

Figure 7 Panaxadiol regulated the activation of MAPK/NF-κB and AMPK/NRF2/NQO1 signaling in mice with dextran sulfate sodium-induced colitis. A: Effect of panaxadiol on the MAPK/NF-κB signaling pathway; B: Effect of panaxadiol on the AMPK/NRF2/NQO1 signaling pathway. Data are expressed as mean ± standard error of the mean (n ≥ 3). ^aP < 0.05; ^bP < 0.01; DSS: Dextran sulfate sodium; PD: Panaxadiol.

NRF2 is a crucial transcription factor that regulates oxidative stress response and inhibits inflammation, with its activation promoting the expression of NQO1, thereby exerting anti-inflammatory and antioxidant effects[25,26]. There is also a potential crosstalk between AMPK and NRF2, where AMPK can enhance the strength and duration of NRF2 signals[27]. In this study, we evaluated the effects of PD on the AMPK/NRF2/NQO1 pathway in colonic tissue homogenates. The results showed that the phosphorylation level of AMPK and the expressions of NRF2 and NQO1 were significantly reduced in the DSS group, but PD treatment reversed this trend (Figure 7B).

These findings suggest that PD may ameliorate DSS-induced colitis in mice, partially through the MAPK/NF-κB and AMPK/NRF2/NQO1 signaling pathways.

DISCUSSION

The complex pathogenesis of UC has led to unsatisfactory therapeutic outcomes, requiring long-term maintenance treatment with medication even surgery, which can have toxic side effects or cause various complications[28]. This situation has created an urgent need for more effective treatment options. TCM has shown promise in treating UC[29], but its complex components and diverse effects make research challenging. Network pharmacology, which applies network analysis of biological systems based on the theory of systems biology, has emerged as a valuable method for studying the components and mechanisms of TCM[30]. While P. ginseng has demonstrated promise and superiority in treating UC[31], a comprehensive understanding of its underlying material basis and molecular mechanisms in treating UC is lacking. To address this gap, network research was conducted to elucidate the regulatory mechanisms of P. ginseng in the treatment of UC, potentially offering more efficient experimental methods and novel ideas for the treatment of UC with TCM.

By integrating the targets of P. ginseng and UC, 223 intersection genes were identified, leading to the construction of a PPI network. From this network, 15 hub genes were identified, namely IL6, AKT1, TNF, PPARG, EGFR, STAT3, CASP3, BCL2, NF-кB1, PTGS2, ESR1, MMP9, ERBB2, SRC, and TLR4, which are primarily associated with inflammation[32], oxidative stress[33], apoptosis[34], and proliferation[35]. GO analyses of these targets revealed their involvement in various biological processes, cellular components, and molecular functions. The KEGG pathway analysis further suggested that P. ginseng may treat UC by influencing the PI3K-AKT signaling, MAPK signaling, and Ras signaling pathways.

Furthermore, the main active ingredients of P. ginseng for treating UC were identified, including PD, Gomisin B, and Ginsenoside-Rh4_qt. Among these, PD, one of the three major ginsenosides found in P. ginseng, has a higher content in P. ginseng and has demonstrated significant anti-inflammatory properties[14]. Research has shown that PD inhibits MAPK activation regulated by ZFP-91, thereby reducing IL-1β secretion[14]. Additionally, studies have revealed its ability to inhibit HIF-1α synthesis through the PI3K and MAPK pathways, as well as STAT3 activation via JAK1, JAK2, and Src pathways in human colon cancer cells[18]. PD has also been found to activate the Nrf2/HO-1 and PI3K/AKT/mTOR signaling pathways, which are associated with ferroptosis in iron-overloaded alkaline mice[36]. Despite these findings, research on the effects of PD on UC remains limited, prompting further investigation into its potential therapeutic mechanisms for the treatment of UC.

The DSS-induced colitis model is widely used to study the function of new drugs and the mechanisms underlying UC owing to its simplicity and many similarities with UC[37]. In this study, we utilized the DSS-induced colitis model to investigate the effects of PD on UC. Typical symptoms, such as weight loss, colon shortening, and elevated DAI scores, are commonly used to evaluate the severity of DSS-induced colitis[38]. Our findings demonstrated that PD effectively mitigated weight loss, prevented colon shortening, and reduced DAI scores in mice with DSS-induced colitis. Additionally, HE staining results of colon tissue and histological scores indicated that PD reduced the intestinal inflammatory response.

The immune response plays an important role in the occurrence, exacerbation, and persistence of UC. Loss of immune tolerance leads to the overproduction of various pro-inflammatory mediators by different cell types, resulting in the proliferation of antigen-specific effectors, triggering the adaptive immune system, and causing local and systemic inflammation[39]. Cytokines, as cell signaling molecules, are major contributors to inflammation and the pathogenesis of UC. In this study, RT-qPCR analysis revealed that PD effectively reduced the DSS-induced increase in mRNA expression levels of several pro-inflammatory cytokines and inflammatory mediators, including TNF-α, IL-6, IL-1β, iNOS, and COX-2.

The intestinal barrier, which consists of physical, chemical, immune, and biological components, is crucial for maintaining intestinal homeostasis, and its disruption plays a key role in the development of UC[40]. TCM has demonstrated advantages in maintaining and repairing the intestinal barrier, primarily by regulating tight junction proteins, mucin production, gut microbiota composition, inflammatory cell and mediator infiltration, and reducing intestinal oxidative stress[41]. In this study, AB/PAS staining indicated that PD increased the expression of mucin, whereas immunohistochemical analysis showed that PD enhanced the expression of tight junction proteins, including ZO1, claudin-1, and occludin. These findings indicate for the first time that PD helps maintain intestinal barrier function in mice with DSS-induced colitis, highlighting its potential as a therapeutic agent for the treatment of UC.

The MAPK signaling pathway, which includes p38, ERK1/2, and JNK1/2, plays a critical role in the occurrence and development of UC by activating transcription factors C-Fos and C-Jun, which regulate the transcription of inflammatory mediators such as IL-6 and TNF-α, contributing to intestinal inflammation[42]. NF-κB, a widely studied downstream transcription factor of MAPK, is activated when MAPK triggers mitogen- and stress-activated protein kinase-1, resulting in the phosphorylation of NF-κB[43]. Under normal conditions, NF-κB exists in the cytoplasm as a complex with the inhibitory protein IκB. Upon activation by extracellular signals, the IκB kinase phosphorylates IκB, causing its dissociation from NF-κB, which then gets activated and participates in the secretion of inflammatory factors, causing intestinal inflammation[44]. In this study, network analysis identified the MAPK signaling pathway as a primary pathway through which P. ginseng exerts its therapeutic effects on UC. Additionally, we found that DSS treatment significantly increased the phosphorylation of NF-κB p65, p38, ERK1/2, and JNK1/2, whereas PD treatment significantly reduced these phosphorylation levels. This suggests that PD alleviates UC, in part, through the MAPK/NF-κB signaling pathway.

NRF2, a stress-responsive transcription factor, maintains cellular homeostasis by regulating antioxidant and anti-inflammatory gene expression. Under normal conditions, NRF2 forms a complex with Keap1 in the cytoplasm. When oxidative or electrophilic stress occurs, Keap1 dissociates from NRF2, allowing NRF2 to translocate to the nucleus and induce gene expression. The NRF2/Keap1 axis is crucial for maintaining normal gastrointestinal function[45]. Studies have shown that NRF2 expression is significantly increased in the intestinal mucosa of patients with UC compared to controls[46], highlighting its role in alleviating intestinal inflammation, mitigating oxidative stress, and promoting maintenance of the intestinal epithelial barrier, making it a potential target for the treatment of UC[47]. AMPK, a crucial enzyme present in eukaryotes, regulates cell growth and energy metabolism and interacts with NRF2. AMPK can directly phosphorylate NRF2, enhancing its ability to defend against oxidative stress, and can also affect NRF2-mediated responses through transcription factor modifications. Conversely, a lack of AMPKα weakens NRF2-mediated defense functions[27]. In this study, PD increased the phosphorylation of AMPK and the expression of NRF2 and NQO1 in mice with DSS-induced colitis, suggesting that PD helps reduce intestinal inflammation and maintain the intestinal barrier partly through the AMPK/NRF2/NQO1 pathway.

CONCLUSION

Network pharmacology is an efficient methodological perspective for studying the mechanism and main components of TCM. By applying this analytical approach, P. ginseng was found to exert multi-component, multi-target, and multi-pathway therapeutic effects on UC, providing a theoretical basis for further research. PD, a key component of P. ginseng, was evidenced for the first time to alleviate colitis and help maintain the intestinal epithelial barrier, effects that were partly mediated through the MAPK/NF-κB and AMPK/NRF2/NQO1 signaling pathways. While these findings suggest that PD is a promising drug for the treatment of UC, further in-depth research is necessary to establish its optimal potential in clinical application.