Peiminine attenuates inflammatory bowel disease by suppressing TLR4 mediated NFƙB pathway: an integrated network pharmacology and experimental validation

Abstract

Ulcerative colitis and Crohn’s disease are two of the complex and recurring inflammatory conditions that comprise inflammatory bowel disease. There are currently relatively few therapeutic alternatives available for its treatment, and those that are available have serious side effects. It is also in a very progressive and prevalent phase. Therefore, it is necessary to find safer and more effective therapeutics for its treatment. Targeting different molecular pathways, peiminine has antioxidant and anti-inflammatory qualities. Integrated network pharmacology and molecular docking depicted various targets, amongst them NFƙB and TNF-α were the targets where peiminine elicited maximum binding affinity. Validating the in silico data, further in in vivo study, mice were randomly allocated into six groups: Normal control (NC), Disease control (DC-TNBS, 2,4,6-trinitrobenzene sulfonic acid), STD (TNBS + sulfasalazine 100 mg/kg, p.o.), PEI1, PEI2 & PEI3 (TNBS + Peiminine 1, 2.5, & 5 mg/kg, p.o.), respectively. A sharp fall in disease activity index (DAI) and changes in histopathological examination revealed the colonoprotective effect of peiminine. It also depicted anti-oxidant activity through reducing the levels of myeloperoxidase (MPO) and nitric oxide (NO) and elevating superoxide dismutase (SOD), and catalase (CAT). It downregulated various pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in in vitro and in vivo studies, depicting its anti-inflammatory activity. Peiminine has also been shown to have strong anti-inflammatory properties by downregulating the gene expression of TLR4 (1.96-fold), NFƙB (1.70-fold), iNOS (1.27-fold), COX-2 (2.40-fold), and IL-1β (0.80-fold). Meanwhile, it increases the expression of anti-inflammatory mediator IL-10 (2.55-fold). Collectively, peiminine likely decreased colon damage and TNBS-induced colitis through the TLR4-mediated NFƙB pathway.

Introduction

Inflammation of the mucosal lining of the gastrointestinal system is a characteristic of inflammatory bowel disease (IBD). This umbrella term includes two main forms: Crohn’s disease (CD) and ulcerative colitis (UC)¹. The hallmark of UC is inflammation, which starts in the rectum and can spread to other parts of the proximal colon. There are multiple factors responsible for the cause and progression of UC. However, the etiology of UC is still unknown. According to the epidemiological studies of IBD, it is anticipated that India will have one of the greatest populations of IBD patients globally². Currently, there is no “gold standard” treatment for IBD. UC is typically treated with 5-aminosalicylic acid (5-ASA), immunosuppressants, TNF-α inhibitors, steroids, and targeted molecular drugs, the use of which varies based on the extent and degree of the disease^3. These medications reduce inflammation, but they come with several unfavourable side effects. Consequently, when UC worsens, people with the illness often have a lower quality of life⁴.

Medicinal herbs have been used for centuries to treat a wide range of diseases⁵. Phytochemicals with anti-oxidant, anti-inflammatory, and neuroprotective properties can be found in botanical sources⁶. Peiminine is an alkaloid compound that naturally occurs in the plant Fritillaria bulbs (family: Liliaceae) from a traditional Chinese medicine⁷. In China, Japan, Turkey, and other Asian nations, it is frequently utilised due to its strong anti-inflammatory effect, and other pharmacological actions like neuroinflammation, atopic dermatitis, ulcer healing, osteoclastogenesis, pulmonary diseases, and mastitis⁸. Notably, peiminine is reportedly having anti-inflammatory properties via acting on various targets, including NFƙB, MAPK & PI3K/Akt and having ulcer healing properties, suggesting its application in GIT disorders.

The implementation of computational pharmacology has become increasingly popular in determining a bioactive’s ability to alleviate a disease⁹. Network pharmacology (NP) is a state-of-the-art method used to find possible therapeutic drugs and learn more about the therapeutic mechanisms behind complex illnesses¹⁰. It has been widely used for newer drug discovery due to its ability to identify relevant genes and target several sites in signalling pathways. NP is essential for the development of herbal medicines as therapeutic agents¹¹. To validate the computational work, the identified hub genes are evaluated in an in-vivo animal model.

The researchers have suggested, nuclear factor kappa B (NFƙB) is a key target responsible for the development and progression of UC. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are two transcriptional enzymes that researchers have discovered¹² are activated by it, potentially leading to colon inflammation¹³. Thus, targeting NFƙB would be favourable in battling against inflammatory complications. Peiminine attenuated LPS-induced acute lung injury in mice by acting through NF-κB pathway. It also showed anti-inflammatory effects in LPS-induced mastitis and lung injury models, eliciting significant suppression of NF-κB and associated signaling pathways in mice. Peiminine has been shown in research to have anti-inflammatory and antioxidant properties in the treatment of acne and myocardial injury^14,15. Researchers have studied the effect of peiminine on IBD through various models. It attenuated colitis by enhancing the function of the intestinal epithelial barrier through Nrf2/HO1 signalling pathway. Peiminine elicited anti-inflammatory activity in acetic-acid induced colitis model by targeting various proinflammatory mediators^16,17.

However, we hypothesized that peiminine could functionally suppress the TLR4–NF-κB inflammatory signaling axis and could alleviate IBD as predicted from the computational network pharmacology approach. The current study is the first study in our knowledge to showcase the targeted relationship of peiminine and IBD through integrated network pharmacology, molecular docking, in vitro and in vivo TNBS-induced colitis model via targeting TLR4-mediated NFƙB pathway. In line with this, various methods were utilised to investigate the potential of peiminine in combating IBD.

Materials and methods

Computational pharmacology

Identification of potential targets

The target fishing for peiminine, as an identified bioactive phytoconstituent can be done by exploring various databases. The simplified molecular-input line-entry system (SMILES) of peiminine was acquired from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/)¹⁸. The canonical SMILES or 2D structure of the query molecule was imported into the databases. Peiminine’s targets had been retrieved using Super-PRED (https://prediction.charite.de/subpages/target_prediction.php) and the Swiss Target Prediction Database (http://www.swisstargetprediction.ch/). Further downstream operations were carried out using the provided targets.

UC and CD-related target prediction

Several databases, such as DisGeNET (https://disgenet.com/), DrugBank (https://go.drugbank.com/), and GeneCards (https://www.genecards.org/), were used to identify the targets implicated in the pathophysiology of IBD¹⁹. Every target was validated using the UniProtKB database (https://www.uniprot.org/). To obtain all of the IBD targets, the combined UC and CD targets were utilized.

Protein–protein interaction network

STRING version 12.0 (https://string-db.org/) was used to process the overlapping data on peiminine and UC target genes in order to obtain the data required for the KEGG pathway, gene ontology, and protein–protein interaction (PPI) studies. To obtain a more visible network diagram²⁰, the PPI values that were obtained from STRING 12.0 were then imported into Cytoscape 3.10.3. The Cytoscape Network Analyzer set up was used to analyze this network. The top ten peiminine intended genes were then processed using the CytoHubba plugin and MNC (Maximum Neighbourhood Component).

Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis

The protein–protein interaction data obtained from STRING 12.0 were further analysed through functional enrichment using DAVID (https://david.ncifcrf.gov/) and INPUT http://cbcb.cdutcm.edu.cn/INPUT/Z). The three categories of Gene Ontology (GO) enrichment were molecular function (MF), cellular component (CC), and biological process (BP). The enrichment analysis encompasses a total of 72 expected targets. Using the Bioinformatics web platform (http://www.bioinformatics.com.cn/), the top 10 phrases from BP, CC, and MF as well as the top 20 KEGG pathways, were chosen for enrichment analysis.

Molecular docking

Molecular docking is a widespread computational technique that predicts a ligand’s capacity to bind with a particular protein at a specific location. The hub genes discovered by network analysis and putative proteins implicated in the pathophysiology of IBD were subjected to molecular docking. Peiminine’s three-dimensional structure was obtained in SDF format from PubChem (https://pubchem.ncbi.nlm.nih.gov/). NFƙB, AKT, and EGFR are among the important targets whose crystal structures were obtained in PDB format from the Protein Data Bank (PDB) database (https://www.rcsb.org/). To determine peiminine’s binding affinity for the target, docking was done via AutoDock Vina using the PyRx platform. A higher ligand-target binding stability is indicated by a more negative Vina score. The docked structures were visualized using BOVIA Discovery Studio Visualizer v21.0.20298 (https://discover.3ds.com/discovery-studio-visualizer-download).

Experimental pharmacology

In vitro assessment of peiminine

From NCCS Pune, the RAW 264.7 mouse monocyte-macrophage cell line was acquired. Raw cells were cultivated at 37°C in a humidified incubator with 5% CO2 and 95% air in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 5% heat-inactivated foetal bovine serum, 100 g/ml streptomycin, and 100U/ml penicillin to prevent bacterial contamination. It was standard procedure to switch the medium every 2 days. After reaching about 80% confluence, the RAW 264.7 cells were passaged²¹.

Cytotoxicity assessment MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) was used to assess the vitality of RAW cells after peiminine treatment. RAW cells were treated in 96-well plates with varying doses of peiminine (10, 20, 40, 80, and 120 μM) and STD (sulfasalazine) as a standard treatment for 24 h. After that, 200 μg/mL of MTT solution was added. The supernatant was removed after an extra 4 h of incubation at 37 °C, and 100 μl of DMSO was used to dissolve the reduced formazan. The absorbance was determined using a microplate reader at 570 nm. Cell viability for the untreated cells was regarded as 100%. Comparing the results to the untreated group, the results are shown as a percentage of cell viability²².

Determination of NO:The cells of RAW 264.7 cell line were grown in 96-well plate plates for 24 h following treatment with different doses of peiminine (10, 20, 40, 80, and 120 μM) as well as 5-ASA as a reference treatment and LPS (1 μg/mL)²¹. After extracting 100 μL of the supernatant, the mixture was mixed with 100 μL of Griess reagent, which is a combination of equal amounts of reagents A and B. Reagent A was 1% sulphanilamide in 5% H3PO4, while Reagent B was 0.1% naphthyl ethylene diamine dihydrochloride. The supernatant was shaken for 20 min at ambient temperature. The nitrite concentrations were then calculated by measuring the absorbance at 540 nm and creating a standard calibration curve. It was expressed as µM of the nitrites that the cells secreted²³.

Determination of TNF-α and IL-6 levels RAW 264.7 cells were seeded in 96-well plates, and they were either pre-treated with peiminine at 10, 20, 40, 80, and 120 μM or left untreated for 24 h before being exposed to 1 μg/mL of LPS for 6 h. TNF-α and IL-6 concentrations in the supernatant were determined with the help of an ELISA kit in accordance with the manufacturer’s instructions.

In vivo assessment of peiminine

Experimental study design Six to eight-week-old Balb/c mice, weighing 20–30 gm, were procured from Zydus Research Centre. The cages held eight animals each, and the bedding was made of corn cobs. Polypropylene cages were utilised to house the animals. Temperature (22–250 °C), humidity (60%), and lighting schedule (12 h of light and 12 h of darkness) were all maintained within a 5% deviation. The research protocol was approved by the Institutional Animal Ethics Committee (IAEC) (RPCP/IAEC/2023-24/R2). All procedures were performed in accordance with the ethical standards of the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, and ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations. The animals were split up into six groups. NC (Normal Control, Saline), DC (Disease Control, only 2,4,6-trinitrobenzene sulfonic acid (TNBS), STD (sulfasalazine 100 mg/kg/day, p.o.), PEI1 (Peiminine 1 mg/kg/day, p.o.), PEI2 (Peiminine 2.5 mg/kg/day, p.o.), and PEI3 (Peiminine 5 mg/kg/day, p.o.).

Induction of IBD through TNBS In animals, the haptenizing agent TNBS in ethanol is found to cause immunogenic reactions in the colon wall that resemble human colitis when given intracolonically²⁴. After 2 days of post-treatment (PEI and Sulfasalazine), the mice were fasted for a full day. The mice were lightly anaesthetized with a cocktail of ketamine (100 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection before induction. 100 μL of TNBS (2.5% TNBS in 50% ethanol) was progressively injected into the descending colon using an 18-gauge cannula that was placed 3.5–4 cm proximal to the anus. The normal control group is provided with only 50% ethanol. On 7th day post TNBS induction the mice were euthanized by inhalation of excess CO₂ and colon samples were collected. Scoring parameters, colon length and weight were measured. For histological analysis, the proximal portion of the colon was preserved in a 10% formaldehyde solution. Colon homogenate was utilised for measuring various pro-inflammatory cytokines and oxidative stress parameters²⁴.

Disease activity index (DAI) scoring Post TNBS administration, weight loss, stool consistency, and rectal hemorrhage were measured on a daily basis. The DAI was calculated by dividing the sum of the stool consistency score, stool occult blood score, and body weight loss score by 3. The DAI was determined by combining these parameters and applying the scoring scheme shown in Table 1²⁵.

Table 1.

The scoring of the disease activity index (DAI).

Body weight loss (%)	Stool consistency	Bleeding	Score
< 0	Normal	Negative	0
1–5			1
6–10	Loose stools	Hemoccult positive	2
11–15			3
> 15	Diarrhea	Gross bleeding	4

Histological assessment of the colon After being embedded in paraffin, the colon samples were kept in a 10% formalin solution. Colon was cut up into tiny pieces, each 5 μm thick, and stained with haematoxylin and eosin (H&E). To determine the extent of inflammatory infiltration, the slices were next examined under a microscope. The injury to the intestinal mucosa was scored²⁶.

Estimation of oxidative stress The samples of colon were homogenized with cold phosphate-buffered saline (PBS), and after centrifugation, the levels of catalase, superoxide dismutase (SOD), nitric oxide (NO), and malondialdehyde (MDA) were measured in the supernatant²⁷. The MDA level was measured at 523 nm as per the description of Ohkawa et al.²⁸. The Griess reagent is used to measure the NO level, according to a study by Green et al. and the results were expressed as μM/g of protein²⁹. Using the technique outlined by Misra et al. the SOD was examined. The results were measured at 450 nm and reported as U/g of tissue³⁰. The activity of catalase was measured spectrophotometrically by recording the changes in absorbance at 240 nm, as units/mg Protein.

Estimation of TNF‑α and IL‑6 levels Colon tissue samples were homogenized with PBS solution and then centrifuged at 12,000 g for 10 min at 4 °C. After that, the supernatant was moved to fresh tubes. TNF-α and IL-6 levels were determined using a standard protocol of the enzyme-linked immunosorbent assay (ELISA) utilizing mice ELISA kits from Krishgen BioSystems, India. The results were expressed as pg/mL.

Quantitative real‑time PCR Colon tissues were preserved in RNA Protector (Takara Bio, USA) and stored at − 80 °C until further processing. The NucleoSpin RNA isolation kit (Takara Bio, USA) was used to purify the total RNA after it was extracted using TRIzol reagent. A QIAxpert nanodrop spectrophotometer was used to measure the concentration and purity of RNA. Then, using the PrimeScript First-Strand cDNA Synthesis Kit (Takara Bio, USA), 2 µg of total RNA was reverse-transcribed into cDNA. As directed by the manufacturer, quantitative real-time PCR was performed using TB Green Premix Ex Taq II (Tli RNase H Plus, Takara Bio, USA). Gene expression levels were analysed by recording cycle threshold (Ct) values, and relative expression was calculated using the 2 − △△CT method with GAPDH serving as the internal control²⁴.

Statistical analysis

GraphPad Prism (version 9.0.0, CA, USA, Trial version) was used for data processing. The disease activity index and histological scoring parameters were analysed using Kruskal-Wallis with Dunn’s multiple comparison test. Whereas, all other parameters were evaluated using one-way analysis of variance (ANOVA), Tukey’s multiple comparison test was employed. The mean ± Standard Error of Mean (SEM) was used to display the data. p < 0.05 was considered a significant level.

Results

Computational pharmacology

Target identification of peiminine

The target fishing of peiminine was done using Super-PRED and Swiss Target Prediction databases. In total, 198 targets of Peiminine were identified. The network was constructed using Cytoscape and was further utilised for finding common targets of peiminine and IBD.

Retrieval of potential targets of IBD

A keyword-based search was used to find a total of 1459 UC-related genes and 1383 CD-related genes from the DisGenet database. A total of 5140 UC-related genes and 5401 CD-related targets were retrieved from GeneCards, OMIM, and TTD databases. With a total of 95 targets from peiminine and 6974 targets to UC and CD, they shared 95 targets in common.

Protein–protein interactions

For protein–protein interaction, 198 targets of peiminine were put into a string database. The PPI network of peiminine comprises of 71 nodes and 114 edges in an interaction network; PPI enrichment p-value was < 1.0e−16 (Fig. 1A). The confidence interval of 0.9% with the highest confidence was selected for the analysis of PPI. The PPI network was analysed using Network Analyzer tool in Cytoscape, and the targets for higher than 7 degrees were selected for further analysis. Figure 1B depicts the top 10 genes targets of peiminine analyzed using CytoHubba plugin and the network analyzer plugin of Cytoscape.

Fig. 1.

Network pharmacology of Peiminine, (A) Protein–protein interaction of targets of peiminine against IBD. (B) Top 10 hub targets of peiminine analyzed using the CytoHubba plugin and the network analysis plugin of Cytoscape. (C) Venn diagram depicting 95 common targets of peiminine and IBD (UC and CD).

Gene ontology and pathway enrichment analysis

To elucidate the primary biological functions of peiminine in ulcerative colitis (UC), GO and KEGG enrichment analyses were performed for the 72 overlapping genes identified between peiminine-associated targets and UC-related targets. GO-Biological Process (BP) enrichment indicated that these genes were mainly associated with responses to diverse stimuli (GO:0048584), cellular responses to chemical stimuli (GO:0070887), responses to oxygen-containing compounds (GO:1901700), and cell-to-cell communication processes (GO:0010646) (Fig. 2A). GO-Cellular Component (CC) analysis showed significant enrichment in vesicle-related components (GO:0031982), plasma membrane regions (GO:0005886), cytoplasmic compartments (GO:0005737), and protein kinase complexes (GO:0032991) (Fig. 2B). GO-Molecular Function (MF) results revealed enrichment in binding-related activities, catalytic functions (GO:0003824), transcription-associated functions (GO:0008134), kinase activity (GO:0,016,301), signalling receptor activity (GO:0038023), and protein binding (GO:0005515) (Fig. 2C).

Fig. 2.

Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis of the predicted targets. (A) Top 20 KEGG pathways. (B) Top 10 enriched Biological Process (BP) terms. (C) Top 10 enriched Cellular Component (CC) terms. (D) Top 10 enriched Molecular Function (MF) terms. GO terms are represented as bar graphs and ranked in order according to p-values. The blue colour bar shows most genes associated with GO terms, followed by magenta and red colours.

To further uncover the potential mechanisms underlying the therapeutic action of peiminine, KEGG pathway enrichment analysis was conducted on 72 predicted targets, from which the top 20 enriched pathways were identified. These pathways included the HIF-1 signalling pathway (hsa04066), pathways in cancer (hsa05200), Toll-like receptor signalling (hsa04620), EGFR tyrosine kinase inhibitor resistance (hsa01521), PI3K-Akt signalling (hsa04151), and the sphingolipid signalling pathway (hsa04071) (Fig. 2D). Additionally, the TLR4/NFƙB pathway is the main signalling cascade with which the targets of peiminine showed higher correlations. Notably, the majority of predicted targets overlapped with these pathways, which are highlighted with pink markers in the pathway map (Fig. 3). Significantly, these pathways are closely linked to the development and course of inflammatory bowel disease (IBD), offering compelling mechanistic evidence for the therapeutic value of peiminine in the treatment of IBD.

Fig. 3.

TLR4/NFƙB pathway. The pink highlights represent targets of Peiminine and its association with the TLR4-mediated NFƙB signalling pathways. KEGG copyright permission was received from Kanehisa laboratories⁴⁸.

Validation by molecular docking

Based on animal studies and network pharmacology findings, peiminine was evaluated for molecular docking with the top ten targets, NFƙB (PDB ID: 4DN5), JAK-2 (PDB ID: 3UGC), MAPK 1 (PDB ID: 4FUX), EGFR (PDB ID: 1M17), CASP3 (PDB ID: 3DEI), AKT (PDB ID: 4FA6), PI3K (PDB ID: 1E7U), and TNF- α (PDB ID: 2AZ5). The binding energy revealed the strength of the interaction between peiminine and their respective target proteins, with stronger interactions being represented by lower (more negative) values. Table 2 showed the binding energies (in kcal/mol) for peiminine with respect to IBD-related targets. Based on network pharmacology and molecular docking studies, the drugs appear to have a greater affinity for NFƙB. Peiminine is expected to work on the NFƙB pathway, which was further evaluated through in vivo animal studies.

Table 2.

Molecular docking score of peiminine with targets.

Target	PDB ID	Docking score	2D interaction
NFƙB	4DN5	− 11.7
JAK-2	3UGC	− 11
MAPK 1	4FUX	− 9.93
EGFR	1M17	− 9.66
CASP3	3DEI	− 9.61
AKT	4FA6	− 9.08
TLR4	3FXI	− 8.90
PI3K	1E7U	− 8.76
TNF-α	2AZ5	− 8.09

Experimental pharmacology

Result of in vitro assessment

Effect of peiminine on MTT assay The MTT test was carried out to assess the impact of peiminine on cell viability. After a 24-h treatment, RAW 264.7 cell viability was unaffected by peiminine at concentrations between 10 to 120 μM. Hence, these concentrations of peiminine were considered suitable for further assays.

Effect of peiminine on LPS-stimulated inflammation LPS dramatically raised the NO level in RAW264.7 cells in only LPS treated group as compared to untreated (Normal control) group. A substantial (p < 0.05) decrease in LPS-inducible NO generation was seen following a 6-h pre-treatment with peiminine and STD (sulfasalazine). This reduction in nitric oxide was in dose-dependent manner, peaking at 120 µM/ml (Fig. 4A).

Fig. 4.

Effect of peiminine on inflammatory mediators, (A) Nitric Oxide concentration, (B) TNF-α concentration, (C) IL-6 concentration produced by LPS-treated RAW 264.7 cells. Values are expressed as the mean ± SEM. NC (normal control), DC  (Disease control, only TNBS), STD (sulfasalazine), PEI1, PEI2, and PEI3 (10, 20, 40, 80 & 120 µM), respectively. ### p<0.0001 compared to NC group. ** p<0.001 , * p<0.01 compared to LPS group.

Effect of peiminine on various pro-inflammatory mediators such as TNF-α and IL-6 in RAW 264.7 cells After RAW 264.7 cells were exposed to LPS for 6 h, an inflammatory state was induced, and the production of TNF-α and IL-6 in the LPS group was increased significantly. Peiminine administration, however, significantly (p < 0.05) decreased the release of pro inflammatory mediators in LPS induced RAW 264.7 cells. (Fig. 4B, C).

Protective effect of peiminine on TNBS-induced IBD model

Effect of peiminine on gross colonic changes against TNBS‑induced colitis Colonic inflammation is indicated by a decrease in colon length and an increase in the colon weight. The colon weight was elevated, and the colon length was significantly reduced (p < 0.0001) in the DC group than in the NC group. However, the treatment groups, PEI3 (5 mg/kg) and STD (sulfasalazine, 100 mg/kg) were able to significantly (p < 0.0001) prevent both the increase in the colon weight and the decrease in colon length (Fig. 5A–C).

Fig. 5.

Peiminine improved the clinical features and reduced macroscopic damage to the colon in mice with TNBS-induced colitis. (A) Representative image of the colon; (B) Colon weight of all experimental groups; (C) Colon length of all experimental groups; (D) Disease-activity index (DAI) score. Values are expressed as the mean ± SEM (n = 8). NC (normal control),  DC ( Disease control, only TNBS), STD (sulfasalazine, 100 mg/kg), PEI1, PEI2, and PEI3 (1, 2.5, and 5 mg/kg), respectively. ### p<0.0001, ## p<0.001 compared to NC; ** p<0.001, * p<0.01 as compared to DC group.

Effect of peiminine on disease activity index (DAI) Score against TNBS‑induced colitis The DC group had a significantly higher DAI score (p < 0.0001) than the NC group following TNBS. Sulfasalazine and peiminine inhibited the TNBS-induced reduction in body weight and bleeding, and improved stool consistency in a dose-dependent manner, as evidenced by a significant (p < 0.001) increase in DAI score compared to the DC group (Fig. 5D).

Effect of peiminine on TNBS‑induced histopathological changes in the colon The NC group’s colonic architecture was intact, as shown in (Fig. 6A). Major mucosal thickening, a decrease in infiltration of goblet cells and inflammatory cells, and a lack of crypt abscesses are all readily noticeable after TNBS treatment. Every indication of TNBS colitis was visible in the DC group’s H&E stain (Fig. 6B). Sulfasalazine and peiminine in dose dependent manner had prevented the colonic architecture, depicting its colonoprotective effect (Fig. 6C–F).

Fig. 6.

Protective effect of peiminine on the colonic damage due to TNBS-induced colitis observed in histology stained with H &E and examined at 40 × and 100 ×. Black arrow: tips of villi. Blue arrow: crypts and goblet cells. Green arrow: immune cell infiltration. Red arrow: red blood cell infiltration. (A) NC (normal control): Exhibits intact colonic architecture, normal-looking crypts and villi, and no signs of immune cell infiltration. (B) DC (Disease control, only TNBS): Loss of crypt, damaged goblet cells, loss of mucosal layer, and a massive accumulation of immune cells inside the crypts, and in the layers of the colon, depicted transmural inflammation. (C) STD (sulfasalazine, 100 mg/kg). (D–F) PEI1, PEI2, and PEI3 Peiminine (1, 2.5, and 5 mg/kg), respectively. Treatment groups demonstrated significantly improved colonic architecture and goblet cells in a dose-dependent manner, while also protecting against immune cell infiltration and villi shortening.

Effect of peiminine on oxidative stress parameters in colon tissues MDA displays lipid peroxidation brought on by free radicals. Oxidative damage is indicated by a high MDA level. The MDA levels in the colon tissues of the DC group were considerably (p < 0.0001) greater than those of the NC group. However, sulfasalazine and peiminine (PEI) prevented the increase in MDA levels (Fig. 7A). The elevated NO level observed in colitis is caused by inducible NO synthase (iNOS), which is generated by inflammatory and smooth muscle cells that induce colon inflammation. As shown in (Fig. 7B), compared to the NC group, the NO level of the DC group was significantly (p < 0.0001) higher. Sulfasalazine and peiminine (PEI, 5 mg/kg) significantly (p < 0.0001) reduced the NO level, indicating their antioxidant activity, and were instructive in reducing oxidative stress. Meanwhile, SOD and catalase is an enzyme that aids in protecting tissue from oxidative damage. In the TNBS model, a lower level of SOD is typically observed, and the current investigation showed comparable findings. As compared to the NC group, the DC group’s SOD and catalase level was significantly (p < 0.0001) lower. However, sulfasalazine and peiminine (2.5 mg/kg and 5 mg/kg) prevented the SOD & catalase level from declining, demonstrating their ability to protect against oxidative damage brought on by TNBS (Fig. 7C, D).

Fig. 7.

Effect of peiminine on oxidative stress parameters, (A) Malondialdehyde (MDA) levels. (B) Nitric oxide (NO) levels. (C) Superoxide dismutase (SOD) levels. And (D) Catalase levels. Values are expressed as the mean ± SEM (n = 8). NC (normal control), DC (Disease control, only TNBS), STD (sulfasalazine, 100 mg/kg), PEI1, PEI2, and PEI3 (1, 2.5, and 5 mg/kg), respectively. ### p<0.0001 compared to NC, ** p<0.001, * p<0.01 compared to DC group.

Effect of peiminine on TNF‑α and IL‑6 levels in colon TNBS leads to release of various pro-inflammatory mediators such as TNF‑α and IL‑6. The DC group had significantly (p < 0.0001) greater levels of TNF-α and IL-6 as compared to the NC group, eliciting inflammation in DC group. Nevertheless, peiminine and sulfasalazine significantly (p < 0.0001) reduced TNF-α and IL-6 levels as compared to the DC group, indicating the anti-inflammatory efficacy of peiminine (Fig. 8A, B).

Fig. 8.

Effect of peiminine on inflammatory mediators, (A) TNF-α concentration. (B) IL-6 concentration. Values are expressed as the mean ± SEM (n = 8). NC (normal control), DC (Disease control, only TNBS), STD (sulfasalazine, 100 mg/kg), PEI1, PEI2, and PEI3 (1, 2.5, and 5 mg/kg), respectively. ### p<0.0001 compared to NC, **p<0.001, p<0.01 compared to DC.

Effect of peiminine in alleviating the inflammatory burden in TNBS-induced colitis In TNBS-induced colitis, the TLR4-driven NFƙB axis plays a key role in the inflammatory cascade. To elucidate the regulatory effect of peiminine (PEI) on this pathway, the mRNA gene expression of key inflammatory mediators, including TLR4, NFƙB, iNOS, COX-2, IL-1β, and IL-10, were quantified. In the disease control (DC) group, TLR4 (4.92-fold), NFƙB (5.20-fold), iNOS (3.18-fold), COX-2 (6.85-fold) and IL-1β (4.08-fold) levels were substantially up regulated as compared to the normal control (NC) group (Fig. 9A–F), reflecting enhanced TLR4 signalling and subsequent NFƙB activation during colonic inflammation.

Fig. 9.

Peiminine downregulated various pro-inflammatory cytokines and enzymes, causing inflammation. The gene expression studies were performed in colon homogenate by real-time quantitative RT-PCR. The relative mRNA expression levels of TLR4, NF-κB, iNOS, COX-2, IL-1β, and IL-10 are shown in Panels (A–F), respectively. Data are presented as mean ± SEM (n = 8). NC (normal control), DC (disease control; TNBS only), STD (standard treatment; sulfasalazine 100 mg/kg), and PEI1, PEI2, and PEI3 (peiminine at 1, 2.5, and 5 mg/kg, respectively) are the experimental groups.

Once activated, NFƙB stimulates the transcription of downstream inflammatory enzymes like COX-2 and iNOS, whose elevated levels are associated with colitis and caused destruction to the mucosa. In comparison to the DC group, treatment with peiminine (5 mg/kg) or the standard drug sulfasalazine (100 mg/kg) significantly (p < 0.0001) reduced the expression of TLR4, NFƙB, iNOS, COX-2, and IL-1β. Peiminine demonstrated a strong downregulation of various pro-inflammatory mediators, TLR4 (1.96-fold), NFƙB (1.70-fold), iNOS (1.27-fold), COX-2 (2.40-fold), and IL-1β (0.80-fold), depicting its strong anti-inflammatory potential (Fig. 9A–F).

In contrast to these pro-inflammatory mediators, IL-10 counteracts excessive immune activation by acting as a crucial anti-inflammatory cytokine. Peiminine significantly raised IL-10 expression (2.55-fold) in contrast to the DC group, indicating its role in restoring immunological stability in the inflammatory colon (Fig. 9F).

Collectively, the profound (p < 0.0001) suppression of NFƙB activation in the PEI-treated groups suggests that peiminine exerts its anti-inflammatory effects by stabilising IκB-α and interfering with upstream TLR4 signalling. This disruption successfully reduces the transcription of several pro-inflammatory mediators, demonstrating the therapeutic potential of peiminine in the treatment of colitis brought on by TNBS.

Discussion

Colon ulcers are caused by ulcerative colitis (UC), a chronic inflammatory disease. Although the precise mechanisms of UC are still unknown³¹. Although there are several categories of drugs for the treatment of IBD, they have several limitations, such as serious side effects and exorbitant prices. Recent pharmacological findings, however, suggest that peiminine-like phytoconstituents are a good alternative for treating UC with fewer side effects. The alkaloid peiminine has several pharmacological properties, including anti-inflammatory and antioxidant effects brought about via the downregulation of specific pro-inflammatory cytokines.

Network pharmacology (NP) has become a useful technique in modern drug discovery and development as it shifts the focus from the traditional “one drug-one target” model to a more realistic “multi-target, multi-pathway” perspective¹⁰. In this study, the top ten hub targets of peiminine were evaluated by using the CytoHubba and Cytoscape network analysis plugins. Accordingly, the molecular docking results revealed that peiminine has a greater binding affinity for IBD targets. Overall, computational network pharmacology matches these potential targets of peiminine to the various components of IBD. KEGG pathway revealed the correlation of TLR4-NFƙB pathway, demonstrating its probable mechanism in attenuating IBD.

Researchers have depicted the correlation of lipopolysaccharide (LPS) and TLR4 as it binds to TLR4 and activates the NFƙB pathway, further releasing various pro-inflammatory mediators and more nitric oxide formation³². The anti-inflammatory effects of peiminine were first examined by exposing RAW 264.7 macrophage cells to 1 μg/mL LPS, which induced inflammatory responses. RAW 264.7 cells were not cytotoxically affected by peiminine, which also successfully reduced inflammation by inhibiting pro-inflammatory cytokines, including NO, TNF-α, and IL-6. Nitric Oxide (NO) is a well-known indicator of both acute and persistent inflammation, and iNOS controls the expression and synthesis of NO³³. Consequently, mononuclear macrophages are encouraged to infiltrate the site of inflammation, and inflammatory mediators such as TNF-α, and IL-6, and pain-producing chemicals can invade³⁴. Nitric oxide plays a vital role in practically every stage of the inflammatory process. Thus, one of the main objectives of anti-inflammatory medications is to inhibit NO. In this study, similar results were observed, and the anti-inflammatory qualities of peiminine were demonstrated by a significant reduction in NO, TNF-α, and IL-6 production in RAW 264.7 cells exposed to LPS.

In an in vitro investigation, peiminine showcased remarkable anti-inflammatory activity. Based on this, the Trinitrobenzenesulfonic acid (TNBS)-induced colitis model was utilized to validate the activity of peiminine through in vivo studies. An increase in neutrophil infiltration and macrophage activation is a hallmark of Th1-mediated immune responses, which are triggered by a hapten molecule known as TNBS that binds to the host protein³⁵. Numerous pro-inflammatory mediators are produced as a result of these interactions, which cause inflammation in the colon. The well-established model has characteristics similar to those of human CD, including severe transmural inflammation, diffuse necrosis, thickening of the large intestine wall, bloody diarrhea, and gradual weight loss³⁶. Similar results were observed in the present study, indicating the successful establishment of the model. However, treatment groups had depicted a colono-protective effect.

TNBS causes immune cells that resemble macrophages to become activated and infiltrate the intestinal mucosa, producing reactive oxygen species (ROS) and eventually NO. This results in oxidative stress and contributes to the pathogenesis of ulcerative colitis. Researchers stated that peiminine has shown antioxidant activity in IBD³⁷ and similar results are observed in our study. It has been shown to have an antioxidant effect by lowering NO levels. SOD is an antioxidant that cells need to defend themselves against ROS and the effects of free radical chain reactions. MDA rises and SOD levels fall in the TNBS-induced colitis model, indicating a rise in ROS production and oxidative damage. Fan et al. reported that osthole has showcased antioxidant activity, reducing MDA levels and elevating SOD levels³⁴. Similar results were observed in this study, peiminine therapy has shielded against oxidative damage and, consequently, ROS production by preventing the reduction of SOD and preventing the rise in MDA levels and nitric oxide levels.

According to the results of a prior study, UC is well known for altering the architecture of the colon in animals with colitis³⁸, which is characterized by a damaged mucosal barrier³⁹ and an enhancement in neutrophil infiltration in the mucosa. The intestinal mucosa produces reactive oxygen species (ROS) as a result of these activated neutrophils⁴⁰, which results in oxidative stress and contributes to the root cause of colitis. Oliveira et al. stated that Bergenine at the dose of 25 mg/kg demonstrated colonoprotective effect by reducing neutrophil infiltration and maintaining the colonic architecture⁴¹. Similar alterations in colon histology have been observed in the current investigation. where peiminine at 5 mg/kg dose have improved the colonic architecture and depicted the reduction in neutrophil infiltration, colonic mucosal damage, and red blood cell accumulation, eliciting its colonoprotective effect.

The precise mechanism by which peiminine protects the colon was uncertain and exploring the signaling pathways implicated in ulcerative colitis has been the main focus of recent research. To detect the presence of certain bacterial antigens, the innate immune system employs a broad family of pattern recognition receptors (PRRs)⁴². TLR4 is a PRR that identifies LPS, a key constituent of gram-negative bacteria’s outer membrane, and triggers the release of pro-inflammatory mediators such as NO, which ultimately leads to production of ROS resulting in the expression of the inflammatory response. Intestinal epithelial cells from IBD patients and the colonic mucosa of animals with experimental colitis also exhibit higher levels of it⁴³ Several studies have shown that inhibiting TLR4 signaling molecules results in the inhibition of pro-inflammatory cytokines and the inactivation of NFƙB¹⁴. Similarly, in our study, peiminine significantly decreased the NO production in LPS-stimulated RAW 264.7 cells and also inhibited various proinflammatory cytokines like TNF-α and IL-6 in the cell supernatant.

NFƙB acts as a central regulator of intestinal inflammation by regulating the transcription of several essential mediators, including iNOS, COX-2, and IL-1β⁴⁴. In order to determine the exact mechanism by which peiminine reduces TNBS-induced colitis, our study evaluated this pathway, as it was predicted via network pharmacology and molecular docking studies. The TLR4-NFƙB signalling pathway was activated, as shown by the experimental results, which showed that the Disease Control (DC) group had considerably greater mRNA levels than the Normal Control (NC) group. When NFƙB is activated, pro-IL-1β is transcriptionally induced and then matures into IL-1β, which intensifies the inflammatory response⁴⁵. Treatment with peiminine efficiently suppressed this signalling pathway and ameliorated inflammatory signals by significantly suppressing the expression of these pro-inflammatory genes. Conversely, IL-10 expression was substantially increased in the peiminine-treated group. Considering IL-10 is a crucial anti-inflammatory cytokine integrally engaged in IBD regulation, as widely reported, its elevation further substantiates the anti-inflammatory activity of peiminine⁴⁶.

The present findings align with earlier research, wherein Yang S et al. demonstrated that chicoric acid (10 mg/kg) mitigates colitis by inhibiting the TLR4/NFƙB signalling axis⁴⁷. Building on this premise, the main objective of our investigation was to discover whether peiminine similarly ameliorates IBD by modulation of this route. Collectively, peiminine showcased the highest binding affinity with NFƙB and TLR4 and in vivo studies supported the prediction and proved the antioxidant, anti-inflammatory, anti-colitic and colono-protective effect of peiminine against TNBS-induced colitis by suppressing TLR4-NFƙB signalling.

While the current work focused on transcriptional regulation of the TLR4/NF-κB pathway, earlier studies have shown that peiminine inhibits nuclear translocation and p65 phosphorylation, which mitigates NF-κB activation at the protein level. As a result, the modifications in gene expression demonstrated in this study coincide with the known molecular mechanism of peiminine. Importantly, the current study employed an integrated experimental strategy combining network pharmacology, molecular docking, biochemical assays, histopathological analysis, and in vivo gene expression profiling. The convergence of computational target prediction with transcriptional and biochemical findings strengthens the mechanistic interpretation of the anti-inflammatory activity of peiminine in combating IBD. Further research utilizing Western blot or immunohistochemical analysis can verify that this signaling pathway is modulated at the protein level.

Conclusion

Network pharmacology analysis identified TLR4 and NF-κB as key hub targets of peiminine, which was substantiated by high network centrality and strong enrichment of TLR and NF-κB signaling pathways in KEGG analysis—both of which are essential to the pathophysiology of inflammatory bowel disease. Molecular docking depicted the highest binding affinity of peiminine to TLR4 and NF-κB. Experimental validation has been offered for these computational predictions. Peiminine markedly decreased nitric oxide, TNF-α, and IL-6 in LPS-stimulated inflammatory models, suggesting functional suppression of TLR4–NF-κB signaling. Peiminine increased IL-10 and decreased colonic gene expression of TLR4, NF-κB, iNOS, COX-2, and IL-1β in TNBS-induced colitis, which is in line with pathway-level suppression anticipated in silico. Taken together, network pharmacology, KEGG pathway enrichment, in vitro cytokine modulation, and in vivo transcriptional regulation created a cohesive mechanistic framework in which peiminine suppresses the TLR4-mediated NF-κB axis to produce anti-colitic effects (Fig. 10). To treat a complicated inflammatory condition like ulcerative colitis, our current study serves as the foundation for the discovery of a peiminine as a novel therapeutic agent.

Fig. 10.

Proposed mechanism of Peiminine in mitigating TNBS-induced colitis in mice through TLR4-mediated NF̙kB pathway. The image was created using BioRender.com.

Abbreviations

IBD

Inflammatory bowel disease

UC

Ulcerative colitis

CD

Crohn’s disease

5-ASA

5-Aminosalicylic acid

PEI

Peiminine

NFƙB

Nuclear factor kappa B

iNOS

Inducible nitric oxide synthase

COX-2

Cyclooxygenase-2

TNBS

2,4,6-Trinitrobenzene sulfonic acid

SMILES

Simplified molecular-input line-entry system

PDB

Protein Data Bank

MD

Molecular docking

DAI

Disease activity index

HE

Hematoxylin and eosin

PBS

Phosphate buffered saline

MDA

Malondialdehyde

NO

Nitric oxide

SOD

Superoxide dismutase

ANOVA

Analysis of variance

Author contributions

Bhagyabhumi Shah: Conceptualization, design of the study, network pharmacology Aditi Sharma: in vivo studies, acquisition of data, data analysis, Mansi Patel: Drafting the manuscript, revising the manuscript Umang Shah: in vitro cell line studies, proof reading of manuscript, Nikunj Parekh: Molecular docking studies, proof reading Nilay Solanki: proofreading.

Data availability

Data will be made available on request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval

The research protocol was approved by the Institutional Animal Ethics Committee (IAEC) (RPCP/IAEC/2023-24/R2). All procedures were performed in accordance with the ethical standards of the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, and ARRIVE guidelines.