Abstract Context Polygonum hydropiper L. (P. hydropiper) has outstanding clinical efficacy in treating both acute and chronic gastroenteritis. However, the definite mechanism remains unclear. Objective This study aimed to explore the potential mechanisms of the total flavonoid of P. hydropiper (FPH) in stress-induced gastric mucosal damage (SGMD) rats through a combination of network pharmacology, molecular docking, and animal experiments. Methods Network pharmacology and molecular docking were utilized to predict the potential mechanisms of FPH against SGMD. In experimental studies, SGMD rat models were established using water-immersion restraint stress (WIRS). FPH was administered at doses of 140, 70, and 35 mg/kg, with ranitidine serving as a positive control, through gavage once daily for 6 consecutive days after model establishment. Stomach and serum specimens were analyzed using HE staining, Western blotting, qPCR, and ELISA to investigate the protective mechanism of FPH in SGMD. Results The network pharmacology analysis identified 16 active ingredients and 183 common targets, with potential pathways including PI3K/Akt, MAPK and Keap1/Nrf2. In vivo experiments demonstrated that FPH intervention alleviated SGMD pathological changes, reduced elevated serum IL-6 and TNF-α levels, and enhanced SOD and GSH activity in rats. Additionally, FPH increased the protein expression of p62, Nrf2, HO-1, PI3K, and p-Akt, along with mRNA levels of Nrf2, p62, and HO-1. Conclusions FPH exerts a gastric mucosal protective effect by upregulating antioxidant gene expression through the PI3K/Akt and Keap1/Nrf2 pathways. This study provides an experimental basis for the potential clinical treatment of SGMD with the traditional Chinese medicine P. hydropiper. Keywords: Polygonum hydropiper, Gastric mucosal damage, Antioxidant, Molecular docking Graphical abstract Image 1 [39]Open in a new tab 1. Introduction Stress-induced gastric mucosal damage (SGMD) is a common acute gastric mucosal lesion characterized by erosion, bleeding, and superficial ulcers. It is often triggered by stressors such as severe trauma, psychological disorders, major surgery, or substances like alcohol and drugs [[40]1]. The mechanisms underlying SGMD are complex and not fully understood, involving factors such as gastric mucosal ischemia, lipid peroxide accumulation, excess oxygen free radicals, and abnormal gastric acid secretion [[41]2]. Current clinical approaches typically involve proton pump inhibitors, antacids, and gastric mucosa protective agents to manage symptoms. However, these treatments have limitations like single-target action, adverse reactions, slow recovery, and potential recurrence. Traditional Chinese medicine (TCM) is increasingly being explored for its multi-target, multi-path effects, good safety, and effectiveness [[42]3,[43]4]. Polygonum hydropiper L. is an annual herb in the Polygonum genus of the Polygonaceae family, contains main components such as quercetin, kaempferol, rutin, and other flavonoids [[44]5]. These compounds exhibit antioxidant, anti-inflammatory, and antibacterial and antihypertensive effects [[45][5], [46][6], [47][7], [48][8], [49][9]]. In clinical practice, P. hydropiper is used effectively for treating acute and chronic gastroenteritis with minimal side effects [[50]10]. Research suggests that flavonoids from plants can help in alleviating peptic ulcers, maintaining intestinal barrier function, regulating enzyme activity and gastric juice secretion, thereby providing gastrointestinal protection [[51]11]. Quercetin, in particular, has been found to reduce oxidative stress damage in GES-1 cells by improving mitochondrial function, cellular barrier integrity, and reducing inflammation [[52]12]. However, the development of SGMD is influenced by multiple factors rather than a single cause. Studies have shown a strong correlation between oxidative stress, inflammatory responses, and the onset and progression of SGMD [[53]13]. Under noxious stress, an excessive production of reactive oxygen species (ROS) can damage the blood vessels and epithelial cells of the gastric mucosa, leading to injury [[54]14]. The Keap1/Nrf2 signaling pathway is recognized as a crucial endogenous antioxidant mechanism, with nuclear factor E2-related factor 2 (Nrf2) serving as a key transcriptional modulator that regulates the transcription and translation of antioxidant genes in response to oxidative stress in vivo [[55]15]. Studies have indicated that water-immersion restraint stress (WIRS) and alcohol exposure can hinder the normal activation and expression of Nrf2 in rats with gastric ulcers, thereby inhibiting the Keap1/Nrf2 pathway and worsening oxidative stress in the gastric mucosa [[56]16,[57]17]. Therefore, enhancing Nrf2 expression and increasing antioxidant enzyme activity through the Keap1/Nrf2 signaling pathway could serve as a promising target for further research in the prevention and treatment of SGMD. Network pharmacology is an interdisciplinary research approach that integrates the unique strengths of TCM and Chinese materia medica to analyze the various compounds present in Chinese medicines [[58]18]. By utilizing computer software and TCM databases, this methodology aims to elucidate the key mechanisms of disease treatment and provide a foundation for clinical research on new drugs. In this particular study, network pharmacology and molecular docking techniques were employed to investigate the potential targets of total flavonoids of P. hydropiper (FPH) in the protection against SGMD. Subsequently, a rat model of SGMD was established using WIRS to validate the potential molecular mechanism. The workflow of this study is illustrated in [59]Fig. 1. Fig. 1. [60]Fig. 1 [61]Open in a new tab Research ideas and research process of this study. 2. Materials and methods 2.1. Collecting of active constituents of P. hydropiper and target prediction The chemical constituents of P. hydropiper were collected by searching PubMed ([62]https://pubmed.ncbi.nlm.nih.gov/), Web of Science ([63]https://webofscience.clarivate.cn/wos/), and China National Knowledge Infrastructure (CNKI, [64]https://www.cnki.net/) databases. Each chemical ingredient was then searched in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, [65]https://old.tcmsp-e.com/tcmsp.php), with only components having oral bioavailability (OB) > 30 % and drug-like properties (DL) > 0.18 being kept. The SIMILE number or SDF format of the compounds was retrieved and saved using the PubChem database ([66]https://pubchem.ncbi.nlm.nih.gov/). The SwissTargetPrediction web-based tool ([67]http://swisstargetprediction.ch/) was utilized to identify potential targets of the candidate compounds, with the resulting CSV files downloaded. Subsequently, gene targets with a probability greater than 0 for each component were retained, and any duplicates were removed to obtain the predicted gene targets for each component. 2.2. The retrieval of gastric mucosal injury-related targets SGMD-related targets were searched for in three public databases using the keywords ‘gastric ulcer' and 'gastritis' on GeneCards ([68]https://www.genecards.org/), DisGeNet ([69]https://www.disgenet.org/), and OMIM ([70]https://www.omim.org/). The SGMD-related targets were then obtained by merging and removing repetitive targets. 2.3. Constructing compound-target networks and interaction networks The intersection of active compounds and SGMD-related genes was analyzed using the bioinformatics database ([71]https://www.bioinformatics.com.cn/). Subsequently, the results were visualized and the common targets were entered into the STRING database ([72]https://cn.string-db.org/), with species limited to ‘Homo sapiens', an interaction score threshold of 0.7, free nodes hidden and other parameters unchanged to construct a protein-protein interaction (PPI) network. The topological parameters of the PPI network were then analyzed to identify key targets of FPH against SGMD. This analysis utilized the CytoHubba plugin to calculate Eccentricity, Closeness, Betweenness, and Degree, ultimately, leading to the identification of hub targets of FPH for treating SGMD. 2.4. GO and KEGG enrichment analysis Gene Ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID, [73]https://david.ncifcrf.gov/). The top 20 biological processes (BP), cellular components (CC), and molecular functions (MF) were selected for visualization in a bubble diagram. Subsequently, the results were further analyzed using the bioinformatics database ([74]https://www.bioinformatics.com.cn/). 2.5. Molecular docking The activation of the PI3K/Akt and Keap1/Nrf2 signaling pathways is thought to significantly contribute to the anti-SGMD effects, as indicated by network pharmacology and literature reviews. To validate this hypothesis, molecular docking of key targets and active ingredients was performed. The 3D structures of essential target proteins (PI3K, Akt, mTOR, p62, Nrf2, HO-1) involved in these pathways were acquired from the Protein Data Bank (PDB, [75]https://www.rcsb.org/). The 3D molecular structures of ligands (quercetin, kaempferol, and isorhamnatin) were retrieved from the TCMSP database. Subsequently, water molecules and protein residues were removed from the proteins using PyMOL software and saved in PDBQT format. The ligands and receptors were then subjected to docking using AutoDock4.2.6 ([76]https://autodock.scripps.edu/) and visualized with PyMOL software. Binding activity was evaluated based on binding energy, with binding considered spontaneous when the energy was below −5 kcal/mol. 2.6. Preparation of P. hydropiper The stems and leaves of P. hydropiper were harvested from Hainan Province, China, and authenticated by Prof. Niankai Zeng of Hainan Medical University. Voucher specimens (No. 20211116) were deposited at the School of Pharmacy, Hainan Medical University. Following this, the plant material was air-dried at room temperature. A specific quantity of dried whole herb of P. hydropiper was measured and subjected to extraction using a material-to-liquid ratio of 1:9, a 60 % ethanol concentration, and an extraction duration of 1 h. The residual material was subjected to two additional extractions under the same conditions, filtered, and the resulting filtrates were combined. This combined filtrate was then passed through macroporous resin. After concentrating the filtrate under reduced pressure to achieve an ointment-like consistency, it was dried to produce the flavonoid extract of P. hydropiper for further utilization. 2.7. Experimental animals Forty-eight Sprague-Dawley rats (weighing 180–230 g, with an equal number of males and females) were sourced from Changsha Tianqing Biotech Co., Ltd. In China (License: SCXX (Xiang) 20220010). The rats, housed at 25 °C, were subjected to a 12-h light/dark cycle and provided with 24-h access to food and water. All animal procedures adhered to the International Guidelines for Care and Use of Laboratory Animals and received approval from the Animal Ethics Committee of Hainan Medical University (HYLL-2022-365). 2.7.1. Animal grouping, models, and administration 48 Sprague-Dawley rats were divided into 6 groups, each consisting of 8 rats: control, model, ranitidine, and high, middle, and low dose groups of FPH (140 mg/kg, 70 mg/kg, 35 mg/kg). Ranitidine and FPH were administered once daily for 6 days. Prior to modeling, rats were fasted for 24 h but had access to water. Following the final administration, all groups, except the control group, were placed in custom-made rat cages with their heads facing upwards and their limbs secured. These cages were then vertically submerged in a thermostatic water tank (19 ± 1 °C) until the water level reached the xiphoid process of the rats, completing the modeling process after 7 h. 2.7.2. Sample collection and processing Following intraperitoneal administration of 10 % chloral hydrate (0.3 mL/100 g) anesthesia, blood samples were collected by making an incision along the ventral median line of the abdominal cavity to expose the abdominal aorta. These samples underwent high-speed centrifugation at 2000 rpm, 4 °C for 15 min. The resulting supernatants were collected and stored at −80 °C for subsequent biochemical analysis. The stomach was incised along the greater curvature, rinsed with normal saline, and excess water from the stomach tissue was removed using filter paper. The ulcer index was then observed and documented. A portion of the antral tissue was preserved at −80 °C for Western blot analysis. 2.7.3. Determination of gastric ulcer index The gastric body was surgically removed and dissected along the greater curvature of the stomach. Stomach contents were washed with normal saline and excess water was removed with filter paper. The dimensions of the gastric mucosa injury were assessed through photographs and vernier calipers. Detailed scoring criteria can be referenced in [77]Table 1. Table 1. Gastric mucosal injury scoring criterion. Damage Degree Damage Length Score spot erosion – 1 linear erosion <1 mm 2 1 ∼ 2 mm 3 2 ∼ 4 mm 4 >4 mm 5 width > 2 mm double [78]Open in a new tab 2.7.4. Gastric mucosal histopathological evaluation Histopathological changes of the stomach were observed by H&E staining. Rat gastric tissues were fixed in 4 % paraformaldehyde, dehydrated, made transparent, immersed in wax, embedded, sectioned, and stained, and then observed under a light microscope. 2.7.5. Anti-inflammatory and antioxidant-related indexes assay The levels of IL-6 and TNF-α in serum were detected using an ELISA kit from Hangzhou Lianke Biotechnology Co., Ltd. Additionally, the activities of GSH, SOD, MPO, and MDA in rat serum were assessed using kits from Nanjing Jiancheng Bioengineering Institute. 2.7.6. Hexosaminidase in gastric tissue and NO in serum The concentration of N-acetylhexosamine (NO) in rat serum was determined using the Nanjing Jiancheng Bioengineering Institute kit. Hexosamine content in gastric tissue was measured by hydrolyzing glycoproteins in the supernatant of mucosal tissue homogenate under acidic and high-temperature conditions, resulting in the formation of acetaminohexose upon reaction with phthalophenone. The acetaminohexose further reacted with p-dimethylaminobenzaldehyde to produce a red compound. The hexosamine content in the supernatant was determined by comparison with a standard tube. Additionally, protein concentration in the supernatant (mg/L) was measured using a BCA protein concentration assay kit. The final result was reported as the amount of hexosamine per mg protein (g/mg protein). 2.7.7. Western blot analysis Gastric tissue weighing 50 mg was cut and subjected to protein lysis solution, followed by mechanical grinding on ice. The resulting mixture was then centrifuged at 12000 rpm, 4 °C for 15 min, and the supernatant was collected for protein quantification and denaturation to prepare samples. Subsequently, blots underwent SDS-PAGE, electrophoresis, transfer to a membrane, blocking with 5 % non-fat milk, and incubation with primary antibodies including p62, Nrf2, and HO-1 (Abcam), β-Actin, Akt, p-Akt, and PI3K (Shanghai Biyuntian Biotechnology Co., Ltd.) overnight at 4 °C. The membrane was then washed, incubated with the corresponding secondary antibody for 1.5 h at room temperature re-washed, and an ECL luminescent solution was added for assay development. Protein bands were analyzed for gray levels using Image J software, and the expression of proteins of interest was determined by dividing the gray level value of the protein by the gray level value of an internal reference protein. 2.7.8. Detection of p62, Nrf2, HO-1 mRNA expression in gastric tissues by RT-qPCR Total RNA was extracted from 50 mg of gastric tissue using TRIzol. Subsequently, the extracted RNA was utilized for cDNA synthesis via reverse transcription (ThermoFisher) The synthesized cDNA was then employed in RT-qPCR analysis of p62, Nrf2, HO-1, and the internal reference gene β-Actin. Cycling parameters were established based on the manufacturer's instructions, with a pre-denaturation step at 95 °C for 2 min, denaturation at 95 °C for 15 s, annealing at 60 °C for 60 s, extension at 65 °C for 5 s, and a total of 40 cycles. The primer sequences are available in [79]Table 2. Amplification curves were recorded, and the relative expression levels were calculated. All primers were manually designed. Table 2. Primer sequence. Primers Forward/Reverse Sequence Nrf2 Forward 5′-GCAGCATACAGCAGGACA-3′ Reverse 5′-GTGGGATTTGAGTCTAAGGA-3′ HO-1 Forward 5′-CGAAACAAGCAGAACCCA-3′ Reverse 5′-CACCAGCAGCTCAGGATG-3′ P62 Forward 5′-GCTTTCAGGCGCACTACCG-3′ Reverse 5′-TCCCACCACAGGCCCATT-3′ β-actin Forward AGGGAAATCGTGCGTGAC Reverse ACCCAGGAAGGAAGGCT [80]Open in a new tab 2.8. Statistical analysis The data were reported as means ± SD. Statistical analyses were conducted using one-way analysis of variance (ANOVA) and the least significant difference (LSD) post hoc test with SPSS 16.0 software (IBM, USA). A significance level of P < 0.05 was used for all cases. 3. Results 3.1. Active constituents and potential targets of P. hydropiper Sixteen active constituents meeting the criteria of OB > 30 % and DL > 0.18 were identified from the TCMSP database. These constituents, which included flavonoids, alkaloids, and phenolics, are known for their beneficial bioactivities. Previous studies have highlighted the therapeutic potential of flavonoids like kaempferol, rutin, and quercetin in protecting gastric mucosa from oxidative damage caused by free radicals like reactive oxygen species and reactive nitrogen [[81]19]. Subsequently, targets of 16 active ingredients were predicted by the SwissTargetPrediction database, leading to the identification of 355 relevant targets ([82]Table 3). Table 3. Information of main active ingredients of Polygonum hydropiper. Molecule ID Compound name Molecular function Molecular weight 2D structure OB DL MOL000098 Quercetin C15H10O7 302.23 Image 1 46.43 0.28 MOL000422 Kaempferol C15H10O6 286.2 Image 2 41.88 0.24 MOL000354 Isorhamnetin C16H12O7 316.26 Image 3 49.60 0.31 MOL000415 Rutin C27H30O16 610.5 Image 4 3.20 0.68 MOL004368 Hyperoside C21H20O12 464.4 Image 5 6.94 0.77 MOL000701 Quercitrin C21H20O11 448.4 Image 6 4.04 0.74 MOL001454 Berberine C20H18NO4+ 336.4 Image 7 36.86 0.78 MOL012920 Sinomenine C19H23NO4 329.4 Image 8 30.98 0.96 MOL000004 Procyanidin β1 C30H26O12 578.5 Image 9 67.87 0.66 MOL005828 Nobiletin C21H22O8 402.4 Image 10 61.67 0.52 MOL002714 Baicalein C15H10O5 270.24 Image 11 33.52 0.21 MOL000392 Formononetin C16H12O4 268.26 Image 12 69.67 0.21 MOL002881 Diosmetin C16H12O6 300.26 Image 13 31.14 0.27 MOL000492 Catechin C15H14O6 290.27 Image 14 54.83 0.24 MOL006504 Catechin gallate C22H18O10 442.4 Image 15 53.57 0.75 MOL001002 Ellagic acid C14H6O8 302.19 Image 16 43.06 0.43 [83]Open in a new tab 3.2. Predicting disease targets for SGMD A total of 3156 SGMD-related targets were compiled by selecting 2930 targets with a relevance score >2.34 from the GeneCards database, 72 targets from the DisGeNet database, and 98 targets from the OMIM database. After removing duplicates, a Venn diagram was created using the 355 active component targets and the 3156 disease targets. This analysis revealed 183 overlapping targets, identified as core genes for further investigation ([84]Fig. 2). Fig. 2. [85]Fig. 2 [86]Open in a new tab Venn diagram of the common targets of FPH and SGMD. FPH, Polygonum hydropiper total flavonoids; SGMD, Stress-induced gastric mucosal damage. 3.3. Network-based compound-disease-targe construction The network diagram was created to analyze the relationship between ingredients, diseases, and targets in TCM ([87]Fig. 3). The findings revealed that the influence of FHP on SGMD was a result of the interactions between multiple active ingredients and targets. The active ingredients were ranked according to their degree, with Baicalein, Diosmetin, Isorhamnetin, Kaempferol, and Quercetin being the top 5 ([88]Table 4). These compounds were identified as potential bioactive ingredients of FPH in combating SGMD. Fig. 3. [89]Fig. 3 [90]Open in a new tab Composition-target network of FPH. The blue circle represents the common target gene, the yellow prism indicates the active ingredient and the red inverted triangle represents the FPH. FPH, Polygonum hydropiper total flavonoids. Table 4. The cytoNCA plug-in screened out the top 5 active ingredient values. Molecule ID Compounds Degree MOL002714 Baicalein 69 MOL002881 Diosmetin 61 MOL000354 Isorhamnetin 60 MOL000422 Kaempferol 59 MOL000098 Quercetin 59 [91]Open in a new tab 3.4. Targets related to SGMD and PPI network of co-targets The PPI network was constructed using the STRING online platform to elucidate the relationship between FPH and diseases ([92]Fig. 4A). Subsequently, a network consisting of 163 nodes and 794 connected edges was created to visualize protein interactions ([93]Fig. 4B). The CytoHubba plugin was utilized to identify core proteins of interest. Proteins meeting specific criteria including Eccentricity >0.22, Closeness >0.002, Betweenness >273.80, and Degree >11.95 were selected, resulting in a refined network map with 32 nodes and 211 edges ([94]Fig. 4C). In the network visualization, nodes with higher degrees are represented as larger and darker in color, signifying their significance. Through this analysis, 10 core targets were pinpointed: AKT1, EGFR, SRC, PIK3CA, PIK3R1, TNF, ESR1, ERBB2, HIF1A, and HSP90AB1, which could serve as crucial targets for the therapeutic application of P. hydropiper in SGMD. Fig. 4. [95]Fig. 4 [96]Open in a new tab PPI network of FPH against gastric mucosal damage. (A) The interactive PPI network was obtained from the STRING database platform; (B) PPI network imported from STRING database to Cytoscape 3.10.0, containing 163 points and 794 edges; (C) Thirty-two core genes were screened using the cytoHubba plugin, and the PPI network contained 32 points and 211 edges. PPI, Protein-protein interaction; FPH, Polygonum hydropiper total flavonoids. 3.5. GO and KEGG pathway enrichment analysis To further investigate the role of FPH in SGMD treatment, GO and KEGG enrichment analyses were conducted using the David database. The 183 intersection targets identified were analyzed, revealing a total of 965 entries across 3 categories with a q-value <0.05: 708 BP, 103 MF, and 154 CC. The top 30 terms in each category were visually presented in a column chart ([97]Fig. 5). A bubble chart was used to depict GO term enrichment, with redder dots indicating lower q-values and higher enrichment. The GO analysis revealed that BP such as protein phosphorylation, negative regulation of apoptotic processes, signal transduction, protein autophosphorylation, and positive regulation of cell proliferation are closely linked to SGMD. Common disease-related terms in cellular components include cytosol, plasma membrane, and nucleus. Additionally, FPH was associated with Molecular Functions such as protein binding, protein serine/threonine/tyrosine kinase activity, and protein kinase activity. Fig. 5. [98]Fig. 5 [99]Open in a new tab The bar plot of GO enrichment analysis includes the top 10 significant enrichment terms of three domains: BP, CC, and MF. GO, Genes Ontology; BP, biological processes; CC, cellular components; MF, molecular functions. KEGG pathway enrichment analysis identified 160 signaling pathways, with the top 20 pathways visualized in a bubble plot based on significant enrichment ([100]Fig. 6). The analysis revealed a strong association between FPH and key pathways such as PI3K/Akt, MAPK, and FoxO signaling pathways. Literature review emphasized the protective role of the Keap1/Nrf2 signaling pathway in SGMD [[101]20], suggesting that FPH may hold therapeutic promise in SGMD treatment by modulating these pathways. Current research attributes SGMD development to an imbalance between gastric mucosal defense and damage factors, with oxidative damage playing a crucial role. Excessive reactive oxygen species can lead to oxidative stress, inflammation, and tissue damage. Organisms have developed intricate defense mechanisms, including the Nrf2-mediated signal pathway, to counteract oxidative stress and maintain redox balance [[102]21]. Studies have indicated that cytokines can activate the PI3K/Akt pathway during gastric ulcer healing, promoting survival and healing of gastric mucosal epithelial cells [[103]22]. Therefore, the integrated analysis of literature mining, PPI, and KEGG pathway enrichment underscores the importance of the Keap1/Nrf2 and PI3K/Akt signaling pathways warranting further animal experimental validation to elucidate the mechanism of action of FPH in the treating of gastric ulcers. Fig. 6. [104]Fig. 6 [105]Open in a new tab KEGG enrichment analysis: Bubble plot of KEGG enrichment pathways. 3.6. Molecular docking analysis By analyzing the co-targets in the PPI network and KEGG enrichment outcomes, Autodock 4.2.6 employed to investigate the binding poses and interactions of three components with six proteins. The binding energies between the protein receptors and active ligands were computed to predict their affinity, with a binding energy < −5.0 kcal/mol signifying a stronger binding capability ([106]Table 5). The interactions between the active components and receptor proteins were visualized using PyMOL software ([107]Fig. 7A−I). The docking results revealed that Quercetin and Kaempferol displayed superior binding abilities with PI3K, Akt, and mTOR proteins, whereas Isorhamnetin exhibited good binding abilities with p62, Nrf2, and HO1 proteins. These results provide partial support for the binding capacities of the small molecule compounds to the target proteins. Table 5. Docking results of three bioactive compounds and core targets. Compounds Binding energy (kcal/mol) __________________________________________________________________ PI3K __________________________________________________________________ Akt __________________________________________________________________ mTOR __________________________________________________________________ Nrf2 __________________________________________________________________ P62 __________________________________________________________________ HO1 __________________________________________________________________ 5jha 3mv5 8er7 7 × 5g 6mj7 1s13 Quercetin −7.16 −7.78 −5.75 −4.69 −5.71 −4.0 Kaempferol −6.6 −7.35 −5.91 −4.68 −4.24 −7.53 Isorhamnetin −7.11 −6.94 – −5.29 −6.04 −5.13 [108]Open in a new tab Fig. 7. [109]Fig. 7 [110]Open in a new tab Binding mode of screened drugs to their targets by molecular docking. (A) Quercetin-PI3K; (B) Quercetin-AKT1; (C) Quercetin-Mtor; (D) Kaempfrol-PI3K; (E) Kaempfrol-AKT1; (F) Kaempfrol-mTOR; (G) Isorhamnetin-p62; (H) Isorhamnetin-Nrf2; (I) Isorhamnetin-HO-1. 3.7. Macroscopic and gross morphology of rat gastric mucosa In the SGMD model constructed using WIRS, rats in the model group displayed severe damage to the gastric mucosa, presenting with mucosal sloughing, edema, congestion, or hemorrhage, and a notably higher ulcer index. Conversely, rats treated with FPH and ranitidine exhibited a reduction in gastric mucosal lesions compared to the model group, showing a significant decrease in ulcer index (P < 0.01). These findings are detailed in [111]Table 6 and [112]Fig. 8A−F. Table 6. Gastric ulcer index of rats in each group (x ± s, n = 8). Group Dose/mg.kg-1 Gastric ulcer index Con – – Mod – 51.5 ± 10.012 Low dose of FPH 35 20.875 ± 4.136^## Medium dose of FPH 70 21.375 ± 3.038^## High dose of FPH 140 29.40 ± 5.666^## Ran – 22.38 ± 5.429^## [113]Open in a new tab Compared with model group, ^#P<0.05 and ^##P<0.01. Fig. 8. [114]Fig. 8 [115]Open in a new tab Morphological observation of gastric mucosa in rats. (A) Contral; (B) Model; (C) Low dose of FPH; (D) Medium dose of FPH; (E) High dose of FPH; (F) Ranitidine group. FPH, Polygonum hydropiper total flavonoids. 3.8. Effect of FPH on pathological changes of gastric mucosa in rats The gastric mucosal surface of rats in the control group appeared smooth and intact, exhibiting normal morphology without any signs of edema or hemorrhage. Conversely, rats in the model group displayed gastric mucosa with evident edema, sloughing off of epithelial cells, and noticeable hemorrhage in the submucosa. While all treatment groups showed improvement in the edema and hemorrhage of the gastric mucosa, the high-dose group of FPH demonstrated the most significant enhancement ([116]Fig. 9A−F). Fig. 9. [117]Fig. 9 [118]Open in a new tab Effect of FPH on pathological changes of gastric mucosa in rats with gastric ulcer. (HE × 20, HE × 100) (A) Contral; (B) Model; (C) Low dose of FPH; (D) Medium dose of FPH; (E) High dose of FPH; (F) Ranitidine group. FPH, Polygonum hydropiper total flavonoids. 3.9. Effect of FPH on the activities of GSH, SOD, MDA, and MPO in the serum of rats Compared to the control group, the serum levels of GSH and SOD decreased significantly in the model group, while MDA and MPO levels increased significantly (P<0.01). In contrast, rats from the high, middle, and low dosage groups of FPH and the ranitidine group showed a substantial increase in GSH and SOD serum levels compared to the model group (P<0.05, P<0.01). Furthermore, there was a significant decrease in MDA levels in the serum of rats from the high, middle, and low dosage groups of FPH and the ranitidine group (P<0.01), along with a notable decrease in MPO expression in the middle and low dosage groups of FPH and the ranitidine group (P<0.05) ([119]Fig. 10A−D). Fig. 10. [120]Fig. 10 [121]Open in a new tab Effects of FPH on the contents of GSH, SOD, MDA, and MPO in rats with SGMD. (A–D) The measurement of GSH, MDA, MPO, and SOD. Compared with control group, ∗P<0.05 and ∗∗P<0.01; Compared with model group, ^#P<0.05 and ^##P<0.01. GSH, Glutathione; MDA, Malondialdehyde; MPO, Myeloperoxidase; SOD, Superoxide Dismutase. 3.10. Effect of FPH on serum IL-6 and TNF-α levels in rats Compared to the control group, the serum levels of IL-6 and TNF-α in rats from the model group significantly increased (P<0.05, P<0.01). Conversely, in the high and medium dose groups of FPH, there was a noticeable decrease in IL-6 and TNF-α levels when compared to the model group (P < 0.05 or P < 0.01) ([122]Fig. 11A and B). Fig. 11. [123]Fig. 11 [124]Open in a new tab Effect of FPH on serum IL-6 and TNF-α in rats with gastric ulcer. (A) The level of IL-6 in the serum of rats; (B) The level of TNF-α in the serum of rats. Compared with control group, ∗P<0.05 and ∗∗P<0.01; Compared with model group, ^#P<0.05 and ^##P<0.01. FPH, Polygonum hydropiper total flavonoids. 3.11. Effect of FPH on hexosamine in gastric tissue and NO in serum of rats Compared to the control group, the stomach tissue of the model group exhibited a significant increase in hexosamine content (P<0.01). There were no significant differences observed among the model group, ranitidine group, and each FPH-treated group. Levels of NO in the high, middle, and low dosage groups of FPH and the ranitidine group were significantly higher compared to the model group (P<0.05) ([125]Fig. 12A and B). Fig. 12. [126]Fig. 12 [127]Open in a new tab Effects of FPH on hexosamine in gastric tissue and NO in serum of rats with gastric ulcer. (A) Levels of hexosamine in gastric tissue; (B) NO level in serum. Compared with control group, ∗P<0.05 and ∗∗P<0.01; Compared with model group, ^#P<0.05 and ^##P<0.01. FPH, Polygonum hydropiper total flavonoids. 3.12. Effect of FPH on PI3K, Akt and P-Akt protein expression in rat gastric mucosa Compared to the control group, the model group exhibited a significant increase in the expression of PI3K and P-Akt proteins (P<0.05). There were no notable differences observed among the Akt protein groups. Additionally, both the groups treated with FPH and the ranitidine group showed a significant increase in PI3K protein expression compared to the model group (P<0.01). The middle and low dosage groups of FPH displayed a significant increase in P-Akt protein expression (P<0.05), while the high dosage group showed an increase in P-Akt protein expression, although it did not reach statistical significance when compared to the model group ([128]Fig. 13A−D). Fig. 13. [129]Fig. 13 [130]Open in a new tab Effects of FPH on the expression of PI3K, Akt, and P-Akt proteins in gastric tissues of rats with SGMD. (A) Effects of FPH on the protein expression of PI3K, Akt, and p-Akt in gastric tissues; (B–D) Quantification of relative protein levels of PI3K, AKT, and p-Akt. Compared with the control group, ∗P<0.05 and ∗∗P<0.01; Compared with the model group, ^#P<0.05 and ^##P<0.01. FPH, Polygonum hydropiper total flavonoids; SGMD, Stress-induced gastric mucosal damage. 3.13. Effect of FPH on the expression of Nrf2, p62, and HO-1 protein and mRNA in rat gastric mucosa The protein expression levels of p62, Nrf2, and HO-1 in the gastric mucosal tissues of model rats were significantly reduced (P < 0.05). Conversely, the groups receiving different dosages of FPH and the ranitidine group exhibited a notable increase in the protein expression levels of p62, Nrf2, and HO-1 compared to the model group (P<0.05 or P<0.01). Among the FPH dose groups, the medium dose group showed the most significant increase in Nrf2 and HO-1 protein expression levels, while the low dose group exhibited the least increase. However, the expression level of p62 protein remained consistent across all three FPH dose groups. Furthermore, the mRNA expression levels of Nrf2, HO-1, and p62 in the FPH-high group were significantly higher than those in the model group (P<0.05). Nrf2 and P62 mRNA expression levels decreased in the model group compared to the control group, with no significant difference observed in p62 mRNA expression between the model and control groups ([131]Fig. 14A−E). Fig. 14. [132]Fig. 14 [133]Open in a new tab Effects of FPH on the expression of Nrf2, P62, HO-1 protein and mRNA in gastric tissues of rats with SGMD. (A) Effects of FPH on Nrf2, p62 and HO-1 protein expression in gastric tissues; (B–D) Quantification of relative protein levels of Nrf2, p62, and HO-1; (E) Relative mRNA expression. Compared with control group, ∗P<0.05 and ∗∗P<0.01; Compared with model group, ^#P<0.05 and ^##P<0.01. FPH, Polygonum hydropiper total flavonoids; SGMD, Stress-induced gastric mucosal damage. 4. Discussion The increasing prevalence of gastric diseases can be attributed to a variety of factors, including high work stress, irregular eating habits, and mental health issues like anxiety and depression [[134][23], [135][24], [136][25], [137][26]]. If left untreated, ongoing damage to the gastric mucosa can result in serious conditions like erosive gastritis, gastric ulcer, gastric perforation, and even cancer [[138]27]. Gastric diseases and associated complications can impact an individual's quality of life and place a substantial economic and public health burden on families and society [[139]27,[140]28]. Therefore, it is imperative to explore effective medications for the prevention and treatment of SGMD. This study investigated P. hydropiper, a traditional Chinese medicine commonly used in China for gastroenteritis and diarrhea treatment [[141]6]. Through network pharmacology, the research identified active ingredients and potential targets of FPH against SGMD, followed by an analysis of the binding activity between these components and targets using molecular docking. A rat model of SGMD induced by WIRS was employed to validate the potential mechanism of action of FPH and to establish a foundation for future clinical investigations. The stomach is the primary organ that responds to stress, with both mental stress and environmental stimuli potentially causing pathological changes [[142]29]. The WIRS model combines physiological factors like hunger, cold, and struggle with psychological factors such as fear and anxiety. Research indicates that WIRS can result in gastric mucosal injury similar to SGMD, including congestion, bleeding, erosion, and superficial ulcers [[143][29], [144][30], [145][31]]. Due to its effectiveness and simplicity, WIRS is commonly used to simulate stress-induced gastric mucosal injury for drug screening [[146]13]. In this study, SD rats were exposed to 19 °C WIRS for 7 h to induce a gastric injury model and investigate the protective effect of FPH against gastric injury. HE staining revealed mucosal edema, epithelial cell shedding, submucosal congestion, hemorrhage, and increased ulcer index in the model group, confirming successful model establishment. Conversely, FPH administration reduced these lesions, with the high-dose group showing the most significant effects, suggesting FPH's potential to alleviate SGMD. Myeloperoxidase (MPO) is an enzyme linked to neutrophils, reflecting neutrophil count and inflammatory levels [[147]2]. TNF-α and IL-6, as pro-inflammatory cytokines, indicate inflammation severity and tissue damage, with elevated levels seen during gastric mucosal injury onset and progression [[148]14]. FPH treatment notably decreased TNF-α and IL-6 release in rats with SGMD, suggesting an anti-inflammatory effect of FPH. Network pharmacology has been widely utilized to identify active compounds and their molecular targets in herbal medicines for treating gastric mucosal damage [[149]32]. For example, the molecular target of Wuji Wan in the management of gastric ulcers was both predicted and confirmed through a network pharmacology approach [[150]33]. In this study, network pharmacology was employed to examine the targets and mechanisms of action of FPH in treating SGMD. A total of 16 compounds and 3156 SGMD-related targets were identified, with 183 being common targets of FPH and SGMD. These results suggest that FPH may alleviate SGMD through these targets. Experimental validation was subsequently carried out based on the network pharmacology analysis and molecular docking results to further explore the mechanism and validate the findings. The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway is a vital cellular signal transduction mechanism that regulates key processes such as cell proliferation, differentiation, migration, and metabolism. PI3K, a lipid kinase, converts phosphatidylinositol (PI) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Activation of PI3K by various extracellular signals triggers downstream activation of effectors like Akt [[151]34]. Akt is recruited and activated at the plasma membrane by PIP3 generated by PI3K, and upon activation, it moves to the cytoplasm or nucleus. By phosphorylating target proteins, Akt regulates cellular functions like proliferation, differentiation, metabolism, and repair. Studies have demonstrated the importance of PI3K/Akt pathway activation in gastric mucosal injury repair [[152]35,[153]36]. Substances like Xiaojianzhong decoction, Rutaecarpine, and methyl palmitate have been shown to alleviate ethanol-induced gastric mucosal injury by activating PI3K/Akt and increasing the expression of PI3K and p-AKT [[154][37], [155][38], [156][39]]. Analysis of PPI identified AKT1, PI3CA, PIK3R1, TNF, and mTOR as core targets for anti-SGMD effects. Additionally, KEGG enrichment analysis and literature mining suggested that primary pathways involved in anti-SGMD may include PI3K/AKT, MAPK, and Keap1/Nrf2 [[157]12,[158]40]. Molecular docking results further indicated that key components of FPH (quercetin, kaempferol and isorhamnetin) exhibit strong binding activities with core targets (PI3K, AKT1 and mTOR), implying that the PI3K/Akt pathway could be a key mechanism through which FPH exerts its anti-SGMD effects. Western blot analysis showed a slight increase in PI3K and p-AKT protein expression in the model group. FPH notably increased the PI3K and p-Akt protein expression in rats with SGMD. Specifically, the high-dose group exhibited a significant increase in PI3K expression (P < 0.01), while the mid-dose group showed a marked increase in p-Akt expression (P < 0.01), suggesting that the regulation of the PI3K/Akt pathway by FPH promotes gastric mucosal epithelial cell proliferation and accelerates mucosal repair, thus improving SGMD. The in vivo experimental results support these findings and align with the predictions made through network pharmacology, substantiating the pivotal role of the PI3K/Akt pathway in the therapeutic efficacy of FPH on SGMD. The Keap1/Nrf2/HO-1 pathway is a vital antioxidant signaling mechanism that protects tissue cells from oxidative stress-induced injury. Keap1, a member of the Kelch family, acts as a negative regulator of Nrf2. Nrf2, a key transcription factor, regulates intracellular antioxidant levels and exerts its effects by regulating the expression of genes encoding antioxidant and detoxification enzymes in the nucleus. HO-1, an antioxidant protein regulated by Nrf2, possesses antioxidant, anti-inflammatory, anti-apoptotic, and cytoprotective properties [[159]41]. Normally, Nrf2 interacts with Keap1 in the cytoplasm, where it remains inactive or subject to degradation through ubiquitination. However, under stress conditions, Nrf2 dissociates from Keap1, moves to the nucleus, binds to the antioxidant response element (ARE), regulates the expression of downstream antioxidant enzymes (HO-1, NQO1, etc.), increases antioxidant capacity, and reduces oxidative stress damage [[160]42]. Molecular docking results indicated strong binding activities between FPH active ingredients and P62, Nrf2, and HO-1. In rats with SGMD, initial decreases in Nrf2 and HO-1 mRNA and protein expression were observed, followed by significant increases post FPH treatment. Particularly, the mid-dose group exhibited a more significant rise in Nrf2 and HO-1 protein levels (P < 0.05), consistent with previous research. Various studies have validated that Sambucus nigra Berry Extract, Xiangshao decoction, and mangiferin can enhance Nrf2 and HO-1 protein expression by modulating Keap1/Nrf2, enhancing antioxidant enzyme activity, and ameliorating mucosal damage in gastric ulcer rats [[161][43], [162][44], [163][45]]. Superoxide dismutase (SOD) and glutathione (GSH) are served as crucial endogenous antioxidant enzymes reflecting free radical levels and the body's antioxidant defense capacity. Conversely, malondialdehyde (MDA) is a significant product of lipid peroxidation, with its levels indirectly indicating cell damage caused by ROS. Our results illustrated that WIRS induced oxidative stress in gastric mucosal epithelial cells, as shown by elevated MDA levels and reduced SOD and GSH activities in the model group compared to the control group. Moreover, FPH decreased the MDA levels and increased SOD and GSH activities, suggesting a deficiency in antioxidant enzymes in WIRS rats, exacerbation of mucosal oxidative damage and the protective role of FPH against WIRS-induced SGMD and reversal of the oxidative stress condition. Sequestosome-1 (p62/SQSTM 1) is a ubiquitin-binding protein that plays a critial role in regulating Nrf2 during oxidative stress, involving processes such as oxidative stress, inflammation, and autophagy [[164]46,[165]47]. The KIR domain of p62 interacts with Keap1, forming the p62-Keap1 complex, which then degrades Keap1 through the ubiquitin-proteasome pathway [[166]47]. Furthermore, p62 can induce Keap1 degradation via autophagy, releasing Nrf2 [[167]48]. Studies have indicated that the PI3K/Akt/mTOR pathway can impact p62 expression [[168]49]. Activation of PI3K/Akt/mTOR inhibits autophagy, leading to p62 accumulation and subsequent binding to Keap1, releasing Nrf2 into the nucleus to activate downstream antioxidant genes [[169]50]. The enhancer of p62 contains ARE and is regulated by Nrf2, creating a positive feedback loop that enhances the body's antioxidant capacity [[170]46]. Ulinastatin alleviates LPS-induced oxidative stress by activating PI3K/Akt to inhibit autophagy, promoting p62 accumulation and interaction with Keap1. This interaction facilitates the translocation of Nrf2 into the nucleus for antioxidant gene expression [[171]51]. Conversely, the ethanol extract of Rosa fruit reduces p62 levels over time, thereby suppressing Nrf2 and HO-1 expression [[172]52]. The crosstalk between the PI3K/Akt and Keap1/Nrf2 pathways is vital for antioxidant effects that protect cells from oxidative damage. Our experimental findings demonstrated that FPH intervention significantly upregulated of PI3K and p-AKT expression, leading to a marked increase in p62 expression (P < 0.05), subsequently enhancing Nrf2 and HO-1 gene and protein expression (P < 0.05). Our investigation revealed that FPH treatment activated the PI3K/Akt pathway, upregulated the expression of PI3K and p-Akt, inhibited autophagy, increased p62 expression, enhanced p62-Keap1 interaction, facilitated Nrf2 dissociation and nuclear translocation, and boosted downstream HO-1 expression, thereby providing antioxidative stress and cell protection against oxidative damage. In summary, FPH has the potential to enhance cellular antioxidant defenses and protect cells from stress-induced damage by promoting the accumulation of p62 through the PI3K/AKT pathway. This process facilitates the release of Keap1 and Nrf2, their translocation to the nucleus, and the subsequent up-regulation of antioxidant genes within the Keap1/Nrf2 pathway. Ultimately, this cascade results in elevated levels of crucial antioxidant enzymes such as SOD and GSH, mitigating the detrimental effects of oxygen radicals on cells. These findings offer a solid foundation for utilizing FPH in the prevention and treatment of SGMD. However, certain limitations should be noted. Further research is required to fully understand the precise mechanism by which p62 aids in Keap1 degradation. Additionally, while some pathways identified by KEGG have been validated, others necessitate confirmation through future experimental studies. 5. Conclusion In conclusion, these data indicate FPH has a protective effect on SGMD induced by WRIS, possibly through the regulation of Keap1/Nrf2 and PI3K/Akt pathways. This protection may involve inhibiting oxidative stress and inflammatory responses, reducing mucosal damage, promoting cell proliferation, and inhibiting apoptosis. These finding offer experimental evidence supporting the use of FPH as a potential treatment for SGMD. Ethics declaration Animal experiments were performed in accordance with the International Guidelines for Care and Use of Laboratory Animals, and the experimental protocol was approved by the Animal Ethics Committee of Hainan Medical University (approval number: HYLL-2022-365). Funding This study was supported by the National Natural Science Foundation of China [No. 82260920, 82474456]. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. CRediT authorship contribution statement Bangpei Chen: Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. Xueqing Huang: Writing – review & editing, Software, Methodology, Investigation. Feifei Zhu: Visualization, Validation, Supervision. Yunyun Zhi: Resources, Project administration, Methodology. Mengyu Mei: Visualization, Validation, Investigation. Yonghui Li: Writing – review & editing, Resources, Project administration. Yiqiang Xie: Investigation, Formal analysis, Data curation. Ye Zhu: Visualization, Validation, Supervision. Shouzhong Ren: Writing – review & editing, Project administration, Funding acquisition, Formal analysis. Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Shouzhong Ren reports financial support was provided by the National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Footnotes ^Appendix A Supplementary data to this article can be found online at [173]https://doi.org/10.1016/j.heliyon.2024.e38629. Contributor Information Yiqiang Xie, Email: xieyiqiang@hainmc.edu.cn. Ye Zhu, Email: hy0204047@hainmc.edu.cn. Shouzhong Ren, Email: hy0207054@hainmc.edu.cn. Appendix A. Supplementary data The following are the Supplementary data to this article: Multimedia component 1 [174]mmc1.xls^ (63KB, xls) Multimedia component 2 [175]mmc2.xls^ (87.5KB, xls) Multimedia component 3 [176]mmc3.xls^ (58.5KB, xls) Multimedia component 4 [177]mmc4.docx^ (5.3MB, docx) figs1. figs1 [178]Open in a new tab figs2. figs2 [179]Open in a new tab figs3. figs3 [180]Open in a new tab figs4. figs4 [181]Open in a new tab figs5. figs5 [182]Open in a new tab figs6. figs6 [183]Open in a new tab figs7. figs7 [184]Open in a new tab figs8. figs8 [185]Open in a new tab figs9. figs9 [186]Open in a new tab figs10. figs10 [187]Open in a new tab figs11. [188]figs11 [189]Open in a new tab figs12. [190]figs12 [191]Open in a new tab figs13. [192]figs13 [193]Open in a new tab figs14. [194]figs14 [195]Open in a new tab figs15. [196]figs15 [197]Open in a new tab figs16. [198]figs16 [199]Open in a new tab References