Research on the core genes and key microRNA screening of Huangqin Huanglian medicine for the treatment of Helicobacter pylori associated peptic ulcers
Peptic ulcer (PU) is one of the most common digestive system diseases in clinical practice, which tends to occur in the stomach and duodenum. The incidence rate of PU varies in different countries and regions. Research shows that about 5%~10% of people in the world have suffered from peptic ulcer in their lifetime. Clinical data shows that patients with peptic ulcer disease account for 10.3% to 32.6% of gastroscopy cases in China. The typical symptom of most PU patients is recurrent rhythmic pain in the upper and middle abdomen; Some patients lack specificity in their symptoms, manifested as gastrointestinal symptoms such as upper abdominal distension, anorexia, and acid reflux; A small number of patients have PU complications such as gastrointestinal bleeding and perforation as the main symptoms. With the growth of China’s economic level, the improvement of people’s quality of life and the accelerated pace of work, the incidence rate of PU is getting higher and higher, which has a serious impact on the physical and mental health and quality of life of patients.

Since Australian scholars Warren and Marshall isolated Helicobacter pylori (Hp) from gastric mucosal biopsy specimens of patients in 1983, Hp has been confirmed to be closely related to the occurrence, development, and recurrence of PU. Peptic ulcers caused by Hp infection are also known as Hp associated PU. The combination of antibiotic antibacterial therapy and proton pump inhibitor acid suppression therapy is currently the basic method for treating Hp related PU. But with the widespread use of antibiotics, the resistance rate of Hp to clarithromycin, metronidazole, and levofloxacin in China has been increasing year by year, leading to a decrease or even failure of triple therapy in eradicating Hp. At present, the preferred treatment for Hp related PU in China is bismuth quadruple therapy with a course of 10 or 14 days. This method also has problems such as increased antibiotic resistance, high adverse reactions, low patient compliance, and heavy economic burden. Therefore, the application of traditional Chinese medicine therapy to expand the treatment plan for Hp related PU is one of the hotspots in gastroenterology research.

Traditional Chinese Medicine believes that Hp related PU can be classified under the categories of “epigastric pain”, “acid swallowing”, “noise”, etc. The basic pathogenesis is the invasion of pathogenic toxins, deficiency of positive and negative energy, and imbalance of qi movement. Hp belongs to the category of “evil qi” and often has pathological properties of heat and toxicity. Ulcers caused by Hp are often manifested as damp heat syndrome. The method of clearing heat, dispelling dampness, and detoxifying is one of the important treatment methods for Hp related PU. Coptis chinensis Franch is a plant in the Ranunculaceae family (also known as Weilian), Sanjiaoye Huanglian C Deltoidea C.Y. Cheng et Hsiao (also known as Yalian) or Yunlian C The dry rhizomes of teeta Wall are cold in nature and bitter in taste. They are found in the meridians of the heart, spleen, stomach, and large intestine, and have the effects of clearing heat, drying dampness, purging fire, and detoxifying. Scutellaria baicalensis Georgi, a plant belonging to the family Lamiaceae, is the dried root of Scutellaria baicalensis Georgi. It has a cold nature and a bitter taste, and is found in the lungs, gallbladder, spleen, large intestine, and small intestine meridians. It has the effects of clearing heat and dampness, purging fire and detoxifying, cooling blood and stopping bleeding. The medicinal pair composed of Scutellaria Radix Coptidis Rhizoma (SRCR) is one of the classic heat clearing and detoxifying drugs in traditional Chinese medicine. The two are often used in combination to treat Hp related PU and have definite clinical efficacy. However, existing reports lack a systematic and comprehensive study on the molecular network regulation mechanisms of their active ingredients, targets, signaling pathways, etc.

Network pharmacology is a new drug research model that integrates theories from multiple disciplines such as bioinformatics, systems biology, and computer network analysis technology. It can explore the relationship between drugs and diseases from the perspective of overall biological network homeostasis. The holistic and systematic characteristics presented by network pharmacology coincide with the principles of TCM holistic view, syndrome differentiation and treatment, and TCM compatibility. This study used network pharmacology and bioinformatics methods to analyze the multi-component, multi-target, and multi pathway characteristics of Huangqin Huanglian medicine. Based on core genes and key microRNAs, the molecular network regulatory mechanism of Huangqin Huanglian medicine in treating Hp related PU was explored, aiming to provide scientific basis for its clinical treatment of Hp related PU and provide ideas and references for subsequent in-depth experimental research.

Firstly, the main active ingredients and potential targets of SRCR were retrieved through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP); Obtain GSE70394 gene expression profile data from GEO database, use R software to homogenize the chip data of differentially expressed genes (DEGs) involved in Hp infected human gastric epithelial cells (GES), and draw DEG volcano and cluster heat maps; Construct a protein interaction network between SRCR component targets and Hp infected human GES cell DEGs using the STRING database; Perform GO functional and KEGG pathway enrichment analysis on the aforementioned characteristic genes using the DAVID database; Then, Cytohuba was used to screen for the core genes involved in SRCR treatment of Hp related PU; Prediction of key microRNAs regulating core genes using Target Scan and miRBase databases.

Peptic ulcer is a disease characterized by inflammatory reactions and necrotic lesions of the mucosa under the action of various pathogenic factors. The lesions can penetrate the mucosal muscle layer or reach deeper levels, with gastric and duodenal mucosa being the most common. Since the discovery of Hp, numerous studies have confirmed that Hp is the most important factor leading to the onset and recurrence of peptic ulcers. Hp related PU refers to the occurrence, development, prognosis, and recurrence of PU that are closely related to Hp infection. The pathogenic mechanism of peptic ulcer caused by Hp infection is very complex and has been a long-term research topic for clinical workers in gastroenterology. Currently, there are four main related theories: ① leaky roof theory; ② Gastrin gastric acid related theory; ③ Gastric metaplasia theory; ④ Media flushing theory. In clinical practice, triple or quadruple therapy is commonly used to inhibit or kill Helicobacter pylori, reduce gastric acid and protect gastric mucosa, thereby treating Hp related PU. Due to the long-term irrational use of antibiotics, the resistance of Hp has been increasing year by year, leading to a decrease or even failure in the cure rate of triple or quadruple therapy. In addition, the impact of Hp eradication programs on the normal gastrointestinal microbiota, such as causing dysbiosis of the microbiota or overgrowth of small intestinal bacteria, has also received widespread attention. Therefore, choosing traditional Chinese medicine comprehensive therapy to solve the problem of Hp resistance, reduce the gastrointestinal effects and other adverse reactions of antibiotics, and control the recurrence rate of ulcers has important clinical significance.

There is no record of the disease name “Hp related PU” in traditional Chinese medicine. Modern doctors classify it as “epigastric pain”, “acid swallowing”, “noise” and other categories based on the main clinical symptoms. Traditional Chinese Medicine believes that there are many causes of epigastric pain, mainly including external pathogens invading the stomach, emotional disorders, irregular diet, and prolonged physical weakness. Hp infection belongs to the category of exogenous pathogenic qi, and Hp related PU often has thermal and toxic properties. When the spleen and stomach are weak and the body’s vital energy is insufficient, Hp takes advantage of the deficiency to invade the gastrointestinal tract, causing disharmony in spleen and stomach function, imbalance in qi movement, and the formation of various pathological products such as qi stagnation and damp heat, damaging the mucosa, rotting the muscles, and causing ulcers. As stated in “The Origin and Source of Miscellaneous Diseases: Rhinoceros Candle · Stomach Diseases”: “Stomach pain is caused by pathogenic dryness in the epigastric region. The stomach is filled with qi that promotes harmony, with abundant qi and blood. When the stomach is strong, evil cannot be dried up, and when it is weak, it becomes a disease. It tends to be cold and hot, with stagnant water and food, all of which are painful due to the struggle with true qi. A large number of clinical research results have shown that Hp related PU is more common in spleen stomach damp heat syndrome, and the treatment of clearing heat and eliminating dampness has important guiding significance for the treatment of this disease. The combination of Huangqin and Huanglian is a classic clinical formula for clearing heat, dispelling dampness, and detoxifying toxins. Both belong to the category of bitter cold and purging heat products, and should be used together to have the effects of clearing heat, drying dampness, purging fire, detoxifying, cooling blood, and stopping bleeding. The combination of Huangqin and Huanglian is also a core drug pair in commonly used formulas for treating Hp related PU, such as Banxia Xiexin Tang, Huanglian Jiedu Tang, Sanhuang Xiexin Tang, etc. Modern pharmacological research has shown that both Scutellaria baicalensis and Polygonatum sibiricum have highly antibacterial effects on Hp, as well as antioxidant, anti-inflammatory, antipyretic, hypoglycemic, anti-tumor, protective of gastric mucosa, and immune regulation effects. Huangqin and Huanglian, two traditional Chinese medicines, have similar efficacy and pharmacological effects. Their combination often has a synergistic effect and is widely used in the clinical treatment of Hp related PU.

This study is based on network pharmacology and bioinformatics to explore the main molecular network regulatory mechanisms of Huangqin Huanglian medicine in the treatment of Hp related PU, and to investigate the core genes and key microRNAs that may play a role in its pharmacological effects. In the TCMSP database, 41 active ingredients of Huangqin Huanglian medicinal pairs were finally screened, among which Huanglian is mainly composed of alkaloids, and Huangqin is mainly composed of flavonoids. Further exploration revealed 216 potential targets and 764 interactions between components and targets. By processing and analyzing the GEO database GSE70394 dataset, 127 significantly differentially expressed genes were obtained between Hp infected human gastric mucosal epithelial cells and normal gastric mucosal epithelial cells, including 77 up-regulated genes and 50 down regulated genes. Using STRING database and Cytoscape software, the PPI network of active ingredient targets in Huangqin Huanglian medicine and the PPI network of significantly differentially expressed genes in Hp infected human gastric epithelial cells were fused, and the intersection of the two was extracted to obtain the PPI network of Huangqin Huanglian medicine for treating Hp related PU. In order to further analyze the possible mechanism of action of Huangqin Huanglian medicine on the treatment of this disease with active ingredients, we conducted GO and KEGG enrichment analysis on 127 specific gene targets in its network.

The GO and KEGG enrichment analysis results involve multiple biological processes and important signaling pathways. The GO analysis results showed that Huangqin Huanglian medicine plays an important regulatory role in biological processes such as cytokine response, positive regulation of protein kinase activity, inflammatory response, immune response, negative regulation of cell proliferation, and positive regulation of NF – κ B transcription factor activity. And these biological processes can be reflected in the signaling pathways involved in the regulation of target genes by Huangqin Huanglian medicine. The KEGG analysis results showed that 35 signaling pathways were closely related to the treatment of Hp related PU with Huangqin Huanglian medicine. The significantly enriched signaling pathways include cytokine cytokine receptor interaction pathway, NOD like receptor signaling pathway, cell adhesion molecule pathway, ErbB signaling pathway, NF – κ B signaling pathway, Jak STAT signaling pathway, etc.

NOD like receptors are pattern recognition receptors located in the cytoplasm, which are of great significance in preventing and treating intracellular pathogen infections and participating in innate immune responses. Although Hp is an extracellular parasite, it can transfer peptidoglycans into host cells and stimulate host defense responses upon contact with NOD1 protein. Research has shown that mice with NOD1 protein deficiency are more susceptible to Hp. NF – κ B is a nuclear transcription factor belonging to the Rel protein family. After activation, it can regulate the transcriptional expression of various genes and widely participate in biological processes such as inflammation, immune response, oxidative stress, cell proliferation, differentiation, and apoptosis in the body. Some studies suggest that activation of the NF – κ B signaling pathway is one of the markers of Hp infection and plays a central role in the development of related inflammatory diseases and cancers. The Jak STAT signaling pathway is a cytokine stimulated signaling pathway that mediates the activation of NF – κ B and the expression of IL8 in Hp infected gastric epithelial cells. From this, it can be seen that a large number of studies have confirmed the close relationship between NOD like receptor signaling pathway, NF – κ B signaling pathway, and Jak STAT signaling pathway and Hp infection, indicating that this study has certain accuracy in predicting the molecular network regulation mechanism of Huangqin Huanglian medicine in the treatment of Hp related PU.

The cytokine cytokine receptor related pathway can regulate cell growth and differentiation, regulate immune response, and participate in the occurrence of various diseases such as inflammation under certain conditions. The cell adhesion molecular pathway is involved in cell attachment and movement, and is the basis of physiological and pathological processes such as immune response, inflammatory response, coagulation, and wound healing. The cytokine cytokine receptor related pathway and cell adhesion molecule pathway are closely related to immune function, but there are currently few reports on their association with Hp infection, which provides new ideas for future research. There are interconnections and intersections between signal pathways, which can expand their respective functions and form complex regulatory networks. Huangqin Huanglian medicine not only acts on these significantly enriched pathways, but also on other pathways related to cancer, jointly exerting therapeutic effects on Hp related PU.

The MCC algorithm of Cytohuba was used to analyze the PPI network of Huangqin Huanglian medicine in the treatment of Hp related PU. This study identified the top 10 core genes: CXCL8 IL10、IL1B、IL1R1、TNFRSF1B、IL6R、IL10RA、CD28、VEGFA、CD40LG。 These core genes are often associated with inflammatory and immune responses. CXCL8, IL1B, and IL10 are inflammatory cytokines. CXCL8, also known as IL8, is a powerful pleiotropic cytokine that can attract neutrophils to the site of inflammation, regulate immune responses, and participate in tumor proliferation, migration, and invasion. Researchers co cultured recombinant Helicobacter pylori antigen HP0986 with gastric epithelial cells and found that HP0986 can induce the secretion of IL8 by gastric epithelial cells in a dose – and time-dependent manner through the NF – κ B pathway. IL1B is an important inflammatory mediator secreted by monocytes/macrophages, promoting T and B lymphocyte proliferation and activating neutrophils. Studies have found that exogenous administration of 10 ng/mL IL1B can stimulate cell proliferation, inhibit Helicobacter pylori induced apoptosis, and suppress parietal cell secretion of H+by downregulating H+- K+- ATPase expression, suggesting that IL1B plays an important role in Hp induced gastric cancer. A large sample meta-analysis of 21 studies also suggests that IL-1B-31C/T gene polymorphism may increase the risk of Hp infection, and IL-1B-511-C/T and IL-8-251T/A gene polymorphisms may be risk factors for Hp associated ulcers. IL10 is an anti-inflammatory cytokine, and clinical studies have found that its expression is significantly higher in Hp positive patients with peptic ulcers and gastritis than in Hp negative healthy individuals. The expression level of IL10 is positively correlated with the degree of Hp infection and the development of chronic inflammation.

IL1R1, TNFRSF1B, IL6R, and IL10RA are all cytokine receptors that bind to inflammatory factors such as IL1, TNF – α, IL6, and IL10. Cytokines exert their effects by binding to corresponding receptors to initiate intracellular signaling pathways. CD28 is the main co stimulatory receptor on T cells, and the co stimulatory signal generated by its binding with B7 plays an important role in T cell activation. CD40LG binds to CD40 as the most important second messenger for B cell activation, promoting B cell differentiation and maturation as well as antibody production. VEGFA is a potent vascular endothelial growth factor that can intervene in angiogenesis and may play a key role in ulcer healing. After Hp infection, these key genes interact with the body, and under the mediation of pathways such as NF – κ B signaling cascade, they form a complex immune inflammation regulatory network. Therefore, Huangqin Huanglian medicine may exert its therapeutic effect on Hp related PU through the direct or indirect action of multiple active ingredients.

MicroRNA is a type of single stranded non coding small RNA that is approximately 18-25 nucleotides long. It binds to the 3 ‘or 5′ UTR region of specific target genes’ mRNA through the principle of complementary base pairing, playing a role in translation inhibition or direct degradation of mRNA. This study aims to further investigate the effect of microRNA mediated gene expression regulation on the core genes of Huangqin Huanglian medicine in the treatment of Hp related PU. The most functional microRNAs were retrieved and screened through the Target Scan and starBase databases. The results showed miR-93-5p, miR-374b-5p, miR-3937, miR-19a-3p, miR-4458, miR-590-5p, miR-6838-5p, and miR-424-5p. The discovery of these microRNAs further elucidated the molecular mechanism of Huangqin Huanglian medicine in treating Hp related PU at the non coding RNA level.

To sum up, based on network pharmacology and bioinformatics, this study explored the main molecular network regulation mechanism of Scutellaria baicalensis Georgi and Coptis chinensis on the treatment of Hp related PU, clarified the core genes and key microRNAs regulated by Scutellaria baicalensis Georgi and Coptis chinensis on the treatment of Hp related PU, and verified the characteristics of the synergistic effect of Scutellaria baicalensis Georgi and Coptis chinensis on multi-component, multi-target, and multi-channel. The research results of this article are consistent with the existing studies on the pathogenesis and pharmacological effects of Hp related PU, which to some extent confirm the reliability of this article. The research results of this article can provide scientific basis for the clinical promotion and application of Huangqin Huanglian medicine in the treatment of Hp related PU, and provide ideas and references for future in-depth experimental research.