| Objective:The study focused on "tonggong points" and combined with the new 4D label-free quantitative proteomics detection technology to investigate the effect of differential protein expression in gastric tissues of rats with Gastric ulcer(GU)model.To investigate the mechanism of action of electroacupuncture homoeopathic points from a protein perspective.Firstly,we evaluated the evidence of clinical application of acupuncture for the treatment of gastric ulcer from the perspective of evidence-based medicine;secondly,we further clarified the biological functions and signaling pathway changes of gastric tissue proteins in rats with electric acupuncture interventions on tonggong points through animal experiments;finally,we verified the differential protein expression in gastric tissues and confirmed the intrinsic connection and biological mechanisms between electric acupuncture interventions on tonggong points and GU regulatory factors,which provided a basis for the study.Finally,we will verify the expression of differential protein effects in gastric tissues and confirm the intrinsic connection between electroacupuncture and GU regulatory factors and biological mechanisms,which will provide new scientific evidence for the study of the commonality of"tonggong points".This will provide evidence and theoretical support for the use of acupuncture "homoeopathic points" in the clinical treatment of GU diseases.Methods:Systematic evaluation and Meta-analysis of clinical effectiveness and safety of acupuncture in the treatment of gastric ulcerChinese and English databases were searched:Pub Med,Embase,Cochrane,VIP,CNKI,CBM,Wanfang,randomized controlled studies that met the criteria for inclusion,study data were extracted,the risk of bias in the literature was evaluated,and Revman 5.4 software was applied to clinical efficacy,ulcer area healing rate,H.pylori conversion rate,ulcer recurrence rate,adverse effects,TCM Meta-analysis of outcome indicators such as symptom scores and inflammatory factors was performed to make a systematic evaluation of acupuncture for gastric ulcer,and also to summarize the acupoint prescriptions included in the study.Experimental partPart Ⅰ:Effect of electroacupuncture intervention at homoeopathic points on gastric histomorphology in GU model rats.Fifty male SD rats of SPF grade.The rats were randomly divided into 6 groups:4 groups of homoeopathic acupuncture points,namely:8 rats each from RN12(Zhongwan group),BL21(Weishu group),PC6(Neiguan group),and ST36(ZuSanli group);8 rats from CON(Blank control group);and 8 rats from Model group(Model group).After respiratory anesthesia,electroacupuncture intervention was performed on single acupuncture points for 30 min/d,respectively,for 2 weeks.The water immersion restraint modeling method was used.Statistical and light microscopic analyses were performed by observing the gastric mucosal damage index and HE staining to determine how the gastric tissue morphology of GU model rats changed by electroacupuncture intervention on the same acupuncture point.Part Ⅱ:Effect of electroacupuncture intervention at homoeopathic points on differential protein expression in GU model rats.Gastric tissues from the above groups of GU model rats were subjected to protein extraction,tryptic digestion,liquid chromatography-mass spectrometry analysis for database search,mass spectrometry quality control assay using 4D label free quantitative proteomics assay technology;the assay results were subjected to statistical analysis,quantitative protein analysis,screening differential protein analysis,Gene Ontology analysis,subcellular localization,COG/KOG functional analysis,KEGG signaling pathway enrichment.Bioinformatic aspects of the differential protein changes and functional enrichment of gastric tissues before and after electroacupuncture intervention on homoeopathic points in GU model rats.Part Ⅲ:Exploring the mechanism of action of electroacupuncture homoeopathic points on GU Model rats based on PI3K/AKT signaling pathwayThe differential protein expression levels in the gastric tissues of GU model rats in the CON group,Model group,and homogeneous cave group were analyzed,and the differential protein expression levels were verified by detecting GGT,PI3K,and AKT factors using Western blot method.The qPCR method was applied to detect the mRNA levels of differential protein PI3K/AKT pathway.To explore the molecular mechanism study of electroacupuncture intervention at tongong points for repairing gastric mucosal damage and reducing inflammatory factors in the treatment of GU.Results:Clinical Literature StudyIn terms of total clinical efficiency,ulcer area cure rate,HP clearance rate,and ulcer recurrence rate,there were significant differences between acupuncture treatment of gastric ulcer compared with conventional western medicine;and compared with conventional western medicine treatment,acupuncture was effective in reducing the inflammatory factors TNF-α,IL-6,GAS,and VEGF in gastric ulcer and in improving clinical symptoms The effect of acupuncture was positive.The most frequently used acupoints in the prescription of acupuncture points were:Zu San Li,Zhong Guan,Nei Guan,and Wei Shu,which provided the basis for the clinical literature study of the "homoeopathic acupuncture points" for gastric ulcer selected for this study.Experimental partPart Ⅰ:The comparison of electroacupuncture intervention at tonggong points with the Model group showed that the gastric mucosa of rats with gastric mucosal injury were repaired to different degrees,mainly by the reduction of gastric mucosal injury index,the improvement of gastric mucosal epithelial cell shedding,neater and more regular arrangement,tissue proliferation of gastric mucosal epithelium,proliferation of gastric glandular cells,which tended to be normal,and the gradual reduction of inflammatory cells;however,the differences between different sites of tonggong points were not significant by the naked eye,ulcer injury index and light microscopic observation.Part Ⅱ:Bioinformatics analysis showed that the differential protein co-regulated by homologous cave was GGT5,and 42 differential proteins related to gastric ulcer were crossscreened by other groups.-multiple species),Focal adhesion,Thl and Th2 cell differentiation,Th17 cell differentiation,Tight junction,FOXO signaling pathway,which act with apoptosis,inflammatory response.Immune response,mucosal repair and other pathways.The GO annotation distribution co-regulates the number of differential proteins in 9 sites of Biological Process,3 sites of Cellular Component and 5 sites of Molecular Function;6 sites of differential proteins co-regulated in subcellular structure annotation classification in the COG/KOG functional classification;8 functions of CELLULAR PROCESSES AND SIGNALING,5 functions of INFORMATION STORAGE AND PROCESSING,and 4 functions of METABOLISM in the COG/KOG functional classification.The number of differential proteins in the functions.Part Ⅲ:Bioinformatics analysis showed that:the differential protein co-regulated by the homologous cave was GGT5,and 42 differential proteins related to gastric ulcer were crossscreened by other groups;the common enriched signaling pathway was PI3K-AKT signaling pathway;7 signaling pathways related to GU were enriched by each group:PI3K-Akt signaling pathway,Apoptosis-multiple species,Focal adhesion,Thl and Th2 cell differentiation(Thl-Akt signaling pathway).pathway,Apoptosis-multiple species,Focal adhesion,Thl and Th2 cell differentiation,Th17 cell differentiation),Tight junction(Tight junction),FOXO signaling pathway(FOXO signaling pathway),where the roles are related to apoptosis,inflammatory response,immune response,mucosal repair and other pathways.The GO annotation distribution co-regulates the number of differential proteins in 9 sites of Biological Process,3 sites of Cellular Component,and 5 sites of Molecular Function;6 sites of differential proteins co-regulated in the subcellular structure annotation classification in the COG/KOG functional classification;8 functions of CELLULAR PROCESSES AND SIGNALING,5 functions of INFORMATION STORAGE AND PROCESSING,and 4 functions of METABOLISM in the COG/KOG functional classification.The number of differential proteins in the functions.Conclusion:1.Through the perspective of evidence-based medicine,the clinical application value of acupuncture and moxibustion for gastric ulcer was clarified,and the most frequently used acupoints in teaching materials and clinical applications were summarized:Zu San Li,Zhong Wan,Nei Guan and Wei Shu,which provided the basis for the literature study on the selection of "homoeopathic points" for gastric ulcer in this thesis.2.The differential protein that can be co-regulated by electroacupuncture GU homologation points is GGT5,and the other groups cross-screened 42 differential proteins related to gastric ulcer,whose effects were related to inhibition of inflammatory cells,reduction of necrosis factor/apoptosis,reduction of cancer cell multiplication and cancer cell markers,promotion of epidermal growth factor and gastrointestinal immune factors;the signaling pathways that were co-enriched to were PI3K-AKT signaling pathway(PI3K-Akt signaling pathway),and seven GU-related signaling pathways were enriched in each group:PI3K-Akt signaling pathway,Apoptosis-multiple species,Focal adhesion signaling pathway,and Thl-Akt signaling pathway.Focal adhesion,Thl and Th2 cell differentiation,Th17 cell differentiation,Tight junction,FOXO signaling pathway),whose roles are related to apoptosis,inflammatory response,immune response,mucosal repair and other pathways.These differential proteins and signaling pathways may be critical "targets" in the study of gastric ulcer mechanisms.3.Electroacupuncture GU tonggong points can regulate the expression of gastric tissue differential proteins,which can down-regulate HP’s toxic protease GGT and inhibit PI3K/AKT signaling pathway activity,reduce gastric cell apoptosis and stimulation,reduce inflammatory factor expression and secretion,repair gastric tissue mucosal damage,thus playing an anti-ulcer effect. |
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