Research On Molecular Mechanism Of Acupuncture At Different Acupoint Combinations Prevents Stress Gastric Ulcer By Regulating The TLRs Signaling Pathway

Objective: This manuscript selects different acupoint combinations(He-Sea-Front-Mu point combination and Back-shu and front-mu point combination)to pre-acupuncture rats with stress gastric ulcer(SGU)to explore their effects on the morphology of gastric mucosa,the effects on the factors related to oxidation and inflammation in serum and gastric tissue,and the effects on TLRs signal pathway in gastric tissue and m RNA of large intestine.The protective effects of different acupoint combinations on the mucosa of SGU rats were analyzed from multiple angles.Discussed the difference in the protective effect of different acupoint combinations on the mucosa,in order to provide an experimental basis for the acupoint combination theory.Methods: 1.36 male Wistar rats were randomly divided into blank group,model group,He-Sea-Front-Mu point combination group and Back-shu and front-mu point combination group,9 rats in each group.The He-Sea-Front-Mu point combination group received electroacupuncture at the RN12 and ST36 acupoints for 10 min every 2 days.The Back-shu and front-mu point combination group received electroacupuncture at the RN12 and BL21 acupoints for 10 min every 2 days.Both groups for 10 days of treatment.The SGU rat model was established by the restraint-water immersion stress method.The morphology of the gastric mucosa of rats in each group was observed macroscopically and hematoxylin-eosin staining,the ulcer index(UI)and histopathological score were calculated.2.Blood was drawn via the abdominal aorta and settled at room temperature for 20 min before separation of the serum by centrifugation at 3000 r/min at 4℃ for 10 minutes;samples were preserved at-20°C until use.These samples were used to detect the levels of GSH-Px,SOD,MDA,MPO,NO,LDH,ET-1,IL-1β,IL-2,IL-6,and TNF-α in serum.Gastric tissue was homogenized in physiological saline(ratio: 1 part tissue to 9 parts saline),then spun by centrifugation(3000 r/min at 4℃ for 10 min)to obtain the supernatant.These samples were used to detect the levels of GSH-Px,NO and MPO.3.Immunohistochemical staining method was used to detect the expression of TLR2,TLR4,MyD88,TRIF protein in gastric tissue of SGU rats.Western blot method was used to detect the relative expression of TLR4,MyD88 and IκB-α protein in gastric tissue of SGU rats.4.Use Fast QC sequencing data and evaluate the quality of the original data,use Trim galore sequencing data for quality control,use HISAT2 reference genome for sequence alignment,Rseqc is used to evaluate the overall quality of the transcriptome,and use String Tie for gene expression quantification and deseq2 for differential gene identification,use WGCNA for WGCNA analysis,use Cluster Profiler for enrichment analysis(GO enrichment analysis,KEGG enrichment analysis).Results: 1.The morphology of the gastric mucosa was observed macroscopically and pathological sections,taking into account the UI and histopathological score,it is found that the pre-acupuncture of He-Sea-Front-Mu point combination and Back-shu and front-mu point combination had the effect of protecting the gastric mucosa of SGU rats.Compared with the model group,there is a significant difference(P<0.0001),moreover,He-Sea-Front-Mu point combination group was better than Back-shu and front-mu point combination group in histopathological score(P<0.05).2.Both He-Sea-Front-Mu point combination group and Back-shu and front-mu point combination group have a regulatory effect on oxidation-antioxidant reactions and inflammatory cytokines.Compared with Back-shu and front-mu point combination group,the SOD(P<0.0001),NO(P<0.001),GSH-Px(P<0.0001)in He-Sea-Front-Mu point combination group was significantly higher,which indicated that He-Sea-Front-Mu point combination group had stronger antioxidant capacity.Compared with He-Sea-Front-Mu point combination group,the MPO(P<0.001),IL-1β(P<0.001)of gastric tissue in Back-shu and front-mu point combination group was significantly reduced,which indicated that the Back-shu and front-mu point combination group had a stronger inhibitory effect on inflammatory injury than He-Sea-Front-Mu point combination group.Therefore,we believed that the reason why oxidative stress can induce SGU is due to the imbalance between the massive production of ROS and the activity of endogenous antioxidants.Acupuncture at He-Sea-Front-Mu point combination can effectively improve the oxidation-antioxidant balance in the stomach,while Back-shu and front-mu point combination tend to inhibit inflammation.3.Compared with the blank group,the expression of TLR4 and MyD88 in the model group was significantly increased(P<0.05),and the expression of IκB-α was reduced(P<0.05).The expression of TLR4,MyD88 and TRIF were significantly reduced in He-Sea-Front-Mu point combination group(P<0.05),and the expression of IκB-α was increased(P<0.05),which indicated that acupuncture at He-Sea-Front-Mu point combination can inhibit the activation of TLR4/MyD88/TRIF/IκB signal transduction pathway.Back-shu and front-mu point combination can significantly reduce TLR4,TRIF and increase IκB-α.This suggested that Back-shu and front-mu point combination may play a role through the TLR4/TRIF/IκB pathway.The difference lies in the amount of MyD88 protein expression.4.The results of KEGG analysis revealed that the genes enriched in the blank group compared with the model group were CASP8,nfkbia,TLR4,ctsk,CXCL10,SPP1,CXCL11,IRF7,TRAF6,which may be the hub genes of stress gastric ulcer.The GO analysis showed that He-Sea-Front-Mu point combination have one common gene named CD36 with Back-shu and front-mu point combination,and two different genes,Tlr1,S100a9,compared with pre acupuncture He-Sea-Front-Mu point combination,and five different genes,Reg3 g,Lbp,Tnf,Tlr5,Nod2,compared with pre acupuncture Back-shu and front-mu point combination,in terms of mucosal protection.These may be the different hub genes in the prevention of mucosal tissue.Conclusion: 1.Pre-acupuncture has a regulatory effect on oxidative stress and inflammatory factor levels in SGU rats,this may be an important initial step in "Balance regulation,prevention and control".Among them,the key factors for the effect difference between different matching acupoints may be SOD,GSH-Px,MPO,IL-1β.2.The protective effect of pre-acupuncture on the gastric tissue of SGU rats may be achieved through TLRs signal pathway transduction,this may be the central link of "Balance regulation,prevention and control".He-Sea-Front-Mu point combination may play a role by inhibiting the TLR1/TLR4/MyD88/TRIF/IκB-α pathway,Back-shu and front-mu point combination may play a role by inhibiting the TLR4/TLR5/MyD88/TRIF/IκB-αpathway,the difference may lie in the TLR1,TLR5,and MyD88 proteins.