Study On The Mechanism Of Down-regulation Of NF-κB/NLRP3 Signaling Pathway By Acupuncture With ST36 And CV12 For Prevention And Treatment Of Exercise-induced Stress Ulcer

ObjectiveStress ulcer(SU)refers to a disease in which symptoms such as acute mucosal erosion,shallow ulcers,and bleeding occur in the digestive tract such as the stomach and duodenum when the body is under stress.Peptic ulcers caused by exercise as a stressor are called exercise-induced stress ulcers(ESU).Pyroptosis is a special type of programmed cell death,which is characterized by the release of a large number of pro-inflammatory factors along with cell expansion and death during the process of pyroptosis,thereby amplifying local and systemic inflammatory responses and causing tissue and cell damage.In order to clarify the role of pyroptosis in the pathogenesis of ESU and the regulation mechanism of ST36 and CV12 pre-acupuncture on pyroptosis,the study was based on the NF-κB/NLRP3 signaling pathway.The network method was used to analyze the mechanism of acupuncture with ESU,and then by constructing an ESU rat model to observe the effect of regulating cell pyroptosis on ESU,and then to clarify the effect of combined acupoints and pre-acupuncture on NFκB/NLRP3 signaling The role of cell pyroptosis mediated by the pathway was revealed,thereby revealing the mechanism of action of pre-acupuncture with ST36 and CV12 in preventing and treating ESU,and providing a basis for clinical application of acupuncture to prevent and treat ESU.Methods1.Analysis of acupoint selection rules and target network research(1)Search the clinical literature on the treatment of peptic ulcer from databases such as Pubmed and Web of science until November 2020,screen the literature according to the inclusion and exclusion criteria,and use the Apriori algorithm of SPSS 22.0 and SPSS Modeler 18.1 to use frequency Analysis,cluster analysis and association rule analysis were used to obtain the common acupoint compatibility of clinical acupuncture and moxibustion in the treatment of peptic gastric ulcer.(2)From 2010 to 2021,CNKI,Wanfang Database and other databases were searched to collect literature on the targets of Zusanli(ST36)and Zhongwan(CV12),and the literatures were screened according to the inclusion and exclusion criteria.Through the UniProt database,the effect targets of acupoints are obtained and then intersected with the disease targets of ESU in the GeneCards database to obtain the action targets.Afterwards,protein interaction network analysis and KEGG enrichment were performed on the targets of ST36 and CV12 in the treatment of ESU,to clarify the mechanism involved in ST36 and CV12 in the treatment of ESU,and to provide a basis for subsequent experimental verification.2.Experimental research(1)Construction of ESU rat model3%weight-bearing swimming to exhaustion was used to establish the model,and 48 Sprague Dawley(SD)rats were randomly divided into blank group,one-week group,twoweek group,and four-week group.Exhaustive swimming training was performed every day,and body weight and food intake were recorded.Basic indicators such as water intake,water intake,and mortality were sacrificed at 1 week,2 weeks,and 4 weeks after the intervention,respectively,and the rats were sacrificed.The expressions of MDA,SOD,IL-18,IL-1β,IL6 and TNF-α in serum of rats in each group were detected.(2)Down-regulation of NF-κB/NLRP3 signaling pathway by ST36 and CV12 inhibits the pyroptosis of gastric mucosal epithelial cells in ESU ratsThe ESU model was constructed by 3%weight-bearing exhaustive swimming,and SD rats were randomly divided into blank group,model group,electronic acupuncture group.inhibitor group and sham acupuncture group.After 1 week of intervention,the rats were sacrificed and the samples were collected.The gross tissue was observed,the ulcer index was calculated,and HE staining The histopathological changes of rat gastric mucosa were observed,the ultrastructural changes of gastric mucosal epithelial cells were observed under electron microscope,and TLR4,NF-κB,p-NF-κB,NLRP3,Myd88,Caspase-1,ASC were detected by PCR,GSDMD,IL-18,IL-1β mRNA expression.The co-localization of NLRP3 and ASC and the expression of cleaved-caspase-1 in rat gastric mucosa were detected by immunofluorescence method under confocal microscope.Western blot was used to detect TLR4,NF-κB and p-NF-κB in rat gastric mucosa.,IκB,p-IκB,NLRP3,caspase-1,cleavedcaspase-1,GSDMD,ASC,HMGB1,p-mTOR,stat3,p-stat3,Myd88,IL-18,IL-1β protein expression,ELISA The contents of SOD,MDA,IL-18,IL-1β,IL-6 and TNF-α in serum of rats were detected by method.Results1.Analysis of acupoint selection rules and target network research(1)An analysis of 222 literatures showed that CV12 and ST36 were most frequently used in clinical acupuncture treatment of PU.In terms of its meridians,the most used is the Ren meridian,followed by the bladder meridian of Foot Taiyin,the stomach meridian of Foot Yangming,the pericardium meridian of Hand Jueyin,and the spleen meridian of Foot Taiyin.The acupoints selected are mainly Weishu,Pishu and Ganshu,which are selected for the PU disease position,and play the role of nourishing qi and strengthening the spleen,soothing the liver and stomach.From the results of association rule analysis,it can be seen that CV12 and ST36 have the highest support as co-motivated acupoints.(2)By analyzing 346 literatures,a total of 317 targets of Zusanli-Zhongwan acupoint effect were obtained,which were mapped with 1601 targets of ESU,and 176 common targets were obtained.The PPI network involves 53 nodes,among which IL-1β,IL-6,TNF,EGFR,TLR4,etc.have high degree values;KEGG pathway analysis obtained 54 pathways,involving inflammatory response,cell proliferation,hormone regulation,immune response,oxidative response Exciter and other pathways.Considering the characteristics of ESU,it is considered that it is mainly related to TNF signaling pathway(TNF signaling pathway),HIF1 signaling pathway(HIF-1 signaling pathway),NOD-like receptor signaling pathway(NOD-like receptor signaling pathway),Toll-like receptor signaling pathway pathway(Tolllike receptor signaling pathway)and other pathways.2.Experimental research(1)Exploration and construction of rat ESU modelCompared with the blank group,the rats in the 1W ESU group had slower weight gain,increased water intake,better swimming performance,higher modeling success rate,and lower mortality.The weight gain of 2W ESU rats slowed down,and there was no difference in water intake,but swimming performance decreased gradually,modeling success rate decreased,and mortality increased.There was no significant difference in weight gain between 4W ESU rats and the blank group,but their swimming performance was poor,and even negative emotions such as depression and anxiety appeared.The success rate of modeling decreased and the mortality rate increased.The gastric mucosal damage in the 1W ESU group was heavier,manifested as acute hemorrhage,heavier ulcer lesions,larger area,deeper ulcers,and obvious necrotic tissue;Few scattered bleeding spots,less necrotic tissue.Compared with blank group,2W ESU group and 4W ESU group,serum SOD level in 1W ESU group was significantly lower than that in blank group,2W ESU group and 4W ESU group(P<0.05);serum MDA level was higher than that in blank group,2W ESU group and 4W ESU group(P<0.05).Compared with blank group,2W ESU group and 4W ESU group,the levels of-1β,IL-18,IL-6 and TNF-α were higher(P<0.05).(2)ST36 and CV12 down-regulation of NF-κB/NLRP3 signaling pathway inhibits cell pyroptosis in ESU ratsAfter intervention,HE staining showed that the gastric mucosal epithelial and interstitial edema of the rats in the EA group and the MCC group was obvious,and inflammatory cells and a large number of red blood cells were observed locally.The situation was significantly improved compared with the MOD group and the SHAM group.UI in EA group and MCC group was lower than that in MOD group and SHAM group(P<0.05).NLRP3 and ASC fluorescence suggested co-localization.The expressions of TLR4,NF-κB,p-NF-κB,NLRP3,Myd88,Caspase-1,ASC,GSDMD,IL-18,and IL-1β mRNA in EA group and MCC group were decreased compared with MOD group and SHAM group(P<0.05).The protein expression of NF-κB,p-NF-κB,IκB,p-IκB,NLRP3,caspase-1,cleaved-caspase-1,GSDMD,ASC,HMGB1,p-mTOR,stat3,p-stat3,Myd88,IL-18,IL-1β,compared with the MOD group and SHAM group,were decreased(P<0.05).The SOD level in serum of EA group and MCC group increased compared with the MOD group and SHAM group(P<0.05),and the content of the MDA,IL-18,IL-1β,IL-6,TNF-α in serum was lower than that of MOD group and SHAM group(P<0.05).Conclusions1.The ESU rat model can be effectively replicated by using 1-week loading exhaustive swimming.2.Pyroptosis exists in the gastric tissue of ESU rats,and inhibition of pyroptosis can prevent the occurrence of ESU.3.ST36 and CV12 can inhibit the pyroptosis and prevent ESU by down-regulating the NF-κB/NLRP3 signaling pathway.4.Pyroptosis can be regarded as the microscopic manifestation of the spleen governing the transport and transformation,and the inhibition of cell pyroptosis is an important way to strengthen the spleen by combining the acupoints and recruiting them.