Influence Of Hot-humid Stress On HSPs Mediated TLR4 Signal Channel In ETEC Induced Diarrhea Mice And Intervention Effect Of Sanren Decoction

Travelers’ diarrhea(TD),as a special type of infectious diarrhea,has epidemiological characteristics that are closely related to the region and time.First,TD occurs closely with the tourist destination.Dupont divides the earth into three categories according to the incidence of dike diarrhea.Most countries in South Asia,Africa and Latin America are high risk areas.Second,the source of the traveler also affects the incidence of TD.If the source of the traveler is similar to the social climate of the destination climate,the incidence of TD is lower.And TD often occurs in the early stages of the stay outside the place,the longer the stay,the lower the incidence and recurrence rate.Similarly,the incidence of TD is also reduced if the recent to the tropical areas are re-visit.Experts speculate that this phenomenon is related to the body’s stress and acclimatization.TD has a variety of factors,but the most important biological factors,of which the main disease to enterotoxigenic Escherichia coli(ETEC),accounting for about 40%.Lipopolysaccharide(LPS)is the main material of the outermost layer of E.coli cell wall.Toll-like receptors(TLRs)are located on the cell surface and play an important role in the immune response.It is also an important receptor for macrophages involved in the inflammatory response and plays a key role in immune defense.When TLR4(Toll like receptor 4)receives LPS(Lipopolysaccharide)stimulation,it can lead to mononuclear-macrophage activation and release of a series of cytokines,so that it can enhance phagocytic and bactericidal ability.MyD88(myeloid differentiation primary response protein 88)is a node position in the TLR signaling pathway,and it can activate the NF-kB signal pathway and plays a critical role in inherent immune defense,signal pathway and disease process.NF-kB is a transcriptional regulator that regulates the expression of inflammation and immune genes,which is activated by a variety of stimuli,such as bacterial lipopolysaccharide,oxidative stress,cytokines and so on.HSP60 and HSP70 belong to the heat shock protein,which is the protein synthesized in vivo when the organism resists the sub-lethal temperature(generally higher than its normal growth temperature 8～12 ℃).when pathogens infected,HSP60(heat shock protein 60)and HSP70(heat shock protein 70)can act as immune antigens to activate the immune system.Therefore,in this study,we investigated the effects of damp-heat stress on the TLR4-related signal pathway in ETEC-infected diarrhea.The pathogenesis and the role of stress in diarrhea were discussed."Strong moisture will cause diarrhea",Damp heat is the main cause of diarrhea.There is also an epidemiological survey that travelers have a higher incidence of diarrhea in the summer and rainy season.Sanren decoction from Wu Jutong’s book"Wen disease differentiation",is the damp heat syndrome common side.Sanren decoction could treat the syndrome of dampness-heat from the triple energizer.In clinical practice,the modified Sanren decoction can treat a variety of damp-related diseases.This study also observed the effect of Sanren decoction on HSPs and TLR4-related signal transduction in ileal tissue of diarrhea mice under the effect of damp-heat stress.To explore the pathogenesis and Sanren decoction prevention and control mechanism.Objective:In this study,the diarrhea mice model under damp-heat stress was induced by compound factors(external damp-heat stress and E.coli ETEC infection).To observe the behavioral changes of Sanren Decoction on ETEC-induced diarrhea in mice under damp-heat stress.And discussion Toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),heat shock protein 60(HSP60),heat shock protein level changes(of HSP70)and nuclear factor-κB(NF-κB)expression.We analyze and discuss the mechanism of heat stress on TLR4 intracellular signal transduction and downstream NF-κB signal transduction,and the mechanism of Sanren decoction.Methods:140 clean BABL/c mice were used and randomized.Through the high temperature and high humidity stimulation,the toxicity of E.coli intraperitoneal injection,comprehensive construction of damp-heat diarrhea diarrhea animal model,given Sanren decoction treatment,given rifaximin as a positive control.The expression of TLR4,MyD88,HSP60,HSP70 and NF-κB were detected by real-time fluorescence quantitative PCR.The pathological changes of ileum were observed by HE staining.Results:1.In the model group,the mice in the damp-heat model group were feverish,infatuated,dull,perianal swelling,pond or mucus.HE staining of ileum tissue in damp-heat model mice showed mucosal congestion,edema,exudation,lymphoid tissue hyperplasia and extensive inflammatory cell infiltration.2.The mRNA levels of TRL4,MyD88,NF-kB,HSP60 and HSP70 in the ileum tissue of the 6th hour model were more increasingly than blank control group,and have significant statistical differences.The mRNA levels of TRL4 and NF-kB in ileum tissue of Sang Ren Tang were significantly decline than those in damp-heat model group.The mRNA level of TRL4 in ileum tissue of Sanren decoction group was significantly decline that of positive control group at the 6th hours.Sanren decoction group compared with the positive control group in the other indicators no significant difference.Conclusion:1.On the basis of the intraperitoneal injection of E.coli,artificial climate incubator,automatically adjust the temperature,humidity,heat diarrhea syndrome model system,reflecting the heat TCM syndrome stomach injuries,then induced the pathogenic characteristics.2.Damp-heat stress and ETEC infection can cause excessive expression of HSP60,HSP70 and TLR4 in ileum tissue of mice at 6th hour,which is the reason for aggravating the inflammatory response.HSP60,HSP70 may modulate the activation of TLR4 and NF-κB.3.Sanren decoction could protect the host cells,inhibit the transmission of LPS-TLR4 signaling pathway and delay the downstream NF-kB expression by maintaining high expression of HSP60 and HSP70.This will control the body inflammation,to achieve the effect of preventive treatment.And Sanren decoction is better than rifaximin,split 1 and split 2.