Effect Of Berberine On Thermoregulation And HSP70-TNFα Expression In Heat Stress

Berberine(BBR)as a natural small molecule drug has the activity of anti-inflammatory and antipyretic.It has been shown that BBR treat the fever caused by inflammation,mainly through its bactericidal and anti-inflammatory.Heat stress caused by hot conditions lead to the increase of body temperature.While in this thesis,we mainly study the effect of berberine on thermoregulation and its possible molecular mechanism in heat stress.The temperature changes of mice were monitored under hot conditions.The results show that BBR could inhibit the increase of body temperature and expression of HSP70 and TNFα caused by hot conditions.KNK437 which can inhibit the expression of HSP70,prevent temperature rise and TNFα expression under heat stress.TNFα as one of the endogenous pyrogens,has been proved to play a role in thermoregulation.Thus it is suggested that HSP70 and TNFα both play an important role in thermoregulation.Our study demonstrated that the expression of TNFα、HSP70 increased with the extension of heat stress,and BBR inhibited their expression in vivo and in vitro.This results show that without neurohumoral regulation,berberine affect thermoregulation through cytokines.In order to explore the relationship between HSP70 and TNFα,engineered cells,TNFα siRNA、TNFα protein were used,and HSP70-TNFα signaling was determined: there is no direct signaling pathway between them.Confocal microscopy,Co-immunoprecipitation and Protein degradation were used to determine the association between HSP70 and TNFα.We found that HSP70 as chaperone protect TNFα protein from rapid degradation.On the basis work,we have demonstrated the inhibition effect of berberine on HSP70 and TNFα.A promoter plasmid reporter assay demonstrated BBR supress the initiation of gene transcription.Results of CHIP,Fluorescence spectrum,EMSA,proved that berberine supress the combination between TBP and “TATA box”,and then inhibit their transcription and expression,finally lowerd the temperature.Site-specific mutagenesis proved that “TATA box” is the exact site of berberine.In conclusion,this study first found berberine could inhibit body temperature rise induced by heat stress directly and the target is “TATA box”,which locate in promoter of HSP70 and TNFα.Our finding benefit to understanding the effect of BBR in thermoregulation especially in hyperthermia and provides an important experimental basis for prevention the high temperature damage caused by heat stress,such as heat stroke.