Inhibiting Action And Mechanism Of BaiHuTang On LPS-induced Pyroptosis

Sepsis is a syndrome in which the host is infected,causing a systemic inflammatory response and multiple organ dysfunction.It is commonly seen as a secondary effect of various infectious diseases,and has a high mortality rate in animals.Baihutang(BHT),a classical prescription,has shown promising clinical effect in treating sepsis,but the micro-molecular mechanism behind its effectiveness remains unclear.Recent studies have highlighted pyroptosis as a crucial factor in the development of animal sepsis.Studies have demonstrated that BHT is effective in treating sepsis,may be through the inhibiting pyroptosis and the resulting systemic inflammation can help prevent damage to multiple organs.Our study aimed to induce a mouse sepsis model using lipopolysaccharide(LPS)and intervene with BHT to explore direct evidence of pyroptosis inhibition.We investigated the inhibitory effect and mechanism of BHT,as well as its main medicinal ingredients mangiferin and glycyrrhizic acid,on LPS-induced pyroptosis using in vitro cytology and molecular biology techniques.The results are as follows:1.High performance liquid chromatography analysis revealed that the four main medicinal ingredients in BHT were present at concentrations of1.079 mg/m L for mangiferin,4.270 mg/m L for timosaponin BII,0.490mg/m L for liquirtin and 0.790 mg/m L for glycyrrhizic acid.Each component met the requirements of the Pharmacopoeia,and the quality of the three batches of decoction remained consistent.2.BHT(10 g/kg·bw)was found to have a significant impact on the survival rate of septic mice,reaching 60% at 96 h.Additionally,BHT was significantly decreased the levels of TNF-α and IL-1β in peripheral blood,and inhibited the migration of lymphocytes in the spleen of septic mice.Furthermore,BHT was found to suppress inflammatory damage and pyroptosis in the spleen and lung.The study also revealed that BHT significantly inhibited the expression and activation of ASC,NLRP3,and caspase-1 in the classical pyroptosis pathway,as well as caspase-11 in the non-classical pyroptosis pathway.Meanwhile,the expression of GSDMD and the production of GSDMD-N-terminal were significantly inhibited by BHT,thus blocking the pyroptosis.3.In an in vitro pyroptosis model,the action of BHT resulted in reduced pyroptosis morphology and inhibited release of IL-1β and LDH in BMDMs.Additionally,BHT significantly inhibited secretion and m RNA expression of TNF-α,IL-6,IL-1β,and IL-18,as well as m RNA expression and protein activation of caspase-1 and GSDMD.In particular,it significantly blocked the cleavage of GSDMD and the production of GSDMD-N,which inhibited the activation of pyroptosis pathway.4.In an in vitro pyroptosis model,the action of mangiferin resulted in reduced pyroptosis morphology and significantly inhibited secretion and m RNA expression of TNF-α,IL-6,and IL-18,as well as m RNA expression and protein activation of IL-1β and caspase-1.And it can inhibited the protein expression and activation of pyroptosis factors(NLRP3,ASC,caspase-1,GSDMD),and blocked the protein cleavage of GSDMD and the production of GSDMD-N,thus preventing the pyroptosis.5.In an in vitro pyroptosis model,the action of glycyrrhetinic acid resulted in reduced pyroptosis morphology and significantly inhibited secretion and m RNA expression of TNF-α,IL-6,and IL-18,as well as m RNA expression and protein activation of IL-1β and caspase-1.And it can inhibited the protein expression and activation of pyroptosis factors(NLRP3,ASC,caspase-1,GSDMD),but did not block the cleavage of GSDMD and the production of GSDMD-N.Conclusion:1.BHT has a significant therapeutic effect on LPS sepsis.The mechanism of action is that it inhibit the activation of classical cell apoptosis pathway by caspase-1-mediated,and block the production and activation of GSDMD-N.BHT can inhibit pyroptosis,prevent inflammatory reaction and tissue inflammatory injury.2.Both mangiferin and glycyrrhizic acid inhibited LPS-induced inflammatory response and caspase-1-mediated classical pyroptosis.And they are the main medicinal ingredients of BHT in inhibiting pyroptosis.However,mangiferin could inhibit the production and activation of GSDMD-N,but glycyrrhizic acid did not.