Objective:To explore the protective effect and the underlying mechanism of silibinin(SIB),one of the active compounds from Silybum marianum(L.)Gaertn in endotoxemia.Methods:Mouse peritoneal macrophage were isolated vi...Objective:To explore the protective effect and the underlying mechanism of silibinin(SIB),one of the active compounds from Silybum marianum(L.)Gaertn in endotoxemia.Methods:Mouse peritoneal macrophage were isolated via intraperitoneally injection of BALB/c mice with thioglycolate medium.Cell viability was assessed using the cell counting kit-8,while cytotoxicity was determined through lactate dehydrogenase cytotoxicity assay.The protein expressions of interleukin(IL)-1α,IL-1β,and IL-18 were determined by enzyme-linked immunosorbent assay.Intracellular lipopolysaccharide(LPS)levels were measured by employing both the limulus amoebocyte lysate assay and flow cytometry.Additionally,proximity ligation assay was employed for the LPS and caspase-11 interaction.Mice were divided into 4 groups:the control,LPS,high-dose-SIB(100 mg/kg),and low-dose-SIB(100 mg/kg)groups(n=8).Zebrafish were divided into 4 groups:the control,LPS,high-dose-SIB(200μmol/L),and low-dose-SIB(100μmol/L)groups(n=30 for survival experiment and n=10 for gene expression analysis).The expression of caspase-11,gasdermin D(GSDMD),and N-GSDMD was determined by Western blot and the expressions of caspy2,gsdmeb,and IL-1βwere detected using quantitative real-time PCR.Histopathological observation was performed through hematoxylineosin staining,and protein levels in bronchoalveolar lavage fluid were quantified using the bicinchoninicacid protein assay.Results:SIB noticeably decreased caspase-11 and GSDMD-mediated pyroptosis and suppressed the secretion of IL-1α,IL-1β,and IL-18 induced by LPS(P<0.05).Moreover,SIB inhibited the translocation of LPS into the cytoplasm and the binding of caspase-11 and intracellular LPS(P<0.05).SIB also attenuated the expression of caspase-11 and N-terminal fragments of GSDMD,inhibited the relative cytokines,prolonged the survival time,and up-regulated the survival rate in the endotoxemia models(P<0.05).Conclusions:SIB can inhibit pyroptosis in the LPS-mediated endotoxemia model,at least in part,by inhibiting the caspase-11-mediated cleavage of GSDMD.Additionally,SIB inhibits the interaction of LPS and caspase-11 and inhibits the LPS-mediated up-regulation of caspase-11 expression,which relieves caspase-11-dependent cell pyroptosis and consequently attenuates LPS-mediated lethality.展开更多
基金Supported by the Guangdong Basic and Applied Basic Research Foundation(No.2023A1515011106)Science and Technology Program of Guangzhou(No.2023A04J1826)。
文摘Objective:To explore the protective effect and the underlying mechanism of silibinin(SIB),one of the active compounds from Silybum marianum(L.)Gaertn in endotoxemia.Methods:Mouse peritoneal macrophage were isolated via intraperitoneally injection of BALB/c mice with thioglycolate medium.Cell viability was assessed using the cell counting kit-8,while cytotoxicity was determined through lactate dehydrogenase cytotoxicity assay.The protein expressions of interleukin(IL)-1α,IL-1β,and IL-18 were determined by enzyme-linked immunosorbent assay.Intracellular lipopolysaccharide(LPS)levels were measured by employing both the limulus amoebocyte lysate assay and flow cytometry.Additionally,proximity ligation assay was employed for the LPS and caspase-11 interaction.Mice were divided into 4 groups:the control,LPS,high-dose-SIB(100 mg/kg),and low-dose-SIB(100 mg/kg)groups(n=8).Zebrafish were divided into 4 groups:the control,LPS,high-dose-SIB(200μmol/L),and low-dose-SIB(100μmol/L)groups(n=30 for survival experiment and n=10 for gene expression analysis).The expression of caspase-11,gasdermin D(GSDMD),and N-GSDMD was determined by Western blot and the expressions of caspy2,gsdmeb,and IL-1βwere detected using quantitative real-time PCR.Histopathological observation was performed through hematoxylineosin staining,and protein levels in bronchoalveolar lavage fluid were quantified using the bicinchoninicacid protein assay.Results:SIB noticeably decreased caspase-11 and GSDMD-mediated pyroptosis and suppressed the secretion of IL-1α,IL-1β,and IL-18 induced by LPS(P<0.05).Moreover,SIB inhibited the translocation of LPS into the cytoplasm and the binding of caspase-11 and intracellular LPS(P<0.05).SIB also attenuated the expression of caspase-11 and N-terminal fragments of GSDMD,inhibited the relative cytokines,prolonged the survival time,and up-regulated the survival rate in the endotoxemia models(P<0.05).Conclusions:SIB can inhibit pyroptosis in the LPS-mediated endotoxemia model,at least in part,by inhibiting the caspase-11-mediated cleavage of GSDMD.Additionally,SIB inhibits the interaction of LPS and caspase-11 and inhibits the LPS-mediated up-regulation of caspase-11 expression,which relieves caspase-11-dependent cell pyroptosis and consequently attenuates LPS-mediated lethality.
