STAT5a Promotes The Information Of Endotoxin Tolerance Via The Negative Regulation Of Pyroptosis In Kupffer Cells

Objective: To investigate the effect of STAT5 a on alleviating pyroptosis and promoting endotoxin tolerance in Kupffer cells.Methods: This study include two parts:1.In vitro experiment:(1)KCs were isolated from livers of male C57BL/6 mice,and LPS was employed for the formation of the endotoxin tolerance model(ET: 10ng/ml+1000ng/ml)and the non-endotoxin tolerance model(NET: 1000ng/ml)in vitro.The levels of pyroptosis-related genes,caspase1 and GSDMD,were evaluated by Western blot;the levels of IL-1?,IL-18 and IL-10 in the supernatant were measured by ELISA.The LDH release percentages were measured to show the pyroptotic cell death.(2)Nigericin was added to the medium of the ET group to strengthen KC pyroptosis.The activation levels of caspase1 and GSDMD,and the release of inflammatory cytokines and LDH were detected.(3)The level of STAT5 a in KCs were detected in the ET group and the NET group.After STAT5a-knockdown lentivirus was transfected into KCs,the effects of STAT5 a knockdown on pyroptosis and the formation of the endotoxin tolerance were observed.(4)The levels of NLRP3 and NF-?B were detected after STAT5 a knockdown.After NF-?B inhibitor,BAY 11-7082,was added to the STAT5 a knockdown group,the changes of KC pyroptosis and the endotoxin tolerance were observed.2.In vivo experiment: LPS was injected intraperitoneally to establish the ET model and the NET model in C57BL/6 mice,and KCs were isolated to detect the levels of STAT5 a.The ET condition was induced in the WT mice and STAT5a-knockdown mice,and the survival and the degree of liver damage were observed.The level of IL-1?,IL-18,IL-10,AST and ALT in serum were detected.The levels of caspase1 and GSDMD in KCs were detected.Results:1.In vitro,the activation levels of caspase1 and GSDMD in KCs and the levels of IL-1?,IL-18 and LDH were significantly increased in the NET group,but the level of IL-10 was decreased.With the treatment of Nigericin,the levels of caspase1 and GSDMD,and the IL-1?,IL-18 and LDH levels were also increased,showing that the formation of the endotoxin tolerance was disrupted.The phosphorylation level of STAT5 a in the ET group was greater than that in the NET group.STAT5 a knockdown induced increased activation of caspase1 and GSDMD,increased production of IL-1?,IL-18 and LDH and decreased production of IL-10,disturbing the endotoxin tolerance.In the STAT5 a knockdown group,the phosphorylation level of NF-?B and the expression of NLRP3 were remarkably increased.The inhibited NF-?B alleviated KC pyroptosis and restored the endotoxin tolerance.2.The phosphorylation level of STAT5 a in KCs in ET mice was significantly greater than that in NET mice.In STAT5 a knockdown mice,the levels of IL-1?,IL-18,AST and ALT in serum were significantly increased,but the level of IL-10 was decreased,indicating that STAT5 a activation was necessary to the formation of the endotoxin tolerance.STAT5 a knockdown led to the failing endotoxin tolerance in vivo,more liver damage and poorer survival in mice.The activation levels of caspase1 and GSDMD in isolated KCs.Conclusions:1.KC pyroptosis inhibits the formation of KC endotoxin tolerance.2.STAT5 a restrains KC pyroptosis and promotes the formation of KC endotoxin tolerance.3.Phosphorylated STAT5 a inhibits NLRP3 inflammasome activation via NF-?B/NLRP3 signaling pathway.