Role Of Bruton’s Tyrosine Kinase In Endotoxin/Lipopolysaccharide-induced Pyroptosis Of Intestinal Cells In Burned Mice

Objective: In patients with extensive burns,due to the infection of Gram-negative bacilli,a large amount of Gram-negative bacilli endotoxin(LPS)is released into the bloodstream,leading to sepsis.The patient has severe systemic inflammation and causes multiple organ failure(MOF).The intestine is the initial organ of MOF,and its damage is related to the pyroptosis induced by the LPS.Pyroptosis is a newly discovered way of cell death.Its occurrence depends on the activation of inflammasomes in the cell and subsequent activation of the caspase protein family.Bruton’s tyrosine kinase(Btk)is a member of the non-receptor tyrosine kinase family.As a component of the Toll-like receptor(TLR)signaling pathway,Btk signaling plays an important role in LPS-mediated inflammation.However,the role of Btk signaling in burns and sepsis-related intestinal injury is not clear.This article aims to explore the role of Btk in LPS-induced intestinal cell pyroptosis in scalded mice.Methods: One hundred and twenty-eight C57BL/6 mice were divided into normal group,burned group,LPS group,A13(3mg/kg)group,A13(10mg/kg)group,and A13(30mg/kg)group.Mice in the normal group were put to 37℃ water bath for 15 s for sham burn and normal saline sham intraperitoneal injection and then sacrificed;the other groups were kept in constant contact with water bath(100℃)on the back of mice for 15 s,and the scald area was calculated as 10% TBSA III degree;then LPS group was intraperitoneally injected with LPS 10mg/kg.This group is a burn sepsis model group.In addition to LPS,the A13(3mg/kg)group,A13(10mg/kg)group,and A13(30mg/kg)group were given different concentrations of the specific Btk inhibitor LFM-A13.Except for the normal group,each group was sacrificed at 0h,12 h,and 24 h after the treatment,with 8 animals at each time point.Collect mouse blood and intestinal tissue for classification and preservation.The immunohistochemical technique was used to detect the expression of p-Btk protein.Western blotting was used to detect the protein content of p-Btk,Caspase 1,Caspase 11 in the mouse intestine.Enzyme-linked immunosorbent assay was used to detect the levels of IL-1β in mouse intestines and serum.One-way analysis of variance and independent sample t test were performed on the data.P<0.05 indicates that the difference is statistically significant.Results:(1)At 12 h,compared with the normal control group,there was no significant activation of Btk in the intestine of the burned group;Btk activation was significantly increased in the LPS group;Btk activation of A13(3mg/kg),A13(10mg/kg)and A13(30mg/ kg)group was gradually decreased with the increase of LFM-A13 concentration.(2)Compared with the normal control group,the p-Btk content in the LPS group increased significantly at 12 h and 24 h,and the difference was statistically significant(P<0.05),but the same phenomenon was not observed at 0h.The A13(3mg/kg),A13(10mg/kg)and A13(30mg/kg)groups showed different degrees of inhibition of Btk activation at 12 h and 24 h,resulting in a decrease in p-Btk content.The inhibition of the A13(30mg/kg)group was the most obvious,and the difference was statistically significant(P<0.05).(3)Compared with the normal control group,the content of Caspase 1 and Caspase 11 spliceosomes in the LPS group increased significantly at 12 h and 24 h,and the difference was statistically significant(P<0.05),but the same phenomenon was not observed at 0h.The A13(3mg/kg),A13(10mg/kg)and A13(30mg/kg)groups all showed different degrees of Caspase 1,Caspase 11 spliceosome content reduction at 12 h and 24 h,and the A13(30mg/kg)group showed the most obvious inhibition,the difference is statistically significant(P<0.05).(4)Compared with the normal control group,the levels of IL-1β in the intestine and serum of the LPS group increased significantly,and the difference was statistically significant(P<0.05).The increase in the content of serum IL-1β was more obvious.The levels of IL-1β in the intestine and serum of the A13(30mg/kg)group were significantly lower than that of the LPS group,and the difference was statistically significant(P<0.05).Conclusion:In burns with sepsis,mouse intestinal Btk is activated,severe inflammation and damage appear in the intestine,and the intestinal epithelial cell pyroptosis increases.Inhibition of Btk activation can inhibit intestinal cell pyroptosis and protect the intestinal tract of mice.It is a potential target for burns and sepsis-related intestinal injury.