| Background and purpose:Diabetic liver injury(DLI)is a chronic liver complication caused by diabetes,which seriously affects the survival and quality of life of diabetic patients.Hyperglycemia causes disorders in liver glucose metabolism and lipid metabolism,leading to diffuse inflammation of the liver.Liver macrophages are closely related to the production of this inflammation and play a key role in the development of diabetic liver injury.NOD like receptor protein 3 inflammasome is a polyprotein complex present in the cytoplasm,which recruits the adaptor protein apoptosis-associated speck-like protein containing a CARD(ASC)from the NLRP3 receptor protein to activate the caspase-1 leads to secretion of mature interleukin 1β(IL-1β)and interleukin 18(IL-18).The study found that NLRP3 inflammasome play an important role in the development of liver disease,inhibiting the expression of NLRP3 inflammasome can reduce liver inflammation and protect the liver.Bruton’s tyrosine kinase(Btk)belongs to the Tec family of non-receptor tyrosine kinases.In recent years,studies have found that Btk is a key signaling molecule for NLRP3 inflammasome activation.In this study,streptozotocin(STZ)-induced diabetic mouse model was used to observe how Btk regulates NLRP3 inflammasome in macrophages and participates in the development of DLI.Methods: Experimental group: normal control group(NC),diabetes group(Diabetic),Btk-/-group(Btk-/-),Btk-/-diabetes group(Btk-/-diabetic);the levels of blood glucose(BG),serum glycated hemoglobin(HbA1c),total cholesterol(TC),triglyceride(TG),free fat acid(FFA),alanine aminotransferase(ALT),aspartate aminotransferase(AST)and liver tissues TC,TG and FFA were detected by kit;the pathological changes of liver tissues were detected by HE staining;the liver tissues of each group were detected by oil red O staining;the expression of cluster of differentiation 68(CD68)and NLRP3 in liver tissue of each group was detected by immunohistochemistry;the co-expressions of CD68 and Btk,CD68 and inducible nitric oxide synthase(iNOS)were detected by laser confocal microscopy;the expressions of Btk,p-Btk,iNOS,IL-1β,tumor necrosis factor-α(TNF-α),monocyte chemoattractant protein 1(MCP-1),NLRP3,ASC and caspase-1 were detected by Western blot;the mRNA level of IL-1β was detected by quantitative real-time PCR(qRT-PCR);the interactions between NLRP3 and Btk,NLRP3 and ASC were examined by immunoprecipitation.Results:(1)Compared with the NC group,BG and HbA1 c were increased in the Diabetic group(P<0.05);compared with the Btk-/-group,BG and HbA1 c were increased in the Btk-/-diabetic group.(2)Compared with the NC group,ALT and AST were increased in the Diabetic group(P<0.05);compared with the Diabetic group,ALT and AST were decreased in the Btk-/-diabetic group(P<0.05).(3)HE staining showed that the lobular structure of the liver tissue of the NC group and the Btk-/-group was intact,the hepatic cord was neatly arranged,in the Diabetic group,the bridged necrosis was observed,a large number of inflammatory cells were infiltrated,and in the Btk-/-diabetic group,hepatocyte edema and scattered necrotic necrosis were observed and a small amount of inflammatory cells infiltrated.(4)Immunohistochemistry showed that the expression of CD68 and NLRP3 was increased in the Diabetic group compared with the NC group(P<0.05),and decreased in the Btk-/-diabetic group compared with the Diabetic group(P<0.05).(5)Confocal microscopy showed that the co-expressions of CD68 with Btk and CD68 with iNOS were significantly enhanced in the Diabetic group compared with the NC group;compared with the Diabetic group,the co-expressions of CD68 with Btk and CD68 with iNOS in the Btk-/-diabetic group was significantly weakened.(6)Western blot showed that compared with NC group,theexpressions of p-Btk/Btk,iNOS,IL-1β,TNF-α,MCP-1,NLRP3,ASC and caspase-1 in Diabetic group was significantly enhanced(P<0.05);compared with the Diabetic group,the expression of each protein in the Btk-/-diabetic group was significantly decreased(P<0.05).(7)qRT-PCR showed that the mRNA level of IL-1β in the Diabetic group was significantly higher than that in the NC group(P<0.05);compared with the Diabetic group,the mRNA level of IL-1β in the Btk-/-diabetic group was significantly down-regulated(P<0.05).(8)Co-immunoprecipitation showed that the interaction between NLRP3 and ASC in the Diabetic group was significantly enhanced compared with NC group;compared with the Diabetic group,the interaction between the Btk-/-diabetic group was significantly attenuated.Conclusion: Btk knockout can significantly reduce liver inflammation caused by diabetes,possibly by down-regulating the formation of NLRP3 inflammasome to reduce the secretion of inflammatory factors,thereby reducing liver damage caused by diabetes. |
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