| Background and ObjectiveDiabetic nephropathy（DN）is one of the most serious complications of diabetics.Recent studies have shown that inflammation is the most important pathological feature of DN,and the invasion and activation of macrophages into kidney tissue can promote inflammatory response.However,the clear molecular mechanism of macrophage activation and pathological effect is not completely revealed.Bruton tyrosine kinase（Btk）,a non-receptor tyrosine kinase,belongs to Tec family.It is one of the main signaling enzymes for cell signal transduction in vivo,and is expressed in myeloid cells such as B lymphocytes,basophils,monocytes,etc.It can catalyze phosphorylation of tyrosine residues of various substrate proteins and plays a very important role in cell growth,proliferation and differentiation.Recent studies have confirmed that it plays a certain role in inflammation-related diseases.inflammasome family in which NLRP3 inflammasome is the most representative one is the key to innate immune inflammatory response.BTK regulates the formation of NLRP3 inflammasome,thus,we hypothesized that Btk affects the activation of macrophages in high glucose environments by regulating NLRP3 inflammasome.In this study,the activation of Btk and NLRP3 inflammasome was detected by high glucose-stimulated BMDM and Btk-/-BMDMt,to explore whether activation of macrophages and pro-inflammatory reaction in high glucose environment were mediated by Btk through regulation of activation of NLRP3 inflammasome.Methods1.Isolation and culture of BMDM from experimental mice（Btk knockout mice and C57BL/6J mice）.2.The purity of BMDM was identified by flow cytometry.3.CCK-8 method was used to detect the cytotoxicity of Btk inhibitor PCI-32765 with different concentrations(10-810-44 mmol/L).4.Experimental grouping:（1）mannitol control,M;（2）control group,C;（3）C+ inhibitor group（C+PCI）;（4）high glucose group（HG）;（5）HG+inhibitor group（HG+PCI）;（6）Btk gene knockout group（BTK-/-）;（7）HG+Btk gene knockout group（HG+Btk-/-）.5.The expression of inducible nitric oxide synthase（iNOS）was detected by cellularimmunofluorescence method to observe the effects of high glucose stimulation,Btk inhibition and gene knockout on BMDM M1 activation.6.The mRNA levels of IL-1β,MCP-1 and TNF-αi were detected by RT-PCR.ELISA was used to detect the IL-1βand MCP-1 in each group.7.Transwell chamber was used to detect the chemotactic function of macrophages in each group.8.Western blot was used to detect the expression of p-Btk,Btk,NLRP3,Pro-caspase-1,caspase-1p20 and ASC proteins in macrophages of different groups stimulated by high glucose at different time points.9.The expression and activation of NLRP3 inflammasome in each group were observed by laser confocal double standard NLRP3 and ASC.Results1.The purity of BMDM was about 85.5%by flow cytometry.2.The concentration of PCI-32765(10-910-55 mmol/L)in this experimental range has no effect on high glucose-induced BMDM activity.（P﹥0.05）.3.The immunofluorescence intensity of macrophage M1 phenotype marker iNOS in high glucose group was higher than that in control group,and the immunofluorescence intensity in Btk gene knockout group was significantly lower than that in high glucose group.4.The levels of IL-1β,MCP-1 and TNF-αmRNA of macrophages in high glucose group are up-regulated,and the secretion of IL-1βand MCP-1 in cell culture supernatant is increased.Btk gene knockout can inhibit the activation of macrophages under high glucose stimulation,making the levels of IL-1β,MCP-1 and TNF-αmRNA down-regulated and the secretion levels of IL-1βand MCP-1 in culture supernatant reduced.5.The chemotactic function of macrophages stimulated by high glucose was significantly enhanced compared with the control group.Btk gene knockout could inhibit the chemotactic function of macrophages stimulated by high glucose.6.The expression levels of p-Btk,Btk,NLRP3,Pro-caspase-1,caspase-1p20,ASC protein in high glucose group increased.Btk gene knockout could reduce the expression of p-Btk,Btk,NLRP3,Pro-caspase-1,caspase-1p20,ASC protein induced by high glucose.7.Through laser confocal,it was observed that high glucose induced the expression of LRP3 and ASC co-localization in BMDM,while Btk knockout inhibited the xpression of NLRP3 and ASC under high glucose stimulation.ConclusionsUnder high glucose stimulation,the expression of iNOS was up-regulated in BMDM,and Btk and NLRP3 inflammasome activated,resulting in increased expression of pro-inflammatory cytokines.Use of Btk inhibitors or gene knockouts can inhibit NLRP3 inflammasome activation and production of inflammatory cytokines. |
