Role Of Bruton’s Tyrosine Kinase In High Glucose-induced Bone Marrow-derived Macrophages Activation

Background and purpose Diabetic nephropathy（DN）is one of the major causes of end stage renal disease（ESRD）.Inflammation plays a key role in the pathogenesis of DN.Macrophages are the main regulate cells in inflammation.The infiltration and polarization of macrophages are closely related to the albuminuria,renal fibrosis and progressive decline of renal function in DN.Bruton’s tyrosine kinase（Btk）,a non-receptor tyrosine kinase,plays a significant role in inflammation and immune response.Although Btk is a critical bioactive molecule in immune regulation,its role in macrophage inflammation response induced by high glucose still needs to be studied.In our study,we observed the morphology change and the secretion levels of monocyte chemoattractant protein-1（MCP-1）,tumor necrotic factor-alpha（TNF-α）,interleukin-1β（IL-1β）of HG-induced bone marrow-derived macrophages（BMMs）under the Btk inhibitor PCI-32765.The expression of nuclear factor-kB（NF-κB）signaling pathways was also investigated to clarify the role of Btk in high glucose-induced macrophage activation.Methods Isolation,culture and identification of the BMMs from C57BL6/J mice.CCK-8 method was used to detect the effects of high glucose stimulation and PCI-32765 in different concentrations on BMMs viability.Optimization of experiment conditions:Western blot analysis was used to detect the expression of inducible nitric oxide synthase（iNOS）and p-Btk in BMMs at different glucose concentrations and the effects of PCI-32765 on the expression of iNOS and p-Btk in high glucose-induced BMMs.The BMMs were divided into four groups as follows:（1）normal control group（LG group）;（2）normal control+Btk inhibitor group（LG+PCI-32765 group）;（3）high glucose group（HG group）;（4）high glucose+Btk inhibitor group（HG+PCI-32765group）.Transwell method was used to detect the effect of PCI-32765 on migration of high glucose-induced BMMs.Confocal microscopy and Western blot analysis were used to detect the effect of PCI-32765 intervention on the activation of high glucose-induced BMMs.TNF-α,IL-1βand MCP-1 in the cell supernatant of each group were detected by ELISA.The expression of TNF-α,IL-1βand MCP-1 mRNA in each group were detected by qRT-PCR.Western blot analysis was used to detect the protein expression of Btk,p-Btk,NF-kBp65,NF-kBpp65,IkB,p-IkB in each group.Confocal microscopy analysis was used to detect the effect of PCI-32765 intervention on nuclear translocation of NF-κBp65 in high glucose-induced BMMs.Results1.The purity of BMMs was 94.5%by flow cytometry analysis.2.High glucose and PCI-32765 within our experimental concentration intervention had no obvious effect on BMMs viability（P>0.05）.3.Compared with LG group,high glucose at the concentration of 30 mmol/L significantly improved p-Btk and iNOS expression（P<0.05）.PCI-32765 at concentration of 10^-6mmol/L remarkably inhibited the expression of p-Btk（P<0.01）.4.High glucose stimulation significantly increased BMMs migration while PCI-32765 intervention markedly inhibited the migration of high glucose-induced BMMs （P<0.05）.5.A high iNOS fluorescence of BMMs was observed under high glucose stimulation,and PCI-32765 intervention decreased the iNOS fluorescence of high glucose-induced BMMs.Western blot analysis showed that the expression of BMMs iNOS protein in HG group was significantly higher compared to LG group（P<0.01）.PCI-32765 significantly inhibited the iNOS protein expression of high glucose-induced BMMs（P<0.05）.6.HG group had higher secretion levels of TNF-α,IL-1βand MCP-1 than that of LG group（P<0.05）.PCI-32765 intervention significantly suppressed the levels of these pro-inflammatory cytokines as compared to HG group（P<0.05）.Similarly,the mRNA expression levels of these cytokines in HG group were higher than that in LG group（P<0.05）,and PCI-32765 intervention inhibited expression levels of these cytokines（P<0.05）.7.The protein levels of p-Btk,NF-κBpp65 and p-IκB were particularly increased in HG group（P<0.01）.PCI-32765 intervention significantly inhibited the protein levels of p-Btk,NF-κBpp65 and p-IκB in high glucose-induced BMMs（P<0.05）.8.The NF-κBp65 marked by green fluorescence in HG group was mainly located in the nucleus region,whereas PCI-32765 intervention inhibited the nuclear translocation of NF-κBp65 in high glucose-induced BMMs.Conclusions1.High glucose stimulation induced the activation of BMMs and increased the expression of pro-inflammatory cytokines include TNF-α,IL-1βand MCP-1.2.High glucose stimulation improved the expression of p-Btk and NF-kappa B signaling pathway in BMMs.3.Btk inhibitor（PCI-32765）inhibited the activation of macrophages in high glucose-induced BMMs,decreased the expression of TNF-α,IL-1β,MCP-1and down regulated the activation of NF-κB signaling pathway,thus alleviated inflammation response.