Role And Mechanism Of TAB1 Mediated Glycolysis In Polarization Of Bone Marrow-Derived Macrophages Induced By High Glucose

Background and Purpose Diabetic nephropathy(DN)is one of the common microvascular complications in patients with diabetes.Studies have shown that macrophage polarization-mediated renal inflammation plays an important role in the development of DN.Therefore,the discovery of specific molecules and signaling pathways that block the polarization of macrophages has important theoretical and clinical implications for further elucidating the mechanism of the development and progression of macrophage inflammation and screening drug targets for the prevention and treatment of DN.In recent years,different energy metabolic pathways have been found to determine the polarization direction of macrophage,but the specific regulation mechanism remains to be clarified.Macrophages have strong phenotypic plasticity.M1 macrophages mainly use glycolysis to provide energy,and secrete a large number of inflammatory factors to mediate DN inflammatory response.TAK1-binding protein 1(TAB1)is a specific protein that interacts with TGF-activated kinase 1(TAK1).TAB1/TAK1 can activate nuclear factor ?B(NF-?B)signaling pathway and play an important role in the regulation of macrophage activation.Studies have shown that activation of NF-?B signaling pathway may up-regulate the transcriptional activity of hypoxia-inducible factor-1(HIF-1?),while high expression of HIF-1? activates transcription level of glycolysis-related enzymes.At present,there is no report about whether TAB1 participates in glycolysis and its mechanism of DN macrophage polarization.In this study,high glucose was used to stimulate mouse bone marrow-derived macrophages (Bone marrow-derived macrophages,BMMs),and knockdown of TAB1 in BMMs using si RNA technology to explore the relationship between glycolysis of TAB1 regulatory macrophages and downstream signaling molecule NF-k B/HIF-1?,and TAB1/NF-k B/ The role of HIF-1? signaling pathway in polarized macrophages by glycolysis and molecular mechanisms provide a new strategy for the prevention and treatment of DN.Methods 1.Extract and culture BMMs of C57BL/6J male mice;2.The purity and maturity of BMMs were determined by flow cytometry.3.Transfect BMMs with si RNA TAB1 and select the optimal transfection band;4.Western blot analyses HK1,PFKFB3,LDHA,TAB1,NF-?B p65 and HIF-1? of BMMs activation and densitometric analysis in a time course;5.Grouping: mannitol control group(D-mannitol),negative control si RNA(control si RNA),blank control(control),hyperglycemic stimulation(HG),TAB1 si RNA(TAB1 si RNA),TAB1 si RNA + high glucose stimulation group(TAB1 si RNA + HG),glycolysis inhibitor group(2DG),glycolysis inhibitor + high glucose stimulation group(2DG + HG).6.The laser confocal microscopy was used to observe the expression of i NOS,HK1,PFKFB3 and LDHA.7.RT-PCR method was used to detect the expression changes of MCP-1,IL-1?,HK1,PFKFB3 and LDHA m RNA in the cells.8.Collect the supernatant of each group of cells and use the ELISA kit to detect the secretion of inflammatory cytokines MCP-1 and IL-1?;9.Lactic acid production kit and glucose absorption kit to detect the lactic acid production and glucose absorption function changes in each group;10.Western blot was used to detect the expression changes of HK1,PFKFB3,LDHA,TAB1,NF-?B p65 and HIF-1? in each group of cells.Results 1.Flow cytometry was used to detect the cultured macrophages.The purity and maturity of macrophages were identified by F4/80 and CD11 b double labeling.The results showed that the success rate of double labeling was 90.6%,which satisfied the experimental requirements.2.Confocal laser scanning results showed that compared with control group,the green fluorescence of HK1,PFKFB3,LDHA and i NOS in HG group was significantly enhanced,while the green fluorescence intensity of the above indicators in TAB1 si RNA group and 2DG group was not significantly different;compared with HG group,in the TAB1 si RNA+HG group and the 2DG+HG group,the green fluorescence intensity of the above indicators were weakened.3.ELISA results showed that compared with the control group,the MCP-1 and IL-1? in the cell culture medium of the HG group were significantly increased(p<0.01),and there was no significant difference in the the TAB1 si RNA group and the 2DG group.Compared with the HG group,the MCP-1 and IL-1? in the cell culture supernatant of the TAB1 si RNA+HG group and the 2DG+HG group were both significantly decreased(p<0.01).4.RT-PCR results showed that compared with the control group,the m RNA expression of HK1,PFKFB3,LDHA,MCP-1,and IL-1? in the HG group were increased significantly(p<0.01).Compared with HG group,m RNA expression of HK1,PFKFB3,LDHA,MCP-1 and IL-1? in TAB1 si RNA+HG group and 2DG+HG group were significantly decreased(p<0.01).5.Lactic acid production and glucose absorption test results showed that compared with the control group,lactate production and glucose uptake in the HG group were significantly higher(p<0.01),but there was no significant difference between the TAB1 si RNA group and the 2DG group.Compared with HG group,lactic acid production and glucose absorption capacity of TAB1 si RNA+HG group and 2DG+HG group were decreased(p<0.01),and the difference was statistically significant.6.Confocal laser scanning of NF-?B p65 into the nucleus was performed.The results showed that the NF-?B p65 in the conrol group did not enter the nucleus,and the nuclear entry of NF-?B p65 in the HG group was obvious.While compared with the HG group,the nuclear entry of NF-?B p65 in the TAB1 si RNA+HG and 2DG+HG group were weakened.7.Western blot results showed that compared with control group,the protein expression of HK1,PFKFB3,LDHA,TAB1,NF-?B p65,HIF-1? in the HG group were significantly increased(p<0.01).Compared with HG group,the protein expression of HK1,PFKFB3,LDHA,TAB1,NF-?B p65,HIF-1? in the TAB1 si RNA+HG group and 2DG+HG group were significantly decreased(p<0.05,p<0.01).Conclusions 1.High glucose can induce the polarization of BMMs to M1 type and increase the expression of MCP-1 and IL-1? secreted by M1 macrophages.TAB1 si RNA can inhibit macrophage polarization to M1 type in high glucose environment.2.High glucose can enhance the glycolysis of M1 macrophages and the expression of glycolytic enzymes HK1,PFKFB3 and LDHA,whose effects may be attenuated by TAB1 si RNA in high glucose conditions.3.Under high glucose conditions,macrophages are polarized to M1 type,and glycolysis is increased,its mechanism may be related to the activation of TAB1/NF-?B / HIF-1? signaling pathway.