TLR4信号通路在高糖诱导骨髓来源巨噬细胞激活中的作用
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摘要
目的通过研究高糖对骨髓来源巨噬细胞(BMDM)Toll受体4(TLR4)表达及其下游信号通路的影响,初步探讨高糖通过TLR4信号通路促进巨噬细胞分泌炎症因子的机制。方法分离获取C57BL/6J及B10Sc NNju(TLR4基因敲除小鼠)BMDM,流式细胞仪检测其纯度;选取不同浓度的高糖及甘露醇,在不同时间点刺激BMDM;后将BMDM分为4组:正常糖浓度(LG)组、高糖刺激(HG)组、TLR4基因敲除(TLR4-/-)组、TLR4基因敲除BMDM高糖刺激(TLR4-/-+HG)组。采用流式细胞术观察BMDM M1表型,细胞免疫荧光法观察BMDM TLR4与诱生型一氧化氮合酶(i NOS)共表达,实时定量PCR法检测各组细胞中促炎细胞因子肿瘤坏死因子α(TNF-α)、单核细胞趋化因子-1(MCP-1)、白细胞介素1β(IL-1β)及iNOS的mRNA表达,Western blot法检测各组总蛋白中TLR4、髓样分化因子88(My D88)、TIR结构域衔接蛋白(Trif)、磷酸化白介素受体相关激酶-1(p-IRAK-1)、干扰素调节因子3(IRF3)、磷酸化(p)-IRF3、核因子κB(NF-κB)p65、NF-κB p-p65、i NOS蛋白的表达,ELISA法测定细胞上清液中促炎因子TNF-α、MCP-1、IL-1β的表达。结果与LG组比较,高糖可使M1型巨噬细胞百分比增加;TNF-α、MCP-1、IL-1β及i NOS的mRNA水平上调;TLR4、My D88、Trif、p-IRAK-1、p-IRF3、IRF3、NF-κB p65、NF-κB p-p65、iNOS蛋白表达水平升高;细胞培养基中分泌的TNF-α、MCP-1、IL-1β水平升高。TLR4基因敲除可消除高糖导致的巨噬细胞激活效应。结论高糖可诱导BMDM向M1型极化;TLR4基因敲除可抑制高糖诱导的巨噬细胞向M1活化及炎症因子的产生。
Objective To investigate the mechanism of high glucose and Toll-like receptor 4( TLR4) signaling pathway in macrophages for promoting the secretion of inflammatory cytokines through observing the effect of high glucose on the expression of TLR4 and its downstream signaling pathway in bone marrow derived macrophages( BMDM). Methods The BMDM were separated from C57 BL /6J and B10 Sc NNju( TLR4 knockout mice),and the purity of which was tested by flow cytometry. Different concentrations of high glucose and mannitol were selected to stimulate BMDM at different time points in order to optimize experimental condition. The BMDM were divided into normal control group( LG),high glucose group( HG),TLR4 knockout group( TLR4- /-) and high glucose stimulated TLR4 knockout BMDM group( TLR4- /-+ HG). The M1 phenotype of macrophages was detected by flow cytometry and the co-expression of TLR4 and macrophage activation marker inducible nitric oxide synthase( i NOS)was observed by immunofluorescence. TNF-α,MCP-1 and IL-1β were assessed by RT-PCR and ELISA,together with the mRNA level of i NOS. Western blot was performed to analyze the protein levels of TLR4,My D88,Trif,pIRAK-1,p-IRF3,IRF3,NF-κB p65,NF-κB p-p65,i NOS. Results Compared with the LG group,high glucose could increase the percentage of M1 macrophages and the mRNA level of TNF-α,MCP-1,IL-1β and i NOS. In addition,the expression of TLR4,My D88,Trif,p-IRAK-1,p-IRF3,IRF3,NF-κB p65,NF-κB p-p65,i NOS protein enhanced either. TLR4 knockout could eliminate the effect of macrophage activation induced by high glucose.Conclusion The study suggests that high glucose can promote BMDM to M1 phenotype polarization and TLR4 knockout can inhibit the M1 activation and the production of inflammatory cytokines induced by high glucose.
Objective To investigate the mechanism of high glucose and Toll-like receptor 4( TLR4) signaling pathway in macrophages for promoting the secretion of inflammatory cytokines through observing the effect of high glucose on the expression of TLR4 and its downstream signaling pathway in bone marrow derived macrophages( BMDM). Methods The BMDM were separated from C57 BL /6J and B10 Sc NNju( TLR4 knockout mice),and the purity of which was tested by flow cytometry. Different concentrations of high glucose and mannitol were selected to stimulate BMDM at different time points in order to optimize experimental condition. The BMDM were divided into normal control group( LG),high glucose group( HG),TLR4 knockout group( TLR4- /-) and high glucose stimulated TLR4 knockout BMDM group( TLR4- /-+ HG). The M1 phenotype of macrophages was detected by flow cytometry and the co-expression of TLR4 and macrophage activation marker inducible nitric oxide synthase( i NOS)was observed by immunofluorescence. TNF-α,MCP-1 and IL-1β were assessed by RT-PCR and ELISA,together with the mRNA level of i NOS. Western blot was performed to analyze the protein levels of TLR4,My D88,Trif,pIRAK-1,p-IRF3,IRF3,NF-κB p65,NF-κB p-p65,i NOS. Results Compared with the LG group,high glucose could increase the percentage of M1 macrophages and the mRNA level of TNF-α,MCP-1,IL-1β and i NOS. In addition,the expression of TLR4,My D88,Trif,p-IRAK-1,p-IRF3,IRF3,NF-κB p65,NF-κB p-p65,i NOS protein enhanced either. TLR4 knockout could eliminate the effect of macrophage activation induced by high glucose.Conclusion The study suggests that high glucose can promote BMDM to M1 phenotype polarization and TLR4 knockout can inhibit the M1 activation and the production of inflammatory cytokines induced by high glucose.
引文
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[12]Mudaliar H,Pollock C,Komala M G,et al.The role of Toll-like receptor proteins(TLR)2 and 4 in mediating inflammation in proximal tubules[J].Am J Physiol Renal Physiol,2013,305(2):F143-54.
[13]Lin M,Yiu W H,Wu H J,et al.Toll-like receptor 4 promotes tubular inflammation in diabetic nephropathy[J].J Am Soc Nephrol,2012,23(1):86-102.
[14]Mudaliar H,Pollock C,Ma J,et al.The role of TLR2 and 4-mediated inflammatory pathways in endothelial cells exposed to high glucose[J].PLo S One,2014,9(10):e108844.
[15]Orr J S,Puglisi M J,Ellacott K L,et al.Toll-like receptor 4 deficiency promotes the alternative activation of adipose tissue macrophages[J].Diabetes,2012,61(11):2718-27.
[16]Lee J J,Wang P W,Yang I H,et al.High-fat diet induces tolllike receptor 4-dependent macrophage/microglial cell activation and retinal impairment[J].Invest Ophthalmol Vis Sci,2015,56(5):3041-50.
[2]Wu C C,Sytwu H K,Lu K C,et al.Role of T cells in type 2 diabetic nephropathy[J].Exp Diabetes Res,2011,2011:514738.
[3]Jialal I,Kaur H.The Role of Toll-like receptors in diabetes induced inflammation:implications for vascular complications[J].Curr Diab Rep,2012,12(2):172-9.
[4]Awad A S,You H,Gao T,et al.Delayed treatment with a small pigment epithelium derived factor(PEDF)peptide prevents the progression of diabetic renal injury[J].PLo S One,2015,10(7):e0133777.
[5]Nguyen D,Ping F,Mu W,et al.Macrophage accumulation in human progressive diabetic nephropathy[J].Nephrology,2006,11(3):226-31.
[6]Xu X X,Qi X M,Zhang W,et al.Effects of total glucosides of paeony on immune regulatory toll-like receptors TLR2 and 4 in the kidney from diabetic rats[J].Phytomedicine,2014,21(6):815-23.
[7]Wang Z,Wei M,Wang M,et al.Inhibition of macrophage migration inhibitory factor reduces diabetic nephropathy in type II diabetes mice[J].Inflammation,2014,37(6):2020-9.
[8]Zhang X L,Guo Y F,Song Z X,et al.Vitamin D prevents podocyte injury via regulation of macrophage M1/M2 phenotype in diabetic nephropathy rats[J].Endocrinology,2014,155(12):4939-50.
[9]Dasu M R,Devaraj S,Zhao L,et al.High glucose induces tolllike receptor expression in human monocytes:mechanism of activation[J].Diabetes,2008,57(11):3090-8.
[10]Lin M,Tang S C.Toll-like receptors:sensing and reacting to diabetic injury in the kidney[J].Nephrol Dial Transplant,2014,29(4):746-54.
[11]Mudaliar H,Pollock C,Panchapakesan U.Role of Toll-like receptors in diabetic nephropathy[J].Clin Sci,2014,126(10):685-94.
[12]Mudaliar H,Pollock C,Komala M G,et al.The role of Toll-like receptor proteins(TLR)2 and 4 in mediating inflammation in proximal tubules[J].Am J Physiol Renal Physiol,2013,305(2):F143-54.
[13]Lin M,Yiu W H,Wu H J,et al.Toll-like receptor 4 promotes tubular inflammation in diabetic nephropathy[J].J Am Soc Nephrol,2012,23(1):86-102.
[14]Mudaliar H,Pollock C,Ma J,et al.The role of TLR2 and 4-mediated inflammatory pathways in endothelial cells exposed to high glucose[J].PLo S One,2014,9(10):e108844.
[15]Orr J S,Puglisi M J,Ellacott K L,et al.Toll-like receptor 4 deficiency promotes the alternative activation of adipose tissue macrophages[J].Diabetes,2012,61(11):2718-27.
[16]Lee J J,Wang P W,Yang I H,et al.High-fat diet induces tolllike receptor 4-dependent macrophage/microglial cell activation and retinal impairment[J].Invest Ophthalmol Vis Sci,2015,56(5):3041-50.
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