褪黑素通过Toll样受体4信号通路抑制高糖诱导的小鼠系膜细胞增殖及炎性因子表达

Melatonin inhibits high glucose-induced cell proliferation and expressions of inflammatory factor via Toll-like receptor 4 signaling pathway in mouse mesangial cells

目的探讨褪黑素(MT)对高糖刺激小鼠系膜细胞(SV40)的增殖、Toll样受体4(TLR4)信号通路及炎性因子表达的影响。方法分组:甘露醇对照组(30mmol/L甘露醇),正常对照组(5mmol/L葡萄糖),对照(5mmol/L葡萄糖)+1000μmol/LMT组,高糖组(25mmol/L葡萄糖),高糖+10、100、1000μmol/LMT组及高糖+TLR4抑制剂(TAK242)组。(1)利用CCK-8细胞毒性试剂盒检测细胞活力,EdU试剂盒测定细胞增殖。利用细胞免疫荧光观察TLR4的表达及核因子κB(NF-κB p65)的入核情况。(2)实时定量PCR检测TLR4 mRNA表达,实时定量PCR及ELISA法分别测定各组细胞下游炎性因子单核细胞趋化因子-1(MCP-1)、白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)的mRNA和蛋白分泌水平;Western印迹检测TLR4通路蛋白TLR4、髓样分化因子88(MyD88)、β干扰素TIR结构域衔接蛋白(Trif)、磷酸化干扰素调节因子3(p-IRF3)和磷酸化NF-κB抑制蛋白(p-IκB)的表达。结果高糖可刺激系膜细胞增殖,促进TLR4表达、NF-κB p65核转移,而MT和TLR4抑制剂均可抑制上述现象,且MT的抑制作用呈浓度依赖性。与正常对照组比较,高糖刺激不仅上调TLR4、MCP-1、IL-1β和TNF-αmRNA的表达(均P<0.05),而且MyD88、Trif、p-IRF3、pI-κB的蛋白表达也明显增加(均P<0.05)。与高糖组比较,高糖+10、100、1000μmol/LMT组及高糖+TAK242组TLR4表达下调的同时,MyD88、Trif、p-IRF3、pI-κB、MCP-1、IL-1β、TNF-α的表达也降低了(均P<0.05),且MT的干预作用呈浓度依赖性。结论高糖刺激增加了系膜细胞的增殖,MT可通过TLR4信号通路抑制高糖诱导的系膜细胞增殖和炎性因子表达。

Objective To investigate effects of melatonin(MT)on high glucose-induced cell proliferation,Toll-like receptor 4(TLR4)signaling pathway and expressions of inflammatory factor in mouse mesangial cells(SV40).Methods SV40 cells were divided into mannitol control group(30 mmol/L mannitol),normal control group(5 mmol/L glucose),control(5 mmol/L glucose)+1000 μmol/L MT group,high glucose group(25 mmol/L glucose),high glucose +10,100,1000 μmol/L MT group and high glucose+TLR4 inhibitor(TAK242)group.(1)The cell viability was measured by CCK-8 cytotoxicity kits,and cell proliferation was measured by EdU kits.The expression of TLR4 and the nuclear translocation of nuclear factor-κB(NF-κB p65)were observed by immunofluorescence.(2)Real-time quantitative PCR was used to detect TLR4 mRNA expression.Real-time quantitative PCR and ELISA were used to determine the mRNA and protein secretion levels of the downstream inflammatory factors,such as monocyte chemoattractant-1(MCP-1),interleukin-1β(IL-1β)and tumor necrosis factor of α(TNF-α);Western blotting was used to detect TLR4 pathway proteins,such as TLR4,myeloid differentiation factor 88(MyD88),β interferon TIR domain adaptor(Trif),phosphorylated interferon regulatory factor 3(p-IRF3)and phosphorylated NF-κB inhibitory protein(p-IκB).Results High glucose stimulated mesangial cell proliferation,promoted TLR4 expression and NF-κB p65 transcription activity.Both MT and TAK242 inhibited the above reactions,and the effects of MT was concentration-dependent.Compared with the normal control group,high glucose group had up-regulated expressions of TLR4,MCP-1,IL-1β and TNF-α mRNA(all P<0.05),but also significantly increased the protein expressions of MyD88,Trif,p-IRF3 and p-IκB(all P<0.05).Compared with those in the high glucose group,the expression of TLR4 was down-regulated in the high glucose+10,100,1000 μmol/L MT group and the high glucose+TAK242 group(all P<0.05),while the expressions of MyD88,Trif,p-IRF3,p-IκB,MCP-1,IL-1β and TNF-α decreased(all P<0.05).The effects of MT was concentration-dependent.Conclusions High glucose stimulates the proliferation of SV40,and MT can inhibit the proliferation of mesangial cells and the expressions of inflammatory factors through TLR4 signaling pathway.