miR-146a经TLR4/MyD88途径加速巨噬细胞迁移所致动脉硬化的作用机制

Mechanism of miR-146a accelerating macrophage migration induced arteriosclerosis via TLR4/MyD88 pathway

目的探讨微小RNA-146a(miR-146a)经Toll样受体4(TLR4)/髓系分化因子88蛋白(MyD88)途径调控巨噬细胞迁移所致动脉硬化的具体机制。方法RAW264.7巨噬细胞分为空白组、ox-LDL组、miR-146a mimic组、miR-146a inhibitor组、shRNA-MyD88组、shRNA-NC组、shRNA-MyD88+miR-146a mimic组、shRNA-MyD88+miR-146a inhibitor组,除空白组,其他各组均用50 mg/L ox-LDL进行干预;采用Western blotting法检测TLR4、MyD88、核因子κB(NF-κB)、白细胞介素-6(IL-6)蛋白表达水平。采用qPCR法检测miR-146a、TLR4、MyD88、NF-κB、IL-6基因表达水平。采用Transwell法检测巨噬细胞迁移能力。结果(1)ox-LDL组细胞内TLR4、IL-6蛋白含量及巨噬细胞迁移率均高于空白组(P均<0.01),miR-146a低于空白组(P<0.01);(2)miR-146a mimic组细胞TLR4 mRNA相对表达量及蛋白含量、巨噬细胞迁移率低于ox-LDL组,miR-146a mRNA相对表达量高于ox-LDL组(P均<0.01);miR-146a inhibitor组细胞TLR4 mRNA相对表达量及蛋白含量、巨噬细胞迁移率高于ox-LDL组,miR-146a mRNA相对表达量低于ox-LDL组(P均<0.01);(3)shRNA-MyD88组MyD88、NF-κB、IL-6蛋白含量、巨噬细胞迁移率低于ox-LDL组(P<0.01),miR-146a、TLR4表达无明显差异(P>0.05);shRNA-MyD88+miR-146a mimic组miR-146a表达高于shRNA-MyD88组,TLR4蛋白含量低于shRNA-MyD88组(P<0.01),MyD88、NF-κB、IL-6蛋白含量表达、巨噬细胞迁移率与shRNA-MyD88组无明显差异(P>0.05);shRNA-MyD88+miR-146a inhibitor组miR-146a表达低于shRNA-MyD88组,TLR4蛋白含量表达高于shRNA-MyD88组(P<0.01),MyD88、NF-κB、IL-6蛋白含量表达、巨噬细胞迁移率与shRNA-MyD88组无明显差异(P>0.05)。结论miR-146a是动脉粥样硬化的保护性因子,通过拮抗TLR4/MyD88途径调控炎症因子分泌进而抑制巨噬细胞迁移所致动脉粥样硬化进程,为动脉粥样硬化的防治提供新的治疗靶点。

Objective To explore the specific mechanism of microRNA-146 a(miR-146 a)regulating macrophage migration induced arteriosclerosis via toll-like receptor 4(TLR4)and myeloid differentiation factor88(MyD88)pathway(TLR4/MyD88).Methods RAW264.7 macrophages were divided into blank group,ox-LDL group,miR-146 a mimic group,miR-146 a inhibitor group,siRNA-MyD88 group,siRNA-NC group,siRNA-MyD88+miR-146 a mimic group,and siRNA-MyD88+miR-146 a inhibitor group.Except the blank group,the other groups were treated with 50 mg/L ox-LDL.The protein expressions of TLR4,MyD88,nuclear factor kappa-B(NF-κB),and interleukin-6(IL-6)were detected with Western blotting.The mRNA expressions of miR-146 a,TLR4,MyD88,NF-κB and IL-6 were detected with qPCR.The migration ability of macrophages was detected with Transwell assay.Results(1)The ox-LDL group had higher protein expressions of TLR4 and IL-6,but lower migration ability of macrophages and lower mRNA expression of TLR4 than the blank group(P<0.01).(2)The miR-146 a mimic group had lower mRNA and protein expressions of TLR4,lower migration ability of macrophages,but higher mRNA expression of miR-146 a than the ox-LDL group(P<0.01).The miR-146 a inhibitor group had higher mRNA and protein expressions of TLR4,higher migration ability of macrophages,but lower mRNA expression of miR-146 a than the ox-LDL group(all P<0.01).(3)The shRNA-MyD88 group had lower protein expressions of MyD88,NF-κB and IL-6,and lower migration ability of macrophages than the ox-LDL group(P<0.01);there was no difference in the expressions of miR-146 a and TLR4(P>0.05).The shRNA-MyD88+miR-146 a mimic group had higher expression of miR-146 a but lower expression of TLR4 than the shRNA-MyD88 group(P<0.01).There were no significant differences in the protein expressions of MyD88,NF-κB,IL-6 and migration rate of macrophages between shRNA-MyD88 group and shRNA-MyD88 group(P>0.05).The shRNA-MyD88+miR-146 a inhibitor group had lower expression of miR-146 a but higher expression of TLR4 than the shRNA-MyD88 group(P<0.01).There were no significant differences in the expressions of MyD88,NF-κB,IL-6 and migration rate of macrophages(P>0.05).Conclusion miR-146 a is a protective factor of atherosclerosis,which can inhibit the process of macrophage migration by regulating the secretion of inflammatory factors by antagonizing TLR4/MyD88 pathway.This can provide a new therapeutic target for the prevention and treatment of arteriosclerosis.