五味子基于miR-124调控TLR4通路介导小胶质细胞表型转化机制  

Mechanism of Schisandra Chinensis-mediated microglia phenotypic transformation by regulation of the TLR4 pathway based on miR-124

作　　者：杨云方 张悦 彭景 吴博 贾英 颜廷旭 YANG Yun-fang;ZHANG Yue;PENG Jing;WU Bo;JIA Ying;YAN Ting-xu(College of Functional Food and Wine,Shenyang Pharmaceutical University,Shenyang 110016,China)

机构地区：[1]沈阳药科大学功能食品与葡萄酒学院,辽宁沈阳110016

出　　处：《药学学报》2023年第2期377-385,共9页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金面上资助项目(82173961).

摘　　要：探讨五味子基于microRNA-124(miR-124)调控Toll样受体4(Toll-like receptor 4,TLR4)通路介导小胶质细胞表型转化的作用机制。利用脂多糖(lipopolysaccharide,LPS)刺激BV2细胞建立模型,不同剂量五味子提取物(Schisandra Chinensis extract,SCE)处理细胞;miR-124抑制剂(miR-124 inhibitor)和阴性对照序列(NC inhibitor)转染至BV2细胞后用SCE处理细胞。MTT法检测细胞活性;NO试剂盒检测NO释放量;ELISA检测白细胞介素-10(interleukin-10,IL^(-1)0)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)含量;免疫荧光染色法检测小胶质细胞标志物离子钙结合接头分子-1(ionized calcium binding adapter molecule-1,IBA-1)、精氨酸酶-1(arginase-1,Arg-1)及下游核转录因子κB(nuclear factor-kappa B,NF-κB)核移位的影响;Western blot检测NF-κB p65、IBA-1、Arg-1、TLR4、骨髓分化蛋白88(myeloid differentiation primary factor 88,MyD88)、核因子抑制蛋白-α(nuclear factor inhibitor protein-α,IκB-α)、IκB激酶-α(inhibitor of nuclear factor-kappa B kinases-α,IKK-α)、IL-10、TNF-α等蛋白的表达。质量浓度为31.25~250μg·mL^(-1)的SCE对于细胞活性无明显影响;SCE作用后,NO释放受到抑制(P<0.001,P<0.01),IL-10释放水平升高(P<0.05),而TNF-α释放水平降低(P<0.001),同时抑制TNF-α、IBA-1、TLR4、MyD88蛋白的表达(P<0.01,P<0.001),升高IL-10、Arg-1、NF-κB p65、IKK-α蛋白表达(P<0.001,P<0.01,P<0.05),SCE还能够促进miR-124的表达(P<0.01);转染miR-124 inhibitor后,TNF-α释放量升高(P<0.001),IL-10释放量减少(P<0.05),TNF-α、IBA-1的mRNA和蛋白表达升高(P<0.05,P<0.01,P<0.001),IL-10、Arg-1 mRNA和蛋白表达降低(P<0.001,P<0.01),同时抑制TLR4、MyD88的激活作用减弱。SCE可能通过上调miR-124抑制TLR4信号通路的激活从而抑制小胶质细胞M1极化,促进小胶质细胞M2极化。To investigate the mechanism by which Schisandra Chinensis mediates the phenotypic transformation of microglia via microRNA-124(miR-124)-based regulation of the Toll-like receptor 4(TLR4)pathway,a model was established using lipopolysaccharide(LPS)stimulation of BV2 cells.Cells were treated with different doses of Schisandra Chinensis extract(SCE).MiR-124 inhibitors and negative control sequences(NC inhibitor)were transfected into LPS-induced BV2 cells and treated with SCE.The MTT assay was used for cell activity detection;an NO kit was used to measure NO release;ELISA kits were used to measure the levels of interleukin-10(IL-10)and tumor necrosis factor-α(TNF-α).Microglia markers,including ionized calcium binding adapter molecule-1(IBA-1)and arginase-1(Arg-1),and the nuclear translocation of nuclear factor-kappa B(NF-κB)were evaluated by immunofluorescent staining.NF-κB p65,IBA-1,Arg-1,TLR4,myeloid differentiation primary factor 88(MyD88),inhibitor of nuclear factor-kappa B kinases-α(IKK-α),IL-10,TNF-αwere detected by immunoblot.SCE at concentrations ranging from 31.25 to 250μg·mL^(-1)had no significant effect on cell activity.SCE treatment significantly inhibited NO release induced by LPS(P<0.001,P<0.01),increased the level of IL-10(P<0.05),and decreased the level of TNF-α(P<0.001).In addition,SCE significantly reduced the expression of TNF-α,IBA-1,TLR4,and MyD88(P<0.01,P<0.001)and elevated the expression of IL-10,Arg-1,NF-κB P65 and IKK-α(P<0.001,P<0.01,P<0.05).SCE treatment could also promote the expression of miR-124(P<0.01).However,transfection with the miR-124 inhibitor increased TNF-α(P<0.001),decreased the level of IL-10(P<0.05),increased the mRNA level and the protein expression of TNF-αand IBA-1(P<0.05,P<0.01,P<0.001),and decreased the mRNA level and protein expression of IL-10 and Arg-1(P<0.001,P<0.01).In addition,the inhibition of TLR4 and MyD88 was attenuated.In conclusion,SCE appears to inhibit the activation of TLR4 signaling pathway by upregulating miR-124 so as to inhibit microgli

关 键 词：五味子提取物 miR-124 脂多糖 BV2细胞 TLR4通路 小胶质细胞表型转化 

分 类 号：R966[医药卫生—药理学]