miR-21在黄芪甲苷保护ox-LDL诱导的内皮细胞炎症损伤过程中的作用

The role of miR-21 in the protective effect of astragaloside on ox-LDL-induced endothelial cell inflammatory injury

目的探讨miR-21在黄芪甲苷保护氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVEC)炎症损伤中的作用及其机制。方法将体外培养的HUVEC分为对照组(未干预)、模型组(以100μmol/L ox-LDL作用24 h)、治疗-低剂量(L)组、治疗-中剂量(M)组和治疗-高剂量(H)组,其中治疗-L、M和H组细胞以100μmol/L ox-LDL作用24 h后,分别加入10、20和40 mg/L的黄芪甲苷作用2 h。MTT法检测细胞的存活率,ELISA法检测细胞上清液中炎症相关因子TNF-α、IL-6和NF-κB的含量,RT-PCR检测细胞内miR-21和TLR4 mRNA的表达,Western blot检测TLR4蛋白的表达。双荧光素酶报告基因实验检测miR-21和TLR4的靶向关系。转染miR-21模拟物和抑制剂构建miR-21过表达和低表达的HUVEC株,观察miR-21对HUVEC炎症因子分泌和TLR4表达的影响;同时,在给予40 mg/L的黄芪甲苷治疗的基础上,观察转染miR-21抑制剂下调miR-21表达对黄芪甲苷作用下HUVEC炎症因子的影响。结果与对照组相比,模型组、治疗-L组、治疗-M组和治疗-H组细胞存活率和细胞中miR-21表达显著降低,而细胞上清液中TNF-α、IL-6和NF-κB的含量以及细胞中TLR4 mRNA、蛋白的表达显著升高(P<0.05);与模型组相比,治疗-L组、治疗-M组和治疗-H组细胞存活率和miR-21表达逐渐升高,而TNF-α、IL-6和NF-κB的含量以及TLR4 mRNA、蛋白的表达逐渐降低,差异具有统计学意义(P<0.05)。双荧光素酶报告基因实验证实TLR4是miR-21的潜在靶基因。miR-21过表达降低HUVEC内炎症因子的分泌和TLR4的表达,反之,则促进HUVEC内炎症因子的分泌和TLR4的表达;下调miR-21表达后可逆转黄芪甲苷对HUVEC炎症因子的调控。结论黄芪甲苷可减轻ox-LDL诱导的HUVEC炎症损伤,其作用机制可能与上调miR-21靶向抑制TLR4表达有关。

Objective To investigate the role and mechanism of miR-21 in the inflammatory injury of human umbilical vein endothelial cells(HUVEC)induced by astragaloside-protected oxidized low density lipoprotein(ox-LDL). Methods HUVEC cultured in vitro were divided into control group(without intervention),model group(treated with 100 μmol/L ox-LDL for 24 h),treatment-low dose group(L),treatment-medium dose group(M)and treatment-high dose group(H). The cells in the treatment group-L,M and H were treated with 100 μmol/L ox-LDL for 24 h,and then added with 10,20 and 40 mg/L of astragaloside for 2 h. Cell viability was detected by MTT assay,the level of inflammatory related factors TNF-α,IL-6 and NF-κB in cell supernatant were measured by ELISA,the expression of intracellular miR-21 and TLR4 was detected by RT-PCR,the expression of TLR4 protein was tested by Western blot,and the targeting relationship between microRNA-21 and TLR4 was checked by dual luciferase reporter gene assay. In addition,HUVEC with over-and low-expression of miR-21 were constructed by transfecting mimic and inhibitor of miR-21,and the effects of miR-21 on the secretion of inflammatory factors and the expression of TLR4 in HUVEC were observed. At the same time,on the basis of 40 mg/L astragaloside treatment,the effects of down-regulation of miR-21 expression by transfection inhibitor of miR-21 on inflammatory factors in HUVEC under the action of astragaloside were observed. Results Compared with the control group,the cell survival rate and the expression of miR-21 in the model group,treatment-L,treatment-M and treatment-H groups were decreased significantly,while the level of TNF-a,IL-6 and NF-kB in cell supernatant and the expression of TLR4 mRNA and protein in cells were increased significantly(P<0.05). Compared with the model group,the cell viability and the expression of miR-21 were increased gradually,while the contents of TNF-a,IL-6 and NF-kB and the expression of TLR4 mRNA and protein were decreased gradually in treatment-L group,treatment-M group and treatment-H group(P<0.05). Dual luciferase reporter assays confirmed that TLR4 was a potential target gene of miR-21. Overexpression of miR-21 reduced the secretion of inflammatory cytokines and the expression of TLR4 in HUVEC. Conversely,it promoted the secretion of inflammatory cytokines and the expression of TLR4 in HUVEC. Down-regulation of miR-21 expression reversed the regulation of Astragaloside on HUVEC inflammatory factors. Conclusion Astragaloside can alleviate the inflammation injury of HUVEC induced by ox-LDL,and its mechanism may be related to up-regulation of the expression of TLR4 targeting by miR-21.