N-乙酰半胱氨酸在缓解氧化应激造成的血管平滑肌细胞损伤和表型转变中的作用

摘要

目的：研究N-乙酰半胱氨酸(NAC)在缓解氧化应激诱导血管平滑肌细胞(VSMC)损伤和表型转变中的作用。方法小鼠VSMC( MOVA细胞)常规培养后随机分为4组：对照组、35μmol/L叔丁基氢过氧化物( t-BHP)处理组、35μmol/L t-BHP+1 mmol/L NAC处理组、1 mmol/L NAC处理组。二氯荧光黄二乙酸酯探针检测t-BHP处理后的细胞内活性氧类( ROS)水平。 CCK-8试剂盒法检测各组细胞的抑制率,实时荧光定量聚合酶链反应技术与Western blot检测血管平滑肌合成型标志物α-肌动蛋白(SMA)和合成型标志物平滑肌胚胎型肌球蛋白重链(SMemb)的mRNA和蛋白表达,同时分析NAC和t-BHP 处理对miR-145表达的影响。结果35μmol/L t-BHP处理后,细胞内ROS水平显著增高,与对照组相比,差异有统计学意义( P<0．05)。与35μmol/L t-BHP处理组相比,35μmol/L t-BHP+1 mmol/L NAC处理组细胞内的ROS水平降低( P<0．05)。与对照组相比,35μmol/L t-BHP处理后细胞抑制率、凋亡率增高,差异有统计学意义( P <0．05)。与35μmol/L t-BHP 处理组相比,35μmol/L t-BHP +1 mmol/L NAC 处理组细胞抑制率和凋亡率均降低( P <0．05)。与对照组相比,35μmol/L t-BHP处理后细胞SMA表达降低,SMemb表达增高,差异有统计学意义(P<0．05)。与35μmol/L t-BHP处理组相比,35μmol/L t-BHP+1 mmol/L NAC处理组SMA表达较高,SMemb表达较低(P<0．05)。与对照组相比,35μmol/L t-BHP处理后miR-145的表达降低,差异有统计学意义( P<0．05)。与35μmol/L t-BHP处理组相比,35μmol/L t-BHP+1 mmol/L NAC处理组miR-145的表达增加(P<0．05)。结论 NAC可以缓解氧化应激诱导的VSMC损伤和表型转变,此过程中伴随miR-145的表达增高。%Objective To explore the role of N-acetyl cysteine( NAC) in attenuating injury and pheno-type shift of vascular smooth muscle cells( VSMC) induced by oxidative stress. Methods Mouse VSMCs (MOVAS cells) were divided into four groups after routine culture,including 35 μmol/L t-BHP treated group,35 μmol/L t-BHP+1 mmol/L NAC treated groups and 1 mmol/L NAC treated group, and control group. Intracellular ROS level in each group was detected by DCFH-DA probe. CCK-8 assay was used to detect the inhibition rate of each group. mRNA expression level of SMemb and SMA in MOVAS cells were analyzed by real-time quantitative PCR,protein expression level of SMemb and SMA were measured by West-ern blot. The effects of NAC and t-BHP on the expression of miR-145 was analyzed as well. Results After treatment with 35μmol/L t-BHP,the intracellular level of ROS increased significantly,compared with the con-trol group,the difference was significant(P <0. 05). Compared with 35 μmol/L t-BH group,in 35 μmol/L t-BHP+1 mmol/L NAC group,the intracellular ROS level was lower (P<0. 05). After the treatment of 35μmol/L t-BHP,cell inhibition rate,apoptosis rate were increased,compared with the control group,the differ-ence was significant(P<0. 05). Compared with 35 μmol/L group t-BHP,in 35 μmol/L t-BHP+1 mmol/L NAC group,the cell inhibition rate and apoptosis were lower(P<0. 05). Compared with control,the expres-sion of SMA was decreased and the expression of Smemb was increased after 35μmol/L t-BHP treatment,the difference was significant(P <0. 05). Compared with 35 μmol/L t-BHP group,in 35 μmol/L t-BHP +1 mmol/L NAC group,the SMA expression was higher and the SMemb was lower(P<0. 05). 35 μmol/L t-BHP treatment can decrease the expression of miR-145,compared with the control group,the difference was significant (P<0. 05). Compared with 35 μmol/L t-BHP group,in 35 μmol/L t-BHP+1 mmol/L NAC group,the miR-145 expression was higher(P<0. 05). Conclusion Oxidative stress induced VSMC damage and phenotype change can be attenuated by NAC,which involves the miR-145 expression increase.