来氟米特通过调节miR-449a在肺纤维化中的机制研究  

Mechanism of leflunomide in regulating pulmonary fibrosis by regulating miR-449a

作　　者：刘冬 赖伟男[2] LIU Dong;LAI Weinan(Department of Pharmacy,Xinqiao Hospital,Army Military Medical University,Chongqing 400037,China;Department of Rheumatology and Immunology,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China)

机构地区：[1]陆军军医大学新桥医院药剂科,重庆400037 [2]南方医科大学南方医院风湿免疫科,广东广州510515

出　　处：《药学实践杂志》2020年第4期296-300,306,共6页Journal of Pharmaceutical Practice

基　　金：广东省自然科学基金资助项目,编号(2017A030313508)。

摘　　要：目的探究来氟米特(leflunomide,LEF)通过调节微小RNA(microRNA,miR)-449a在肺纤维化中的机制研究。方法将人肺成纤维细胞MRC-5分为6组,即对照组、LEF组、LEF+mimic组、mimic组、LEF+inhibitor组和inhibitor组。通过质粒转染miR-449a mimic或inhibitor来过表达或沉默miR-449a,在5 mg/L LEF的条件下培养48 h。分别通过CCK-8法、克隆形成实验和流式细胞术检测各组细胞活力、细胞增殖能力和凋亡率。使用免疫荧光染色检测α平滑肌肌动蛋白(αsmooth muscle actin,α-SMA)胶质蛋白I(collagen I,col I)。分别使用qPCR和Western blot检测miRNA和蛋白的水平。结果 mimic组miR-449a水平显著高于对照组(P<0.05)。LEF组和inhibitor组的miR-449a水平显著低于对照组(P<0.05)。LEF+mimic组的miR-449a的表达水平显著高于LEF组,LEF+inhibitor组的miR-449a水平显著低于LEF组(P<0.05)。LEF组和inhibitor组的细胞活力和细胞增殖能力显著高于对照组(P<0.05)。mimic组的细胞活力和细胞增殖能力显著低于对照组(P<0.05)。LEF+mimic组的细胞活力和细胞增殖能力显著低于LEF组而LEF+inhibitor组的细胞活力显著高于LEF组(P<0.05)。LEF组和inhibitor组的细胞凋亡率低于对照组(P<0.05),mimic组的细胞凋亡率显著高于对照组(P<0.05)。LEF+mimic组的细胞凋亡率显著高于LEF组而LEF+inhibitor组的凋亡率显著低于LEF组(P<0.05)。LEF组和inhibitor组的α-SMA和Col I蛋白的荧光强度显著高于对照组(P<0.05),mimic组的相对荧光强度低于对照组(P<0.05)。LEF+mimic组的α-SMA和Col I蛋白相对荧光强度显著低于LEF组,LEF+inhibitor组的α-SMA和Col I蛋白相对荧光强度显著高于LEF组(P<0.05)。LEF组和inhibitor组的p-JNK/JNK水平高于对照组(P<0.05),mimic组的p-JNK/JNK水平显著低于对照组(P<0.05),LEF+mimic组中p-JNK/JNK水平显著低于LEF组而LEF+inhibitor组的p-JNK/JNK水平显著高于LEF组(P<0.05)。结论 LEF可能通过抑制肺成纤维细胞中miR-449a的表达激活JNK途径,Objective To investigate the mechanism of leflunomide(LEF) in regulating pulmonary fibrosis by regulating microRNA(miR)-449 a. Methods Human lung fibroblasts MRC-5 were divided into 6 groups: control group, LEF group,LEF+mimic group, mimic group, LEF+inhibitor group and inhibitor group. MiR-449 a was overexpressed or silenced by plasmid transfection with miR-449 a mimic or inhibitor and ncubate for 48 h at 5 mg/L LEF. The cell viability, cell proliferation ability and apoptotic rate of each group were measured by CCK-8 method, clone formation experiment and flow cytometry. Immunofluorescent staining was used to detect α smooth muscle actin(α-SMA) and collagen I(col I). The levels of miRNA and protein were detected using qPCR and Western blot, respectively. Results The miR-449 a level in the mimic group was significantly higher than that in the control group(P<0.05). The level of miR-449 a in LEF group and inhibitor group was significantly lower than that in control group(P<0.05). The expression level of miR-449 a in LEF+mimic group was significantly higher than that in LEF group, and the level of miR-449 a in LEF+inhibitor group was significantly lower than that in LEF group(P<0.05). The cell viability and cell proliferation ability of the LEF group and inhibitor group were significantly higher than those of the control group(P<0.05). The cell viability and cell proliferation ability of the mimic group were significantly lower than those of the control group(P<0.05).The cell viability and cell proliferation ability of the LEF+mimic group were significantly lower than those of the LEF group,while the cell viability of the LEF+inhibitor group was significantly higher than that of the LEF group(P<0.05).The apoptosis rate of LEF group and inhibitor group was lower than that of control group(P<0.05).The apoptosis rate of mimic group was significantly higher than that of control group(P<0.05).The apoptosis rate of LEF+mimic group was significantly higher than that of LEF group,while the apoptosis rate of LEF+inhibito

关 键 词：来氟米特 肺纤维化 成纤维细胞 Α平滑肌肌动蛋白 微小RNA-449a C-JUN氨基末端激酶 

分 类 号：R971.1[医药卫生—药品]