温阳通络方基于miR-26b-3p调控类风湿关节炎滑膜成纤维细胞的机制研究

Mechanism of Wenyang Tongluo Prescription in Regulating Synovial Fibroblasts in Rheumatoid Arthritis Based on MiR-26b-3p

目的:探讨温阳通络方基于微小RNA(miR)-26b-3p调控类风湿关节炎滑膜成纤维细胞增殖、迁移、侵袭的作用。方法:SD大鼠随机分为温阳通络方组和空白血清组。温阳通络方组大鼠给药剂量为4.05 g·kg^(-1)·d^(–1),2次/d,间隔12 h,连续给药3 d,末次给药后1 h腹主动脉取血分离血清;细胞计数试剂盒-8(CCK-8)实验筛选温阳通络方含药血清最佳浓度和干预时间;在人类风湿关节炎滑膜成纤维细胞MH7A中分别转染inhibitor NC、miR-26b-3p inhibitor、mimic NC、miR-26b-3p mimic片段,并用温阳通络方含药血清干预,设置正常关节成纤维样滑膜细胞为对照组,实时定量聚合酶链式反应(qRT-PCR)检测MH7A细胞中miR-26b-3p的表达;CCK-8实验检测各组细胞的增殖情况;Transwell实验检测各组细胞的迁移、侵袭情况。结果:不同浓度温阳通络方含药血清均能上调miR-26b-3p的表达(P<0.05),miR-26b-3p的表达呈浓度依赖性升高。温阳通络方含药血清干预MH7A细胞后,细胞的增殖、迁移、侵袭能力减弱(P<0.05)。miR-26b-3p过表达后,MH7A细胞的增殖、迁移、侵袭能力减弱(P<0.05);干扰miR-26b-3p的表达后,MH7A细胞的增殖、迁移、侵袭能力增强(P<0.05)。在干扰miR-26b-3p表达的基础上,温阳通络方含药血清干预MH7A细胞后,细胞的增殖、迁移、侵袭能力减弱(P<0.05)。结论:温阳通络方通过上调miR-26b-3p的表达抑制类风湿关节炎滑膜成纤维细胞的增殖、迁移、侵袭。

Objective:To investigate the regulatory effect of Wenyang Tongluo Prescription on the proliferation,migration,and invasion of synovial fibroblasts in rheumatoid arthritis based on miR-26b-3p.Methods:SD rats were randomly divided into a Wenyang Tongluo Prescription group and a blank serum group.The Wenyang Tongluo Prescription group was given Wenyang Tongluo Prescription at 4.05 g·kg^(-1)·d^(–1),twice daily,with a 12-hour interval,for 3 consecutive days.Blood was collected from the abdominal aorta 1 hour after the last administration,followed by serum isolation.The Cell Counting Kit-8(CCK-8)assay was used to determine the optimal concentration and intervention time of Wenyang Tongluo Prescription-containing serum.Human rheumatoid arthritis synovial fibroblasts(MH7A)were transfected with inhibitor NC,miR-26b-3p inhibitor,mimic NC,and miR-26b-3p mimic,respectively,and treated with Wenyang Tongluo Prescription-containing serum.Human fibroblast-like synovial cells(HFLS)were set as the blank control group.Real-time quantitative PCR(qRT-PCR)was used to detect the expression of miR-26b-3p in MH7A cells.The proliferation of cells in each group was detected by the CCK-8 assay.The migration and invasion of cells in each group were detected by the Transwell assay.Results:The expression of miR-26b-3p was upregulated by serum containing Wenyang Tongluo Prescription at different concentrations(P<0.05),and the expression of miR-26b-3p increased in a concentration-dependent manner.After the intervention with the drug-containing serum of Wenyang Tongluo Prescription,the proliferation,migration,and invasion abilities of MH7A cells were weakened(P<0.05).After miR-26b-3p overexpression,the proliferation,migration,and invasion of MH7A cells were weakened(P<0.05).After interfering with the expression of miR-26b-3p,the proliferation,migration,and invasion of MH7A cells were enhanced(P<0.05).On the basis of interfering with the expression of miR-26b-3p,the proliferation,migration,and invasion of MH7A cells were weakened after intervention with the drug-containing serum of Wenyang Tongluo Prescription(P<0.05).Conclusion:Wenyang Tongluo Prescription can inhibit the proliferation,migration,and invasion of synovial fibroblasts in rheumatoid arthritis by upregulating the expression of miR-26b-3p.