杜仲皮水提取物调节circFADS2靶向miR-491-5p干预类风湿性关节炎滑膜成纤维样细胞生长与凋亡  

Water extract of Eucommiae cortex regulates circFADS2 targeting miR-491-5p to interfere with viability and apoptosis of rheumatoid arthritis fi⁃broblast-like synoviocytes

作　　者：郑国洪 孙斌 王晶晶 杨正明[4] ZHENG Guo-hong;SUN Bin;WANG Jing-jing;YANG Zheng-ming(Department of Emergency Orthopedics and Traumatology,Zhejiang Zhoushan Hospital of Traditional Chinese Medicine,Zhoushan 316000,China;Department of Osteoarthritis,Zhejiang Zhoushan Hospital of Traditional Chinese Medicine,Zhoushan 316000,China;Department of Orthopedics and Traumatology,Ningbo Longsai Hospital,Ningbo 315299,Chi-na;Department of Orthopedics,The Second Affiliated Hospital of Medical College of Zhejiang University,Hangzhou 310009,China)

机构地区：[1]浙江省舟山市中医院急诊骨伤科,浙江舟山316000 [2]浙江省舟山市中医院骨关节科,浙江舟山316000 [3]宁波龙赛医院骨伤科,浙江宁波315299 [4]浙江大学医学院附属第二医院骨科,浙江杭州310009

出　　处：《中国病理生理杂志》2021年第11期2044-2049,共6页Chinese Journal of Pathophysiology

基　　金：舟山市科技计划项目(No.2019c31113)。

摘　　要：目的:探讨杜仲皮水提取物(water extract of Eucommia cortex,WEEC)对类风湿性关节炎滑膜成纤维样细胞(rheumatoid arthritis fibroblast-like synoviocytes,RA-FLS)生长与凋亡的影响及分子机制。方法:采用不同浓度WEEC(0.1、0.3、0.9和2.7 g/L)处理RA-FLS,CCK-8法检测细胞活力,选取最适WEEC浓度进行后续实验。细胞分别转染pcDNA-circFADS2和/或anti-miR-491-5p,探讨WEEC对RA-FLS生长与凋亡的影响及分子机制:用CCK-8法检测细胞活力;流式细胞术检测细胞凋亡率;Western blot法检测cleaved caspase-3的蛋白水平;RT-qPCR检测环状RNA-FADS2(circular RNA-FADS2,circFADS2)和miR-491-5p的表达水平;双萤光素酶基因报告实验检测circ-FADS2和miR-491-5p的靶向关系。结果:不同浓度WEEC处理后,RA-FLS的活力降低,凋亡率升高,cleaved cas-pase-3蛋白水平升高,circFADS2表达水平降低,miR-491-5p表达水平升高(P<0.05)。过表达circFADS2或抑制miR-491-5p表达后,WEEC处理的细胞活力升高,凋亡率及cleaved caspase-3蛋白水平降低(P<0.05)。circFADS2靶向调控miR-491-5p,过表达miR-491-5p逆转了circFADS2质粒对WEEC处理的RA-FLS活力与凋亡的影响。结论:杜仲皮水提取物通过调节circFADS2/miR-491-5p抑制类风湿性关节炎滑膜成纤维样细胞生长并促进其凋亡。AIM:To explore the effect of water extract of Eucommia cortex(WEEC)on the viability and apop-tosis of rheumatoid arthritis fibroblast-like synoviocytes(RA-FLS)and the underlying mechanism.METHODS:RA-FLS was treated with different concentrations of WEEC(0.1,0.3,0.9 and 2.7 g/L),and then cell viability was detected by CCK-8 assay,and the optimal concentration of WEEC was selected for follow-up experiments.The RA-FLS were transfect-ed with pcDNA-circular RNA-FADS2(circFADS2)or/and anti-miR-491-5 respectively,to explore the effect of WEEC on the growth and apoptosis of RA-FLS and the underlying molecular mechanism.CCK-8 assay was used to detect viability,apoptotic rate was analyzed by flow cytometry,the protein levels of cleaved caspase-3 were determined by Western blot,the expression levels of circFADS2 and miR-491-5p were detected by RT-qPCR.Dual luciferase report experiment was used to detect the targeting relationship of circFADS2 and miR-491-5p.RESULTS:After treatment with WEEC at differ-ent concentrations,the viability of RA-FLS was decreased,the apoptotic rate was increased,the protein levels of cleaved caspase-3 were increased,circFADS2 level was decreased,and miR-491-5p level was increased(P<0.05).Under the condition of WEEC treatment,circFADS2 over-expression or knock-down of miR-491-5p expression increased the viabili-ty,decreased the apoptosis and the protein levels of cleaved caspase-3 in RA-FLS(P<0.05).circFADS2 targeted and regulated miR-491-5p expression.miR-491-5p over-expression counteracted the effect of circFADS2 plasmid on the activi-ty and apoptosis of RA-FLS treated by WEEC.CONCLUSION:WEEC inhibits the viability of RA-FLS and promotes its apoptosis by regulating circFADS2/miR-491-5p.

关 键 词：杜仲皮水提取物 环状RNA-FADS2 微小RNA-491-5p 类风湿性关节炎滑膜成纤维样细胞 细胞活力 细胞凋亡 

分 类 号：R593.22[医药卫生—内科学] R363.2[医药卫生—临床医学]