摘要
目的:探索雷公藤甲素(TPT)通过miR-23a-3p/PTEN/PI3K/AKT/mTOR改善强直性脊柱炎(AS)滑膜成纤维细胞(FLS)作用机制。方法:Transwell小室培养CD_(4)^(+)T细胞与AS-FLS,TPT作用24、48和72 h后,采用CCK-8法检测细胞的变化并选取最佳浓度;RT-qPCR和Western Blot分别检测miR-23a-3p、PTEN、PI3K、AKT、mTOR,ELISA法检测IL-1β、IL-6、IL-10、TNF-α表达水平。结果:TPT为100μg/mL其抑制细胞活力的作用最为明显。用TPT处理后导致miR-23a-3p RNA,PI3K、AKT、mTOR基因及蛋白水平,p-PI3K、p-AKT、p-mTOR蛋白水平表达,TNF-α、IL-1β、IL-6显著降低,PTEN基因及蛋白,IL-10显著升高(P<0.01)。在过表达miR-23a-3p的情况下,TPT素干预后miR-23a-3p、PI3K、AKT、mTOR基因及蛋白水平,p-AKT、p-mTOR蛋白水平表达显著降低,PTEN基因及蛋白,IL-10显著升高(P<0.01)。结论:TPT可抑制AS-FLS活力,并可改善细胞炎症,其机制可能与下调mi R-23a-3p/PTEN/PI3K/Akt/m TOR信号通路有关。
Objective:To explore the mechanism of triptolide in improving synovial fibroblasts in ankylosing spondylitis through miR-23a-3p/PTEN/PI3K/AKT/mTOR.Methods:CD:T cells were cultured in a transwell chamber and AS-FLS were treated with triptolide for 24,48 and 72 hours,and the changes of cells were detected by CCK8 method and the optimal concentration was selected;RT-qPCR and Western Blot were used to detect miR-23a-3p,PTEN,PI3K,AKT,mTOR,IL-1β,IL-6,IL-10,TNF-αdetected by ELISA.Results:The results of CCK-8 detection showed that triptolide had the most obvious inhibitory effect on cell viability at 100μg/mL.Treatment with triptolide resulted in miR-23a-3p RNA,PI3K,AKT,mTOR gene and protein levels,p-PI3K,p-AKT,p-mTOR protein levels,The levels of TNF-α,IL-1β,and IL-6 were significantly reduced,and the levels of PTEN gene and protein,and IL-10 were significantly increased(P<0.01).In the case of overexpression of miR-23a-3p,miR-23a-3p,PI3K,AKT,mTOR gene and protein levels,p-AKT,p-mTOR protein levels were significantly reduced after triptolide intervention,PTEN gene and protein,IL-10 increased significantly(P<0.01).Conclusion:Triptolide can inhibit the activity of AS-FLS and improve cell inflammation,and its mechanism may be related to the down-regulation of miR-23a-3p/PTEN/PI3K/Akt/mTOR signaling pathway.
作者
丁香
刘健
陈晓露
张先恒
周琴
DING Xiang;LIU Jian;CHEN Xiaolu;ZHANG Xianheng;ZHOU Qin(The First Affiliated Hospital of Anhui University of CM,Hefei 230031,China)
出处
《中华中医药杂志》
CSCD
北大核心
2023年第12期6024-6029,共6页
China Journal of Traditional Chinese Medicine and Pharmacy
基金
国家自然科学基金项目(No.82104817,No.82205054)
安徽省高校自然基金(No.KJ2021A0558)。