Yang Fan, Shen Junyi, Cai Hui
Nanjing University Of Chinese Medicine, Department of Integrated Medicine, Jinling Hospital, Nanjing 210002, China.
Department of Integrated Medicine, Jinling Hospital, Nanjing 210002, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2022 Aug;38(8):692-698.
Objective To explore the effect and mechanism of paeoniflorin (PAE) on apoptosis of fibroblast synovial cells derived from rheumatoid arthritis tissues. Methods Rheumatoid arthritis fibroblast-like synovial cells (RA-FLSs) were cultured under different concentrations of PAE (20, 40, 80 μmol/L). Survival rate of cells was determined at different incubation time-points (24, 48, 72 hours) by MTT assay. Flow cytometry was performed to determine apoptosis rate of cells. Cells were transfected with 3 small interfering RNAs(siRNAs) fragments of long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1(lncRNA MALAT1) for 48 hours. Real-time quantitative PCR was performed to determine if MALAT1 knockdown was successful. Cells were assigned into control group, PAE group, si-NC group, si-MALAT1 group and PAE combined with si-MALAT1 group. Cell apoptosis rate was determined by flow cytometry after knockdown of MALAT1 expression. mRNA expression levels of Wnt1, β-catenin, caspase-3, caspase-9, B-cell lymphoma 2(Bcl2) and Bcl2-associated X protein (BAX) were determined using real-time quantitative PCR. Western blot analysis was performed to determine protein expression levels of Wnt1, β-catenin, caspase-3, caspase-9, Bcl2 and BAX in each group. Results The findings showed that incubation of RA-FLSs with PAE at the tested concentration decreased their viability in a time-dependent manner. Expression level of lncRNA MALAT1 was lower in RA-FLSs group compared with the level in NC-FLSs group. Analysis showed that apoptosis rate was higher in cells treated with PAE. si-MALAT1 group had the lowest apoptosis rate, whereas the PAE combined with si-MALAT1 group showed a significantly higher apoptosis rate compared with si-MALAT1 group. mRNA levels of Bcl2, Wnt1 and β-catenin were lower in PAE group compared with the level in control group, whereas mRNA expression levels of caspase-3, caspase-9 and BAX were higher in PAE group compared with the levels in control group. Expression level of Bcl2 was significantly higher in si-MALAT1 group compared with the level in the control group. Expression level of BAX, caspase-3 and caspase-9 were higher in PAE combined with si-MALAT1 group compared with the level in the control group. Conclusion PAE promotes apoptosis and inhibits the Wnt1/β-catenin pathway in RA-FLSs by upregulating expression of lncRNA MALAT1.
目的 探讨芍药苷（PAE）对类风湿关节炎组织来源的成纤维样滑膜细胞凋亡的影响及机制。方法 将类风湿关节炎成纤维样滑膜细胞（RA-FLSs）置于不同浓度（20、40、80 μmol/L）的PAE中培养。在不同孵育时间点（24、48、72小时）通过MTT法测定细胞存活率。采用流式细胞术测定细胞凋亡率。用3条长链非编码RNA转移相关肺腺癌转录本1（lncRNA MALAT1）的小干扰RNA（siRNAs）片段转染细胞48小时。通过实时定量PCR检测MALAT1敲低是否成功。将细胞分为对照组、PAE组、si-NC组、si-MALAT1组和PAE联合si-MALAT1组。敲低MALAT1表达后，通过流式细胞术测定细胞凋亡率。采用实时定量PCR检测Wnt1、β-连环蛋白、半胱天冬酶-3、半胱天冬酶-9、B细胞淋巴瘤-2（Bcl2）和Bcl2相关X蛋白（BAX）的mRNA表达水平。通过蛋白质印迹分析检测各组中Wnt1、β-连环蛋白、半胱天冬酶-3、半胱天冬酶-9、Bcl2和BAX的蛋白表达水平。结果 结果显示，用测试浓度的PAE孵育RA-FLSs可使其活力呈时间依赖性降低。RA-FLSs组lncRNA MALAT1的表达水平低于正常对照成纤维样滑膜细胞（NC-FLSs）组。分析表明，PAE处理的细胞凋亡率更高。si-MALAT1组的凋亡率最低，而PAE联合si-MALAT1组的凋亡率明显高于si-MALAT1组。PAE组中Bcl2、Wnt1和β-连环蛋白的mRNA水平低于对照组，而PAE组中半胱天冬酶-3、半胱天冬酶-9和BAX的mRNA表达水平高于对照组。si-MALAT1组中Bcl2的表达水平明显高于对照组。PAE联合si-MALAT1组中BAX、半胱天冬酶-3和半胱天冬酶-9的表达水平高于对照组。结论 PAE通过上调lncRNA MALAT1的表达促进RA-FLSs凋亡并抑制Wnt1/β-连环蛋白通路。
