二甲双胍对类风湿性关节炎患者滑膜细胞增殖及凋亡的影响

Effect of metformin on proliferation and apoptosis in synoviocytes isolated from rheumatoid arthritis patients

目的探讨二甲双胍对类风湿性关节炎患者滑膜细胞增殖与凋亡的影响。方法取类风湿性关节炎患者滑膜组织,用胰酶消化法分离其滑膜细胞,分别用流式细胞术和CCK-8法检测三种浓度的二甲双胍(10、20、30 mmol/L)对滑膜细胞增殖的影响,TUNEL和流式细胞仪检测滑膜细胞凋亡情况,ELISA法检测二甲双胍处理的滑膜细胞上清中的炎性因子和抗炎性因子。结果三种浓度(10、20、30 mmol/L)的二甲双胍分别干预滑膜细胞24、48、72 h,均不同程度抑制了滑膜细胞的增殖,且成时间-浓度依赖性。CCK-8法检测结果显示20、30 mmol/L二甲双胍处理显著抑制了滑膜细胞的增殖(P<0.05),流式细胞术结果表明20 mmol/L二甲双胍处理48 h后,G0/G1期滑膜细胞的百分比与对照组相比显著增加了约20%(P<0.05),S期和G2/M期的细胞比例分别显著下降了9%和11%(P<0.05)。凋亡结果表明,20、30 mmol/L二甲双胍处理48、72 h,较对照组显著促进滑膜细胞的凋亡(P<0.05),流式细胞术显示早期和晚期的滑膜细胞凋亡率约17.5%。检测炎性因子发现,20、30 mmol/L二甲双胍处理后显著降低炎症因子IL-6、IL-1β和TNF-α的水平(P<0.05),增加抗炎因子IL-10水平约66%和76%(P<0.05)。结论二甲双胍可显著抑制体外培养的人滑膜细胞的增殖,诱导其凋亡,可能与二甲双胍降低炎性因子TNF-α、IL-6、IL-1β水平和增加抗炎因子IL-10水平有关。

Objective To determine the effect of metformin on the proliferation and apoptosis in the synoviocytes isolated from the patients with rheumatoid arthritis. Methods The synovial tissues were collected from rheumatoid arthritis patients,and then digested by trypsin to isolated synoviocytes. CCK-8 assay and flow cytometry were employed to detect the cell proliferation in the synoviocytes after treated by metformin at the concentrations of 10,20 and 30 mmol / L. TUNEL assay and flow cytometry were used to measure the cell apoptosis after treatment. The contents of inflammatory factors and anti-inflammatory factors（ TNF-α,IL-6,IL-1β and IL-10） in the supernatant were determined by ELISA. Results Metformin treatment（ 10,20,30 mmol / L） for 24,48 or 72 h inhibited the proliferation of synoviocytes in concentration- and timedependent manners. CCK8 assay indicated that 20 and 30 mmol / L metformin significantly inhibited the proliferation of synoviocytes（ P〈0. 05）. Flow cytometry showed that 20 mmol / L metformin treatment for 48 h increased the percentage of the cells at G0/ G1 phase by 20% when compared with the control group（ P〈0. 05）,while decreased the percentages of synoviocytes at G2/ M and S phases by 9% and 11% respectively（ P〈0. 05）. Metformin treatment（ 20 and 30 mmol/L） for 48 and 72 h significantly enhanced the apoptotic rate（ by 17. 5%） compared with the control group（ P〈0. 05）. The contents of TNF-α,IL-6 and IL-1β wasmarkedly suppressed after metformin treatment of 20 and 30 mmol / L（ P〈0. 05）,and the treatment elevated the IL-10 level by 66% and 76% respectively（ P〈0. 05）. Conclusion Metformin can suppresse the proliferation and induce the apoptosis of synoviocytes isolated from rheumatoid arthritis patients,and these effects may contribute to the decreases of TNF-α,IL-6,IL-1β and the elevation of IL-10.