来源：风湿病与关节炎,2022,11（7）:7-11,23.

苯甲酰乌头原碱对类风湿关节炎患者外周血单个核细胞凋亡、炎症因子的影响（基础研究）

邵　鑫，蒋先虹，王　瑞

　　【摘　要】目的：探究苯甲酰乌头原碱（BAC）对类风湿关节炎（RA）患者外周血单个核细胞（PBMC）凋亡、炎症因子的影响及具体凋亡机制，为RA的临床治疗提供参考。方法：募集24例高度活动期RA患者（DAS28评分 > 5.1分），同期纳入性别、年龄匹配的16例健康体检者为正常对照组（HC组）。采用梯度离心法分离PBMC；分别检测加药前及最终质量浓度为0，150，300 μmol·L^-1 BAC干预RA组、HC组PBMC细胞24 h后，实时荧光定量PCR与Western blot法检测细胞中凋亡相关基因及蛋白的表达水平；流式细胞仪检测细胞凋亡率；酶联免疫吸附法（ELISA）检测细胞培养液中炎症因子白细胞介素（IL）-1、IL-6、IL-17、肿瘤坏死因子-α（TNF-α）水平。结果：RA组PBMC细胞中Bcl-2 mRNA和蛋白相对表达量增加，Bax、Caspase-3/Cleaved Caspase-3 mRNA和蛋白相对表达量减少，与HC组比较，差异均有统计学意义（P < 0.05）。加入BAC干预后，150，300 μmol·L^-1 BAC加药组PBMC凋亡率增加，与对照组比较，差异均有统计学意义（P < 0.05）。150，300 μmol·L^-1 BAC加药组PBMC中Bcl-2 mRNA和蛋白相对表达量明显降低，Bax、Caspase-3/Cleaved Caspase-3 mRNA和蛋白相对表达量明显增加，与0 μmol·L^-1 BAC组比较，除150 μmol·L^-1 BAC组Bax mRNA和蛋白表达外，其余差异均有统计学意义（P < 0.05）。同时BAC可以降低PBMC培养液中的炎症因子IL-1、IL-6、IL-17、TNF-α水平，300 μmol·L^-1 BAC组最为显著，与0 μmol·L^-1 BAC组比较，差异有统计学意义（P < 0.05）。结论：RA患者血清中炎症因子IL-1、IL-6、IL-17、TNF-α水平升高，PBMC凋亡减少，BAC可能通过Bcl-2/Bax、Caspase-3途径诱导PBMC细胞凋亡，并能降低PBMC中炎症因子水平。

　　【关键词】　类风湿关节炎；苯甲酰乌头原碱；外周血单个核细胞；凋亡；炎症因子

Effects of Benzoyl Aconitine on Apoptosis of Peripheral Blood Mononuclear Cells and Inflammatory Factors in Patients with Rheumatoid Arthritis

SHAO Xin,JIANG Xian-hong,WANG Rui

　　【ABSTRACT】Objective:To explore the effects of benzoyl aconitine（BAC）on apoptosis of peripheral blood mononuclear cells（PBMC）and inflammatory factors in patients with rheumatoid arthritis（RA）and the specific apoptosis mechanism,so as to provide reference for the clinical treatment of RA.Methods:Twenty patients with highly active RA（DAS28 score > 5.1）were collected,and 16 healthy physical examiners matched by gender and age were included in the same period as the normal control group（HC group）.PBMC was separated by gradient centrifugation.PBMC cells in the RA group and HC group were detected before medication and till the final mass concentrations being 0 μmol·L^-1,150 μmol·L^-1 and 300 μmol·L^-1.Twenty-four hours later,the expression levels of apoptosis related genes and proteins were detected by real-time fluorescent quantitative PCR and Western blot.The apoptosis rate was detected by flow cytometry.Enzyme linked immunosorbent assay（ELISA）was used to detect the inflammatory factors IL-1,IL-6,IL-17 and tumor necrosis factor（TNF-α）in cell culture medium.Results:The relative expressions of Bcl-2 mRNA and protein in PBMC cells of the RA group increased,while the relative expressions of Bax,Caspase-3/Cleaved Caspase-3 mRNA and protein decreased.Compared with the HC group,the difference was statistically significant（P < 0.05）.After BAC intervention,the apoptosis rate of PBMC with mass concentrations of 150 μmol·L^-1 and 300 μmol·L^-1 increased,and the difference was statistically significant compared with the control group（P < 0.05）.The relative expressions of Bcl-2 mRNA and protein in PBMC with mass concentrations of 150 μmol·L^-1 and 300 μmol·L^-1 were significantly reduced,and the relative expressions of Bax,Caspase-3/Cleared Caspase-3 mRNA and protein were significantly increased.Compared with the BAC group with mass concentration of 0 μmol·L^-1,except for the expression of Bax mRNA and protein in the BAC group with mass concentration of 150 μmol·L^-1,the other differences were statistically significant（P < 0.05）.At the same time,BAC can reduce the levels of inflammatory factors IL-1,IL-6,IL-17 and TNF-α in PBMC culture medium,especially in BAC group with mass concentration of 300 μmol·L^-1.Compared with the BAC group with mass concentration of 0 μmol·L^-1,the difference was statistically significant（P < 0.05）.Conclusion:The levels of inflammatory factors IL-1,IL-6,IL-17 and TNF-α in serum of RA patients increase,while apoptosis decreases.BAC may induce PBMC apoptosis through Bcl-2/Bax and Caspase-3 pathways and reduce the level of inflammatory factors in PBMC.

　　【Keywords】 rheumatoid arthritis;benzoyl aconitine;peripheral blood mononuclear cells;apoptosis;inflammatory factors