类叶牡丹提取物对TNF-α诱导的人类风湿性关节炎成纤维滑膜细胞因子的影响

Effects of Caulophyllum robustum Maxim Extract on Cytokines Induced by TNF-α in Human Rheumatoid Arthritis Fibroblasts

目的利用肿瘤坏死因子-α(TNF-α)诱导的人类风湿性关节炎成纤维滑膜细胞(MH7A)对细胞因子分泌的影响来探讨类叶牡丹提取物(Caulophyllum robustum Maxim Extract,CRME)抗类风湿性关节炎(RA)的分子机制。方法采用噻唑蓝(MTT)法检测CRME对MH7A细胞活力的影响,选取半数抑制浓度(IC_(50))以下的药物浓度作为干预剂量;ELISA法检测CRME(50,100,500μg·mL^(-1))剂量对TNF-α(20 ng·mL^(-1))诱导MH7A细胞释放白介素-6(IL-6)、白介素-4(IL-4)、血管内皮生长因子(VEGF)、核因子κB受体活化因子配体(RANKL)、促凋亡因子Bax、Fas L和抗凋亡因子Bcl-2的影响。结果 CRME在质量浓度为50μg·mL^(-1)~500μg·mL^(-1)对细胞活力均无影响,IC_(50)值为645.32μg·mL^(-1)。与空白组比较,模型组细胞上清中IL-4、IL-6、VEGF、RANKL、Bax、Bcl-2和Fas L含量均显著提高(P<0.05,P<0.01)。与模型组比较,CRME各剂量组均能降低IL-6水平及升高IL-4水平(P<0.01);CRME 100,500μg·mL^(-1)剂量组能明显降低VEGF的表达(P<0.05),且具有浓度依赖性;CRME 500μg·mL^(-1)剂量组能明显降低RANKL含量(P<0.01);CRME 100μg·mL^(-1)剂量组可促进Fas L的表达(P<0.01);CRME 50μg·mL^(-1)剂量组可降低Bcl-2的表达(P<0.05);CRME各剂量组均可促进Bax的表达(P<0.01)。结论减轻炎症、降低血管翳形成、增加骨保护,促进异常增生的滑膜细胞的凋亡可能是CRME抗类风湿性疾病的机理之一。

Objective To investigate the molecular mechanism of anti-rheumatoid arthritis（RA） of Caulophyllum robustum Maxim Extract（CRME） by studying its effect on the secretion of cytokines in human rheumatoid arthritis fibroblast synovial cells（MH7A） induced by tumor necrosis factor-α（TNF-α）. Methods The effect of CRME on the viability of MH7A was detected by Methyl thiazolyl tetrazolium（MTT） assay and the drug concentration below the half inhibitory concentration（IC_（50）） value was chosen as the intervention dose. Contents of interleukin-6（IL-6）,interleukin-4（IL-4）,vascular endothelial growth factor（VEGF）,receptor activator of nuclear factor kappa B ligand（RANKL）, pro-apoptotic factor（Bax, Fas L） and anti-apoptotic factor（Bcl-2） in MH7A cells stimulated by TNF-α（20 ng·mL^（-1）） with CRME（50,100,500 μg·mL^（-1）） intervention were measured by ELISA.Results CRME showed no significant effect on the cell viability in MH7A cells at doses range from 50 to 500μg·m L^（-1）. IC_（50） of CRME to MH7A cells was 645.32 μg · mL^（-1）. Compared with the blank group,the contents of IL-4,IL-6,VEGF,RANKL,Bax,Bcl-2 and Fas L in the cell supernatant of the model group were significantly increased（P〈0.05,P〈0.01）. Compared with the model group,each dose group of CRME reduced the level of IL-6 and increase the level of IL-4（P〈0.01）;CRME decreased the expression of VEGF significantly（P〈0.05） at the concentration of 100,500 μg · mL^（-1） in a dose-dependent manner. The content of RANKL in CRME500 μg · mL^（-1） dose group was significantly decreased（P〈0.01）. Expression of Fas L was promoted in the CRME100 μg · mL^（-1） dose group（P〈0.01）;expression of Bcl-2 was reduced in the CRME 50 μg · mL^（-1） dose group（P〈0.05）. All doses of CRME promoted the expression of Bax（P〈0.01）. Conclusion One of the mechanisms that CRME against rheumatoid diseases may be through suppressing inflammation and angiogenesis,increasing bone protection and promoting apoptosis of abnormal proliferating synovial cells.