化痰通络方对IL-1β刺激的RA滑膜成纤维细胞增殖及TNF-α和aFGF的影响

Effects of Huatan Tongluo Recipe on IL-1β-induced Proliferation of Rheumatoid Arthritis Synovial Fibroblasts and the Production of TNF-α and aFGF

目的探讨化痰通络方对IL-1β刺激的类风湿关节炎滑膜成纤维细胞(rheumatoid arthritis synovial fibroblast,RASFB)增殖及肿瘤坏死因子-α(TNF-α)和酸性成纤维细胞生长因子(acidic fibroblast growth factor,a FGF)分泌的影响。方法体外培养RASFB细胞株,加入终浓度为1、5、10、20μg/L的IL-1β24、48 h以WST-1法检测RASFB增殖率;然后选择20μg/L的IL-1β为诱导剂量为IL-1β组。在此基础上加入终浓度(V/V)为5%、2%、1%的高、中、低浓度化痰通络方水煎液为高、中、低浓度化痰通络方组,培养24、48 h,并设空白对照组。比较RASFB的增长率。ELISA法检测各组TNF-α和a FGF含量,RT-PCR检测TNF-α和a FGF mRNA表达。结果24、48 h时,与IL-1β1μg/L比较,IL-1β5、10、20μg/L RASFB增殖率升高(P<0.01);与IL-1β5μg/L比较,IL-1β10、20μg/L RASFB增值率升高(P<0.01),且IL-1β20μg/L RASFB增值率高于IL-1β10μg/L(P<0.01)。48 h时各浓度的RASFB增值率高于24 h(P<0.01)。与高浓度化痰通络方组比较,中、低浓度化痰通络方组RASFB增殖率降低,且中浓度化痰通络方组对IL-1β刺激的RASFB增殖率明显降低(P<0.01)。与空白对照组比较,IL-1β组24、48 h时TNF-α、a FGF mRNA表达及含量均升高(P<0.05);与IL-1β组比较,除24 h低浓度化痰通络方组TNF-αmRNA表达外,24、48 h高、中、低浓度化痰通络方TNF-α、a FGF mRNA表达及含量均降低(P<0.05);与高浓度化痰通络方组比较,24、48 h时中、低浓度化痰通络方TNF-α、a FGF mRNA表达升高,24 h低浓度化痰通络方TNF-α含量以及24、48 h中、低浓度化痰通络方TNF-α、a FGF含量升高(P<0.05)。结论化痰通络方治疗RA的机理可能涉及抑制RASFB的增殖,降低TNF-α和a FGF mRNA的表达及其蛋白的分泌。

Objective To observe the effects of Huatan Tongluo Recipe （HI-FLR） on the prolifer- ation of IL-1β induced rheumatoid arthritis synovial fibroblast （RASFB） and secretion of necrosis factor （TNF-a） and acidic fibroblast growth factor （aFGF） in vitro. Methods RASFB cell line was cultured in vitro and stimulated by IL-113. The proliferation of RASFB was detected using WST-1 after adding IL-1β with final concentrations of 1,5, 10, 20 μg/L for 24 and 48 h respectively. Then 20 μg/L IL-1β recruited as in- duction dose was set up as IL-1β group. High, middle, low dose HTILR groups were set up by adding HT- TLR decoction with final concentration of 5%, 2%, 1% （V/V）, respectively for 24 and 48 h. A blank con- trol group was also set up. The proliferation rates were compared. Contents of TNF-β and aFGF were de- tected in each group using ELISA. mRNA expressions of TNF-β and aFGF were detected using RT-PCR. Results The proliferation rates of RASFB at 24 h and 48 h were lower at 1 μg/L IL-1β than at 5, 10, 20 μg/L IL-1β（P 〈0.01 ）. The proliferation rate of RASFB was higher at 10 and 20 μg/L IL-1βthan at 5 μl/L IL-1B （P 〈0.01 ）. Besides. the oroliferation rate of RASFB was hiaher at 20 μa/L IL-1 P, than μg/L IL-1β （P 〈0.01）. The proliferation rate of RASFB was higher at 48 h than at 24 h （P 〈0.01 ）. Com- pared with the high dose HI-I-LR group, the proliferation rate of RASFB was lowered in middle and low dose HTTLR groups （P 〈 0.01 ）. Besides, IL-1β induced proliferation rate of RASFB was obviously re- duced in the middle dose HTTLR group （P 〈0.01 ）. Compared with the blank control group, mRNA ex- pressions of TNF-α and aFGF and their contents were elevated in the IL-1β group at 24 and 48 h （P 〈 0.05）. Compared with the IL-1β group, mRNA expressions of TNF-α and aFGF and their contents, except TNF-α mRNA expression in the low dose HI-FLR group at 24 h, were all obviously lowered in 3 dose HT- TLR groups at 24 h and 48 h （P 〈0.05）. Compared with the high dose HI-rLR group, mRNA expressions of TNF-α and aFGF increased in middle and low dose HI-FLR groups at 24 h and 48 h; TNF-α content in the low dose HTTLR group at 24 h ; contents of TNF-α and aFGF in middle and low dose HTI-LR groups at 24 h and 48 h all increased （P 〈0.05）. Conclusion The mechanism of HI-I-LR treatment for RA might be re- lated to inhibiting RASFB proliferation, and decreasing mRNA expressions of TNF-α and aFGF as well as their protein secretion.