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[摘要]
目的 探讨雷公藤甲素治疗类风湿关节炎的作用机制,并分析安全性。方法 将60只大鼠随机分为对照组、模型组、甲氨蝶呤(0.4 mg/kg)组和雷公藤甲素低、中、高剂量(0.1、0.2、0.4 mg/kg)组,每组10只。除对照组外,其余各组大鼠以Ⅱ型胶原诱导类风湿关节炎模型。待造模成功后,从第3周开始,各组分别ig给药,每天1次,连续4周。比较各组大鼠足爪肿胀情况,采用流式细胞仪检测CD4+CD25+和CD4+Foxp3+调节性T细胞(Treg)百分比,检测血清中白细胞介素-10(IL-10)、IL-17、肿瘤坏死因子-α(TNF-α)、γ干扰素(IFN-γ)、转化生长因子-β(TGF-β)、血管内皮生长因子(VEGF)、丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)水平,镜下观察踝关节组织病理形态,并采用免疫组化法检测组织IL-10、IL-17、TNF-α、VEGF、IFN-γ、TGF-β表达水平。结果 病理切片显示模型组大鼠踝关节内滑膜细胞显著增生,有单核细胞、淋巴细胞等大量炎性细胞浸润、新生毛细血管,骨小梁变细,软骨表面侵蚀严重。其余各组组织内病变程度较模型组明显减轻。给药后,甲氨蝶呤组、雷公藤甲素各剂量组大鼠关节肿胀度和关节炎指数较给药前明显减少(P<0.05)。与模型组比较,甲氨蝶呤组、雷公藤甲素各剂量组CD4+Foxp3+Treg和CD4+CD25+Treg均升高(P<0.05)。与模型组比较,甲氨蝶呤组、雷公藤甲素各剂量组大鼠血清IL-10、TGF-β水平升高,IL-17、TNF-α、VEGF、IFN-γ水平降低(P<0.05)。与模型组比较,甲氨蝶呤组、雷公藤甲素各剂量组大鼠踝关节组织IL-10、TGF-β表达明显增多,IL-17、TNF-α、VEGF、IFN-γ表达明显减少(P<0.05)。与对照组或模型组比较,甲氨蝶呤组、雷公藤甲素各剂量组血清ALT、AST水平差异均无统计学意义(P > 0.05)。甲氨蝶呤组与雷公藤甲素高剂量组比较前述各项指标差异均无统计学意义(P > 0.05)。结论 雷公藤甲素能够明显改善Ⅱ型胶原诱导类风湿关节炎大鼠的关节肿胀情况,作用机制与促进IL-10、TGF-β表达,增加Treg细胞比例,抑制IL-17、TNF-α、VEGF、IFN-γ表达有关,并且无明显肝毒性。
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[Abstract]
Objective To explore the mechanism of triptolide in the treatment of rheumatoid arthritis (RA) and analyze its safety. Methods A total of 60 rats were randomly divided into control group, model group, methotrexate (MTX) group and triptolide (TP) low, medium and high dose groups with 10 rats in each group. In addition to the control group, the rats in the other groups were established type Ⅱ collagen-induced RA model. After the successful establishment of the model, rats in MTX group were given 0.4 mg/kg MTX by gavage from the 3rd week. Rats in TP high, middle, and low dose groups were given 0.1, 0.2, and 0.4 mg/kg TP by gavage. Rats in control group and model group were given equal volume distilled water once a day for 4 weeks. The paw swelling of rats in each group was compared. The percentage of CD4+CD25+ and CD4+Foxp3+ Treg was detected by flow cytometry. The levels of IL-10, IL-17, TNF-α, VEGF, IFN-γ, TGF-β, ALT, and AST in the serum were detected. The pathological morphology of ankle joint was observed under microscope. The expression levels of IL-10, IL-17, TNF-alpha, VEGF, IFN-γ, and TGF-β were detected by immunohistochemistry. Results Pathological sections showed that synovial cells were proliferated significantly in the ankle joint of rats in the model group, with infiltration of a large number of inflammatory cells such as monocytes and lymphocytes, new capillaries, thinning of bone trabeculae and serious erosion of cartilage surface. The degree of pathological changes in other groups was significantly less than that in model group. After treatment, the degree of joint swelling and arthritis index in MTX and TP groups were significantly decreased compared with those before treatment (P < 0.05). Compared with model group, CD4+Foxp3+ Treg and CD4+CD25+ Treg were increased in MTX group and TP all dose groups (P < 0.05). Compared with the model group, the serum levels of IL-10, TGF-β in MTX and TP all dose groups were increased, while the levels of IL-17, TNF-α, VEGF and IFN-γ were decreased (P < 0.05). Compared with model group, the expressions of IL-10 and TGF-β in ankle joint tissue of rats in MTX and TP all dose groups were increased significantly, while the expressions of IL-17, TNF-α, VEGF and IFN-γ were decreased significantly (P < 0.05). Compared with control group or model group, there was no significant difference in serum ALT and AST levels between MTX group and TP all dose groups (P > 0.05). There was no significant difference between MTX group and TP high dose group (P > 0.05). Conclusion TP is effective in treating type Ⅱ collagen-induced arthritis in rats, which can significantly improve joint swelling. Its mechanism is related to promoting the expression of IL-10 and TGF-β, increasing the proportion of Treg cells, inhibiting the expression of IL-17, TNF-α, VEGF and IFN-γ, and has no obvious hepatotoxicity.
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