风湿祛痛胶囊对Ⅱ型胶原诱导性关节炎大鼠滑膜血管新生的影响

Effect of Fengshi Qutong Capsules on synovial angiogenesis in rats with type Ⅱ collagen induced arthritis

观察风湿祛痛胶囊对大鼠主动脉环血管生成的影响以及对Ⅱ型胶原诱导性关节炎(CIA)大鼠关节滑膜血管新生的作用。取正常SD大鼠胸主动脉环,经血管内皮生长因子(VEGF)20μg·L^(-1)体外诱导,风湿祛痛胶囊(0.02,0.1,0.5μg·L^(-1))连续作用9 d,观察血管环芽出的新生血管数目、长度和面积;同时,采用SD大鼠建立CIA模型,以甲氨蝶呤(0.2 mg·kg^(-1)·d^(-1))为阳性对照,风湿祛痛胶囊(0.25,0.5,1 g·kg^(-1)·d^(-1))连续治疗19 d,组织病理学检查(HE)观察炎症关节滑膜中血管形态和分析血管密度,免疫组化检测滑膜中血管内皮细胞标志物血小板-内皮细胞黏附分子(CD31)、VEGF和VEGF受体2(VEGFR_2)的表达情况,免疫荧光双染观察滑膜中CD31和α平滑肌肌动蛋白(αSMA)的表达,酶联免疫吸附法检测血清中TGF-β,PDGF和VEGFR_2的含量。结果显示,VEGF诱导后都能显著增加大鼠胸主动脉环芽出的血管数目、分支长度和面积,风湿祛痛胶囊作用后可显著降低VEGF诱导升高的血管环芽出的血管数量、长度和面积。模型组CIA大鼠炎症关节滑膜中血管密度、CD31阳性表达量、CD31^+/αSMA^-不成熟及总血管阳性表达灰度值(IOD)均较正常组显著升高,滑膜中VEGF和VEGFR_2以及血清中VEGFR2,TGF-β和PDGF含量也明显增加;风湿祛痛胶囊各组治疗后降低关节滑膜血管密度,抑制CD31阳性表达量、CD31^+/αSMA^-不成熟血管和总血管阳性表达IOD,对CD31^+/αSMA^+成熟血管则无明显影响,同时负向调节滑膜和/或血清中VEGF,VEGFR_2,TGF-β和PDGF的含量,且中、高剂量作用显著,甲氨喋呤的作用与高剂量组相近。研究结果提示,风湿祛痛胶囊具有抑制CIA大鼠关节滑膜组织血管新生以及离体血管环生成的作用,这一作用可能和降低促血管新生调控因子的含量有关。

To observe the effect of Fengshi Qutong Capsules(FSQTC) on angiogenesis of rat aortarings and in knee joint synovium of type Ⅱ collagen-induced arthritis(CIA) rats. The blood vessel of aorta rings of normal SD rats were induced by vascular endothelial growth factor(VEGF) 20 μg·L^-1 in vitro, and were treated with FSQTC(0.02, 0.1 and 0.5 μg·L^-1) continuously for 9 days. The number, length and area of neovascularization of the vascular ring were measured. SD rats were immunized to establish collagen-induced arthritis. CIA rats were treated with FSQTC(0.25, 0.5, 1 g·kg^-1·d^-1) and methotrexate(0.2 mg·kg^-1·d^-1) daily for 19 days. Histopathological examination(HE) was performed to observe the vascular morphology and vascular density in the synovial membrane of the inflamed joint. Immunohistochemistry was performed to observe the expression of platelets-endothelial cell adhesion molecule(CD31), VEGF and VEGF receptor 2(VEGFR2)in the synovium. Immunofluorescence was performed to observe the expression of CD31 and α smooth muscle actin(αSMA) in synovial membrane.TGF-β, PDGF and VEGFR2 in serum were detected by enzyme-linked immunosorbent assay. The number, branch length and area of blood vessels of aorta rings were significantly increased induced by VEGF, and FSQTC could significantly reduce the number, branch length and area of blood vessels. Compared with the normal group, the vascular density, CD31 positive expression, CD31^+/αSMA^- immature and total vascular positive expression in the synovial membrane of the model group were significantly increased, and so as VEGF and VEGFR2 in the synovium. The VEGFR2, TGF-β and PDGF in sera were also significantly increased in model group. FSQTC reduced the synovial vascular density and inhibited the positive expression of CD31, CD31^+/αSMA^- immature blood vessels and total vascular. FSQTC has no significant effect on CD31^+/αSMA^+mature blood vessels. FSQTC also negatively inhibited the expression of VEGF, VEGFR2, TGF-β and PDGF in synovial membrane and/or sera. The effect of methotrexate is similar with to the high dose group. Our results demonstrated that FSQTC could inhibit the angiogenesis of synovial tissue in CIA rats and of aortaring in rats, which is related to the reduction of angiogenesis regulatory factor.