Objective: To explore the inhibitory effect of Sidaxue (SX), a traditional Guizhou Miao herbal medicine formula, on necrotic apoptosis and synovial angiogenesis in rats with collagen-induced arthritis (CIA) and the role of the RIPK1/RIPK3/MLKL pathway in mediating this effect.
Methods: Forty-two SD rats were randomized into 6 groups (n=7), including a normal control group, a CIA model group, 3 SX treatment groups at low (10 g/kg), moderate (20 g/kg) and high (40 g/kg) doses, and a GTW treatment group. CIA rat models were established by subcutaneous injections of bovine type II collagen, and the treatments were administered daily by gavage for 21 days. The rats were observed for swelling of the hind limb joints, which was rated using the arthritis index (AI) score on a weekly basis. Serum levels of TNF-α, IL-1β and IL-17 in the rats were detected using ELISA, and the pathological changes in the synovium were observed with HE staining. Real-time PCR was performed to detect the mRNA expression levels of VEGF, MMP-9, Ang-1, RIPK1, RIPK3, and caspase-8 in the synovial tissues, and the protein expressions of VEGF, MMP9, Ang-1, Stat-3, RIPK1, RIPK3, MLKLl, p-MLKL and caspase-8 were detected using Western blotting.
Results: Compared with those in CIA model group, the rats receiving treatment with GTW and SX showed milder swelling of the hind limb joints with significantly lower AI scores (P < 0.05). In CIA model group, a large number of inflammatory cells were observed in the synovium with obvious damages of the tissue structure. In the drug treatment groups, inflammatory cell infiltration, synovial angiogenesis and synovial hyperplasia were alleviated, and the therapeutic effects were obviously enhanced as SX dose increased. Compared with those in the model group, the rats treated with GTW and high-dose SX showed significantly decreased serum levels of IL-1β, IL-17 and TNF-α (P < 0.05), lower mRNA and protein expressions of RIPK1, RIPK3, VEGF, Ang-1, and MMP9 (P < 0.05), higher expressions of caspase-8 (P < 0.01), and obviously lowered expression of Stat-3 protein and phosphorylation level of MLKL (P < 0.05).
Conclusion: SX can improve synovial angiogenesis in CIA rats possibly by inhibiting the activation of RIP1/RIP3/MLKL signaling pathway and down-regulating the expression of the vascular growth factors VEGF, Ang-1, MMP9, and Stat-3.
目的: 探究贵州苗医验方“四大血”（SX）对胶原诱导型关节炎（CIA）大鼠滑膜组织坏死性凋亡和滑膜血管新生的作用机制。
方法: 将42只SD大鼠均分为空白组（Nor）、模型组（Mod）、雷公藤多苷片阳性对照组（GTW，40 g/kg）和SX高、中、低剂量组（SX 40 g/kg、SX 20 g/kg、SX 10 g/kg），7只/组。构建CIA大鼠模型。建模后测量大鼠双后肢足跖肿胀程度并进行关节炎指数评分。ELISA法检测血清中TNF-α、IL-1β和IL-17变化，HE染色法观察大鼠关节滑膜病理变化，Real-time PCR检测滑膜组织中VEGF、MMP-9、Ang-1、RIPK1、RIPK3、Caspase-8 mRNA表达水平，Western blot检测滑膜组织中VEGF、MMP9、Ang-1、STAT-3、RIPK1、RIPK3、MLKL、p-MLKL、Caspase-8蛋白表达水平。
结果: GTW组和SX高、中、低剂量组足跖肿胀度及关节炎指数评分均小于Mod组（P < 0.05）。在光镜下观察大鼠关节滑膜病理组织切片，Mod组可见大量炎性细胞浸润，各层组织结构混乱；而药物治疗组中，炎性细胞浸润、滑膜血管新生和滑膜增生程度均降低。且随SX剂量增高，治疗效果呈逐渐增强的趋势。与Mod组相比，GTW组和SX 40 g/kg组大鼠血清中TNF-α、IL-1β和IL-17含量，滑膜组织中RIPK1、RIPK3、VEGF、Ang-1、MMP9 mRNA和蛋白表达均降低（P < 0.05），Caspase-8 mRNA和蛋白表达水平增高（P < 0.05）并下调Stat-3蛋白表达和MLKL的磷酸化水平（P < 0.05）。
结论: SX可以改善CIA大鼠滑膜血管新生，其机制可能与抑制RIP1/RIP3/MLKL信号通路激活，并下调促血管生长因子VEGF、Ang-1、MMP9、STAT-3表达有关。
Keywords: Miao medicine Sidaxue formula; RIPK1/RIPK3/MLKL pathway; inflammation; pannus; rheumatoid arthritis.