膝痹宁Ⅱ方抑制TGF-β/Smad3介导巨噬细胞-肌成纤维细胞的转化改善KOA滑膜纤维化

Xibining Ⅱ Formula Inhibits TGF-β/Smad3-Mediated Macrophage-to-Myofibroblast Transformation to Improve Synovial Fibrosis in Knee Osteoarthritis

    摘要
  • 摘要:
    目的 探讨膝痹宁Ⅱ方对膝骨关节炎（KOA）大鼠滑膜纤维化的改善作用，并研究其通过TGF-β/Smad3介导巨噬细胞-肌成纤维细胞的转化机制。
    方法 将25只SPF级SD大鼠随机分为假手术组、模型组、膝痹宁Ⅱ低剂量组、膝痹宁Ⅱ高剂量组以及塞来昔布组，每组5只。前交叉韧带横断术（ACLT）构建KOA模型，ACLT术后第14天开始灌胃干预，给药28 d后提取各组滑膜组织。用Masson和天狼星红染色评估滑膜纤维化程度；Western blot检测各组组织TGF-β、Collagen Ⅰ、α-SMA蛋白相对表达。采用RAW264.7细胞，并分为空白组、TGF-β组、膝痹宁Ⅱ50 mg·L-1组、膝痹宁Ⅱ100 mg·L-1组以及Galunisertib组。免疫荧光双标检测RAW264.7细胞的CD68、α-SMA的表达；流式细胞术检测细胞中共表达CD68和α-SMA的细胞群体；细胞收缩实验检测各组巨噬细胞-肌成纤维细胞的转化情况；Western blot检测各组细胞的TGF-β、p-Smad3、Collagen Ⅰ、α-SMA的蛋白相对表达；RT-qPCR检测各组细胞的TGF-β、Collagen Ⅰ、α-SMA的mRNA相对表达。
    结果 与模型组相比，膝痹宁Ⅱ高剂量组的滑膜纤维化病变减轻，组织中可见大量致密胶原纤维沉积下降（P<0.05，P<0.01），Collagen Ⅰ、TGF-β、α-SMA的蛋白相对表达减少（P<0.05，P<0.01）。与TGF-β组相比，膝痹宁Ⅱ100 mg·L-1组的细胞CD68和α-SMA的共表达减少（P<0.01），Collagen Ⅰ、TGF-β、α-SMA、p-Smad3的蛋白相对表达减少（P<0.05，P<0.01），TGF-β、Collagen Ⅰ、α-SMA的mRNA相对表达减少（P<0.05，P<0.01）；细胞收缩能力减弱（P<0.01）。
    结论 膝痹宁Ⅱ方能抑制TGF-β/Smad3介导巨噬细胞-肌成纤维细胞的转化改善KOA滑膜纤维化。

     

    Abstract:
    OBJECTIVE To investigate the ameliorative effects of Xibining (XBN)Ⅱ formula on synovial fibrosis in rats with knee osteoarthritis (KOA), and to examine its mechanism of action through TGF-β/Smad3-mediated macrophage-to-myofibroblast transdifferentiation(MMT).
    METHODS Twenty-five SPF-grade SD rats were randomly divided into five groups of five rats each: a sham-operated group, a model group, a low-dose XBNⅡ group, a high-dose XBNⅡ group, and a celecoxib group. Anterior cruciate ligament transection (ACLT) was employed to establish a KOA model. Gavage intervention commenced on day 14 post-ACLT surgery, with synovial tissue extracted from each group 28 days after drug administration. The degree of synovial fibrosis was assessed by Masson staining and Sirius red staining; the relative expression of TGF-β, Collagen Ⅰ, and α-SMA proteins in each group was detected by Western blot. RAW264.7 cells were used and divided into the following groups: control group, TGF-β group, XBNⅡ50 mg·L-1 group, XBNⅡ100 mg·L-1 group, and Galunisertib group. Immunofluorescence double labeling was used to detect the expression of CD68 and α-SMA in RAW264.7 cells; flow cytometry was used to detect cell populations co-expressing CD68 and α-SMA; cell contraction assays were employed to detect the macrophage-myofibroblast transformation in each group; Western blot was adopted to detect the relative protein expression of TGF-β, p-Smad3, Collagen Ⅰ, and α-SMA in each group; and RT-qPCR was used to detect the relative mRNA expression of TGF-β, Collagen Ⅰ, and α-SMA in each group.
    RESULTS Compared with the model group, the high-dose group of XBNⅡ showed reduced synovial fibrosis lesions, with a significant decrease in the abundance of dense collagen fiber deposits visible in the tissue (P<0.05, P<0.01). The relative expression levels of Collagen Ⅰ, TGF-β, and α-SMA proteins were also reduced (P<0.05, P<0.01). Compared with the TGF-β group, the XBNⅡ100 mg·L-1 group exhibited reduced co-expression of CD68 and α-SMA (P<0.01), decreased relative protein expression of Collagen Ⅰ, TGF-β, α-SMA, and p-Smad3 (P<0.05, P<0.01), while the relative mRNA expression of TGF-β, Collagen Ⅰ, and α-SMA reduced (P<0.05, P<0.01). Cell contractility also reduced (P<0.01).
    CONCLUSION XBNⅡ suppresses TGF-β/Smad3-mediated macrophage-to-myofibroblast transdifferentiation, thereby improving fibrotic changes in the synovium of knee osteoarthritis.

     

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