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OA

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 Mas‑son 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 as‑says were employed to detect the macrophage-myofibroblast transformation in each group;Western blot was adopted to detect the rela‑tive 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.