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

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.