冲击波通过TGF-β/Smad2/3信号通路改善软骨细胞形态及促进Ⅱ型胶原表达的机制研究OA

Objective The aim of this study was to investigate the effects of focused extracorporeal shock wave(F-ESW)on the chondrocyte morphology in rats with knee osteoarthritis(KOA)injury,and to explore its regulatory role on type Ⅱ collagen through the TGF-β/Smad2/3 signaling pathway.Methods One-week-old Sprague-Dawley(SD)rats were selected,and bilateral knee articular cartilage was isolated after euthanasia by cervical dislocation for primary chondrocyte isolation and culture.Third-passage chondrocytes were divided into a blank control group,an osteoarthritis model group(OA group),and a shock wave intervention group(F-ESW group).Chondrocytes were stimulated with 10 ng/mL interleukin-1β(IL-1β)for 24 h to establish an OA model.The expression of interleukin-6(IL-6)was measured by enzyme-linked immunosorbent assay(ELISA)to determine whether the inflammatory model had been successfully established.Toluidine blue staining was used to observe chondrocyte morphology under a microscope.Immuno-fluorescence was used to assess the expression of type Ⅱ collagen(Col2a1).The mRNA expression levels of TGF-β,Smad2,and Smad3 were measured by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR),and the protein expression levels of TGF-β,Smad2,Smad3,p-Smad2,and p-Smad3 in chondrocytes from each group were detected by Western blotting(WB).Results ELISA results showed that the IL-6 level in the supernatant of the OA group was significantly higher than that in the control group(P<0.001),indicating that the in vitro OA inflammatory model was successfully established.Toluidine blue staining showed that,compared with the control group,most cells in the OA group were elongated spindle-shaped or irregular in shape.Compared with the OA group,cells in the F-ESW group were morphologically similar to those in the blank control group,largely restored to normal morphology.Immunofluorescence analysis showed that Col2a1 expression in the F-ESW group was significantly higher than that in the OA group(P<0.01).The mRNA expression levels of TGF-β,Smad2,and Smad3 in the OA group were lower than those in the control group(P<0.05),whereas the expression levels of these indicators in the F-ESW group were higher than those in the OA group(P<0.05).Similarly,the protein expression levels of TGF-β,Smad2,and Smad3 in the OA group were lower than those in the control group(P<0.05),while those in the F-ESW group were higher than those in the OA group(P<0.05).The protein expression levels of p-Smad2 and p-Smad3 showed similar trends.Conclusion F-ESW can improve the morphology of chondrocytes after inflammatory stimulation and activate the TGF-β signaling pathway.The underlying mechanism may involve the upregulation of TGF-β expression,increased expression of Smad2 and Smad3,and enhanced phosphorylation of Smad2 and Smad3,thereby improving chondrocyte homeostasis and delaying the progression of osteoarthritis.