BACKGROUND:Chondrocyte apoptosis is an important factor in the development of osteoarthritis,and Complanatoside A has a flavonoid effect,which can inhibit apoptosis of various cells,but its effect on chondrocyte apoptosis and the mechanism of action are not clear. OBJECTIVE:To investigate the intrinsic association and mechanism of Complanatoside A in chondrocyte apoptosis based on the Wnt/β-catenin signaling pathway. METHODS:(1)The cartilage tissues of the femur and tibia transected during knee arthroplasty were collected,and chondrocytes were isolated,cultured in vitro,and identified.(2)Cell counting kit-8 was used to detect the optimal intervention concentration of Complanatoside A in the concentration range of 0-160 μmol/L.(3)Chondrocytes were divided into blank group,sodium nitroprusside(1.5 mmol/L)-induced group,and sodium nitroprusside(1.5 mmol/L)+Complanatoside A(5 μmol/L)group.The viability and apoptosis rate of the cells in each group were detected by cell counting kit-8 and flow cytometry.The expression of type Ⅱ collagen and SOX9 was detected by immunofluorescence staining.The expression of apoptosis-related proteins and Wnt/β-catenin pathway proteins was detected by western blot assay. RESULTS AND CONCLUSION:The cells extracted in vitro were cultured and stained,and were clearly identified as chondrocytes.Complanatoside A had no obvious cytotoxicity to chondrocytes in the concentration range of 0-80 μmol/L,and significantly improved the chondrocyte viability in the concentration range of 2.5-10 μmol/L,especially when the concentration was 5 μmol/L.The apoptotic rate of chondrocytes was higher in the sodium nitroprusside-induced group than the blank control group,while the apoptotic rate was lower in the sodium nitroprusside+Complanatoside A group than the sodium nitroprusside-induced group.The fluorescence intensity of type Ⅱ collagen and SOX9 in chondrocytes was weaker in the sodium nitroprusside-induced group than the blank control group,while the fluorescence intensity of type Ⅱ collagen and SOX9 in the sodium nitroprusside+Complanatoside A group was higher than that of the sodium nitroprusside-induced group.In the sodium nitroprusside-induced group,the protein expression of Bax,Caspase-3,matrix metalloproteinase 13,Wnt3a,Wnt5a and β-catenin was higher than that of the blank control group,while the protein expression of Bcl-2 was lower than that of the blank control group.In the sodium nitroprusside+Complanatoside A group,except for the protein expression of Bcl-2 which was higher than that of the sodium nitroprusside-induced group,the expression of the other aforementioned proteins was lower than that of the sodium nitroprusside-induced group.To conclude,Complanatoside A has a certain inhibitory effect on chondrocyte apoptosis,which could regulate apoptosis-related proteins and promote the expression of chondrocyte regulatory factors,and presumably might play a role through inhibiting the Wnt/β-catenin signaling pathway.

OBJECTIVE：To study the mechanism of enhancement effects of Astragalus complanatus polysaccharides（ACP） on the proliferation of meniscal fibrochondrocytes cells in rabbits. METHODS ：The meniscal fibrochondrocytes cells were isolated from 1-month-old New Zealand white rabbits. The meniscal fibrochondrocytes cells were divided into normal control group （PBS）， positive control group （glucosamine sulfate ，10 mg/mL）and ACP high-dose，medium-dose and low-dose groups （40，20，10 mg/mL）. The morphology of meniscal fibrochondrocytes cells were observed under microscope. Cell proliferation rate was detected by MTT assay. Cell cycle was observed with flow cytometry. ELISA assay was used to detect relative expression of medium collagen type Ⅱ（Col Ⅱ）and alkaline phosphatase protein （ALP）in meniscal fibrochondrocytes cells. RT-qPCR and Western blotting assay were adopted to detect mRNA and protein expression of transforming growth factor β 1 （TGF-β 1） and bone morphogenetic protein 2（BMP-2）. RESULTS ：After cultured for 72 h，meniscal fibrochondrocytes cells were fused into a single layer，and most of them were slender type in appearance. Compared with normal control group ，the proliferation rate of meniscal fibrochondrocytes cells and the percentage of cells at G 1/G0 phase were decreased significantly in positive control group and ACP high-dose，medium-dose and low-dose groups （P＜0.05）；the percentage of cells at S phase ，protein expression of Col Ⅱ and ALP，mRNA and protein expression of TGF-β1 and BMP- 2 were increased significantly （P＜0.05）. Compared with positive control group，inhibitory rate of meniscal fibrochondrocytes cells proliferation and the percentage of cells at G 1/G0 phase were decreased significantly in ACP high-dose group （P＜0.05），while the percentage of cells at S phase ，protein expression of Col Ⅱ and ALP ， mRNA and protein expression of TGF-β1 and BMP- 2 were increased significantly （P＜0.05）. The inhibitory proliferation rate of meniscal fibrochondrocytes cells and the percentage of cells at G 1/G0 phase were increased significantly in ACP low-dose group （P＜ 0.05），while the percentage of cells at S phase ，protein expression of Col Ⅱ and ALP ，mRNA and protein expression of TGF-β1 and BMP- 2 were decreased significantly （P＜0.05）. There was no statistical significance in above indexes of ACP medium-dose group. CONCLUSIONS ：ACP can promote the proliferation of meniscal fibrochondrocytes cells ，reduce the percentage of cells at G1/G0 phase，promote cell transformation to S phase ；the mechanism of which may be related to up-regulating TGF-β1，BMP-2 mRNA and protein expression ，promoting Col Ⅱ and ALP protein expression enhancement.