Objective To investigate the effects of Bushen Huoxue Formula(BSHXF)on chondrocyte apoptosis in rats with osteoarthritis(OA)through the c-Jun N-terminal kinase(JNK)signaling pathway.Methods An in vitro OA model was constructed using interleukin-1 β(IL-1β)to induce apoptosis in rat chondrocytes cultured ex vivo.Different concentrations of BSHXF were used for intervention.Cell viability was determined using MTT assay;CCK-8 assay was used to examine cell proliferation ability;flow cytometry was employed to assess cell apoptosis;Western blot was used to check the levels of apoptosis-related proteins,including cleaved cysteine protease-3(Cleaved Caspase-3),B-cell lymphoma 2(Bcl-2),and Bcl-2 associated X protein(Bax),as well as the levels of JNK and phosphorylated c-Jun N-terminal kinase(p-JNK)proteins.The reagent kit was used to measure the levels of reactive oxygen species(ROS),malondialdehyde(MDA),and glutathione(GSH)in cells;ELISA was used to check the content of pro-inflammatory factor of tumor necrosis factor-α(TNF-α)and IL-6 in the cell supernatant.Results(1)Compared with the control group,the model group showed a significant decrease in chondrocyte viability and a significant increase in cell apoptosis rate(P＜0.01);the protein expression levels of Cleaved Caspase-3,Bax,and p-JNK in the model group significantly increased(P＜0.01),while the expression of Bcl-2 significantly decreased(P＜0.01).Compared with the model group,the BSHXF treatment group showed an increase in chondrocyte viability(P＜0.05)and a significant decrease in apoptosis rate(P＜0.01);the protein expression levels of Cleaved Caspase-3,Bax,and p-JNK were significantly reduced(P＜0.01)and the protein expression level of Bcl-2 increased(P＜0.01)in the BSHXF treatment group.(2)Compared with the control group,the relative content of ROS and MDA in the model group cells significantly increased(P＜0.01),while the relative content of GSH significantly decreased(P＜0.01);the content of TNF-α and IL-6 in the model group cells significantly increased(P＜0.01).Compared with the model group,the BSHXF treatment group showed a significant decrease in the relative content of ROS and MDA(P＜0.01)and a significant increase in the relative content of GSH(P＜0.01);the content of TNF-α and IL-6 in cells treated with BSHXF was significantly reduced(P＜0.01).(3)Compared with the solvent control group,the activator control group showed a significant upregulation of p-JNK,Cleaved Caspase-3,and Bax protein levels(P＜0.05),a significant reduction in Bcl-2 protein expression(P＜0.01),a decrease in cell proliferation ability(P＜0.05),and a significant increase in cell apoptosis rate(P＜0.01);there were no significant differences in cell proliferation,apoptosis rate,and expressions of apoptosis-related proteins(Cleaved Caspase-3,Bax,and Bcl-2)between the BSHXF treatment group and the solvent control group(P＞0.05).(4)Compared with the solvent control group,the activator control group exhibited a significant increase in ROS and MDA levels(P＜0.01)and a significant decrease in GSH level(P＜0.01),with increased content of TNF-α and IL-6(P＜0.05);there were no significant differences in the relative content of ROS and the levels of MDA and GSH between the BSHXF treatment group and the solvent control group(P＞0.05).Conclusion BSHXF inhibits IL-1β-induced apoptosis in OA chondrocytes by suppressing the activation of JNK signaling pathway,thereby relieving oxidative stress and inflammatory responses.
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