Effects of Bushen Huoxue Formula on chondrocyte apoptosis in rats with osteoarthritis based on JNK pathway
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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