基于JNK通路探讨补肾活血方对大鼠骨关节炎软骨细胞凋亡的影响

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

目的 探讨补肾活血方(Bushen Huoxue Formula,BSHXF)通过c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)信号通路对大鼠骨关节炎(Osteoarthritis,OA)软骨细胞凋亡的影响。方法 采用白细胞介素-1β (interleukin-1β,IL-1β)诱导体外分离培养的大鼠软骨细胞构建体外OA模型,并使用不同浓度的BSHXF干预处理。使用MTT法测定细胞活力;CCK-8法检测细胞增殖能力;流式细胞术评估细胞凋亡情况;Western bolt检测凋亡相关蛋白裂解的胱天蛋白酶-3(Cleaved Caspase-3)、B淋巴细胞瘤-2(B-cell lymphoma 2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)水平和JNK、磷酸化c-Jun氨基末端激酶(phosphorylated c-Jun N-terminal kinase,p-JNK)蛋白水平。试剂盒检测细胞中活性氧(reactive oxygen species,ROS)、丙二醛(malondialdehyde,MDA)、谷胱甘肽(Glutathione,GSH)水平;ELISA法检测细胞上清液中促炎因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)含量。结果 (1)与对照组相比,模型组软骨细胞活力显著降低,细胞凋亡率显著升高(P<0.01);模型组Cleaved Caspase-3、Bax、p-JNK蛋白表达水平显著升高(P<0.01),Bcl-2蛋白表达水平显著降低(P<0.01)。与模型组相比,BSHXF治疗组软骨细胞活力升高(P<0.05),凋亡率显著降低(P<0.01);BSHXF治疗组Cleaved Caspase-3、Bax、p-JNK蛋白表达水平显著降低(P<0.01),Bcl-2蛋白表达水平升高(P<0.01)。(2)与对照组相比,模型组细胞中ROS和MDA的相对含量显著升高(P<0.01),GSH的相对含量显著降低(P<0.01);模型组细胞中TNF-α和IL-6的含量均显著升高(P<0.01)。与模型组相比,BSHXF治疗组细胞中ROS和MDA的相对含量显著降低(P<0.01),GSH的相对含量显著升高(P<0.01);BSHXF治疗组细胞中TNF-α和IL-6的含量均显著降低(P<0.01)。(3)与溶剂对照组相比,激活剂对照组的p-JNK、Cleaved Caspase-3、Bax蛋白水平显著上调(P<0.05),Bcl-2蛋白表达水平显著降低(P<0.01),细胞增殖能力下降(P<0.05),细胞凋亡率显著增加(P<0.01);BSHXF治疗组和溶剂对照组两组间细胞增殖、凋亡率和凋亡相关蛋白表达(Cleaved Caspase-3、Bax、Bcl-2)情况差异均无统计学意义(P>0.05)。(4)与溶剂对照组相比,激活剂对照组的ROS和MDA水平显著升高(P<0.01),而GSH水平显著降低(P<0.01),TNF-α、IL-6含量升高(P<0.05);BSHXF治疗组和溶剂对照组两组间ROS相对含量、MDA、GSH水平差异均无统计学意义(P>0.05)。结论 BSHXF通过抑制JNK信号通路激活抑制IL-1β诱导的OA软骨细胞凋亡,改善氧化应激和炎症反应。

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-1β(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 proinflammatory 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.