酪氨酸激酶2/信号转导与转录激活因子3通路介导机械性软骨细胞凋亡

Janus kinase 2/signal transducer and activators of transcription 3 signaling mediate mechanical apoptosis of chondrocytes

目的探索酪氨酸激酶2(JAK2)/信号转导与转录激活因子3(STAT3)信号通路在机械性软骨细胞凋亡过程中的作用机制。方法使用大鼠软骨细胞,分为对照组和机械性损伤组,干预后行膜联蛋白V(Annexin V)-异硫氰酸荧光素(FITC)/碘化丙锭(PI)双染色以确定细胞凋亡程度,4’,6-二脒基-2-苯基吲哚(DAPI)染色现实细胞核形态,蛋白质印迹法(Western blot)检测半胱氨酰天冬氨酸特异性蛋白酶-3(Caspase-3)蛋白表达,以及JAK2蛋白、STAT3蛋白的磷酸化/非磷酸化激活比例。第二项实验中,将软骨细胞分组为机械性损伤组,以及机械性损伤+AG490组,分别检测软骨细胞凋亡比例、实时定量反转录聚合酶链反应(RT-qPCR)检测B细胞淋巴瘤/白血病-2(bcl-2)/bcl-2相关X蛋白(bax)基因的表达比值,Western blot检测Caspase-3蛋白表达和核因子-κB(NF-κB)p65磷酸化激活比例,组间比较采用非配对t检验。结果体外培养的软骨细胞凋亡检测中,机械性损伤组的凋亡比例高于对照组[(37.5±7.9)%比(8.4±6.8)%,t=4.815,P<0.05],DAPI染色证实细胞核凋亡性改变,同时,机械性损伤组的Caspase-3蛋白表达高于对照组(倍数为14.2±7.3,t=8.390,P<0.05)。同时,机械性损伤组的JAK2蛋白及STAT3蛋白的磷酸化激活比例均高于对照组(倍数分别为2.2±1.1和1.6±0.4,t=2.886、4.652,P<0.05)。第二项实验中,机械性损伤+AG490组的凋亡比例减低显著低于机械性损伤组[减低至(11.9±3.2)%,t=5.144,P<0.05]。机械性损伤组的bcl-2/bax的基因表达比值高于对照组(比值为0.31±0.07,t=2.789,P<0.05)和机械性损伤+AG490组(比值为0.61±0.12,t=3.690,P<0.05)。机械性损伤+AG490组的Caspase-3蛋白低于机械性损伤组[变化倍数为(1.7±1.2)倍,t=8.273,P<0.05]。此时,机械性损伤组的NF-κB p65蛋白的磷酸化比例高于对照组[变化倍数为(6.7±1.4)倍,t=5.098,P<0.05]和机械性损伤+AG490组[变化倍数为(3.3±0.2)倍,t=2.875,P<0.05]。结论JAK2/STAT3信号通路介导了机械性软骨细胞凋亡,JAK2特异性抑制剂AG490可通过抑制NF-κB p65活化调控凋亡反应。

Objective To explore the mechanism of janus kinase 2(JAK2)/signal transducer and activators of transcription 3(STAT3)signaling in the mechanical regulation of chondrocyte apoptosis.Methods Rat chondrocytes were used.They were firstly divided into a control group and a mechanical injury group.After intervention,Annexin V-fluoresceine isothiocyanate(FITC)/propidium iodide(PI)double staining was performed to determine the prevalence of apoptotic cells.The 4′,6-diamidino-2-phenylindole(DAPI)staining was used to reveal the nuclear morphology.Western blotting was used to detect the expression of apoptotic protein-3(Caspase-3)protein,as well as the phosphorylation/non-phosphorylation activation ratio of JAK2 protein and STAT3 protein.Secondly,chondrocytes were divided into control group,mechanical injury group and mechanical injury+AG490 group.The frequency of chondrocyte apoptosis was measured,and the ratio of B cell lymphoma/leukemia-2(bcl-2)/bcl-2 associated X protein(bax)gene was detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR).The expression of Caspase-3 protein and phosphorylation of nuclear factor-κB(NF-κB)p65 was detected by Western blotting.Unpaired t test was used for statistical analysis.Results The apoptosis results from chondrocytes in vitro showed that the apoptosis in mechanical injury group was greater than control group[(37.5±7.9)%vs.(8.4±6.8)%,t=4.815,P<0.05].DAPI staining confirmed changes in nuclear apoptosis,while Caspase-3 protein in mechanical injury group was significantly higher than control group(fold changes of 14.2±7.3,t=8.390,P<0.05).Meanwhile,the phosphorylation activation ratio of JAK2 protein and STAT3 protein in mechanical injury group was increased as compared with control group(fold changes of 2.2±1.1 and 1.6±0.4,t=8.390,t=2.886,P<0.05;t=4.652,P<0.05).In the second experiment,the proportion of apoptosis from AG490+mechanical injury group was significantly lower than the mechanical injury group[frequency decreased to(11.9±3.2)%,t=5.144,P<0.05].The gene expression ratio of bcl-2/bax in the mechanical injury group was significantly higher than the control group(ratio of 0.31±0.07,t=2.789,P<0.05)and the mechanical injury+AG490 group(ratio of 0.61±0.12,t=3.690,P<0.05)as well.The expression of Caspase-3 protein from AG490+mechanical injury group was significantly lower than mechanical injury group(fold changes of 1.7±1.2,t=8.273,P<0.05).At this point,the phosphorylation ratio in protein expression as the crucial factor in the p65 NF-κB singling was higher in the control group(fold changes of 6.7±1.4,t=5.098,P<0.05)and the mechanical injury+AG490 group(fold changes of 3.3±0.2,t=2.875,P<0.05).Conclusion JAK2/STAT3 signaling pathway mediated mechanical chondrocyte apoptosis.The JAK2 specific inhibitor AG490 inhibited the activation of NF-κB p65 thus regulating cellular apoptosis.