Janus kinase 2/signal transducer and activators of transcription 3 signaling mediate mechanical apoptosis of chondrocytes
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 in-jury group.After intervention,Annexin V-fluoresceine isothiocyanate(FITC)/propidium iodide(PI)doub-le staining was performed to determine the prevalence of apoptotic cells.The 4',6-diamidino-2-phenylin-dole(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 apop-tosis 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 con-trol 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+mechani-cal 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.
ChondrocytesMechanical injuryApoptosisJanus kinase 2Signal trans-ducer and activators of transcription 3Nuclear factor-κB p65