两面神激酶2/信号传导及转录激活因子3信号通路在地塞米松诱导成骨细胞MC3T3-E1凋亡中的作用

Role of Janus kinase 2/signal transducer and activator of transcription 3 signal transduction pathway in dexamethasone induced osteoblasts MC3T3-E1 apoptosis

目的探讨两面神激酶2/信号传导及转录激活因子3(JAK2/STAT3)信号通路在地塞米松(DEX)诱导成骨细胞MC3T3-E1凋亡中作用机制。方法将成骨细胞MC3T3-E1分为3组,分别为对照组、DEX组、DEX+AG490组。对照组只加入正常培养基,DEX组加入200μmol/L的DEX培养,DEX+AG490组预先给予50μmol/L的AG490处理后加入200μmol/L的DEX培养。采用细胞计数试剂盒(CCK-8)法检测细胞活力,流式细胞术检测细胞凋亡率,蛋白印迹法检测通路蛋白及凋亡蛋白的表达。组间比较采用t检验。结果对照组、DEX组、DEX+12.5、25、50、75μmol/L的AG490的细胞存活率分别为(97.74±1.45)%、(52.05±5.50)%、(54.98±3.77)%、(70.99±4.15)%、(81.53±6.43)%、(79.16±7.35)%。表明DEX明显使成骨细胞MC3T3-E1活力降低(t=11.364,P<0.001),50μmol/L JAK2/STAT3通路抑制剂(AG490)明显抑制了DEX对成骨细胞MC3T3-E1活力的降低作用(t=4.928,P<0.01)。对照组、DEX组、DEX+AG490组的细胞凋亡率分别为(6.067±0.545)%、(26.233±2.631)%和(8.463±1.179)%。表明DEX明显诱导成骨细胞MC3T3-E1凋亡(t=12.999,P<0.001),经AG490处理后,DEX诱导的成骨细胞MC3T3-E1凋亡明显受到抑制(t=10.675,P<0.001)。蛋白质印迹法(Western blot)检测正常组、DEX组、DEX+AG490组p-JAK2蛋白表达分别为1.000±0.323、2.839±0.640、0.286±0.068,p-STAT3蛋白表达分别为1.000±0.245、3.471±0.157、0.618±0.078,bax蛋白表达分别为1.000±0.083、6.571±0.405、3.048±0.905,bcl-2蛋白表达分别为1.000±0.086、0.207±0.040、0.563±0.083,cleaved Caspase-3蛋白表达分别为1.000±0.192、5.685±0.699、3.411±0.247。DEX组较对照组p-JAK2、p-STAT3、cleaved Caspase-3、bax的表达明显增加,bcl-2的表达明显减少(t=4.429、14.912、11.100、23.561、14.565,P<0.05),AG490+DEX组较DEX组p-JAK2、p-STAT3、cleaved Caspase-3、bax的表达明显减少,bcl-2的表达明显增加(t=6.860、28.404、5.262、6.185、6.721,P<0.01)。结论DEX通过JAK2/STAT3信号通路,调节cleaved Caspase-3、bax、bcl-2的表达,诱导成骨细胞MC3T3-E1凋亡。

Objective To investigate the role of Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)signal transduction pathway in dexamethasone induced osteoblasts MC3T3-E1 apoptosis.Methods The osteoblasts MC3T3-E1 were divided into three groups:control group,DEX group and DEX+AG490 group.The control group was treated with routine medium.DEX group was treated with 200μmol/L DEX to culture the cells.DEX+AG490 group was treated with 50μmol/L AG490 in advance and then treated with 200μmol/L DEX to culture the cells.Cell counting kit-8(CCK-8)assay was used to detect the cell viability.The apoptosis of osteoblasts was detected by flow cytometry.Western blotting was used to detect the protein levels of pathways related genes and apoptosis related genes.Univariate anova was used to compare the mean values.T test was used for comparison between groups.Results The cell viability of control group,DEX group and DEX+12.5,25,50,75μmol/L AG490 group were(97.74±1.45)%,(52.05±5.50)%,(54.98±3.77)%,(70.99±4.15)%,(81.53±6.43)%and(79.16±7.35)%respectively.These results showed that DEX significantly decreased the cell viability of osteoblasts MC3T3-E1(t=11.364,P<0.001).50μmol/L JAK2/STAT3 pathway inhibitor(AG490)significantly inhibited the decreased cell viability of osteoblasts MC3T3-E1 induced by DEX(t=4.928,P<0.01).The apoptosis rates of the control group,DEX group and DEX+AG490 group were(6.067±0.545)%,(26.233±2.631)%and(8.463±1.179)%respectively.These results showed that DEX significantly induced apoptosis of osteoblasts MC3T3-E1(t=12.999,P<0.001).After treated with AG490,the apoptosis of osteoblasts MC3T3-E1 induced by DEX was significantly inhibited(t=10.675,P<0.001).Western blotting showed the expression of p-JAK2 protein in control group,DEX group and DEX+AG490 group was 1.000±0.323,2.839±0.640 and 0.286±0.068,that of p-STAT3 protein was 1.000±0.245,3.471±0.157,0.618±0.078,that of bax protein was 1.000±0.083,6.571±0.405,3.048±0.905,that of bcl-2 protein was 1.000±0.086,0.207±0.040,0.563±0.083,and that of cleaved Caspase-3 protein was 1.000±0.192,5.685±0.699,3.411±0.247.Compared with control group,the expression of p-Jak2,p-STAT3,cleaved Caspase-3,and bax were significantly increased,and that of bcl-2 was significantly decreased in DEX group(t=4.429,14.912,11.100,23.561,14.565,P<0.05).Compared with DEX group,the expression of p-JAK2,p-STAT3,cleaved Caspase-3,and bax were significantly decreased,and that of bcl-2 significantly was increased in AG490+DEX group(t=6.860,28.404,5.262,6.185,6.721,P<0.01).Conclusion DEX regulate cleaved Caspase-3,bax,and bcl-2 expression through JAK2/STAT3 signal transduction pathway,inducing apoptosis of osteoblasts MC3T3-E1.