磷酸化PKC-δ对地塞米松诱导大鼠成骨细胞凋亡的影响

Effect of phosphorylated PKC-δ on Dexamethasone-induced osteoblast apoptosis of rats

目的观察磷酸化PKC-δ对地塞米松(Dex)诱导的大鼠成骨细胞凋亡的影响。方法取新生SD大鼠颅骨第3代成骨细胞,分为对照组、Dex组(1×10-7mol/L Dex)、PMA预处理组(1×10-7mol/L Dex+100 nmol/L PMA)、Rottlerin预处理组(1×10-7mol/L Dex+2μmol/L Rottlerin),培养24 h后采用MTT法检测细胞增殖活性,采用Annexin-V-FITC/PI染色流式细胞术和吖啶橙/溴乙锭(AO/EB)荧光染色法检测细胞凋亡,采用Western blotting法检测凋亡相关蛋白Bcl-2、Bax、Caspase-3及磷酸化PKC-δ。结果与对照组相比,Dex组细胞增殖活性低,细胞凋亡多,磷酸化PKC-δ、Caspase-3、Bax表达高,Bcl-2表达低(P均<0.05)。与Dex组相比,PMA预处理组细胞增殖活性低,细胞凋亡多,磷酸化PKC-δ、Caspase-3、Bax表达高,Bcl-2表达低(P均<0.05)。与Dex组相比,Rottlerin预处理组细胞增殖活性高,细胞凋亡少,磷酸化PKC-δ、Caspase-3、Bax表达低,Bcl-2表达高(P均<0.05)。结论磷酸化PKC-δ可促进Dex诱导的大鼠成骨细胞凋亡。

Objective To observe the effect of phosphorylated protein kinase C-δ（ PKC-δ） on Dexamethasone（ Dex）-induced osteoblast apoptosis of rats. Methods The osteoblasts of the third generation from newly born SD rats＇ calvaria were divided into four groups： the control group,Dex group（ 1 × 10-7mol / L Dex）,PMA preconditioning group（ 1 ×10-7mol / L Dex ＋ 100 nmol / L PMA） and Rottlerin preconditioning group（ 1 × 10-7mol / L Dex ＋ 2 μmol / L Rottlerin）.The cells of each group were incubated for 24 h. Then,the cell proliferation activity was measured by MTT assay,the apoptosis was detected by Annexin Ⅴ-FITC / propidium iodide（ PI）-flow cytometry（ FCM） and acridine oringe / ethidium bromide（ AO / EB） staining,and the expression of the apoptosis-related proteins（ Bcl-2,Bax and Caspase-3） and phosphorylated PKC-δ was detected by Western blotting. Results Compared with the control group,the proliferation activity was reduced,the apoptosis was increased,the expression of phosphorylated PKC-δ,Caspase-3 and Bax was increased,while the Bcl-2 expression was decreased in the Dex group,and the difference was statistically significant（ all P 0. 05）. Compared with the Dex group,the cell proliferation activity was reduced,the apoptosis was increased,the expression of phosphorylated PKC-δ,Caspase-3 and Bax was increased,while the Bcl-2 expression was decreased in the PMA preconditioning group,and the difference was statistically significant（ all P 0. 05）. Compared with the Dex group,the cell proliferation activity was obviously higher,the apoptosis was decreased,the expression of phosphorylated PKC-δ,Caspase-3 and Bax was decreased,while the Bcl-2 expression was increased in the Rottlerin preconditioning group,and the difference was statistically significant（ all P 0. 05）. Conclusion The phosphorylated PKC-δ can promote the Dex-induced osteoblast apoptosis of rats.