过表达脾酪氨酸激酶降低氧化应激诱导的小鼠成骨细胞凋亡

目的探究脾酪氨酸激酶(SYK)对氧化应激(OS)诱导的小鼠成骨细胞MC3T3-1凋亡的影响及其机制。方法过氧化氢(H2O2)孵育MC3T3-1细胞,建立OS模型;实时荧光定量PCR检测SYK的mRNA表达量;Western blot检测SYK、AKT、p-AKT和UCP2蛋白表达量;构建重组质粒pcDNA.3.1-SYK或pcDNA.3.1-UCP2,分别转染MC3T3-1;AKT抑制剂HIMO抑制AKT磷酸化;annexin-V FITC/PI法检测细胞凋亡;caspase-3荧光检测等试剂盒检测caspase-3活性和ROS水平。结果OS降低MC3T3-1中SYK的表达(P<0.01);过表达SYK降低OS诱导的细胞凋亡(P<0.05)、caspase-3活性(P<0.01)和ROS水平(P<0.01);过表达SYK提高AKT的磷酸化水平(P<0.05)和UCP2的表达量(P<0.01);抑制AKT磷酸化后可消除过表达SYK对细胞凋亡和UCP2的表达的影响(P<0.01)。在过表达SYK、抑制AKT的MC3T3-1细胞中,过表达UCP2,细胞凋亡(P<0.05)和ROS水平(P<0.01)均显著降低。结论过表达SYK可通过调控AKT/UCP2降低OS对小鼠成骨细胞的伤害。

Over-expression of spleen tyrosine kinase inhibits of apoptosis mouse osteoblasts induced by oxidative stress

Objective To investigate the protective effect of spleen tyrosine kinase(SYK)on oxidative stress(OS)-induced cell apoptosis in mouse osteoblasts MC3T3-1 and the mechanism.Methods Osteoblast MC3T3-1 was incubated with hydrogen peroxide(H 2O 2)to establish OS model;the mRNA expression of SYK was measured by RT-qPCR;the protein expression of SYK,AKT,p-AKT and UCP2 was detected by Western blot.Recombinant plasmids pcDNA.3.1-SYK and pcDNA.3.1-UCP2 were constructed and separately transfected into MC3T3-1;AKT phosphorylation was inhibited by AKT inhibitor HIMO;cell apoptosis was measured by annexin-V FITC/PI method;caspase-3 activity and reactive oxygen species(ROS)level were detected by kit methods.Results SYK was significantly decreased by OS in osteoblasts(P<0.01).The over-expression of SYK significantly down-regulated cell apoptosis(P<0.05),caspase-3 activity(P<0.01),ROS level(P<0.01)induced by OS.Over-expression of SYK obviously increased AKT phosphorylation(P<0.05)and UCP2 expression(P<0.01).The inhibi-tion of AKT phosphorylation could abolish the effects of SYK on cell apoptosis and UCP2 expression(P<0.01).UCP2 was over-expressed in MC3T3-1 with SYK over-expression and AKT inhibition,and the cell apoptosis(P<0.05)and ROS level(P<0.01)were both markedly decreased.Conclusions The over-expression of SYK can prevent osteoblasts from apoptosis induced by OS via regulating AKT/UCP2.