肝细胞生长因子预处理对过氧化氢诱导大鼠神经干细胞凋亡的保护作用

Protective effect of hepatocyte growth factor pre-treatment on rat neural stem cell apoptosis induced by hydrogen peroxide

目的研究肝细胞生长因子(HGF)对神经干细胞(NSC)凋亡的保护作用及其作用机制,为HGF用于NSC移植提供实验基础。方法分离培养大鼠NSC。细胞分为正常对照、模型(H2O2100μmo.lL-1)、HGF+H2O2(HGF15,30及60μg.L-1预处理24h后,再加入H2O2100μmol.L-1处理4h),LY294002(PI3K/Akt通路抑制剂)+HGF+H2O2(先加入LY29400220μmol.L-1处理30min,再加入HGF60μg.L-1处理24h,最后再加入100μmo.lL-1H2O2培养4h)组。MTT法检测细胞存活率;TUNEL法检测细胞凋亡率;比色法检测半胱氨酸天冬氨酸蛋白酶(caspase)-3活性;Western印迹分析Bcl-2,Bax蛋白表达。结果MTT检测发现,随着HGF浓度的增加,NSC细胞的存活率也增加。与模型组〔(63.5±2.4)%〕比较,HGF15,30及60μg.L-1预处理组细胞存活率明显升高〔(79.1±7.5)%,(83.8±6.1)%和(86.6±8.2)%;n=3,P<0.05〕。TUNEL法检测发现,HGF预处理组凋亡细胞明显减少,模型组的凋亡率为(43.5±6.2)%,HGF预处理组则分别为(34.2±8.6)%,(21.7±3.8)%及(19.4±4.0)%。Caspase-3活性检测表明,与模型组相比,HGF预处理组细胞caspase-3活性降低。Western印迹分析结果显示,与模型组比较,HGF预处理使细胞的Bcl-2蛋白表达升高,但Bax蛋白表达不受影响;HGF的抗凋亡效应可被PI3K/Akt通道阻滞剂LY294002阻断。结论HGF可减轻H2O2所诱导的大鼠NSC凋亡,且呈一定的浓度依赖关系,其作用机制可能与NSC的PI3K/Akt通路激活和Bcl-2表达增强有关。

AIM To investigate the protective effect of hepatocyte growth factor（HGF） pre-treatment on rat neural stem cells（NSC） apoptosis induced by hydrogen peroxide（H2O2） in culture,and to provide the experimental data for NSC transplantation.METHODS Cultured NSC isolated from Sprague-Dawley rats were randomly divided into 6 groups：normal control,model group（H2O2 100 μmol·L^-1）,HGF＋H2O2 groups（cells were preconditioned with HGF 15,30,and 60 μg·L^-1 for 24 h,and then exposed to 100 μmol·L^-1 H2O2 for 4 h,respectively）,LY294002〔1-phosphatidylinositol-3 kinase/preotein kinase B（PI3K/Akt） pathway inhibitor〕＋HGF＋H2O2 group（cells were pretreated with LY294002 20 μmol·L^-1 for 30 min,then exposed to HGF 15,30 and 60 μg·L^-1 for 24 h followed by 100 μmol·L^-1 H2O2 treatment for 4 h,respectively）.The cell viability of NSC was measured by MTT assay.Assay for neural apoptosis was performed by using TUNEL staining,and caspase-3 activity assay,respectively.The expressions of Bcl-2 and Bax proteins were determined by Western blotting analysis.RESULTS The cell viability of NSC was improved with the increasing concentration of HGF.Compared with model group [（63.5±2.4） %],15,30,and 60 μg·L^-1 HGF significantly increased the cell viabilities [（79.1±7.5）%,（83.8±6.1）%,and（86.6±8.2）%,n=3,P〈0.05].The results of TUNEL staining revealed that HGF pretreatment could significantly reduce the apoptotic rate of NSC.The apoptotic rate of model group was（43.5±6.2）%,while those of HGF＋H2O2 groups were（34.2±8.6） %,（21.7±3.8）% and（19.4±4.0）%,respectively.The data from caspase-3 activity assay indicated that HGF precondition could also remarkably decrease the caspase-3 activity for NSC.In addition,HGF could increase the expression level of Bcl-2 protein,whereas the expression of Bax protein was not affected.Moreover,the effect of HGF could be blocked by a PI3K/Akt pathway inhibitor,LY294002.CONCLUSION HGF has the protective effect on rat NSC apoptosis induced by H2O2,which may be related to activation of PI3K/Akt pathway,and thus enhancement of the expression of Bcl-2 protein.