过表达Homer1a对神经元机械性损伤模型的保护作用及可能机制

目的:探讨过表达Homer1a对神经元机械性损伤模型细胞凋亡和AMP活化蛋白激酶(AMPK)表达的影响。方法:分离培养大鼠大脑皮层神经元,随机分组为对照组、模型组、空载组和Homer1a过表达(Exp-Homer1a)组。构建神经元机械性损伤模型,转染Homer1a过表达载体。采用MTT法检测各组细胞活力,qPCR检测各组细胞Homer1a的mRNA表达水平,使用乳酸脱氢酶(LDH)测试盒测定各组细胞上清液LDH活性,流式细胞术检测各组细胞凋亡水平,Western blot检测各组Hormer1a、cleaved caspase-3、Bax、Bcl-2、p-AMPKα和AMPKα蛋白的表达。结果:与对照组比较,机械性损伤神经元的活力显著降低,上清液LDH活性和神经元凋亡率显著增加(P<0.05),且Homer1a mRNA和蛋白表达水平显著升高(P<0.05);与模型组比较,Exp-Homer1a组的上清液LDH活性和神经元凋亡率显著降低,神经元中cleaved caspase-3和Bax的表达水平显著降低(P<0.05),Bcl-2和p-AMPKα的表达水平显著升高(P<0.05)。结论:过表达Homer1a可能通过促进激活AMPKα磷酸化及抑制神经元凋亡来提高机械性损伤神经元的存活率。

Effects of Homer1a over-expression on apoptosis and AMPK protein expression in mechanically injured neurons

AIM: To investigate the effects of Homer1 a over-expression on the apoptosis and AMP-activated protein kinase(AMPK) protein expression in mechanically injured neurons. METHODS: The rat cortical neurons were isolated and cultured in vitro, and then ramdomly divided into control group, model group, empty vector group, and Exp-Homer1 a group. Neuron models with mechanical injury were constructed and infected with the Homer1 a over-expression vector. The mRNA expression of Homer1 a was detected by qPCR. The cell viability in each group was detected by MTT assay. The activity of lactate dehydrogenase(LDH) in the supernatant of each group was measured by LDH test kit. The apoptosis level was analyzed by flow cytometry. The protein levels of Hormer1 a, cleaved caspase-3, Bax, Bcl-2, p-AMPKα and AMPKα were determined by Western blot. RESULTS: Compared with control group, the viability of mechanically injured neurons was significantly decreased, the LDH activity in the supernatant and neuronal apoptotic rate were significantly increased(P<0.05), and Homer1 a expression at mRNA and protein levels was significantly increased(P<0.05). Compared with model group, the LDH activity in the supernatant and neuronal apoptotic rate in Exp-Homer1 a group were significantly decreased, the protein levels of cleaved caspase-3 and Bax were significantly decreased(P<0.05), and the protein levels of Bcl-2 and p-AMPKα were significantly increased(P<0.05). CONCLUSION: Over-expression of Homer1 a may increase the viability of mechanically injured neurons and inhibit their apoptosis by promoting the activation of AMPKα phosphorylation.