星形胶质细胞来源的GJA1-20k在氧化应激后参与神经元保护作用的机制

Protective effects and mechanism of astrocytic GJA1-20k on neurons in oxidative stress injury

目的:观察星形胶质细胞(astrocyte)通过上调神经元(neuron)内源性缝隙连接蛋白α1截短单体-20k(gap junction protein alpha 1 truncated monomer-20k,GJA1-20k)参与氧化应激损伤后神经保护作用的机制。方法:采用C57BL/6胎鼠以原代培养法获取神经元及星形胶质细胞,建立共培养模型,给予神经元过氧化氢(H2O2)损伤,及星形胶质细胞胰岛素样生长因子1(insulin-like growth factor-1,IGF-1)受体阻滞剂AG1024,分别设立Neuron组、Neuron+Stress组、Neuron+Astrocyte+Stress组及Neuron+Astrocyte+Stress+AG1024组,通过Western blot测定神经元GJA1-20k、去磷酸化(non-phosphorylated,NP)-Cx43的表达,谷氨酸转运酶(glutamate transporter-1,GLT-1)、线粒体功能相关蛋白(PGC-1α、mtTFA、Tom20、CoxⅣ)、凋亡相关蛋白(Bcl-2、Bax、Caspases-9)的变化,采用酶联免疫荧光分析(ELFA)及ELISA法分别检测氧化应激因子NAPDH氧化酶活性和白介素(interleukin,IL)-1β、IL-6、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)等炎性因子含量的变化,采用Annexin V-FITC/PI测定神经元凋亡。结果:星形胶质细胞共培养可明显上调在神经元氧化损伤后内源性GJA1-20k和NP-Cx43表达,抑制PGC-1α、mtTFA、Tom20、CoxⅣ的下调,上调凋亡抑制因子Bcl-2表达,下调凋亡促进因子Bax、Caspase-9表达,降低NAPDH氧化酶活性,降低炎性产物IL-1β、IL-6和TNF-α的水平及抑制神经元的凋亡(P<0.05)。在给予AG1024后,可明显抑制与以上因素相关的星形胶质细胞对神经元的保护作用(P<0.05)。结论:与IGF-1相关的星形胶质细胞对神经元的保护机制可能与增加神经元内源性GJA1-20k的含量及线粒体功能的保护有关。

Objective:To observe the neuroprotective mechanism of astrocytes after oxidative stress injury by up-regulating endogenous gap junction protein alpha 1 truncated monomer-20 k(GJA1-20 k)of neurons.Methods:Neurons and astrocytes were obtained from C57 BL/6 fetal mice by primary culture method,and co-culture model was established.Neurons were injured by hydrogen peroxide(H2 O2),insulin-like growth factor-1(IGF-1)receptor blocker AG1024 was given to astrocytes,respectively.Thus,Neuron+Astrocyte+Stress group and Neuron+Astrocyte+Stress+AG1024 group were established.Meanwhile,Neuron(Neuron alone without treatment)group and Neuron+Stress group(separately cultured Neuron given with H2 O2)were also set up as control groups.The changes of GJA1-20 k,non-phosphorylated(NP)-Cx43,glutamate transporter-1(GLT-1),mitochondrial function-related proteins(PGC-1α,mtTFA,Tom20,CoxⅣ),apoptosis-related proteins(Bcl-2,Bax,Caspases-9)were measured by Western blot.The oxidative stress factor NAPDH oxidase activity,inflammatory factors interleukin(IL)-1β,IL-6 and tumor necrosis factor-α(TNF-α)were detected by ELFA and ELISA,apoptosis of neurons was measured by Annexin V-FITC/PI assay,respectively.Results:Astrocytes co-culture significantly up-regulated the expression of endogenous GJA1-20 k and NP-Cx43,reversed the down-regulation of PGC-1α,mtTFA,Tom20 and CoxⅣ,up-regulated apoptotic inhibitor Bcl-2,down-regulated apoptotic promoter Bax,Caspase-9,and reduced the expression of NAPDH oxidase activity and inflammatory products IL-1β,IL-6 and TNF-α(P<0.05).The protective effect of astrocytes on neurons was significantly inhibited by AG1024(P<0.05).Conclusion:The protective mechanism of astrocyte on neuron associated with IGF-1 may be related to the increase of endogenous GJA1-20 k and protection of mitochondria function in neurons.