与缝隙连接43蛋白相关的星形胶质细胞通过外泌体对神经元保护作用的机制研究

目的:观察缝隙连接43(Cx43)蛋白参与星形胶质细胞来源的外泌体对神经元保护作用的机制.方法:采用C57BL/6胎鼠以原代培养法获取星形胶质细胞及神经元,建立共培养(Co-Culture)模型,给予神经元过氧化氢(H2O2)损伤,星形胶质细胞给予Cx43特异性阻断剂Gap26,分别建立(Co-Culture+H2O2)组及(Co-Culture+H2O2+Gap26)组,同时设单纯神经元损伤(Neuron+H2O2)组,通过蛋白免疫印记(WB)法,测定神经元骨架相关的紧密连接蛋白(ZO-1)、α-肌动蛋白(α-actin)、谷氨酸转运蛋白-1(GLT-1)、外泌体标记蛋白CD63和凋亡相关的Bax/Bcl-2比例的变化,采用高效液相色谱仪(HPLC)观察神经元谷氨酸(Glu),采用ELFA法检测氧化应激因子腺嘌呤二核苷酸磷酸(NAPDH)氧化酶活性.结果:与(Neuron+H2O2)组相比,(Co-Culture+H2O2)组的神经元ZO-1、α-actin、GLT-1和CD63的表达明显上调,而Bax/Bcl-2比例、NAPDH氧化酶活性则明显降低(P<0.05).在给予Gap26后,可明显抑制以上星形胶质细胞对神经元的此种作用(P<0.05).结论:Cx43蛋白可促进星形胶质细胞的外泌体被神经元吸收,可产生稳定细胞形态结构、加强兴奋性氨基酸转运及降低神经元凋亡和氧化应激损伤等神经保护功能.

Involvement Cx43 in the Protective Effects and Mechanism of Astrocytic Exosome on Neurons

Objective:To investigate Cx43 protein join in the protective effects and mechanism of exosme derived from astrocytes to neurons against oxidative stress injury.Methods:Primary neurons and astrocytes were procured from C57BL/6 pregnant rats and clutured in Neurobasal or DMEM-F12.Co-culture model was established with Transwell.H₂O₂ was added to neuronal medium,and astrocytes were treated with Gap26,a kind of Cx43 inhibitor.Co-culture+H₂O₂ and Co-culture+H₂O₂+Gap26 groups were established,Neuron without co-culture(Neuron)and was set as control group.ZO-1、α-actin,GLT-1,CD63、Bcl-2 and Bax in neurons were measured by western blot.Glu and NAPDH oxidase activity were assayed by HLPC and ELFA,respectively.Results:ZO-1、α-actin,GLT-1,CD63 were upregulated by co-culture with astrocytes,compared with Neuron+H₂O₂ group.Anti-apoptosis factor Bcl-2 was increased,and apoptosis promoter Bax,was reduced after cocultured with astrocytes.Glu density and NAPDH oxidase activity after co-culture were lower than that in Neuron+H₂O₂ group.Simultaneously,the above effects were blocked by Cx43 special inhibitor Gap26.Conclusion:CX43 can promote the absorption of astrocyte by neurons,stabilize cell morphology,enhance excitatory amino acid transport,and reduce neuronal apoptosis and oxidative stress injury.