Nrf2参与CaMKII抑制引起的神经细胞毒性作用

Nrf2 participates in neurocytotoxicity caused by CaMKII inhibition

目的探讨Ca^(2+)/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKII)的特异性抑制剂KN93对神经细胞的作用及其与Nrf2通路之间的关系。方法培养小鼠脑神经瘤细胞N2a,CCK8检测确定给药12h后KN93对细胞的毒性作用与浓度的相关性;应用5μmolKN93给药12h后进行荧光NeuN染色,检测细胞存活率;应用ELISA试剂盒检测KN93对炎症因子TNF-α、IL-1β表达的影响;Westernblot检测给药KN93后Nrf2蛋白表达的变化;ELISA试剂盒检测KN93与Nrf2拮抗剂ML385同时处理时,炎症相关因子TNF-α、IL-1β表达的变化。结果与对照组相比,5μmol KN93给药12h后,KN93给药组中NeuN染色的阳性细胞明显减少;TNF-α和IL-1β表达明显增加;Nrf2蛋白表达显著增加;与KN93给药组相比,KN93和Nrf2拮抗剂ML385同时处理组炎症相关蛋白表达下降,即Nrf2的抑制剂ML385能逆转KN93诱发的炎症反应。结论Nrf2通过诱发炎性反应,参与CaMKII的抑制剂KN93引起的神经细胞毒性作用。

Objective To investigate the effect of Ca^(2+)/calmodulin-dependent protein kinaseⅡ(CaMKII)specific inhibitor KN93 on nerve cells and its relationship with Nrf2 pathway.Methods Mouse brain neuroma cell N2a was cultured,and the correlation between the toxicity of KN93 on cells and the concentration after 12 h was determined by CCK8 detection;5μmol KN93 was applied for 12 h after administration,and the survival rate of cells was detected by fluorescent NeuN staining;the effect of KN93 on the expression of inflammatory factors TNF-αand IL-1βwas detected by ELISA kit;the change of Nrf2 protein expression after administration of KN93 was detected by Western blot;the change of inflammation-related factors TNF-αand IL-1βexpression was detected by ELISA kit when KN93 and Nrf2 antagonist ML385 were treated at the same time.Results Compared with the control group,5μmol KN93 was applied for 12 h,and the positive cells of NeuN staining in the KN93 group were significantly reduced;the expression of TNF-αand IL-1βwas significantly increased;the expression of Nrf2 protein was significantly increased;compared with the KN93 group,the expression of inflammation-related proteins in the KN93 and Nrf2 antagonist ML385 simultaneous treatment group was decreased,that is,the inhibitor of Nrf2 ML385 could reverse the inflammatory response induced by KN93.Conclusion Nrf2 is involved in theneurocytotoxicity caused by CaMKII inhibitor KN93 by inducing inflammatory response.