脑震宁含药血清对Erastin诱导HT22细胞铁死亡的影响

Effects of Naozhenning Medicated Serum on Ferroptosis of HT22 Induced by Erastin

目的观察脑震宁含药血清对Erastin诱导的小鼠海马神经元细胞HT22铁死亡的影响,基于JNK信号通路探讨其作用机制。方法采用Erastin诱导HT22细胞铁死亡模型,将细胞分为空白组、模型组、Fer-1组、1%脑震宁含药血清组、5%脑震宁含药血清组、10%脑震宁含药血清组、抑制剂+1%脑震宁含药血清组、抑制剂+5%脑震宁含药血清组、抑制剂+10%脑震宁含药血清组,分别加入相应药物培养。TBA法检测细胞丙二醛(MDA)含量,WST-1法检测细胞超氧化物歧化酶(SOD)活性,DCFH-DA荧光探针检测细胞活性氧(ROS)含量,比色法检测细胞内铁离子含量,RT-PCR检测细胞谷胱甘肽过氧化物酶4(GPX4)、铁蛋白重链(FTH)、铁蛋白轻链(FTL)、c-Jun氨基末端激酶(JNK)mRNA表达,Westernblot检测细胞GPX4、FTH、FTL、p-JNK、JNK蛋白表达。结果与空白组比较,模型组细胞MDA、ROS含量显著增加,SOD活性显著降低,Fe^(2+)含量显著增加,GPX4、FTH、FTL、JNK mRNA和蛋白表达显著升高,p-JNK蛋白表达显著升高(P<0.01);与模型组比较,Fer-1组、抑制剂+5%脑震宁含药血清组、抑制剂+10%脑震宁含药血清组细胞MDA、ROS含量显著减少,SOD活性显著升高,Fe^(2+)含量显著减少(P<0.01),GPX4、FTH、FTL、JNK mRNA和蛋白表达显著降低,p-JNK蛋白表达显著降低(P<0.05)。结论脑震宁含药血清可能通过抑制JNK信号通路激活,降低HT22细胞氧化应激水平,减少细胞内Fe^(2+)含量,改善Erastin诱导的细胞铁死亡。

Objective To observe the effects of Naozhenning medicated serum on the ferroptosis of HT22 cells induced by Erastin;To explore the mechanism of action based on the JNK signaling pathway.Methods The ferroptosis of HT22 cells was induced by Erastin and divided into blank group,model group,Fer-1 group,1%Naozhenning medicated serum group,5%Naozhenning medicated serum group,10%Naozhenning medicated serum group,inhibitor+1%Naozhenning medicated serum group,inhibitor+5%Naozhenning medicated serum group,and inhibitor+10%Naozhenning medicated serum group.The content of MDA was detected by TBA method,the activity of SOD was detected by WST-1 method,the concentration of ROS was detected by DCFH-DA fluorescence probe,the content of ferric ion in cells was detected by colorimetry,the mRNA expressions of GPX4,FTH,FTL and JNK were detected by RT-PCR,the expressions of GPX4,FTH,FTL,p-JNK and JNK protein in cells were detected by Western blot.Results Compared with the blank group,the contents of MDA and ROS significantly increased in the model group,the activity of SOD significantly decreased,the content of Fe^(2+)significantly increased,the mRNA and protein expressions of GPX4,FTH,FTL,and JNK significantly increased,and the protein expression of p-JNK significantly increased(P<0.01).Compared with the model group,the contents of MDA and ROS in Fer-1 group,inhibitor+5%Naozhenning medicated serum group and inhibitor+10%Naozhenning medicated serum group significantly reduced,the activity of SOD significantly increased,the content of Fe^(2+)significantly reduced(P<0.01),the mRNA and protein expressions of GPX4,FTH,FTL,JNK significantly decreased,and the expression of p-JNK protein significantly decreased(P<0.05).Conclusion Naozhenning medicated serum can reduce the level of oxidative stress of HT22 cells,reduce the content of Fe^(2+)in cells and improve the ferroptosis of HT22 cells induced by erastin by inhibiting the activation of JNK signaling pathway.