右美托咪定通过调控Nrf2减轻HT22细胞缺氧/复氧引起的铁死亡

Dexmedetomidine attenuates ferroptosis through regulation of Nrf2 on HT22 cells with hypoxia/reoxygenation

目的基于铁死亡探讨右美托咪定(dexmedetomidine,DEX)对HT22细胞缺氧/复氧(hypoxia/reoxygenation,H/R)损伤的保护作用及机制。方法采用HT22细胞制备H/R损伤模型。为了探究DEX对细胞H/R损伤的保护作用的最适浓度,HT22细胞分为5组:对照组(Control)、缺氧复氧组(H/R)、低浓度DEX组(H/R+DEX_(2.5),2.5μmol·L^(-1))、中浓度DEX组(H/R+DEX_(5),5μmol·L^(-1))、高浓度DEX组(H/R+DEX_(10),10μmol·L^(-1)),MTT法检测细胞的存活率。为了探讨DEX对HT22细胞H/R保护作用机制,将HT22细胞分为4组:Control组、H/R组、H/R+DEX_(5)组、H/R+DEX_(5)+ML385(Nrf2抑制剂)组。MTT法检测细胞的存活率;使用FerroOrange荧光探针检测细胞内Fe^(2+)水平的变化;C11 BODIPY 581/591检测细胞中Lipid ROS的变化;使用丙二醛和还原型谷胱甘肽试剂盒检测细胞中MDA及GSH含量。蛋白免疫印记法检测细胞中核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、转铁蛋白受体1(transferrin receptor 1,TFR1)和胱氨酸/谷氨酸逆向转运蛋白溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11)的表达。结果与Control组相比,H/R损伤后细胞的存活率、GSH含量、Nrf2、GPX4和SLC7A11蛋白表达明显降低(均P<0.05),Lipid ROS、MDA、Fe^(2+)含量和TFR1蛋白表达明显升高(均P<0.01);与H/R组相比,低、中、高浓度DEX均可使H/R损伤后细胞的存活率、GSH含量、Nrf2、GPX4和SLC7A11蛋白表达明显升高(均P<0.05),Lipid ROS、MDA、Fe^(2+)含量和TFR1蛋白表达明显降低(均P<0.05);在DEX的基础上使用ML385后,与H/R+DEX 5组相比,细胞存活率、GSH的含量、Nrf2、GPX4和SLC7A11蛋白表达明显降低(均P<0.05),Lipid ROS、MDA、Fe^(2+)含量和TFR1蛋白表达明显升高(均P<0.05)。结论DEX可通过抑制铁死亡减轻HT22细胞H/R损伤,这种保护作用可能与其促进Nrf2的表达有关。

Aim To investigate the effects of Dexmedetomidine(DEX)on HT22 cells with hypoxia/reoxygenation based on ferroptosis and the underlying mechanism.Methods HT22 cells were used to prepare H/R injury model.In order to investigate the optimal concentration of DEX,cells were divided into five groups:control group,H/R group,low concentration(H/R+DEX_(2.5),2.5μmol·L^(-1)),medium concentration(H/R+DEX_(5),5μmol·L^(-1)),high concentration(H/R+DEX_(10),10μmol·L^(-1))DEX intervention H/R group,and the survival rates of cells were detected by MTT assay.To investigate the mechanism of the protective effects on HT22 cells with H/R injury,HT22 cells were divided into four groups:control group,H/R group,H/R+DEX_(5) group,and H/R+DEX_(5)+ML385 group.The survival rates of cells were detected by MTT assay;the levels of Fe^(2+)were detected by FerroOrange fluorescent probe;the C11BODIPY581/591 was used to detect the change of lipid ROS on the cells;MDA and reduced glutathione kits were used to detect the content of MDA and GSH of cells respectively.The expressions of Nrf2,GPX4,TFR1 and SLC7A11 were detected by Western blot.Results Compared with control group,the survival rate of cells,the content of GSH,and the expression of Nrf2,GPX4 and SLC7A11 all significantly decreased(all P<0.05),the level of lipid ROS,the content of MDA,the level of Fe^(2+),and the expression of TFR1 all significantly increased in H/R group(all P<0.01).Compared with H/R group,the survival rate of cells,the content of GSH,and the expression of Nrf2,GPX4 and SLC7A11 significantly increased(all P<0.05),the level of lipid ROS,the content of MDA,the level of Fe^(2+),and the expression of TFR1 significantly decreased with the treatment of DEX(all P<0.05).Compared with H/R+DEX group,the survival rate of cells,the content of GSH,and the expression of Nrf2,GPX4 and SLC7A11 markedly decreased(all P<0.05),the lipid ROS,MDA and Fe^(2+),and the expression of TFR1 significantly increased in H/R+DEX+ML385 group(all P<0.05).Conclusions DEX can reduce H/R injury on HT22 cells by inhibiting ferroptosis,and the mechanism might be related to the promotive expression of Nrf2.