铁死亡在脂多糖诱导巨噬细胞损伤小鼠模型中的增强作用

Enhancement of ferroptosis induced by lipopolysaccharide in a mouse model of mitochondrial injury of macrophage

目的评价铁死亡在脂多糖(LPS)诱导小鼠巨噬细胞线粒体损伤模型中的作用。方法常规培养小鼠RAW264.7细胞,按随机数字表法将培养的细胞分为4组(n=6):对照组(Con组)、脂多糖组(LPS组)、脂多糖+铁死亡激动剂Erastin组(LPS+E组)和脂多糖+铁死亡抑素Ferrostatin-1组(LPS+F组)。LPS组给予终浓度为1μg/ml的LPS进行孵育6 h;LPS+E组给予终浓度为1μg/ml的LPS和浓度为1 mmol/L的含Erastin培养液进行孵育6 h;LPS+F组给予终浓度为1μg/ml的LPS和浓度为1 mmol/L的含Ferrostatin-1培养液进行孵育6 h。采用Clark氧电极法及JC-1法分别测定线粒体呼吸控制率(RCR)及线粒体膜电位(MMP),透射电镜下观察细胞内线粒体改变情况,采用Western blot法测定细胞内铁死亡相关蛋白谷胱甘肽过氧化物酶4(GPX4)、长链酯酰辅酶A合成酶4(ACSL4)的表达,采用荧光探针法测定活性氧族(ROS)和Fe^(2+)含量,采用硫代巴比妥酸(TBA)法测定丙二醛(MDA)含量。结果与Con组比较,LPS组RCR和MMP均降低(P均<0.05),GPX4表达下调(P<0.05),ACSL4表达上调(P<0.05);线粒体出现线粒体膜增厚、线粒体皱缩、线粒体嵴消失等铁死亡表现,细胞内Fe^(2+)含量升高(P<0.05),ROS含量升高(P<0.05),MDA含量升高(P<0.05)。与LPS组比较,LPS+E组RCR和MMP降低(P<0.05),GPX4表达下调(P<0.05),ACSL4表达上调(P<0.05),Fe^(2+)含量、ROS含量和MDA含量均升高(P均<0.05)。LPS+F组RCR和MMP升高(P<0.05),GPX4表达上调(P<0.05),ACSL4表达下调(P<0.05),出现铁死亡变化的线粒体增加(P<0.05),Fe^(2+)含量、ROS含量和MDA含量均降低(P均<0.05)。结论铁死亡增强LPS致小鼠巨噬细胞线粒体损伤。

Objective To evaluate the role of ferroptosis induced by lipopolysaccharide(LPS)in mitochondrial model of mouse macrophages.Methods RAW264.7 cells were routinely cultured in mice,and the cultured cells were randomly divided into 4 groups(n=6):control group(Con group),lipopolysaccharide group(LPS group),lipopolysaccharide+iron death excitement Erastin group(LPS+E group)and lipopolysaccharide+Ferrostatin-1 group(LPS+F group).LPS group was given LPS with a final concentration of 1μg/ml for 6 h.LPS+E group was given LPS with a final concentration of 1μg/ml and 1 mmol/L of Erastin-containing culture medium for 6 h.LPS+F group was given LPS with a final concentration of 1μg/ml and Ferrostatin-1 nutrient solution with a concentration of 1 mmol/L for 6 h.The mitochondrial respiratory control rate(RCR)and mitochondrial membrane potential(MMP)were measured by Clark oxygen electrode method and JC-1 method respectively,and the changes of intracellular mitochondria were observed under transmission electron microscope.To determine the expression of glutathione peroxidase 4(GPX4)and long-chain acyl-CoA synthetase 4(ACSL4)in cells,iron death-related proteins fluorescence probe method for the determination of ROS and Fe^(2+),and thiobarbituric acid(TBA)method for the determination of malondialdehyde(MDA).Results Compared with the Con group,RCR and MMP in the LPS group were all reduced(P<0.05),GPX4 expression was down-regulated(P<0.05),ACSL4 expression was up-regulated(P<0.05).Mitochondria shrank,mitochondrial membranes thickened,and mitochondria crista shrank as the expression of ferroptosis.And the intracellular Fe^(2+)content increased(P<0.05),ROS content and MDA content both increased(both P<0.05).Compared with LPS group,RCR and MMP in LCR+E group decreased(P<0.05),GPX4 expression was down-regulated(P<0.05),ACSL4 expression was up-regulated(P<0.05),Fe^(2+)content,ROS content and MDA content all increased(all P<0.05);RCR and MMP in LPS+F group increased(P<0.05),GPX4 expression was up-regulated(P<0.05),ACSL4 expression was down-regulated(P<0.05),and mitochondria with ferroptosis increased(P<0.05),Fe^(2+)content,ROS content,and MDA content decreased(all P<0.05).Conclusion Ferroptosis enhanced LPS-induced mitochondrial damage in mouse macrophages.