摘要目的: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1 μg/ml的LPS进行孵育6 h；LPS+E组给予终浓度为1 μg/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1 μg/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。

abstractsObjective: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 1 μg/ml for 6 h. LPS+E group was given LPS with a final concentration of 1 μg/ml and 1 mmol/L of Erastin-containing culture medium for 6 h. LPS+F group was given LPS with a final concentration of 1 μg/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.