ATRA通过抑制铁死亡缓解脂多糖诱导的小鼠急性心肌损伤

ATRA alleviates lipopolysaccharide-induced acute myocardial injury in mice by inhibiting Ferroptosis

目的研究全反式维甲酸(ATRA)对脂多糖(LPS)诱导C57BL/6小鼠急性心肌损伤的影响及机制。方法20只C57BL/6雄性小鼠随机均分成正常组、模型组、ATRA组、ferrostatin-1组。ATRA组和ferrostatin-1组小鼠分别腹腔注射ATRA[15 mg/kg·d]、ferrostatin-1[2 mg/(kg·d)],正常组和模型组给予溶剂,持续给药一周后,模型组、ATRA组、ferrostatin-1组腹腔注射LPS(6 mg/kg),6 h后处死所有小鼠。检测小鼠血清中丙二醛(MDA)、还原型谷胱甘肽(GSH)含量;qPCR检测心脏组织IL-6、TNF-α的mRNA水平;苏木精-伊红(HE)染色观察小鼠心脏组织变化;透射电子显微镜(TEM)观察小鼠心肌线粒体的结构;蛋白质印迹法(Western blot)检测铁死亡标志物[谷胱甘肽过氧化酶4(GPX4)、铁蛋白重链1(FTH1)、溶质载体家族7成员11(SLC7A11)、长链脂酰辅酶A合成酶4(ACSL4)]以及相关调控蛋白[核因子E2相关因子2(NRF2)、kelch样ECH关联蛋白1(KEAP1)]的表达。结果与正常组小鼠相比,模型组小鼠血清中MDA含量上升、GSH含量下降、还原型谷胱甘肽和氧化型谷胱甘肽的比值(GSH/GSSG)下降,ATRA组和ferrostatin-1组逆转以上变化;与正常组小鼠相比,模型组小鼠心脏组织IL-6、TNF-α的mRNA水平增高,与模型组相比ATRA组和ferrostatin-1组mRNA水平降低;各组小鼠心肌组织HE染色无明显差异;TEM显示与正常组相比,模型组心肌线粒体出现嵴减少或消失的现象,与模型组相比,ATRA组和ferrostatin-1组心肌线粒体嵴数量增多;Western blot显示,与正常组相比,模型组小鼠心肌组织中GPX4、FTH1、SLC7A11、NRF2蛋白表达减少,ACSL4、KEAP1蛋白表达增加,ATRA组、ferrostatin-1组逆转以上变化。结论ATRA通过抑制铁死亡缓解脂多糖引起的小鼠急性心肌损伤。

Objective To investigate the effect and mechanism of all-trans retinoic acid(ATRA)on lipopolysaccharide(LPS)-induced acute myocardial injury in C57BL/6 mice.Methods Male mice of C57BL/6 strain were randomly divided into normal group,model group,ATRA group,and ferrostatin-1 group.Mice in the ATRA group were injected intraperitoneally with ATRA 15mg/(kg·d),ferrostatin-1 group received ferrostatin-12 mg/(kg·d),the normal group and the model group were given solvent.After one week of continuous administration,the model group,ATRA group,and ferrostatin-1 group were intraperitoneally injected with LPS 6 mg/kg.All mice were sacrificed after 6 hours.The contents of malondialdehyde(MDA)and glutathione(GSH)in serum of mice were detected.qPCR was used to detect mRNA levels of interleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-α)in heart tissue.Hematoxylin-eosin(HE)staining was used to observe the changes of heart tissue in mice.Transmission electron microscopy(TEM)was used to observe the structure of mouse myocardial mitochondria.Western blot was used to detect the expression of ferroptosis markers glutathione peroxidase 4(GPX4),ferritin heavy chain 1(FTH1),Solute carrier family 7 member 11(SLC7A11),acyl-CoA synthetase long-chain family member 4(ACSL4)and related regulatory proteins,Nuclear factor erythroid 2-related factor 2(NRF2),kelch-like ECH-associated protein 1(KEAP1).Results Compared with the normal group,the MDA content in the serum of the model group increased and the GSH content decreased,the above changes were reversed in the ATRA group as well as in the ferrostatin-1 group.Compared with normal group,the mRNA levels of IL-6 and TNF-αin the heart tissue of model group increased steeply,the above changes were relieved in the ATRA group and the ferrostatin-1 group.There was no significant difference in HE staining of myocardial tissue among the groups of mice.Compared with the normal group,myocardial mitochondria in the model group showed the phenomenon of cristae reduction or disappearance under TEM,while myocardial mitochondrial injury was alleviated in the ATRA group and the ferrostatin-1 group.Western blot showed that GPX4,FTH1,SLC7A11,and NRF2 expression were reduced in the myocardial tissue of mice in the model group compared with the normal group,ACSL4 and KEAP1 expression increased.The above changes were reversed in the ATRA group as well as in the ferrostatin-1 group.Conclusion ATRA alleviates lipopolysaccharide-induced acute myocardial injury in mice by inhibiting ferroptosis.