鸢尾素上调Akt/Nrf2抑制铁死亡减轻阿霉素诱导的心肌损伤

Irisin Upregulates Akt/Nrf2 to Inhibit Ferroptosis Alleviating Doxorubicin-induced Myocardial Injury

目的探讨鸢尾素对阿霉素(doxorubicin,DOX)诱导的心脏损伤的作用及其机制。方法选取C57BL/6J雄性小鼠30只,将其随机分为空白组、DOX组和鸢尾素治疗组,每组各10只。每5天腹腔注射1次DOX(5mg/kg),总计20mg/kg诱导心肌损伤;空白组给予等比例0.9%氯化钠溶液;鸢尾素治疗组提前1周每天腹腔注射鸢尾素[1mg/(kg·d)],之后DOX处理的同时注射鸢尾素(1mg/kg)。实验过程中记录小鼠生存率,存活的小鼠在第21天检测心功能,计算心脏重量与胫骨长度比值。酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测血浆肌酸激酶同工酶(creatine kinase isoenzyme,CK-MB)、乳酸脱氢酶(lactate dehydrogenase,LDH)和肌钙蛋白(cardiac troponin,cTnT)水平;苏木精-伊红(hematoxylin-eosin,HE)染色观察心肌结构;Masson染色观察心肌纤维化;试剂盒检测心肌丙二醛(malondialdehyde,MDA)、还原型谷胱甘肽(reduced glutathione,GSH)/氧化型谷胱甘肽(oxidized glutathione,GSSG)含量及超氧化物歧化酶(superoxide dismutase,SOD)活性;实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)检测心脏炎性细胞因子白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)及肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)表达;Western blot法检测蛋白激酶B(Akt)及其磷酸化水平、核转录相关因子2(nuclear transcription-related factor 2,Nrf2)、谷胱甘肽氧化酶4(glutathione oxidase 4,GPx4)及酰基辅酶A合成酶长链家族成员4(acyl-CoA synthetase long chain family member 4,ACSL4)水平。结果DOX组小鼠生存率降低,左心室射血分数和短轴缩短率明显低于空白组,左心室舒张末期容积增大,心脏重量与胫骨长度比值下降,血浆CK-MB、LDH和cTnT上升,并伴组织水肿、炎性浸润及纤维增生。同时,DOX组心脏MDA和GSSG含量高于空白组,GSH水平和SOD活性则低于空白组,伴随高水平炎症和氧化应激。鸢尾素治疗组小鼠生存率提高,心脏损伤和功能障碍减轻,氧化应激和炎性损伤减轻并伴Akt磷酸化水平上升,Nrf2及GPx4表达上调,ACSL4表达抑制。结论鸢尾素上调Akt/Nrf2抑制铁死亡,减轻DOX诱导的心脏炎症和氧化应激,改善心脏损伤。

Objective To investigate the role of irisin on doxorubicin(DOX)-induced cardiac injury and its mechanism.Methods Thirty male C57BL/6J mice were selected and randomly divided into blank group,DOX group and irisin treatment group,with 10mice in each group.Myocardial injury was induced by intraperitoneal injection of DOX(5mg/kg)every five days,with a total of 20mg/kg;the blank group was given an equal proportion of normal saline;the irisin treatment group was injected intraperitoneally with irisin[1mg/(kg·d)]every day for one week in advance,and irisin(1mg/kg)was injected at the same time as the DOX treatment.The survival rate was recorded during the experiment,and the surviving mice were tested for cardiac function on the twenty-first day,and the ratio of heart weight to tibia length was calculated.Enzyme-linked immunosorbent assay(ELISA)was used to detect the plasma creatine kinase isoenzyme(CK-MB),lactate dehydrogenase(LDH),and cardiac troponin(cTnT)levels;hematoxylin-eosin(HE)staining was used to observe the myocardial structure;Masson staining was used to observe the myocardial fibrosis;and kit was used to detect myocardial malondialdehyde(MDA),reduced glutathione/oxidized glutathione(GSH/GSSG)levels and superoxide dismutase(SOD)activity;real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect cardiac inflammatory factors interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)expression;Western blot was used to detect protein kinase B(Akt)and its phosphorylation(p-Akt)level,nuclear transcription-related factor 2(Nrf2)and glutathione oxidase 4(GPx4)and acyl-CoA synthetase long chain family member 4(ACSL4)levels.Results The survival rate of DOX group was decreased,left ventricular ejection fraction and short-axis shortening rate were significantly lower than those in the blank group,left ventricular end-diastolic volume was increased,the ratio of heart weight to tibia length was decreased,and the plasma CK-MB,LDH,and cTnT were increased,accompanied by tissue edema,inflammatory infiltration,and fibroplasia.Meanwhile,cardiac MDA and GSSG contents were higher in the DOX group than those in the blank group,while the GSH levels and SOD activity were lower than those in the blank group,accompanied by high levels of inflammation and oxidative stress.In the irisin treatment group,the survival rate was increased,cardiac injury and dysfunction were alleviated,oxidative stress and inflammatory injury were reduced,accompanied by an increase in the level of p-Akt,the expression of Nrf2 and GPx4 was up-regulated,and the expression of ACSL4 was inhibited.Conclusion Irisin up-regulates Akt/Nrf2,inhibits ferroptosis,alleviates DOX-induced cardiac inflammation and oxidative stress,and improves cardiac injury.