【摘要】 目的 探讨利伐沙班调控核因子E2相关因子2(Nrf2)信号通路和铁死亡在大鼠心肌缺血-再灌注损伤（MIRI）中的作用及可能机制。方法 雄性SPF级SD大鼠50只，体质量240～260g，随机分为假手术组、MIRI组、利伐沙班组、利伐沙班+ML385组、利伐沙班+ferrostatin-1组各10只。利伐沙班组每天灌胃20mg/kg利伐沙班，利伐沙班+ML385组每天灌胃20mg/kg利伐沙班并腹腔注射30mg/kg ML385，利伐沙班+ferrostatin-1组每天灌胃20mg/kg利伐沙班并腹腔注射5mg/kg ferrostatin-1,MIRI组、假手术组每天灌胃等体积生理盐水，连续1周后，MIRI组、利伐沙班组、利伐沙班+ML385组、利伐沙班+ferrostatin-1组构建MIRI模型，假手术组造模时用丝线穿过左前降支但不结扎。再灌注4h后，5组大鼠行超声心动图检查，记录左室射血分数（LVEF）、左心室缩短分数（FS）；采用ELISA法检测血清肌酸激酶同工酶（CK-MB）、心肌肌钙蛋白I(cTnI)、乳酸脱氢酶（LDH）水平；行心肌组织病理检查，采用HE染色观察心肌组织形态学改变，应用透射电子显微镜观察心肌组织线粒体形态；采用亚铁嗪比色法检测心肌组织铁水平，采用硫代巴比妥酸法检测心肌组织丙二醛（MDA）水平，采用黄嘌呤氧化酶法检测心肌组织超氧化物歧化酶（SOD）水平，采用二氯荧光素双醋酸盐法检测心肌组织活性氧（ROS）水平，采用Western blot法检测心肌组织Nrf2、长链酯酰辅酶A合成酶4(ACSL4)、谷胱甘肽过氧化物酶4(GPX4)、核受体共激活因子4(NCOA4)、铁蛋白重链1(FTH1)蛋白相对表达量。结果 5组LVEF、FS及血清CK-MB、cTnI、LDH水平比较差异均有统计学意义（F=79.471～114.613,P均<0.05）。假手术组、利伐沙班+ferrostatin-1组LVEF[(83.65±8.09)%、（75.87±7.09）%]、FS[(45.98±4.21)%、（39.02±3.31）%]均高于MIRI组[（42.67±4.21）%、（23.87±2.09）%]、利伐沙班组[（63.98±6.12）%、（31.35±2.78）%]、利伐沙班+ML385组[（48.90±4.32）%、（26.78±2.42）%](P<0.05)，血清CK-MB、cTnI、LDH水平均低于MIRI组、利伐沙班组、利伐沙班+ML385组（P<0.05）；利伐沙班组LVEF、FS均高于MIRI组、利伐沙班+ML385组（P<0.05），血清CK-MB、cTnI、LDH水平均低于MIRI组、利伐沙班+ML385组（P<0.05），以上指标假手术组与利伐沙班+ferrostatin-1组、MIRI组与利伐沙班+ML385组比较差异均无统计学意义（P>0.05）。假手术组心肌细胞排列规则，横纹清晰；MIRI组心肌纤维紊乱、肿胀，大量炎症细胞浸润；利伐沙班组心肌纤维紊乱、肿胀及炎症细胞浸润程度较MIRI组降低；利伐沙班+ML385组心肌组织病理表现介于MIRI组与利伐沙班组之间；利伐沙班+ferrostatin-1组心肌组织病理表现介于假手术组与利伐沙班组之间。假手术组线粒体形态正常；MIRI组线粒体结构紊乱，线粒体嵴密度较假手术组降低；利伐沙班组线粒体嵴密度较MIRI组增加；利伐沙班+ML385组线粒体形态介于MIRI组与利伐沙班组之间；利伐沙班+ferrostatin-1组线粒体形态介于假手术组与利伐沙班组之间。5组心肌组织铁、ROS、MDA、SOD水平及Nrf2、ACSL4、GPX4、NCOA4、FTH1蛋白相对表达量比较差异均有统计学意义（F=112.146～465.212,P均<0.05）；假手术组、利伐沙班+ferrostatin-1组、利伐沙班组、利伐沙班+ML385组、MIRI组心肌组织铁、ROS、MDA水平及ACSL4、NCOA4蛋白相对表达量均依次升高（P<0.05）,SOD水平及GPX4、FTH1蛋白相对表达量均依次降低（P<0.05）；利伐沙班+ferrostatin-1组、利伐沙班组、利伐沙班+ML385组、MIRI组、假手术组Nrf2蛋白相对表达量依次降低（P<0.05）。结论 利伐沙班可减轻大鼠MIRI，其机制可能与利伐沙班激活Nrf2信号通路、抑制铁死亡有关。更多还原

【Abstract】 Objective To explore the role and potential mechanism of rivaroxaban in regulating nuclear factor E2-related factor 2(Nrf2)signaling pathway and ferroptosis in rat myocardial ischemia-reperfusion injury(MIRI).Methods Fifty male SPF-grade SD rats,weighing 240 to 260g,were randomly assigned to five groups:the sham operation group,the MIRI group,the rivaroxaban group,the rivaroxaban+ ML385 group,and the rivaroxaban+ferrostatin-1 group,with10 rats in each group.The rivaroxaban group was given 20 mg/kg rivaroxaban by gavage daily,the rivaroxaban +ML385 group was given 20 mg/kg rivaroxaban by gavage and 30 mg/kg ML385 by intraperitoneal injection daily,the rivaroxaban + ferrostatin-1 group was given 20 mg/kg rivaroxaban by gavage and 5 mg/kg ferrostatin-1 by intraperitoneal injection daily,and the MIRI group and the sham operation group were given the same volume of normal saline by gavage daily.After one week of continuous administration,the MIRI models were established in the MIRI,rivaroxaban,rivaroxaban+ ML385,and rivaroxaban+ferrostatin-1 groups.The rats in the sham operation group only received threading a suture through the left anterior descending artery without ligation.Four hours after reperfusion,echocardiography examination was done to record left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(FS);the serum levels of creatine kinase isoenzyme(CK-MB),cardiac troponin I(cTnI)and lactic dehydrogenase(LDH)were detected with ELISA.Myocardial tissue was subjected to histopathological examination to observe the morphological changes using HE staining and to observe the morphology of mitochondria using transmission electron microscopy.Ferrozine colorimetric method was used to detect the iron level,thiobarbituric acid method was used to detect the malonaldehyde(MDA)level,xanthine oxidase method was used to detect the superoxide dismutase(SOD)level,and 2,7-dichlorofluorescein diacetate method was used to detect the reactive oxygen(ROS)level in myocardial tissue.Western blot was used to detect the relative expressions of Nrf2,long-chain acyl-CoA synthetase 4(ACSL4),glutathione peroxidase 4(GPX4),nuclear receptor coactivator 4(NCOA4),and ferritin heavy chain 1(FTH1)proteins.Results The comparisons of LVEF,FS,and serum levels of CK-MB,cTnI and LDH all showed statistically significant differences among the five groups(F=79.471-114.613,all P<0.05).The LVEF and FS were both higher in the sham operation group[(83.65±8.09)%,(45.98±4.21)%]and the rivaroxaban+ferrostatin-1 group [(75.87±7.09)%,(39.02±3.31)%]than those in the MIRI group [(42.67±4.21)%,(23.87±2.09)%],the rivaroxaban group[(63.98±6.12)%,(31.35±2.78)%],and the rivaroxaban+ML385 group [(48.90±4.32)%,(26.78±2.42)%](P<0.05),while the serum levels of serum CK-MB,cTnI,and LDH were all lower(P<0.05).The LVEF and FS were higher in the rivaroxaban group than those in the MIRI group and the rivaroxaban+ML385 group(P<0.05),while the serum levels of CK-MB,cTnI,and LDH were all lower(P<0.05).There were no statistically significant differences in the above indicators between the sham operation group and the rivaroxaban + ferrostatin-1 group,and between the MIRI group and the rivaroxaban+ ML385 group(P>0.05).The myocardial cells were arranged regularly and the striations were clear in the sham operation group.The myocardial fibers were disordered and swollen,with a large number of inflammatory cells infiltrating in the MIRI group,which decreased in degree in the rivaroxaban group.The myocardial tissue pathology in the rivaroxaban+ ML385 group was intermediate between the MIRI group and the rivaroxaban group,and the myocardial tissue pathology in the rivaroxaban+ferrostatin-1 group was intermediate between the sham operation group and the rivaroxaban group.The mitochondrial morphology was normal in the sham operation group;the mitochondrial structure was disordered,and the density of mitochondrial cristae decreased in the MIRI group compared with the sham operation group;the density of mitochondrial cristae was higher in the rivaroxaban group than that in the MIRI group;the mitochondrial morphology in the rivaroxaban+ML385 group was intermediate between the MIRI group and the rivaroxaban group;the mitochondrial morphology in the rivaroxaban+ferrostatin-1 group was intermediate between the sham operation group and the rivaroxaban group.There were statistically significant differences in the levels of iron,ROS,MDA and SOD,as well as the relative expressions of Nrf2,ACSL4,GPX4,NCOA4 and FTH1 proteins among the five groups(F=112.146-465.212,all P<0.05).The levels of iron,ROS and MDA as well as the relative expressions of ACSL4 and NCOA4 proteins increased successively in the sham operation group,the rivaroxaban+ferrostatin-1 group,the rivaroxaban group,the rivaroxaban+ ML385 group and the MIRI group(P<0.05),while the level of SOD and the relative expressions of GPX4 and FTH1 proteins decreased successively(P<0.05).The relative expression of Nrf2 protein decreased successively in the rivaroxaban+ferrostatin-1 group,the rivaroxaban group,the rivaroxaban+ ML385 group,the MIRI group,and the sham operation group(P<0.05).Conclusion Rivaroxaban can alleviate MIRI in rats,which may be correlated with the activation of the Nrf2 signaling pathway and the inhibition of ferroptosis by rivaroxaban.更多还原