鹿红方调控Nrf2因子保护缺血/再灌注损伤心肌细胞的机制研究  

The Protective Effect of Luhong Recipe on Ischemia/Reperfusion Injury of Myocardial Cells Regulated by Nrf2

作　　者：蔡婉[1] 葛文[1] 王婧 周华[1] 池浩[1] CAI Wan;GE Wen;WANG Jing;ZHOU Hua;CHI Hao(Shuguang Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200120,China)

机构地区：[1]上海中医药大学附属曙光医院,上海200120

出　　处：《中西医结合心脑血管病杂志》2022年第23期4257-4264,共8页Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease

基　　金：国家自然科学基金资助项目(No.8210151929)。

摘　　要：目的探究鹿红方防治心肌缺血/再灌注损伤的机制。方法选取无特定病原体(SPF)级Wistar雄性大鼠40只,随机分为对照组、模型组、鹿红方低剂量组、鹿红方中剂量组、鹿红方高剂量组,根据鹿红方不同的剂量,连续灌胃7 d后,结扎左前降支30 min后复灌24 h制备心肌缺血再灌注损伤模型。将H9c2心肌细胞分为对照组、模型组、鹿红方低剂量组、鹿红方中剂量组、鹿红方高剂量组。鹿红方低剂量组、鹿红方中剂量组、鹿红方高剂量组分别加入6%、12%、18%鹿红方含药血清预处理,经液体石蜡封闭12 h后复氧2 h制备心肌缺氧/复氧模型。造模成功后取材,观察心肌梗死面积,苏木精-伊红(HE)染色观察心肌组织形态,检测心肌组织中血清超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽(GSH)含量,DNA断裂的原位末端标记(TUNEL)染色观察心肌细胞凋亡,蛋白免疫印迹法(Western Blot)检测心脏组织中Kelch样环氧氯丙烷相关蛋白-1(Keap-1)及核因子相关因子-2(Nrf2)蛋白含量。流式细胞观察H9c2心肌细胞凋亡率,检测心肌细胞及培养基中SOD、MDA、LDH含量,Western Blot检测心肌细胞中Keap-1及Nrf2蛋白含量。结果与对照组比较,模型组心肌梗死面积较大,心肌细胞凋亡率较高,心肌细胞出现水肿、坏死的程度较重,MDA浓度明显升高,GSH和SOD含量明显下降(P<0.05),鹿红方低、中、高剂量组可以明显改善上述情况,以中、高剂量组最优。与对照组比较,模型组H9c2心肌细胞凋亡率较高,心肌细胞培养基上清液中LDH含量明显升高,心肌细胞中MDA浓度升高,SOD含量明显降低(P<0.05),而鹿红方低、中、高剂量可以有效改善上述指标,以中、高剂量组效果最优。在大鼠心肌缺血再灌注模型及H9c2心肌细胞缺氧/复氧模型中,与对照组比较,模型组Nrf2蛋白表达下降,鹿红方可以上调Nrf2蛋白表达,以中、高浓度效果最明显。此外,与对照组比较,�Objective To explore the mechanism of Luhong recipe in preventing and treating myocardial ischemia/reperfusion injury.Methods Forty male Wistar rats without specific pathogen(SPF)grade were randomly divided into control group,model group,Luhong recipe low dose group,medium dose,high dose group.According to different doses of Luhong recipe,the myocardial ischemia-reperfusion injury model was prepared by continuous intragastric administration for 7 days,followed by ligation of the left anterior ligation for 30 minutes and reperfusion for 24 h.H9c2 cardiomyocytes were divided into control group,model group,Luhong recipe low dose,medium dose,and high dose group.Luhong recipe low dose group,medium dose group and high dose group were pretreated with 6%,12%and 18%Luhong recipe medicated serum,respectively to prepare myocardial hypoxia/reoxygenation model.After successful modeling,the sample was taken and the myocardial infarction area was observed by Hematoxylin-eosin(HE)staining.The contents of serum superoxide dismutase(SOD),malondialdehyde(MDA)and glutathione(GSH)in myocardial tissue were detected.The apoptosis of cardiomyocytes was observed by TUNEL staining of DNA breaks.The protein contents of Keap-1 and Nrf2 in heart tissue were determined by Western Blot.The apoptosis rate of H9c2 cardiomyocytes were observed by flow cytometry.The contents of SOD,MDA and LDH in cardiomyocytes and culture medium were detected.The protein contents of Keap-1 and Nrf2 in cardiomyocytes were detected by Western Blot.Results Compared with the control group,the myocardial infarction area of the model group was larger,the myocardial cell apoptosis was higher,the degree of myocardial cell edema and necrosis were more serious,the concentration of MDA was significantly increased,and the contents of GSH and SOD were significantly decreased(P<0.05).The low,medium and high dose groups of Luhong recipe could significantly improve the above indexes,and that in the medium and high dose groups were the best.Compared with that in the control group

关 键 词：心肌缺血再灌注损伤 鹿红方 氧化应激 Keap-1/Nrf2/ARE信号通路 铁蓄积 

分 类 号：R285.5[医药卫生—中药学]