异莲心碱促进Nrf2/HO-1通路减轻缺氧/复氧致心肌细胞凋亡的机制研究  

Mechanism of isoliensinine in reducing myocardial cell apoptosis induced by hypoxia/reoxygenation by promoting Nrf2/HO 1 signaling pathway

作　　者：张翔 张博方[2] ZHANG Xiang;ZHANG Bofang(Department of Pharmacy,Jingzhou Central Hospital,Hubei,Jingzhou 434020,China;不详)

机构地区：[1]湖北省荆州市中心医院药学部,434020 [2]武汉大学人民医院心内科

出　　处：《河北医药》2023年第11期1611-1615,1620,共6页Hebei Medical Journal

基　　金：湖北省自然科学基金面上项目(编号:2021CFB110)。

摘　　要：目的探究异莲心碱(Isoliensinine,IL)对心肌缺血再灌注损伤(myocardial ischemia/reperfusion injury,MI/RI)的作用及其机制。方法提取新生大鼠原代心肌细胞并鉴定,在离体状态下建立心肌细胞缺氧/复氧(hypoxia/reoxygenation,H/R)模型,CCK-8法检测不同浓度异莲心碱对H/R处理后细胞活力的影响并确定最佳药物浓度。随后,将心肌细胞随机分为对照组、H/R组及H/R+IL组,每组6只。检测3组乳酸脱氢酶(LDH)和肌酸激酶同工酶(CK-MB)的水平评估细胞损伤,TUNEL染色观察心肌细胞凋亡情况。RT-PCR及蛋白免疫印迹法检测活化半胱氨酸蛋白酶3(Caspase-3)、B淋巴细胞瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、核因子E2相关因子2(Nrf2)和血红素加氧酶-1(HO-1)的mRNA和蛋白表达情况。结果荧光显微镜下观察原代细胞,α-actinin染色阳性为心肌细胞(红色),其纯度高。异莲心碱处理6、12 h时,相对于H/R组,1μg/ml和5μg/ml异莲心碱均能明显升高细胞活性,且5μg/ml效果更为显著(P<0.05),而在作用24 h时,两种浓度效果无明显差异。此外,10μg/ml异莲心碱会导致细胞活力下降,综合考虑实验时长因素,选用5μg/ml为最佳作用浓度用于后续实验。与CON组比较,H/R组培养基中LDH和CK-MB释放量显著升高(P<0.05);与H/R组比较,H/R+IL组LDH和CK-MB释放量显著降低(P<0.05)。经过H/R处理后,心肌细胞凋亡率显著升高(P<0.001),TUNEL染色阳性的细胞数目明显增加。而异莲心碱能减轻H/R诱导的细胞凋亡,与H/R组相比,H/R+IL组TUNEL染色阳性的细胞比例明显减少(P<0.01)。与CON组比较,H/R组心肌细胞Bax及Caspase-3的mRNA表达量和蛋白表达水平显著升高(P<0.001),Bcl-2的mRNA和蛋白表达明显降低(P<0.01),Bcl-2/Bax值下降;与H/R组比较,H/R+IL组心肌细胞Bax及Caspase-3的mRNA和蛋白表达明显下调(P<0.05),Bcl-2表达显著增高(P<0.05),Bcl-2/Bax值升高。与CON组比较,H/R组心肌细胞Nrf2和HO-1的mRNA和蛋白表达量显著下降(P<0.Objective To investigate the effect and mechanism of isoliensinine(IL)in myocardial ischemia/reperfusion injury(MI/RI).Methods Primary cardiomyocytes were extracted from neonatal rats and identified,and a hypoxia/reoxygenation(H/R)model of cardiomyocytes was established in vitro.Cell Counting Kit-8(CCK-8)method was used to detect the effect of different concentrations of IL cell viability after H/R treatment,and to determine the optimal drug concentration.Subsequently,cardiomyocytes were randomly divided into control group,H/R group and H/R+IL group,with 6 in each group.The levels of lactate dehydrogenase(LDH)and creatine kinase isoenzyme(CK-MB)in the three groups were measured to assess cell damage,and the apoptosis of myocardial cells was observed by terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)staining.The real-time reverse transcription-polymerase chain reaction(RT-PCR)and Western blotting were used to detect the mRNA and protein expressions of activated cysteine proteinase 3(Caspase-3),B lymphoblastoma 2(Bcl-2),Bcl-2 related X protein(Bax),nuclear factor E2 related factor 2(Nrf2)and heme oxygenase 1(HO-1).Results Primary cardiomyocytes were observed under a fluorescence microscope,andαpositive actinin staining indicated myocardial cells(red),with high purity.After treatment with IL for 6 and 12h,compared with H/R group,1μg/ml and 5μg/ml of IL could significantly increase cell activity,and the effect of 5μg/ml of IL was more significant(P<0.05).However,there was no significant difference between the two concentrations after 24h of treatment(P>0.05).In addition,10μg/ml of IL could lead to a decrease in cell viability.Therefore,5μg/ml of IL was the optimal concentration for subsequent experiments.Compared with control group,the release of LDH and CK-MB in H/R group significantly increased(P<0.05).Compared with H/R group,the release of LDH and CK-MB in H/R+IL group significantly decreased(P<0.05).After H/R treatment,the apoptosis rate of myocardial cells increased obviously(P<0.001),a

关 键 词：异莲心碱 MI/RI 细胞凋亡 Nrf2/HO-1 

分 类 号：R541.4[医药卫生—心血管疾病]