摘要
目的探讨和厚朴酚(HK)对心肌缺血/再灌注损伤(MIRI)小鼠心脏的保护作用及其机制。方法2022年6月—2023年7月于陕西中医药大学基础医学院中医诊断实验中心进行实验。78只雄性C57BL/6J小鼠随机分为假手术(Sham)组、MIRI组和MIRI+HK组,每组26只。术前对MIRI+HK组小鼠给予HK(0.2 mg·kg-1·d-1)腹腔注射,Sham组和MIRI组给予等量溶剂,连续14 d。造模术中Sham组仅穿线不结扎,其余2组均结扎左前降支30 min后,解开缝线再灌注2 h,构建MIRI模型,再灌注结束后立即取血,剥离心脏。比较各组血清中肌酸激酶同工酶(CK-MB)和心肌肌钙蛋白T(cTnT)水平;超声心动图检测心功能;2,3,5-三苯基氯化四氮唑染色法计算心肌梗死面积;原位末端标记技术检测细胞凋亡水平;电镜观察线粒体结构损伤情况;Western-blot检测线粒体凋亡和磷酸肌醇-3激酶/蛋白激酶B(PI3K/AKT)通路相关蛋白表达。结果与Sham组比较,MIRI组小鼠血清CK-MB、cTnT和左心室收缩末期内径(LVESD)水平及心肌梗死面积、细胞凋亡率升高,左心室射血分数(LVEF)和左心室短轴缩短率(LVFS)降低(P<0.001),心肌细胞线粒体大多肿胀,内外双层膜模糊,嵴畸形且减少或缺失,线粒体凋亡相关蛋白细胞色素(Cyto)-C、Bcl-2相关X蛋白(Bax)和活化半胱氨酸蛋白酶(Cleaved Caspase-9)表达升高,B淋巴细胞瘤2基因(Bcl-2)、p-AKT/AKT和p-PI3K/PI3K表达降低(P<0.001);与MIRI组比较,MIRI+HK组小鼠血清CK-MB、cTnT和LVESD水平及心肌梗死面积、细胞凋亡率降低,LVEF和LVFS增加(P<0.001),心肌细胞线粒体超微结构有所好转,结构较为完整,Cyto-C、Bax和Cleaved Caspase-9蛋白表达降低,Bcl-2、p-AKT/AKT和p-PI3K/PI3K表达升高(P<0.001)。结论HK可通过激活PI3K/AKT信号通路抑制MIRI诱导的心肌细胞线粒体凋亡,从而发挥心肌保护作用。
Objective To investigate the protective effect and mechanism of honokiol(HK)on the heart of mice with myocardial ischemia/reperfusion injury(MIRI).Methods From June 2022 to July 2023,the experiment was conducted at the Traditional Chinese Medicine Diagnosis Experimental Center of the School of Basic Medicine,Shaanxi University of Chinese Medicine.78 male C57BL/6J mice were randomly divided into sham group,MIRI group,and MIRI+HK group,with 26 mice in each group.Before surgery,the MIRI+HK group mice were given HK(0.2 mg·kg-1·d-1)intraperitoneal injection,while the Sham and MIRI groups were given equal amounts of solvent for 14 consecutive days.The Sham group only threaded without ligation,while the other groups ligated the left anterior descending branch for 30 minutes,then opened the suture and reperfusion for 2 hours to construct a MIRI model.After reperfusion,blood was immediately taken and the heart was stripped.Compare the levels of serum creatine kinase isoenzyme(CK-MB)and cardiac troponin T(cTnT)in each group.Echocardiography detects heart function.Calculate myocardial infarction area using 2,3,5-triphenyl tetrazolium chloride staining method;In situ end labeling technology was used to detect the level of cell apoptosis;Electron microscopy observation of mitochondrial structural damage.Western blot was used to detect mitochondrial apoptosis and protein expression related to the phosphoinositol-3 kinase/protein kinase B(PI3K/AKT)pathway.Results Compared with the Sham group,the MIRI group mice showed higher levels of serum CK-MB,cTnT,left ventricular end systolic diameter(LVESD),myocardial infarction area,and cell apoptosis rate,while the left ventricular ejection fraction(LVEF)and left ventricular short axis shortening rate(LVFS)decreased(P<0.001).Most myocardial mitochondria were swollen,with blurred inner and outer bilayers,reduced or absent cristae,and mitochondrial apoptosis related protein cytochrome(Cyto-C)The expression of Bcl-2 related X protein(Bax)and activated cysteine protease(Cleared Caspase-9)increased,while the expression of B lymphocyte tumor 2 gene(Bcl-2),p-AKT/AKT,and p-PI3K/PI3K decreased(P<0.001).Compared with the MIRI group,the serum CK-MB,cTnT,and LVESD levels,myocardial infarction area,and cell apoptosis rate of the MIRI+HK group mice decreased,while LVEF and LVFS increased(P<0.001).The mitochondrial ultrastructure of myocardial cells improved and the structure was more complete.The expression of Cyto-C,Bax,and Cleared Caspase-9 proteins decreased,while the expression of Bcl-2,p-AKT/AKT,and p-PI3K/PI3K increased(P<0.001).Conclusion HK can inhibit MIRI induced mitochondrial apoptosis in cardiomyocytes by activating the PI3K/AKT signaling pathway,thereby exerting a cardioprotective effect.
作者
王青瑛
王相东
邢文文
闫颖
王郁金
Wang Qingying;Wang Xiangdong;Xing Wenwen;Yan Ying;Wang Yujin(School of Basic Medicine,Shaanxi University of Chinese Medicine,Shaanxi Province,Xianyang 712046,China)
出处
《疑难病杂志》
CAS
2024年第6期729-735,共7页
Chinese Journal of Difficult and Complicated Cases
基金
陕西省自然科学基础研究计划一般项目(面上)(2019JM-420)
陕西省科技厅自然科学基础研究计划一般项目(青年)(2019JQ-011)
陕西省中医药大学科学研究计划(2017QN25)。
