吉林大学学报(医学版) ›› 2023, Vol. 49 ›› Issue (3): 580-589.doi: 10.13481/j.1671-587X.20230305

• 基础研究 • 上一篇    下一篇

薏苡附子散对小鼠心肌缺血和血管内皮功能损伤的保护作用及其机制

李畅1,2,马梓珊1,2,黄汕梅1,2,银帮巧2,陈枝凡1,2,聂莎1,2,张子谦3,李力3,刘鹰1(),唐耀平2,4()   

  1. 1.广西中医药大学研究生院,广西 南宁 530200
    2.广西中医药大学附属瑞康医院心血管内科,广西 南宁 530200
    3.广西中医药大学科学实验中心 ;广西 南宁 530200
    4.广西壮族自治区高发传染病 中西医结合转化医学重点实验室,广西 南宁 530200
  • 收稿日期:2022-07-10 出版日期:2023-05-28 发布日期:2023-06-20
  • 通讯作者: 刘鹰,唐耀平 E-mail:xdtly001@126.com;tangyp2014@gxtcmu.edu.cn
  • 作者简介:李 畅(1996-),女,湖北省监利市人,在读硕士研究生,主要从事中西医结合防治心血管疾病方面的研究。
  • 基金资助:
    国家自然科学基金项目(81774115);广西中医药大学博士科研启动基金项目(2020BS035);广西高校中青年教师科研基础能力提升项目(2022KY0303)

Protective effect of Yiyi Fuzisan on myocardium ischemia and vascular endothelial function injury in mice and its mechanism

Chang LI1,2,Zishan MA1,2,Shanmei HUANG1,2,Bangqiao YIN2,Zhifan CHEN1,2,Sha NIE1,2,Ziqian ZHANG3,Li LI3,Ying LIU1(),Yaoping TANG2,4()   

  1. 1.Graduate School, Guangxi University of Traditional Chinese Medicine, Nanning 530200, China
    2.Affiliated Ruikang Hospital, Guangxi University of Traditional Chinese Medicine, Nanning 530011, China
    3.Center of Scientific Research, Guangxi University of Traditional Chinese Medicine, Nanning 530200, China
    4.Key Laboratory of Translational Medicine for Treating High-Incidence Infectious Diseases with Integrative Medicine, Guangxi Zhuang Autonomous Region, Nanning 530200, China
  • Received:2022-07-10 Online:2023-05-28 Published:2023-06-20
  • Contact: Ying LIU,Yaoping TANG E-mail:xdtly001@126.com;tangyp2014@gxtcmu.edu.cn

摘要:

目的 探讨薏苡附子散对小鼠心肌缺血和血管内皮功能损伤的保护作用,阐明其可能的作用机制。 方法 60只雄性SPF级C57BL/6J小鼠,随机分为空白组、假手术组、急性心肌缺血(AMI)组、低剂量薏苡附子散组、中剂量薏苡附子散组和高剂量薏苡附子散组,每组10只;另取40只C57BL/6J小鼠随机分为生理盐水组、低剂量薏苡附子散组、中剂量薏苡附子散组和高剂量薏苡附子散组,每组10只。采用冠脉左前降支(LAD)结扎法建立AMI小鼠模型后进行相应的药物干预处理28 d。采用Western blotting法检测小鼠心肌组织中一氧化氮合酶(eNOS)蛋白表达水平,动脉张力检测法检测各组小鼠离体胸主动脉血管张力和舒张率,总一氧化氮(NO)检测试剂盒检测各组小鼠血清中NO水平,氯化三苯基四氮唑(TTC)染色观察各组小鼠心肌组织缺血面积,HE染色观察各组小鼠心肌组织病理形态表现,观察各组小鼠术后存活率。人冠脉内皮细胞(HCAECs)随机分为空白组、缺氧组、缺氧+低剂量薏苡附子散组、缺氧+中剂量薏苡附子散组和缺氧+高剂量薏苡附子散组,除空白组外,其余各组细胞采用三气培养箱培养24 h建立缺氧模型。Griess实验检测各组HCAECs中NO水平,荧光染色法检测各组HCAECs中线粒体膜电位(MMP)和活性氧(ROS)水平。 结果 与建立AMI模型前比较,建立AMI模型后小鼠心电图ST段明显抬高。Western blotting法检测,与空白组比较,注射N-硝基-L-精氨酸甲酯(L-NAME)后假手术组、AMI组和低、中及高剂量薏苡附子散组小鼠心肌组织中eNOS蛋白表达水平降低(P<0.05)。与生理盐水组比较,低、中和高剂量薏苡附子散组小鼠胸主动脉舒张率升高(P<0.05)。与空白组和假手术组比较,AMI组小鼠血清中NO水平降低(P<0.05),高剂量薏苡附子散组小鼠血清中NO水平升高(P<0.05);与AMI组比较,低、中和高剂量薏苡附子散组小鼠血清中NO水平升高(P<0.05)。TTC染色观察,与空白组和假手术组比较,AMI组和低、中及高剂量薏苡附子散组小鼠心肌组织均有不同程度心肌缺血;与AMI组比较,低、中和高剂量薏苡附子散组小鼠心肌组织缺血面积减少,心肌组织缺血面积百分率降低(P<0.05)。HE染色,空白组和假手术组小鼠心肌细胞排列整齐,胞核清晰完整,大小均匀,未见炎性细胞浸润;AMI组小鼠可见明显的心肌组织损伤,心肌细胞排列紊乱、破裂坏死,有炎性细胞浸润;低、中和高剂量薏苡附子散组小鼠可见心肌组织病理性损伤恢复。药物干预第28天,空白组和假手术组小鼠生存率为100%;与AMI组比较,低、中和高剂量薏苡附子散组小鼠生存率升高(χ2=15.03,P=0.010 2)。Griess实验检测,与空白组比较,缺氧组和缺氧+低剂量组薏苡附子散组HCAECs中NO水平降低(P<0.05);与缺氧组比较,缺氧+中剂量薏苡附子散组和缺氧+高剂量薏苡附子散组HCAECs中NO水平升高(P<0.05)。荧光染色法检测,与空白组比较,缺氧组、缺氧+低剂量薏苡附子散组和缺氧+中剂量薏苡附子散组HCAECs中MMP水平降低(P<0.05),ROS水平升高(P<0.05);与缺氧组比较,缺氧+中剂量薏苡附子散组和缺氧+高剂量薏苡附子散组HCAECs中MMP水平升高(P<0.05),缺氧+低剂量薏苡附子散组、缺氧+中剂量薏苡附子散组和缺氧+高剂量薏苡附子散组HCAECs中ROS水平降低(P<0.05)。 结论 薏苡附子散可通过增强NO生物活性,增加主动脉血管活性,改善心肌缺血病理状态,恢复MMP,减少ROS的生成,改善线粒体及血管内皮细胞功能障碍。

关键词: 薏苡附子散, 一氧化氮, 心肌缺血, 人冠脉内皮细胞, 线粒体

Abstract:

Objective To discuss the protective effect of Yiyi Fuzisan on the myocardium ischemia and vascular endothelial dysfunction of the mice, and to clarify its possible mechanism. Methods Sixty male SPF grade C57BL/6J mice were randomly divided into blank group, sham operation group, acute myocardial ischemia (AMI) group, and low,medium and high doses of Yiyi Fuzisan groups, and there were 10 mice in each group;another forty C57BL/6J mice were randomly divided into normal saline group and low, medium, and high doses of Yiyi Fuzisan groups, and there were 10 mice in each group. The left anterior descending coronary artery (LAD) ligation method was used to establish the AMI mouse model, and corresponding drug intervention treatment was performed for 28 d. Western blotting method was used to detect the expression levels of endothelial nitric oxide synthase (eNOS) protein in myocardium tissue of the mice in various groups;arterial tension detection method was used to detect the tension of isolated aortic and relaxation rates of the mice in various groups;total nitric oxide(NO) detection kit was used to detect the levels of NO in serum of the mice in various groups;triphenyltetra zolium chloride(TTC) staining was used to observe the ischemia areas of myocardium tissue of the mice in various groups; HE staining was used to observe the pathomorphology of myocardium tissue of the mice in various groups;the postoperative survival rates of the mice in various groups were observed. The human coronary endothelial cells (HCAECs) were randomly divided into blank group, hypoxia group,hypoxia+ low dose of Yiyi Fuzisan group, hypoxia+medium dose of Yiyi Fuzisan group, and hypoxia+high dose of Yiyi Fuzisan group. Except for blank group, the cells in the other groups were cultured in the three gas incubator for 24 h to establish the hypoxia models. Griess test was used to detect the levels of NO in the HCAECs in various groups;fluorescence staining was used to detect the levels of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) in the HCAECs in various groups. Results Compared with before establishing the AMI model, the ST segment of electrocardiogram of the mice was significantly elevated after establishing the AMI model. The Western blotting results showed that compared with blank group, the expression levels of eNOS protein in myocardium tissue of the mice in sham operation group, AMI group,and low, medium, and high doses of Yiyi Fuzisan groups were decreased after injection of N-nitro-L-arginine-methylesterhydro chloride(L-NAME)(P<0.05). Compared with saline group, the relaxation rates of thoracic aorta of the mice in low, medium, and high doses of Yiyi Fuzisan groups were increased (P<0.05). Compared with blank group and sham operation group, the serum NO level of the mice in AMI group was decreased (P<0.05), the serum NO level of the mice in high dose of Yiyi Fuzisan group was increased (P<0.05); compared with AMI group, the serum NO levels of the mice in low, medium, and high doses of Yiyi Fuzisan groups were increased (P<0.05). The TTC staining observation showed that compared with blank group and sham operation group, there were various degrees of myocardium ischemia of the mice in myocardium tissue of the mice in AMI group and low, medium, and high doses of Yiyi Fuzisan groups; compared with AMI group,the ischemia areas of myocardium tissue of the mice in low, medium, and high doses of Yiyi Fuzisan groups were decreased (P<0.05),and the percentages of ischemia areas of myocardium tissue were decreased.The HE staining results showed that the cardiomyocytes of the mice in blank group and sham operation group were neatly arranged, with clear and complete nuclei and uniform sizes,and no inflammatory cell infiltration was observed;the cardiomyocytes of the mice in AMI group showed significant myocardial tissue damage, with disordered arrangement,rupture and necrosis of cardiomyocytes, and infiltration of inflammatory cells;the myocardium tissue of the mice in low, medium, and high doses of Yiyi Fuzisan groups showed pathological recovery of myocardial tissue damage. On the 28th day after drug intervention, the survival rates of the mice in blank group and sham operation group were 100%; compared with AMI model group, the survival rates of the mice in low, medium, and high doses of Yiyi Fuzisan groups were increased(χ2=15.03,P=0.0102).The Griess experiment results showed that compared with blank group, the levels of NO in the HEAEs in hypoxia group and hypoxia+low dose of Yiyi Fuzisan group were decreased (P<0.05); compared with hypoxia group, the levels of NO in the HEAECs in hypoxia+medium dose of Yiyi Fuzisan and hypoxia+high dose of Yiyi Fuzisan groups were increased (P<0.05). The fluorescence staining results showed that compared with blank group, the levels of MMP in the cells in hypoxia group, hypoxia group+low dose of Yiyi Fuzisan group, and hypoxia +medium dose of Yiyi Fuzisan group were decreased (P<0.05),while the levels of ROS were increased (P<0.05); compared with hypoxia group, the levels of MMP in the HEAECs in hypoxia+low dose of Yiyi Fuzisan,hypoxia+medium dose of Yiyi Fuzisan,and hypoxia+high dose of Yiyi Fuzisan groups were increased (P<0.05), while the levels of ROS in the HCAECs in hypoxia+low dose of Yiyi Fuzisan,hypoxia+medium dose of Yiyi Fuzisan,and hypoxia+high dose of Yiyi fuzisan groups were decreased (P<0.05). Conclusion Yiyi Fuzisan can improve the pathological state of myocardium ischemia, restore the MMP, reduce the production of ROS, and improve the dysfunction of mitochondria and vascular endothelial cells by enhancing the biological activity of NO and increasing the vascular activity of aorta.

Key words: Yiyi Fuzisan, Nitric oxide, Myocardium ischemia, Human coronary artery endothelial cells, Mitochondria

中图分类号: 

  • R541