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