Research On Active Ingredients Of Chinese Medicine And Molecular Mechanisms Based On The Myocardial Protective Effect

Cardiovascular disease, with increasingly high morbidity and mortality worldwide, is currently the leading threat to human health. Therefore, it is imperative to improve the prevention and treatment levels of cardiovascular disease. Multifactor process may result in cardiovascular disease, including myocardial ischemia/reperfusion injury, hyperlipidemia, arterial atherosclerosis, chemotherapeutic agent-induced side effects and so on. With the development of modern medicine, great progress has been made in the prevention, diagnosis, and treatment of cardiovascular disease. However, because of the high incidence, low cure rate and lots of complications, it is still very difficult for the treatment of this disease. In recent years, the application of Chinese traditional medicine on cardiovascular disease has caused the extensive concern of the medicine. Sceening traditional Chinese medicine and compound with definite curative effect on cardiovascular disease to research the underlying mechanism and clear its fundamental experimental basis and function characteristics will be undoubtedly benefit to human health and development.In the present study, we aim to screen the myocardial protective activities from Guanxi Danshen formula and flavonoid, and further investigate the underlying mechanisms. There are two parts of this study as follows:Part I. Active ingredient screening and molecular mechanism research of Guanxin Danshen Formula on myocardial protectionObjective:Guanxin Danshen Formula is an effective prescription of traditional Chinese medicine for coronary heart disease treatment. It is consisted of Salvia miltiorrhiza Bunge, Panax notoginseng and Lignum Dalbergiae Odoriferae. Guanxin Danshen Formula shows the effects of promoting blood circulation by removing blood stasis and promoting qi circulation and relieving pain in clinical and used to treat cardiovascular diseases. Although the protection efficacy and fundamental experimental basis of Guanxin Danshen Formula have been investigated widely, it is still difficult to elucidate the active ingredients and action targets because of the complex composition of the Chinese herbal compound prescription. Therefore, the aim of this study is to screen the myocardial protective activities from Guanxi Danshen formula and flavonoid, and further investigate the underlying mechanisms to provide the candidate drugs for prevention and treatment of cardiovascular disease and pre-clinical research data, as well as fundamental experimental basis for the explanation of the effectiveness and mechanisms of traditional Chinese formula by modern molecular biological methods.Methods:Screen the active ingredient with myocardial protective effect among9main components from Guanxi Danshen formula on hypoxia-reoxygenation model in H9c2cardiomyocytes. Then the possible mechanisms of Ginsenoside RK3were investigated by multiple detection methods such as Hoechst33342/PI and TUNEL assay, fluorescence spectrophotometer analysis of caspase-3activity and western blot assay.Results:1. All the components of Guanxin Danshen Formula in this study showed protective effect on hypoxia-reoxygenation induced cell injury after12hours of pretreatment. Ginsenoside RK3showed significant protective effects. Pretreatment with RK3(6.25μg/ml to25μg/ml) increased cell viability in a dose-dependent manner. Higher RK3concentration (up to50μg/ml) did not improve cell viability.2.25Ψg/ml Ginsenoside RK3pretreatment significantly reduced the TUNEL apoptotic rate and increased hypoxia-reoxygenation induced expression of anti-apoptotic protein Bcl-2while decreased the expression of pro-apoptotic protein Bax. Our data also demonstrated that the myocardial protection effect of Ginsenoside RK3was associated with PI3K/AKT and MAPK signal transduction pathway.Conclusion:Saponins, especially Ginsenoside RK3from Guanxin Danshen Formula in this study showed significant protective effects on hypoxia-reoxygenation induced myocardial cell injury. The underlying mechanism of this cytoprotection was connected with AKT and MAPK pathways. Part Ⅱ. Active ingredient screening and molecular mechanism research of flavonoids on myocardial protectionObjective:Flavonoids is used for the treatment and prevention of cardiovascular disease as one of the important effective components in traditional Chinese medicine. Epidemiological studies have shown that dietary flavonoids intake can reduce the risk of cardiovascular which shown that flavonoids has potential application value and great medicinal development prospects in prevention and treatment of cardiovascular disease. Our laboratory has found that flavonoids is a main active site of Guanxin Danshen Formula with significant antioxidant activity.Therefore, the aim of this study is to screen the myocardial protective activities from9flavonoids, and further investigate the underlying mechanisms to provide the scientific evidence for the application of flavonoids.Methods:Screen the active ingredient with myocardial protective effect among10flavonoids components on doxorubicin model in H9c2cardiomyocytes. Pathology technology (HE staining and immunohistochemistry), serum biochemical (myocardial enzyme, lipid peroxidation products and antioxidant enzymes), fluorescence stainin (H33342/PI、TUNEL、ROS and JC-1)and molecular cell biology technology (western blot) were adopted to assess the protection capacity and the underlying mechanism of isorhamnetin against doxorubicin induced myocardial cell injury at molecular level, cell level and animal level.Results:1. Isorhamnetin showed significant protective effects against Dox induced H9c2cell injury among the tested flavonoids. Pretreatment with isorhamnetin (3.125μg/ml to12.5μg/ml) increased cell viability in a dose-dependent manner. Higher concentration (up to25μg/ml) did not improve cell viability.2. Isorhamnetin on12.5μg/ml caused a reduction of cardiac index, a decrease of cardiac enzymes in serum and an amelioration of heart vacuolation in vivo. In vitro studies on H9c2rat cardiomyocytes showed that isorhamnetin can effectively reduce Dox induced cell toxicity and apoptosis. A further mechanism study indicated that isorhamnetin pretreatment can counteract Dox induced oxidative stress and suppress the activation of mitochondrion-dependent apoptotic pathway and mitogen-activated protein kinase pathway. Isorhamnetin potentiated the anti-cancer activity of Dox in MCF-7, HepG2and Hep2cells.Conclusion:Isorhamnetin elicited a typical cardioprotective effect on Dox induced cardiotoxicity in vivo and in vitro.This protective effect was correlated with the inhibition of oxidative stress and subsequent suppression of the mitochondrial apoptotic pathway and MAPK pathway. Thus, isorhamnetin can be a novel candidate for the combination with Dox to protect against Dox induced cardiotoxicity.