Study On The Anti-myocardial Ischemic Efficacy And Mechanism Of Tibetan Medicine Corydalis Dwarfi And Meconopsis Spinulos

With the economic development,people’s living standards are getting higher,however,it also cause large amounts of people with cardiovascular diseases.Acute myocardial infarction(AMI)is one of the most common coronary heart diseases(CHD),due to its high incidence,high cost of treatment,therefore,greatly affected people’s health.The aim of this study is to evaluate the pharmacological effects of two Tibetan medicines,Corydalis hendersonii HemsL and Meconopsis horridula,against ischemic heart disease(IHD),and their underlying mechanisms.C.hendersonii Hemsl.(CH),belonging to the family Papaveraceae,is mainly distributed in the northern temperate regions and is called ’ai-zi-jin’ in Chinese,and‘ri-gun-zi-ma’ or ’ri-gun’ in Tibetan.As one of characteristic Tibetan folk medicine,CH has been used for hundreds of years in China to mainly treat HAPC,named "plethora" in Tibetan medicine,lower blood pressure,and cure fever,hepatitis,edema,gastritis,and cholecystitis.Previous studies have shown that alkaloids especially isoquinolines are the main ingredients in the extracts of CH.M.horridula(MH),belonging to the family Papaveraceae,have been used as traditional Tibetan medicine for more than 1000 years.Phytochemical studies revealed the occurrence of major ingredients in MH is isoquinoline alkaloids and flavonoids.Modern pharmacological research have demonstrated that the extracts and ingredients exhibited antitumor,analgestic,antimicrobial and anti-myocardial ischemia.This thesis is composed of the following parts.Review charpter describes the progress of the MH in the chemical and pharmacological effects over the past two decades,along with a pharmacological advance of oleracein E(OE),one of bioactive alkaloid from MH.Anti-myocardial ischemic evaluation of CH:the ICR mice model was prepared by ligating the left anterior descending coronary artery.After 24 h,the mice were treated with ethanol extract(100,200,400 mg·kg-1)for one week.Heart function was evaluated by measuring EF and FS by echocardiography.The levels of CK-MB and LDH in the serum,AngⅡ,IL-6,TNF-α in the plasma,MMP-2 and MMP-9 in the cardiac tissue homogenate,the expression of MMP-2 and MMP-9 proteins in the heart tissues,as well as the signal-transducing proteins,p65 and STAT3,were also measured by western-blot analysis.Inflammatory cell infiltration and changes in collagen deposition in the myocardial ischemic heart tissues were observed by pathological examination.Platelet aggregation in vitro was also investigated.Intragastric CH treatment resulted in a dose-dependent protection of cardiac function.It significantly reduced the LVEDs and LVEDd,improved EF and FS compared to the model group,inhibited the elevation of CK-MB,LDH in serum,AngⅡ,TNF-α and IL-6 in plasma,MMP-2 and MMP-9 in the cardiac tissue homogenates,and down-regulated the expression of p-p65,p-IκBα,p-JAK2,p-STTA3,MMP-2,and MMP-9 in AMI mice.Also,an obvious reduction in the infiltration of inflammatory cells into the infarcted myocardial tissue was found in each CH treated group.Besides,CH also inhibited platelet aggregation induced by AA,ADP,and THR.Anti-myocardial ischemia effect of MH in mice:C57BL/6 mice were used to evaluate the anti-myocadial ischemia effect of MH.The mice were opertated by ligation of LAD coronary artery,and then intragastric adiministrated for seven days consecutively.Echocardiography was used to evaluate the cardiac function of mice in each group after one week.The levels of CK-MB and LDH in the serum of mice were determined.Bax,Bel-2,p53,and caspase-3/8/9 were detected by Western blotting experiments.HE and MASSON stainings were employed to detect the degree of myocardial inflammatory infiltration and myocardial fibrosis were observed in each group.Intragastric MH treatment resulted in a dose-dependent protection of cardiac function,including that significantly reduced the LVEDs and LVEDd,improved EF and FS compared to the model group,inhibited the elevation of CK-MB,LDH in serum,greatly improved the activity of SOD in plasma and decreased MDA,and down-regulated the expression Bax,p53,caspase-3,caspase-8 and caspase-9 in AMI mice.The expression of Bax/Bcl-2 on the mitochondrial membrane inhibits the endogenous apoptotic pathway induced by caspase9,and inhibited the apoptosis of cardiomyocytes,suggesting that the ethanol extract of MH can significantly improved the decline left ventricular function,and decrease the occurrence of cardiomyocyte apoptosis.In vitro assays of oxidative injury:H9c2 cardiomyocytes were induced by H2O2,and the single compounds from MH were tested.The results showed that compounds 2,18,and 23 possess the protective active against injury to H9c2 cardiomyocytes.In vitro anti-inflammatory assay:The cell supernatant from LPS-induced RAW264.7 cells was used as a conditional medium to stimulate H9c2 cell,which established a myocardial inflammation injury model.MTT(3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-diphenyl tetrazolium bromide method was used to detect the viability of H9c2 cell.The bioassay results showed that compounds 7 and 23 have the protective effect against the injury to H9c2 cardiomyocytes.In addition,the chemical constituents of the bioactive extract of CH was characterized by using a HPLC-MS-IT-TOF method.In general,CH effectively improved the cardiac function of ischemic mice to exert a protective effect against ischemic myocardium.It reduced the severity of inflammatory cells infiltrated in myocardium,reduce the inflammatory factors,and it potential mechanism was related to the regulation of NF-κB and JAK2-STAT3 signalling pathways.MH can significantly improve the decline left ventricular function,reduce the incidence of inflammatory cell infiltration,oxidative damage and inhibit the apoptosis of cardiomyocytes in acute myocardial infarction mice,suggesting a mechanism of MH underlying cardioprotective effect through the anti-oxidative and anti-cardio myocyte apoptosis ways.The present study will expand the clinical application of CH and MH as ethno medicines,further excavate valuable traditional medicinal resources,enrich the basic research of the traditional Chinese medicine for the treatment IHD,and enhance the national drug innovative research.