| From the foundation of free radical theory in 1920s,the research about free radicals injury continuously deepened.A large number of researches indicate that many diseases are related with oxidative injury,there is no doubt about the impacts and importance of free radicals to life and health,so it is vital to look for potent antioxidants.At present,synthetic antioxidants such as BHT,BHA et al,they always have complicated synthetic technology with high cost,moreover,synthetic antioxidants have relatively higher toxicity even carcinogenic effects,so people nowadays disbelieve and refuse to use synthetic antioxidants,the clinical use of synthetic antioxidants is greatly limited.Natural antioxidants are safer and with lower toxicity which meet the health needs of people in new century,so finding antioxidants with high effect,low toxicity and low price from natural compounds has become a important research direction of this field,studies about the mechanisms underlying their antioxidant effects are of great significance in the further development of natural antioxidants. PART ONE Protective effects of grape seed proanthocyanidins against cardiac cells oxidative damage and the mechanisms underlying itProanthocyanidins or Procyanidins(PC) is a group of polyphenols which occur widely in plant.At first,they are put into the category of condensation tannins or flavanols.With the development of isolation and identification technology,at the same time,with deeper researches and understanding of these kinds of compounds, now they have become a large group of compounds called proanthocyanidins.For many years,people have done a lots of studies on PC extracted from different plants. As a ideal source of PC,Grape seed proanthocyanidins extract(GSP) has received the deepest and widest attention.GSP is polymerized by different amounts of catechin or epicatehin.The simplest proanthocyanidins is the dimer formed by catechin and epicatehin,besides this,there are trimer,tetramer till decamer.Sized by polymerization level,dimer to tetramer are called oligomer,more than 5 are called high polymer.A predominant proportion of GSP is type B,because the differences in conformation and biding sites in dimmers,there are several isomers,8 have been isolated and identified,called B1～B8.GSP have many important bioactivities,it has become a hot research topic.Cardiovascular disease remains a leading cause of death worldwide.It comprises its own set of pathologies,chief among which are atherosclerosis,hypertension, congestive heart failure,cardiomyopathy,coronary heart disease,hypertrophy, arrhythmias,ventricular fibrillation(VF),ventricular tachycardia(VT),myocardial infarction,and stroke.Strong evidence for the role of oxidative stress in the pathogenesis of heart failure has been provided by studies on experimental animals as well as humans.When the equilibrium between flee-radical production and cellular antioxidant defenses is disturbed in favour of more flee radicals,it causes oxidative stress which can promote cellular injury.Free radicals and oxidative stress also appear to be a common mediator of apoptosis,directly or via the formation of lipid peroxidation and lipid hydroperoxides.Free radical scavengers and antioxidants have been found to be cardioprotective against various ischemic heart diseases.Sectionâ… Grape seed proanthocyanidins protect cardiac cells from apoptosis via induction of endogenous antioxidantGrape seed extract(GSE) has been reported to exert protective effects on various forms of cardiac disorders.The cardiovascular protective effects of GSE are believed to be ascribed to its antioxidative properties.A series of studies have demonstrated that polyphenols are instrumental for the antioxidative properties of GSE.This study was undertaken to investigate whether two major polyphenols isolated from GSE(catechin and proanthocyanidin B4) could increase the endogenous antioxidant enzymes in cardiomycytes,and whether such increased cellular defenses could provide protection against oxidative cardiac cell injury.The results are showed below:1.Incubation of cardiac H9C2 cells with catechin or proanthocyanidin B4 resulted in a significant induction of cellular antioxidant enzymes in a concentration-dependent fashion.2.Catechin or proanthocyanidin B4 pretreatment led to a marked reduction in xanthine oxidase(XO)/xanthine -induced intracellular reactive oxygen species (ROS) accumulation and cardiac cell apoptosis.These results indicated that grape seed proanthocyanidins could protect against cardiac cell apoptosis via the induction of endogenous antioxidant enzymes.This may be an important mechanism underlying the protective effects of GSE observed with various forms of cardiovascular disordersSectionâ…¡Protection effects of grape seed proanthocyanidins against doxorubicin-induced toxicity in cardiomyocytes and the mechanisms underlying itThe clinical use of doxorubicin,a highly active anticancer drug,is limited by its severe cardiotoxic side effects.Grape seed extract(GSE) has been reported to exert protective effects on doxorubicin cardiotoxicity.This study was designed to investigate whether two major polyphenols isolated from GSE(catechin and proanthocyanidin B4(pc B4)) had protective effects against doxorubicin-induced cardiotoxicity and the proper mechanisms underlying it.The results are showed below:1.Catechin and pc B4 pretreatment would decrease doxorubicin-induced ROS generation in cardiac cells.2.Catechin and pc B4 pretreatment would decrease doxorubicin-induced decrease the number of apoptotic cells and prevent DNA fragmentation in cardiac cells.3.Catechin and pc B4 pretreatment would regulate the levels of expression of the pro-apoptotic protein Bax-αand the anti-apoptotic protein Bcl-2 in cardiac cells.4.Catechin and pc B4 pretreatment would inhibit apoptotic signaling pathways in cardiac cells.Because grape seed proanthocyanidins are instrumental for the bioactive properties of GSE,above results may be important mechanisms underlying the protective effects of GSE observed in doxorubicin cardiotoxicity.Thereby,we provide the mechanical basis of GSE and grape seed proanthocyanidins in clinical application to protect patients from doxorubicin induced cardiotoxicity.PART TWO Protection effects of oridonin against arsenic-induced cell toxicity and apoptosis and the mechanisms underlying itArsenic is one of the major environmental pollutants.It exists in soil and minerals and readily enters the ground water system,contaminating drinking water.The concentration of arsenic in the ground water varies significantly in different geographic areas.Arsenic concentrations are highest in East Asia,including Bangladesh,West Bengal,India,and China.Many efforts have been made in an attempt to reduce arsenic damage as exemplified by the guideline for arsenic in drinking water set by the World Health Organization and by local governments. Nevertheless,a large number of populations are still at risk of arsenic exposure and are suffering from arsenic-induced adverse effects,such as hypertension, arteriosclerosis,diabetes,hyperkeratosis,neuropathy,and cancer in the skin,liver, bladder,and lung.Clearly,the best way to protect humans from arsenic-induced damage is to reduce arsenic intake.However,it is not always practical,because many people have no choice but to live off from drinking water and rice that are heavily contaminated with arsenic,as these are their only sources of food and water. Therefore,an alternative choice,of equal importance,is to subvert the detrimental effects of arsenic by modulating the body's defense system.Nrf2 is a critical transcription factor that regulates a cytoprotective response.Many of its downstream target genes are important in maintaining the cellular antioxidant response as well as xenobiotic metabolism.Nrf2 controls the fate of cells through transcriptional upregulation of antioxidant response dement(ARE)-bearing genes,includingthose encoding endogenous antioxidants,phaseâ…¡detoxifying enzymes,and transporters. Expression of the Nrf2-dependent proteins is critical for ameliorating or eliminating toxicants/carcinogens to maintain cellular redox homeostasis.As a result,activation of the Nrf2 pathway,by naturally-occurring compounds or synthetic chemicals at sub-toxic doses,confers protection against subsequent toxic/carcinogenic exposure. Thus,the use of dietary compounds or synthetic chemicals to boost the Nrf2-dependent adaptive response to counteract environmental insults has emerged to be a promising strategy for cancer prevention.Recently,upregulation of the Nrf2-dependent defense response has proved to be beneficial in reducing arsenic-induced toxicity in a cell culture model.So using Nrf2 activators is of great significance to resist arsenic-induced toxicity.Oridonin is a kind of ent - kaurene diterpenoid purified from the Chinese medicinal herb Rabdosia rubescens.The main plant sources of oridinin are:R.am ethystoides,R.nervosa,R.rabdosia,R.downsis, R.rubescens,Isodon japonicus et al.Currently the major research focus on oridonin is in its antiproliferation and antitumor activities.The anticancer activity of oridonin is thought to rely on its ability to inhibit cell growth,reduce angiogenesis,and enhance apoptosis.This study was designed to test the protection effects of oridonin against arsenic-induced cell toxicity and apoptotic.We identified oridonin as representing a novel class of Nrf2 activators and illustrated the mechanism by which the Nrf2 pathway is activated.Furthermore,we demonstrated the feasibility of using natural compounds targeting Nrf2 as a therapeutic approach to protect humans from various environmental insults that may occur daily.Sectionâ… Efficacy of oridonin in protecting against Arsenic-induced Urosta cells toxicity and apoptosisThe chemopreventive activity of oridonin was demonstrated using a previously established arsenic-UROtsa cell model.The results are showed below:1.Oridonin treatment resulted in a significant increase in the glutathione level2.Treatment with 30μM As(â…¢) for 24 hr increased the level of ROS significantly, whereas 5.6μM oridonin itself had no effect.Pretreatment of cells with several doses of oridonin for 24 hr and further cotreatment with As(â…¢) for an additional 24 hr resulted in a significant reduction of ROS levels,especially with 5.6μM oridonin.3.By using Annexin V-FITC flow cytometry and Hoechst staining,we demonstrated that 1.4μM oridonin can inhibit As(â…¢)-induced cell apoptosis.4.Oridonin can induce Nrf2 in a dose-dependent fashion,from 1.4 to 14μM. Doses more than 14μM appeared to be toxic,and induction of Nrf2 was decreased5.By using both MTT and colony formation assays,we have demonstrated that pretreatment with1.4μM oridonin in UROtsa for 24h can significantly improved cell survival which were treated with As(â…¢) for 48 hr first.When using Nrf2-siRNA transfected UROtsa cells,oridonin lost its protection against As(â…¢) toxicity.Together,these results demonstrate that a low dose of oridonin is able to protect cells from As(â…¢)-induced damage,as illustrated by increased cellular antioxidant ability, inhibited As(â…¢)-induced oxidative stress,increased cell survival in response to As(â…¢) and decreased cell apoptosis,moreover,the protection effect was attributed to the activation of Nrf2 by oridonin.Sectionâ…¡Oridonin is an Nrf2 activator.Studies have showed that the activity of ARE-luciferase reporter gene is closely related with Nrf2 protein level.So testing effect of oridonin on ARE-luciferase reporter gene's activity can show us its effect on Nrf2. 1.Using highthroughput stable ARE luciferase reporter system established in our laboratory,we found out that oridonin dose-dependently increased the activity of luciferase.2.To confirm oridonin activation of Nrf2 using the high-throughput screening method,we also performed a dual luciferase reporter gene assay.Consistent with the data obtained from the high-throughput screening,oridonin induced the ARE-dependent luciferase activity in a dose-dependent manner Above results demonstrated that oridonin is an Nrf2activator.Sectionâ…¢Oridonin activated the ARE-dependent response primarily through up-regulation of the Nrf2 protein level.This section is about oridonin's effects on Nrf2 protein level and the mRNA level of Nrf2 downstream genes,the results are showed below:1.We used the same cell lysates from the dual luciferase reporter gene assay for immunoblot analysis for detection of Nrf2,Keap1,andβ-actin.Oridonin enhanced the levels of Nrf2 protein in a dose-dependent manner,with the highest induction at 14μM.2.We tested Nrf2 protein levels in response to high doses of oridonin.At doses more than 28μM,Nrf2 protein levels decreased in a dose-dependent manner, whereas the expression of Keap1 andβ-actin had no significant change.3.We measured mRNA expression of Nrf2 and its target genes,NQO1 and HO-1, in response to oridonin using real-time RT-PCR.Nrf2 mRNA increased slightly in a dosedependent manner in response to oridonin,mRNA of NQO1 and HO-1 were induced significantly by oridonin in a dose-dependent manner.These results demonstrate that oridonin is able to induce the Nrf2 signaling pathway mainly through upregulation of Nrf2 at the protein level. Sectionâ…£Oridonin blocked Nrf2 ubiquitination and enhanced Keap1 ubiquitinationtBHQ enhances the Nrf2 protein level by interfering with the Keap1-dependent ubiquitin conjugation process.Therefore,we tested the ability of oridonin in modulating Nrf2 ubiquitination.The results are showed below:1.Oridonin suppressed Nrf2 ubiquitination in MDA-MB-231cells and UROtsa cells.2.Oridonin treatment was also effective in enhancing ubiquitination of Keap1 MDA-MB-231cells and UROtsa cells.3.We measured the half-life of Nrf2 in the absence or presence of oridonin. Half-life of the endogenous Nrf2 protein in MDA-MB-231 cells was 19 min, whereas treatment with oridonin increased the half-life to 51 min.The half life of Nrf2 in UROtsa was increased from 10 min in the untreated condition to 16 min in response to oridonin treatment.Above results indicate that oridonin activates the Nrf2 pathway by inhibiting ubiquitination and degradation of Nrf2,leading to an increase in Nrf2 protein level and activation of the Nrf2-dependent response.
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