Protective Effect Of Three Monomers Extracted From Traditional Chinese Medicine On Oxidative Damage To Hepatocytes And Cardiomyocytes

Cell damage caused by elevated levels of oxidative stress is considered to be an important factor leading to aging and disease.In recent years,the research on the antioxidant effect of traditional Chinese medicine has attracted wide attention,especially the protective effects and mechanisms of active components isolated from natural drugs on cell damage have become important research trends.However,the multi-components,multi-targets and multi-pathways features of traditional Chinese medicine and the complex structure of active natural product have led to more difficulties and challenges in their further development and application.Based on the above status,In order to explore the potential value of more active components of traditional Chinese medicine to prevent and cure diseases caused by oxidative stress,this paper has carried out three parts of work.In Chapter 1,through cell-level activity screening,it was found that four diterpenoids obtained from Ganoderma lucidum have a great protective effect on H2O2-induced liver cell and myocardial oxidative danage,further in-depth studies on molecular level revealed the potential mechanism of this effect.In chapter 2,it was found that a series of triterpenoids isolated from Anchusa italica Retiz.had potent protective effects on cardiomyocytes cell ischemia-reperfusion injury,and the mechanism was preliminary investigated on molecular level;In chapter 3,five unusual dimers of ent-labdane diterpenoids isolated from Andrographis paniculata were studied and the absolute configuration of key positions(C-12 and C-15’)were revealed for the first time.The microscale thermophoresis(MST)was used to detect the binding ability between these compounds and TRPV channels.It was revealed that the compounds could selectively activate TRPV channels,and their protective effect on cardiomyocytes was verified at the cellular level.Reactive oxygen in the body is a class of single electron reduction product of oxygen.Excessive level of reactive oxygen in the body can cause oxidative damage to biological macromolecules such as nucleic acids and proteins.Damaged DNA may break,mutate,etc.,which affects the normal transcription and translation of genetic information and other processes,further leading to aging,inflammation and various chronic diseases.Ganoderma sinense.was first recorded in Shennong’s compendium of Materia medica.It has various functions such as regulating immunity,anti-inflammatory,anti-tumor and anti-oxidation.As valuable traditional Chinese medicine,its active components have received widely attention from pharmacists in recent years.Four meroterpenoids,applanatumols F(1),applanatumols H(3)and I(2),and lingzhiol(4)were isolated from 95%ethanol extracts of Gan.oderma lucidum based on early laboratory results.Compounds 1,2 and 4 existed as racemic mixtures(+)1a,2a,4a;(-)1b,2b,4b,while 3 is a single enantiomer.We found that compound 1-4 may have a potential protective effect on apoptosis induced by hydrogen peroxide(H2O2)via cell-level activity screening,and the potential molecular mechanism was further explored in human normal liver L02 cells.Among them,compound 2α could effectively protect L02 cells from H2O2-induced cell damage and apoptosis.H2O2 treatment could increase the ROS content in L02 cells and reduce the mitochondrial membrane potential,while caspase-3 and PARP are activated and the cells undergo apoptosis;compound 2a could reverse this apoptosis process by reducing ROS content,improving mitochondrial membrane potential,and reducing the expression levels of cleavedcaspase 3 and cleaved-PARP.In addition,up-regulation of Nrf2,HO-1,phosphorylated Akt,and antioxidant enzymes was detected in 2a-treated cells,these results indicated that the antioxidant effect of 2a on H2O2-induced L02 cell injury may protect cells from oxidative damage through PI3K/Akt-mediated activation of the Nrf2/HO-1 pathway.In addition,compound 2a showed potential protective effect on cardiomyocyte from ischemia/reperfusion injury.The above experimental results provided a certain research basis of Ganoderma lucidum on the prevention and treatment of oxidative damage related diseases.Cardiovascular disease is one of the major diseases that seriously threaten human life and health in today’s society.Anchusa italica Retz.is a traditional Uyghur medicine used to treat cardiovascular and cerebrovascular diseases.It has the effects of regulating abnormal black gall,relieving cold and replenishing heart,relieving cough and asthma.Modern pharmacologic researches have shown that the bugloss has a variety of pharmacological effects such as scavenging free radicals,anti-oxidation,and regulating the expression of apoptotic genes.In the previously research work in our laboratory,six new triterpenoids(1-6)and 22 known triterpenoids(7-28)were isolated from the part of aerial parts of Anchusa italica Retz..In view of the efficacy of bugloss in the treatment of cardiovascular diseases,in this chapter we screened compounds 1-28 at the cellular level for the protective effect of hypoxia/reoxygenation(H/R)induced myocardial cell injury.Among them,compounds 4,6-17,21-22,26-28 showed remarkable protective effects on cardiomyocyte injury.In addition,the representative new compound 6 could significantly inhibit H/R-induced apoptosis and autophagy of neonatal rat primary cardiomyocytes,reverse the downregulated expression of Bcl-2 and upregulated expression of Bax and Beclin-1 in neonatal rat cardiomyocytes.Compound 6 could decrease CK-MB and LDH levels in cardiomyocytes after H/R treatment.In addition,compound 6 also alleviated H/R-induced phosphorylation of p38 MAPK in neonatal rat cardiomyocytes.To sum up,this study revealed that the series of triterpenoids in the Anchusa italica Retz.have a protective effect on H/R-induced myocardial cell damage on the cellular and molecular levels,which provides reference and guidance for its in-depth study on the treatment of cardiocerebrovascular diseases.Ischemic heart disease is caused by changes in coronary circulation leading to myocardial ischemia and hypoxia.Restoring blood perfusion in ischemic myocardium is crucial for effectively treatment of patients with ischemic heart disease.However,changes in the level of oxidative stress in myocardial cells may lead to myocardial ischemia-reperfusion injury.At present,the drugs used for the treatment of ischemic heart disease are mainly chemical drugs.Their pharmacological effects and mechanisms are complicated,and adverse reactions are unavoidable.Traditional Chinese medicine has unique characteristics and advantages in the treatment of cardiovascular diseases.Andrographis paniculate(Burm.f)Nees,a traditional Chinese medicine,has the functions of clearing heat and detoxifying,cooling blood and stopping bleeding.It is mainly used to treat various infectious diseases and cardiovascular diseases such as hypertension.In recent years,researches on its active components and potential applications have received extensive attention from natural pharmaceutical chemists.Based on preliminary experimental research,five rare andrographolide dimers were isolated from the Andrographis paniculata,including two bisandrogrographolides E(1)and F(2),and three known stereoisomers A-C(3-5).However,the absolute configuration of the two newly formed chiral centers(C-12 and C-15’)in the aggregation site in its structure has not yet been resolved for many years,which brought obstacles for further activity evaluation and structure-activity relationship research.This part of the research is based on the results of our previous researches.Propylene derivatization method was used to change the physical properties of the compound and three dimer single crystals were successfully cultivated to reveal the absolute configuration of the key positions(C-12 and C-15’).Previous electrophysiological experiments results showed that this series of compounds could activate TRPV channels.Therefore,in this chapter,MST was used to determine the dissociation constants of compounds and TRPV proteins,and it was further revealed that these compounds can protect cardiomyocytes from hypoxia/reoxygenation-induced damage.There were great differences of activities between different dimers with chemical structures or absolute configurations on the activation and cardiac protection of TRPV 1-4 channels,which provide guidance and reference for further structure-activity relationship and in-depth study of mechanism.In summary,based on the preliminary work,we developed a systematic research framework for anti-oxidative stress and cell protection evaluation of active natural products and applied it to the natural active components isolated from Ganoderma lucidum,Anchusa italica Retiz and Andrographis paniculata.We systematically studied the protective effects and mechanism on cell damage of different active components from traditional Chinese medicine on the cellular level and molecular level,providing a new strategy for the research on the protection and mechanism of natural products.In addition,we revealed the structural characteristics and diversity of some complex molecules.We hope these studies can promote the extensive researches and application of active natural products in the field of cardiovascular and cerebrovascular diseases,and promote the development of the Chinese medicine industry in our country.