Discovery Of Natural Molecules With Potential Therapeutic Effect Against Chronic Obstructive Pulmonary Disease And Investigation Of Their Mechanism

Exposure of cells and tissues to the environmental oxidants and toxicants(e.g.heavy metals,xenobiotics,pathogen,free radicals,drugs,and ionizing radiation)gives rise to oxidative stress and inflammation,which is characterized by the overproduction of reactive oxygen species(ROS)and inflammatory mediators.Excessive oxidative stress and inflammation generally account for the aetiology of chronic obstructive pulmonary disease(COPD).Therefore,inhibition of oxidative stress and inflammation effectively intervenes the pathogenesis of COPD.The nuclear transcription factor erythroid 2-related factor(Nrf2)plays a vital role in maintaining intracellular redox homeostasis.When the intracellular redox imbalance is triggered by exogenous oxidants and toxicants,Nrf2 is activated and promotes the transcriptions of cytoprotective genes,such as intracellular redox balancing proteins and phase ? detoxifying enzymes.Nuclear factor ?B(NF-?B)is a redox-sensitive transcription factor,and plays a critical role in the regulation of genes in response to inflammatory stimuli.Inhibition of the NF-?B pathway is capable of suppressing the inflammatory response.On the basis of biological functions of Nrf2 and NF-?B,characterization of molecules that activate Nrf2-mediated defensive responses and inhibit NF-KB-regulated inflammatory response,is a meaningful strategy for discovering potential preventive agents against COPD.Natural products have made important contributions to the history of human drug development.Searching for lead compounds and drugs from natural products is an important way to research and development of new drugs.Focusing on the direction of 'Discovery of natural molecules with potential therapeutic effect against chronic obstructive pulmonary disease and investigation of their mechanism',my work has completed the three parts of research1.Discovery of natural flavonoids as activators of Nrf2-mediated defense systemFlavonoids are a group of important natural antioxidants,which have aroused extensive attention attributed to their direct free radical scavenging effect.However,there is no systematic study on the antioxidant effect of flavonoids through up-regulating the antioxidant system.Given this limitation,a natural flavonoid library was established and tested for their potential Nrf2 inducing effects.Based on QR inducing effect of flavonoids,7-O-methylbiochanin(7-MBA)were firstly identified to be potential activators of Nrf2-mediated defensive response.Then,7-MBA was further investigated for its capability on the Nrf2 activation and prevention against oxidative insults in human lung epithelial cells.Further studies indicated that 7-MBA activated Nrf2 signaling pathway and protected human lung epithelial Beas-2B cells against sodium arsenite[As(?)]-induced cytotoxicity in an Nrf2-dependent manner.Activation of Nrf2 by 7-MBA upregulated intracellular antioxidant capacity,which was produced by enhancement of Nrf2 stabilization,blockage of Nrf2 ubiquitination,as well as Nrf2 phosphorylation by mitogen-activated protein kinase(MAPK),protein kinase C(PKC),protein kinase R-like endoplasmic reticulum kinase(PERK),and phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K).Taken together,7-MBA is a novel isoflavone-type Nrf2 activator displaying potential preventive effect against oxidative damages in human lung epithelial cells.2.Investigation of the anti-inflammatory constituents from Cinnanmomum camphora in lipopolysaccharide-stimulated RAW 264.7 macrophagesCinnamomum camphora has been used for the traditional medicine as a therapeutic agent in the treatment of inflammation-related diseases for a long history.However,the correlations between chemical constituents and anti-inflammatory applications of C.camphora,as well as their underlying anti-inflammatory mechanism of purified constituents still remains unknown.Given this limitation,lipopolysaccharide(LPS)-stimulated RAW 264.7 macrophages was adopted for evaluating anti-inflammatory function.The nitric oxide(NO)production assay were used to screen the constituents with anti-inflammatory property.Eight constituents,covering phenylpropanoid,lignan,flavonoids,coumarin,and terpenoid significantly inhibited LPS-stimulated NO production with maximum inhibition rates(MIRs)of?80%,and thus were verified to be the anti-inflammatory substances of this ethnomedical plant.The lignan(+)-episesaminone(SMO)and 3S-(+)-9-oxonerolidol(NLD)are two new anti-inflammatory molecules through inhibiting NF-?B regulated inflammatory response.SMO and NLD blocked NF-?B activation via inducing I?B?expression.Moreover,these two constituents inhibited production of tumor necrosis factor-?(TNF-?);interleukin-6(IL-6),and prostaglandin E2(PGE2),and alleviated increased mRNA and protein levels of inducible nitric oxide synthase(iNOS),cyclooxygenase(COX-2),and matrix metallopeptidase-9(MMP-9)in LPS-stimulated RAW 264.7 macrophages.Our results verified the anti-inflammatory substances of C.camphora,and identified SMO and NLD as two new anti-inflammatory molecules.Beaides,the anti-inflammatory mechanisms of bioactive constituents has been illustrated and the potential of two chemical components to prevent COPD by inhibiting the inflammatory response has been identified.In above mentioned parts of the work,we found and explored the mechanism of new active ingredients and the potential of intervention in COPD from anti-oxidative stress and anti-inflammatory aspects.Next,we will further search for more active ingredients from dual inhibitions of oxidative stress and inflammation,which will lay the foundation for the research and development of drugs to prevent and treat COPD,3.Ingredients from Litsea garrettii as potential preventive agents against oxidative insult and inflammatory responseOur previous study indicated that the EtOH extract of Litsea garrettii protected cells against oxidative insult and inflammatory response via the activation of Nrf2 and the dysregulation transcriptional activity of the NF-?B.However,no investigation on its phytochemical aspects has been reported,and accordingly the bioactivites composition of this plant remains unknown.In the present study,a systemic phytochemical investigation of L.garrettii led to the isolation of twenty-one chemical ingredients,which were further evaluated for their inhibitions on oxidative stress and inflammation using NAD(P)H:quinone reductase(QR)assay and nitric oxide(NO)production assay.Of which,3-methoxy-5-pentyl-phenol(MPP)was identified as an Nrf2 activator and an NF-?B inhibitor.Further studies demonstrated that:(i)MPP upregulated the protein levels of Nrf2,NAD(P)H:quinone oxidoreductase 1(NQO-1),and glutamate-cysteine ligase regulatory subunit(GCLM),enhanced the nuclear translocation and stabilization of Nrf2,and inhibited arsenic[As(?)]-induced oxidative insult in normal human lung epithelial Beas-2B cells;and(?)MPP suppressed the nuclear translocation of NF-?B p65 subunit,inhibited the lipopolysaccharide(LPS)-stimulated increases of NF-?B p65 subunit,COX-2,iNOS,TNF-? and IL-1?,and blocked the LPS-induced biodegrade of I?B-? in RAW 264.7 murine macrophages.Taken together,MPP displayed potential preventive effects against inflammation and oxidative stress related diseases.In summary,MPP,2,5-dihydroxybenzoic acid methyl ester,kaempferol,and quercetin possessed potential dual inhibitions against oxidative stress and inflammation.MPP is identified to be an activator of Nrf2 and an inhibitor of NF-?B,and protects cells against As(?)-induced oxidative insult,and LPS-induced inflammatory response for the first time.These data implied the potential uses of MPP as preventive agent against the pathogenesis of oxidative stress and inflammation-induced COPD.