Investigations On Natural Antioxidants Targeting Nrf2 Signaling Pathway

Oxidative stress is an imbalance of the oxidants/antioxidants tilting toward an oxidative status.Oxidative stress has been shown to be closely related to the pathogenesis of lung diseases.Since the direct exposure to the oxidants and toxicants,human lung tissue is apt to be harmed to give rise to acute and chronic oxidative insults.Nuclear factor erythroid 2-related factor 2(Nrf2)is an important transcription factor that regulates cellular redox balance.Studies have confirmed that Nrf2 activators can inhibit lung tissue oxidative stress and interfere with the pathological processes of lung diseases.Traditional Chinese mdicines(TCMs)and natural medicines are important sources of Nrf2 activators,lung disease prevention drugs and lead compounds.In this research,we investigated TCMs and natural medicines targeting Nrf2 signaling pathway,to find TCM extracts and active molecules with antioxidant activities and Nrf2 inducing effects,explore their mechanisms,and reveal the effectiveness of their prevention lung cells from oxidative damage.A total of 196 TCM extracts were collected from our research group.Their anti-oxidative activities were evaluated by the quinone reductase(QR)induction assay.NO production inhibitory assay was used to evaluate their anti-inflammatory activities.TCM extracts with MQI ≥ 1.3 under the tested concentrations were regared to be antioxidant extracts.TCM extracts with MIR>30%under the tested concentrations were regared to be anti-inflammatory extracts.39 kinds of TCM extracts displayed antioxidant activity;56 kinds of TCM extracts had anti-inflammatory activity;20 kinds of TCM extracts showed dual inhibiting effects against oxidative stress and inflammatory response.The establishment of a TCM extracts library laid the foundation for the systematic investigations of natural Nrf2-induced active molecules.Cinnamomum chartophyllum H.W.Li is a plant belongs to the family of Lauraceae.In our previous study,it was found that the EtOH extract of C.chartophyllum demonstrated Nrf2 inducing acitivity,and thirty chemical constituents were isolated from it.In the present research,investigations were performed as follows:(1)A high-throughput QR induction assay was used to evaluate the Nrf2 inducing effect of isolated compounds.The results showed that a sesquiterpenoid,3S-(+)-9-oxonerolidol(NLD,1),a biphenyl,3,3’,4,4’-tetrahydroxydiphenyl(THD,2),a lignan,and three flavonoids demonstrated dose-dependent QR inducing effects.(2)We adopted MDA-MB-231 cell line to confirm the inductions of Nrf2-mediated defensive system by NLD and THD.The results show that NLD and THD upregulated Nrf2 transcription,and actived Nrf2 and its downstream genes,NQO1 and y-GCS.NLD and THD are firstly found to be Nrf2 activators.(3)Experiments in vitro were performed using human bronchial epithelial Beas-2B cells to evaluate the Nrf2 inducing effects of NLD and THD.We have found that NLD and THD significantly activated Nrf2 and its downstream genes,NQO1 and γ-GCS,but had no effect on the protein expression of Keapl.Meanwhile,12.5 μM NLD and THD induced the translocation of Nrf2 and prolonged the half-life from 15 min to 25.8 min and 31.7 min respectively.(4)We found THD had three binding sites with Keap1 protein through molecular docking analysis.The three interactions were hydrogens at 1,2,1’-hydroxyl groups of THD to Leu365,Ser508,and Leu557 in Keapl,with the distances of 1.6 A,2.4 A,and 1.7 A.(5)As(Ⅲ)-induced oxidative damage model in Beas-2B cells was used to evaluate the protective effects of NLD and THD.The results showed that cell viability was significantly increased pretreatment with NLD and THD.NLD and THD treatments increased the levels of reduced GSH in Beas-2B cells and reduced the As(Ⅲ)-induced ROS levels,while NLD and THD themselves had no on ROS production.The results of AO/EB double staining and Annexin V-FITC/PI flow cytometry showed that NLD and THD had protective effects on As(Ⅲ)-induced Beas-2B cells injury.Our results implied the potential applications of NLD and THD against oxidative stress-related diseases(e.g.COPD,diabetes,cardiovascular diseases).Flavonoids possess diverse biological activities,such as antioxidant,anti-cancer,anti-inflammatory,cardioprotective,and neuropretective activities.We have collected 74 flavonoids.The results showed that eight flavones and flavonols,six dihydroflavones and dihydroflavonols,ten chalcones and dihydrochalcones,and an isoflavone demonstrated QR inducing effect.Of these,20 compounds were firstly discovered to be activators of Nrf2 pathway.In this experiment,(2S)-5,6,7,3’,4’-pentamethoxyflavanone(PMF)was selected as a representative of flavonoids and subjected to further investigations.(1)The mechanism of activating Nrf2 signaling pathway was investigated using normal human bronchial epithelial Beas-2B cells.The results showed that PMF could induce the protein expressions of Nrf2,NQO1 and y-GCS,upregulate the mRNA levels of NQO1 and GCLM,and enhance the nuclear translocation and stabilization of Nrf2.(2)The As(III)and CSE(cigarette smoke extracts)induced cell-based models were established to examine the capability of PMF against oxidative insults in Beas-2B cells.Cell viability was significantly increased pretreatment with PMF,and PMF reduced the ROS levels induced by As(III)and CSE.The results of flow cytometry with Annexin V-FITC/PI showed that PMF could protect Beas-2B cells injury induced by As(III)and CSE.Finally,we evaluated whether the protection of PMF against As(III)-induced cytotoxicity is an Nrf2-dependent manner.Immunoblot analysis verified the effectiveness of Nrf2 small interfering RNA(Nrf2-siRNA)on silencing the expression of Nrf2.Results showed that pretreatment with PMF significantly improved the survival of Beas-2B cells compared with the indicated doses of As(III)and CSE treatment alone;however,the PMF-mediated protection vanished in Beas-2B cells transfected with Nrf2-siRNA.These results suggested that PMF activated the Nrf2 pathway and had a potential preventive and therapeutic effect on lung injury induced by oxidative stress(such as COPD).Oxidative stress plays an important role in the pathological process of lung diseases,and activation of Nrf2 pathway is an effective mean to resist exogenous oxidative damage in lung tissue.In this study,we established a library of TCM extracts with Nrf2 inducing activity,investigated the Nrf2 inducing activity of components from Cinnamomum chartophyllum,illustrated the mechanism of Nrf2 activation by flavonoids,and tested the effectiveness of active molecules in inhibiting oxidative damage in human bronchial epithelial cells.The discovery of TCM extracts and molecules with inhibitory effect against oxidative stress provides directions for prevention and therapy of lung diseases targeting Nrf2 signaling pathway.