The Therapeutic Effect And Mechanisms Of Quercetin On The Hypoxia Induced Pulmonary Hypertension

Part I:The therapeutic effect of quercetin on the hypoxia induced pulmonary hypertensionObjective:In order to seek new ways for pulmonary hypertension treatment and therapeutic targets, experiments in vivo were engaged to explore the role of quercetin alleviate hypoxic pulmonary hypertension.Methods:30 male Sprague-Dawley rats were randomly divided into normal, hypoxia and hypoxia+quercetin group(each group of 10). Last hypoxia for four weeks, under the physiological permeameter monitoring, which through the determination of right heart floating catheter to detect rats systemic pressure, pulmonary arterial pressure, and the extent of right ventricular hypertrophy [Rv/(left ventricle plus septum)]; The expression of PCNA, Ki67 and TUNEL of each rat lung small artery were assessed by immunohistochemistry.Results:In this study, we found that quercetin prevented the development of PH, right ventricular hypertrophy, and vascular remodeling after exposure to hypoxia. Quercetin inhibited PASMC proliferation and increased the apoptosis of PASMCs in vivo.Conclusions:Our data suggest that quercetin is a potential candidate for the treatment of hypoxia-induced PH.Part II:Quercetin Reverses Hypoxia Induced Pulmonary Hypertension by Modulating the TrkA/AKT PathwayObjective:Further explore the underlying molecular mechanisms that quercetin inhibited hypoxic pulmonary arterial smooth muscle cell (PASMC) proliferation and induce cell apoptosis thus alleviated hypoxic pulmonary hypertension (PH), and is aim to clear whether autophagy have been involved.Methods:Isolated and cultured PASMCs, then setted up cell hypoxia model by Oxycycler model C21 hypoxic incubator. IC50 of quercetin were determined by MTT; Flow cytometry were uesed to detect the change of cell cycle and the number of apoptotic cells, Western-blot detect the cycle and apoptotic proteins relatively; Tanswell checked the effect of quercetin on migration of PASMCs in hypoxia; Using genome-wide microarray analysis filtrated PASMCs proliferation, apoptosis and migration of relevant regulation genetic changes under hypoxia condition.Results:In the present study, In vitro, quercetin significantly inhibited hypoxia-induced PASMC proliferation, arrested cells in G1/G0 and inhibited cell migration in a dose-dependent manner. Moreover, our results showed that quercetin increased cyclinD1 protein levels and decreased the protein expression of cyclinB1 and Cdc2. Additionally quercetin altered the Bax/Bcl-2 ratio and reduced MMP2, MMP9, CXCR4, integrin ?1, and integrin a5 expression. Using genome-wide microarray analysis, we found that factors regulating proliferation, apoptosis, cell cycle, and migration were related to the tyrosine receptor kinase A (TrkA) pathway. In addition, activation of the TrkA/AKT signaling cascade during hypoxia was inhibited by quercetin in a dose-dependent manner. Moreover, quercetin alone inhibited the TrkA/AKT signaling pathway, resulting in decreased PASMC migration, cell cycle arrest and the induction of apoptosis.Conclusions:Taken together, the mechanisms of quercetin reverseing PH may be by inhibiting TrkA/AKT signaling pathways, which regulating proliferation, apoptosis, cell cycle, and migration of PASMCs.Part III:Quercetin Induce Autophagy Via FOXO1-Depentent Pathways And Autophagy Suppression Enhances Quercetin-induced apoptosis of PASMCs in HypoxiaObjective:Further explore the underlying molecular mechanisms that quercetin inhibited hypoxic pulmonary arterial smooth muscle cell (PASMC) proliferation and induce cell apoptosis and is aim to clear whether autophagy have been involved.Methods:Isolated and cultured PASMCs, then setted up cell hypoxia model by Oxycycler model C21 hypoxic incubator. Flow cytometry, Western Blot and TUNEL cell immunofluorescence assessed the pro-apoptosis effects of quercetin; Electron microscopy, LC3 cell immunefluorescence observed cell morphological changes of autophagy before and after quercetin processing; Builded mRFP-GFP-LC3 double fluorescent lentivirus to observe the effects of quercetin on autophagy flow; Using siRNA technology targeted FOXO1 and SESN3 silenceing,3MA inhibited autophagy, to detect the effectsof quercetin on autophagy and the effects of autophagy in hypxia induced PASMC proliferation. Finally, Sprague-Dawley rats PH model established, on the basis of gavageing quercetin, feeding with autophagy inhibitors, and observed whether have superimposition effect.Results:In the present study, we found that quercetin promoted PASMC apoptosis and increased the expression of apoptosis related proteins. At the same time, quercetin aggrandized the autophagy which aroused by hypoxia, as well as expression level of autophagy regulating proteins. Further, it did enhance the formation of autophagy flux. Also, quercetin increased PASMC FOXO1 expression and transcription and activity in hypxoia. When inhibited FOXO1 by si-FOXO1, the phosphorylation of mTOR and its downstream target protein P70-S6K,4E-BPI increased, and significantly weaken the autophagy induced by quercetin. In addition, we observed that FOXO1-mediated autophagy is achieved via suppression of mTOR via elevating expression of the SESN3 gene not Rictor, inhibit SESN3 can effectively restrain the quercetin enhanced autophagy. Finally, we found that inhibition of autophagy markedly enhanced quercetin-mediated PASMCs apoptosis in vivo and in vitro.Conclusions:Taken together, our studies support the notion that combining quercetin and autophagy inhibitors may be a novel therapeutic strategy in PAH therapy.