| Stress-induced aging is premature aging,such as cigarette smoke-induced and doxorubicin-induced premature aging which was the common stimulus of stress-induced premature aging in vivo and in vitro,respectively.Chronic obstructive pulmonary disease(COPD)is an aging-related disease.Pulmonary vascular remodeling(PVR)which is closely related to aging is an important complication of COPD.Fisetin is one kind of flavonoids and has a wide range of biological effects.Numerous studies have confirmed that Fisetin has various effects of antioxidant and anti-aging so that it can alleviate the process of various diseases.This study was to investigate the relationship between stress-induced premature aging and pulmonary vascular remodeling,and the effect of anti-aging of Fisetin on pulmonary vascular remodeling.Part 1 To explore the relationship between stress-induced premature aging and pulmonary vascular remodeling ObjectiveIn the study of lung diseases in vivo,smoking is a common cause of stress-induced premature aging in lung tissue which involved a variety of molecular mechanisms of DNA damage.The Nrf2/ARE pathway is the most important antioxidant pathway by inhibiting oxidative stress and protecting DNA from damage.Cigarette stimulation can lead to changes in pulmonary vascular structure.Therefore,we explore the relationship between stress-induced premature aging and pulmonary vascular remodeling in this part of experiment and the expression of Nrf2/ARE.Methods Between the group of current smokers and the group of lifelong non-smokers,there was no significant difference of age and the pulmonary function.The degree of proliferation of smooth muscle cells in the current smokers was significantly higher than that in the lifelong non-smokers.The expression of gene of Nrf2/ARE was detected by total RNA and QPCR methods in clinical trials.The expression of P53,P21,P16 and Nrf2/ARE were detected by Western blot in the lung tissue of experimental rats.Results Western blot analysis indicated that the expression of P53,P21 and P16 in the lung tissue of the smoking rats was significantly higher than that in the untreated group and the expression of protein of Nrf2/ARE was decreased in the smoking rats.In addition,QPCR showed that the expression of antioxidant factors of Nrf2 and NQO-1 were decreased in lung tissue of current smokers,while there was no significantly change of the expression of HO-1 in the study of human clinical specimens.Conclusion The development of pulmonary vascular remodeling was associated with stress-induced premature aging and the decrease of Nrf2/ARE.Part 2 To study the anti-aging effect of Fisetin in pulmonary vascular remodelingObjective Fisetin is one kind of flavonoids and has a wide range of biological effects,mainly exists in a large numbers of vegetables and fruits.Numerous studies have confirmed that Fisetin has various effects of antioxidant and anti-aging.Aging-associated secretory phenotypes(SASP),which is derived from aging cells,contains all kinds of inflammation and cell growth factors and can promote the proliferation of pulmonary artery smooth muscle cells so that the pulmonary vascular was remodeled.Doxocubicin,which is more stable and effective than cigarette smoke extract,is more widely used in the establishment of aging model of cells and is a stimulus of premature aging as same as the cigarette smoke extract.Therefore,we study Fisetin-induced inhibition of doxorubicin-induced premature aging in pulmonary vascular remodeling in this part of experiment.Methods Human pulmonary arterial endothelial cells(HPAECs)and human pulmonary artery smooth muscle cells(HPSMCs)were cultured.The effects of Fisetin(5?M,10?M,20?M,30?M,50?M,100?M)on the proliferation of HPAECs were detected by CCK-8.HPAECs elicited senescence response after exposure to doxorubicin.The senescence-related ?-galactosidase activity staining,the protein levels of P53/P21 and P16 and the cell cycle observed by flow cytometry were used to analysis the senescence activity in HPAECs.The expression of HO-1,the nuclear transfer of Nrf2 and the phosphorylation of P38 MAPK pathway were detected by Western blot.The molecular mechanism of Fisetin on anti-aging effect of cells was analyzed by HO-1 transient silencing si RNA and P38 inhibitor SB203580.Finally,HPASMCs were cultured in conditioned medium of different treatment groups of HPAECs,and the proliferation of HPASMCs was measured by CCK-8.Results The results of CCK-8 showed that Fisetin had no obvious influence on cellular viability or proliferation when the concentration was less than or equal to 20?M in human pulmonary artery endothelial cells,the difference was not statistically significant(P>0.05).In addition,the assay of senescence-related ?-galactosidase staining confirmed that Fisetin reduced doxorubicin-induced senescence staining in a dose-dependent manner.Flow cytometry showed that the percentage of S phase in pre-stimulation of Fisetin group was significantly lower than that in doxorubicin group.And Fisetin decreased the protein expression of P53/P21 and P16 in a certain extent.In addition,Fisetin promoted the phosphorylation of P38 and the protein expression of Nrf2 and HO-1.The transient silencing of HO-1 also reversed the anti-aging effect of Fisetin in HPAECs.At the same time,the P38 MAPK inhibitor SB203580 partially eliminates anti-aging effect induced by Fisetin.Finally,the conditioned medium of Fisetin-treated groups reduced the proliferation of human pulmonary artery smooth muscle cells(HPASMCs)compared to the conditioned medium of the doxorubicin-treated group in HPAECs.Conclusion The effect of Fisetin on the senescence of pulmonary artery endothelial cells reverse the premature senescence of cells through the promotion of P38 MAPK pathway and the activity of antioxidant gene of Nrf2/HO-1 so that the proliferation of pulmonary artery smooth muscle cells was declined and the process of pulmonary vascular remodeling was alleviated.This provides a potential reference value for the further study of aging-related pulmonary vascular diseases. |
PDF Full Text Request