Effect Of Resveratorl On The Aging Of THP-1 Macrophages Induced By Hydrogen Peroxide And The Mechanism Of Autophagy

1.BackgroundAccording to the newly sixth census of China in 2010 year,the ageing populaion more than 60 years old has accounted for 13.26%of the total population in China.with the development of aging population,the cost of medical care for diseases which the older people are more likely to suffer from has increased.At present,The former 3 diseases with higher incidence are cardiovascular and cerebrovascular disease,being characteristic of atherosclerosis(AS),which has become the first killer for its higher incidence,higher fatality rate and higher disability rate to threaten the health of the ageing population.Epidemiology indicates that age is one of the independent risk factors for AS,and the incidence of AS increases with age evidently.Aging vascular changes include,mainly due to various cellular senescence,vascular elasticity decline,vascular structural changes,and the formation of AS plaques.However,the specific mechanisms of these aging changes are not yet clear.In recent years,many studies have been carried out by scientists both at home and abroad,and many theories of aging have been put forward.The changes of atherosclerosis mainly exist in various internal and external environmental factors in vassel,especially the damage of free radical damage.So we discuss the theory of free radical damage,represented by the damage of reactive oxygen species(ROS).ROS is a kind of active oxygen metabolite and its derivatives generated after single electron reduction of molecular oxygen,including hydrogen peroxide(H2O2),superoxide anion(O2-),hydroxyl(OH)and lipid metabolites.A large number of studies have shown that excessive ROS causes vascular lesions to play an important role in AS.Previous studies of ROS on cell senescence effect mainly focused on vascular endothelialcells,smooth muscle cells and fibroblasts.Macrophages play an important role in the development of atherosclerotic plaques,the aging changes of macrophage and the impact on AS research is relatively small.Recent studies have shown that removing aging macrophages in AS plaque of mouses can slow the progression of AS,we want to further investigate whether the ROS can accelerate the aging of macrophages or not.What kind of biology changes associated to the aging of macrophages and whether these aging cells may be liable to develop to AS?Resveratrol(Res)is a polyphenols plant antitoxin and has a wide range of biological activities and pharmacological effects.It can resist or delay diseases related to aging and oxidation.Many studies have shown that resveratrol can protect vascular endothelial cells,smooth muscle cells and cardiac muscle cells.And it can delay aging,reduce inflammatory factors release,and fight AS lesions.Resveratrol is an activator of silencing regulatory protein 1(sirtuin type 1,Sirtl).Sirtl protein is a mammalian homologue of yeast chromatin silencing factor Sir2.It has been proved to play an important role in maintaining gene stability,repairing DNA damage,and anti-aging,apoptosis and other life activities.Sirtl is also a NAD+dependent deacetylase,which can induce autophagy by regulating autophagy related proteins by deacetylation.Can resveratrol regulate the signal transduction pathway between aging and autophagy through Sirtl?At present,studies have found that Sirtl regulates the autophagy process of mang cells.Resveratrol can delay the aging of many cells.It is obvious that veratrol can indeed delay aging by autophagy.However,it is still necessary to further explore the molecular mechanism of resveratrol on autophagy and aging.Therefore,this research aims to find the effect of resveratrol on the aging of THP-1 macrophages induced by hydrogen peroxide and the mechanism of autophagyAnd we need further researches about inhibition of autophagy related signal pathways associated to aging and animal experiments.This may be a new way to treat atherosclerosis and other diseases of aging.2.Methods2.1 The effects of resveratrol on the viability of THP-1 macrophages induced by H2O2 and experimental groupsThe viability of THP-1 macrophages were determined by CCK-8 assay.After culturing the THP-1 cells with H2O2 of different concentrations for 24h,48h and 72h,the viability of THP-1 macrophages was measured by absorbance at 450nm by a multifunctional microplate reader spectrophotometrically.The terminal concentrations of H2O2 group was determinated according to the results.Before culturing the THP-1 cells with the terminal H2O2,resveratrol was added for 60min in advance.And the resveratrol concentration was determined according to the results.Experiments were carried out according to the following groups:(1)control group;(2)H2O2 group;(3)Res+H2O2 group;(4)Res group.2.2 Senescent detection of THP-1 macrophageThe degree of senescent THP-1 macrophages can be detecte by the related beta galactosidase(SA-β-GA)staining.It can revealed that senescent cells are stained blue but normal cells.2.3 The expression of senescent proteins by Western blots.We examined the expression of senedcence-related proteins by Western blots,including Rb protein and dephosphorylation Rb protein.Before measurament of tagert protein,totle protein needed to be extracted using protein issue extract and protein concentration also needed to be measured by BCA method.2.4 The expression of autophagy-related proteins by Western blots.We examined the expression of autophagy-related proteins by Western blots,including LC3 protein,Beclin 1 protein,p62 protein.Before measurament of tagert protein,totle protein needed to be extracted using protein issue extract and protein concentration also needed to be measured by BCA method.2.5 The relative fluorescence of LC3 using confocallaser-scanning microscopy.we evaluated the relative fluorescence of LC3 using confocallaser-scanning microscopy.LC3-stained punctate spots represente the formation of phagophore when LC3 proteins attached to it.2.6 Secretion of Inflammatory Factors in THP-1 Macrophages.The cell supernatant of each group individually was extracted.The secretion of the inflammatory factors were detected by ELISA kits,including TNF-α、IL-1β、IL-6.2.7 Statistical Analysis.All experiments were replicated at least three times independently.SPSS 22.0 statistical software was used.The data were presented as the mean±standard deviation and were analyzed using one-way analysis of variance(ANOVA).Statistical significance was defined as<0.05.1.Results3.1 The viability of THP-1 macrophages induced by H2O2CCK8 results showed that it may cause due to the high concentration cell apoptosis or necrosis;while low concentrations(50 umol/L,100 umol/L,200 umol/L)for 48h did not cause cell growth change comparing to control groups.This study selected 300 umol/L H2O2 for hours further to observe the possibility of THP-1 macrophage senescence.3.2 The effects of resveratrol on the viability of THP-1 macrophages induced by H2O2 and experimental groupsCCK8 results showed that 20 umol/L,40 umol/L,60 umol/L resveratrol all increased the viability of THP-1 macrophage,obviously 40 umol/L resveratrol.But the 80 umol/L resveratrol had the opposite result,which indicated that the appropriate concentration of resveratrol decreased the viability of THP-1 macrophage induced by H2O2 and delayed its senescence.Therefore,the experiment was carried out according to the following groups:(1)the control group;(2)300 umol/L H2O2 group;(3)40 umol/L Res+300 umol/L H2O2 group;(4)40 umol/L Res group.3.3 SA-β-gal staining of THP-1 macrophageCompared to the control group,the positive rate of SA-β-gal staining in H2O2 group increased significantly.After resveratrol intervention,the positive rate of SA-β-gal staining decreased,which indicated that resveratrol could improve cellular aging induced by H2O2.3.4 The expression of senescent proteins by Western blots.Compared to the control group,the expression of p-Rb protein incresaed and the expression of Rb protein in THP-1 macrophages decreased after H2O2 treatment.After the treatment of 40 mol/L Res in advance,the expression of pRb protein up-regulated and the expression of Rb protein were down-regulated.3.5 The expression of autophagy-related proteins by Western blots.Compared to the control group,the expression of Beclin 1 protein and the quantifications of the ratio decresaed and the expression of p62 protein increased after H2O2 treatment.After the treatment of 40 mol/L Res in advance,results were opposite.3.6 The relative fluorescence of LC3 using confocallaser-scanning microscopy.Compared to the control group,there was a greater number of LC3-stained punctate spots than those in H2O2 group,After the treatment of 40 mol/L Res in advance,there was a greater number of LC3-stained punctate spots than other groups.3.7 Secretion of Inflammatory Factors in THP-1 Macrophages.Compared to other groups,pretreatment with 40 mol/L Res decreased the secretion of TNF-α、IL-1β、IL-6,which shows resveratrol inhibited inflammatory reaction.4.Conclusions4.1 Hydrogen peroxide induced aging of THP-1 macrophages through oxidative stress and increased the secretion of inflammatory factors.4.2 Resveratrol may inhibit aging of THP-1 macrophages induced by hydrogen peroxide,reduce inflammatory reactin and delay the progression of atherosclerosis by promoting autophagy.