A Study On Rejuvenating The Internal Environment To Ameliorate The Heart Aging In Elder Mice And The Mechanism Analysis Of Cytokines Affecting Heart Aging

Objective:Cardiovascular disease is the main cause of health problems in aging society.Aging is a major risk factor of cardiovascular disease,which exists for a long time in the occurrence and development of cardiovascular disease.The typical characteristics of cardiac aging are cardiomyocyte hypertrophy,myocardial interstitial fibrosis,inflammatory cell infiltration and persistent myocardial stiffness;These changes are caused by the basic functional changes of myocardial cells and myocardial interstitial cells.It is very important to detect the key facors in the occurrence and pathogenesis of cardiac aging.To explore the key factors in the process of cardiac aging may provide useful clues for clinical treatment.Our study found that the internal environment has an important impact on the cardiac aging process.Changes of the internal environment can ameliorate or reverse the cardiac aging process.However,the effect and mechanism of young internal environment on aging myocardium are still unclear.On the base of our previous research,we put forward a scientific hypothesis:there may be some humoral factors that rejuvenate or reverse organ aging in the internal environment,which can ameliorate or reverse organ aging by regulation the expression of autophagy and inflammatory cytokines genes.We used parabiosis animal models to establish the shared blood circulation between old and young mice,further verify whether the youthful internal environment can ameliorate the heart aging of old mice,and explore the mechanism of humoral cytokines affecting heart aging.Methods:Part 1:On the basis of previous work,C57BL/6 mice aged 3 months and 22 months were selected to establish the young-old parabiosis animal model and the old-old parabiosis animal model.Groups in the research of the internal environment on cardiac aging:Group 1 was the young single control 8 mice(Y-con);Group 2 was the old single control 8 mice(O-con);Group 3 was the old-old parabiosis 8 pairs(isochronic parabiotic,O-IP)(n=16);Group 4 was the young-old parabiosis 8 pairs(heterochronic parobiotic,Y-O HP).After 5 weeks of parabiosis,all mice were sacrificed.Peripheral blood samples and heart tissue samples were collected.The myocardium tissures were PAS stained?Massion stained and WGA stained.Autophagy indicators LC3,p62 and inflammatory indexes IL-1,IL-6,pNF?B and NF?B were measured by western blot.We observe the effect of youthful internal environment changes on cardiac aging.Part 2:Using high-throughput screening methods in the blood circulation system cytokine chip to analyze the serum of 3-5 mice in the above groups,we filter out the significant different cytokines;then further explore the mechanism of cytokines affecting heart aging.We have found out significant different circulation cytokines that may be closely associatied with heart aging,included IGF-1,SCF,Thrombopoietin(TPO),Eotaxin-1(CCL11),GM-CSF,CD30Ligand.We choose SCF to further explore the key factors in the occurrence and the pathogenesis of aging and elderly heart disease.Stem cell factor(SCF),the ligand of the c-KIT receptor,actively participates in the organ reconstruction and fibrosis associated with various diseases,including cardiac disease.We selected 6-week-old c-KIT mutant mice(c-KIT^MUT)and C57BL/6 mice of the same week age to receive continuous subcutaneous infusion of Ang? or saline for 4 weeks.The experimental animals were divided into four groups:Group 1 was the C57BL/6 angiotensin ?(Ang?)pump embedding group(n=8);Group 2 was the C57BL/6 control group with saline pump embedding group(n=5);Group 3 was the c-KIT^MUT Ang? group(n=8);Group 4 was the c-KIT^MUT saline pump control group(n=5).Blood pressure and cardiac function were measured at 0?2 and 4 weeks respectively.After 28 days of Ang? or saline infusion,blood and myocardial tissue samples were collected.The myocardium tissure protein expression was measured by western blot,included that AMPK?,IGF-1R,mTOR,LC3 and p62.Results:Part 1:1.With the influence of youthful systemic milieu,cardiomyocyte hypertrophy and myocardial fibrosis in the young-old parabiosis animal model group were significantly ameliorated.Under the electron microscope,autophagosomes increased significantly in the young-old parabiosis group,autophagy level increased and inflammatory response decreased in the young-old parabiosis model group;The expression of autophagosome and LC3 were increased,p62 was down regulated,and the expression of NF-?B and IL-6 decreased(P<0.01).Part 2:1.In the previous study,six kinds of differential circulation factors were screened out by cytokine microarray.Stem cell factor was selected for the deeply study.We selected 6-week-old c-KIT gene mutant mice and C57BL/6 mice of the same age to complete the implantation of Angiotensin ? or saline micropump,and observed the changes of blood pressure?cardiac function and myocardial tissue protein in the four groups.2.Compared with C57BL/6 mice of the same age,c-KIT gene mutant mice had increased blood pressure and decreased cardiac function at 2 and 4 weeks after Ang ? infusion(P<0.01).3.Compared with C57BL/6 mice of the same age,the degree of cardiomyocyte hypertrophy and myocardial fibrosis in c-KIT^MUT mice were significantly aggravated after Ang ? infusion for 28 days(P<0.01).4.Compared with C57BL/6 mice in Ang ?group,the expression of LC3?/? was decreased and the expression of autophagy degradation marker P62 was increased in c-KIT^MUT Ang? group.Due to the loss of c-KIT protein function,the activation level of AMPK? decreased,and the expression activity of mTOR and IGF-1R increased.Conclusion:1.We used the established parabiosis mouse model to study the aging of conjoined mouse hearts.We found that the youthful systemic milieu environment can significantly enhance the autophagy of myocardial tissue,low down the level of apoptosis and inflammation,reduce the hypertrophy of myocardial cells and myocardial interstitial fibrosis 2.After silencing SCF/c-KIT signaling pathway,cardiomyocyte hypertrophy and myocardial fibrosis were significantly aggravated(P<0.01).We speculate that the youthful systemic milieu environment can increase the expression of SCF/c-kit in the aged heart,thus delaying and alleviating the myocardial injury in the ageing mice.3.SCF/c-kit pathway delays cardiac aging by up regulating AMPK-? expression and inhibiting mTOR pathway and IGF1R signaling pathway.