| BackgroundInflamm-aging is a phenomenon that aging promotes the development of chronic,persistent low-grade inflammation in many of the body’s tissues and organs.Inflamm-aging is believed to be an important contributor to the adverse outcomes of aging,including the development of age-related diseases such as arthritis,diabetes,sarcoma,cardiovascular disease(CVD),cancer and dementia.Although there are some strong epidemiological evidences that high levels of pro-inflammatory markers in the elderly are associated with the risk of most typical aging-related diseases and a variety of chronic diseases,the specific mechanisms of the pro-inflammatory state in the elderly and the link between inflammation and chronic disease are not yet clear.In our study,inflammation is taken as the core mechanism of aging.The role of inflammatory pathway PGF2α in cardiac interstitial fibrosis through FP receptor was discussed,so as to clarify the specific causes of the occurrence and development of cardiovascular diseases caused by aging.In addition,we further studied the molecular biological mechanism of Inflamm-aging by taking adipose tissue,the birthplace of inflammatory cytokines,as the research object,in order to provide new ideas for promoting the health of the elderly and intervening in the aging process.Firstly,epidemiological studies show that with the acceleration of population aging,the incidence of cardiovascular diseases are increasing year by year worldwide.The latest research shows that the aging of the Chinese population is the main driving factor for the increase of cardiovascular disease mortality(64.11%).Heart tissue is mainly composed of cardiomyocytes and fibroblasts while cardiac fibroblasts account for 2/3 of the total number of cells.The first manifestation of cardiac aging is cardiac fibroblast proliferation.Cardiac fibroblasts are the key cells for the maintenance of cardiac structure and function.In healthy adult hearts,collagen secretion and production by cardiac fibroblasts is a homeostasis process,while in elderly hearts,cardiac fibroblasts are activated and acquire a pro-fibrotic phenotype,leading to interstitial fibrosis,ventricular stiffness and diastolic dysfunction,which may lead to the development and progression of heart failure.Autophagy,as an important mechanism for effectively removing and replacing damaged proteins and organelles,is very important for maintaining cell homeostasis and prolonging cell life.It has been reported that the level of autophagy decreased significantly in the aging heart,and the increase of autophagy inhibited age-induced apoptosis,cardiac hypertrophy and fibrosis,so as to delay cardiac aging.Relevant studies have confirmed that activation of autophagy in cardiac fibroblasts can effectively inhibit inflammation and cardiac fibrosis induced by Angiotensin Ⅱ(Ang Ⅱ).Intraperitoneal injection of rapamycin improved AngⅡ-induced cardiac fibrosis and cardiac dysfunction.Therefore,increased levels of autophagy can inhibit inflammation and reduce excessive deposition of collagen in the heart,thus playing a key role in delaying the process of cardiac fibrosis and cardiac aging.Prostaglandin(PG)is a metabolite of arachidonic acid under the action of cyclooxygenase,including PGD2,PGE2,PGF2α,PGI2 and thromboxane A2(TXA2).Related studies have shown that the plasma levels of PGF2α in patients with sepsis increase and inhibiting the PGF2α-specific cell membrane surface receptor FP can reduce systemic inflammation by enhancing the production of the anti-inflammatory factor Interleukin-10(IL-10).In addition,PGF2α binding to FP receptor has been shown to directly act on lung fibroblasts to promote their proliferation and collagen synthesis independently of the TGF-βclassical pathway,leading to pulmonary interstitial fibrosis.Relevant studies have confirmed that with the increase of age,the level of PGF2α in human body also increases.Previous studies in our group have confirmed that FP receptor can promote vascular smooth muscle aging by up-regulating Src/PAI-1 signaling pathway,but the role of FP receptor in cardiac aging has not been reported.Therefore,our study proposed that in the hearts of aging mice,activation of FP receptor promotes the decrease of autophagy level of heart cells,aggravates the degree of cardiac fibrosis,leading to cardiac aging and damage to cardiac structure and function.In view of the above hypotheses,this study explored the specific role and mechanism of PGF2α,an inflammatory pathway,in cardiac aging at the global and cellular levels through FP receptor.Further relevant studies have confirmed that visceral fat inflammation exists in the aging body,which may be closely related to abnormal aggregation of macrophages in adipose tissue.Adipose tissue macrophages(ATM)is composed of at least two different phenotypes,namely classically activated M1 macrophages and selectively activated M2 macrophages.M1 macrophages can produce high-level proinflammatory factors,reactive nitrogen and reactive oxygen intermediates,and have strong bactericidal and tumor killing activities.On the contrary,M2 macrophages have strong phagocytosis,high expression of scavenger molecules,and expression of mannose and galactose receptors,which can produce high levels of anti-inflammatory factors.M1 and M2 macrophages can be interconverted,Compared with the simple increase in the number of macrophages,the transformation of adipose tissue M2 to M1 macrophages and the increase of M1/M2 macrophage ratio further aggravate the visceral adipose tissue inflammation.Growth arrest-specific protein 6(Gas6)is a secretory protein encoded by growth arrest-specific gene 6,Axl is widely distributed in a variety of tissues,organs and cells,and has the strongest binding force to Gas6,so it is often referred to as the Gas6/Axl pathway.Previous studies have found that Gas6/Axl pathway is closely related to inflammation in adipose tissue in overweight and obese adults.Previous studies of our research group have also confirmed that Gas6/Axl pathway plays a key role in the delay of vascular aging by testosterone.As an important downstream signaling molecule of Gas6/Axl pathway,NF-κB has been demonstrated to promote the development of macrophage polarization in the pathogenesis of immune-mediated glomerulonphritis.Therefore,by constructing WT and Axl-/-natural aging mouse models,this study observed the effect of Axl gene knockout on fat aging,and explored the role of Gas6/Axl pathway in improving fat aging by reducing fat inflammation and the possible mechanism.Dissertation ⅠEffects and Mechanism of FP Receptor in Aging HeartObjectives1.To investigate whether FP receptor affects cardiac fibrosis and cardiac senescence.2.To investigate the possible mechanism of FP receptor on cardiac fibrosis and cardiac aging.3.To investigate the effect of FP receptor on collagen synthesis in cardiac fibroblasts.4.To investigate the possible mechanism of FP receptor promoting collagen synthesis in aging CFs.Methods1.Group of experimental animals6-week-old male C57BL/6J wild-type(WT)mice were randomly divided into four groups:Young,Old,Old+vehicle,Old+FP receptor shRNA(short hairpin RNA,shRNA).After being reared to 12 months of age,the mice were injected with lentivirus into the tail vein to induce gene silencing to establish an Old+vehicle group and an Old+FP receptor shRNA group.The mice in the young group were raised to the age of 3 months,and the mice in the old group were raised to the age of 18 months.2.Group of vitro experimentsMouse primary cardiac fibroblasts were extracted,and the aging model of CFs induced by the optimal concentration of D-galactose was established at the optimal time point.CFs were divided into four groups:Control,Old,Old+AL8810(FPrecptor inhibitor,AL8810),Old+AL8810+MHY1485(mTOR activator,MHY1485),Old+AL8810+740Y-P(PI3K activator,740Y-P).3.The changes of blood pressure,cardiac structure and function were detected by blood pressure meter and echocardiography.4.The changes of relative telomere length in mouse heart tissue were detected by real-time PCR.5.The changes of cardiac morphology and fibrosis level were detedcted by HE staining,Masson staining,Sirius red staining and Immunohistochemistry.6.Cardiac fibroblasts were identified by vimentin staining.7.The changes of aging index,autophagy level,fibrosis level and signal pathway in mouse heart tissue and CFs were detedcted by Western blotting.8.SPSS 25.0 statistical software was used for statistical analysis.Results1.Establishment of natural aging mouse modelCompared with the Young group,the mice of Old group had sparse hair,dull color,slow movement,and an upward trend in body weight,and the protein expression levels of GLB-1,P53,P21 and P16 in heart tissue were significantly increased and the relative telomere length was significantly shortened,which proved that the natural aging mouse model was successfully established.2.The blood pressure and systolic and diastolic functions of heart of aging mice increased significantlyCompared with the Young group,the systolic blood pressure(SBP)and mean blood pressure(MBP)of mice in the Old group were significantly increased.Compared with the Young group,for the mice of Old group,their left ventricular end diastolc diameter(LVEDd)was significantly increased,the left ventricular ejection fraction(LVEF)and fractional shortening(FS)were significantly decreased,and the E/e’ was significantly increased.3.Cardiac interstitial fibrosis is significantly increased in aging miceCompared with Young group,a series of pathological changes occurred in the heart of mice of Old group,including left ventricular dilation,widening of cell space and disordered arrangement of cardiomyocytes.The volume fraction of collagen and the contents of type I and type III collagen were significantly increased.4.FP receptor expression levels are associated with cardiac interstitial fibrosis in aging miceCompared with the Young group,the expression level of FP receptor protein in the heart tissue of the mice of Old group was significantly increased.The collagen volume fraction in mice heart tissue was positively correlated with the expression level of FP receptor protein.5.FP receptor gene silencing can reduce cardiac aging markers and interstitial fibrosis in miceCompared with the Old+vehicle group,the protein expression levels of GLB-1,P53,P21,and P16 in the heart tissue of mice of the Old group+FP receptor shRNA group were significantly decreased,and the relative telomere length was significantly prolonged.Compared with the Old+vehicle group,the collagen volume fraction,type I and type III collagen contents in the heart tissue of mice in the Old group+FP receptor shRNA group were significantly decreased.6.FP receptor gene silencing can improve blood pressure and cardiac function in aging miceCompared with the Old +vehicle group,the systolic blood pressure(SBP)and mean blood pressure(MBP)of mice in the Old+FP receptor shRNA group were significantly decreased.Compared with the Old+vehicle group,in the mice of the Old+FP receptor shRNA group,the left ventricular end diastolc diameter(LVEDd)of was significantly decreased,and the left ventricular ejection fraction(LVEF)and fractional shortening(FS)were significantly increased,and E/e’ was significantly decreased.7.FP receptor gene silencing may delay cardiac aging in mice by increasing autophagy levelCompared with the Young group,in the heart tissue of mice of the Old group,the LC3B/A ratio was significantly decreased,and the P62 protein expression level was significantly increased.Compared with the Old+vehicle group,in the heart tissue of mice of the Old+FP receptor shRNA group,LC3B/A ratio was significantly increased,and the P62 protein expression level was significantly decreased.8.FP receptor gene silencing may regulate autophagy through PI3K/AKT/mTOR signaling pathwayCompared with the Young group,the phosphorylation levels of PI3K,AKT and mTOR in the heart tissue of mice in the Old group were significantly increased.Compared with the Old+vehicle group,the phosphorylation levels of PI3K,AKT and mTOR in the heart tissue of mice in the Old+FP receptor shRNA group were significantly decreased.9.Effect of FP receptor inhibitor AL8810 on the expression of FP receptor in aging CFsSince the protein expression levels of GLB-1 and P21,the aging indexes of CFs,were significantly increased at 10g/L and 48h stimulation,the optimal concentration and time of D-galactose induced CFs aging were taken as the optimal concentration and time of D-galactose.Compared with the control group,the protein expression level of CFs FP receptor in aging group was significantly increased.Compared with aging group,the protein expression level of CFs FP receptor in aging+AL8810 stimulation group was significantly decreased.10.Effect of FP receptor inhibitor AL8810 on collagen expression and autophagy level in aging CFsCompared with the control group,the protein expression levels of type I and III collagen in CFs of aging group were significantly increased.Compared with aging group,the protein expression levels of type I and III collagen in CFs of aging+AL8810 stimulation group were significantly decreased.Compared with the control group,the LC3B/A ratio of CFs in aging group was significantly decreased,and the expression level of P62 protein was significantly increased.Compared with the aging group,the LC3B/A ratio of CFs in the aging+AL8810 stimulation group was significantly increased,and the expression level of P62 protein was significantly decreased.11.Effect of FP receptor inhibitor AL8810 on PI3K/AKT/mTOR signaling pathway in aging CFsCompared with the control group,the phosphorylation levels of PI3K,AKT and mTOR in CFs of aging group were significantly increased.Compared with the aging group,the phosphorylation levels of PI3K,AKT and mTOR in CFs in the aging+AL8810 stimulation group were significantly decreased.12.The role of mTOR activator MHY1485 and PI3K activator 740Y-P in regulating PI3K/AKT/mTOR phosphorylationCompared with aging+AL8810 stimulation group,the phosphorylation levels of PI3K,AKT and mTOR in aging+AL8810+MHY1485 stimulation group were significantly increased.Compared with the aging+AL8810 stimulation group,the phosphorylation levels of PI3K,AKT and mTOR in the aging+AL8810+740Y-P stimulation group were significantly increased.13.The role of PI3K/AKT/mTOR signaling pathway in regulating autophagy level and collagen expression in aging CFsCompared with aging+AL8810 stimulation group,aging+AL8 810+MHY1485 stimulation group showed significantly increased protein expression levels of type I and III collagen.Compared with aging+AL8 810 stimulation group,aging+AL8810+740Y-P stimulation group showed significantly increased protein expression levels of type I and III collagen.Compared with the aging+AL8810 stimulation group,the LC3B/A ratio of CFs in the aging+AL8810+MHY1485 stimulation group was significantly decreased,and the level of P62 protein surface was significantly increased.Compared with the aging+AL8810 stimulation group,the LC3B/A ratio of CFs in the aging+AL8810+740Y-P stimulation group was significantly decreased,and the level of P62 protein surface was significantly increased.Conclusions1.The expression of FP receptor was significantly increased,autophagy level was decreased,interstitial fibrosis was increased,and left ventricular systolic and diastolic functions were significantly decreased in aged mice.2.FP receptor gene silencing significantly inhibited cardiac aging and interstitial fibrosis,and significantly improved left ventricular systolic and diastolic functions in aging mice.FP receptor gene silencing may up-regulate CFs autophagy level by inhibiting PI3K/AKT/mTOR signaling pathway,reduce cardiac interstitial fibrosis,and delay cardiac aging.Dissertation ⅡEffects and Mechanism of Gas6/Axl Pathway in Macrophage Polarization of Aging Adipose TissueObjectives1.Establish a mouse model of natural aging,and explore the role of inhibiting Gas6/Axl pathway in delaying visceral adipose tissue inflammation and aging.2.To determine whether inhibition of Gas6/Axl pathway can inhibit visceral adipose inflammation and adipose senescence by regulating macrophage polarization in visceral adipose tissue.3.To clarify the molecular mechanism of Gas6/Axl pathway in regulating the polarization of macrophages in visceral adipose tissue in aging state.Methods1.Establishment of mouse aging model40 male WT mice and 40 male Axl-/-mice were randomly divided into 2 groups:young group(n=20,3 months old),old group(n=20,18 months old).The mice of young group were fed to 3 months old,and the mice of old group were fed to 18 months old.2.HE staining and immunohistochemical techniques were used to detect the changes of adipose tissue morphology,macrophages,pro-inflammatory factors and anti-inflammatory factors of mouse epididymis.3.Western blotting technique was used to detect the changes of aging indexes and signaling pathways in mouse epididymal adipose tissue.4.SPSS 25.0 statistical software was used for statistical analysis.Results1.Alteration of Gas6 and Axl expression in visceral fat of aging miceCompared with the WT and Axl-/-young groups,the protein expression level of Gas6 in epididymal adipose tissue of mice of the WT and Axl-/-old groups was significantly decreased.Compared with the WT young group,the protein expression level of Axl in epididymal adipose tissue of mice in the WT old group was apparently decreased.The protein expression of Axl in epididymal adipose tissue of mice in Axl-/-gene knockout groups were at low levels.2.Axl gene knockout aggravate visceral fat senescence in aging miceCompared with the WT and Axl-/-young groups,the protein expression levels of senescence indexes,GLB-1,P21,and P16 in epididymal adipose tissue of mice in the WT and Axl-/-old groups were markedly increased.Compared with the WT old group,the protein expression levels of GLB-1,P21 and P16 in epididymal adipose tissue of mice in the Axl-/-old group were considerably increased.3.Axl gene knockout aggravate visceral fat morphology alteration in aging miceCompared with WT and Axl-/-young groups,epididymal adipocytes in mice of the WT and Axl-/-old group were different in size and arranged in disorder.Compared with the WT old group,the size of epididymal adipocytes in mice of the Axl-/-old group was different,and the disorderly arrangement was more obvious.4.Axl gene knockout aggravate visceral adipose macrophage polarization in aging miceCompared with WT and Axl-/-young groups,the protein expression levels of macrophages,F4/80,CD11c and CD206 in epididymal adipose tissue of mice in the WT and Axl-/-old group were significantly increased,and the M1/M2 ratio was obviously increased,however,the degree of increased expression of CD206 in adipose tissue in each group was less than that of CD11c.Compared with the WT old group,the protein expression levels of F4/80 and CD11c in the epididymal adipose tissue of mice in the Axl-/-old group were greatly increased,and the M1/M2 ratio increased more significantly.5.Axl gene knockout aggravate visceral fat inflammation in aging miceCompared with WT and Axl-/-young groups,the protein expression levels of pro-inflammatory factors,MCP-1,TNF-α,IL-1 and IL-6 and anti-inflammatory factor IL-10 in epididymal adipose tissue of mice of the WT and Axl-/-old groups were significantly increased,but the increase of IL-10 expression in adipose tissue in each group was less than that of the pro-inflammatory factors.Compared with WT old group,the protein expression levels of pro-inflammatory factors,MCP-1,TNF-α,IL-1 and IL-6 in epididymal adipose tissue of mice in the Axl-/-old group increased more significantly.6.Axl gene knockout may regulate visceral fat senescence in aging mice via NF-κB signaling pathwayCompared with WT and Axl-/-young groups,NF-κB phosphorylation levels of epididymal adipose tissue in mice of the WT and Axl-/-old group were obvioulsy increased.Compared with the WT old group,the NF-κB phosphorylation level of epididymal adipose tissue in mice of the Axl-/-old group was greatly increased.Conclusions1.The proteins expression levels of Gas6 and Axl in visceral adipose tissue of aging mice decreased.2.The expression of F4/80,CD11c,and CD206 in the visceral adipose tissue of aging mice increased,and the ratio of M1/M2 increased,and this changes were enhanced after Axl gene knockout,suggesting that the Gas6/Axl pathway is involved in macrophage polarization in visceral adipose tissue of aging mice.3.Axl gene knockout promotes visceral adipose tissue macrophage polarization and enhances visceral adipose tissue inflammation in aging mice,and it works possibly by affecting NF-κB signaling pathway.Dissertation ⅢInteraction of SUMO4 Gene SNP rs237025 and Weight Management on the Risk of MetSObjectives1.Through the collection of clinical data of the interviewed population and the detection of gene mutation at rs237025 locus,the effect of SNP rs237025 on the risk of MetS disease was studied,and the effect of SNP rs237025 on the risk of each component of MetS in the population was studied.2.Through a 5-year follow-up of the interviewed population,the effect of SNP rs237025 on the changes of clinical data of the interviewed population was studied,and the interaction between weight management and rs237025 and its impact on MetS were analyzed.MethodsIn this study,the Han population in Shandong Province,China was selected as the respondents.The target population was interviewed from January to December 2007.We first randomly selected 1 village and 1 city from Shandong Province,and randomly selected 2 communities in the selected village and city respectively,and then randomly selected an adult individual from each family in the community for investigation.A total of 1047 control group respondents and 1008 MetS group respondents were finally recruited.The diagnosis of MetS was carried out with reference to the diagnostic criteria jointly developed by IDF and AHA/NHLBI in 2009.Questionnaire survey,physical sign measurement,and blood sample collection were all carried out by professional investigators.The questionnaire included detailed information such as date of birth,past medical history,medication history,smoking and drinking history.Height,weight and waist circumference were measured repeatedly by 2 investigators and averaged.The blood pressure of the right arm after a 5-minute rest in the sitting position was measured by using an Omron HEM-7011 electronic sphygmomanometer,and for each respondent,3 consecutive values were recorded and the average was calculated.The fasting blood samples of the respondents in the morning were collected,and immediately sent the samples to a standardized laboratory for testing blood sugar,blood lipids and other biochemical indicators,and DNA extraction was performed.The genotype of rs237025 was detected by the Sequenom MassArray Genotyping System.We provide clinical advice and guidance to patients diagnosed with MetS and carrying risk factors for MetS during the survey.The follow-up survey method after 5 years was the same as before.A total of 269 respondents in the control group and 871 respondents in the MetS group participated in the follow-up after 5 years.The difference of each index in follow-up after 5 years was calculated by subtracting the cross-sectional value from the follow-up value and recorded as "Δ".Continuous variables are expressed as mean plus or minus standard deviation(x±s)and tested by independent sample t or compared by one-way ANOVA.The χ2 goodness-of-fit test was used to test the Hardy-Weinberg equilibrium at rs237025 locus of SUMO4 gene.The risk factors of MetS and MetS components were tested by multivariate logistic regression analysis.Cross-over analysis was used to analyze the effect of the interaction between two independent variables on the dependent variable.All statistical analyses were performed by using SPSS 26.0.A two-tailed P value less than 0.05 was considered statistically significant.Results1.Baseline comparison between control group and MetS groupThere were no significant differences in gender and age between subjects in the control group and MetS group.Compared with the control group,in the MetS group,body weight,waist circumference(WC),body mass index(BMI),systolic blood pressure(SBP),diastolic blood pressure(DBP),triglycerides(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-c),fasting plasma glucose(FPG)levels were significantly increased,and high density lipoprotein cholesterol(HDL-c)level was greatly decreased.The distributions of AA,AG,GG genotypes and A and G alleles at rs237025 in the control group,MetS group and overall subjects were in line with Hardy-Weinberg genetic equilibrium.2.Comparison of clinical data of SNP rs237025 genotypesThe TG of subjects with GG genotype at rs237025 of SUMO4 gene in the control group was significantly higher than that of subjects with AG/GG genotypes,while in the MetS group,WC and FPG of subjects with GG genotype were significantly higher than those of AG/GG genotype subjects.3.Relationship between SNP rs237025 and MetS and MetS components riskThe AA/AG and GG genotype distribution of rs237025 of SUMO4 gene were significantly different between the control group and the MetS group.The five components of MetS were studied separately,the genotypes of AA/AG and GG genotype at rs237025 were found that there are obvious differences between the WC normal group and the WC increased group,between the TG normal group and the TG elevated group,and between the FPG normal group and the FPG elevated group.Further studies showed that the rs237025 mutation was an independent risk factor for MetS,and the rs237025 mutation was also an independent risk factor for increased WC and increased FPG.4.Changes in baseline characteristics after 5 years of follow-up subjectsBecause Mets patients received lifestyle intervention and drug treatment in the past five years,compared with the control group,the decline range of weight,WC,SBP,DBP,TG,TC,LDL-c,HDL-c and FPG of MetS group increased significantly after five years.The rs237025 mutation had no significant difference in the changes of new MetS and MetS components.5.Interaction of SNP rs237025 with weight managementThe interaction of SNP rs237025 with weight management is an independent risk factor for new-onset MetS.The changes of MetS components were studied separately,and the interaction between SNP rs237025 and weight management was an independent risk factor for new-onset WC increase,blood pressure increase,HDL-c decrease and FPG increase.The cross analysis under the logistic regression model showed that the synergistic effect of SNP rs 237025 and weight management on WC,TG and HDL-c was negative.Conclusions1.SUMO4 gene rs237025 polymorphism is an independent risk factor for MetS,and the rs237025 mutant carries a significant increase in the number of MetS components.2.SUMO4 gene rs237025 polymorphism is an independent risk factor for increased WC,increased TG and increased FPG.3.The interaction between the SUMO4 gene rs237025 polymorphism and weight management is an independent risk factor for new-onset MetS,as well as an independent risk factor for increased WC,increased blood pressure,decreased HDL-c,and increased FPG.rs237025 wild-type carriers were more likely to have increased WC,increased blood pressure,decreased HDL-c,and increased FPG in poor weight management. |
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