Intervention Effect Of Testosterone On The Cardiac Fibrosis During Aging Process Of Heart

With aging,aging-related biochemical and cell-biological changes of heart lead to pathophysiological conditions,Cardiac aging being a complex process which includes aging of cardiac fibroblasts and cardiomyocyte,abnormal accumulation of extracellular matrix in the myocardial interstitial and perivascular leads to cardiac fibrosis.Cardiac fibrosis not only is one of important mechanisms on the myocardial remodeling of hypertension,coronary heart disease and heart failure,but also is an important feature of cardiac aging.Cardiac fibrosis could cause myocardial structure disordered,stiffness increased,cardiac compliance decreased,morbidity of arrhythmia and severe refractory heart failure increased.So it is urgent to develop studies on pathogenesis and prevention strategy of cardiac fibrosis.Mechanism studies on cardiac aging related fibrosis in the heart first involve studies on mechanism of aging.Now there are tens of theories associated to mechanism for aging.Generally they can be summarized as the following two types:1.Environment injury theory of aging,representative by injury of reactive oxygen species(ROS),glycosylation,etc.;2.Genetic factor programming theory of aging, representative by telomere shortening,aging gene,cell cycle regulatory factors,etc. These two theories clarify possible mechanisms for aging organism,deepening study on aging to cellular,molecular and gene level.ROS-based oxidative stress theory for the pathogenesis of cardiac fibrosis on which we can focus is the most widely recognized among theories of aging for the heart. In the elderly,besides the age-related remodeling of heart another risk factor why the cardiac fibrosis liable to happen in the aged is the age-related change of sex hormone balance.In the progress of aging,corresponding change happen in endogenous sex hormone balance,among which changes in sex hormone level after climacteric period show an important role in the organism.Endogenous testosterone and bioactive testosterone level are significantly decreased in men more than 50 years. Is it any relation between the age-related change of endogenous testosterone level and the age-related heart remodeling in men? At present,there is few research about it, and it is lack of the research about the effects of testosterone on the senescence of cardiac fibroblast and its mechanism of action.So we induced cardiac fibroblast senescence by hydrogen peroxide(H₂O₂) at low concentration,then detected a few senescence biomakers of cardiac fibroblast induced by H₂O₂,such as the proliferaton of cells and senescence associatedβ-galactosidase,the level of the intracellular oxidative stress,the cell cycle and the expression ofα-smooth muscle actin(α-SMA) protein,TGF-β1mRNA, P16mRNA,cyclinD1 mRNA,and approached the aging mechanism of cardiac fibroblast then intervention with testosterone and its receptor antagonist,and detected the effects of testosterone at physiological concentration on the senescence of cardiac fibroblast induced by ROS and approached its possible mechanism of action.So as to identify the effect of testosterone on the invention of cardiac aging,and provide new theory and experiment evidence to its mechanism of acting,it may provide new target for the prevention and cure of age-related heart diseases.[Methods]1.Culture cardiac fibroblasts,the cells of passages of 4～6 were used in the experiments.Inducing cardiac fibroblasts senescence by 60μmol/L H₂O₂ for 72h.2.Groups:â… ) group A:cultured with low serum(2%) medium.2) group B: cultured with low serum medium and H₂O₂(60μmol/L).3) group C:cultured with testosterone at physiological concentration(30nmol/L) for 30min,then added H₂O₂(60μmol/L).4) group D:cultrued with androgen receptor antagonist Flutamide (1μmol/L) for 30min,and added testosterone(30nmol/L) for 30min,then added H₂O₂(60μmol/L).3,The expression of SA-β-gal in each group was detected through senescence-associatedβ-gal staining and the proliferation of cells was detected by MTT method,the cell cycle distribution was analyzed by a FACS Coulter Counter,α-smooth muscle actin(α-SMA) protein level was determined by immunostaining and MPIAS-500 multimedia colorful pathological graphic analysis system Meanwhile 2,7-dichlorofluorescein diacetate(DCFH-DA) was used to determine intracellular reactive oxygen species(ROS).The changes of expression of TGF,P16 and cyclinD mRNA were measured by RT-PCR.Statistical analysisResults are expressed as means±SD and analyzed by SPSS13.0 Statistical software.Statistical analysis was analyzed with ANOVA followed by LSD multiple comparison test(Equal Variances Assumed) or Dunnett's T3 multiple comparison test (Equal Variances Not Assumed).A value of P＜0.05 was considered significant.Result1,cell proliferationComparing with control group,H₂O₂(60μmol/L,72h) depressed proliferation of cardiac fibroblasts(P=0.000 versus control).Testosterone at physiological concentrations had the trend of delaying depression of proliferation of cardiac fibroblasts stimulated by H₂O₂(P=0.000 versus H₂O₂ group),whereas the androgen antagonist Flutamide at concentration of 1μmol/L inhibited the delaying effect induced by testosterone at physiological concentrations(P=0.000 versus testosterone group).2,the expression of SA-β-gal activityComparing with control group,H₂O₂(60μmol/L,72h) stimulated the expression of SA-β-gal activity(P=0.000 versus control).Testosterone at physiological concentrations attenuated these effects of H₂O₂ exposure(P=0.000 versus H₂O₂ group),whereas Flutamide administered prior to testosterone pretreatment partly antagonized the protective effects of testosterone against H₂O₂ exposure(P=0.000 versus testosterone_group).3,Intracelluar ROS levelThe Intracelluar ROS level demonstrated a signifcant difference among the four groups(One Way ANOVA F=1381.57,P=0.000).Exposure to H₂O₂ caused an increase in intracellular ROS accumulation in the cultured cardiac fibroblasts (P=0.000).Pretreatment with Testosterone normalized this effect(P=0.000);the use of an AR antagonist abrogates the effects of Testosterone(P=0.000).4,a SMA immunochemical staining4.1 Control group,cardiac fibroblasts were slightly immunostained with no obvious structural positioning features;H₂O₂ group,a SMA immunostaining present strongly positive;although staining intensity in different cells is not completely the same,the structural positioning is obvious.Physiological testosterone inhibited the expression of a SMA,also the cardiac fibroblasts had no apparent structural positioning features.Flutamide partly antagonized the protective effects of testosterone against H₂O₂ exposure.4.2 Comparing with the control group,H₂O₂ significantly increased the expression of a-SMA content within cytoplasm(P=0.000).Physiological testosterone decreased a-SMA content o within cytoplasm(P=0.000),and Flutamide can block the protective effect of testosterone,the difference is also significant(P= 0.000).5,Cell cycle analysisThe proportion of G₀/G₁ phase is significantly different(F=116.955,P=0.000). Most of cells in H₂O₂ treated group stagnating in G₀/G₁ phase caused cell growth arrest and cell proliferation decreasing significantly.Physiological testosterone reduced the proportion of G₀/G₁ phase with statistical significance(P= 0.000),whereas Flutamide weakened these effects with statistical significance(P= 0.000).There is no significant difference between Group Flu and Group H₂O₂(P= 0.393).6,The expression and analysis of TGF mRNA,P16mRNA,cyclinD1 mRNA The electrophoresis showed the different density of integral value after the reverse transcription and amplification of TGF mRNA,P16mRNA,cyclinD1 mRNA. Comparing with the P16mRNA,cyclinD1 mRNA,TGF mRNA in control group, H₂O₂ up-regulated the expression of the mRNA,whereas the physiological testosterone can reversed these effects(P=0.000).Flutamide administered prior to testosterone pretreatment partly block the protective effects of testosterone,the difference is also significant(P=0.000).Conclusion1 60μmol/L H₂O₂ can significantly induce premature senescence of cardiac fibroblasts.2 Physiological testosterone can inhibit the expression ofβ-galactosidase activity in cardiac fibroblasts induced by active oxygen.3 Physiological testosterone can decrease the ROS in cardiac fibroblasts.4 Physiological testosterone can inhibit the expression of TGF mRNA,P16mRNA, cyclinD1 mRNA in cardiac fibroblasts induced by active oxygen.5 Physiological testosterone can inhibit the stagnation of cell cycle in cardiac fibroblasts induced by active oxygen.6 Physiological testosterone can reverse the phenotype conversion of cardiac fibroblasts to cardiac myofibroblast induced by active oxygen.7 All the protective effect of testosterone on cardiac fibroblasts is AR-dependent.