| 1.BackgroundsDelaying aging process is a great challenge for modern gerontology and geriatrics.Free radical theory of aging considers aging as a result of accumulative oxidative damage of cell components,which is supposed to be caused mainly by reactive oxygen species(ROS) generated by mitochondria according to the theory of free radical-mitochondria.Several studies on animal myocardial mitochondrial metabolism have shown mitochondria play critical role in the generation of oxidative products and modulation of cell death.The decrease of myocardial mitochondria function may be one of the causes of decrease of the cardiac function in the elderly.Recent studies showed that melatonin can clear oxidative free radicals and has anti-oxidation and anti-aging effects.However,few studies have addressed the protective effect of melatonin on aging of myocardial mitochondria.2.ObjectivesTo observe the changes in myocardial mitochondria function and integrity on aging rodent models induced by D-gal and investigate the protective effect on myocardial mitochondria and to explore the possible mechanisms.3.Methods3.1 Animal modelsSixty young SD rats of 3 months(30 males and 30 females) were randomly divided into 3 groups.1) Control group(C group,n=20).2) Accelerated aging group(D group,n=20).Rats were treated with D-galactose(125mg/kg/d,sc for 6 weeks) alone.3) Melatonin treated group(M group,n=20).Rats were treated with D-galactose(125mg/kg/d,sc) combined with melatonin(10mg/kg/d,sc) for 6 weeks.The natural aging group(N group) included 20 rats of twenty-four months(10 male and 10 female rats in each group).3.2 Measurements of ATP,ADP and AMP ATP,ADP and AMP in left ventricular myocyte in each group were determined by high performance liquid chromatography,and total adenylic acid number(TAN=ATP+ADP+AMP) was calculated.Energetic state of myocytes was evaluated with energy charge(EC=1/2ADP+ATP/TAN).3.3 Mitochondrial examinationsIn order to study the changes of myocardial cytochrome c levels,left ventricular muscle samples were prepared for microscopic, immunohistochemical and immunofluorescence examination.Hematoxylin-eosin (HE) staining,immunohistochemical and immunofluorescence staining of cytochrome c,voltage-dependent anion channel(VDAC),adenine nucleotide transporter(ANT),Bcl-2 and Bax were performed.Western blot technique was used to analyze their respective protein level semi-quantitatively.Gene expression of VDAC,ANT,cytochrome c was also measured by fluorescent quantitative polymerase chain reaction.3.4.Statistical analysisAll data are shown as means±SD.One-way ANOVA(SPSS 14.0;SPSS, Chicago,IL) were used to determine the significance between the means of multiple groups with the least-significant difference test for equal and Dunnett test for unequal variances.The paired and unpaired t tests were used to compare the continuous variables of two specific groups.Statistical significance was defined as P＜0.05.4.Results4.1 Effect of melatonin on cardiac energetic metabolism in D-galactose pretreated ratsEnergetic metabolism status of left ventriculum in each group was observed in this part of experiment.There were significant difference among levels of ATP, TAN in the M group,while all of them were significantly higher than those in natural aging group and those in D-gal treated group(all p＜0.05).AMP was significantly higher and ADP,ATP,TAN and EC were significantly lower in N group than those in the other three groups,there were no differences of AMP and ADP levels among the other three groups.4.2 Changes of myocardial cytochrome c in mitochondria of rats treated by both D-galactose and melatonin4.2.1 HE staining results of left ventricle tissueHE staining showed more myocardiocytes,less intercellular matrix and more adventitial vessels in the left ventricle in rats treated by D-galactose and melatonin than those in the accelerated aging rats treated by D-galactose as well as those in the natural aging rats.4.2.2 Immunohistochemical results of mitochondrial ANT,VDAC and Bcl-2 in rats treated by D-galactose and melatoninANT staining is more intensive,while VDAC and cytochrome c staining are less intensive in control group and Melatonin treated group than those in accelerated aging rats treated by D-galactose and those in natural aging group (p＜0.05).There is no difference of Bax staining among 4 groups,but the staining is more intensive at adventitia in each group.Bcl-2 staining is rather weak and there is no difference among 4 groups.4.2.3 mRNA expression of mitochondrial ANT,VDAC and Bcl-2 in rats treated by D-galactose and melatoninmRNA expression of ANT significantly increased while the mRNA expression of VDAC and cytochrome c significantly decreased in accelerated aging rats treated by D-galactose and in natural aging rats in comparasion with those in control group and in melatonin treated group(both P＜0.05).4.2.4 Protein expression of mitochondrial and cytoplasmic ANT,VDAC and Bcl-2 in rats treated by D-galactose and melatoninProtein expression of cytochrome c in mitochondria and cytoplasma tended to but not significantly higher in accelerated aging rats treated by D-galactose than those in other three groups.No differences were found between the other three groups.Ratio of of cytoplasmic cytochrome c to mitochondria cytochrome c was significnatly increased in accelerated aging rats treated by D-galactose and natural aging rats compared with which in adult rats and rats treated by D-galactose and melatonin(both P＜0.05).The levels of Bcl-2 was higher in the control group than those in the other three groups.Mitochondrial Bcl-2/Bax ratio was significantly increased in the control group and melatonin treated group,compared with that in the accelerated aging rats treated by D-galactose and that in the natural aging group(both P＜0.05).ANT in mitochondria tended to but not significantly higher in the control group and the melatonin treated group,compared with that in the accelerated aging rats treated by D-galactose and that in the natural aging group. VDAC protein was the lowest in rats treated by D-galactose and melatonin among the four groups(p＜0.05).5.Conclusion5.1 Myocardial energetic metabolism is higher in rats treated by D-galactose and melatonin than that in the other three groups,suggesting the protective effect of melatonin on the function of mitochondria.5.2 Melatonin may maintain electron transfer chain,ensure normal oxidative phospholization,maintain production of ATP,prevent leakage of electron and formation of free radicals.One of the underlying mechanisms might be that melatonin can decrease the translocation of cytochrome c from mitochondria to cytoplasma,increase cytochrome c level in mitochondria,prevent cell apoptosis and protect the function of mitochondria.5.3 Melatonin can modulate the expression of Bcl-2 family,which might be also one of the mechanisms for the prevention of translocation of cytochrome c from mitochondria to cytoplasma. |
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