The Research Of The Expressions And Anti-apoptosis In Response To Oxidative Stress In Cardiac Myocytes Between Resveratrol And SIRTs Of Longevity Genes

Background and Objectives Review:Cardiovascular diseases (CVD) is one of leading causes of Mortality in the world. More and more evidences show that detecting the survival and death of cardiomyocytes has the meaningful significance for the long time period following up the situations and functional changes to the groups of patients who suffering from CVD.The mortality of CVD is closed related to the age of patients,research the main mechanisms of decline aged will have the meaningful theoretical study when considering future developing of new CVD drugs.Calorie restriction (CR) is believed can extend the average life to most mammalian animals including of yeast, worms, flies and rodents.,etc. The results of the designed experimants of Clinical Trials of CR almost has the similar results as animal models. CR was noted could also prevent the occurance of CVD, most malignant diseases, certain kinds of Degenerative diseases and two types of DM.Reducing the mortality of CVD and strokes were also noted by taking CR through the past researches. Massive studies show that the extension of life via CR was mediated and enhanced bv Sir2. Sir2 is an NAD-dependent histone deacetylase with the multiple functions of cellular processes, including of gene silencing, rDNA recombination, life span extension in response to caloric restriction, and DNA damage repairme.ntsMammals have seven Sir2 homologus genes named Sirtuins (Sirtl-7). SIRT1 is the most homologous gene of Sir2 and has been broadly studied. The proteins of SIRT1 have been linked to the control of the metabolic processes in adipose, liver and muscle tissues through the regulation of the nuclear receptor PPARγand its transcriptional co-activator PGC-1α. p53 is a kind of the tumor suppressors, transcription factors of the FOXO family and NF-κB transcription factor are another kinds of non-histone substrates of SIRT1 which are belived can regulate the cell survival, proliferation and stress responses. SIRT1 can regulate the cell survival by the response of deacetylation via the DNA repair factor Ku70 which believed an inhibitor of Bax-mediated apoptosis. SIRT1 can also control the myocardial development and decrease the stress and aging-associated myocardial dysfunction in the heart depends on the processes of deacetylation by p53 and FoxO1 (forkhead box O1). SIRT1 was also observed can promote vasodilatation and improve the regenerative functions in smooth and endothelial muscle cells of the vascular wall through modulating the activities of FoxO1, and p53, eNOS (endothelial nitric oxide synthase)_and the expressions of AT1R (angiotensinⅡtype 1 receptor) respectively.Sirt7 is also one of the essential regulators on tissue homeostasis to the heart according to the current study. Sirt7-deficient (genes knocked out) mice were noted reduction of mean and maximum lifespans and could finally caused cardio hypertrophy and other cardiomyopathy. Primary cardiomyocytes of Sirt7-deficiency revel around 200% increase in basal apoptosis and diminished resistance to oxidative and genotoxic stress significantly suggesting a essential role of Sirt7 in the regulation of stress responses and cell death in the heart. While to other Sirtuins(SIRTs), for examples:SIRT 2,3 and 5 may have the same NAD-dependent deacetylases activity as SIRT1, meanwhile due to SIRT4 and SIRT6 are ADP-ribosyl-transferase, and their functions in the heart are still uncleared till now.Resveratrol is a kind of chemical compound of polyphenols. Resveratrol was treated as phytoalexin of plants.It is believed with the effects of anti-aging,anti-cancer,also can be treated as Herbal estrogen can treat most of the symptoms to the most Menopausal syndroms. Besides, Resveratrol is proved with the effects and functions as:anti-free radicals, Anti-oxidations, anti-inflammations, prevent genes mutations, prevent the occurance of oncogenesis,could be used as protections agents to cardio-vascular diseases,reduce the onsets of myocardial infarctions,damages of cardio re-perfusions, improve the vaso-dilatations .Resveratrol is also improved can help the symtoms and over all situations of Osteoporoisis,DM,Senile Dementia,Depressions,...etc. The main mechanisms of protection of Resveratrol are:(1)As intracellular agents (2)As Anti-inflammatory agents (3)NOS formation (4)Improve angiogenesis (5)Improve neurons growth (6)Enhance the activities of MitochondriaTo investigate the expressions of the 7 different Sirtuins (SIRT1-7) of mammalian cardiomyocytes in vitro in response to oxidative stress in order to have an well and more clear understandings about whether each or any of the 7 SIRTs members plays a role in the heart, and whether each or any of them could be activated by the SIRT1 activator Resveratrol is parts of our purpose to this research. MethodsThe H9c2 embryonic heart-derived cells of rats were cultured in DMEM (Dulbecco's modifided Eagle's medium) which was supplemented with 100ml/L Fetal Bovine Serum (FBS). Initially, the H9c2 embryonic heart-derived cells were trypsinized, plated (10000-12000cells/cm2) in culture dishes containing different kinds density of H2O2 dilution (0,10,50,100,200μM) for 6 h to observe and check viability and apoptosis of the H9c2 embryonic heart-derived cells via the assignment of the method of MTT. The H9c2 embryonic heart-derived cells were also placed in the media of containing a series of different kinds density of Resveratrol respectively(0,10, 20,50,100μM) for 24 h to analyze their effects of the viability on the H9c2 embryonic heart-derived cells. After the above processes of the experiment, the H9c2 embryonic heart-derived cells were subsequently divided into three experimental groups randomly after culturing for 48 h, (1) Normal control group (Nil any treatments nor interference to the H9c2 embryonic heart-derived cells) (2) Oxidative stress group(the H9c2 embryonic heart-derived cells were incubated in DMEM which containing 50μM H2O2; (3) Resveratrol 20μmol/L (Res group):(the H9c2 embryonic heart-derived cells were added 20μM Resveratrol for 30 min then exposure to H2O2.solution for 6 h later). The viability of the H9c2 embryonic heart-derived cells was evaluated by MTT assay. The methods of TUNEL staining and Flow Cytometry (FCM) were applied to check the apotosis of the H9c2 embryonic heart-derived cells. The methods of RT-RCR and Western blot were also applied to detect the expressions of SIRT1-7 mRNA and their proteins. The method of Western blot was also applied to check the expressions of p53 detected. Results1. H9c2 embryonic heart-derived cells were exposed to the different concentration of H2O2 solutions (10,50, and 100μM) for 6 h and the viability of H9c2 embryonic heart-derived cells were assessed by the MTT assay. Via the MTT results of the different concentration of H2O2 solutions showed that certain concentration of H2O2 solutions inhibits cell viability in response to different concentration of H2O2 solutions.14.6% (P> 0.05),39.1% (P< 0.05) and 81.2% (P< 0.01) of the reductions in cell viability were observed by the different concentration of H2O2 solutions as 10,50 and 100μM concentration of H2O2 solutions groups respectively. When the concentration of H2O2 solution reached the concentration of 50μM was noted starting showed the results with the statistically significance of the survival inhibitory effects on the H9c2 embryonic heart-derived cells. Under the concentration of H2O2 solution was treated as oxidative stress stimulator in the following tests.2. The H9c2 embryonic heart-derived cells were treated with the different concentration of Resveratrol by 0,10,20,50 and 100μM respectively for 24 h. The results of the experiments revealed the optical density (OD) which were taking by MTT methods were observed increasing in 10μM and 20μM groups, especially in 20μM (P＜0.05). Meanwhile the values of the results were observed decreasing in 50μM and 100μM, in 100μM higher notability were observed, and which was significantly lower than Normal Control groups(P<0.05).20μM Resveratrol was applied in the following tests to observe its functions of cellular protection against oxidative stress and the expression of SIRT1-7 in H9c2 embryonic heart-derived cells.3. The proportions of TUNEL-positive cells significantly increased in H2O2 group compared with the Normal Control group (Control:1.56±0.26% vs. H2O2:16.98±2.65% ,ρ< 0.05). Meanwhile, pretreatment with 20μM Resveratrol were observed could prevent majority of the increasing of apoptosis which caused by H2O2 (Resveratrol:8.65±1.28% vs. H2O2,ρ0.05). Similar results were obtained with PI staining. However, a significant increase in cell apoptosis was observed when the Sirtuins inhibitor (20 mM nicotinamide) was added on before the application of Resveratrol (Nam: 18.23±2.21% vs. Res; P< 0.05).4. As H9c2 embryonic heart-derived cells cells were treated with 50μM concentration of H2O2, the expressions of endogenous SIRT1,3,4,7 mRNA were observed decreasing. Resveratrol pretreatment caused SIRT1,3,4,7 mRNA significant up-regulation statistically, but SIRT2,5,6 were unaffected.5. The expressions of endogenous SIRT1,3,4,7 proteins were consisted with the mRNA. The expressions of endogenous SIRT1,3,4,7 proteins were observed decreasing when H9c2 embryonic heart-derived cells were treated with 50μM H2O2, Meanwhile, Resveratrol pretreatment caused SIRT1,3,4,7 proteins statistically significant up-regulation were observed, but SIRT2,5,6 were unaffected.6. p53 expression increasing was observed in cells incubated with 50μM H2O2 for 6 h (ρ< 0.05), and it was succsesfully prevented by pretreatment of Resveratrol for 30 min (ρ< 0.05). Conclusion:1. The expressions of SIRT1,3,4,7 in H9c2 embryonic heart-derived cells was decreased in vitro in reponse to the oxidative stress of H2O2 which induced cell apoptosis, suggesting that the apoptosis induced by oxidative stress may be associated with the decrease of the expressions of SIRT1,3,4,7.2. Resveratrol has direct cellular protective effects on H2O2-induced apoptosis in H9c2 embryonic heart-derived cells which might be implemented by the increasing of the expressions of SIRT1,3,4,7.3. Resveratrol may also be the activator of SIRT3,4,7 besides SIRT1. However, SIRT2,5,6 may not affected by Resveratrol in H9c2 embryonic heart-derived cells.