Protection Of Loureirin A Against Oxidative Stress Induced Damage Of H9c2 Myocardial Cells And Its Underling Mechanisms

Background and Objective :Cardiovascular disease is one of most common C hronic non-communicable disease(NCD).The risk for developing this disease in creases with age,with a peak between 35 and 50 years. It is three- four times more common in female than male between the turn of life.Since the beginning of the 21 st century, accounting for 30% of global deaths,it is responsible for over 97,000,000 annual deaths worldwide each year.Moreover, cardiovascular disease patients have the highest lifetime treatment cost per patient of all types of diseases, which has brought a serious economic burden to people.One of oxidative stress(OS) as the pathogenesis of many cardiovascular diseases, and interact with other pathophysiological mechanisms affect, directly or indirectly involved in the myocardial damage occurs, cardiac arrhythmia, cardiac hypertrophy and other cardiovascular diseases. Natural antioxidants get constantly attention.Flavonoids are remarkable ones which are remarkable by the natural active. Loureirin A, a major flavanone present in plants, is the main active substance of Resina Draconis(Dragon’s Blood in C hinese). Modern pharmacological studies indicate that Resina Draconis also has cardiovascular protection,antibacterial, immune suppression Blood hemostasis way adjustment, anti- inflammatory and analgesic, lowering blood glucose and lipid, antibacterial, and promote skin repair and anti-oxidative. For the above reasons, the protective effects of Loureirin A from oxidative stress in vitro of H9c2 cells can be studied from the dissertation,then the cellular and molecular responses of H9c2 cells to the treatments were analyzed by multiple approaches,which is expected to provide preclinical proof-of-principle for Resina Draconis in clinical treatment of cardiovascular diseases.Methods:(1) CCK-8 assay was used to detect the inhibition of loureirin A on TBHP –induce decrease of H9c2 cells viability.Camparision of the antioxidant effects of loureirin A and N-acetylcysteine(NAC).(2) The intracellular and serum MDA content and SOD enzymatic activities were detected by UV spectrophotometry to evaluate the regu lation of Loureirin A on TBHP- induced cellular redoxidative level.(3) TUNEL Apoptosis Detection Kit(FITC) assay and Annexin V-FITC Apoptosis Detection K it assay by flow cytometry was used to detect the effect of Loureirin A on the cell apoptotic ratio in TBHP- induced H9c2 cells.(4) The regulation of Loureirin A on apoptosis-related Bax and Bcl-2 genes expressions were obtained by PCR.(5) Western blotting method was used to detect the protein expressions of Bax, Bcl-2.Result:(1) Oxidative stress damage in vitro experimental model was set up by 0.4μM TBHP stimulusing H9c2 cells 6 hours. Loureirin A at the concentration of less 500 n M had little cytotoxity on H9c2 cells.(2) Concentration range in 125,250,500 nM Loureirin A and NAC, acting on TBHP injury H9c2 cells 6,12,24 hours, with the increase of drug concentration, cell viability was significantly higher mortality rate decreased significantly.(3) Loureirin A(125,250,500 n M) inhibited significantly the decrease of cell viability and cellular enzymatic activities of SOD and increase of MDA generation in TBHP- induced H9c2 cells in a dose-dependent manner.(4) TUNEL Apoptosis Detection Kit(FITC) assay demonstrated that Loureirin A at the concentrations of 0,125,250,500 n M attenuated significantly TBHP- induced H9c2 cells apoptotic radio from 49.8±2.1%to,40.1±3.5%,29.7±2.4%,respectively.Annexin V-FITC Apoptosis Detection K it assay demonstrated that Loureirin A at the concentrations of 0,125,250,500 n M attenuated significantly TBHP- induced H9c2 cells apoptotic radio from 52±0.1% to 31±0.5%,18±1.4%,10±1.1%.(5) Western blotting and PCR analysis showed that Loureirin A of 500 n M inhibited significantly TBHP- induced increase of Bax m RNA and protein expression and the decrease of Bcl-2 m RNA and protein expression in a dose-dependent manner.Conclusions:(1) Loureirin A through antioxidant effects can significantly reduce TBHP-induced apoptosis in H9C2.(2) All the results indicated that Loureirin A decreased the concentration of MDA and enhanced the activity of SOD.(3) Loureirin A inhibited oxidative stress induced apoptosis through regulating Bcl-2 family protein. Loureirin A can significantly inhibit inhibited significantly TBHP- induced increase of Bax protein expression and and the decrease of Bcl-2 protein expression in a dose-dependent manner,which is related to the biological effect of Bax and Bcl-2 relative content in H9c2 cells.