Protective Effects And Potential Mechanisms Of SalB Against Doxorubicin-induced Cardiotoxicity

Part one:ObjectiveThis article establish rat model of adriamycin-induced cardiotoxicity and doxorubicin in vitro stimulation H9C2 cardiomyocytes, while applicating SalB intervention to improve further exploring of the role of doxorubicin cardiotoxicity and make mechanisms possible for further clarify the adverse effects of doxorubicin mechanism and Provide experimental evidence for clinical use of anthracycline chemotherapy in cardioprotective agent.Part two:Experimental Research1.Preparation and determination modelThe whole experiment:In this study with dose escalation manner (1,1.5,2,2.5,3rng · kg-1) according to body weight to 1ml ·100g-1 every 2 days by intraperitoneal injection of doxorubicin, Rat HF model. After about four weeks extrement weight loss was observed that rats move slowly, MAP physiological recording instruments to detect left ventricular systolic blood pressure less than the normal value of 2/3 is LVSP successful model.Part Two:SalB protective effect against doxorubicin-induced cardiotoxicity in rats.l.hemodynamics.Compared with the control group, the blood flow in rats group dynamics parameters LVSP, (+) dp/dt, ASP, HR decreased significantly, LVDP and a significant increase in (-) dp/dt (P<0.01). Each treatment group compared with the model group, with increasing duration of administration, the rats LVSP the treatment groups, (+) dp/dt, HR, ASP were increased, (-) dp/dt, LVDP all lower, and has a difference (P<0.05).2.Effect of Sal B on adriamycin induced cardiotoxicity tissue morphology in rats. Pathology of myocardial tissue sections were HE, Masson and 8-OHdG immunohistochemical staining effect SalB of muscle fibers, interstitial form, inflammatory infiltration and myocardial collagen libers. Pathology results showed:SalB significantly improve myocardial cell morphology, inflammatory infiltration, reduced collagen fiber, reduce the incidence of ventricular remodeling (P<0.05). Model of myocardial tissue showing brown, indicating high expression in the model group 8-OHdG, which was significantly increased compared with the normal range, SalB can significantly reduce the expression of a large number of 8-OHdG, improve doxorubicin-induced oxidative DNA damage (P<0.05).3.Effect of Sal B on adriamycin induced cardiotoxicity NO, TNOS, TSH, SOD, MDA Using the kit to detect serum and heart tissue of nitric oxide (NO), total nitric oxide synthase (TNOS), malondialdehyde (MDA), superoxide dismutase (SOD) and total sulfhydryl (TSH) content change. The results showed that:each treatment group compared with the model group, with increasing duration of administration of different doses of SalB significantly reduced MDA content in rat heart tissue model group decreased serum TNOS activity, inhibit NO production, reduce free radical damage to the heart. SalB also enhance TSH, SOD activity and increase the body’s ability to clear free radicals, protect against myocardial cells (P<0.01).4.Investigated the effect of SalB on adriamycin induced with inflammatory cytokines TNF-a^ IL-6. By ELISA serum TNF-a, IL-6 activity, the effect of different doses of SalB on Rat Serum TNF-a, IL-6 activity of the. The results show:SalB 25.4 and 12.5mg·kg-1dose group can significantly reduce generation of doxorubicin-induced TNF-α (P<0.05, P<0.01), while SalB different doses can inhibit rat serum IL-6 activity (P<0.01). Above indicate SalB could significantly inhibit the generation of inflammatory cytokines, thereby reducing inflammation induced myocardial injury, has protective effects on the heart.5.Investigated the effect of SalB on adriamycin induced about apoptotic factor NF-kB NF-kB protein expression using Western Blot detection of cardiac tissue, the effects of different doses of SalB of NF-kB protein expression. The results showed that the model group adriamycin induced myocardial cell NF-kB lot of expression, apoptosis. SalB different doses significantly reduced NF-kB expression in heart tissue, decreased cell apoptosis (P<0.01).6.The impact SalB on doxorubicin-induced cardiotoxicity in rats downstream SIRT1 protein expression. Using the Western Blot method to detect heart tissue in rats SIRTl.SalB influence to doxorubicin-induced cardiotoxicity P-FOXO3a,MnSOD protein expression. Western Blot results showed that:the model group doxorubicin down cardiomyocytes SIRT1, P-FOXO3a, MnSOD, SalB different doses significantly increased SIRT1, P-FOXO3a, protein expression of MnSOD (P <0.01). Tips, SalB reduce doxorubicin cardiotoxicity and SIRT1 proteins.Part Three:SalB on adriamycin induced effect H9C2 cardiomyocytes1.MTT screen the best doxorubicin and SalB concentrations. Using MTT cell proliferation and cytotoxicity screening kits and SalB optimum concentration of doxorubicin. MTT results showed that:different concentrations of doxorubicin on H9C2 cardiomyocytes exhibit inhibitory activity.2umol·L-1 concentrations of doxorubicin stimulation 12h, significantly inhibited H9C2 cardiomyocytes viability (P<0.01). The SalB each treatment group were on H9C2 cardiomyocytes proliferation of different degrees, showed a dose-effect relationship, the strongest cell proliferation after 60umol·L-1 concentrations SalB stimulus 12h (P<0.01).2.Investigated the effect of SalB on adriamycin induced protective effect H9C2 cardiomyocytes oxidative injury. In H9C2 cardiomyocytes for the study, doxorubicin and nicotinamide intervention, using an inverted microscope SalB effect on adriamycin induced cardiomyocyte morphology, myocardial cells by flow cytometry in mitochondrial membrane potential size; use DCFH changes in the content DA fluorescent probe to detect ROS in cardiomyocytes. The results show significantly improved abnormal SalB adriamycin induced cardiac cell morphology, reduced cell death (P<0.01), reducing the increase in myocardial cells ROS content (P<0.01), resulting in increased doxorubicin It decreased mitochondrial membrane potential (P<0.01). Protective effects of nicotinamide inhibits SalB thereof (P<0.01).3.Investigated the effect of SalB on adriamycin induced oxidative stress markers of SIRT1 protein in H9C2 cardiac cytotoxicity. Using the Western Blot method to detect SalB on adriamycin induced toxicity H9C2 cardiomyocytes oxidative stress markers SIRT1 protein, FOXO3a phosphorylated protein expression levels of MnSOD protein. Western Blot results showed that:SalB could significantly increase the expression of SIRT1 protein levels (P<0.01), a significant increase in protein phosphorylation levels of FOXO3a (P<0.01), significantly increased MnSOD protein expression (P<0.01). SIRT1 inhibitor nicotinamide significantly blocked SalB activation (P<0.01) of SIRT1 protein. Above tips, SalB can increase oxidative stress markers SIRT1 protein levels. On adriamycin induced cardiac oxidative injury in rats.Part Four:conclusionSalB improve the doxorubicin-induced cardiotoxicity in rats, the mechanism may be SalB reduce oxidative stress markers SIRT1 proteins.