Anti-apoptotic And Anti-inflammatory Effects Of GYY4137in Rat Myocardial Cells Infected By Coxsackie Virus B₃

Viral myocarditis (VM) is the most common cardiovascular diseases in children’s diseases, which are usually caused by coxsackievirus B3type (Coxsackie virus B3, CVB3), due to the continuous improvement and improvement of treatment, the cure rate significantly improved compared with the previous, but some still are not sensitive to the presence of children in the treatment or relapse, poor clinical prognosis. Clinical studies have shown that evidence-based medicine and exercise, Coxsackie B virus infection caused by a virus on myocardial cell injury and subsequent continuous direct immune injury is caused by viral myocarditis is difficult to completely cure the root cause, but also some children repeatedly relapse root knot, this pathology and pathophysiology of the changes in our clinic for treatment is a serious challenge. Therefore, to continuously explore new drugs and new treatments to give children a purpose, targeted therapy to improve the cure rate in children is important.GYY4137is a new Hydrogen sulfide (H2S) release agent having a solubility in water, can slowly release hydrogen sulfide gas (H2S), which is a morpholino phosphorodithioate dichloromethane complex ((p-methoxyphenyl) morpholino-phosphinodithioic acid), has anti-inflammatory, anti-apoptotic variety of biological effects, protecting blood vessels, known as nitric oxide is the second (Nitric oxide, NO) and carbon monoxide (Carbon monoxide, CO) after the third gaseous messenger molecule. Foreign scholars found, GYY4137in LPS (Lipopolysaccharide, LPS) murine macrophage-induced inflammation model, showing the inhibition of the secretion of cytokine expression, regulation of inflammation-related genes and proteins play an anti-inflammatory effect of reaction. At the same time, research has found that in a number of organ ischemia rat model of hypoxia-reperfusion injury, GYY4137not only showing the effect of inhibiting the inflammatory response, but also play the biological effects of anti-apoptotic. The VMC is a direct myocardial cell damage caused by the Coxsackie virus and subsequent ongoing immune damage and apoptosis of cardiomyocytes accompanied eventually lead to changes in cardiac structure and function. Accordingly, we speculate GYY4137might be has a protective effect on viral myocarditis caused by the Coxsackie virus.This study was first established in vitro models of viral myocarditis, and verifies the success and reliability of the model from the morphological, physiological and biochemical. On the basis of this model gives a new hydrogen sulfide release agent GYY4137, by measuring physiological and biochemical indicators and detect the expression of inflammation-related factors and their upstream regulatory proteins, to explore GYY4137of neonatal rat cardiomyocytes infected with Coxsackie virus cytoprotective; and to further explore the relationship between the protective effect and cell signal transduction pathway; to further elucidate the mechanism for the protective effect of VMC.PartⅠEstablishment and identification of viral myocarditis in vitro models. Objective： The purpose of this study is to establishment and identification of viral myocarditis in vitro models. To initially explore the pathogenesis of viral myocarditis from the perspective of molecular biology and pathophysiology.Methods: Dual enzymatic digestion was used cultured neonatal rat primary cardiomyocytes, established of viral myocarditis in vitro model. The beating rate and cell morphology of cardiomyocytes was observed by Inverted microscope. Identification of myocardial cell purity was using immunofluorescence method. Cardio-myocyte Damage-related Enzyme Activities, such as lactate dehydrogenase (LDH), creatine kinase MB (CK-MB) was measured by the ELISA kits. CVB3-specific gene expression was measured by multiplex RT-PCR.Results: After infected CVB3, the typical cytopathic effect (CPE) was being observed and the beating rate was decreased and even stopped when the damage was very serious. The levels of LDH、CK-MB was significantly increased, CVB3Specific band was amplified by RT-PCR.Conclusion： The model of viral myocarditis in vitro was successfully constructed; this model can be used to studies of viral myocarditis. Part ⅡProtective effects of GYY4137on rat myocardial cells infected by Coxsackie virus B3and its effect on inflammatory cytokine secretionsObjective:To initially explore the Protective effects of GYY4137on rat myocardial cells infected by Coxsackie virus B3and its effect on inflammatory cytokine secretions, provide a theoretical basis for the clinical treatment of viral myocarditis.Methods Ⅰ The cultured myocardial cells of neonatal Sprague-Dawley rats were randomly treated with CVB3, CVB3+GYY4137(10umol/150umol/1100umol/1),normal myocardial cells. After treatment, the beating rate of myocardial cells and the cellular morphology were observed. Cell viability was ascertained with CCK-8assay. TNF-α IL-1β、IL-6. levels in the supernatants were evaluated by sandwich ELISA. The levels of LDH, CK-MB was measured by the ELISA kits.Results： The Cell viability5days after CVB3infection in GYY4137-treated group was higher than that in the untreated CVB3infection group (p<0.01); the levels of LDH、 CK-MB and levels of TNF-α IL-1β、IL-6in the GYY4137-treated CVB3infection group was significantly reduced when compared with untreated CVB3infection group(p<0.01, p <0.05) respectively.Conclusion:GYY4137is not Cytotoxic on Cardiomyocytes, it does can provide protective effects on rat myocardial cells infected by CVB3; GYY4137also can inhibit CVB3induced inflammatory factor secretions from cardiomyocytes. Part ⅢEffects of GYY4137on NF-κB and MAPK signal transduction pathway and mechanism of sensitivity in Coxsackie virus B3infected rat myocardial cells Objective:This study will investigate the NF-κB and MAPK signal transduction pathway and mechanism of sensitivity in Coxsackie virus B3infected rat myocardial cells, To initially explore the Effects of GYY4137on NF-κB and MAPK signal transduction pathway and mechanism of sensitivity in Coxsackie virus B3infected rat myocardial cells.Methods:The cultured myocardial cells of neonatal Sprague-Dawley rats were randomly treated with CVB3, CVB3+GYY4137(10umol/1、50umol/1、100umol/1),normal myocardial cells. After reach the appropriate point in time, total protein、 n nuclear proteins and membrane proteins were extracted in each groups, the effects of GYY4137on CVB3-induced nuclear factor-kappaB (NF-κB) activity by Western blot analysis, Immunofluorescence analysis and Electrophoretic Mobility Shift Assay (EMSA). Mitogen-activated protein kinase (MAPK) signaling protein was detected by Western blot.Results:Treatment with GYY4137prevent the IκBαa degradation induced by CVB3(p <0.05), the p65was expression mainly in the cytoplamic before infected CVB3(p<0.05),Incubated with CVB3caused the accumulation of p65in the nucleus (p<0.05); The DNA binding activity of NF-κB was increased significantly in CVB3group (p<0.05), while treatment with different concentrations GYY4137markedly reduced the CVB3-induced DNA binding activities of NF-κB in a dose-dependent manner (p<0.05) Compared to normal cells, intracellular P65transferred from the cytoplasm to the nucleus in CVB3-infected cells. P-P38, P-ERK1/2and P-JNK1/2in cells was significantly increased (p<0.05) after2h treatment with Incubated with CVB3, and the treatment of GYY4137(10-100μM) abolished the P38, ERK1/2, JNK1/2phosphorylation induced by CVB3(p<0.05).Conclusion:The nuclear translocation of p65was suppressed by GYY4137in a concentration-dependent manner; GYY4137may reduce CVB3-induced inflammatory factors by suppressing the phosphorylation of P38, ERK1/2and JNK1/2pathway. Part IVProtective effect of GYY4137on myocardial apoptosis of rat myocardial cells with coxsackievirus B3infection and its mechanismObjective: This study will investigate the Apoptotic signaling pathways associated protein in Coxsackie virus B3infected rat myocardial cells, to further explore the effect of GYY4137on Apoptotic signaling pathways associated protein in Coxsackie virus B3infected rat myocardial cells.Methods: The cultured myocardial cells of neonatal Sprague-Dawley rats were randomly treated with CVB3, CVB3+GYY4137(50umol/1), normal myocardial cells and normal myocardial cells+GYY4137(50umol/1). Hoechst33342staining and Annexin V-FICT/PI double staining was used to detected the apoptotic of myocardial cells, the activity of superoxidedismutase (SOD) and the levels of malondialdehyde (MDA) in all the media samples were measured by Commercial kits, apoptosis-associated proteins such as Bcl-2-、 Bax and Cleaved-caspase3was detected by Western blot.Results： After infected CVB3, the percentage of apoptotic cells was increased markedly (p<0.05), while treatment with GYY4137reduced the CVB3-induced Cardiomyocyte apoptosis. the levels of SOD in the GYY4137-treated CVB3infection group was significantly increased when compared with CVB3infection group (p<0.01, p<0.05) respectively, the levels of MDA was in the GYY4137-treated CVB3infection group was significantly decreased when compared with CVB3infection group (p<0.01, p<0.05) respectively. The ratio of Bax/Bcl-2and Cleaved-caspase3in CVB3infection group was significantly increased (p<0.01, p<0.05, p<0.05), while treatment with GYY4137can abolish the above responses (p<0.01, p<0.05, p<0.05) respectively.Conclusion： GYY4137can significantly improve the apoptotic of cardiomyocytes induced by Coxsackie virus B3; the mechanism might involve of inhibition Bcl-2/Bax/Cleaved-caspase3signal pathway activation and antioxidative stress mechanisms.