Study On Novel Small Molecule AF-HF001in Inhibiting The Oxidative Stress-induced Cardiomyocyte Apoptosis

Heart failure is the final destination of a variety of cardiovascular diseases, which can be characterized with the high incidence, the high mortality and a heavy economic burden and constitutes a major threat on the national health. By screening zebrafish heart failure model, we discovered and confirmed a novel small molecule AF-HF001 which improved the heart enlargement and hemodynamics and efficiency, indicating the potential in curing heart failure.Ventricular remodeling is the basic mechanism of heart failure duration, along with hypertrophy and loss of cardiomyocytes. It is an important incentive for heart failure that myocardial ischemia/hypoxia-induced oxidative stress leads to apoptosis. In this thesis, we used hydrogen peroxide– and cobalt chloride– induced apoptosis model. With western blot, ELISA and reactive oxygen species sensitive green fluorescence probe 6-carboxy-2’, 7’- dichlorofluoroscein diacetate, AF-HF001 was detected in protecting myocardial cells from apoptosis and reactive oxygen species production under oxidative stress. With the help of m RNA microarray analysis and quantitative PCR, we explored preliminarily the candidate with AF-HF001 against oxidative stress induced apoptosis of H9c2 cells.The results revealed that: AF-HF001 could significantly reduce the caspase3 cleavage and DNA fragment by oxidative stress, and showed the anti-apoptotic effect in 50 n M and completely reversed apoptosis in 500 n M; Within 50–500 n M concentration range, the greater the concentration of AF-HF001, the more obvious anti-apoptotic effect. In addition, DCFH-DA fluorescence detection indicated that pretreatment with AF-HF001 significantly reduced the amount of intracellular reactive oxygen species in H9c2 cells under oxidative stress. Microarray results analysis showed that AF-HF001 reversed the change in the gene expression profile of hydrogen peroxide–treated H9c2 cells: such as Ndufs2, Ndufs3, TPM1, Ry R1 and TIMP4 which are related to ROS production, cardiac contractility and extracellular matrix remodeling; such as Araf, MAP2K5, Bax, Hsp27, Apaf1, casp3 and casp7 which are involved in the MAPK family and intrinsic mitochondrial pathway.Further experiments exposed that AF-HF001 increased early JNK activation(p-T183/Y185) and inhibited later P38 activation(p-T180) to regulate the anti-apoptosis in cells. Microarray results analysis and identification found that pretreatment AF-HF001 significantly decreased expression levels of MDSP(dual specificity phosphatase 13A) under hydrogen peroxide stimulation. To confirm the role of MDSP in the AF-HF001 mediated anti-apoptosis effect, quantitative PCR results found that hydrogen peroxide significantly increased MDSP gene expression in a time-dependent manner and reached the peak after the hydrogen peroxide stimulation for 36 h; JNK inhibitor(SP600125) significantly led to the upregulated expression level in MDSP of H9c2 cells in condition of oxidative stimuli. Using p EGFP plasmid-mediated overexpression of exogenous GFP-MDSP fusion protein significantly increased DNA cleavage; Gene silencing against MDSP in H9c2 rat cardiomyocytes significantly reduced the increased caspase3 lysis in hydrogen peroxide-induced apoptosis with reduced endogenous MDSP. Consequently, pretreatment AF-HF001 activated JNK and significantly reversed the increased expression of MDSP in hydrogen peroxide stimulation to defend against cell apoptosisTo sum up: We first discovered and confirmed a novel small molecule AF-HF001 concentration-dependently inhibited the apoptosis of myocardial cells under oxidative stress and reduced intracellular reactive oxygen species production; We also found that AF-HF001 decreased the expression levels of MDSP to inhibit hydrogen peroxide-induced rat myocardial cells apoptosis. Thus, the depth study of AF-HF001 related to regulatory mechanism against apoptosis in cardiocytes, not only could provide a theoretical basis for promoting AF-HF001 to be clinical drug in treatment of heart failure, but also may provide new therapeutic targets or direction for heart failure.