Potential Use Of A Novel Small-molecular Compound,ZLN005,in Treatment Of Cardiovascular Diseases

The aggravating trend of aging population is emerging globally and in China.Aging leads to impaired physiological function and increased vulnerability to a number of human pathologies,including diabetes and cardiovascular disorders.Diabetes mellitus is one of the main threats to human health,with a worldwide prevalence of 366 million individuals in 2011 and a constantly growing incidence in recent years.More than 50% of the mortality rate in diabetic patients is due to cardiovascular complications.On the other side,ischemic heart disease remains the leading cause of mortality globally.Calorie restriction(CR)is the most robust non-genetic intervention to delay aging,and has been implicated benefits in many aging related diseases.SIRT1,a NAD+-dependent deacetylase,is involved in cellular calorie restriction response.Studies have shown that SIRT1 functions in a wide array of cellular processes,including cell survival,senescence,autophagy,and metabolism,by deacetylating histones and a number of transcriptionalregulators,including NF-κB,p53,Fox Os and PGC-1α.Small molecule ZLN005,also known as 2-(4-tert-Butylphenyl)benzimidazole,is a novel PGC-1α transcriptional regulator shown to have beneficial metabolic regulating effects in diabetic mice.However,whether ZLN005 has protective effects in heart diseases has not been studied.Aims 1.To investigate whether ZLN005 protects cardiomyocytes from high glucose induced cardiomyocytes injuries;2.To investigate whether ZLN005 modulates macrophage polarization and cholesterol metabolism;3.To study mechanisms involved in effects of ZLN005 and make preliminary feasibility evaluation for drug development.Methods 1.Neonatal mouse cardiomyocytes were isolated and cultured in high glucose circumstances.ZLN005 was added in intervention groups.Cell morphology was observed;cell viability,cell oxidative stress,level of autophagy and rate of apoptosis were detected by proper methods respectively.2.Macrophages were cultured in high cholesterol circumstances with intervention of ZLN005.The formation of foam cells were observed.Cellular cholesterol level was measured.3.Macrophages were treated with LPS and cell viability assay was carried with intervention of ZLN005,as well as measurement of the expression of macrophage M1 and M2 markers.4.The effect of ZLN005 on cell SIRT1 expression was evaluated.The impacts of specific inhibition of SIRT1 in ZLN005 treated groups on cell viability,autophagy,apoptosis,macrophage polarization and cholesterol metabolism were evaluated with respective method.5.Molecular docking was carried to predict the way of ZLN005 on SIRT1 activation.The combination ability of ZLN005 and SIRT1 was evaluated simultaneously and compared with other SIRT1 activators.Results 1.In cultured neonatal mouse cardiomyocytes,33 m M glucose incubation lead to decreased cell viability,downregulated autophagy,increased oxidative stress and apoptosis.Administration of ZLN005 increased autophagy level and reduced cell stress,leading to less cell injuries under high glucose circumstance.2.Macrophages derived from Thp-1 cells were induced to foam cell by ox LDL-c,while ZLN005 was seen to reduce the amount of lipid contents in cells.Further investigation found that ZLN005 decreased cell cholesterol contents.3.LPS downregulated cell viability of M2 macrophages derived from Thp-1 cells and lead to M1 differentiation.Administration of ZLN005 protected cells from viability decrease and downregulated M1 inflammatory cytokines expression.4.ZLN005 promoted SIRT1 m RNA and protein expression in cardiomyocytes.Specific inhibition of SIRT1 eliminated upregulation of autophagy related proteins expression by ZLN005 and restrained the protective effect of ZLN005 against high glucose caused apoptosis.5.In Thp-1 derived macrophages,specific SIRT1 inhibition negatively regulated the protective effects of ZLN005 on decreased cell viability induced by LPS and increased foam cell formation induced by high lipid,and caused elevated cholesterol levels in ZLN005-treated groups.Specific SIRT1 inhibition antagonized the effects of ZLN005 on macrophage polarization and promoted M1 marker expression.ZLN005 affected ABCA1 and ABCG1 expression by SIRT1 activation.6.The results of molecular docking provided information concerning possible binding way of ZLN005 and SIRT1,including specific binding site and its relationship with the structure of ZLN005.The docking results suggested that the binding force of ZLN005 to SIRT1 was stronger than several other SIRT1 activators,and it has shown preliminary possibilities to make structural modifications based on ZLN005 and to make further drug development researches.Conclusions 1.ZLN005 protects neonatal cardiomyocytes from high glucose-induced cell injury through SIRT1 pathway and possibly by restoring autophagy activity and reducing apoptosis.2.ZLN005 regulates cholesterol metabolism of Thp-1 derived macrophages via SIRT1 pathway,possiblely involving upregulation of ABCA1 and ABCG1 expression,and reduces lipid content in macrophage derived foam cells.3.ZLN005 modulates polarization of Thp-1 macrophages,inhibiting M1 differentiation in a SIRT1-dependent manner.4.ZLN005 promotes SIRT1 transcription and translation in cardiomyocytes.Molecular docking results showed that ZLN005 is able to directly activate SIRT1 by an allosteric mechanism and the predicted biding force of which is stronger than several other SIRT1 activators.