ABSTRACT
Phosphorylated-cyclic adenosine monophosphate response element-binding protein (Phospho-CREB) has an important role in the pathogenesis of myocardial ischemia. We isolated the iridoid glycoside cornin from the fruit of Verbena officinalis L, investigated its effects against myocardial ischemia and reperfusion (I/R) injury in vivo, and elucidated its potential mechanism in vitro. Effects of cornin on cell viability, as well as expression of phospho-CREB and phospho-Akt in hypoxic H9c2 cells in vitro, and myocardial I/R injury in vivo, were investigated. Cornin attenuated hypoxia-induced cytotoxicity significantly in H9c2 cells in a concentration-dependent manner. Treatment of H9c2 cells with cornin (10 µM) blocked the reduction of expression of phospho-CREB and phospho-Akt in a hypoxic condition. Treatment of rats with cornin (30 mg/kg, iv) protected them from myocardial I/R injury as indicated by a decrease in infarct volume, improvement in hemodynamics, and reduction of severity of myocardial damage. Cornin treatment also attenuated the reduction of expression of phospho-CREB and phospho-Akt in ischemic myocardial tissue. These data suggest that cornin exerts protective effects due to an increase in expression of phospho-CREB and phospho-Akt.
Subject(s)
Animals , Male , Myocardial Ischemia/drug therapy , Verbena/chemistry , CREB-Binding Protein/metabolism , Iridoid Glycosides/pharmacology , Fruit/chemistry , Phytotherapy , Troponin/blood , Cell Line/drug effects , Cell Survival/drug effects , Blotting, Western , Rats, Sprague-Dawley , Creatine Kinase/blood , Disease Models, Animal , CREB-Binding Protein/drug effects , Iridoid Glycosides/isolation & purification , Hypoxia/drug therapyABSTRACT
Objective: We tested the possibility of the use of reverse-phase high performance liquid chromatography (RP-HPLC), a sensitive, simple and cost effective technique, for evaluation of global DNA methylation alteration in stem cells. Methods: We detected genomic methylation in four cell lines of mouse embryonic stem cells and in the cell at various developmental stages, including mouse embryonic fibroblast (n=5) and mouse tissues (n=9), using RP-HPLC. The samples were extracted for single nucleosides and investigated for the concentrations of deoxy-cytidine (dC), deoxy-guanosine (dG) and deoxy-methyl-cytidine (5-dmC) using RP-HPLC. The methylation levels were obtained by a ratio of the entire genomic 5m-C to dC. Results: Our results demonstrated that the RP-HPLC technique can be used for analysis of global methylation on the stem cell genome. It can display the discrepancy of methylation levels among the cells at different degrees of cell differentiation. Conclusion: We present the evidence that the RP-HPLC technique can be used for the entire-genomic methylation detection in stem cells and should be further developed as a strategy for monitoring the global DNA methylation of stem cells in advanced research.