| Objective:In recent years, studies have shown that fargesin have a role in protecting the ischemic myocardium, but the specific drug targets and mechanism of action is unclear. In our study,the targets of fargesin were studied using myocardial cell membrane chromatography(CM/CMC) and high expression of β1-adrenergic receptor cell membrane chromatography(β1-AR/CMC); and tested fargesin inhibition of β1-AR through the establishment of isoproterenol(ISO) induced β1-AR/CHO-S cell model; further, we evaluated cardioprotective effect on rats and possible action mechanisms on the establishment of myocardial ischemia-reperfusion injury model.Methods:CM/CMC and β1-AR/CMC method were used to scan and analyze the chromatographic retention peak of fargesin. The CM/CMC and β1-AR/CMC models(the transfected CHO-S cells with high β1AR expression level were used to prepare the cell membrane stationary phase) were successfully established and used. The suitability and reliability of the CM/CMC and β1-AR/CMC methods were assessed using metoprolol as positive control, and these methods were then applied to screen bioactive components.The high expression β1 adrenergic receptor Chinese hamster ovary-S(β1-AR/CHO-S)cells were cultured with ISO(50 μM) and then cultured in metoprolol(10 μM) and fargesin for 24 h and then incubated ISO(50 μM) for 12 h. The concentrations of cyclic adenosine monophosphate(c AMP) and protein kinase A(PKA) were detected by ELISA.We explored the protective role of fargesin in myocardial ischemia/reperfusion injury.Rats were treated once with fargesin(15 μmol/kg) through the tail vein after 30 min of ischemia, followed by 2 h reperfusion. Myocardial infarction size was estimated with Evans Blue/triphenyl tetrazolium chloride(TTC) staining. Serum biochemical markers were spectrophotometrically determined. Histopathological changes and apoptosis of ischemic myocardium were observed. The levels of reactive oxygen species(ROS), cytochrome coxidase(COX), caspase-3 in heart homogenates were measured by ELISA.Results:Our CM/CMC and β1-AR/CMC methods can be applied for screening anti-myocardial ischemia components. And we found that fargesin displayed similar chromatographic retention peak to metoprolol in the CM/CMC and β1-AR/CMC methods. This finding suggested their potential antagonistic effect on β1-AR and cardioprotective effect.To provide more biological information about fargesin, we investigated the effects of fargesin on isoproterenol-(ISO-) induced cells injury in the high expression β1 adrenergic receptor /Chinese hamster ovary-S(β1-AR/CHO-S) cells and occluding the left coronary artery-(LAD-) induced myocardial ischemia/reperfusion(MI/R) injury in rats. The results in vitro showed that ISO-induced canonical cyclic adenosine monophosphate(c AMP) and protein kinase A(PKA) levels were decreased by fargesin in β1-AR/CHO-S cells. Fargesin attenuated the serum creatine kinase(CK), lactate dehydrogenase(LDH), and improved histopathological changes of ischemic myocardium compared with the I/R rats. Similar results were obtained with Evans Blue/TTC staining, in which fargesin notably reduced infarct size.Moreover, compared with the I/R group, fargesin increased COX release and the activities of some endogenous antioxidative enzymes including superoxide dismutase(SOD), catalase(CAT), glutathione peroxidase(GSH-Px), but suppressed malondialdehyde(MDA), and intracellular ROS release. Additionally, terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL) assay demonstrated fargesin suppressed myocardial apoptosis, which may be related to inhibition of caspase-3 activity.Conclusion:Taken together, the present study provided proof of concept that fargesin was successful found through the CM/CMC and β1-AR/CMC models. Fargesin is a potential β1-AR blocker candidate by downregulation of c AMP and PKA in vitro and has ability to protect myocardium from I/R damage in vivo, which is associated with its anti-oxidative and anti-apoptosis actions. Although the precise mechanisms underlying the protection remain to be clarified, this finding suggests that fargesin may be a promising agent for treatment of cardiovascular disease. |
PDF Full Text Request