| BackgroundIschemic heart disease is one of the major diseases that threaten human health. Shortening ischemia time of the tissue and recovering blood flow early are the most effective measures to reduce ischemia injury. Thrombolysis, percutaneous transcoronary angioplasty and coronary artery bypass graft efficiently recover blood flow of ischemic tissue, but these therapies also lead to myocardial ischemia/reperfusion (MI/R) injury. It has been reported that that endogenous nitric oxide (NO) decreases in the early stage of MI/R, which results in neutrocyte activation and myocardial damage. Previous evidences from our and other laboratories have shown that NO relaxes small vessels and improves myocardial function after ischemia/reperfusion, so nitrate drugs are used as the exogenous NO donor to reduce MI/R injury.The role of reactive oxygen species (ROS) in MI/R injury has been extensively studied. Experimental evidence demonstrated that a burst of ROS generated during the early stage of reperfusion. The increase in ROS would lead to lipid peroxidation to the cell resulting in the damage myocardial cell membrane, and this may be the main reason of myocardial injury. Besides, ROS may induce vascular endothelial injury and reduce endothelial NO synthesis, which exacerbates myocardial injury. Moreover, increased membrane permeability, intracellular Ca2+ overload and inflammatory mediators production are associated with an effect of ROS. Experimental results show that in MI/R, the activity of endogenous free radical scavenging enzyme system is reduced. Thus, free radical scavenger such as ferulic acid (FA) could be used for the treatment of IHD.Because both decreased endothelial NO generation and increased ROS level are involved in MI/R injury, the aim of the present study was to design and synthesiz a new drug named acetyl ferulic acid isosorbide mononitrate (AFI) from ROS scavenger FA and NO donor ISMN and to test the hypothesis that AFI has a stronger cardioprotective effect than ROS scavenger, NO donor or their combined administration does.Aims1. To synthesize a new compound which both releases NO and eliminates ROS from ferulaic acid (FA) and isosorbide mononitrate (ISMN).2. To investigate the cardioprotective effect of the new compound in myocardial ischemia/reperfusion.Methods1. After acetylation of FA and acetic anhydride, the intermediate product was esterify with ISMN, monitoring with thin-layer chromatography, after finishing the reaction, filtered the suspension then dried by R/E. It was the end production acetyl ferulaic isosorbide (AFI) after purified by chromatograph. (This part of work is achieved by Department of Chemistry and us.)2. Male Sprague-Dawley rats, subjected to 30 minutes of myocardial ischemia and 3 hours of reperfusion, randomly received one of the following treatments respectively: SHAM; I/R (MI/R+solvent); SF (MI/R+SF, 40 mg/kg, ig); ISMN (MI/R+ISMN, 30 mg/kg, ig); SF+ISMN (MI/R+SF+ISMN, 40 mg/kg+30 mg/kg, ig) and AFI (MI/R+AFI, 10 mg/kg, ig). Left ventricle developed pressures (LVDP) and the maximal first derivative of developed pressure (±dP/dtmax) were monitored throughout the experiments. Myocardial infarction size, serum creatine kinase (CK) activity, lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity, hydrogen peroxide (H2O2), malondialdehyde (MDA) and nitric oxide (NO) production were determined at the end of reperfusion.Results1. We got 2.99 g (73 mmol) the end product AFI from 10 mmol material FA, total yield was 73%. Melting point of AFI was 162.5 ~ 163℃. The results of infrared and nuclear magnetic resonance indicated that the end product was conformity with objective structure.2. The concentration for 50% of maximum effect (EC50) coming from regression equation was 7.02 mg.3. AFI improved myocardial functionI/R significantly depressed LVDP and±dP/dtmax in MI/R rats compared with SHAM (n = 8, P < 0.01); SF (n = 8, P < 0.05), ISMN (n = 8, P < 0.01) or SF+ISMN (n = 8, P < 0.01) treatment significantly improved LVDP and±dP/dtmax compared with I/R; AFI treatment further improved LVDP and±dP/dtmax in MI/R rats compared with SF (n = 8, P < 0.05), ISMN (n = 8, P < 0.05) or SF+ISMN did(n = 8, P < 0.05). These data showed that AFI significantly improved myocardial function in MI/R rats and AFI had a stronger cardioprotective effect than SF and ISMN combined administration did.4. AFI reduced myocardial injuryThere were no differences in area-at-risk (AAR/LV %) among SHAM, MI/R, SF, ISMN, SF+ISMN and AFI. SF(39±5) %, ISMN(36±7) % or SF+ISMN (36±5) %significantly reduced myocardial infarct size compared with I/R [ (56±6) %, n = 8, all P < 0.01]; AFI [(21±8) %]treatment further decreased myocardial infarct size compared with SF, ISMN or SF+ISMN. These data showed that AFI significantly diminished myocardial injury in MI/R rats and AFI has a stronger cardioprotective effect than SF and ISMN combined administration did. Compared with SHAM, I/R both increased CK activity and LDH activity in MI/R rats (n = 8, P < 0.01); SF (n = 8, P < 0.05), ISMN (n = 8, P < 0.01) or SF+ISMN (n = 8, P < 0.01) significantly reduced CK activity and LDH activity compared with I/R; AFI treatment further decreased the activities of CK and LDH compared with SF or ISMN (n = 8, P < 0.01); AFI treatment significantly decreased CK activity compared with SF+ISMN (n = 8, P < 0.05). The results showed that AFI significantly reduced death rate of ischemic myocardial cells and AFI has a stronger cardioprotective effect than SF and ISMN combined administration did.5. AFI increased NO contentThe increase of NO was detected in SF (n = 8, P < 0.05), ISMN (n = 8, P < 0.01), SF+ISMN (n = 8, P < 0.01) or AFI (n = 8, P < 0.01) compared with I/R; AFI significantly increased NO content compared with ISMN which is NO donor drug (n = 8, P < 0.01) when the molar drug dose was only one of eighth of ISMN.6. AFI eliminated ROSI/R significantly decreased SOD activity in MI/R rats compared with SHAM (n = 8, P < 0.01); SF or SF+ISMN treatment significantly increased SOD activity compared with I/R (n = 8, P < 0.05); AFI treatment further increased SOD activity in MI/R rats compared with SF (n = 8, P < 0.01) or SF+ISMN (n = 8, P < 0.05).I/R both increased H2O2 and MDA contents in MI/R rats compared with SHAM (n = 8, P < 0.01); SF or SF+ISMN treatment significantly decreased H2O2 and MDA contents compared with I/R (n = 8, P < 0.01); AFI treatment further decreased H2O2 and MDA contents in MI/R rats compared with SF or SF+ISMN (n = 8, P < 0.05). These data showed that AFI significantly improved antioxidase system activity in MI/R rats and AFI has a stronger cardioprotective effect than SF and ISMN combined administration did.These data from NO content, SOD activity, H2O2 and MDA content showed that the effect of AFI might protect myocardium by releasing NO and eliminating ROS.Conclusions1. AFI is synthesized from FA and ISMN, yield is 73%. The results of infrared and nuclear magnetic resonance indicate that the end product is conformity with objective structure. The synthesis method is simple and easy to realize. The reaction can finish in short time and the cost is low, so it is suitable to industrial production.2. The new compound AFI synthesized from ISMN (NO donor drug) and SF (ROS scavenger) both releases NO during MI/R and eliminates ROS generation during reperfusion. AFI has a stronger cardioprotective effect against MI/R injury than SF, ISMN or their combined administration does.
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