| Myocardial ischemia/reperfusion injury (I/RI) refers to the reversible, resurvival damage induced by cell ischemia, the ischemic injury will be worsened after retrieval of ischemia, moreover, it induces cell death or further functional impairment. Ischemia/reperfusion injury (I/RI) is common in the process of myocardial infarction (MI) thrombolytic therapy, coronary interventional, cardiopulmonary resuscitation,coronary artery bypass grafting, cardiac transplantation, etc. I/RI has a very important impact on post-MI infarct size and cardiac function. It has been reported that the outbreak of oxygen free radical is closely related to the mechanism in myocardial ischemia/reperfusion injury. Edaravone (EDRV) is a novel free radical scavenger, which can capture the activity of hydroxyl radical, inhibit lipid peroxidation, and transform the free radical into an inert one. It has been reported that EDRV is mainly used in patients with acute cerebral infarction clinically, It has been also reported that EDRV can protect ischemic heart disease.The cloride ion (Cl-) is the most abundant anions in vivo. The activated Cl- channels were demonstrated by generous evidence playing an crucial role in apoptosis volume decrease (AVD). In our previous studies in cells and whole animal level, we confirmed the effectiveness of the chloride channel inhibitor (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, DIDS) to protect the ischemia/ reperfusion injury (I/RI). Therefore, the purpose of this study is to compare the effect and difference on acute ischemia/reperfusion injury (I/RI) myocardium and the possible mechanisms between chloride channel inhibitor (4,4'-diisothiocyanostilbene -2,2'-disulfonic acid, DIDS) and free radical scavenger (edaravone, EDRV).Aim1. In the whole animal level to compare the effect of chloride channel inhibitor(4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and free radical scavenger-edaravone (EDRV) on acute ischemia/reperfusion injure (I/RI) myocardium.2. What is the mechanism of the protection of the heart between chloride channel inhibitor (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, DIDS) and free radical scavenger, (edaravone, EDRV).Methods1. The preparation of rat heart ischemia-reperfusion infarction model and experiment group:Male Sprague- Dawley rats, subjected to 30 minutes of myocardial ischemia and 4 hours of reperfusion, received one of the following treatments separately: sham-operation group, I/RI group, DIDS group, EDRV group and DIDS + EDRV group, with 8 rats in each group. The rats were administered by program controlled micro pump injection of DIDS [4ml (14mg/kg/h) 2h] at the beginning of reperfusion, Edaravone (10 mg/kg) injected intra arteria carotis 5 min before reperfusion.2. Measurement of hemodynamics:The changes of Left ventricular systolic pressure (LVSP) and the maximal first derivative of developed pressure (±dp/dtmax) were monitored incessantly from the moment of myocardial ischemia to the finishing point of reperfusion and analyzed and given csore.3. Measurement of myocardial infarction, apoptosis and damage: Measurement of myocardial infarct size : Evan`s blue-TTC double staining method after reperfusion for 4 hours. Detection of cardiomyocyte apoptosis by TUNEL-method at the end of reperfusion. Measurement of serum creatine kinase (CK) activity or lactate dehydrogenase (LDH) activity: Colorimetric method at the end of reperfusion.4. Measurement of ROS of the cardiac muscle and Serum: Measurement of ROS : Spectrofluorophotometric method ,take the cardiac muscle in vivo at once at the end of reperfusion; Measurement of OH- or O2- : Colorimetric method, take the cardiac muscle in vivo at once at the end of reperfusion; Measurement of Serum superoxide dismutase (SOD) activity or malondialdehyde (MDA) concentration: Colorimetric method at the end of reperfusion.Results1. There was no statistical difference in heart rate among every group from the ponit of myocardial ischemia to the finishing point of reperfusion except Sham. Both of them decreased and there was no difference in LVSP and±dP/dtmax during the period of myocardial ischemia except Sham (P>0.05). However, after reperfusion, it is significantly depressed LVSP and±dP/dtmax in I/RI, DIDS, EDRV or DIDS + EDRV rats compared with Sham (P<0.05) after reperfusion. DIDS, EDRV or DIDS + EDRV treatment significantly improved LVSP and±dP/dtmax compared with I/RI (P<0.05); there were no differences in DIDS and EDRV(P>0.05); DIDS + EDRV treatment significantly improved LVSP and±dP/dtmax compared with DIDS or EDRV(P<0.05). The data showed that DIDS and EDRV significantly improved myocardial function in I/RI rats and DIDS + EDRV combined administration had a stronger cardioprotective effect than DIDS or EDRV did.2. There were no differences in area-at-risk (AAR/LV%) among Sham, I/RI, DIDS, EDRV and DIDS + EDRV. There was an evident I/RI myocardial infarct (MI) size [(47.15±3.85)%] after subjected to 30 minutes of ischemia and 4 hours of reperfusion; DIDS [(32.7±2.53)%], EDRV [(27.62±1.71)%] or DIDS + EDRV [(20.17±1.94)%] treatment significantly reduced myocardial infarct (MI) size compared with I/RI [(47.15±3.85)%] , (P<0.05). There were no differences in DIDS and EDRV(P>0.05); DIDS + EDRV treatment further decreased myocardial infarct compared with DIDS or EDRV (P<0.05). These data showed that DIDS and EDRV significantly diminished myocardial infarct in I/RI rats and DIDS + EDRV combined administration had a stronger cardioprotective effect than DIDS or EDRV did.3. The levels of serum creatine kinase (CK) activity and lactate dehydrogenase (LDH) activity both increased in I/RI compared with Sham (P<0.05). DIDS,EDRV or DIDS + EDRV treatment significantly reduced reatine kinase (CK) activity and lactate dehydrogenase (LDH) activity compared with I/RI (P<0.05); DIDS + EDRV treatment further reduced CK activity and LDH activity compared with DIDS or EDRV (P<0.05).The results showed that DIDS and EDRV significantly reduced death rate of ischemic myocardial cell and DIDS + EDRV combined administration had a stronger cardioprotective effect than DIDS or EDRV did.4. I/RI significantly decreased SOD activity in I/R rats compared with Sham ( P<0.05), but the contents of MDA concentration significantly increased (P<0.05). DIDS, EDRV or DIDS + EDRV treatment significantly increased SOD activity in I/R rats compared with I/RI (P<0.05) but the contents of MDA concentration significantly decreased (P<0.05).; DIDS significantly decreased SOD activity in I/R rats compared with EDRV, but the contents of MDA concentration further increased of DIDS than that of EDRV (P<0.05); DIDS + EDRV treatment further increased SOD activity in I/RI rats compared with EDRV ( P<0.05); but the contents of MDA concentration further decrease than that of EDRV (P<0.05). These date from SOD activity and MDA contents showed that the effect of DIDS might protect myocardium by reducing free radical and the effect of DIDS reduce free radical weaker than free radical scavenger- EDRV.5. I/RI, DIDS, EDRV or DIDS + EDRV treatment significantly increased myocardial infarct (MI) and myocardial apoptotic index compared with Sham (P<0.05). DIDS, EDRV or DIDS + EDRV treatment significantly reduced myocardial apoptotic index compared with I/RI (P<0.05). There were no differences in DIDS and EDRV(P>0.05); DIDS+EDRV treatment further decreased myocardial apoptotic index compared with DIDS or EDRV (P<0.05).6. I/RI significantly increased ROS contents,O2-·contents and OH·contents in I/R rats compared with Sham ( P<0.05). DIDS, EDRV or DIDS + EDRV treatment significantly decreased ROS contents,O2-·contents and OH·contents in I/R rats compared with I/RI (P<0.05); EDRV significantly decreased ROS contents,O2-·contents and OH·contents in I/RI rats compared with DIDS (P<0.05); DIDS + EDRV treatment further decreased ROS contents ,O2-·contents and OH·contents in I/R rats compared with EDRV ( P<0.05). These date from ROS contents,O2-·contents and OH·contents showed that the effect of DIDS might protect myocardium by reducing free radical and the effect of DIDS reduce free radical weaker than free radical scavenger EDRV, and DIDS + EDRV combined administration had a stronger cardioprotective effect than DIDS or EDRV did and combined administration possess synergies action.Conclusion1. DIDS and EDRV has the effectiveness by improving cardiac, reducing infarct size and reducing apoptosis to protect the I/RI heart.2. It is not through a common path that DIDS and Edaravone function as the protection of the heart.3. DIDS and EDRV protects the heart mainly through inhibition of ROS activity levels, chloride channel blockers DIDS has the same effect of reducing the activity of ROS, the combined administration of its protective effect can be enhanced. The results show that it is not through a common path that DIDS and Edaravone function as the protection of the heart and DIDS may inhibit produce of ROS while EDRV clear ROS. |
PDF Full Text Request