The Effects And Mechanisms Of Hydroxytyrosol On Myocardial Ischemia Reperfusion Injury In Rats

Objective:Acute myocardial infarction?AMI?is one of the leading causes of death or disability in the world.Myocardial ischemia,the timely and effective myocardial ischemia myocardial reperfusion is considered to be the best way to stop myocardial necrosis.However,reperfusion itself may exacerbate post-ischemic myocardial damage,namely myocardial ischemia-reperfusion injury?MIRI?.The mechanism of MIRI is not yet completely clear.It is generally believed at the moment that it is related to excessive reactive oxygen species?ROS?,calcium overload and abnormal pH.At present,many studies have proposed ways to prevent and reduce MIRI.Among them,ischemic preconditioning?IPC?and post-ischemic postconditioning?IPOC?have been shown to protect MIRI in many animal models.However,these two methods are clinically complicated and have some limitations.Therefore,drug prevention of MIRI may be of great significance.Hydroxytyrosol?HT?is a phenolic compound found in Mediterranean dietary virgin olive oil.HT has a variety of biological effects,such as regulating blood lipids,anti-oxidation,anti-inflammatory,anti-tumor,etc.,and it has the most obvious protective effect on the cardiovascular system in particular.Recent study has shown that in the vivo MIRI model of SD rats,HT was shown to reduce MIRI in rats by injecting it intraperitoneally 5 min befor reperfusion.It has been reported that the opening of mitochondria permeability transition pore?MPTP?is one of the important mechanisms of MIRI.When MIRI occurs,MPTP is over-opened and releases Cytochrome C into the cytoplasm,activating aspartate cysteine-specific protease?caspase 9 and caspase 3?,leading to apoptosis.Previous studies have shown that cyclosporin A?CsA?can reduce myocardial necrosis and acute coronary-related complications,which are caused by reperfusion.MPTP over-opening is caused by the depletion of intracellular ATP,excessive ROS and Ca²⁺overload.HT can reduce ROS,thereby reducing oxidative stress-related damage.Therefore,we hypothesize that the effect of HT on the opening of MPTP may be an important entry point to study its role in alleviating MIRI.This study carried out the following two aspects based on previous literature reports:?1?Constructing rat MIRI model to observe the protective effect of HT on MIRI;?2?Comparing the opening status of MPTP in MIRI group and HT+MIRI group,As well as protein expression changes of MPTP downstream apoptotic pathway and MPTP-related regulatory factors;with the application of MPTP-specific opener atractyloside?ATR?,observing whether ATR can cancel the protective effect of HT on myocardial MIRI in rats,and whether HT played a protective effect on rat heart MIRI by inhibiting the MPTP opening.Research methods:1.Isolated rat heart MIRI model?IR group?The hearts of healthy male Wistar rats weighing 250-400 g were selected,and the isolated heart MIRI model was prepared by using Langendorff isolated cardioplegia device for 30 min after ischemia and 120 min of reperfusion.2.Pharmacological interventionTwenty-five minutes before ischemia,HT was administered via perfusion device for 15 minutes with a drug delivery rate at 8 ml/min.The isolated hearts were perfused for 5 minutes with buffer to ensure the residual HT was rinsed.Thirty minutes before the heart was removed,the ATR was injected intraperitoneally into rats at a ratio of 5 mg/kg.3.HE staining observing changes in myocardial cell structure4.TTC staining observing infarct size5.spectrophotometric detection of MPTP open6.TUNEL staining observing myocardial cell apoptosis7.Using Western blot to observe the protein expression of Cytochrome C,cleaved caspase 9 and cleaved caspase 3,and to detect the protein expression of Bcl-2 and Bax.8.Statistical analysis with the application of SPSS17.0 statistical software.The two groups were compared using the test of independent samples t;multiple groups were compared using One Way ANOVA;LSD test was used between two groups;P<0.05was considered statistically significant.Results:1.In the administration groups with three concentrations of HT?10?M,100?M,1000?M?,it was showed by HE staining that myocardial injury in each group were reduced compared with IR group:in 100?M HT group,the myocardial injury significantly reduced if compared with 10?M HT group,but there was no significant difference when compared with 1000?M HT group.TTC staining showed that myocardial infarct size in each group decreased by 4.9%,23.4%and 22%respectively compared with IR group.In addition,100?M HT group further reduced myocardial infarction area compared with 10?M HT group,but compared with 1000?M HT group,there was no significant difference.Therefore,we chose to use a concentration of 100?M HT for follow-up experiments.Moreover,the myocardial damage in 100?M HT group was lighter than that in IR group,and there was a significant reduction in apoptosis rate as well as the myocardial infarction area.2.HT treatment can reduce the sensitivity of MPTP opening induced by Ca2+,suggesting that HT can inhibit the opening of MPTP.3.The expression of downstream apoptotic proteins,Cytochrome C,cleaved caspase9,cleaved caspase 3 in MPTP in HT group decreased compared with IR group,and the apoptosis rate decreased by 16.5%in HT group.4.Compared with IR group,the Bcl-2 protein expression increased,the Bax protein expression decreased,and the ratio of Bax/Bcl-2 decreased in HT group.5.By given atractyloside Intervention,myocardial infarction area and the myocardial apoptosis rate increased,suggesting that the protective effect of HT on myocardial ischemia-reperfusion injury was canceled by atractyloside.Conclusion:1.HT can effectively reduce MIRIin rats.2.HT can inhibit the over-opening of MPTP by overexpressing Bcl-2 protein and suppressing Bax protein expression,thereby inhibiting MPTP over-opening and its downstream apoptotic pathway activation,reducing myocardial ischemia-reperfusion injury.