The Protective Effect And Its Related Mechanism Research Of Vitamin C Postconditioning To Myocardial Ischemia-reperfusion Injury In Vivo Rats

Objective:The coronary heart disease（CHD）is a leading cause of death world wide.Timely restoring coronary blood flow is the basic measure to save the acute ischemic myocardium,but the reperfusion in myocardium can also lead to myocardial reperfusion injury at the same time,that is so-called myocardial ischemia-reperfusion injury.Therefore,to find an effective measure to alleviate ischemia-reperfusion injury has a important theoretical and practical significance in clinic.Vitamin C is a widely used clinical antioxidant,can reduce the production of oxygen free radicals,but there is less studies on the myocardial protection and its mechanism.This studyis aim to observing whether vitamin C ischemia postconditioning can alleviate the myocardialischemia-reperfusioninjuryinvivoratsandfrom ischemia-reperfusion myocardial environmental disorder and apoptosis two aspects to explore its possible mechanisms.Method:48 healthy male SD rats（280-320g）which through ligating the anterior descending coronary artery to make ischemia-reperfusion mode were randomly divided into four groups:Sham group,I/R group,VC group,HCL group（PH=2.5,that is the same with VC solution）.The electrocardiogram（ECG）and heart function indexs of rats including heart rate,HR),left ventricular end-diastolic pressure（LVEDP）and left ventricular systolic pressure（LVSP）,left ventricular pressure maximum change rate（±dp/dtmax）were recorded by Powerlab multichannel physiologic recorder.The concentration changes of reactive oxygen species and the gas signal molecule NO in myocardial were recorded by TBR4100 gas signal molecule and free radicals detector within one hour during reperfusion.After reperfusion,blood was keeped from carotid artery to measure the LDH and SOD activity;the myocardial infarction was measured by TTC staining method,heart frozen sections were made HE staining to observe the cellular structure changes;Western Blot was used to detect the expression of apoptosis related proteins.Data were expressed as the mean±SD.All data were analyzed by one-way ANOVA（SPSS 22.0 statistic software）.when the beginning of F test of variance choosen the LSD test,otherwise choosen the Tamhane,s T2 test.The change H₂O₂,gas signal molecule NO variance were analyzed by repeated measure ANOVAs.The differences were considered statistically significant when P<0.05.Results:Hemodynamic Measurements:The HR（excpect 1min point）and the LVEDP index of each point in time have no statistical difference among groups（P>0.05）.Compared with the I/R group,the±dp/dtmax,LVSP increased obviously in VC group（P<0.05）;At the early ischemia/reperfusion,the LVSP at 1 min and 30 min pionts increased obviously（P<0.05）,and the+dp/dtmax increased significantly at 1 min point in HCL group（<0.05）.Serum enzymology:Compared with I/R group（374.66±21.44U/L）,the activity of LDH in VC group（305.54±46.56 U/L）reduced significantly（P<0.05）,the activity of LDH in HCL group（366.05±37.67U/L）had no statistical difference（P>0.05）.Compared with I/R group（433.00±61.34U/L）,the activity of SOD in VC group（566.67±62.46U/L）increased significantly（P<0.05）,the activity of SOD in HCL group（382.67±35.93U/L）had no statistical difference（P>0.05）.VC solution administration significantly decreased infarct size（21.38±2.74%vs 41.65±2.62%）than that in the I/R group（P<0.05）.In contrast,HCL solution administration had no significant difference with the I/R group（36.80±2.41%vs 41.65±2.62%;P>0.05）.In cell morphological aspects:After VC solution administration,the arrangement of myocardial fibers structure and the degree of swelling,and inflammatory cells infiltration,and the size of the focal necrosis had a apparent ease.With HCL solution administration,it has a certain degree of myocardial injury degree of ease,but the reduce degree is weaker than the VC group.The changes of H₂O₂and NO concentration in myocardium:The changes of H₂O₂ concentration:Compared with Sham group,the myocardial H₂O₂ content over time appeared significantly higher in I/R group（P<0.05）.Compared with I/R group,the changes of H₂O₂ decreased significantly in VC group and HCL group（P<0.05）,but HCL group compared with VC group the degree of myocardial H₂O₂ content reduce smaller（P<0.05）.The changes of NO concentration:Compared with Sham group,the myocardial NO content over time significantly reduced in I/R group（P<0.05）.Compared with I/R group,the reduction of NO concentration decreased significantly in VC group（P<0.05）,even the NO concentration increased compared with baseline;the reduction of NO concentration had no significant difference in HCL group（P>0.05）.Apoptosis proteins:Compared with I/R group,the expression of Cyto-C,Caspase-9,Bax and P-ERK decreased significantly in VC group（P<0.05）,the expression of Bcl-2 and the Bcl-2/Bax ratio increased significantly in VC group（P<0.05）;the expression of Cyto-C,Caspase–9,Bax,Bcl-2,and the Bcl-2/Bax ratio had no significant difference in HCL group（P>0.05）,the expression of P-ERK decreased significantly in HCL group（P<0.05）,but the expression are less than VC group.Conclusion:Vitamin C postconditioning may by directly inhibiting the burst of ROS,improving myocardial NO concentration,indirectly inhibiting the abnormal of PH and calcium overload to improve the internal environment disorder,and inhibiting ERK signaling pathway,reducing the expression of Cyto-C,Bax,Caspase-9,at the same time increasing the expression of Bcl-2 to alleviate myocardial ischemia-reperfusion injury in rats.The protection of vitamin C to cardiac ischemia-reperfusion injury is mainly related to its antioxidation,and has certain relevance to it weak acid.