Effect Of HGF On Myocardial Ischemia Reperfusion Injury In Rats And Its Mechanism

BackgroundGlobally,cardiovascular disease remains a serious health hazard that can cause nearly 20 million deaths each year,more than half of that are acute myocardial infarctions.In addition to drug therapy,myocardial reperfusion therapy,percutaneous coronary intervention(PCI)and coronary artery bypass grafting(CABG),have received much attention.Coronary revascularization can effectively save ischemic myocardial tissue,reduce infarct size,improve cardiac function,reduce the incidence of pump failure and hospital mortality.By mechanically reperfusion of the myocardium,it is inevitable that myocardial ischemia reperfusion injury(MIRI)may occur.After a certain time of ischemia,myocardial tissue may be deficient in restoring blood supply.The phenomenon of blood damage is further aggravated.MIRI has a great influence on cardiac function,which can lead to myocardial stunning,myocardial systolic and diastolic function;myocardial electrophysiological changes,decreased fibrillation threshold and reperfusion arrhythmia;myocardial cell membrane and myofibrils structural damage causes changes in myocardial ultrastructure.At present,the mechanism of MIRI has not been completely clarified.It is recognized as the classical theory of free radical damage,vascular endothelial injury theory and neutrophil infiltration,but it still cannot explain all the phenomena of IRI.Therefore,domestic and foreign scholars have done a lot of research on the mechanism of ischemia/reperfusion injury.Evidence suggests that cardiomyocyte apoptosis is caused by myocardial infarction and systolic dysfunction after ischemia/reperfusion injury.The main cause of this is that apoptosis occurs within 24 hours,causing extensive or sub-wide damage to the myocardium,making myocardial dysfunction more likely to occur.Therefore,if cardiomyocyte apoptosis can be inhibited,cardiac pathophysiological changes,dysfunction,and ischemia-reperfusion injury caused by myocardial infarction will be reduced.Activation of inflammatory cytokines is another important pathway for myocardial ischemia-reperfusion injury,including the interleukin family(IL-1,IL-6,IL-8 and IL-10,etc.),tumor necrosis factor-(TNF-a),neutrophil activating protein(NAP)and other factors are involved in the development of MIRI,and the inflammatory response is closely related to the formation and instability of coronary atherosclerotic plaque,so the intervention of inflammatory response is also reduced An important method of MIRI.Hepatocyte Growth Factor(HGF)is a glycoprotein and a multifunctional cytokine derived from mesenchymal cells.HGF plays an important role in cell division,angiogenesis,cell movement and growth.In recent years,it has been found that it is also a kind of cardiotrophic factor,and it is also a specific type of endothelial cell growth factor,which has strong vascular endothelial cell mitosis,inhibits apoptosis and tissue remodeling,and repairs endothelial damage.C-met is a protein product encoded by the proto-oncogene c-met and is a tyrosine kinase receptor.c-met is mainly expressed in epithelial cells and plays an important physiological role.HGF is a natural ligand for c-met,and HGF mediates a variety of downstream effector proteins through c-met to promote cell proliferation and survival.Previous studies have confirmed that the expression level of HGF and its receptor c-met is up-regulated in the rat model of ischemia-reperfusion,at least in coronary endothelial cells,and other studies have confirmed that whether it is purified myocardial endothelial cells The expression of HGF and HGF/c-met receptors were detected in mature cardiomyocytes,suggesting that HGF plays an important physiological role in cardiomyocytes or endothelial cells.Whether HGF interferes with myocardial ischemia-reperfusion injury by binding to c-met receptor has not been reported.whether HGF interferes with MIRI by reducing cardiomyocyte apoptosis and inflammation has not been reported.In this study,different doses of HGF were used to intervene in the MIRI rat model,and c-met inhibitors were added for control.The myocardial enzymes and myocardial infarct size were measured.The role of HGF/c-met pathway in HGF intervention MIRI was observed.PI3K/AKT protein inhibitor control,detection of cardiomyocyte apoptosis-related factors and other indicators,observation of PI3K/AKT signaling pathway-mediated HGF anti-apoptosis intervention MIRI role;by adding HSP70 inhibitors,detection of related inflammatory factors and NF-?B pathway-associated protein,observed the mechanism of action of HSP70/NF-?B pathway-mediated HGF anti-inflammatory intervention MIRI.This study is divided into three parts:Part I Effect of HGF on myocardial ischemia-reperfusion injury in ratsPart II:PI3K/Akt pathway-mediated HGF inhibits cardiomyocyte apoptosis in rats with myocardial ischemia-reperfusionPart III HSP70/NF-?B pathway mediates HGF anti-inflammatory intervention in myocardial ischemia-reperfusion injuryPart I Effect of HGF on myocardial ischemia-reperfusion injury in ratsObjective1.Effects of HGF on myocardial enzymology and myocardial infarct size in rats with ischemia-reperfusion injury2.Possible mechanism of HGF intervention in myocardial ischemia-reperfusion injuryMethodsobjective:To investigate the effects of different doses of HGF on myocardial enzymes and myocardial infarct size in rats with myocardial ischemia-reperfusion injury,and to investigate the effect of HGF on myocardial ischemia-reperfusion injury.Role and mechanism.1.Grouping and processing of experimental animals:40 male Wistar rats were randomly divided into 5 groups,8 in each group.only.Sham-operated group(Sham group):only threaded under the left anterior descending artery of the animal,no ligation;myocardial ischemia-reperfusion group(MIRI group):sutured the upper 1/3 of the anterior descending branch of the rat heart The silicone tube was ligated together,and then ligated for 30 minutes and then reperfused for 180 minutes.In the HGF group,the operation was the same as the MIRI group.The HGF was administered via the jugular vein at the same time as the anterior descending branch;the HGF double dose group(HGF-Group D):HGF(2mg/Kg)was injected into the jugular vein while ligation of the left anterior descending artery;C-met inhibitor group(ARQ197 group):before ligation of the left anterior descending artery,ie before HGF intervention 2 The c-met inhibitor ARQ197(20 mg/Kg)was administered intravenously every hour.Rats in each group were reperfused for 180 minutes and then tested as follows:2.Automated biochemical analyzer was used to detect serum CK-MB and cTNI levels in each group.3.Myocardial infarct size was measured by myocardial staining in each group.4.HE staining was used to observe the pathological changes of myocardium.Results1.Establishment of rat myocardial ischemia-reperfusion modelThe MI segment of the MIRI rats showed an increase in the ST segment of the archback 30 minutes after ligation,suggesting that the ligation was successful,the ligature was released,and after ventricular reperfusion,ventricular arrhythmia and even ventricular fibrillation may occur.Reperfusion arrhythmias were reduced in HGF intervention group and HGF-D group.Rats in the C-met inhibitor group had an increased proportion of reperfusion arrhythmias.2.Serum CK-MB and cTNI levels in each groupThe levels of serum CK-MB and cTNI in the MIRI group were significantly higher than those in the sham group,while the HGF group and the HGF-D group were significantly lower than the MIRI group,which was higher than the sham group,but there was no significant difference between the two groups;ARQ 197 group serum The levels of CK-MB and cTNI were significantly higher than those of the HGF group,and there was no significant difference compared with the MIRI group.3.Myocardial infarct size in each groupMyocardial infarct size was significantly higher in the MIRI group than in the sham group.The myocardial infarct size in the HGF and HGF-D groups was significantly lower than that in the MIRI group,but still higher than that in the sham group.The myocardial infarct size in the ARQ197 group was higher than that in the HGF group.There was no significant difference compared with the MIRI group.4.Myocardial pathological changes in rats in each groupThe pathological changes of myocardial morphology were observed by HE staining.The myocardial structure of the sham group was intact,the myocardial fibers were arranged regularly,and there was no obvious inflammatory cell infiltration in myocardial tissue.In the MIRI group,the myocardial fibers were disordered,the cells were degenerated and necrotic,interstitial edema,myolysis was obvious,and large inflammatory cells infiltrated in the myocardial tissue.The myocardial structure destruction of HGF group was significantly reduced compared with MIRI group.The cells were degenerated and necrotic,the interstitial edema was relieved,and a small amount of inflammatory cells infiltrated.The pathological changes of myocardial histology in HGF-D group were similar to those in HGF group.In the ARQ197 group,the degeneration and necrosis of myocardial cells increased,interstitial edema,myolysis,and inflammatory cell infiltration were obvious.Conclusions1.HGF can alleviate myocardial ischemia-reperfusion injury in MIRI rat model.2.HGF reduces myocardial ischemia-reperfusion injury and the effect of c-met receptor binding.Part ?:PI3K/Akt pathway-mediated HGF inhibits cardiomyocyte apoptosis in rats with myocardial ischemia-reperfusionObjectiveBy constructing a rat model of myocardial ischemia-reperfusion and applying HGF intervention,the anti-cardiomyocyte apoptosis effect of HGF and related signaling pathways were observed.Methods1.Experimental animals and grouping:32 male Wistar rats were randomly divided into 4 groups(n=8).Including sham group:only threading without ligation;MIRI group:ligation of left anterior descending artery for 30 minutes,reperfusion for 180 minutes;HGF group:simultaneous ligation of rat anterior descending artery with HGF(1mg/Kg);PI3K/Akt inhibitor group(LY294002 group):LY294002 was administered intravenously(0.3mg/Kg)while HGF intervention was given.2.Detect serum CK-MB levels in each group3.Tunel method to measure the apoptosis index of myocardial cells in each group4.Immunohistochemistry was used to measure the expression of Caspase-3 protein and Bax expression in each group.5.Pathological observation(HE staining)6.Echocardiography to measure changes in cardiac function in each group7.Western blot was used to measure the expression levels of PI3K and pAkt signaling proteins in each group.Results1.Establishment of rat myocardial ischemia-reperfusion modelIn the MIRI group,the ST segment of the ST-segment was raised upwards 30 minutes after the ligated anterior branch,suggesting that the ligation was successful,the ligature was released,and the ventricular arrhythmia,ie,reperfusion arrhythmia,may occur after reperfusion.Reperfusion arrhythmia decreased in the HGF intervention group,and the proportion of reperfosion arrhythmias increased in the LY294002 group.2.Various rat serum CK-MB levelsThe level of serum CK-MB in the MIRI group was significantly higher than that in the sham group.The level of CK-MB in the HGF group was significantly lower than that in the MIRI group,but still higher than that in the sham group.The serum CK-MB level in the LY294002 group was higher than that in the HGF group.Still below the MIRI group.3.Cardiomyocyte apoptosis index of each groupThe apoptotic index of myocardial cells in the MIRI group was significantly higher than that in the sham group,while the HGF group was significantly lower than the MIRI group,which was higher than the sham group.The apoptotic index of the LY294002 group was higher than that of the HGF group and still lower than the MIRI group.4.Expression of Caspase-3 and Bax protein in each groupThe expression levels of Caspase-3 and Bax protein in the MIRI group were significantly higher than those in the sham group.The expression levels of Caspase-3 and Bax protein in the HGF group and the LY294002 group were lower than those in the MIRI group,while the LY294002 group was myocardial Caspase-3.And Bax protein expression level is higher than HGF group.5.Myocardial pathological changes in rats in each groupThe pathological changes of myocardial morphology were observed by HE staining.The myocardial structure of the sham group was intact,the myocardial fibers were arranged regularly,and there was no obvious inflammatory cell infiltration in myocardial tissue.In the MIRI group,the myocardial fibers were disordered,the cells were degenerated and necrotic,interstitial edema,myolysis was obvious,and large inflammatory cells infiltrated in the myocardial tissue.The myocardial structure destruction in the HGF group was significantly reduced compared with the MIRI group,a small amount of degeneration and necrosis of the cells,a reduction in interstitial edema,and a small amount of inflammatory cells.In the LY294002 group,the degeneration and necrosis of myocardial cells increased,interstitial edema,myolysis,and inflammatory cell infiltration were obvious.6.Comparison of cardiac function indexes in each groupCompared with the sham group,the LVIDs,LVIDd,LVEDV and LVESV indexes in the MIRI group were significantly increased,but the LVEF was significantly decreased;the LVIDs,LVIDd,LVEDV and LVESV indexes in the HGF group were lower than those in the MIRI group,and the LVEF was higher than the MIRI group;LV294s in the LY294002 group,The LVIDd,LVEDV and LVESV indicators were higher than the HGF group,still lower than the MIRI group,and the LVEF was lower than the HGF group,which was higher than the MIRI group.7.Expression of PI3K and pAkt protein in myocardium of each groupThe expression of PI3K and pAkt protein in the MIRI group was higher than that in the sham group.The HGF group was significantly higher than the MIRI group.The expression of PI3K and pAkt protein in the LY294002 group was lower than that in the MIRI and HGF groups,which was different from the sham group.No significantness.Conclusions1.HGF reduces myocardial ischemia-reperfusion injury by inhibiting cardiomyocyte apoptosis.2.The PI3K/Akt signaling pathway mediates the mechanism of action of HGF against apoptosis.Part III HSP70/NF-?B pathway mediates HGF anti-inflammatory intervention for myocardial ischemia-reperfusion injuryObjective1.To observe the changes of inflammatory factors IL-6,IL-1?,TNF-a and HMGB1 by constructing a rat model of ischemia-reperfusion and using HGF intervention.2.To explore the regulatory mechanism of HSP70/NF-?B pathway in HGF anti-inflammatory intervention in myocardial ischemia-reperfusion injury.Methods1.Experimental animals and subgroups:32 male Wistar rats were randomly divided into 4 groups(n=8),including sham group:only threaded without ligation;MIRI group ligated the left anterior descending artery for 30 minutes,then Infusion for 180 minutes;HGF group:HGF(1 mg/Kg)was administered to the tail vein while ligating the anterior descending artery;HSP70 inhibitor group(VER-155008 group):VER-155008 was given intravenously 2 hours before the HGF intervention.(25mg/Kg).2.Serum CK-MB levels were measured in each group.The levels of inflammatory factors IL-6,IL-1?,TNF-a and HMGB1 in each group were measured by ELISA.3.Conventional pathological staining to observe the inflammatory changes of myocardial tissue under the microscope.4.Immunohistochemistry was used to measure HSP70 protein expression.5.The expression levels of I?Ba protein and NF-?Bp65 protein in each group were measured by western blot.6.Echocardiography was used to measure the changes of cardiac function in each group.Results1.Establishment of rat myocardial ischemia-reperfusion modelThe Sham group was a normal electrocardiogram.In the MIRI group,the ST-segment was raised upwards 30 minutes after ligation of the anterior descending branch,suggesting that the ligation was successful,the ligation line was loosened,and the ventricular arrhythmia,ie,reperfusion,may occur after the perfusion was restored.Arrhythmia.Reperfusion arrhythmia was reduced in the HGF intervention group.and the proportion of reperfusion arrhythmias increased in the VER-155008 group.2.Serum CK-MB levels in each groupThe serum CK-MB level in the MIRI group was significantly higher than that in the sham group and the HGF group.The myocardial enzymes in the HGF group were lower than those in the MIRI group,which was still higher than that in the sham group.The serum CK-MB level in the VER-155008 group was higher than that in the HGF group.,still below the MIRI group.3.Serum levels of IL-6,IL-1?,TNF-a and HMGB1 in each group The levels of serum IL-?,IL-1b,TNF-a and HMGB1 in the MIRI group were significantly higher than those in the sham group.The levels of IL-6,IL-1?,TNF-a and HMGB1 in the HGF group were lower than those in the MIRI group,still higher than sham.The levels of serum IL-6,IL-1 ?,TNF-a and HMGB1 in the VER-155008 group were higher than those in the HGF group,which was still lower than that in the MIRI group.4.Myocardial pathological changes of rats in each group The pathological changes of myocardial morphology were observed by conventional HE staining.The myocardial structure of the sham group was intact,the myocardial fibers were arranged regularly,and there was no obvious inflammatory cell infiltration in the myocardial tissue.The MIRI group had disordered myocardial fibers,cell degeneration and necrosis,interstitial edema.Myolysis was obvious,and macroscopic inflammatory cells infiltrated in myocardial tissue;myocardial structural destruction in HGF group was significantly reduced compared with MIRI group,small amount of degeneration and necrosis,decreased interstitial edema,and a small amount of inflammatory cell infiltration;VER-155008 group of rat cardiomyocytes Increased degeneration and necrosis,interstitial edema,myolysis,and inflammatory cell infiltration were significantly increased compared with the HGF group,but still reduced compared with the MIRI group.5.The level of HSP70 protein in rat myocardiumThe expression of HSP70 protein in myocardial tissue of HGF group was significantly higher than that of MIRI group and sham group.The expression of HSP70 protein in HGF group and MIRI group was significantly higher than that in VER-155008 group;VER-155008 group rat myocardial tissue There was no significant difference in the expression level of HSP70 protein compared with the control group,suggesting that VER-155008 fully inhibited the expression of HSP70 protein.6.Levels of myocardial I?Ba and NF-?KBp65 tested by Western blot analysis 6.1 Effect of HGF on the expression of I?Ba protein in each groupThe expression of I?Ba protein in myocardial tissue of HGF group was significantly higher than that of control group and MIRI group.The level of IKBa protein in myocardial tissue of MIRI group was lower than that of control group;the level of IKBa protein in myocardial tissue of VER-155008 group was higher than that of MIRI group.There was no statistically significant difference in the level of I?Ba protein in the myocardial tissue of the HGF group compared with the control group.6.2 Comparison of NF-KBp65 protein expression in myocardial tissue of each groupThe expression of NF-?Bp65 protein in myocardial tissue of rats in MIRI group was significantly higher than that in control group.The level of NF-KBp65 protein in myocardial tissue of HGF group was lower than that in MIRI group and VER-155008 group,which was still higher than that of control group.The level of NF-KBp65 protein in myocardial lung tissue of VER-155008 group was still lower than that of MIRI group.7.Comparison of cardiac function indexes in each groupCompared with the sham group,the LVIDs,LVIDd,LVEDV,and LVESV indexes in the MIRI group were significantly increased,but the LVEF was significantly decreased.The LVIDs,LVIDd,LVEDV and LVESV indexes in the HGF group were lower than those in the MIRI group,and the LVEF was higher than the MIRI group.The LVIDs,LVIDd,LVEDV and LVESV indexes in the VER-155008 group were higher than those in the HGF group,still lower than the MIRI group,and the LVEF was lower than the HGF group,which was higher than the MIRI group.Conclusions1.HGF can reduce serum inflammatory factor levels in rats with myocardial ischemia-reperfusion and reduce the inflammatory response of myocardial ischemia-reperfusion injury.2.HSP70/NF-?B pathway mediates the mechanism of HGF anti-inflammatory alleviation of myocardial ischemia-reperfusion injury.