| BackgroundAlthough restoring blood perfusion after acute coronary artery occlusion is the most effective way to save ischemic myocardium,it can also lead to the aggravation of myocardial tissue damage,which is defined as myocardial ischemia/reperfusion injury(MIRI).MIRI is the main cause of disability or even death in patients with ischemic heart disease.Therefore,how to avoid or alleviate the organ or tissue IRI is always an important challenge that clinician faces.Diabetes is a common complication of patients with ischemic heart disease,and it is associated with high mortality after acute myocardial infarction(AMI).Available evidences have shown that diabetes can increase myocardial susceptibility to IRI,and it can affect the severity of MIRI by destroying intracellular signaling transduction which plays an important role in cardioprotective therapy against cell death.The pro-survival protein kinases cascade formed by PI3K/Akt signaling transduction pathway is an important part of the reperfusion injury salvage kinase(RISK)pathway,so the activation of PI3K/Akt signaling transduction pathway can protect myocardium against lethal IRI.The study has found that glycogen synthase kinase-3?(GSK-3?)is an important downstream component of PI3K/Akt signaling transduction pathway.In diabetic MIRI model,the decrease or disappearance of the protective effect of the relevant interventions is mainly attributed to the dysfunction of RISK pathway.Insulin deficiency or resistance is also involved in RISK pathway alterations.Insulin can lead to the activation of Akt by phosphorylating Thr308 and Ser473,and then activated Akt can inhibit the opening of mitochondrial permeability transition pore(mPTP)by GSK-3? phosphorylation.Therefore,insulin deficiency or resistance can impact PI3K/Akt/GSK-3? signaling transduction pathway.PI3K/Akt/GSK-3? signaling transduction pathway may play an important role in diabetic MIRI,but specific mechanisms are still unclear and further studies are needed to illustrate the mechanisms.Apelin/APLN is a polypeptide produced by the APLN gene on human chromosomes and is an endogenous ligand of APJ.Apelin and its receptor APJ are widely expressed in various tissues,especially in the cardiovascular system.Apelin is cleaved into shorter biologically active C-terminal fragments in the endoplasmic reticulum.Different fragments of apelin have different activities in different metabolic pathways.Apelin-13 is a most widely studied active polypeptide at present.It has been shown that,in normal rat Langendorff isolated heart,the administration of Apelin-13 can ameliorate MIRI by inducing ERK1/2 and Akt phosphorylations and increasing the expression of eNOS.In addition,in in vivo rat model of MIRI,Apelin-13 can inhibite endoplasmic reticulum stress-induced apoptosis through PI3K/Akt,AMPK and ERK signaling transduction pathways in MIRI.Apelin-13 also inhibits the decrease of mitochondrial membrane potential and protects the heart against MIRI through the PI3K/Akt/GSK-3?/mPTP signaling transduction pathway.These results suggested that Apelin-13 may play an important role in normal MIRI through many pathways.Existing studies have shown that Apelin is closely related to diabetes.Apelin can improve diabetic cardiac function by increasing myocardial angiogenesis,glucose uptake and insulin sensitivity,indicating that Apelin has a certain therapeutic potential in diabetic cardiac dysfunction.However,there are few studies involved in the effects of Apelin-13 on diabetic MIRI.As we all know,risk factors associated with ischemic heart disease,such as hypertension,hypercholesterolemia,diabetes and aging,can significantly affect the protective effects of various interventions on MIRI.Although studies have shown that Apelin-13 can exert significant protective effect against normal MIRI,there is no study to determine whether Apelin-13 has protective effect on diabetic MIRI.Considering that the myocardial protective effect of Apelin-13 should be validated in pathological animal models with ischemic heart disease risk factors before it can be effectively applied in clinic,we designed this randomized controlled experiment.By comparing the effects of Apelin-13 on MIRI,cardiomyocyte apoptosis,myocardial histology and related signaling transduction pathway in normal and diabetic rats,the object of this study was to evaluate the protection and related mechanisms of Apelin-13 against diabetic MIRI,especially the specific role of apoptosis and PI3K/Akt/GSK-3? signaling transduction pathway in the protective effect of Apelin-13 on diabetic MIRI.This experiment is divided into four parts:Part 1 Establishment of a Diabetic Rat ModelThis part experiment was designed to establish a diabetic rat model.The male Sprague Dawley(SD)rats were treated with an intraperitoneal injection of streptozotocin and were bred with the high fat and high sugar diet.Both the body weights and blood glucose levels during the establishment of diabetic rat model were measured by continuous observation and monitoring.The food intake,water consumption,urine volume,mental state and the gloss of the fur of the rats were observed.And the above indicators were used to test whether the diabetic rat model was successfully established.Normal rats were bred for 1 w with the normal diet,the body weights and blood glucose levels of the rats were recorded as baseline values.They were fasted for 12 h,but kept drinking water,and the rats were treated with an intraperitoneal injection of citric acid buffer,which volume was the same as streptozotocin in diabetic rats.Then the rats were continued to feed for 8 w,and their body weights and random blood glucose levels were recorded at 48 h,2 w,4 w,6 w and 8 w after baseline values were recorded.Diabetic rat model was established according to the method reported previously.Eight-week-old male SD rats were bred for 1 w with the high fat and high sugar diet.The body weights and blood glucose levels of the rats were recorded as baseline values.Afterwards,the rats were fasted for 12 h,but kept drinking water,then they were treated with an intraperitoneal injection of streptozotocin(STZ,50 mg/kg).Diabetic condition was verified 48 h later by evaluating blood glucose levels.Rats with blood glucose levels of 16.65 mmol/L or greater were considered to be diabetic condition.The diabetic rats were bred with the high fat diet for an additional 8 w,and their body weights and random blood glucose levels were recorded at 48 h,2 w,4 w,6 w and 8 w after baseline values were recorded.The random blood glucose levels of diabetic rats were maintained at or above 16.65 mmol/L during feeding.The results showed that the body weights of normal and diabetic rats have been increasing during feeding.The weights of normal rats at 2 w were significantly higher than baseline value,and the weights of normal and diabetic rats at 4 w,6 w and 8 w were significantly higher than baseline values.The random blood glucose levels of normal rats at 48 h,2 w,4 w,6 w,and 8 w were not significantly different compared with baseline values.But the random blood glucose levels of diabetic rats at 48 h,2 w,4 w,6 w and 8 w were significantly higher than baseline values and those of normal rats.Part 2 Experimental study on protection of Apelin-13 against diabetic myocardial ischemia/reperfusion injuryThe object of this part of the experiment was to evaluate the protective effect of Apelin-13 on diabetic MIRI by comparing the effects of Apelin-13 on normal and diabetic MIRI.Thirty healthy male SD rats and thirty diabetic rats with in vivo rat model of MIRI were randomly divided into 6 groups(10 in each group):normal blank control group(C group),normal IRI group(IRI group),normal Apelin-13 group(A group),diabetic blank control group(DC group),diabetic IRI group(DIRI group)and diabetic Apelin-13 group(DA group).After left thoracotomy in all rats,a silk ligature was placed around the left anterior descending coronary artery(LAD)and encircled with a suture.In all rats except those in C and DC groups,the LAD was ligated for 30 min followed by a 120-min reperfusion.In the C and DC groups,no additional intervention was performed.In the IRI and DIRI groups,an in vivo rat model of MIRI was established.In the A and DA groups,Apelin-13(0.1 mg/kg)was injected intravenously immediately before a 120-min reperfusion.Throughout the experiment,heart rate(HR),mean arterial pressure(MAP),and a lead II electrocardiogram(ECG)were continuously monitored.Blood samples were taken at 120min of reperfusion for measuring serum concentrations of cardiac troponin I(cTnI)and creatinine kinase isoenzyme MB(CK-MB)by the kits specifically for rats.At the end of experiment,infarction size(IS)was assessed from excised hearts by Evan's blue and triphenyltetrazolium chloride(TTC)staining.The results showed no significant differences in body weights between normal and diabetic rats.The blood glucose levels in diabetic rats were significantly higher than that in normal rats.There was no significant difference between baseline values and hemodynamic parameters after 15 min ischemia.The HR of IRI,DIRI,A and DA groups at 1 to 5 min during myocardial ischemia were significantly higher than baseline values,whereas MAP and RPP of IRI,DIRI,A and DA groups at 1 to 5 min during myocardial ischemia were significantly lower than baseline values.There were no significant differences in the number of rats with VT and VF between IRI and DIRI groups,A and DA groups,whether during ischemic period or the early reperfusion.Compared with the C group,serum cTnI and CK-MB concentrations and IS were significantly increased in the IRI and A groups.Compared with the IRI group,serum cTnI and CK-MB concentrations and IS were significantly decreased in the A group,whereas significantly increased in the DIRI group.Compared with the A group,IS was significantly increased in the DA group.Compared with the DC group,serum cTnI and CK-MB concentrations and IS were significantly increased in the DIRI and DA groups.Compared with the DIRI group,serum cTnI and CK-MB concentrations and IS were significantly decreased in the DA group.Part 3 Experimental study on the effect of Apelin-13 on apoptosis in diabetic myocardial ischemia/reperfusion injuryThe object of this part experiment was to observe the effect of Apelin-13 on apoptosis and myocardial histology in diabetic MIRI,and to explore the role of apoptosis in the protection of Apelin-13 against diabetic MIRI.Thirty healthy male SD rats and thirty diabetic rats with in vivo rat model of MIRI were randomly divided into 6 groups(10 in each group),the grouping and processing methods were same as the second part experiment.At the end of reperfusion,myocardial tissue samples were collected from the ischemic area.Apoptotic index(AI)in ischemic area was assessed through Tunel assay.The optical microscope and electron microscope were used to observe the cardiomyocyte morphology.The results showed that,there were no significant differences in body weights between normal and diabetic rats.The blood glucose levels in diabetic rats were significantly higher than that in normal rats.Compared with the C group,the AI was significantly increased in the IRI,A and DC groups.Compared with the IRI group,the AI was significantly decreased in the A group,whereas significantly increased in the DIRI group.Compared with the A group,the AI was significantly increased in the DA group.Compared with the DC group,the AI was significantly increased in the DIRI and DA groups.Compared with the DIRI group,the AI was significantly decreased in the DA group.Under the optical microscope,the cardiomyocytes in the A group were sparsely arranged,cardiomyocytes degeneration and myocardial tissue edema occurred,myocardial fiber arrangement was slightly disordered,and some inflammatory cells infiltrated,the pathological changes in the A group were significantly less severe than that in the IRI group.The number of cardiomyocytes was decreased in the DA group compared to the A group,cardiomyocytes degeneration and myocardial tissue edema were observed,myocardial fiber arrangement was slightly disordered,and some inflammatory cells infiltrated in the DA group.The pathological changes were significantly less severe in the DA group than that in the DIRI group.Under the electron microscope,the morphology of cardiomyocytes in the A and DA groups was basically normal.The myofibrils were structurally intact,the Z-line arrangement was roughly tidy.Most of the mitochondria were normal in morphology and structurally intact,and the cristae in mitochondria was clear and well arranged.Part 4 Experimental study on role of PI3K/Akt/GSK-3p signaling transduction pathway in protection of Apelin-13 against diabetic myocardial ischemia/reperfusion injuryIn this part experiment,PI3K inhibitors were used in normal and diabetic MIRI models.By comparing the myocardial protection of Apelin-13 and its effects on the expressions of Akt and GSK-3? in myocardium,the object of this part experiment was to explore the role of PI3K/Akt/GSK-3? signaling transduction pathway in protection of Apelin-13 against diabetic MIRI.Sixty healthy male SD rats and sixty diabetic rats with in vivo rat model of MIRI were randomly divided into 12 groups(10 in each group):normal blank control group(C group),normal IRI group(IRI group),normal Apelin-13 group(A group),normal Apelin-13+LY294002 group(A+L group),normal LY294002 group(L group),normal 0.2%DMSO group(DMSO group),diabetic blank control group(DC group),diabetic IRI group(DIRI group),diabetic Apelin-13 group(DA group),diabetic Apelin-13+LY294002 group(DA+L group),diabetic LY294002 group(DL group)and diabetic 0.2%DMSO group(DDMSO group).After left thoracotomy in all rats,a silk ligature was placed around LAD and encircled with a suture.In all rats except those in C and DC groups,the LAD was ligated for 30 min followed by a 120 min reperfusion.In the C and DC groups,no additional intervention was performed.In the IRI and DIRI groups,an in vivo rat model of MIRI was established.In the A and DA groups,Apelin-13(0.1 mg/kg)was injected intravenously immediately before a 120-min reperfusion.In the A+L and DA+L groups,LY294002(0.3 mg/kg)and Apelin-13(0.1 mg/kg)were injected intravenously immediately before a 120-min reperfusion.In the L and DL groups,LY294002(0.3 mg/kg)was injected intravenously immediately before a 120-min reperfusion.In the DMSO and DDMSO groups,0.2%DMSO was injected intravenously immediately before a 120-min reperfusion.At 120 min of reperfusion,blood samples were taken for measuring serum cTnI and CK-MB concentrations by ELISA kits,and IS was assessed from excised hearts by Evan's blue and TTC staining.Phosphorylated Akt and GSK-3? in ischemic myocardium were assessed through western blotting.And the expressions of Akt and GSK-3? mRNA were assessed through real time PCR.The results showed that,compared with the C group,serum cTnI and CK-MB concentrations and IS were significantly increased in the IRI,A,A+L,L and DMSO groups.Compared with the IRI group,serum cTnI and CK-MB concentrations and IS were significantly decreased in the A group,whereas significantly increased in the DIRI group.Compared with the A group,IS was significantly increased in the DA group.Compared with the DMSO group,serum cTnI and CK-MB concentrations and IS were significantly increased in the DDMSO group.Compared with the DC group,serum cTnI and CK-MB concentrations and IS were significantly increased in the DIRI,DA,DA+L,DL and DDMSO groups.Compared with the DIRI group,serum cTnI and CK-MB concentrations and IS were significantly decreased in the DA group.Compared with the C group,p-Akt/Akt and p-GSK-3p/GSK-3p ratios were significantly decreased in the IRI,A+L,L,DMSO and DC groups.Compared with the IRI group,p-Akt/Akt and p-GSK-3?/GSK-3? ratios were significantly increased in the A group,whereas significantly decreased in the DIRI group.Compared with the DC group,p-Akt/Akt and p-GSK-3?/GSK-3? ratios were significantly decreased in the DIRI,DA+L,DL and DDMSO groups.Compared with the DIRI group,p-Akt/Akt ratio was significantly increased in the DA group,however,there was no significant difference in p-GSK-3?/GSK-3? ratio between DIRI and DA groups.Compared with the DA group,p-Akt/Akt ratio was significantly decreased in the DA+L and DL groups,p-GSK-3?/GSK-3? ratio was significantly decreased in the DL group,whereas there was no significant difference in p-GSK-3?/GSK-3? ratio between DA and DA+L groups.Compared with the C group,myocardial expressions of Akt and GSK-3? mRNA were increased significantly in the IRI,A,A+L and DMSO groups,myocardial expression of Akt mRNA was increased significantly in the DC group,and myocardial expression of GSK-3? mRNA was decreased significantly in the DC group.Compared with the IRI group,the myocardial expressions of Akt mRNA in A and DIRI groups were significantly increased,while the myocardial expressions of GSK-3? mRNA were significantly decreased in A and DIRI groups.Compared with the A group,the myocardial expression of Akt mRNA was significantly increased in the DA group.Compared with the DC group,the myocardial expressions of Akt and GSK-3P mRNA in DIRI group,DA group and DDMSO group were increased significantly,while myocardial expressions of GSK-3? mRNA in the DA+L and DL groups were significantly increased.Compared with the DIRI group,the myocardial expression of Akt mRNA was significantly increased in the DA group,while myocardial expressions of Akt and GSK-3? mRNA were significantly decreased in the DA+L and DL groups.Compared with the DA group,myocardial expressions of Akt and GSK-3? mRNA were significantly decreased in the DA+L and DL groups.ConclusionsBased on the results of all experiments,the following conclusions can be drawn:1.The diabetic rat model can be successfully established by intraperitoneal injection of STZ combined with high-fat and high-sugar diet.2.Apelin-13 can provide protection against normal and diabetic MIRI.3.Apelin-13 can significantly inhibit cardiomyocyte apoptosis in normal and diabetic MIRI,and significantly improve myocardial cell morphology,suggesting that inhibition of apoptosis is one of the important mechanisms for protection of Apelin-13 against MIRI.4.Apelin-13 can inhibit the activity of GSK-3? by activating PI3K/Akt signaling transduction pathway and exert protection on normal MIRI.Although Apelin-13 can exert protection on diabetic MIRI by activating PI3K/Akt signaling transduction pathway,the protective effect does not depend on the inhibition of GSK-3? activity.5.There is no significant difference in the protection of Apelin-13 between normal and diabetic MIRI.It may be that the protection of Apelin-13 against diabetic MIRI does not depend on the activation of the intact PI3K/Akt/GSK-3? signaling transduction pathway. |
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