| BackgroundAcute myocardial infarction(AMI)is one of the most common causes of death in humans,and both ischemia and subsequent reperfusion can cause myocardial injury when AMI occurs."Time is myocardium."Although reperfusion as soon as possible is the best treatment for ischemic heart disease,it will cause tissue damage and destruction abnormally,namely ischemia/reperfusion injury(IRI).The final myocardial infarction size caused by reperfusion injury can reach up to 50%when AMI occurs.Decades of studies have confirmed that ischemia preconditioning(IPC)can reduces myocardial infarct size,which indicates that IPC has a strong and definite protective effect on myocardial IRI.As IPC needs to be performed before myocardial ischemia occurs,its clinical application is extremely limited.In contrast,pharmacological postconditioning has a greater clinical value due to its unique operational convenience.It is well known that the tissue injury caused by IRI results from the interaction of many factors,among which the inflammatory response is a crucial mechanism of this pathological process and exits in the whole process of myocardial IRI.In recent years,the cholinergic anti-inflammatory pathway(CAP)has attracted much attention for its inflammatory immune response,which plays a role through the important nicotinic acetylcholine receptor,namely α7 nicotinic acetylcholine receptor(α7nAChR).The main mechanism is that body senses the signal of inflammatory stimulus firstly,then the signal is afferent by vagus nerve,integrating through the central nervous system and efferenting to the nerve endings to release acetylcholine(ACh),which acts on the α7nAChR on the cell membrane such as macrophages to inhibit the release of pro-inflammatory cytokines.Therefore,it plays an important role in the anti-inflammatory process of the body.Phosphatidylinositol-3-kinase(PI3K)and its downstream protein kinase B(Akt),namely PI3K/Akt,are widespread intracellular signaling pathways involved in inflammatory responses.Endothelial nitric oxide synthase(eNOS)is a downstream effector of Akt,widely distributed and continuously expressed in mammalian cardiomyocytes.Activation of PI3K/Akt signaling pathway can upregulate eNOS expression and increase nitric oxide(NO)synthesis,thus exerting a protective effect on myocardial IRI.However,knockout of eNOS gene specifically can significantly aggravate myocardial IRI in mice.Available evidence suggests that the PI3K/Akt/eNOS signaling pathway plays an important role in the mechanism of myocardial IRI and the myocardial protective effects of various interventions,which is closely related to the inflammatory response.Hypercholesterolemia is an independent risk factor for ischemic heart disease and can significantly affect the protective effect of various interventions on myocardial IRI.However,there are no studies on whether hypercholesterolemia affects the protective effect of α7nAChR agonist postconditioning on myocardial IRI and the role of CAP and PI3K/Akt/eNOS signaling pathways in the myocardial protection of α7nAChR agonist postconditioning by hypercholesterolemia.Therefore,we designed this randomized controlled experiment in vivo to confirm the effect of hypercholesterolemia on the protective efficacy of IPC and PNU282987 postconditioning and its possible mechanism by comparing the cardioprotective effects,inflammatory response and PI3K/Akt/eNOS signaling pathway of IPC and PNU282987 postconditioning on normal and hypercholesterolemic myocardial IRI.In addition,this experiment sought to determine whether exogenous supplementation with tetrahydrobiopterin(BH4,cofactor of eNOS)could affect the cardioprotective effects of IPC and PNU282987 postconditioning on myocardial IRI in hypercholesterolemic rats,with the aim of providing an effective and feasible cardioprotective interventions.This experiment was performed with the support of Young Innovation Project Fund of Plastic Surgery Hospital,Chinese Academy of Medical Sciences(grant no.Q2017002)and was divided into four parts.Part I Establishment of a rat model of hypercholesterolemiaThe purpose of this experiment is to establish a rat model of hypercholesterolemia.Male Sprague Dawley(SD)rats were fed with high-cholesterol diet for 8 weeks.The body mass of the rats was recorded before and after 8 weeks of feeding.The serum total cholesterol(TC),triacylglycerol(TG),low-density lipoprotein(LDL)and high-density lipoprotein(HDL)level were measured by automatic biochemical analyzer to determine whether the model was successfully established.Twenty normal rats(body mass 130-150g)were acclimatized for 1 week and randomly divided into 2 groups:normal group(NC group,n=10)and high cholesterol group(HC group,n=10).We recorded basal body mass and measured serum TG,TC,LDL and HDL levels by blood sampling from caudal vein.Rats in normal group were fed a normal diet for 8 weeks,while rats in high cholesterol group were fed a highcholesterol diet containing 2%cholesterol and 0.5%bile salt for 8 weeks,and then blood was collected from the tail and the serum TG,TC,LDL and HDL levels were measured.The results showed that there was no statistically significant difference in basal body mass between the NC group and HC group(P>0.05).No significant statistical differences were noted in basal serum TG,TC,LDL and HDL levels between the NC group and the HC group(P>0.05).After 8 weeks of feeding,there was no significant difference in body mass between the NC group and HC group(P>0.05).Compared with the NC group,there was no statistically significant difference in serum TG levels(P>0.05);compared with the NC group,serum TC and LDL levels were significantly higher in the HC group(P<0.05),and serum HDL level was significantly lower in the HC group(P<0.05).Part Ⅱ Experimental Study on the role of inflammation and PI3K/Akt/eNOS signaling pathway in the protection of normal myocardium against ischemia/reperfusion injury by ischemic preconditioning and PNU282987 postconditioningThe purpose of this part was to assess the protective effects of ischemic preconditioning and PNU282987 postconditioning on myocardial IRI in normal rats and to evaluate the alterations in the inflammation and P13K/Akt/eNOS signaling pathway with the aim of determining the role of inflammation and PI3K/Akt/eNOS signaling pathway in the cardioprotective effects of the two interventions.Forty normal rats(body mass 130-150 g)were fed adaptively for 1 week,and then fed with normal chow for 8 weeks.After anesthetized,all rats were divided equally into four groups at random:normal control group(NC group,n=10),normal IRI group(NI group,n=10),normal ischemia preconditioning group(NIPC group,n=10),and normal PNU282987 postconditioning group(NPNU group,n=10).After thoracotomy,all rats were threaded below the left anterior descending coronary artery(LAD)and stabilized for 10 min.In the NC group,no further operation was performed except that 1ml saline was administered via the internal jugular vein after 30 min of observation.In the NI group,LAD was blocked for 30 min and 1 ml saline was injected intravenously before reperfusion for 120 min.In the IPC group,3 cycles of intervention were performed before blocking LAD;Each cycle was composed of 5-min occlusion and 5-min reperfusion of the LAD,then the LAD was blocked for 30 min;lml saline was administered intravenously before reperfusion for 120 minutes.In the NPNU group,LAD was blocked for 30 min,then PNU282987(2 mg/kg,total volume 1 ml)was injected intravenously before reperfusion and the LAD was recanalized for 120 min.At the end of the experiment,blood samples were taken from the carotid artery in each group.Serum TC and TG concentrations were measured by oxidase assay,and serum creatine kinase isoenzyme(CK-MB)concentration was measured by Immunosuppression assay,and lactate dehydrogenase(LDH)and cardiac troponin I(cTnI)concentration were measured by the rate method,and the concentrations of Tumour Necrosis Factor α(TNF-α)and Interleukin 6(IL-6)were measured by the ELISA method.The ischemic left ventricular myocardium was analyzed by Western blotting for Akt,p-Akt and eNOS.We determined the infarct size(IS%)by Evans blue staining and 2,3,5-triphenyltetrazolium chloride(TTC)staining.The results at 120 minutes of reperfusion showed that serum LDH,CK-MB and cTnI concentrations were significantly higher in the NI,NIPC and NPNU groups compared with the NC group(P<0.05);serum LDH,CK-MB and cTnI concentrations were significantly lower in the NIPC and NPNU groups compared with the NI group(P<0.05);compared with the NIPC group,serum CK-MB and cTnI levels were obviously reduced in the NPNU group(P<0.05).Serum TNF-α and IL-6 concentrations were significantly higher in the NI,NIPC and NPNU groups compared with the NC group(P<0.05);serum TNF-α and IL-6 concentrations were significantly lower in the NIPC and NPNU groups compared with the NI group(P<0.05);serum TNF-α and IL-6 concentrations were significantly higher in the NPNU group compared with the NIPC group(P<0.05).Since the NC group did not experience ischemic conditioning and reperfusion injury,myocardial infarction was not observed.IS%was significantly smaller in the NIPC and NPNU groups compared with the NI group(P<0.05)and increased in the NPNU group compared with the NIPC group(P<0.05).In the normal rats,compared with the NC group,the myocardial p-Akt/Akt and eNOS/GAPDH ratios were obviously increased in the NI,NIPC and NPNU groups(P<0.05);compared with the NI group,the myocardial p-Akt/Akt and eNOS/GAPDH ratios were significantly higher in the NIPC and NPNU groups(P<0.05);compared with the NIPC group,the myocardial p-Akt/Akt ratio was obviously decreased in the NPNU group(P<0.05),but myocardial eNOS/GAPDH ratio was not significantly changed in the NPNU group(P>0.05).Part III Experimental Study on the role of inflammation and PI3K/Akt/eNOS signaling pathway in the protection of hypercholesterolemic myocardium against ischemia/reperfusion injury by ischemic preconditioning and PNU282987 postconditioningThe purpose of this part of experiment was to observe the myocardial protection of ischemic preconditioning and PNU282987 postconditioning on myocardial IRI in hypercholesterolemic rats and to evaluate changes in the inflammatory response and P13K/Akt/eNOS signaling pathway,with the aim of determining whether hypercholesterolemia affects the cardioprotective effects of ischemic preconditioning and PNU282987 postconditioning by altering the inhibition of the inflammatory response and activation of the P13K/Akt/eNOS signaling pathway.Forty normal rats(body mass 130-150 g)were fed adaptively for 1 week,and then fed with high cholesterol chow for 8 weeks.After anesthetized,they were randomly divided into 4 groups:hypercholesterolemic control group(HC group,n=10),hypercholesterolemic IRI group(HI group,n=10),hypercholesterolemic ischemia preconditioning group(HIPC group,n=10),hypercholesterolemic PNU282987 postconditioning group(NPNU group,n=10).After the chest was opened,all rats were threaded below the LAD and stabilized for 10 min.In the HC group,none was performed except that 1ml saline was administered via the internal jugular vein after 30 min of observation.In the HI group,LAD was blocked for 30 min and 1ml saline was injected intravenously before reperfusion for 120 min.In the HIPC group,3 cycles of intervention were performed before blocking LAD,and each cycle was composed of 5-min occlusion and 5-min reperfusion of the LAD;then the LAD was blocked for 30 min,and 1ml saline was administered intravenously before reperfusion for 120 minutes.In the HPNU group,LAD was blocked for 30 min;PNU282987(2 mg/kg,total volume 1ml)was injected intravenously before reperfusion and the LAD was recanalized for 120 min.At the end of the experiment,blood samples were drawn from the carotid artery in each group.Serum TC,LDL,LDH,CK-MB,cTnI,TNF-α and IL-6 levels were measured.Left ventricular myocardium were analyzed by Western blotting for Akt,p-Akt and eNOS.The IS%was determined by double staining with Evans blue and TTC.The results showed that serum TC and LDL levels after 8 weeks were significantly higher compared with the basal values(P<0.05).Compared with the HC group,serum LDH,CK-MB and cTnI concentrations were significantly higher in the HI,HIPC and HPNU groups(P<0.05);compared with the HI group,serum LDH,CK-MB and cTnI concentrations in the HIPC group decreased dramaticlly(P<0.05),and serum LDH concentration was significantly lower in the HPNU group(P<0.05),but the differences in CK-MB and cTnI concentrations were not statistically significant in the HPNU group(P>0.05);compared with the HIPC group,the difference in serum LDH concentration in the HPNU group was not statistically significant(P>0.05),but serum CK-MB and serum cTnI concentrations were significantly higher in the HPNU group(P<0.05).Serum TNF-α and IL-6 concentrations were significantly higher in the HI,HIPC,and HPNU groups compared with the HC group(P<0.05);compared with the HI group,serum TNF-α and IL-6 levels were significantly reduced in the HIPC group(P<0.05);compared with the HIPC group,serum TNF-α and IL-6 levels were obviously elevated in the HPNU group(P<0.05).Since the HC group did not experience ischemic conditioning and reperfusion injury,myocardial infarction was not observed.Compared with the HI group,the IS%was significantly reduced in the HIPC group(P<0.05),but the difference in IS%in the HPNU group was not statistically significant(P>0.05);compared with the HIPC group,the the IS%was significantly increased in the HPNU group(P<0.05).Compared with the HC group,p-Akt/Akt and eNOS/GAPDH ratios were significantly higher in the HI,HIPC and HPNU groups(P<0.05);there was no significant difference between the HI,HIPC and HPNU groups in p-Akt/Akt and eNOS/GAPDH ratios(P>0.05).Part Ⅳ Experimental study on the effects and its mechanisms of administrating tetrahydrobiopterin on myocardial protection of ischemic preconditioning and PNU282987 postconditioning in hypercholesterolemic ratsThe purpose of this part was to observe the effect of ischemic preconditioning and PNU282987 postconditioning on myocardial protection by applying tetrahydrobiopterin(BH4)to hypercholesterolemic rats,aiming to to determine whether the application of BH4 could improve the cardioprotective effect of both interventions on myocardial IRI and to investigate the role of PI3K/Akt/eNOS signaling pathway,inflammatory response,and apoptosis in the cardioprotective effects.Forty normal rats(body mass 130-150 g)were fed adaptively for 1 week,and then fed with high cholesterol chow for 8 weeks.After anesthetized,they were randomly divided into 4 groups:hypercholesterolemic IRI group(HI group,n=10),BH4 postconditioning of hypercholesterolemic IRI group(HI+B group,n=10),BH4 postconditioning together with IPC of hypercholesterolemic group(HIPC+B group,n=10),and BH4 combined with PNU282987 post conditioning of hypercholesterolemic IRI group(HPNU+B group,n=10).After the chest was opened,all rats were threaded below the LAD and stabilized for 10 min.In the HI group,LAD was blocked for 30 min and 1 ml saline was injected intravenously before reperfusion for 120 min.In the HI+B group,LAD was blocked for 30 min and BH4(10 mg/kg,total volume 1 ml)was injected intravenously before reperfusion for 120 min.In the HIPC+B group,3 cycles of ischemic preconditioning were performed before LAD blockade;each cycle was composed of 5-min occlusion and 5-min reperfusion of the LAD,followed by 30 min of LAD blockade,then BH4(10 mg/kg,total volume 1 ml)was administrated intravenously before reperfusion for 120 min.In the HPNU+B group,LAD blockade was performed for 30 min;PNU282987 and BH4(total volume 1 ml)was administrated intravenously before reperfusion for 120 min.At the end of the experiment,blood specimens were taken from the carotid artery,and serum levels of LDH,CK-MB,cTnI,TNF-α and IL-6 were measured;left ventricular myocardium were analyzed by Western blotting for Akt,p-Akt and eNOS.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect myocardial Akt mRNA,eNOS mRNA,Bax mRNA and Bcl-2 mRN expression.The IS%was determined by double staining with Evans blue and TTC.Compared with the HI group,serum LDH concentration were significantly lower in the HI+B,HIPC+B and HPNU+B groups(P<0.05);compared with the HI+B group,serum LDH concentration were significantly lower in the HIPC+B group(P<0.05),but did not obviously change in the HPNU+B groups(P>0.05);compared with the HIPC+B group,serum LDH level was evidently increased in the HPNU+B group(P<0.05).Compared with the HI group,the differences in serum CK-MB and cTnI concentrations between the HI+B and HPNU+B groups were not statistically significant(P>0.05),but serum CK-MB and cTnI concentrations were dramatically decreased in the HIPC+B group(P<0.05);compared with the HI+B group,serum CK-MB and cTnI levels were evidently reduced in the HIPC+B group(P<0.05),while the differences in serum CK-MB and cTnI concentrations in the HPNU+B group were not statistically significant(P>0.05);compared with the HIPC+B group,serum CK-MB and cTnI concentrations were significantly higher in the HPNU+B group(P<0.05).The concentrations of serum TNF-α and IL-6 in HI,HI+B and HPNU+B groups had no statistical significance.(P>0.05).Compared with the HI group,serum TNF-α and IL-6 concentrations group were significantly lower in the HIPC+B(P<0.05);compared with the HI+B group,serum TNF-α and IL-6 levels were evidently reduced in the HIPC+B group(P<0.05);compared with the HIPC+B group,serum TNF-α and IL-6 levels were significantly higher in the HPNU+B group(P<0.05).In the hypercholesterolemic rats,compared with the HI group,IS%did not obviously change in the HI+B and HPNU+B groups(P>0.05),but dramatically lessen in the HIPC+B group(P<0.05).Compared with the HI+B group,IS%was significantly lower in the HIPC+B group(P<0.05);nevertheless,there was no statistically significant difference in IS%between the HI+B and HPNU+B group(P>0.05).Compared with the HIPC+B group,IS%was significantly higher in the HPNU+B group(P<0.05).There was no significant difference in myocardial Akt mRNA expression among the HI,HI+B,HIPC+B and HPNU+B groups(P>0.05).Compared with the HI group,myocardial eNOS mRNA expression was significantly higher in the HI+B,HIPC+B and HPNU+B groups(P<0.05);however,there were not significantly difference in the myocardial eNOS mRNA expression between the HI+B,HIPC+B and HPNU+B groups(P>0.05).There was no significant difference in myocardial p-Akt/Akt ratio among the HI,HI+B,HIPC+B and HPNU+B groups(P>0.05).Compared with the HI group,myocardial p-Akt/Akt ratio was significantly higher in the HI+B,HIPC+B and HPNU+B groups(P<0.05),while there was no significant difference in myocardial p-Akt/Akt ratio among HI+B,HIPC+B and HPNU+B groups(P>0.05).There was no significant difference in myocardial Bcl-2/Bax ratio in the HI,HI+B and HPNU+B groups(P>0.05).Compared with the HIPC+B group,the myocardial Bcl-2/Bax ratio was significantly lower in the HI,HI+B and HPNU+B groups(P<0.05).ConclusionsBased on the results of all experiments,the following conclusions can be drawn:1.Hypercholesterolemic rat model can be successfully established when normal rats were fed high cholesterol diet containing 2%cholesterol and 0.5%bile salt for 8 weeks.2.Both IPC and α7nAChR agonist postconditioning have myocardial protective effect in normal rats,but the myocardial protective effect of IPC is greater than that ofα7nAChR agonist postconditioning.Inhibition of inflammatory response and activation of PI3K/Akt/eNOS signaling pathway are involved in the process of myocardial protective effect of α7nAChR agonist postconditioning and IPC in normal rats.3.Hypercholesterolemia can aggravate myocardial IRI,weaken the myocardial protective effect of IPC,and eliminate the myocardial protective effect of α7nAChR agonist postconditioning.Enhanced inflammatory response and decreased activation of myocardial PI3K/Akt/eNOS signaling pathway are involved in the process of hypercholesterolemia affecting the myocardial protective effect of α7nAChR agonist postconditioning and IPC.4.Exogenous supplementation of BH4 could not activate the PI3K/Akt signaling pathway but could increase the level of eNOS.However,increased expression of eNOS did not restore the cardioprotective effect of α7nAChR agonist postconditioning.BH4 enhanced the myocardial protective effect of IPC in hypercholesterolemic rats,which may be associated with inhibition of inflammation and anti-apoptosis. |
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