| BackgroundStudies have shown that the mortality rate attributed to myocardial ischemia reperfusion injury (IRI) is still up to10%after prompt restoration of blood flow in the infarct-related coronary. In addition, many animal researches have shown that the IRI is responsible for up to50%of the final infarct size during acute myocardial ischemia. Although the ischemic preconditioning (IPC) remains the most powerful cardioprotective measure, its clinical application has been hampered by the requirement of intervention before onset of acute myocardial ischemia, which is clearly impossible in the setting of acute myocardial infarction. Ischemic postconditioning (IPOC) can be triggered during the clinically applicable period of myocardial reperfusion, which has already been shown cardioprotective. However, it can cause dangerous complications including rupture of diseased coronary artery and fall off of atheromatous plaque, which attribute to the requirement of repeated occlusion of coronary artery. Thus, the clinical application of IPOC is quite limited too. In contrast, limb remote ischemic postconditioning (LRIPOC) can be operated away from the heart and it has definitely cardioprotective effects. In addition, LRIPOC is easy to operate, safe and low-cost. As a result, LRIPOC is really a good choice to treat myocardial IRI.Endogenous inflammatory response is a key factor in formation and progression of myocardial IRI. Recently, the immunoregulation of vagus nerve has been demonstrated, which can modulate inflammation by the cholinergic anti-inflammatory pathway when activated. During inflammatory diseases such as endotoxemia, colonitis, rheumatic arthritis and IRI, vagus nerve stimulation can provide protection by inhibiting inflammatory cells recruitment and inflammatory cytokines release. Moreover, it has been demonstrated that vagus nerve stimulation can reduce the infarct size caused by myocardial IRI. However, there has been no research observing the effects of vagus nerve stimulation postconditioning (POES) on local myocardial and systemic inflammation during myocardial IRI.Combining different interventions to obtain an augmented cardioprotection is always one of the most popular research focuses in the area of myocardial IRI. Obviously, POES and LRIPOC might be triggered by different mechanisms. As a result, this randomized, controlled animal experimental study was designed and the aims of the present study were:1) to investigate the validity, usability and optimal intervention time of cardioprotection provided by vagus nerve stimulation postconditioning during myocardial IRI;2) to determine whether there was a synergistic cardioprotection by combination of POES and LRIPOC;3) to assess roles of both PI3K/Akt and JAK/STAT signal pathways in the cardioprotection of combined POES and LRIPOC, in order to explore the inherent mechanism of the interaction between POES and LEIPOC. This study was divided into three parts.Part1Experimental study on cardioprotective and anti-inflammatory effects and optimal intervention time of vagus nerve stimulation postconditioning in rat with myocardial ischemia reperfusion injury in vivoIn this part of the experiment, an in vivo rat model of myocardial IRI was used to compare the cardioprotections and anti-inflammatory effects of vagus nerve stimulation postconditioning at different time points, and to identify the optimal intervention time of vagus nerve stimulation postconditioning to provide a maximal cardioprotection.One hundred and forty anesthetized male Sprague Dawley rats (weighed290to320g) were randomly divided equally into seven groups (n=20in each group):sham group (S group), control group (C group), ischemic preconditioning group (IPC group), vagus nerve stimulation postconditioning at15min of myocardial ischemia period group (POESI15group), vagus nerve stimulation postconditioning immediately before myocardial reperfusion group (POESR0group), vagus nerve stimulation postconditioning at30min of myocardial reperfusion period group (POESR30group) and vagus nerve stimulation postconditioning at60min of myocardial reperfusion period group (POESR60group). After left thoracotomy in all rats, a6-0silk ligature was placed around the left anterior descending coronary artery (LAD) and encircled with a suture. In the groups other than S group, the LAD was ligated for30min followed by a120-min reperfusion. In C group, no additional intervention was performed. In IPC group, rats underwent three consecutive5-min LAD occlusion followed by a5-min reperfusion, which was performed before a30-min LAD ligation. In POESI15, POESR0, POESR30and POESR60groups, the right cervical vagus nerve was stimulated electrically at15min of ischemia, the time immediately before reperfusion,30min of reperfusion and60min of reperfusion and lasted for30min respectively. Throughout the experiment, the heart rate (HR), mean arterial pressure (MAP), and a lead II electrocardiogram were continuously monitored. The rectal temperature was maintained at36.5-37.5℃. Then, rats in each group were randomly divided equally into subgroup A and subgroup B. In subgroup A, blood samples were taken at30min,60min and120min of reperfusion for measuring serum concentrations of troponin I (TnI), myocardial-bound creatine kinase (CK-MB), tumor necrosis factor-a (TNF-a), high mobility group box1protein (HMGB1), intercellular adhesion molecule1(ICAM1), interleukin-1(IL-1), interleukin-6(IL-6) and interleukin-10(IL-10) by the kits specifically for rat. At the end of experiment, the infarct size (IS%) was assessed from excised hearts by Evans blue and triphenyltetrazolium chloride (TTC) staining. In subgroup B, the myocardial contents of TNF-a, HMGB1, ICAM1, IL-1, IL-6and IL-10were measured at the end of the experiment.The results showed that rat's body weight, body temperature and baselines of HR, MAP and rate-pressure product (RPP) did not differ among the seven groups (P>0.05).During the periods of ischemia and reperfusion, the HR was significantly lower in POESI15group than in S, C or IPC group. And during the period of reperfusion, the HR was significantly lower in POESR0, POESR30and POESR60groups than in S, C or IPC group. Compared to S group, the MAP and RPP were significantly decreased in C, IPC, POESI15, POESR0, POESR30and POESR60goups during the periods of both ischemia an reperfusion. Compared to IPC group, the number of rats suffering ischemic arrhythmia and arrhythmia score were significantly increased in C, POESI15, POESR0, POESR30and POESR60groups. Comparing to C group, the number of rats suffering reperfusion ventricular arrhythmia and arrhythmia score were significantly decreased in IPC, POESI15and POESR0groups.The IS%and serum concentrations of cTnI and CK-MB were significantly higher in C group than in IPC, POESI15, POESR0, POESR30and POESR60groups. Compared to IPC group, the IS%was significantly increased in POESI15, POESR0, POESR30and PORSR60groups. Compared to POESI15group, the IS%, serum concentrations of cTnI and CK-MB were significantly increased in POESR60group. Compared to POESR0group, the IS%, serum concentrations of cTnI and CK-MB were increased significantly in POESR60group, but there was no difference in the IS%, secrum concentrations of cTnI and CK-MB between POESR0and POESR30goups. And there was no difference in the IS%and serum concentration of CK-MB between POESR30and POESR60groups.Compared to S group, the serum concentration of TNF-a at30min,60min and120 min of reperfusion in C group, the serum concentration of HMGB1at60min of reperfusion in C, IPC and POESI15groups, the serum concentration of HMGB1at120min of reperfusion in C, POESR0, POESR30and POESR60groups, the serum concentration of ICAM1at120min of reperfusion in C, POESI15, POESRO, POESR30and POESR60groups, the serum concentration of IL-1at120min of reperfusion in C, IPC, POESRO, POESR30and POESR60groups, the serum concentration of IL-6at120min of reperfusion in C, IPC, POESR30, POESR60groups, the serum concentration of IL-10at120min of reperfusion in POESI15group were all significantly increased. Compared to C group, the serum concentration of TNF-α at30min and60min of reperfusion in both IPC and POESI15groups, the serum concentrations of TNF-α, HMGB1, ICAM1, IL-1and IL-6at120min of reperfusion in IPC, POESI15, POESR0, POESR30and POESR60groups were all significantly decreased. Compared to IPC group, the serum concentration of TNF-α at60min of reperfusion in POESI15group, the serum concentration of TNF-α at120min of reperfusion in POESR60group, the serum concentrations of HMGB1and ICAM1at120min of reperfusion in POESR0, POESR30and POESR60groups were all significantly increased. However, the serum concentration of IL-1at120min of reperfusion in POESI15group, the serum concentration of IL-6at120min of reperfusion in both POESI15and POESR0groups were all significantly decreased. Compared to POESI15group, the serum concentrations of HMGB1and ICAM1at120min of reperfusion in POESR0, POESR30and POESR60groups, the serum concentrations of IL-1, IL-6and TNF-α at120min of reperfusion in POESR60group were all significantly increased. Compared to POESR0group, the serum concentrations of HMGB1, IL-6and TNF-α at120min of reperfusin in POESR60group, the serum concentration of ICAM1at120min of reperfusion in both POESR30and POESR60groups were all significantly increased. The serum concentrations of TNF-α, HMGB1and ICAM1at120min of reperfusion were significantly increased in POESR60group than in POESR30group.Compared to S group, the ischemic myocardial contents of TNF-α and HMGB1in C group, the non-ischemic myocardial content of TNF-α in both C and POESR60groups, the non-ischemic myocardial content of HMGB1in C group, the ischemic myocardial contents of ICAM1, IL-1and IL-6in C, POESI15, POESR0, POESR30and POESR60groups, the non-ischemic myocardial contents of ICAM1and IL-6in C and POESR60groups, the non-ischemic myocardial content of IL-1and both ischemic and non-ischemic myocardial contents of IL-10in C, IPC, POESI15, POESR0, POESR30 and POESR60groups were all significantly increased. However, the ischemic myocardial content of TNF-a in IPC group, the non-ischemic myocardial content of HMGB1in IPC, POESI15, POESRO and POESR30groups, the non-ischemic myocardial content of ICAM1in IPC, POESI15and POESRO groups, the non-ischemic myocardial content of IL-6in both IPC and POESI15groups were all significantly decreased comparing to S group. Compared to C group, both ischemic and non-ischemic myocardial contents of TNF-a, HMGB1, IL-1and IL-6in IPC, POESI15, POESRO, POESR30and POESR60groups were all significantly decreased, but both ischemic and non-ischemic myocardial contents of IL-10in POESI15group were significantly increased. Compared to IPC group, the ischemic myocardial content of TNF-a in POESI15, POESRO, POESR30and POESR60groups, the non-ischemic myocardial content of HMGB1in POESR60group, the ischemic myocardial content of ICAM1in POESI15, POESRO, POESR30and POESR60groups, the non-ischemic myocardial content of ICAM1and the ischemic myocardial content of IL-6in both POESR30and POESR60groups, the ischemic and non-ischemic myocardial contents of IL-1, the non-ischemic myocardial content of IL-6in POESRO, POESR30and POESR60groups were all significantly increased, but the non-ischemic myocardial content of HMGB1in POESI15group was significantly decreased. Compared to POESI15group, both ischemic and non-ischemic myocardial contents of TNF-a, the non-ischemic myocardial content of ICAM1, ischemic myocardial content of IL-1in both POESR30and POESR60groups, the ischemic myocardial contents of HMGB1and IL-6in POESR60group, the ischemic myocardial content of ICAM1, the non-ischemic myocardial contents of HMGB1, IL-1and IL-6in POESRO, POESR30and POESR60groups were significantly increased, but both ischemic and non-ischemic myocardial contents of IL-10in POESR60group were significantly decreased. Compared to POESRO group, the ischemic myocardial contents of TNF-a, HMGB1, IL-1and IL-6in POESR60group, the non-ischemic myocardial contents of HMGB1, IL-1and IL-6in POESR60group, the ischemic and non-ischemic myocardial contents of ICAMl in both POESR30and POESR60groups were all significantly increased. The ischemic and non-ischemic myocardial contents of ICAM1, IL-6and non-ischemic myocardial content of HMGB1were all significantly higher in POESR60group than in POESR30group.Part2Experimental study on the protective effect of combined vagus nerve stimulation postconditioning and limb remote ischemic postconditioning against myocardial ischemia reperfusion injury in rats in vivoBased on the results from the first part of the experiment, we designed this part to oberve whether combine vagus nerve stimulation postconditioning and limb remote ischemic postconditioning would gain an augmented cardioprotective effect.One hundred and twenty anesthetized male SD rats (weighed290to320g) were randomly allocated into the six groups (n=20in each group):S group, C group, IPC group, POES group, limb remote ischemic postconditioning (LRIPOC) group and combined vagus nerve stimulation postconditioning and limb remote ischemic postconditioning (POES-LRIPOC) group. The procedures of C, IPC and POES groups were as same as those in the first part experiment, and the vagus nerve was stimulated at15min of myocardial ischemia at this part of experiment. In LRIPOC group, the bilateral hind legs were ligatured with an elastic tourniquet at20min of myocardial ischemia. In POES-LRIPOC group, rats received not only the same POES protocol as that of the POES group, but also the same LRIPOC protocol as that of the LRIPOC group. Then, rats in each group were futher randomly allocated into two subgroups. All testing variables in this part were as same as those in the first part of the experiment.The results showed that rat's body weight, body temperature and baselines of HR, MAP and RPP did not differ among the six groups (P>0.05).During the periods of ischemia and reperfusion, HR was significantly lower in POES and POES-LRIPOC groups than in S, C or IPC group. During the periods of ischemia and reperfusion, MAP and RPP were significantly lower in C, IPC, POES, LRIPOC and POES-LRIPOC groups than in S group. During the period of ischemia, the number of rats suffering ventricular arrhythmia and the score of arrhythmia were significantly increased in C, POES, LRIPOC and POES-LRIPOC groups comparing to IPC group. During the period of reperfusion, the number of rats suffering ventricular arrhythmia and the score of arrhythmia were significantly reduced in IPC, POES, LRIPOC and POES-LRIPOC groups comparing to C group. And during the period of reperfusion, the number of rats suffering ventricular arrhythmia and the score of arrhythmia were significantly reduced in IPC, POES and POES-LRIPOC groups comparing to LRIPOC group.Compared to C group, the IS%, serum concentrations of cTnI and CK-MB were significantly decreased in IPC, POES, LRIPOC and POES-LRIPOC groups. Compared to IPC or POES-LRIPOC group, the IS%in POES and LRIPOC groups was significantly increased. Compared to POES group, the IS%and serum concentration of cTnI in LRIPOC group were significantly increased, but the serum concentration of CK-MB was significantly decreased in LRIPOC group. The serum concentrations of cTnI and CK-MB were significantly decreased in IPC and POES-LRIPOC groups than in LRIPOC group.Compared to S group, the serum concentration of TNF-a at30min of reperfusion in C and LRIPOC groups, the serum concentration of TNF-a at60min and120min of reperfusion in C group, the serum concentration of HMGB1at60min of reperfusion in C, IPC, POES, LRIPOC and POES-LRIPOC groups, the serum concentration of HMGB1at120min of reperfusion in C and LRIPOC groups, the serum concentration of ICAM1at120min of reperfusion in C, POES and LRIPOC groups, the serum concentrations of IL-1and IL-6at120min of reperfusion at C, IPC and LRIPOC groups, the serum concentration of IL-10at120min of reperfusion in POES and POES-LRIPOC groups were all significantly increased, but the serum concentration of TNF-a at60min of reperfusion in IPC and POES-LRIPOC groups, the serum concentration of TNF-a at120min of reperfusion in IPC, POES, LRIPOC and POES-LRIPOC groups were all significantly decreased. Compared to C group, the serum concentration of TNF-a at30min,60min,120min of reperfusion and the serum concentrations of HMGB1, ICAM1, IL-1, IL-6at120min of reperfusion in IPC, POES, LRIPOC and POES-LRIPOC groups, the serum concentration of HMGB1at60min of reperfusion in IPC group were all significantly decreased, but the serum concentration of IL-10at120min of reperfusion in POES-LRIPOC group was significantly increased. Compared to IPC group, the serum concentration of TNF-a at60min of reperfusion in POES and LRIPOC groups, the serum concentrations of HMGB1and ICAM1at120min of reperfusion in LRIPOC group were all significantly increased, but the serum concentrations of IL-1and IL-6in POES and POES-LRIPOC groups were all significantly decreased. Compared to POES group, the serum concentration of TNF-a at60min of reperfusion, the serum concentrations of HMGB1, ICAM1, IL-1and IL-6at120min of reperfusion in LRIPOC group were all significantly increased, but the serum concentration of ICAM1in POES-LRIPOC group was significantly decreased. The serum concentrations of TNF-a at30min,60min and120min of reperfusion, the serum concentrations of HMGB1, ICAM1, IL-1and IL-6at120min of reperfusion were significantly decreased in POES-LRIPOC group than in LRIPOC group.Compared to S group, the ischemic myocardial contents of TNF-a and HMGB1, the non-ischemic myocardial contents of TNF-a, HMGB1and ICAM1in C group, the ischemic myocardial content of ICAM1in C, POES, LRIPOC and POES-LRIPOC groups, the ischemic myocardial contents of IL-1and IL-6in C, POES and LRIPOC groups, the ischemic myocardial content of IL-10, the non-ischemic myocardial contents of IL-1and IL-10in C, IPC, POES, LRIPOC and POES-LRIPOC groups, the non-ischemic myocardial content of IL-6in C and LRIPOC groups were all significantly increased, but the ischemic myocardial content of TNF-a in IPC and POES-LRIPOC groups, the non-ischemic myocardial content of HMGB1in IPC, POES, LRIPOC and POES-LRIPOC groups, the non-ischemic myocardial contents of ICAM1and IL-6in IPC, POES and POES-LRIPOC groups were all significantly decreased. Compared to C group, the ischemic and non-ischemic myocardial contents of TNF-a, HMGB1, ICAM1, IL-1and IL-6in IPC, POES, LRIPOC and POES-LRIPOC groups were all significantly decreased, but the ischemic and non-ischemic myocardial contents of IL-10in POES and POES-LRIPOC groups were significantly increased. Compared to IPC group, the ischemic myocardial contents of TNF-a, ICAM1and IL-1in POES and LRIPOC groups, the ischemic and non-ischemic myocardial contents of IL-10in POES-LRIPOC group, the ischemic myocardial content of IL-6and the non-ischemic myocardial contents of ICAM1, IL-1and IL-6in LRIPOC group were all significantly increased, but the ischemic myocardial content of HMGB1in POES-LRIPOC group, the non-ischemic myocardial content of HMGB1in POES and POES-LRIPOC groups were all significantly decreased. Compared to POES group, the ischemic myocardial contents of TNF-a, ICAM1and IL-1in POES-LRIPOC group, the ischemic myocardial content of IL-10in LRIPOC group were all significantly decreased, but the non-ischemic myocardial content of IL-10in POES-LRIPOC group, the non-ischemic myocardial contents of HMGB1, ICAM1, IL-1and IL-6, ischemic myocardial contents of ICAM1, IL-1and IL-6in LRIPOC group were all significantly increased. Compared to LRIPOC group, the ischemic myocardial contents of TNF-a, ICAM1, IL-1and IL-6, the non-ischemic myocardial contents of HMGB1, ICAM1, IL-1and IL-6in POES-LRIPOC group were all significantly decreased, but the ischemic and non-ischemic myocardial contents of IL-10were significantly increased.Part3Roles of the PI3K/Akt and JAK/STAT signal pathways in the cardioprotections of vagus nerve stimulation postconditioning and combined vagus nerve stimulation postconditioning and limb remote ischemic postconditioningThe aims of this part of the experiment were to explore the modulating roles of PI3K/Akt and JAK/STAT signal pathways in cardioprotective effects of vagus nerve stimulation postconditioning and combined vagus nerve stimulation postconditioning and limb remote ischemic postconditioning.Twenty anesthetized male SD rats (weighed290to320g) were randomly equally divided into four groups (n=5in each group):control group, POES group, LRIPOC group and combined POES and LRIPOC group. After60min of reperfusion, the myocardial tissues from the area at risk in the left ventricle and non-ischemic area in the right ventricule were harvested from excised hearts. Total RNA and total protein were extracted from all myocardial samples, respectively. The real time quantitative polymerase chain reaction (RQ-PCR) was used to quantify the mRNA expression of Akt and STAT3genes in all groups. Also, phosphorylated Akt and phosphorylated STAT3in all samples were assessed by Western-blotting technique.Compared to C group, ischemic and non-ischemic myocardial phosphorylated Akt (p-Akt) and phosphorylated STAT3(p-STAT3) were all significantly increased in POES, LRIPOC and POES-LRIPOC groups. The ischemic and non-ischemic myocardial p-Akt and p-STAT3were significantly increased in POES-LRIPOC group than in POES or LRIPOC group.The results of RQ-PCR demonstrated that the mRNA expression of ischemic and non-ischemic myocardial Akt and STAT3genes were significantly enhanced in POES-LRIPOC group compared to C group. Also, the mRNA expression of Akt and STAT3genes were significantly higher in POES-LRIPOC group than in POES or LRIPOC group.ConclusionBased on the results of all experiments, the following conclusions can be drawn:1. Vagus nerve stimulation postconditioning performed at15min of myocardial ischemia, the time immediately before myocardial reperfusion,30min of myocardial reperfusion and60min of myocardial reperfusion can all significantly reduce the infarct size, decrease serum concentrations of cTnI and CK-MB during myocardial ischemia reperfusion injury. Moreover, the cardioprotection provided by vagus nerve stimulation postconditioning at60min of myocardial reperfusion is weakest, while the cardioprotection provided by vagus nerve stimulation postconditioning at15min of myocardial ischemia is strongest but still weaker than ischemic preconditioning.2. Ischemic preconditioning and vagus nerve stimulation postconditioning performed at different time points can all significantly reduce the ventricular arrhythmias during myocardial reperfusion period. Among vagus nerve stimulation postconditionings performed at different time points, vagus nerve stimulation postconditioning at15min of myocardial ischemia can provide the strongest anti-arrhythmic effect.3. Combined vagus nerve stimulation postconditioning and limb remote ischemic postconditioning can provide a stronger cardioprotection which is comparable to ischemic preconditioning.4. Ischemc preconditioning, vagus nerve stimulation postconditioning performed at different time points, limb remote ischemic postconditioning and combined vagus nerve stimulation postconditioning and limb remote ischemic postconditioning can all provide cardioprotections by inhibiting inflammation during myocardial ischemia reperfusion injury.5. Protein phosphorylation upregulation of related genes involved in both PI3K/Akt and JAK/STAT signal pathways mediates the cardioprotections of vagus nerve stimulation postconditioning and limb remote ischemic postconditoning. And the mRNA expression upregulation of related genes involved in both PI3K/Akt and JAK/STAT signal pathways mediates the cardioprotection of combined vagus nerve stimulation postconditioning and limb remote ischemic postconditioning. Also, the protein phosphorylation upregulation of related genes involved in both PI3K/Akt and JAK/STAT signal pathways probably contributes to the cardioprotection of combined vagus nerve stimulation postconditioning and limb remote ischemic postconditioning... |
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