| BackgroundIschemia reperfusion injury (IRI) is a main cause of mortality after acute myocardial infarction. It has been shown that inflammatory response is a key cause of IRI and regulation of inflammatory response is proved to afford cardioprotection. The cholinergic anti-inflammatory pathway (CAP) is a newly found endogenous mechanism consisiting of vagus nerve, acetylcholine and Ω7 nicotinic acetylcholiner receptor (α7nAChR). It has been demonstrated that postconditioning with the vagal stimulation can significantly attenuate the local and systemic inflammatory responses by myocardial IRI. The α7nAChR agonist also has the ability to activate the CAP and has widely been applied to treat various diseases associated with inflammation. Thus, we chose the specific α7nAChR agonist to observe its cardioprotective effect.The mitochondrial permeability transition pore (MPTP) is an important tunnel-like protein complex localized in the membrane of mitochondrial which is crucial determinants of cardiomyocyte fate following an episode of acute myocardial ischemia reperfusion injury. In particular, reperfusing acutely ischemic myocardium induces the opening of the MPTP, an event that mediates cardiomyocyte death. The glycogen synthase kinase-3p (GSK-3β) is a widely existing serine threonine kinase. The inactivation of GSK-3β’s activity is associated with the mechanism of many cardioprotective interventions. The nuclear transcription factor κB (NF-κB) is a key component of the inflammatory response and affects expression of many cytokines. Recent study has shown that vagus nerve stimulation can decrease myocardial infarct size after IRI on rats by attenuate the expression of GSK-3β. Besides, genetically knock out of the gene coding GSK-3β can elevate the threshold of MPTP, which indicate that GSK-3β facilitates the MPTP opening.Combining different interventions to obtain an augmented cardioprotection is always one of the most popular research focuses in the myocardial IRI. Thus, this randomized controlled experiment was designed to observe whether combining α7nAChR agonist postconditioning and limb remote ischemic postconditioning can provide a synergistic cardioprotection, and to assess the role of interactions between inflammatory response, MPTP and GSK-3β in the cardioprotections of a7nAChR agonist postconditioning, limb remote ischemic postconditioning and combined a7nAChR agonist postconditioning and limb remote ischemic postconditioning. This study was divided into three parts:Part 1 Experimental study of cardiaoprotective and anti-inflammatory effects of PNU282987 postconditioning and limb remote ischemic postconditioning in rats with myocardial ischemia reperfusion injuryIn this part of study, an in vivo rat model of myocardial IRI was used to compare the cardioprotection of PNU282987 and limb remote ischemic postconditioning, and to determine whether there was synergistic infarct-sparing effect by combining the two treatments.Sixty male SD rats were randomly divided into 6 groups (10 in each group): sham group (S group), control group (C group), ischemia preconditioning group (IPC group), PNU282987 postconditioning group (P group), lime remote ischemic postconditioning group (L group), combined PNU282987 postconditioning and lime remote ischemic postconditioning group (P+L 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 the groups other than S group, the LAD was ligated for 30 min followed by a 120 min reperfusion. In C group, no additional intervention was performed. In IPC group, rats underwent three consecutive 5-min LAD occlusion followed by a 5-min reperfusion, which was performed before a 30-min LAD ligation.In P group, PNU282987(2.0 mg/kg) was injected intravenously immediately before a 120-min reperfusion. In L group, after 20 min of LAD ligation, blood flow in the bilateral hind limbs was stopped for 10 min and opened before reperfusion. In P+L group, rats received the same protocols as those of the L and P groups. Throughout the experiment, heart rate (HR), mean arterial pressure (MAP), and a lead II electrocardiogram were continuously monitored. Blood samples were taken at 30 min and 180 min of reperfusion for measuring serum concentrations of troponin I (TnI), myocardial-bound creatine kinase (CK-MB), tumor necrosis factor-a (TNF-α), high mobility group box 1 protein (HMGB-1), interleukin-6 (IL-6) and interleukin-10 (IL-10) by the kits specifically for rats. At the end of experiment, the infarct size (IS%) was assessed from excised hearts by Evans blue and triphenyltetrazolium chloride (TTC) staining.The results showed that rats’body weight, body temperature and baselines of HR, MAP and RPP did not differ among 6 groups. Also, HR, MAP and RPP did not differ among these groups after 15 min ischemia.Compared to C group, the number of rats suffering reperfusion ventricular arrhythmia and arrhythmia score were significantly decreased in IPC group.Compared to C group, the IS% and serum cTnI and CK-MB concentrations were significantly decreased in IPC, P, L and P+L groups. Compared to IPC group, the IS% and serum cTnI concentration were significantly higher in P, L and P+L groups. Compared to P and L group, the IS% and serum cTnI and CK-MB concentrations were significantly decreased in P+L group.At 120-min of reperfusion, serum TNF-a, IL-6 and HMGB1 concentrations were significantly increased in C group in S group. Compared to C group, the serum TNF-a, IL-6 and HMGB1 concentrations were significantly decreased in IPC, P, L and P+L group. Compared to IPC and L groups, the serum TNF-a, IL-6 and HMGB1 concentrations were significantly decreased in P and P+L groups.Part 2 The roles of MPTP in the cardioprotective and anti-inflammatory effects of PNU282987 postconditioning and limb remote ischemic postconditioningBased on the results of above part 1, this part experiment was designed to assess the roles of MPTP in the cardioprotective and anti-inflammatory effects of PNU282987 postconditioning and limb remote ischemic postconditioning by using the MPTP opening inhibitor, cyclosporine (CSA) and MPTP opening activator, atractyloside (ATR).One hundred and twenty male SD rat were randomly divided into 12 groups (10 in each group):control group (C group), PNU282987 postconditioning group (P group), limb remote ischemic postconditioning group (L group), combined PNU282987 postconditioning and limb remote ischemic postconditioning group (P+L group), control group with CSA treated during the ischemic period (CSA+C group), PNU282987 postconditioning group with CSA treated during the ischemic period (CSA+P group), limb remote ischemic postconditioning group with CSA treated during the ischemic period (CSA+L group), combined PNU282987 postconditioning and limb remote ischemic postconditioning group with CSA treated during the ischemic period (CSA+P+L group), control group with ATR treated during the ischemic period (ATR+C group), PNU282987 postconditioning group with ATR treated during the ischemic period (ATR+P group), limb remote ischemic postconditioning group with ATR treated during the ischemic period (ATR+L group), combined PNU282987 postconditioning and limb remote ischemic postconditioning group with ATR treated during the ischemic period (ATR+P+L group). All testing variables in this part were as same as those in the first part.The results showed that compared to C group, the IS% and serum cTnI and CK-MB concentrations were significantly decreased in P, L, P+L, CSA+C, CSA+P, CSA+L and CSA+P+L groups. Compared to CSA+C group, the IS% and serum cTnI and CK-MB concentrations were significantly decreased in CSA+P, CSA+L and CSA+P+L groups. Compared to CSA+P and CSA+L groups, the IS% and serum cTnI and CK-MB concentrations were significantly decreased in CSA+P+L group. There were no significant difference in the IS% and serum cTnI and CK-MB concentrations between C, ATR+C, ATR+P, ATR+L, and ATR+P+L groups.Compared to C group, the serum TNF-a, IL-6 and HMGB1 concentrations were significantly decreased in P, L, P+L, CSA+P, CSA+L, CSA+P+L, ATR+P, ATR+L, and ATR+P+L groups and were significantly increased in ATR+C group. Compared to CSA+C group, the serum TNF-a, IL-6 and HMGB1 concentrations were significantly decreased in CSA+P, CSA+L and CSA+P+L groups. Compared to CSA+P group, the serum TNF-a, IL-6 and HMGB1 concentrations were significantly increased in CSA+L group. Compared to CSA+L group, the serum TNF-a, IL-6 and HMGB1 concentrations were significantly decreased in CSA+P+L group. Compared to ATR+C group, the serum TNF-a, IL-6 and HMGB1 concentrations were significantly decreased in ATR+P, ATR+L and ATR+P+L groups. Compared to ATR+P group, the serum TNF-a, IL-6 and HMGB1 concentrations were significantly increased in ATR+L group. Compared to ATR+L group, the serum TNF-a, IL-6 and HMGB1 concentrations were significantly decreased in ATR+P+L group.Part 3 The roles of GSK-3β and inflammatory response in the cardioprotection of PNU282987 postconditioning and limb remote ischemic postconditioning and their interactionsSixty male rats were randomly divided into 4 groups (15 in each group):control group (C group), PNU282987 postconditioning group (P group), limb remote ischemic postconditioning group (L group), combined PNU282987 postconditioning and limb remote ischemic postconditioning group (P+L group). After 120 min of reperfusion, myocardial tissues from area at risk in the left ventricle were harvested from excised hearts. The function of MPTP was assessed by calcium retention capacity (CRC) test. Real-time quantitative polymerase chain reaction (RQ-PCR) analysis and TUNEL apoptosis test were used to quantify the expression of genes associated with apoptosis and cardiomyocyte apoptosis index. The Western blot analysis was used to assess phosphorylated GSK-3β and phosphorylated NF-κB.The results of CRC test showed that compared to C group, calcium retention capacity of mitochondria in P, L, P+L groups were significantly increased. Compared to P and L groups, the calcium retention capacity of mitochondria in P+L group were significantly increased.The results of RQ-PCR showed that compared to C group, expression of Bcl-2 mRNA was significantly enhanced in P, L and P+L groups compared to C group. Expression of Bax mRNA was significantly desreased in P, L and P+L groups. The results of TUNEL apoptosis test showed that cardiomyocyte apoptosis index was significantly decreased in P, L and P+L groups compared to C group.The results of western blot demonstrated that the expression of p-GSK-3β (Ser9) was significantly increased in P, L, P+L groups compared to C group. Compared to P group, the expression of p-GSK-3(3 (Ser9) was significantly decreased in L group. Compared to L group, the expression of p-GSK-3β(Ser9) was significantly increased in P+L group. The expression of p-NF-κBp65 (Ser536) were significantly decreased in P, L, P+L groups compared to C group. Compared to P group, the expression of p-GSK-3β (Ser9) was significantly increased in L group. Compared to L group, the expression of p-GSK-3β (Ser9) was significantly desreased in P+L group.ConclusionsBased on the results of all experiments, the following conclusions can be drawn:1. Ischemia preconditioning, PNU282987 postconditioning and limb remote ischemic postconditioning all can produce obvious cardioprotective effects. Although cardioprotection induced by ischemia preconditioning is stronger than that of PNU282987 postconditioning and limb remote ischemic postconditioning, the combination of PNU282987 postconditioning and limb remote ischemic postconditioning can provide a significantly enhanced cardioprotective effect.2. Ischemia preconditioning, PNU282987 postconditioning, limb remote ischemic postconditioning and combined PNU282987 postconditioning and limb remote ischemic postconditioning all can attenuate inflammatory response caued by IRI. The anti-inflammatory effects of PNU282987 postconditioning and combined PNU282987 postconditioning and limb remote ischemic postconditioning are stronger than that of ischemia preconditioning or limb remote ischemic postconditioning alone.3. The MPTP opening inhibitor, CSA, does not affect the cardioprotective and anti-inflammatory effects of PNU282987 postconditioning, limb remote ischemic postconditioning and combined PNU282987 postconditioning and limb remote ischemic postconditioning, whease the MPTP opening activator, ATR, can abolish cardioprotective effects of these interventions but do not affect their anti-inflammatory effects.4. PNU282987 postconditioning and limb remote ischemic postconditioning can decrease the cardiac apoptosis index by suppressing MPTP opening. Combination of them can enhance the MPTP opening suppressing effect. This effect might be associated with the depression of activity of GSK-3β, which is a key regulator of MPTP.5. PNU282987 postconditioning and limb remote ischemic postconditioning can down-regulate inflammatory response by depressing the activity of GSK-3p, which can directly affect NF-κB. |
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