| BackgroundMany animal studies have shown that after acute myocardial infarction, the ischemia reperfusion injury (IRI) is responsible for up to 50%of the final infarct size. Although the ischemia preconditioning (IPC) remains the most powerful cardioprotective measure, but 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. Ischemia postconditioning (IPOC) can be triggered during the clinically applicable period of reperfusion. However, it has the similar limitation as the IPC, i.e. requirement of an invasive protocol. Thus, the IPOC can only be utilized in the patients undergoing percutaneous coronary intervention (PCI) or special surgery for acute myocardial ischemia. Meanwhile, operation at the "culprit coronary artery" can produce new lesion of vessel or plaque fragmentation, which increase the risk of complications. Due to the limitations of the IPC and IPOC, pharmacological postconditioning protocol may be of more clinically feasible, by which cardioprotective drugs can be administered after ischemia insult. Now pharmacological preconditioning and postconditioning have been improved to substitute the IPC and IPOC effectively. The merits of pharmacological postconditioning include no limitation of myocardial ischemia, low cost, noninvasive and convenience.Endogenous inflammatory response is a key factor in myocardial IRI, characterized with the local infiltration of inflammatory cells and excessive production and release of cytokines. Recently, cholinergic anti-inflammatory pathway has been demonstrated, by which vagus nerve can inhibit production of cytokines to avoid excessive inflammatory response through the specificα7 subunit of the nicotinic acetylcholine receptor (a7nAChR). Studies have identified that vagus nerve stimulation can decrease blood and tissue levels of tumor necrosis factor a (TNF-a), interleukin 6 (IL-6) and high mobility group box 1 (HMGB1), providing a protection against myocardial IRI. Because a7nAChR agonists can produce same effects as vagus nerve stimulation, they are potential treatments for myocardial IRI as noninvasive and effective pharmacological postconditioning. Although the renoprotective effect of a7nAChR agonist has been identified, there has still been no study evaluating its cardioprotection.Combining different interventions to obtain an augmented cardioprotection is always one of the most popular research focuses in the area of myocardial IRI. Because IPOC andα7nAChR agonist postconditioning might be triggered by different mechanisms, we designed this randomized, controlled animal experimental study. The aims of the present study were:1) to investigate cardioprotection of the IPOC and a7nAChR agonist postconditioning; 2) to determine whether there was the synergistic cardioprotection by a combination of IPOC and a7nAChR agonist postconditioning; 3) to verify the best implementation time of a7nAChR agonist postconditioning; 4) to assess roles of both PI3K/Akt and JAK/STAT signal pathways in the cardioprotection of combined a7nAChR agonist postconditioning and IPOC, in order to explore the inherent mechanism of the interaction betweenα7nAChR agonist postconditioning and IPOC. This study was divided into the three parts.Part 1 Experimental study on cardioprotective and anti-inflammatory effects of PNU282987 postconditioning and ischemia postconditioning in rat with myocardial ischemia reperfusion injury in vivoIn this part experiment, an in vivo rat model of myocardial IRI was used to compare the cardioprotection of PNU282987 and ischemia postconditioning, and to identify whether there was synergistic infarct-sparing effect by combining the two treatments.Sixty anesthetized male Sprague Dawley rats (weighed 290 to 320 g) were randomly divided equally into six groups (n=10 in each group):sham group (S group), control group (C group), ischemia preconditioning group (IPC group), ischemia postconditioning group (IPOC group), PNU282987 postconditioning group (P group)and combined PNU282987 postconditioning and ischemia postconditioning group (PPOC group). After left thoracotomy in all rats, a 6-0 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 180-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 IPOC group, animals were subjected to three cycles of a 10-sec reperfusion followed by a 10-sec LAD reocclusion, which was executed immediately at the onset of a 180-min reperfusion. Furthermore, the rats in the sham, control and IPOC groups were injected intraperitoneally with 1.5 ml normal saline at the end of ischemia. PNU282987 (2.4 mg/kg) was injected intraperitoneally immediately before a 180-min reperfusion in P group. In PPOC group, rats received not only the same IPOC protocol as that of the IPOC group, but also the same PNU282987 injection as that of the P group. Throughout the experiment, heart rate (HR), mean arterial pressure (MAP), and a lead II electrocardiogram were continuously monitored. The rectal temperature was maintained at 36.5-37.5℃. Blood samples were taken at 30 min and 180 min of reperfusion for measuring serum concentrations of troponin I (Tnl), TNF-a, HMGB-1 and IL-6 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.The results showed that rat's body weight, body temperature and baselines of HR, MAP and RPP did not differ among the seven groups (P>0.05). Also, HR, MAP and RPP did not differ among these groups after 15 min ischemia (P>0.05).During the period of reperfusion, the rate-pressure product (RPP) was significantly higher in S group than in C group. Also, MAP was significantly lower in C group than in S, IPC, IPOC, P and PPOC groups. Compared to IPC group, the number of rats suffering ischemic arrhythmia and arrhythmia score were significantly increased in C, IPOC, P and PPOC groups. Comparing to C group, the number of rats suffering reperfusion ventricular arrhythmia and arrhythmia score were significantly decreased in IPOC and PPOC groups.The IS%and serum concentration of cTnl were significantly higher in C group than in IPC, IPOC, P and PPOC groups. Compared to IPC group, the IS%was significantly increased in IPOC and P groups, but the serum concentration of cTnl was significantly decreased in P and PPOC groups. There was no difference in the IS%between the IPC and PPOC groups. Compared to IPOC group, the IS%was significantly reduced in PPOC group, and serum concentration of cTnl was significantly reduced in P and PPOC groups. There was no difference in the IS%between the IPOC and P groups. The results of Factorial design ANOVA showed that there was no synergistic effect when combining ischemic postconditioning and PNU282987 postconditioning.Compared to S group, the serum concentrations of TNF-a and IL-6 at 30 min of reperfusion and TNF-a, HMGB1 and IL-6 at 180 min of reperfusion were significantly increased in C group. Compared to C group, the serum concentrations of TNF-a and HMGB1 at 180 min of reperfusion were significantly decreased in IPC, IPOC, P and PPOC groups. The serum concentrations of TNF-a and IL-6 at 30 min of reperfusion were significantly higher in IPOC group than in C group, but the serum levels of TNF-a and IL-6 at 30 min of reperfusion were significantly lower in IPC, P and PPOC groups than in C group. The serum concentration of IL-6 at 180 min of reperfusion was significantly higher in IPOC group than in C group, however, the serum concentration of IL-6 at 180 min of reperfusion was significantly reduced in P and PPOC groups compared to C group. Compared to IPC group, the serum concentrations of TNF-a and IL-6 at 30 min of reperfusion and TNF-a, IL-6 and HMGB1 at 180 min of reperfusion were significantly increased in IPOC group. The serum concentrations of TNF-a, IL-6 and HMGB1 at 180 min of reperfusion and TNF-a at 30 min of reperfusion were significantly decreased in P group comparing to IPC group. However, the serum concentration of IL-6 at 30 min of reperfusion was significantly increased in P group comparing to IPC group. The Serum concentrations of TNF-a and IL-6 at 30 min of reperfusion and IL-6 and HMGB1 at 180 min of reperfusion were significantly lower in PPOC group than in IPC group. Compared to IPOC group, the serum concentrations of TNF-a and IL-6 at 30 min of reperfusion and those of TNF-a, IL-6 and HMGB1 at 180 min of reperfusion were significantly lower in P and PPOC groups.Part 2 The best time of PNU282987 postconditioning attenuating myocardial ischemia reperfusion injury in ratsBased on the results from the first part experiment, we designed this part experiment to identify what time PNU282987 postconditioning was implemented during period of myocardial ischemia reperfusion injury process can produce the best cardioprotection.Seventy anesthetized male SD rats (weighed 290 to 320 g) were randomly allocated into the six groups (n=10 in each group):S group, C group, IPC group, P group, PNU282987 postconditioning at 30 min reperfusion group (P30 group), PNU282987 postconditioning at 60 min reperfusion group (P60 group) and PNU282987 postconditioning at 90 min reperfusion group (P90 group). The procedures of C, IPC and P groups were as same as those in the first part experiment. The treatments of P30, P60 and P90 groups were similar to that of P group, except PNU282987 were administrated intraperitoneally at 30 min,60 min and 90 min of reperfusion, respectively. All testing variables in this part were as same as those in the first part. However, the serum concentrations of inflammatory cytokines were assayed only at 180 min of reperfusion.The results showed that rat's body weight, body temperature and baselines of HR, MAP and RPP did not differ among the seven groups (P>0.05). Also, HR, MAP and RPP after 15 min of ischemia did also not differ among six groups (P>0.05). During the period of reperfusion, MAP was significantly lower in C group than in S, IPC, P, P30 and P60 groups. During the period of ischemia, the number of rat suffering ventricular arrhythmias and the score of arrhythmia were significantly reduced in IPC group compared to C, P, P30, P60 and P90 groups. Compared to IPC group, moreover, the arrhythmia score at onset of reperfusion was significantly increased in C, P30, P60 and P90 groups.Compared to C group, the serum concentration of cTnl at 180 min of reperfusion and IS%were significantly decreased in IPC, P, P30, P60 and P90 groups. The serum concentration of cTnI at 180 min of reperfusion was significantly lower in P, P30, P60 and P90 groups than in IPC group, but the IS%was significantly higher in P, P30, P60 and P90 groups than in IPC group. Compared to P30 group, the IS%was significantly increased in P90 group.Compared to S group, serum concentrations of TNF-a, IL-6 and HMGB1 at 180 min of reperfusion were significantly increased in C group. Compared to C group, the serum levers of TNF-a and HMGB1 at 180 min of reperfusion were significantly decreased in IPC, P, P30, P60 and P90 groups, and the serum concentration of IL-6 at 180 min of reperfusion were also significantly decreased in P, P30, P60 and P90 groups. Compared to IPC group, the serum concentrations of HMGB 1 and IL-6 at 180 min of reperfusion were significantly reduced in P, P30, P60 and P90 groups. Although the serum concentration of TNF-a at 180 min of reperfusion was significantly lower in P group than in IPC group, serum concentration of TNF-a at 180 min of reperfusion was significantly higher in P30 and P60 groups than in IPC group. Compared to P group, the serum levels of TNF-a and HMGB1 at 180 min of reperfusion were significantly increased in P30, P60 and P90 groups, but the serum concentration of IL-6 at 180 min of reperfusion was significantly reduced in these groups. The serum concentrations of TNF-a and HMGB1 at 180 min of reperfusion were significantly lower in P60 and P90 groups than in P30 group, however, the serum concentrations of IL-6 at 180 min of reperfusion were significantly increased in P60 and P90 group compared to P30 group.Part 3 Roles of the PI3K/Akt and JAK/STAT signal pathways in the cardioprotection of combined PNU282987 postconditioning and ischemia conditioningThe aims of this part experiment were to explore the modulating roles of PI3K/Akt and JAK/STAT signal pathways in cardioprotective effects of combined PNU282987 postconditioning and IPOC.Twenty five anesthetized male SD rats (weighed 290 to 320 g) were randomly divided equally into five groups (n=5 in each group):control group, IPC group, IPOC group, PNU282987 postconditioning group and combined PNU282987 postconditioning and IPOC group. After 60 min of reperfusion, the myocardial tissues from the area at risk in the left ventricle 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 gene expression of Akt and STAT3 in all groups. Also, phosphorylated Akt and phosphorylated STAT3 in all samples were assessed by Western-blotting technique.Compared to C group, p-Akt in IPC group, and p-STAT3 and p-Akt were significantly increased in IPOC group. P-Akt and p-STAT3 in P group, and p-STAT3 in PPOC group were significantly lower than those in IPC group. Compared to IPOC group, p-Akt and p-STAT3 in P group and p-STAT3 in PPOC group were significantly reduced.The results of RQ-PCR demonstrated that the expression of STAT3 gene was significantly enhanced in IPC group compared to C and P groups. Also, the expression of Akt gene was significantly higher in IPOC group than in PPOC group.ConclusionBased on the results of all experiments, the following conclusions can be drawn:1. Cardioprotections induced by PNU282987 postconditioning and IPOC are not same. Although there is no significant difference between the two groups'effects of infarct size-sparing, IPOC inhibits reperfusion arrhythmia more effectively than PNU292987 postconditioning.2. Combination of PNU282987 postconditioning and IPOC can provide a stronger cardioprotection, evidenced by reduction of infarct size. Also, inability of PNU282987 postconditioning related to reperfusion arrhythmia is eliminated by their combination. 3. In the present study, IPC, IPOC, PNU282987 postconditioning and combination of IPOC and PNU282987 postconditioning can inhibit the inflammatory response induced by the myocardial ischemia reperfusion injury. Also, the inflammatory inhibitory effect is strongest when combining PNU282987 postconditioning and IPOC. In addition, the intensity and pattern of inhibiting inflammatory response are different among the IPC, IPOC and PNU282987 postconditioning.4. Compared to PNU282987 postconditioning at the onset of reperfusion,60 min and 90 min of reperfusion, PNU282987 postconditioning at 30 min of reperfusion can produce the best cardioprotection.5. Although the cardioprotection of the IPC and IPOC may be contributed to activation of both PI3K/Akt and JAK/STAT signal pathways, these signal pathways are not the critical roles in the cardioprotection induced by PNU282987 postconditioning or the conbination of PNU282987 postconditioning and IPOC. The mechanisms of PNU282987 postconditioning against myocardial ischemia reperfusion injury may be different from those of the IPC and IPOC.
|
PDF Full Text Request