| Since the phenomenon of ischemia-reperfusion injury was first reported by Jennings in the year of1960, it has been perplexing researchers for more than half a century. In order to attenuate ischemia reperfusion injury (IRI) and finally reduce infarct size of myocardium, lots of interventions have been invented. Ischemic preconditioning-the most powerful therapeutic strategy is confined to laboratory study because of the unpredictability of an acute myocardial infarction. Besides the classical stimulus of short-term ischemia, there are several stimuli that may induce a preconditioning-like effect. The cardioprotection of sevoflurane has been proven by laboratory and clinical trials and may be a tool to win our battle against the ischemia-reperfusion injury. While remote ischemic postconditioning is elicited by one or cycles of non-lethal ischemia-reperfusion to an organ or tissue remote from the heart. Its cardioprotective effect has been translated into the clinical setting with the discovery that the remote stimulus can be non-invasively induced using a standard blood pressure cuff placed on the upper arm or leg.Hypoxia inducible factor1is an important transcriptive factor that regulates the expression of more than a hundred of genes related to hypoxia adaptation. Its downstream genes heme oxygenase1and inducible nitric oxide synthase have been proven involved in cardioprotection. Akt and ERK1/2are important components of the reperfusion injury salvage kinase (RISK) pathway, and the convergent point of many irrelevant measures. Activation of the RISK signaling pathway can exert strong protective effect against IRI.Multi-model therapy has become a research focus in the IRI cardioprotection field in an attempt to augment the beneficial effect of strategies with different mechanism. This experiment was designed to investigate:1) cardioprotection of sevoflurane postconditioning and remote ischemic postconditioning;2) whether there is synergistic cardioprotection by combining the two strategies;3) role of HIF-1α, HO-1and iNOS in the two strategies;4) role of Akt and ERK1/2in the two strategies. This study was divided into three parts.Part One Experimental study on cardioprotection of combining sevoflurane postconditioning and remote limb ischemic postconditioning against ischemia reperfusion injury in rat heart in vivoSixty healthy male Sprague Dawley rats were randomly allocated into six groups:(1) control group (CON);(2) ischemia reperfusion group (IR);(3) ischemic preconditioning group (IPC);(4) limb remote ischemic postconditioning group (LRIPOC);(5) sevoflurane postconditioning group (SEVPOC);(6) combination of limb remote ischemic postconditioning and sevoflurane postconditioning group (COMBINE). After left thoracotomy, a5-0silk ligature was passed below a main branch of the left coronary artery. The ends of the ligature were passed through a propylene tube to form a snare. Rats except the ones in the control group endured regional ischemia reperfusion injury by occlusion of the LAD for30min and then loosening for120min. In IR group, no additional intervention was performed. In IPC group, rats underwent three consercutive5-min LAD occlusion followed by5-min reperfusion before the30-min LAD ligation. In LRIPOC group, after15min of LAD ligation, blood flow in the bilateral hind limbs was stopped for10min and opened at5min before reperfusion. In SEVPOC group, rats received a10-min episode of1MAC sevoflurane5min before reperfusion and5min after reperfusion. In COMBINE group, after15min of LAD ligation, blood flow in the bilateral hind limbs was stopped for10min and opened at5min before reperfusion, and rats began a10-min episode of1MAC sevoflurane just at the time when the remote ischemia stimulus was stopped. The rectal temperature was sustained at37℃～38℃. Heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and lead Ⅱ electrocardiogram were continuously monitored. Arterial blood sample were taken at120min of reperfusion, and the concentration of serum LDH, CK-MB and cTnI were measured by rat ELISA kits. At the end of reperfusion, infarct size (IS%) was assessed by Evans Blue and triphenyltetrazolium chloride staining.Forty-nine rats were finally included in this experiment (9in the CON group,8in any of the rest groups) and results showed that rat's body weight, rectal temperature and the hemodynamic parameter did not differ among the six groups (P>0.05).Compared with IPC group, the number of rats suffering ischemic arrhythmia and arrhythmia score were significantly increased in IR group, LRIPOC group, SEVPOC group and COMBINE group. Sevoflurane could postphone ischemic arrhythmia.Serum concentration of LDH and cTnI in the IR group, LRIPOC group, SEVPOC group, COMBINE group and IPC group were significantly higher when compared with CON group. Compared with IR group, they were significantly lower in the LRIPOC group, SEVPOC group, COMBINE group and IPC group. They were similar in the LRIPOC group and SEVPOC group, and both were significantly higher than in COMBINE group. They were similar in COMBINE group and IPC group. Serum concentration of CK-MB in the IR group, LRIPOC group and SEVPOC group was higher when compared with CON group. Compared with IR group, it was significantly lower in LRIPOC group, SEVPOC group, COMBINE group and IPC group. It was higher in LRIPOC group and SEVPOC group than in IPC group but was not statistically higher than COMBINE group.IS%in the IR group, LRIPOC group, SEVPOC group, COMBINE group and IPC group was significantly higher when compared with CON group. Compared with IR group, it was significantly lower in LRIPOC group, SEVPOC group, COMBINE group and IPC group. It was similar in LRIPOC group and SEVPOC group, and both were significantly higher than COMBINE group. It was similar in COMBINE group and IPC group.Part Two Experimental study on the expression of HIF-la, HO-1and iNOS in limb remote ischemic postconditioing and sevoflurane postconditioningTwenty-five healthy male SD rats were randomly allocated into five groups:(1) ischemia reperfusion group;(2) ischemic preconditioning group;(3) limb remote ischemic postconditioning group;(4) sevoflurane postconditioning group;(5) combination of limb remote ischemic postconditioning and sevoflurane postconditioning group. After left thoracotomy, a5-0silk ligature was passed below a main branch of the left coronary artery. The ends of the ligature were passed through a propylene tube to form a snare. Rats endured regional ischemia reperfusion injury by occlusion of the LAD for30min and then loosening for60min. Hearts were excised at the termination of experiments. The part of the anterior wall of the left ventricular myocardium near the cardiac apex was separated as the ischemic myocardium, and the right atrium was separated as the non-ischemic myocardium, and skeletal muscle of the hind limb was excised as the remote ischemic tissue and skeletal muscle of the fore limb was also excised as the remote non-ischemic tissue. Real time quantitative polymerase chain reaction (RQ-PCR) was used to quantify the gene expression of hypoxia inducible factor1αand heme oxygenase1and inducible nitrix oxide synthase in these tissues.In the non-ischemic myocardium, the expression of HIF-1αmRNA was escalated in an order of IR group, LRIPOC group, SEVPOC group, COMBINE group and IPC group. HIF-1αin IR group was significantly lower than in the other groups. The mRNA expression was significantly higher in COMBINE group than in IR group, LRIPOC group and SEVPOC group, and significantly lower than in IPC group. There was no difference between LRIPOC group and SEVPOC group. There is no difference between COMBINE group and IPC group in the expression of HO-1mRNA, and they were higher than in IR group, LRIPOC group and SEVPOC group. There is no difference between the expression of HO-1mRNA in LRIPOC group and SEVPOC group and they were higher than in IR group but without statistical differences. The expression of iNOS mRNA was significantly lower in IR group than in other groups. It was significantly higher in COMBINE group than in IR group, LRIPOC group, SEVPOC group, but significantly lower than in IPC group. There was no difference between LRIPOC group and SEVPOC group.In the ischemic myocardium, the expression of HIF-lamRNA was significantly lower in IR group than in other groups and it was significantly higher in IPC group than in other groups. There was no difference among LRIPOC group, SEVPOC group and COMBINE group. The expression of HO-1mRNA was significantly lower in IR group than in the other groups. There was no significant difference among LRIPOC group, SEVPOC group, COMBINE group and IPC group. The expression of iNOS mRNA was significantly lower in IR group than in other groups and it was significantly higher in LRIPOC group than in other groups. There was no difference among SEVPOC group, COMBINE group and IPC group.In the fore limb, the HIF-lamRNA expression was similar in IR group, LRIPOC group and SEVPOC group, and they were significantly lower than in IPC group and COMBINE group. There was no significant difference between IPC group and COMBINE group. The expression of HO-1mRNA was escalated in an order of IR group, SEVPOC group, LRIPOC group, IPC group and COMBINE group. However, there was no significant difference among IR group, SEVPOC group and LRIPOC group. There was no significant difference among SEVPOC group and LRIPOC and IPC group. The expression of iNOS was significantly higher in COMBINE group than in other groups. There was no difference between the other groups.In the hind limb, the expression of HIF-1αmRNA was escalated in an order of IR group, LRIPOC group, SEVPOC group, IPC group and COMBINE group. There was no difference between LRIPOC group and SEVPOC group. There was no difference between SEVPOC group and COMBINE group. No differences existed between COMBINE group and IPC group. The expression of HO-1mRNA was escalated in in an order of IR group, SEVPOC group, LRIPOC group, IPC group and COMBINE group. There was no difference between SEVPOC group and LRIPOC group. It was significantly lower in IPC group than in COMBINE group, but significantly higher than in IR group, SEVPOC group and LRIPOC group. The expression of iNOS mRNA was escalated in an order of IR group, LRIPOC group, SEVPOC group, IPC group and COMBINE group. There was no significant difference between IR group and LRIPOC group. There was no significant difference between IPC group and COMBINE group. Part Three Experimental study on the expression of Akt and ERK in limb remote ischemic postconditioing and sevoflurane postconditioningTwenty-five healthy male Sprague Dawley rats were randomly allocated into five groups:(1) ischemia reperfusion group;(2) ischemic preconditioning group;(3) limb remote ischemic postconditioning group;(4) sevoflurane postconditioning group;(5) combination of remote ischemic postconditioning and sevoflurane postconditioning group. After left thoracotomy, a5-0silk ligature was passed below a main branch of the left coronary artery. The ends of the ligature were passed through a propylene tube to form a snare. Rats endured regional ischemia reperfusion injury by occlusion of the LAD for30min and then loosening for60min. Hearts were removed at the termination of experiments. The part of the anterior wall of the left ventricular myocardium near the cardiac apex was separated as the ischemic myocardium, and the right atrium was separated as the non-ischemic myocardium. Western-Blot was used to examine the expression of Akt, p-Akt, ERK1/2and p-ERK1/2and caculated ratios of p-Akt/Akt and (p-ERK1/2)/(ERK1/2).In the ischemic myocardium, the ratio of p-Akt/Akt was escalated in an order of LRIPOC group, SEVPOC group, COMBINE group and IPC group. There was no significant difference between RIPOC group and SEVPOC group. While in the non-ischemic myocardium, the p-Akt/Akt ratio was also escalated in an order of LRIPOC group, SEVPOC group, COMBINE group and IPC group. There was no difference between COMBINE group and IPC group.In the ischemic myocardium, the ratio of (p-ERK1/2)/(ERK1/2) was escalated in an order of LRIPOC group, SEVPOC group, COMBINE group and IPC group. There was no significant difference between LRIPOC group and SEVPOC group. There was no significant difference between COMBINE group and IPC group. While in the non-ischemic myocardium, the (p-ERK1/2)/(ERK1/2) ratio was also escalated in an order of IR group, LRIPOC group, SEVPOC group, COMBINE group and IPC group. There was no significant difference between LRIPOC group and SEVPOC group. There was no significant difference between COMBINE group and IPC group.ConclusionsBased on the results of all experiments, the following conclusion can be drawn:1. Remote limb ischemic postconditioning and sevoflurane postconditioning provides cardioprotection against ischemia-reperfusion injury in rat hearts in vivo. Combination of the two interventions can produce an augmented protective effect. 2. Both limb remote ischemic postconditioning and sevoflurane postconditioning can trigger system response to ischemia reperfusion injury. They provoke gene expression of HIF-la, HO-1and iNOS in ischemic myocardium, non-ischemic myocardium, hind limb skeletal muscle and fore limb skeletal muscle.3. Both limb remote ischemic postconditioning and sevoflurane postconditioning can induce the phosphorylation of Akt and ERK1/2, so the RISK signaling pathway participates in the cardioprotection of the two interventions.
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