The Combination Of Pitavastain And Ischemic Postconditioning Attenuates Myocardial Ischemic-Reperfusion Injury In Impaired Glucose Tolerance Rat In Vivo

Background: It has become consensus that ischemia postconditioning is able to alleviate myocardial ischemia-reperfusion injury. Recent studies have confirmed that statins also has a cardioprotective effect in myocardial ischemia-reperfusion injury through anti-oxidation and activation of phosphatidylinositol -3 kinase - protein serine threonine kinase-endothelial nitric oxide synthase (PI3K-Akt-eNOS) signaling pathway and other mechanisms. Recently, experiments show that myocardial ischemia-reperfusion injury (MIRI) can be alleviated by ischemia post-conditioning (IPC) and/or statin post-conditioning (SPC), and their combination.However, it is unclear that the cardio-protection works in impaired glucose tolerance (IGT) state since IGT significantly abolishes intrinsic myocardial self-protections, and if does so, what mechanisms are involved in?Objective: Our study was in order to establish the experimental model of myocardial ischemia-reperfusion on impaired glucose tolerance rats; in following, this study was to investigate the cardio-protective effects and possible mechanisms by combination of SPC and IPC in the IGT rats.Methods: (1) 117 clean male Wistar rats were allocated randomly into two groups: control group (A group, n=12); impaired glucose tolerance group (B group, n=105). B group were feed with normal diet after injection of STZ, monitoring their weight, fasting plasma glucose (FPG); measuring glucose in the time of 0.5h, 1h, 2h, after determined by the OGTT test; the contents of triglycerides and total cholesterol were detected by automatic biochemical analyzer; at last enzyme-linked immuno sorbent assay (ELISA) was used to measure blood insulin level; the pancreatic tissue of two groups were sliced for HE staining. (2) IGT model rats were randomly allocated into six groups (n=12 per group): Sham group, treated with open chest operation but without myocardial ischemia as controls; I/R group, with ischemia 30min and reperfusion 2h in LAD territory but without other interventions; IPC group, treated with initial ischemia 30min, then 3 consecutive runs of 10s reperfusion/10s ischemia, and final reperfusion 2h; SPC group, with initial ischemia 30min, then pitavastatin (0.1mg/kg) intravenously 3 mins before reperfusion, and final reperfusion 2h; ISPC group, with initial ischemia 30min, then combination of 3 consecutive runs of 10s reperfusion/10s ischemia and pitavastatin 3 mins before reperfusion, and final reperfusion 2h; ISPC+LY294002 group, with initial ischemia 30min, then combination of 3 consecutive runs of 10s reperfusion/10s ischemia, pitavastatin and PI3-K inhibitor LY294002 (0.3mg/kg, intravenously) 3 and 15 mins before reperfusion, respectively. At last ,measuring the infarct size by TTC, observing the ultrastructure of mitochondria by electron microscopy, detecting the expression of total Akt, phosphorylate Akt, total eNOS and phosphorylate eNOS of myocardial tissue by Western-blot in the different groups.Results: (1) Compared with normal group, the FPG is similar in the impaired glucose tolerance group(3.14±1.40mmol/L vs 3.92±0.97mmol/L,P>0.05),but weight increased significantly(305.37±19.30g vs 352.84±43.24g,P<0.05).Meanwhile there are no significant difference in the serum fasting insulin levels between two groups (25.62±3.54mIU/ml vs 23.45±2.40mIU/ml, P> 0.05),but 2h insulin levels of B group was significantly higher than A group (67.28±2.43mIU/ml vs.39.86±5.98 mIU/ml, P<0.05). (2)Compared with sham group, I/R group had larger infarct size (70.1±3.1% vs 0±0%, P<0.05), and higher mitochondria score (3.24±0.74 vs 0.00±0.00, P<0.05). (3) Compared with I/R group, IPC group, SPC group, and ISPC group all reduced myocardial infarct size (P<0.05, each) and CK-MB level (P<0.05, each), alleviated mitochondria injuries(P<0.05, each), and enhanced PI3K activation by up-regulated expression of phosphorylate Akt (P<0.05, each) and phosphorylate eNOS (P<0.05, each). (4)Compared with IPC and SPC groups, ISPC group further reduced myocardial infarct size (33.4±6.5% vs 45.3±4.6%, P<0.05; vs 43.2±4.1%, P<0.05), CK-MB level(P<0.05, each) and mitochondria score (1.23±0.68 vs 2.28±0.77, P<0.05; vs 2.33±0.79, P<0.05) with more activation of PI3K evidenced by higher expression of phosphorylate Akt (P<0.05, each) and eNOS (P<0.05, each). (5)However, compared with IPC, SPC and ISPC groups, phosphorylated Akt expression and phosphorylation levels of eNOS was significantly lower or undetectable in ISPC+LY294002 group (P<0.05, each), indicating that PI3K inhibiter LY294002 could completely block the PI3K-Akt-eNOS signaling pathway, resulting in abolishment of cardio-protection induced by IPC, SPC and their combination.Conclusions: (1) It needs four weeks to establish impaired glucose tolerance rats successfully through intravenous injection of streptozotocin (STZ), and we also establish the myocardial ischemia reperfusion model based on the impaired glucose tolerance rats by ligating left anterior descending coronary artery. Experimental methods mentioned above could provides a reliable animal model to study pathophysiological mechanisms and its associated kinase signaling pathway in myocardial ischemia-reperfusion injury on basic of impaired glucose tolerance. (2) The combination of pitavastain and ischemic postconditioning enhances the cardioprotection against myocardial ischaemia-reperfusion injury in impaired glucose tolerance rats, and PI3K-Akt-eNOS may be the major signaling pathway mediated this cardioprotection.