The Influence And Function Mechanism Of Hyperbaric Oxygen Pretreatment On Myocardial Ischemia Reperfusion(MIR) Injury In Diabetic Rats

Objective:Established the type2diabetic rat model by high-fat diet and STZ-induced,using Langendorff isolated heart perfusion model to observe the effects ofhyperbaric oxygen pretreatment in normal and diabetic rats ischemia/reperfusion injury and to explore the mechanisms of ischemia/reperfusioninjury in diabetic rats,look for a new way to protect diabetic myocardialischemia/reperfusion injury.Methods:The first part of the experiment:75adult male Wistar rats weighing250-300g were randomly divided into normal group (N30): give the basis offeeding, diabetic experimental group(DM45): giving a high fat diet feeding.Given2%STZ intraperitoneal injection (40mg/kg) after4weeks feeding,measure the blood sugar of the tail blood after1week, bloodglucose>16.7mmol/L for the model of diabetes. Rats continue regulardiet for2months.Normal rats(N group) and diabetic rats(D group)are divided into threegroups respectively, each group of10rats, the groups were normal controls(N-Cgroup),normal ischemia/reperfusion group(N-I/R group), normal hype-rbaric oxygen preconditioning group (N-P group), diabetic control group(D-Cgroup), diabetes ischemia/reperfusion group(D-I/R group), diabetes hyperbaricoxygen preconditioning group (D-P group). Record hemodynamic indexes ofbiological and functional test systems, including HR, LVSP,+dp/dtmax,detection the cardiac enzymes from the coronary effluent. Determinate the activity of SOD and MDA formation using myocardial tissue kits. UnderwentTTC staining after the perfusate, Determinate the myocardial infarct size andobserve the changes in myocardial ultrastructure by myocardial HE staining.The second part of the experiment: Modeling the Type2diabetes animalmodel using the same method with the first part of the experiment.Increase thewortmannin in the intevention group. Group as follows: normal controlgroup(N-C group), normal ischemia/reperfusion group(N-I/R group), Normalhyperbaric oxygen preconditioning group(N-P group),normal the hyperbaricoxygen preconditionding plus wortmanmin group (N-W group),diabetic controlgroup(D-C group),diabetes ischemia/reperfusion group(D-I/R group),diabeteshyperbaric oxygen preconditio-ning group(D-P group),the diabetes hyperbaricoxygen preconditionding plus wortmanmin group (D-W group). Preparation ofisolated rat heart perfusion (the Langendorff) model the same way as the firstpart of the experiment. Using TUNEL to detect myocardial apoptosis,calculated the apoptotic index. Detect the myocardial cells of caspase-3,Bcl-2, Bax,cytochrome C,Akt,P-pAkt, GSK3and P-GSK3byimmunohistochemical methods. Measure the protein by Western blot of tissueAkt (of pAkt) and GSK3(PGSK3) protein content.Statistical analysis: Data Processing Application Spss13.0statisticalsoftware for measurement data are expressed as mean±standard deviation(x±s),count data with the χ2test,between the two groups by t test, multiplesets of data using analysis of variance.Results:(1)High fat diet fed rats for28days, the weight of the high-fat group,serum triglyceride (TG),total cholesterol(TC) were significantly higher thanthe normal control group (P<0.01). Listlessness, high-fat diet diabetic ratsgiven2%STZ intraperitoneal injection significantly increased in the yellowcoat color, dull, water intake, urine output compared with administration. Seventh day of intraperitoneal injection, diabetic rats, body weight decreased,blood glucose was significantly higher than the normal group (P <0.01).(2) N-I/R group,D-I/R group by ischemia30min and reperfusion10min,30min,60min,120min when LVSP,+dp/dtmax,-dp/dtmax compared with thecorresponding control group were significantly lower(P<0.05), in which D-I/Rgroup compared with N-I/R group decreased more significantly at each timepoint；N-P group,D-P group LVSP,+dp/dt max,-dp/dtmax graduallyincreased at each time point compared with the corresponding I/R group, inwhich N-P group than D-P group increased more significantly at each timepoint.(3) The content of LDH, CK of N-I/R group and D-I/R group at120minof reperfusion were significantly increased (P<0.05) compared with thecorresponding control group, in which the D-I/R group than the N-I/R groupincreased more significantly (P<0.05; The content of LDH, CK of N-P groupand D-P group at120min of reperfusion were significantly decreased (P<0.05)compared with the corresponding I/R group, in which the N-P group than theD-P group decreased more significantly (P<0.05).(4) N-I/R group and D-I/R group compared with the corresponding controlgroup, myocardial tissue SOD activity decreased, MDA content increased (P<0.05).in which the D-I/R group compared with the N-I/R group SODactivity decreased, MDA content increased more significantly(P <0.05); N-Pgroup and D-P group compared with the corresponding I/R group,myocardialtissue SOD activity increased, MDA content decreased (P <0.05).in which theN-P group compared with the D-P group SOD activity increased, MDA contentdecreased more significantly(P <0.05).(5) N-I/R group and D-I/R group compared with the corresponding controlgroup, myocardial infarct size was significantly increased(P<0.05), in whichthe D-I/R group compared with the N-I/R group the myocardial infarct size was larger more significantly (P<0.05);N-P group and D-P grpup compared with thecorresponding I/R group, myocardial infarct size was significantly reduced (P<0.05), in which N-P group myocardial infarct size decreased moresignificantly (P <0.05).(6) Histopathological changes: I/R group myocardial cells arranged inirregular,cell swelling, cytoplasmic eosinophilic enhancement, inflammatorycell invasion. D-I/R group ischemia-reperfusion injury severer than normal rats.The hyperbaric oxygen group myocardial cells have a little swelling,cytoplasmic eosinophilic enhancement,a little inflammatory cell invasion. N-Pgroup ischemia-reperfusion injury milder than D-P group.(7)N-I/R group and D-I/R group compared with the corresponding controlgroup, apoptosis index (AI) increased (P<0.01), in which the D-I/R group thanthe N-I/R group AI increased more significantly (P <0.01).;N-P group and D-Pgroup compared with the corresponding I/R group AI decreased (P<0.01), inwhich the N-P group than the D-P group AI decreased more significantly (P<0.01).Give Wortmanmin reperfusion N-W group and D-W group comparedwith the corresponding I/R group AI has no significant difference, withcorresponding P group has significantly difference (P<0.01).(8) N-I/R group and D-I/R group compared with the corresponding controlgroup, the expression of caspase-3, Bax, cytochrome C increased, theexpression of Bcl-2decreased (P<0.01). In which the D-I/R group than theN-I/R group the expression of caspase-3,Bax, cytochrome C increased moresignificantly and the expression of Bcl-2expression decreased moresignificantly(P<0.01); N-P group and D-P group compared with thecorresponding I/R group,the expression of caspase-3,Bax,cytochrome Cdecreased and the expression of Bcl-2increased (P<0.01), in which the N-Pgroup than the D-P group the expression of caspase-3,Bax, cytochrome Cdecreased more significantly and the expression of Bcl-2expression increased more significantly(P<0.01); Give Wortmanmin reperfusion N-W group andD-W group compared with the corresponding I/R group the expression ofapoptotic proteins has no significant difference, with corresponding P grouphas significantly difference (P<0.01).(9) Immunohistochemistry and Western-blot analysis: each group isolatedheart after30min ischemia and120min reperfusion, the myocardial tissue ofAkt, of GSK3β protein expression was no significant difference (P>0.05). Butthe N-P group and the D-P group compared with the corresponding I/R groupthe phosphorylation of Akt (p-Akt Ser473) and phosphorylated GSK-3βexpression was significantly enhanced (P <0.01). Which the N-P group than theD-P group phosphorylated Akt (p-Akt Ser473) and phosphorylated GSK-3βexpression increased significantly (P <0.01). Give Wortmanmin reperfusionN-W group and D-W group compared with the corresponding I/R group theexpression of phosphorylation of Akt(p-of Akt Ser473) and phosphorylation ofGSK-3has no significant difference, with corresponding P group hassignificantly difference (P<0.01).Conclusion:1. Hyperbaric oxygen pretreatment in normal and diabetic rats withmyocardial ischemia/reperfusion injury have a protective effect, showingsignificant improvement of cardiac hemodynamics,reduction of myocardialinfarct size, reduction of myocardial enzyme release,increase of the myocardialantioxidant enzyme activity, but the pretreatment with hyperbaric oxygenationhave stronger protective effect in normal rats.2. Hyperbaric oxygen pretreatment inhibits apoptosis in normal anddiabetic rat myocardium, its role may be through upregulation of Bcl-2proteinexpression and at the same time reduced the expression of Bax and Caspase-3and cytochrome C, presenting stronger protective effect of hyperbaric oxygenpretreatment on the myocardial cells in normal rats. 3. The protective effect of hyperbaric oxygen pretreatment on themyocardium of diabetic rats in ischemia reperfusion injury is through activationof PI3K-Akt-of GSK-3β signaling pathway.