Role Of PI3K/Akt/eNos Signaling Pathway In Attenuation Of Brain Injury By Sevoflurane Postcondition In A Rat Model Of Hemorrhagic Shock And Resuscitation

Objective To investigate the role of PI3-Kinase-Akt-endothelial nitric oxide synthase(PI3K-Akt-e NOS)signaling pathway in the attenuation of brain injury by sevoflurane postconditioning in a rat model of hemorrhagic shock and resuscitation.Methods Ninety healty adult male Sprague-Dawleg rats,weighing300 ~350g,were divided into 5 groups randomly(n=18 each): sham operation group(group Sham)、hemorrhagic shock and resuscitation group(group HSR)、sevoflurane postconditioning group(group SP)、 sevoflurane postconditioning + PI3 K inhibitor wortmannin group(group SP+WT)and wortmannin group(group WT).For group Sham,rats were just made catheterization of arterio-vein;for group HSR,rats were subjected to the treatment of hemorrhagic shock and resuscitation;for group SP,rats were subjected to hemorrhagic shock for 60 min and resuscitation with inhalation of 2.4% sevoflurane for 30 minutes meanwhilely;for group SP+WT and WT,30 min before rats subjected to the blood withdrawing,Wortmannin(0.6mg/kg)was administrated via the left jugular vein,then all rats were subjected to the modle of hemorrhagic shock and resuscitation and sevoflurane postconditioning.The process of hemorrhagic shock and resuscitation could roughly summed up as follows:approximately 40% of the total blood volume was withdrawed from the right common carotiod artery in 30 min,and 1h later the animals were resuscitated with infusion of the shed blood via the left jugular vein over30 min.The MAP was recorded at intervals of 10 minutes during the process and we drawed some blood samples from the common carotid artery to arterial blood gas analysis and recorded lactate concentration 、 BE and PH at several time points,including before blood withdrawing(T0),immediately at the end of blood drawing(T1),at 30 min after the end of withdrawing blood(T2),before infusion of the shedblood(T3)and immediately after the end of infusion(T4).24 h after the completion of modle,6 rats were randomly selected from each group and subjected to decapitation,their brain tissuses were immediatedly removed.We use TTC staining to determine and analysis the cerebral infarct volume;we use HE staining to observe the hippocampus cell morphology and the immuohistochemical stainin to analysis the level of caspase-3 in hippocampal region and for the rest six rats,we use Western-blot to detect the expression of Akt、Phosphorylated Akt(p-Akt)and e NOS and calculate the ration of p-Akt/Akt.Results1.Hemodynamics parameter At the time point T0,there was no statistically significant difference in five groups of rats in the MAP value comparison(P > 0.05);At the time point T1-T2,compared with group S,the MAP in group HSR 、 SP 、 SP+WT and WT was significantly decreased(P < 0.05);At the time point T3,the blood pressure in group HSR、SP 、SP+WT and WT was gradually recovered to the level of pre-operation,there was no statistically significant difference in four groups of rats in the MAP value(P > 0.05).2.Analysis of arterial blood gas At the time point T0,there was no statistically significant difference in five groups of rats in the Lac value(P > 0.05);At the time point T1-T2,compared with group Sham,the Lac was significantly increased while the BE and PH was significantly decreased in group HSR、SP、 SP+WT and WT(P < 0.05);At the time point T3,the blood pressure in group HSR、SP、 SP+WT and WT was gradually recovered to the level of pre-operation,there was no statistically significant difference in four groups of rats in the Lac、BE and PH value(P > 0.05).3.The infarct volume of brain Compared wih the group Sham,the infarct volume of brain significantlly increased in group HSR,difference between the two groups has statistical significance(P < 0.05). Compared wih the group HSR,the infarct volume of brain in group SP significantlly decreased(P < 0.05);Compared wih the group SP,the infarct volume of brain in group SP+WT significantlly increased(P < 0.05).4.The HE staining and expression of apoptosis protein Caspase-3 in hippocampus Compared wih the group Sham,cell shape of HE staining is irrugular、nucleus is pyknotic,and the expression of Caspase-3 in hippocampus in group HSR、SP+WT and WT significantly increased,their difference was statistically significant(P < 0.05);Compared wih the group HSR,cell morphology obviously improved and the expression of Caspase-3 in hippocampus significantly decreased in group SP;Compared wih the group SP,hippocampal cell is atrophied and the expression of Caspase-3 in hippocampus significantly increased in group SP+WT(P < 0.05).5.The expression of Akt、p-Akt and e NOS Compared wih the group Sham,there were no significantlly changes in the ration of p-Akt/Akt、e NOS in group HSR、SP+WT and WT(P > 0.05).Compared with the group HSR,the ration of p-Akt/Akt and the expression of e NOS in group SP were upregulated(P < 0.05),but these changes haven’t found in group SP+WT(P > 0.05).Compared with the group SP,the ration of p-Akt/Akt and the expression of e NOS in group SP+WT were downregulated(P < 0.05).Conclusion PI3K-Akt-e NOS signaling pathway mediates the attenuation of brain injury by sevoflurane postconditioning in hemorrhagic shock and resuscitation rats.