The Neuroprotective Effect Of PI3K/Akt Channel By Sevoflurane Postconditioning In Diabetic Rats With Cerebral Ischemia

Research BackgroundCerebral stroke is a group of diseases which are caused by various inducing factors such as occlusion and rupture of the internal cerebral artery,resulting in cerebral blood circulation disorder and brain tissue damage.It can be divided into two types:ischemic and hemorrhagic.Stroke is one of the most deadly diseases in the world,with 10.3 million new cases every year.Recently,this disease trends to occur among young people,thereafter bringing a great burden to the society,families and patients.Furthermore,many other diseases will directly or indirectly lead to the occurrence of stroke.For example,diabetes is an independent risk factor of cerebrovascular disease.According to foreign data,the probability of cerebrovascular disease in patients with diabetes history at the same time has reached 30%-40%,and the patients with diabetes and stroke are more than twice as many as those without diabetes.Ischemic stroke accounts for 80%of the patients with diabetes mellitus and stroke,and the incidence rate of diabetes mellitus complicated with stroke is increasing rapidly.Therefore,how to design and develop safe and effective stroke treatment compounds,especially diabetic stroke,is an urgent problemMany studies have shown that pretreatment or post-treatment with inhaled anesthetics can reduce ischemic brain damage.It has been found that sevoflurane pretreatment can significantly reduce the volume of cerebral infarction and apoptotic cells caused by artery occlusion.In a number of animal experiments of cerebral ischemia-reperfusion injury,sevoflurane pretreatment can effectively reduce the apoptosis of nerve cells,improve the volume of cerebral infarction,neurological score,inflammatory response and other aspects to protect the brain nerves.However,it is still unclear whether diabetes will affect the neuroprotective effect of sevoflurane postconditioning,and the mechanism of sevoflurane’s effect remains to be studied.Some scholars have found that diabetic rats can improve their learning and memory ability by restoring their hippocampal synaptic function and Na+,K+-ATPase activity,and the mechanism may be related to the PI3K/Akt phase signal pathway;Yang and colleagues have found that lilalutide can activate the PI3K/Akt signal pathway in diabetic rats,promote autophagy,and ultimately provide protection for learning and memory disorders related to chronic T2DM;other studies have confirmed that sugar The expression of PI3K/Akt signaling pathway related proteins in the cerebral cortex cells of rats with uremia decreased significantly.Therefore,it is necessary to study the effect of sevoflurane on neurologic damage after cerebral ischemia in diabetic rats,in order to find out whether sevoflurane treatment is effective on neurologic damage after cerebral ischemia-reperfusion,and the mechanism of PI3K/Akt signal pathway in the postconditioning of sevoflurane,so as to provide a theoretical basis for reducing the neurological impairment after cerebral ischemia and promoting the recovery of neurological function after stroke in diabetic population in the later stage.ObjectiveIn this study,we constructed a rat model of cerebral ischemia and diabetes mellitus to evaluate the effects of sevoflurane on brain injury,oxidative stress and apoptosis,and to explore the molecular mechanism of sevofluraneMaterials and Methods1.Subjects and grouping:288 healthy Wistar rats,weighing(260±30)g,were housed in a clean environment with room temperature of 20±2℃ and relative humidity of 50%-70%.The rats were fed with food and water freely and fed adaptively for one week.First,240 rats were randomly divided into 6 groups(40 in each group):Control group,no treatment;DM,diabetes model rats;CI,the middle cerebral artery blood flow of healthy rats was blocked for 2h and then rats were subjected to reperfusion;DMCI,middle cerebral artery blood flow of diabetic rats was blocked for 2h before reperfusion;SCI,at the beginning of ischemia-reperfusion,the CI rats were given 2.6%sevoflurane for 30 minutes;SDMCI,the DMCI rats were given 2.6%sevoflurane for 30min immediately at the beginning of ischemia-reperfusion.The rats in Control,DM,CI and DMCI groups were given 100%oxygen at the beginning of reperfusion for 30min,and the corresponding indexes were detected 2h after reperfusion.2.Establishment of rat model of cerebral ischemia and diabetes:rats were fed with a high-fat and high-sugar diet.After 5 weeks of feeding,intraperitoneal injection of STZ(streptozotocin,dissolved in citrate buffer,25mg/kg)was injected intraperitoneally once a day for 2 consecutive days.After 72h,the blood was collected from the tail vein to measure fasting blood glucose.The model of diabetes mellitus was successfully established when blood glucose ≥16.7mmol/l,accompanied with polyphagia,polydipsia and polyuria.The methods of constructing the rat model of cerebral ischemia were as follows:rats were fasted 12h before operation.Rats were anesthetized by intraperitoneal injection of 10%chloral hydrate and fixed in supine position.The right common carotid artery was exposed by operation.The internal carotid artery and external carotid artery were separated in bifurcations.The middle cerebral artery was blocked by 4/0 nylon wire.After 2h,nylon wire was drawn out to open the middle cerebral artery and recover the perfusion of ischemic area.The successful establishment of the model was marked by:awake after operation,hemiplegia of the left limb,especially the left upper limb;curling and flexion of the left upper limb after tail lifting suspension;abnormal straight down,left turning and falling.3.Intraventricular injection of LY294002:To study the effect of sevoflurane on PI3K/Akt pathway in cerebral ischemia reperfusion,rats in the sevoflurane treatment group were pretreated with LY294002,a specific inhibitor of PI3K/Akt.The remaining 48 rats were divided into 6 groups(8 rats in each group).The normal cerebral ischemia plus normal saline group(NS-CI),diabetes mellitus with cerebral ischemia plus normal saline group(NS-DMCI),sevoflurane post-treatment cerebral ischemia plus normal saline group(NS-SCI),sevoflurane post-treatment diabetic cerebral ischemia plus normal saline group(NS-SDMCI),sevoflurane post-treatment cerebral ischemia plus inhibitor group Group(LY-SCI)and group(LY-SDMCI)were treated with sevoflurane.LY294002 was dissolved in DMSO and ethanol before operation,and the final concentration was 10 mM.Rats were anesthetized with 10%chloral hydrate(350mg/kg,intraperitoneally).In the LY-SCI group and LY-SDMCI group,10 μl LY294002 solution was injected into the ischemic ventricles 30 minutes before the establishment of the ischemic model,and the rats in the other groups were injected with the same amount of normal saline as the control.The rats in NS-SCI group,NS-SDMCI group,LY-SCI group and LY-SDMCI group were given 2.6%sevoflurane inhalation for 30min after 2h of ischemia,and the rats in NS-CI group and NS-DMCI group were given 100%oxygen for 30min at the beginning of reperfusion.4.Indicator testing:For neurological assessment,Longa’s 5-point scale was used to score and record neurological deficit symptoms 24h after anesthesia was awake;for cerebral infarction volume assessment,TTC staining method and ImageJ software were used for calculation;For the brain water content of rats,the dry and wet method was used for calculation;For the morphological changes of rat brain tissue,HE staining was used to observe under light microscope;For the detection of neurological learning ability of rats,Morris water maze experiment was used.The determination of S100B,Bal-2,Bax,GSH-PX,Caspase-3 and SOD in rat serum was carried out by ELISA kit;For NOS activity and NO content in rat serum,colorimetry was used.Moreover,detection of phosphorylation of PI3K/Akt pathway-related proteins in brain tissue by Western blot assay.5.Statistics:All the data were analyzed by SPSS 19.0,and all the quantitative data were expressed as mean ± standard deviation(SD).Two comparisons were evaluated by t-test,multiple comparisons were evaluated by ANOVA,and post hoc analyses were evaluated by LSD-t test.Two variables Pearson was used to analyze the correlation between the two groups.P<0.05 was considered significant.Results1.Sevoflurane postconditioning attenuates ischemic brain injury in rats with cerebral ischemia.Compared with the control group,the infarct volume,Longa neurological deficit score and brain water content of rats in each cerebral ischemia group were increased,and the above indexes of rats in DMCI group were further increased compared with those in CI and DM group(P<0.05).Sevoflurane postconditioning reduced infarct volume,longa score and brain water content in SCI and SDMCI groups(P<0.05).The results showed that sevoflurane postconditioning attenuated the ischemic brain injury in SCI and SDMCI groups and had protective effect on the nervous system2.Sevoflurane postconditioning improves spatial learning and memory in rats with cerebral ischemia.Morris water maze was used to evaluate the spatial learning and memory function of rats.The results showed that compared with the Control group,the escape latency time of other cerebral ischemia groups was significantly longer(P<0.05),and the number of crossing platform was significantly reduced(P<0.05).Compared with CI group and DMCI group,the escape latency time of sevoflurane inhaled rats(SCI group and SDMCI group)at the beginning of reperfusion was significantly reduced,the number of platform crossing was significantly increased(P<0.05),and the platform could be found in the navigation test in a shorter time(P<0.05).These results showed that sevoflurane postconditioning improved the spatial learning and memory ability of diabetic rats with cerebral ischemia.3.Sevoflurane postconditioning mitigates morphological damages in SCI and SDMCI rats.The results of HE staining showed that the brain tissue of normal Control group was composed of healthy nerve cells,without cell swelling,membrane blistering,nuclear concentration and broken dissolution.Compared with the Control group,the brain sections of Cl group showed necrosis,neuron loss,nuclear pyknosis,neuron contraction and astrocyte swelling.In addition,DMCI group showed more severe injury than CI group.After sevoflurane postconditioning,the tissue damage in SCI group and SDMCI group was significantly improved4.Sevoflurane postconditioning reduces oxidative stress in rats with cerebral ischemia.In order to investigate whether the neuroprotection of sevoflurane is related to its antioxidant properties,we measured the activities of SOD and GSH-PX,as well as the levels of NOS and NO.Compared with the Control group,the level of SOD and GSH-PX in CI group decreased(P<0.05),while the expression level of both in DMCI group further decreased(P<0.05).Compared with CI and DMCI,SOD and GSH-PX contents in SCI and SDMCI groups increased significantly(P<0.05).Sevoflurane significantly increased the expression of SOD and GSH-PX in SCI and SDMCI groups(P<0.05).On the other hand,the expression level of NOS and NO increased in CI group(P<0.05),and further increased in DMCI group(P<0.05),but the expression level of both decreased significantly after sevoflurane postconditioning(P<0.05).These results show that sevoflurane can protect rats from oxidative damage in SCI and SDMCI groups.5.Sevoflurane postconditioning inhibited the apoptosis of brain tissue in rats with cerebral infarction.After ischemia-reperfusion,the expression of Bax and caspase-3 was up-regulated in CI and DMCI groups(P<0.05),but down regulated in sevoflurane postconditioning group(P<0.05).On the other hand,the expression level of anti-apoptotic factor Bcl-2 was lower than that of the Control group(P<0.05),and the expression level of Bcl-2 was further reduced in DMCI group(P<0.05).Compared with CI and DMCI,sevoflurane can significantly increase the expression of Bcl-2(P<0.05).These results indicate that sevoflurane postconditioning can reduce ischemic brain damage by inhibiting apoptosis in the brain tissue of rats with cerebral infarction.6.The protein level of S100B in sevoflurane postconditioning group was significantly reduced.Compared with the Control group,the concentration of S100B in serum of rats in other groups was significantly higher(P<0.05),and the content of S100B in DMCI group was higher than that in CI group and DM group(P<0.05),indicating that diabetes mellitus can add brain damage caused by ischemia-reperfusion in rats.After sevoflurane inhalation,S100B levels in SCI and SDMCI groups were significantly lower than those in CI and DMCI groups(P<0.05).7.The correlation analysis between S100B protein level and oxidative damage index.Results showed that S100B protein expression was positively correlated with longa neurological deficit score,infarct volume,water content,NOS,NO,Bax and Caspase-3(P<0.01),and negatively correlated with SOD,GSH-PX and Bcl-2(P<0.01).It is suggested that serum S100B can be used as a reliable index to evaluate cerebral ischemia injury in rats,and sevoflurane postconditioning can reduce the degree of brain injury caused by stroke in rats.8.Sevoflurane postconditioning significantly increased PI3K/Akt pathway activity.Western blot results showed that the phosphorylation levels of PI3K and Akt protein in cerebral ischemic rats were significantly lower than those in Control group(P<0.05).The contents of p-PI3K and p-Akt in DMCI group were lower than those in CI group and DM group(P<0.05).Compared with CI and DMCI groups,the proportion of p-PI3K/PI3K and p-Akt/Akt increased significantly after sevoflurane treatment(P<0.05).It suggests that sevoflurane may inhibit apoptosis induced by oxidative stress through activation of PI3K/Akt signaling pathway.9.Blocking the PI3K/Akt signaling pathway reduces the protective effect of sevoflurane on oxidative stress and apoptosis.The levels of oxidative stress and apoptosis related factors in serum were measured by injection of LY294002,a PI3K/Akt pathway inhibitor,into rats.Sevoflurane can reduce the levels of Bax and caspase-3,and increase the protein content of Bcl-2 in the serum of rats with cerebral ischemia and diabetes mellitus combined with cerebral ischemia(P<0.05).Similarly,inhalation of sevoflurane at the beginning of reperfusion in rats with cerebral ischemia and diabetes mellitus combined with cerebral ischemia ameliorated the ischemia-induced decrease in the levels of antioxidant factors SOD and GSH-PX(P<0.05),resulting in a substantial increase in their expression,and the levels of NOS and NO,representing the degree of oxidation,were also decreased compared with those in rats without sevoflurane treatment(P<0.05).However,the introduction of LY294002 attenuated this therapeutic effect of sevoflurane,making the serum SOD,GSH-PX,NOS and NO levels close to those of ischemia-reperfusion rats treated with non-inhaled sevoflurane.This indicates that blocking the PI3K/Akt signaling pathway can inhibit the therapeutic effect of sevoflurane,further verifying that sevoflurane plays a protective role by activating the PI3K/Akt pathway.ConclusionSevoflurane postconditioning can significantly improve the brain injury induced by ischemia-reperfusion in diabetic rats and possibly play a protective role in cerebral ischemia-reperfusion injury by activating PI3K/Akt signaling pathway,inhibiting oxidative stress response and cell apoptosis in brain neurons.