Diabetes Mellitus Attenuates The Neuroprotection By Sufentanil Postconditioning And The Role Of Glycogen Synthase Kinase-3β

Objective Sufentanil, a μ opioid agonist, is gaining popularity in clinical use, and alsohas been demonstrated to provide neuroprotection against ischemia/reperfusion(IR)injury. The underlying mechanism may be mediated through promoting neuronalsurvival signaling pathways. Our previous research found that sufentanilpostconditioning at a dosage of1μg/kg generate the strongest protection against focaltransient focal cerebral ischemia/reperfusion injury(CIRI) compared to the dosage of0.3μg/kg and3μg/kg. Epidemiological studies have demonstrated that diabetesmellitus is a leading risk factor of ischemic cerebrovascular diseases. Diabetes mellitusis one of the important mediating factors that influence the effect of drug conditioning.Here, we explored the roles of GSK-3β on attenuation of the neuroprotection bysufentanil against transient focal CIRI in diabetic rats. Methods The first stage Healthy adult male SD rats weighing250~300g were randomlydivided into five groups. All rats in IR group,SP1group,SP2group and SP3group were subjectedto the right middle cerebral artery occlusion(MCAO)for90min and24h reperfusion. At5minbefore reperfusion, the rats were intravenously injected sufentanil0.3μg/kg(SP1group),1μg/kg(SP2group),3μg/kg (SP3group) or normal saline(sham group and IR group). The neurologicaldeficit scores(NDS)were evaluated at24h after reperfusion，then all the animals were sacrificed toevaluate the infarct volume of the brain by2,3,5-triph-enyltetrazolium (TTC)staining. Thesecond stage Healthy male adult SD rats were choosed to conduct diabetes mellitusmodel. Diabetes mellitus model was established by intraperitoneal injection of50mg/kg streptozotocin and was confirmed by blood glucose＞16.7mmol/L two daysafter injection. Healthy male adult SD rats, weighing250-280g which diabetes mellitusmodel was successfully established were randomly divided into4groups: diabeticsham operation group(DM-sham), diabetic ischemia reperfusion group(DM-IR),diabetic sufentanil postconditioning group(DM-SP) and diabetic TDZD-8group(DM-TD).Another non-diabetic rats were randomly divided into4groups：non-diabetic sham operation group（NDM-sham），non-diabetic ischemia reperfusiongroup（NDM-IR），non-diabetic sufentanil postconditioning group(NDM-SP), andnon-diabetic TDZD-8group(NDM-TD). All rats in IR group, SP group and TD groupwere subjected to the right MCAO for90min and then followed by24h of reperfusion.Sufentanil postconditioning was induced by intravenously injection of sufentanil1μg/kg at5min before reperfusion. The rats in IR group were intravenously injectednormal saline at5min before reperfusion. The rats in sham group received the samesurgical procedure without occlusion. The NDS were evaluated at24h afterreperfusion，then all the animals were sacrificed．Rats in each group were sacrificed toevaluate the infarct volume of the brain, to calculate the water content of the ischemichemisphere, to detect the neuron apoptosis index through TUNEL staining and to assaythe GSK-3β and p-GSK-3β expressions in the ischemic hemicerebrum by Western blot. Infarct volume，as a percentage of volume at normal cerebral hemisphere，wasTTCstaining.The water content of the right cerebral hemisphere was assessed toevaluate the brain edema.Results The first stage Neurological Deficit Score and the cerebral infarctvolume：At24h after reperfusion, the NDS and the cerebral infarct volume of SP2group and SP3group were significantly higher than those of IR group(p <0.05).The second stage Characteristic of rats: After an intraperitoneal injection ofstreptozotocin，the blood glucose of rats were higher than that of the non-diabetic rats(p <0.05). Neurological Deficit Score: At24h after reperfusion，the neurologicaldeficit score in NDM-SP and NDM-TD group was lower than that in NDM-IR group(p <0.05). The neurological deficit score in DM-SP group was higher than that inNDM-SP group (p <0.05). The neurological deficit score in DM-TD group was lowerthan that in DM-IR group (p <0.05). The infarct volume, water content and cellapoptosis: Sufentanil postconditioning significantly decreased the infarct volume, thewater content of the right cerebral hemisphere and the apoptosis index in the ischemicpeunumbra in non-diabetic rats at24h after reperfusion(P<0.05),but had no significanteffect on these in diabetic rats(P>0.05). The infarct volume, the water content of theright cerebral hemisphere and the apoptosis index in the ischemia peunumbra weresignificantly higher in DM-SP group than those in NDM-SP group(P<0.05). Theinfarct volume, the water content of the right cerebral hemisphere and the apoptosisindex in the ischemia peunumbra were significantly lower in DM-TD group than thosein DM-IR group(P<0.05). The p-GSK-3β and GSK-3β expression in theischemia cortex: The expressions of the total GSK-3β did not change between all thegroups(p>0.05). The p-GSK-3β protein level（p-GSK-3β/GSK-3β） of NDM-SPgroup and NDM-TD group was higher than that of the NDM-IR group (p <0.05).However, there was no significant difference between DM-IR and DM-SP group.p-GSK-3β protein expression significantly decreased in DM-SP group compared to NDM-SP group,(p <0.05). p-GSK-3β protein level was higher in DM-TD group thanthat in DM-IR group (p <0.05).Conclusions Diabetes mellitus ameliorates the neuroprotection of sufentanil againsttransient focal cerebral ischemia/reperfusion injury in diabetic rats, which is associatedwith the down regulation of the phosphorylation of GSK-3β. Our data also suggestthat direct inhibition of GSK-3β may provide a novel strategy to protect non-diabeticand diabetic brains from ischemia reperfusion injury.