Sevoflurane Postconditioning Alleviates Hypoxic-ischemic Brain Injury In Neonatal Rats By Modulating ER Stress Mediated Autophagy Via IRE1/JNK/beclin1 Pathway

Objective: Neonatal hypoxic ischemic encephalopathy,the major cause of infant death and neurological sequelae,is a common complication in the neonatal period and caused by many reasons..Patients with severe hypoxic-ischemic brain injury(HIBI)die during the neonatal period,and ninety percents of survivors have long-term neurological and cognitive dysfunction,leading a heavy burden on families and society.At present,the treatment mainly focuses on the symptomatic treatment in the acute phase and the rehabilitation treatment after the injury,and the effect is not satisfactory.Therefore,there is an urgent need to develop new methods for the treatment of patients with HIBI.With the widespread use of inhaled anesthetics in infant surgery,the protection in brain of inhaled anesthetics has received attention.As an inhalation anesthetic,sevoflurane has the advantages of rapid induction,quick recovery,and easy adjustment of anesthesia depth.Studies have shown that sevoflurane post-treatment can alleviate HIBI in neonatal rats,reduce infarct volume,inhibit neuronal apoptosis,and play a neuroprotective role,but its mechanism has not been fully elucidated.Previous studies have shown that sevoflurane plays a neuroprotective role by reducing autophagy and neuronal apoptosis via PI3K/Akt/mTOR and MAPK/ERK/mTOR signaling pathways in a model of HIBI.Autophagy is a defensive reaction of the body.With the further related research,it is found that autophagy is not only related to apoptosis,proliferation,differentiation,migration,but also has an intrinsic connection with inflammatory mediators,mitochondrial oxidative stress,etc.Studies have shown that unfolded protein response can activate autophagy when endoplasmic reticulum stress(ERS)happened,and autophagy can reduce endoplasmic reticulum load by degrading misfolded or unfolded proteins,inhibiting excessive activation of endoplasmic reticulum stress,and simultaneously the degradation products also provide raw materials for the synthesis of new proteins in the body cells,the reconstruction of cell structures,and the production of ATP.However,overactivated endoplasmic reticulum stress-autophagy can aggravate cell damage and even lead to cell death.Studies have shown that endoplasmic reticulum stress-mediated autophagy is associated with neurodegenerative diseases.There are granular vacuolar degeneration in the brain of AD patients,which contains the ERS marker protein,and the autophagy marker protein such as LC3 is also increased in the neurons.In a model of cerebral ischemia-reperfusion injury,ERS can activate autophagy via the PERK and IRE1 pathways,and autophagy can be inhibited by 4-PBA,while 3-MA can only inhibit autophagy without significant effect on ERS;Melanin can reduce brain damage by inhibiting ERS and autophagy,while tunicamycin and RAPA have antagonistic effects.It can be seen that ERS and autophagy are involved in cerebral hypoxia-ischemia,and how endoplasmic reticulum stress regulates autophagy,and whether sevoflurane plays a protective role by regulating endoplasmic reticulum stress-autophagy in HIBI remains to be confirmed.Materials and Methods: 1.The experimental animals were selected from SD rats,7 days old and weighing 12-16 g,which were provided by Benxi Medical Research and Education Development Base of Shengjing Hospital affiliated to China Medical University.The experiment was in strict accordance with international ethical guidelines and laboratory animals of National Institutes of Health.The nursing and use guidelines were carried out and approved by the Medical Ethics Committee of Shengjing Hospital affiliated to China Medical University(2017PS020K).2.The model of HIBI in neonatal rats was operated according to the previous experiment.Endoplasmic reticulum stress-related protein GRP78 and autophagy-related protein LC3-II expression levels in the left hippocampus of newborn rats was detected by Western blot after 3 hours,6 hours,12 hours and 24 hours.3.The endoplasmic reticulum stress agonist TM,endoplasmic reticulum stress inhibitor 4-PBA and autophagy inhibitor 3-MA were injected into the left ventricle of 7-day-old SD newborn rats 30 minutes before the HIBI model operation,meanwhile the other groups were injected with the same amount of vehicle.After 24 hours of modeling,Western blot was used to detect the endoplasmic reticulum stress-related protein GRP78 and autophagy-related proteins LC3-II and p62 in the left hippocampus;Double immunofluorescence staining of GRP78 and Neuron,p62 and Neuron in the left hippocampal CA1 and CA3 regions;TUNEL was used to detect the number of neuronal apoptosis in the above regions.4.The left ventricle of SD newbornrats was injected with TM,TM + IRE1 inhibitor,and the other groups were injected with the same amount of vehicle 30 minutes before HIBI.Immediately after HIBI,2%sevoflurane delivered by 30% O2+70% N2 humidified gas mixture was inhaled for 30 min,and the remaining two groups were inhaled with a humidified gas mixture without sevoflurane.GRP78,LC3-II,p62,and IRE1/JNK/beclin1 pathway protein expression levels in the left hippocampus were detected by Western blot 24 hours after HIBI.5.Neonatal rats were also tested for long-term exercise activity,emotional changes,and spatial learning and memory function in the newborn rats by the open field test and Morris water maze test 28 days after the above treatment.Then number of neurons in the hippocampal CA1 and CA3 regions were counted by Nissl staining after the water maze test.Results: 1.Endoplasmic reticulum stress and autophagy were both activated in the HIBI model: GRP78 increased at 3th hour after HIBI and reached a peak at 6th hour;LC3-II increased 6th hour after HIBI and reached a peak at 24 th hour.2.Endoplasmic reticulum stress regulates autophagy and mediates neuronal apoptosis in neonatal rats with HIBI:Treatment with endoplasmic reticulum stress agonist TM,increased the protein expression levels of GRP78,LC3-II,decreased the level of p62,and added the number of TUNEL-positive cells.While endoplasmic reticulum stress inhibitor 4-PBA,decreased the levels of GRP78 and LC3-II,increased the expression of p62,and reduced the number of TUNEL-positive cells.The expression of GRP78 was not changed when treated with autophagy inhibitor 3-MA,however,the levels of LC3-II and the number of TUNEL-positive cells decreased,the expression of p62 increased.3.Sevoflurane postconditioning reduced the expression of endoplasmic reticulum stress-autophagy in the HIBI model via IRE1/JNK/beclin1 signaling pathway: Compared with the HIBI group,the levels of GRP78,LC3-II,p-IRE1,p-JNK,beclin1 decreased in the sevoflurane postconditioning(SPC)group,while the expression of p62 increased;Compared with SPC group,the expression levels of GRP78,LC3-II,p-IRE1,p-JNK,beclin1 were elevated and p62 decreased in SPC+TM group;The levels of GRP78,LC3-II,p-IRE1,p-JNK,beclin1 decreased,and p62 increased in the SPC+TM+IRE1 inhibitor group compared with the former.4.Sevoflurane postconditioning improve long-term learning and memory function and increase the number of neurons in CA1 and CA3 area via theIRE1/JNK/beclin1 signaling pathway: Compared with the HIBI group,the SPC group shortened the escape latency,increased the times of crossing platforms,and the number of neurons in CA1 and CA3 area;Compared with the SPC group,the escape latency was lengthened,the times of crossing platforms and the number of neurons in the CA1 and CA3 area was reduced in the SPC+TM group;Compared with the SPC+TM group,the SPC+TM+IRE1 inhibitor group shortened the escape latency,increased the number of crossing platforms,and the number of neurons in the CA1 and CA3 regions.There was no significant difference in the number of feces,total distance,and time in central region among the groups in open field.Conclusion: Endoplasmic reticulum stress and autophagy could be activated in neonatal rats with HIBI;Endoplasmic reticulum stress regulates autophagy and mediates neuronal apoptosis.Sevoflurane postconditioning can improve long-term learning and memory function and increase the number of neurons in CA1 and CA3 by decreasing the level of endoplasmic reticulum stress-autophagy,which may be related to the IRE1/JNK/beclin1 signaling pathway.