| Objective:Every year thousands of infants and young children receive general anesthesia.Whether general anesthesia has adverse effects on children’s neurological development and cognitive function is a topic of great concern in recent years,and there are also controversies.Many preclinical studies have shown that anesthetics can cause nerve damage,synaptic plasticity,neuronal apoptosis during development,and affect adult learning and memory function.More and more clinical and experimental studies have shown that anesthetics can cause cognitive dysfunction and morphological changes in the brain,especially when they are used in immature and elderly brains.Although several possible mechanisms have been proposed,including oxidative damage and neuronal apoptosis,the mechanism of neurotoxicity remains unclear.Many studies have also been looking for ways to reduce the neurotoxicity of anesthetics.Hemin is a product of heme oxidation,also known as heme chloride,porphyrin iron chloride and ferroheme,which can induce heme oxidase activity and has a wide range of biological functions.It is generally believed that hemin not only has neuroprotective effects,but also induces the expression of neuroglobin（Ngb）.Neuroglobin also has neuroprotective effects,resisting ischemia/hypoxia injury and inhibiting neuronal apoptosis.Hemin’s neuroprotective effect has become a hot topic in cerebral ischemia and traumatic brain injury.However,the effect and mechanism of hemin on neurotoxicity induced by anesthetics have not been elucidated.Although studies have shown that sevoflurane can induce changes in mitochondrial dynamics,the effect of hemin on sevoflurane-induced mitochondrial dynamics is not clear.Therefore,in this experiment,we studied the mitochondrial mechanism of sevoflurane neurotoxicity and whether hemin can alleviate cognitive impairment induced by sevoflurane exposure in neonatal rats.Hemin is the substrate and inducer of HO-1,and HO-1 also has neuroprotective effect.Hemin exerts neuroprotective effects by inducing Nrf2-dependent antioxidants such as heme oxygenase-1（HO-1）,thioredoxin-1（TRX-1）and peroxidoredoxin-2（PRX-2）.Hemin can prevent the damage caused by free radicals,reduce the toxicity of excitatory amino acids,and play an important role in protecting important organs from hypoxic-ischemic injury.Whether hemin participates in the protection of sevoflurane neurotoxicity in neonatal rats via PI3K/Akt pathway remains unclear.So we studied whether PI3K/AKT pathway is involved in hemin neuroprotection by intraventricular injection of LY294002（LY）,a specific inhibitor of PI3K/AKT.Western blot was used to detect the changes of Ngb,caspase-3,Akt,pAkt,HO-1 and SOD to observe the other possible mechanisms of hemin’s protective effect on sevoflurane toxicity.Methods:Eighty-four 7-day-old healthy SD rats weighing 16-18 g were randomly divided into four groups:（1）control group（group C）:only saline was injected intraperitoneally,and 40%oxygen and 60%nitrogen were inhaled.（2）group H:Hemin was injected intraperitoneally on the 5th and 6th day after birth in group H;（3）group S:3%sevoflurane was inhaled for 4 hours mixed with 40%oxygen and 60%nitrogen.（4）In SH group,3%sevoflurane was inhaled for 4 hours,and hemin(50mg.kg-1)was injected intraperitoneally on the 5th and 6th day after birth.Inhibitor group:24 7-day-old healthy SD rats weighing 16-18 g were randomly divided into four groups:（1）control group（group C）:only saline was injected intraperitoneally,and 40%oxygen and 60%nitrogen were inhaled.（2）group H:Hemin was injected intraperitoneally on the 5th and 6th day after birth in the hemin administration group（H group）;（3）3%sevoflurane was inhaled for 4hours in the SH group,and hemin was injected intraperitoneally on the 5th and 6th day after birth.（4）In SHI group,3%sevoflurane was inhaled for 4 hours,hemin was injected intraperitoneally on the 5th and 6th day after birth(50mg kg-1),and LY294002 3ul was injected into the lateral ventricle two hours before anesthesia.Cleaved caspase-3 was detected by Western blot and TUNEL for hippocampal cell apoptosis,and cytochrome C for mitochondrial function.Immunohistochemistry was used to detect neuroglobin expression,immunofluorescence double staining was used to detect the distribution and localization of neuroglobin expression,and Western blot was used to detect neuroglobin,Akt and pAkt.The effects of sevoflurane on pAkt and whether hemin acts through PI3K/Akt pathway were observed.The expression of HO-1 and SOD was detected,and the neurotoxicity of sevoflurane and the protective effect of hemin were observed from the perspective of oxidative stress.Morris water maze test was used to detect the cognitive function of rats 30 days after birth and the expression of PSD-95 and GAP-43 was detected by Western blot.Results:1 Sevoflurane induced apoptosis in hippocampus,mitochondrial disfunction,oxidative stress and cognitive impairment in rats after exposure to 3%sevoflurane for 4hours.Compared with the control group,the expressions of cleaved caspase-3,cytochrome c,HO-1,Drp1 and neuroglobin in the hippocampus of neonatal SD rats in sevoflurane group increased,while the expressions of Mfn2,pAkt and SOD decreased,and time in target quadrant residence time decreased（P<0.05）.Compared with group S,the number of apoptotic cells in SH group decreased by TUNEL（P<0.05）.There was no significant difference in the number of apoptotic cells between SH group and C group（P>0.05）.2Hemin increased the expression of neuroglobin,reduced the apoptosis of hippocampal cells induced by sevoflurane exposure,improved mitochondrial dynamics,reduced oxidative stress injury,and improved cognitive impairment caused by sevoflurane exposure.Compared with sevoflurane group,cleaved caspase-3,cytochrome c and Drp1expression decreased,Mfn2,pAkt and SOD expression increased and time in target quadrant prolonged in sevoflurane+hemin group（P<0.05）.Immunohistochemical results showed that the optical density of Ngb in hippocampal CA1 region of neonatal rats in the other three groups increased after hemin administration compared with group C（P<0.05）.Immunofluorescence double staining showed that Ngb was mainly localized in neurons and a few in astrocytes.3 Hemin alleviates apoptosis induced by sevoflurane exposure in neonatal SD rats through PI3K/Akt pathway.Compared with the other three groups,pAkt/Akt in inhibitor group decreased significantly（P<0.05）.The expression of cleaved caspase-3 increased significantly（P<0.05）.4 Compared with group C and group S,we found that the expression of PSD-95 in group H and SH increased significantly（P<0.05）.Compared with group C,the expression of GAP-43 in group S was lower,and the expression of GAP-43 in group SH was higher than that in group S（P<0.05）.5 The inhibitor Ly294002 didn’t affect the expression of neuroglobin.Conclusion:Hemin can reduce the apoptosis of hippocampal cells in neonatal SD rats,improve mitochondrial dynamics,improve the neurocognitive impairment and the change of Gap-43 induced by sevoflurane exposure in neonatal SD rats.In addition,Hemin alleviated the decrease of SOD level induced by sevoflurane inhalation by up-regulating the expression of heme oxygenase（HO-1）,thereby enhancing the antioxidant defense response of neonatal SD rats and protecting them from oxidative stress injury caused by sevoflurane exposure.Hemin may protect neurons through PI3K/Akt signaling pathway.In addition,the expression of neuroglobin was not affected,suggesting that PI3K/Akt may be the downstream signal transduction pathway of neuroglobin. |
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