| With the development of neonatal intensive care medical,survival rate of preterm children improved significantly.Oxygen therapy is one of the common methods used in intensive care unit to treat emergency,but inhaled high concentration oxygen for a long time has been confirmed in animal experiment and clinical research that can cause bronchopulmonary dysplasia(BPD),retinopathy of prematurity(ROP),brain damage.Many studies show that exposure to hyperoxia not only affects brain development,but also can lead to cerebral edema,reduces density of nerve connection,activity of oligodendrocyte and survival signaling pathways of neurotrophic factor,decreases the content of neurotrophic factor.Also it increased cell apoptosis of inflammatory cytokines,neuron and vascular endothelial cells.Meanwhile,the problems of brain injury induced sequel bring serious burden on children and their families,society,and aroused concern of the majority of pediatricians.Hippocampus is an important conformation of limbic system,which is related to learning,memory and emotion and immunoregulation.Its damage plays a vital role in various stress injuries.Endoplasmic reticulum stress(ERS)is closely related to the occurrence of many diseases,and relationship between ERS and hyperoxia-induced lung injury has been paid much more attention.The studies show that the endoplasmic reticulum peroxidation can influence the correct folding of the protein,resulting in the unfolded protein response(UPR),UPR react through the membrane PERK,IRE1 and ATF6 and other three transmembrane protein stress signal transduction,however,the role of UPR in hyperoxia-induced brain injury and its mechanism is unclear.Insulin-like growth factor(IGF-1)as one of the members of the insulin-like growth factor family,is a basic,single-chain protein of molecular,which is composed of 70 amino acids,plays an important role in growth and development of fetal.IGF-I can promote cell DNA synthesis and metabolism,impel cells into G1 phase,thereby facilitating cell proliferation.Therefore,we made hyperoxic brain injury model using neonatal rats to explore the role and targets of the ERS and UPR signaling pathway in the hyperoxic-induced hippocampal cell injury.With this as basis,to further clarify the protection role of exogenous IGF-2 on ERS reactive signal in hyperoxia-induced brain injury.This will effectively reveal the role of ERS in hyperoxic-induced hippocampal cell injury and its molecular mechanism,and provide scientific and theoretical basis for the clinical applications of IGF-1 in prevention and treatment of hyperoxic-induced hippocampal cell injury.Part one The role of ERS pathway on hyperoxia-induced hippocampus cell injuryObjective:To investigate changes of ERS signaling pathways in hyperoxia-induced hippocampal cell apoptosis and its molecular mechanism.Methods:Prepare full-term newborn Wistar rats to make hyperoxia-induced brain injury model,after modeling,wet and dry ratio of brain tissues was detected on the 1st,3rd,7th and 14th day.The brain development and morphopathological changes was observed using by HE staining,the ultrastructure changes of hippocampal cells were observed by electron microscope,hippoc ampal cell apoptosis was observed by tunel staining.Expression levels of TNF-??IL-1??IL-6 in the brain tissue were detected by ELISA,levels of Bcl-2/BaX,GRP78,CHOP,caspase-12 were detected by western blotting method.Results:1.Compared with the control group,the weight decreased on the 3rd.7th and 14th day after exposure to high oxygen in hyperoxia group,There were was significant differences(P<0.05),but there was no statistical significance compared with the control group on the 1st day.2.Compared with control group,brain wet/dry(W/D)proportion of hyperoxia group increased on the 3rd,7th and 14th day after exposure to high oxygen(P<0.05),the differences were statically significant There was no statistical significance compared with control group on the 1st day.3.HE staining results:In hyperoxia group,we observed that arrangement disorder of brain cell,vacuolation formation in cytoplasm,part of neuron swelling,uneven distribution,nuclear pycnosis,disappearance of nucleoli.Compared with the hyperoxia group,the morphomogy of neurons in hippocampus of control group was normal.4.Transmission electron microscopy results showed that in hyperoxia group double-deck membranes of mitochondria were disintegrated,the number of mitochondria and synapses were reduced.So was the density of synapse.As hyperoxia prolonged we detected disruption of cristae and vacuolated mitochondria.In control group,we observed complete construction of mitochondrial and synaptic,also increased number of synapses.5.TUNEL staining results showed that,compared with control group,on 3rd day(5.6±1.1),7th day(5.3±0.9),14th day(4.5±0.7),hippocampus apoptotic index of hyperoxia group was significantly higher on 3rd day(25.6±0.9),7th day(36.3±1.4),14th day(38.4±2.8).6.Levels of TNF-?,IL-1?,IL-6 by ELASA method:Compared with control group,levels of TNF-?,IL-1?,IL-6 in high oxygen group on 1st,3rd,7th and 14th day were significantly higher(p<0.05).With high oxygen exposure time prolonged,levels of TNF-?,IL-1?,IL-6 gradually increased,also reached its peak on 7th day.Meanwhile,we detected these levels reduced to the 14th day.7.Western blotting results showed that,BaX expression in hyperoxia group on 3rd day were higher than control group,with high oxygen exposure time prolonged,expression of BaX were gradually increased.On 3rd day,Bc12 expressions in hyperoxia group were lower than control group,and the expression were gradually reduced as oxygen exposure time prolonged.The ratio of Bc12/BaX in hyperoxia group on 3rd days was lower than control group,and the expression were gradually reduced as oxygen exposure time prolonged.GRP78,CHOP,caspase-12 expression in hyperoxia group were higher than control group,when the hyperoxia time extended,the protein content became higher,while th.ere was no significant difference in the hyperoxia group on 1st day.Conclusion:1.High concentrations of oxygen can induce cerebral edema,inflammatory response.2.ERS involved in hyperoxia-induced hippocampus cell apoptosis.Part two IGF-? ameliorates hyperoxia-induced hippocampus cell injury by inhibiting apoptosis and ERS Signaling pathwayObjective:To observe ERS signal molecular mechanism and interventional effect of IGF-1 in hyperoxia-induced hippocampal cell apoptosis.Methods:Prepare full-term newborn Wistar rats to make hyperoxia-induced brain injury model,then divided into three groups ? room air control group(RA);?The hyperoxia group(85%O2);?hyperoxia+IGF-1(85%O2+rh-IGF-1)group.The hyperoxia+IGF-1 received intraperitoneal injection of IGF-1.We Observed wet and dry ratio of brain tissues on the 1st,3rd,7th and 14th day,and brain development and morphopathological changes with HE staining.Changes in the ultramicrostructure of hippocampal cells were observed by electron microscope,hippocampal cell apoptosis was observed by Tunel staining.Expression levels of TNF-??IL-1??IL-6 in the brain tissue were detected by ELISA,levels of caspase-12,JNK?p-JNK?IRE1?CHOP were detected by western blotting method.Results:1.Compared with hyperoxia group,weight of hyperoxia+IGF-1 group increased on the 3rd,7th and 14th day(P<0.05),the differences was statically significant.Compared with control group,weight of hyperoxia+IGF-1 group decreased on the 3rd,7th and 14th day(P<0.05),but there was no statistical significance compared with control group on the 1st day.2.Compared with hyperoxia group,brain wet/dry(W/D)proportion of hyperoxia+IGF-1 group decreased on the 3rd,7th and 14th day,the differences was statically significant(P<0.05).Compared with control group,brain W/D proportion of hyperoxia+IGF-1 group increased on the 3rd,7th and 14th day(P<0.05).There was no statistical significance compared with control group on the 1st day.3.HE staining results:Compared with hyperoxia group,we observed that hippocampal cell swelling was reduced and cell morphology was significantly improved in hyperoxia+IGF-1 group.In hyperoxia group,we observed that arrangement disorder of brain cell,vacuolation formation in cytoplasm,part of neuron swelling,uneven distribution,nuclear pycnosis,disappearance of nucleoli.In control group,the morphomogy of neurons in hippocampus was normal.4.Transmission electron microscopy results showed that increased number of synapses and mitochondrial,also complete construction of mitochondrial in hyperoxia+IGF-1 group.In hyperoxia group double-deck membranes of mitochondria were disintegrated,the number of mitochondria and synapses were reduced.So was the density of synapse.As hyperoxia prolonged we detected disruption of cristae and vacuolated mitochondria.In control group,we observed complete construction of mitochondrial and synaptic,also increased number of synapses.5.TUNEL staining results showed that,compared with hyperoxia group,hippocampus apoptotic index of hyperoxia+IGF-1 group was significantly decreased(p<0.05).Compared with control group,hippocampus apoptotic index of hyperoxia+IGF-1 group was significantly higher,At 3 days(5.6±1.1)7 days(5.3±0.9),14 days(4.5±0.7),the differences was statically significant(p<0.05).6.Levels of TNF-?,IL-1?,IL-6 by ELASA method:Compared with hyperoxia group,expression levels of TNF-?,IL-1?,IL-6 in hyperoxia+IGF-1 group at 1st day,3rd,7th and 14th day were significantly decreased(p<0.05).With high oxygen exposure time prolonged,levels of TNF-?,IL-1?,IL-6 gradually increased,also reached its peak on 7th day.Meanwhile,we detected these levels reduced to the 14th day.7.Western blotting results showed that,Compared with hyperoxia group.expression of caspase-12,JNK,pJNK,IRE1,CHOP in hyperoxia+IGF-1 group were lower than hyperoxia group.Compared with control group,these expressions were increased at same day,which indicate that IGF-1 can inhibit protein expression.Conclusion:1.IGF-1 can reduce hyperoxia-induced cerebral edema,inflammatory response,and inhibit hippocampus apoptosis.2.Protective effect of IGF-1 in hyperoxia-induced hippocampus injury was correlated with down-regulating of ERS relative protein expression(caspase-12,IRE1,JNK,pJNK,CHOP,which indicated that IGF-1 can protect hyperoxia-induced brain injury through ERS,JNK signaling pathway. |
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