| Objective: To explore the protective effect and mechanism of R18 peptide combined with sub-hypothermia on neurons after traumatic brain injury by inhibiting autophagy.Methods: 1)Primary neurons from cortical tissues of E1-19 Sprague-Dawley rats was isolated and cultured according to precious reports,and then neurons were randomly divided into two groups,which were control group and glutamic acid group.Immunofluorescence staining was used to verify the primary neurons;Cell viability was detected by MTS method.TBI model of SD rats was established by weight-drop impact under anesthesia.Neurons loss of 72 h and 12 days after TBI was explored by Nissl staining;ethology of rats was investigated by treadmill balance test from the first day after the TBI model;neuronal function was evaluated by modified neurological severity scores as well;2)Primary neuron cells were divided into 5 groups,that were control group,glutamic acid group,sub-hypothermia treatment group(32.0?0.5 ?),R18 peptide treatment group and combined treatment group.Control group represented no treatments,glut groups represented neurons injury induced by glutamic acid,R18 group,sub-hypothermia group and R18 united sub-hypothermia group represented that neurons were treated with corresponding treatments when glutamic acid was added to neurons.And peptide had four levels,which were 0.2?M,0.5?M,1?M and 2?M respectively.MTS assay was performed to detect cell viability of neurons after 72h;intracellular calcium influx was monitored using Fura-2 AM methods to determine the relative change in intracellular calcium before and after treatments and glutamic acid exposure;the expression of Beclin-1,LC3,caspase-3 and Bcl-2 were determined by western blot and RT-q PCR;3)SD rats were divided into 5 groups,8 rats for each group,they were control group,TBI model group,sub-hypothermia treatment group,R18 peptide treatment group and combined treatment group.Control group represented non-TBI normal rats,TBI grouprepresented TBI model rats,R18 peptide group,sub-hypothermia group and combined group represented that rats were treated with corresponding treatment after TBI;subhypothermia treatment was performed by using ice packs to keep the body temperature of rats at hypothermia,R18 peptide treatment(300nmol/kg)was administered intravenously through the right internal jugular vein using infusion pump 30 min post-impact.72 hours later,neuronal function was evaluated by modified neurological severity scores(m NSS);Water content of brain was determined by the ratio of dry/wet method.HE staining and Bielschowsky's silver staining of brain tissues: 72 h after treatment,3 rats of control group and TBI group were randomly chosen,then the brain tissues of chosen rats were extracted to do HE staining to evaluate hemorrhagic state and Bielschowsky's silver staining to evaluate axonal injury.Expression of Beclin-1 and LC3 in brain tissues was explored by Immunohistochemistry(IHC);Hippocampal tissues were isolated from rats,and then apoptosis positive cells of hippocampal tissues were investigated by Tunnel method.Results: 1)Immunoflurorescence displayed that isolated cells were Tubulin positive,which meant the isolated cells were neurons.Cell viability was obviously decreased after glutamic acid induction(P<0.01),which meants glutamic acid induced neuronal injury.Nissl staining showed that neurons of 72 h post-TBI was reduced(5542/mm3 vs 4124/mm3,P < 0.01);neurons of 12 d post-TBI recovered(5613mm3 vs 5109mm3,P < 0.01).Ethology examination demonstrated that compared with control group,number of slides were incrased in TBI group since the first day post-TBI;compared with control rats,m NSS scores was the highest after 3d,and returned back since 3d;2)Cell viability of neurons was decreased after glutamic acid exposure(P<0.01);compared with glut group,sub-hypothermia and R18 peptide all elevated cell viability of neurons in a dose-dependent manner(P<0.05),and combined treatment also increased cell viability of neurons in as dose-dependent manner(P<0.05).Fura-2 AM real-time monitoring showed that compared with control group,intracellular calcium influx was obviously elevated after glutamic acid exposure,R18 peptide treatment,sub-hypothermia treatment and combined treatment all decreased intracellular calcium influx,and the inhibitory degrees was combined treatment(29)R18 peptide treatment(29)sub-hypothermia treatment,the result of microscope was the same as real-time monitoring.The expression of autophagy-related protein,Beclin-1 and LC3,were increased after glutamic acid exposure,the expression of caspase-3 was elevated,and Bcl-2 was decreased;R18 peptide treatment,sub-hypothermia treatment and combined treatment all reduced the expression of Beclin-1,LC3 and caspase-3 and increased the expression of Bcl-2,besides,the effect of combined treatment was better than single treatment;3)TBI induced neuronal dysfunction and increased m NSS score(P<0.01),m NSS score was decreased after sub-hypothermia treatment,R18 peptide treatmen and combined treatment(P<0.05),and the score of combined treatments was the lowest.Hemorrhage and disorder of cell arrangement could be observed after TBI;in the absence of injury,axons within the corpus callosum appeared smooth and well-organized,with oligodendrocyte nuclei arranged parallel to axons,following TBI,the architecture of callosal axons showed disorganization and undulation,with loss of the normal longitudinal orientation of oligodendrocyte nuclei.Compared with control group,water content of brain was obviously elevated after TBI(86.20?1.21% vs 71.12?1.32%,P<0.05),compared with TBI group,the water content of brain tissue from sub-hypothermia treatment(80.34?0.91%),R18 peptide treatment(76.23?0.87%)and combined treatment(73.02?1.09%)were decreased(P<0.05).IHC showed that the expression of Beclin-1 and LC3 in brain tissues was increased after TBI,compared with TBI group,sub-hypothermia treatment,R18 peptide treatment and combined treatment all decreased the expression of Beclin-1 and LC3,and the effect of combined treatment was most obvious.Tunnel assay showed apoptosis-positive cell was obviously increased after TBI(P<0.01),compared with TBI group,sub-hypothermia treatment,R18 peptide treatment and combined treatment all inhibited cell apoptosis of hippocampal tissues(P<0.05),and inhibitory effect of combined treatment was better than single treatment(P<0.01).Conclusion: Combination sub-hypothermia with R18 peptide could inhibit glutamic acid-induced damages of neuron cells via elevating cell viability,reduced intracellular calcium concentration and inhibits Cell autophagy and apoptosis of neurons,and the inhibitory effect of combined treatment is stronger than single treatment.Combination sub-hypothermia with R18 peptide could protect TBI rats from brain damage via promoting recovery of neuronal function,decrease the degree of edema,inhibit cell autophagy of neurons,and the combined treatment was better than single treatment. |
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