The Effects Of Activation Of PI3K/AKT/Nur77/RXRα Pathway On Neuronal Apoptosis After Spinal Cord Injury

Objectives:1.In vivo experiments,we studied the effects of Hypoxia Preconditioning（HPC）activating the PI3K/AKT/Nur77/RXRαpathway on neuronal apoptosis in female SD rats with spinal cord injury.2.In vitro experiments,the H₂O₂-induced PC12 cell injury model was used to explore the effects and related mechanisms of Insulin-like Growth Factor-1（IGF-1）preconditioning activating the PI3K/AKT/Nur77/RXRαpathway on apoptosis.3.The combined results of vivo experiments and vitro experiments laid a solid theoretical foundation for the further researches of detecting changes of some items of the blood and cerebrospinal fluid of rats with spinal cord injuries and injured rats pretreated by hypoxia.Methods:1.Hypoxia preconditioning models were first established,with those rats held in a sealed box with oxygen concentration of 8%（about 8125 meters above the sea level）for2 hours per day and for 7 consecutive days.After that,spinal cord injury models were made by using a specific instrument.Then,their hindlimb motion functions were evaluated by BBB scale in each group,and HE staining and immunofluorescence staining were used to detect spinal cords and related neuronal apoptosis in each group respectively at 1d,3d,7d,14d,21d,and 28d after SCI.The expressions of related factors at protein level were observed using WB method.2.Well-differentiated PC12 cells were used to establish the model of damage.Different concentrations of H₂O₂ were used to damage those cells as an injury factor with those cells being treated for different durations.After that,cellular viability of each group was detected at 3h,6h,12h,and 24h after injury.The same method was used to determine the dose and durations of action of IGF-1,one agonist of PI3K,and LY294002,one depressor of PI3K.The administration concentrations of IGF-1 were 25μg/L,50μg/L and100μg/L,and the cell viability was measured at 12h and 24h after pretreatment.LY294002was administered at concentrations of 10μM,25μM and 50μM,and cell viability was measured at the same points with IGF-1 after preconditioning.Then,the concentrations and durations of action of H₂O₂,IGF-1 and LY294002 were determined according to cell viability.After the model was successfully established,the viability of cells in each group were measured and compared by Cell Counting Kit-8（CCK-8）,and Giemsa staining was performed to observe morphological changes of those PC12 cells.The apoptotic changes of each group were observed by Hoechst33258 vital cell staining.Then Western-Blot method was used to measure the expressions of related proteins on molecular level and to explore the effects of IGF-1 pretreatment on apoptosis after PC12 cells were injured.Results:1.The conditions of functional recovery of rats pretreated by hypoxia are better than that of rats in SCI group.The BBB scores in SCI group and HPC+SCI group were similar,and there was no significant difference between the two groups at 1d and 3d after operation.The motion ability of rats in HPC+SCI group promoted significantly at 7d after the operation,which was significantly improved compared with 1d and 3d after operation.And their motional abilities remained at high levels at 14d,21d,and 28d,approaching the level of rats in Sham group.Since the change（increasing）of BBB scoring in the postoperative period from 3d to 7d most significantly increased compared with the results in other time periods,we speculated that hypoxia pretreatment had the greatest effects on rats at 7d after operation.So,we used the rats at 7d after the operation for subsequent experiments.Compared with the rats in SCI group,the size of injured area of spinal cords in the HPC+SCI group was smaller,and the extent of damage was milder,with more surviving motor neurons.After pretreated by hypoxia,the changes on apoptosis of neurons were less obvious than those cells just undergone injury,and the expressions of apoptosis-related proteins,including Bax,Caspase-3,Nur77 and RXRα,were also less.The expression of hypoxia inducible factor-1αat protein level increased significantly in rats with hypoxia preconditioning compared with that in rats in SCI group.2.IGF-1,like hypoxic pretreatment,can also affect the PI3K/AKT/Nur77/RXRαpathway to produce corresponding effects on the progress of apoptosis.In this study,it was discovered that cell viability of PC12 cell pretreated by IGF-1 was higher than that in H₂O₂-induced group.And cells keep morphological integrity in IGF-1 pretreated group,and the influences of apoptotic death were also less obvious.Then,the expressions of apoptosis-related proteins,like Caspase-3,Nur77 and RXRα,in PC12 cells decreased and some factors,including PI3K and AKT,improving the activity of PC12 cells enormously increased after those cells were pretreated with IGF-1.Conclusions:1.Neuronal apoptosis after spinal cord injury or nerve cell injury is significant.2.The activation of PI3K/AKT/Nur77/RXRαpathway can inhibit apoptosis and play a neuroprotective role.3.Hypoxia and/or IGF-1 pretreatment can activate the PI3K/AKT/Nur77/RXRα pathway to exert neuroprotective effects.