| ObjectiveTo evaluate the role of lnc RNA NEAT1 on neuronal function recovery and neuronal apoptosis after traumatic brain injury(TBI)in mice and To explore the possible mechanism.MethodsExperiment 1: the effects NEAT1 on neurological functions after TBI in miceThe mice’s motor functions were measured by Neurologic severity score(NSS)scoring and the rotarod test on 1st day before impact and 1st,3rd,7th,14 th,21st,and 28 th days after TBI.The Morris water maze was used to measure the spatial memory and learning ability on the 20 th day after TBI when mice have finished the learning and have measured on the15 th,16th,17 th,18th and 19 th days after TBI.Experiment 2: the effects of NEAT1 on apoptosis in TBI miceAt 3 day after TBI,mice were anaesthetized by isoflurane and transcardially perfused with normal saline 50 ml and 4% paraformaldehyde30 ml.Brain samples were soaked orderly in 4% paraformaldehyde and30% sucrose solution.One-step TUNEL apoptosis detection kit(Servicebio,China)was used for the detection of cell apoptosis to determine the relationship between apoptosis and behavior.Experiment 3: expression of Pidd1,caspase2,caspase9 and caspase3 after TBIwestern blot was used to detect the expression of Pidd1,caspase2,caspase9 and caspase3 at different points(6h,1d,3d and 7d after TBI)to determine the point as the observation point for the follow-up experiments.Experiment 4: the effects of NEAT1 on the expression of Pidd1,caspase2,Cyt c(mitochondria and cytoplasm)and caspase3 in TBI miceOn the 3rd days after TBI,the expression of Pidd1 was detected by immunofluorescence through overexpression and knockdown of NEAT1.Western blot was used to detect the expressions of apoptosis-related proteins(Pidd1,caspase2,Cyt c and caspase3)in mice brain.ResultsExperiment 1: NSS showed that the scores of mice in NEAT1-over-expression group were significantly lower than those in the blank control group,while the scores of mice in the NEAT1 knockdown group significantly increased.The rotarod test showed that,compared with the blank control group,the scores of mice in NEAT1-over-expression group significantly increased,while the scores of mice in NEAT1 knockout group were significantly decreased.Through the learning on day 16-19 after TBI,mice in NEAT1-over-expression group spent less time in locating hidden platform than those in blank control,while NEAT1 knockdown mice resulted in longer time for proper location.The hidden platform was removed on 20 th day after TBI.In quadrant 3,moving distance and time both decreased in NEAT1 knockdown group.Time and moving distance obviously increase in NEAT1-over-expression group.Experiment 2: TUNEL results showed that,compared with blank control group,the number of apoptotic cells in NEAT1-over-expression group significantly reduced,and the number in NEAT1 knockdown group increased.Experiment 3: Western blot showed that the expressions of Pidd1 and caspase2 were significantly increased at 1 day after impact,while the expressions of caspase9 and caspase3 were significantly increased at 3 days after CCI.Experiment 4: Immunofluorescence and Western blot both showed that on the 3rd days after TBI,compared with the blank control group and the no-load virus group,the expressions of Pidd1,caspase2,cytoplasmic Cyt c and caspase3 in the NEAT1 overexpression group were significantly decreased,and the expressions of Cyt c in mitochondria were increased.After the knockdown of NEAT1,the expressions of Pidd1,caspase2,intracytoplasmic Cyt c and caspase3 were significantly increased,while the expressions of Cyt c in mitochondria were decreased.ConclusionsThe expression of NEAT1 can change the prognosis of traumatic brain injury by affecting neuron apoptosis.The mechanism might be as follows:NEAT1 binds to Pidd1,and then inhibits the activation of capsase2,thereby inhibiting the activation of mitochondrial apoptosis pathway,which reduce neuron apoptosis,and improve the prognosis of TBI. |
