LncRNA-Neat1 Improves Prognosis Of TBI Through PIDD1-caspase-2 Pathway

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PROTECTIVE EFFECT AND MECHANISM OF LONG CHAIN NON-CODING RNA NEAT1 ON TRAUMATIC BRAIN INJURY IN MICEObjectiveTo observe and evaluate the effect of Neat1 on neural function recovery and neuronal apoptosis of mice after traumatic brain injury(TBI);To further explore the related molecules mechanism of Neat1 in the apoptosis pathway of TBI mice.Methods60 healthy C57BL/6J male mice(age:10-12months;Body weight :18-20g)were the objects of this experiment.The selected mice were randomly divided into four groups: normal(sham operation)group,control group,Neat1 gene over-expression group,Nea1 gene down-regulation group,each group of 15 subjects.Neat1 gene over-expression or interference(down-regulation)adenovirus was injected into the lateral ventricle,the control group was injected with the same amount of isosmotic saline.One week after transfection,control cortical impact(CCI)was constructed to simulate moderate TBI mice.After successful modeling,following experiments were carried out:Experiment 1: To evaluate the differences of motor,learning and memory function in each group after CCIThe Neural Severity Score(NSS),the scratching line test and the Morris water maze test were used to evaluate the neural function injury,motor function,learning and memory ability of mice,respectively.Experiment 2: The changes of PIDD1,caspase-2 and cleaved caspase-3 expression in CCI mice at different time points were detectedThe total brain tissue protein around the wound foci was collected for Western-Blot detection at six hours,D1(day1),D3 and D7,after CCI respectively.The changes of PIDD1,caspase-2 and cleaved caspase-3expression at different time points were analyzed,and the most significant time point was selected as the sampling time of the procedure experiment.Experiment 3: Immunofluorescence semi-quantitative assay to detect the differences of PIDD1 between the control group and the Neat1over-expression or Neat1 down-regulation groupThe difference between the PIDD1 of Neat1 over-expression group,down-regulation group,normal group and control group was detected by immunofluorescence semi-quantitative assay according to the time point determined in experiment 2.Experiment 4: Western-Blot to detect differences of PIDD1,caspase-2,cleaved caspase-3 and cytochrome C between the Neat1over-expression group or down-regulated group and the control groupThe differences of PIDD1,caspase-2,cleaved caspase-3 and cytoplasmic cytochrome C in the Neat1 over-expression group and down-regulated groups were detected according to the time points determined by experiment 2;The expression of mitochondrial cytochrome C in Neat1over-expression group and down-regulation group was detected by Western-Blot experiment according to the time point determined by experiment 2.ResultsExperiment 1: compared with the control group,NSS in the Neat1 gene over-expression group was significantly lower,and the score of Neat1 gene down-regulation group was significantly higher,indicating that Neat1 was beneficial to improve the neural function of mice.The scores of grabbing line experiment in Neat1 over-expression group was significantly higher than the control group,while the scores in the down-regulation group were significantly lower than those in the control group,indicating that Neat1 was helpful to improve the line-catching ability of mice.The time of finding underwater platform in the Neat1 gene over-expression group was significantly shorter than that in the control group in the Morris water maze experiment on 15-19 days after CCI,while that in the Neat1 gene down-regulation group was significantly longer.Experiment 2: Western-Blot experiment results show that the expression of PIDD1 and caspase-2 began to increase significantly on the first day after modeling,and remained at a higher level on the third and seventh days after modeling.The expression of cleaved caspase-3 began to increase significantly on the third day after CCI.The third day after modeling was selected as the sampling time point of the subsequent experiment.Experiment 3: The results of immunofluorescence semi-quantitative experiment showed that the PIDD1 expression in the Neat1 gene over-expression group was significantly lower than that in the control group,while that in the Neat1 down-regulation group was significantly higher than that in the control group.Experiment 4: Western-Blot results showed that the cleaved caspase-3 and cytosolic cytochrome C in the Neat1 over-expression group were significantly lower than those in the control group(p<0.05),while the expression of cytochrome C in mitochondria was significantly higher than that in the control group(p < 0.05).The mice in the Neat1 gene down-regulation group had the opposite results(p<0.05).ConclusionsNeat1 may change the prognosis of traumatic brain injury mice by inhibiting the expression of PIDD1 and capsase-2 and the activation of mitochondrial apoptosis pathway.This is consistent with the previous research Neat1 which may inhibit the apoptosis pathway by combining with PIDD1.PART ? NEAT1 DECREASES NEURONAL APOPTOSIS AFTER OGD BY INFLUENCING PIDD1-CASPASE-2PATHWAYObjective:By culturing primary neurons in vitro and establishing an oxygen glucose deprivation(OGD)model to simulate traumatic brain injury in mice,the effects of Neat1 on neuronal apoptosis and the related mechanism of mitochondrial apoptosis pathways were explored.Methods:The primary neurons of mouse cortex were cultured in vitro to establish a model of OGD to simulate traumatic brain injury.Neat1 gene over-expression and interference adenovirus were constructed and to infect the primary neurons respectively.Four experimental groups were established,namely the normal group,the control group,the Neat1 gene over-expression group and the Neat1 gene down-regulation group.Carry out the following experiments respectively:Experiment 1: Establish an oxygen and glucose deprivation model and compare the changes of Neat1's expression levels in neuron for different modeling time:Transfection of adenovirus was carried out within 24 hours after neuron plating.After successful transfection,OGD was performed for 2 hours,3hours,4 hours,5 hours and 6 hours respectively.After successful modeling,the total RNA of neurons in the normal group and the control group were extracted at each time point and RT-PCR,the time point where Neat1 changed the most was selected as the modeling time for subsequent experiments.Experiment 2: Measurement of neuronal cell viabilityAfter the OGD model was established,the cell counting kit 8(CCK-8)was used to measure the changes in the cell viability of the neurons in each hole in each group(6 holes in each group),and the OD value of the neurons in each hole was measured by a microplate reader at a wavelength of 540 nm and take the average value for comparison.Experiment 3: Determination of neuronal cell apoptosis rateAfter the OGD model was successfully established,the apoptosis rate of each group of neurons was measured by Terminal d UTP nick-end labeling(TUNEL)method.Experiment 4: Western-Blot determination of apoptosis-related proteins:After the OGD model was established,the total protein of each group of neurons was extracted,and the expression levels of neuronal apoptosis-related proteins PIDD1,caspase-2,cytoplasmic cytochrome C and cleaved caspase-3 were measured by Western-Blot method.Mitochondrial protein was extracted,and Western-Blot was used to determine the difference in cytochrome C protein expression in the mitochondria of each group of neurons.Results:Experiment 1: Determine the changes of Neat1 expression after different time of oxygen and glucose deprivation in the control group:In this experiment,the total RNA of neurons in the control group and the normal group was extracted,and RT-PCR was performed.It was found that the Neat1 expression of the control group was significantly higher than that of the normal group after 2 hours and 3 hours of oxygen glucose deprivation,especially 3 hours of OGD.So 3 hours after OGD was selected as modeling time for subsequent experiments.Experiment 2: Measurement of neuronal cell viabilityAmong the four groups,the Neat1 gene down-regulation group had the lowest optical density(OD),indicating that the Neat1 gene down-regulation group had the lowest neuronal viability.The OD value of the Neat1 gene over-expression group was significantly higher than that of the control group(p<0.05),but lower than that of the normal group.The neuron viability of the Neat1 gene down-regulation group was significantly lower than that of the control group(p<0.05),but higher compared with the normal group.Experiment 3: Determination of neuronal cell apoptosis rateThe positive rate of TUNEL staining in control group was higher than that in the normal group(p<0.05),while the positive rate of TUNEL staining in the Neat1 gene over-expression group was significantly lower than that in the control group(p<0.05),and the positive rate of TUNEL staining in the Neat1 gene down-regulation group was significantly higher than that in the control group(p<0.05).Experiment 4: Western-Blot determination of apoptosis-related proteinsThe results of Western-Blot experiments showed that the expression levels of PIDD1,caspase-2,and cleaved caspase-3 in the Neat1 gene over-expression group were significantly lower than those of the control group(P<0.05).The expression levels of PIDD1,caspase-2 and cleaved caspase-3 in Neat1 down-regulation group were significantly higher than those in the control group(P<0.05).In addition,the cytoplasmic cytochrome C of the control group was significantly higher than that of the normal group(P<0.05).The cytosolic cytochrome C of the Neat1 gene over-expression group was lower than that of the control group(P<0.05).The cytosolic cytochrome C level of the Neat1 down-regulation group was significantly higher than that of the control group(P<0.05).Conclusion:Neat1 may inhibit neuronal apoptosis after oxygen and glucose deprivation by affecting PIDD1-caspase-2-cytochrome C pathway,which is consistent with the results of animal experiments.