The Role And Mechanism Of Epac2/p38MAPK Signaling Pathway In Traumatic Brain Injury

Objective Traumatic brain injury(TBI)is a major cause of mortality and disability world wide.TBI-induced neuronal apoptosis is one of the main contributors to the secondary injury process.The aim of this study is to investigate the involvement of Exchange protein directly activated by cAMP 2(Epac2)on TBI.Methods 1.Free-fall impact method was used to make rat model of traumatic brain injury.we collected the rat's brains at 6 hrs,12 hrs,24 hrs,48 hrs and 72 hrs after TBI,then we examined the protein levels and the expression and location of Epac2 in the cortex tissue of each group by western blot and immunofluorescence.2.Rats were randomly divided into sham group,TBI group,vehicle group,and Epac2 inhibitor(ESI-05)group.The vehicle group and the ESI-05 group were respectively injected into lateral ventricles with 1% DMSO and ESI-05(10 mg/kg,dissolved in 1% DMSO)30 min before TBI,and the neurobehavioral scores of each group were measured 12 hours after injury.Then collect brain tissue from each group.3.The expression levels of P38,p-P38 and caspase-3 in brain tissue of each group were detected by Western Blot.The expression level and localization of caspase-3 in brain tissue of each group were detected by immunofluorescence staining.FJB and Tunel were used to detect the neuronal apoptosis of rats in each group.Wet and dry weights were used to measure the brain tissue water content in each group.4.Finally,we used statistical software for statistical analysis of all experimental data.Result 1.The expression level of Epac2 was significantly increased at 12 hours after TBI and was co-localized with neurons.2.The neurological behavior scores of TBI group were significantly higher than the sham group(higher neurological score indicates more severe neurological impairment),indicating that the rats of TBI group had a significant neurological function deficits compared with sham group.ESI-05 treatment could significantly decrease the neurological behavior scores.These findings suggest that Epac2 inhibition improved neurological behavioral impairment following TBI.Decrease of Epac2 expression level contributed to alleviate neurological deficits after TBI.3.We found that the brain water content was significantly increased in TBI model rats,and ESI-05 treatment could significantly reduce it while vehicle group couldn't.This shows that inhibition of Epac2 helps to improve brain edema after TBI.4.Both of the western blot and immunofluorescence staining results showed that the expression of caspase-3 was significantly decreased in the ESI-05 treated group.Few FJB-positive and TUNEL-positive apoptotic cells was found in the sham group.And the number of apoptotic cells was dramatically higher in TBI group.The FJB and TUNEL staining results also demonstrated that inhibition of Epac2 could significantly prevent the increase of cell death.The expression of P38 has no significance between each experimental groups.But the expression of p-P38 was markedly increased after TBI and treatment with ESI-05 could reverse this change.This suggests that inhibition of Epac2 helps reduce neuronal death.Conclusions 1.Western blot and immunofluorescence staining results have shown that the expression of Epac2 was dramatically increased at 12 h after TBI.The brain water content measurement showed that reduction of Epac2 alleviated encephaledema in TBI model.The neurological behavioral test demonstrated that decrease of Epac2 improved neurobehavioral outcome after TBI.The immunohistochemistry,FJB,TUNEL,western blot were used to show that inhibition of Epac2 significantly attenuated the neuronal cell death after TBI.Phosphorylation of P38 may be involved in this process.2 Inhibition of Epac2 may play a neuroprotective role in TBI management through attenuating neural cell death,alleviating brain edema and improving neurological deficits,implying that Epac2 could be a new targets for treatment of secondary neuronal injury after TBI.