Study On The Mechanism Of P38 Inhibitor Upregulates DJ-1 For Nerve Cells In Rats With Subarachnoid Hemorrhage

BackgroundSubarachnoid hemorrhage(SAH)is an acute cerebrovascular disease caused by sudden rupture of blood vessels in the brain or spinal cord and blood entering the subarachnoid space.Up to now,there is still no effective treatment for it.SAH has two pathological stages,namely,early brain injury(EBI)in 72 h and delayed cerebral vasospasm(DCVS).EBI plays a leading role in prognosis.Recent studies on autophagy in EBI phase suggest that mitochondrial dysfunction and abnormal autophagy after SAH lead to neuronal death,but its mechanism is not clear.Activated p38 mapk(a mitogen-activated protein kinase)is not only closely related to the occurrence and development of CVS after SAH,but also involved in EBI by regulating autophagy and mitochondrial function.SB203580 is a p38 inhibitor of pyridazolidazole,which selectively inhibits the phosphorylation of p38 by directly acting on p38 itself.In this study,PC12 cells with sympathetic neurons and SD rats were used to establish SAH model in vitro.We observed the effects of p38 inhibitors on DJ-1 protein,autophagy related proteins(Atg5,LC3-II,LC3-I and p62),mitochondrial function and neural function after SAH,and explored the effect and mechanism of SB203580 on neurons from the angle of regulating autophagy and mitochondrial function.ObjectiveTo investigate the protective effect and mechanism of that p38 inhibitors regulated DJ-1 after SAH.Methods1.PC12 cells were used to establish SAH models in vitro by oxyhemoglobin(Oxy Hb).MTT method was used to observe the best time of Oxy Hb to PC12 cells(24h).The optimal protective concentration of p38 inhibitor to PC12 cells(20u M)was selected by MTT.2.PC12 cells were randomly divided into four groups: sham group,SAH group,SAH+DMSO group and p38 inhibitor group.The changes of mitochondrial membrane potential in each group were observed by fluorescent probe JC-1.3.24 adult male SD rats were selected as the prechiasmatic cistern made SAH model,and then divided into four groups according to the random number table: sham group,SAH group,SAH+DMSO group and p38 inhibitor group.Western blot was used to detect the changes of p38,p-p38,DJ-1,Atg5,p62,LC3-I and LC3-II in each group.4.The nerve function injury of SD rats was evaluated by Garcia nerve function score.Results1.Oxy Hb had a time-dependent effect on cell growth and viability.Although Oxy Hb stimulated 12 h growth,cell viability increased significantly,but it was still at a low level compared with 24 h and 48h(P < 0.01),there was little difference in cell vitality between 24 h and 48h(P > 0.05).The activity of 24 h and 48 h cells increased significantly after SAH(P < 0.01 vs sham group).The effect of SB203580 on the viability of PC12 cells was concentration dependent: compared with the SAH group,1 M SB203580 24 h treatment decreased the cell viability(P < 0.05),48 h had no difference(P > 0.05);Similarly,10 M SB203580 treatment for 24 h,cell viability decreased(P < 0.05),48 h had no difference(P > 0.05);But when the SB203580 concentration reached 20 M,the activity of cell which managed 24 h and 48 h were significantly lower than that of SAH group(P < 0.01,P < 0.05),close to the normal level(P > 0.05 vs sham group).2.p38 inhibitor protected mitochondrial membrane potential after SAH by inhibiting p38 activity,compared with sham group,mitochondrial membrane potential decreased after SAH(P < 0.01),and mitochondrial membrane potential in group SAH+DMSO further decreased(P < 0.01),but mitochondrial membrane potential in p38 inhibitor group increased(P > 0.05 vs sham group,P < 0.01 vs SAH group).3.p38 inhibitor inhibited the expression of p-p38: compared with sham group,the expression of p38 increased gradually in SAH group,SAH+DMSO group and p38 inhibition group(P < 0.01).The expression of p-p38 in SAH group and SAH+DMSO group was gradually up-regulated(P < 0.01 vs sham group),but after treatment with p38 inhibitor,it returned to the normal level(P = 0.136 vs sham group,P < 0.01 vs SAH group,P < 0.01 vs SAH+DMSO group).p38 inhibitors up-regulated the expression of DJ-1: the expression of DJ-1 in the sham group was very low,and the expression increased after SAH(P < 0.01 vs sham group),and the expression of DJ-1 in SAH+DMSO group continued to increase(P < 0.01 vs sham group,P < 0.01 vs SAH group).After treatment with p38 inhibitor,the expression level of DJ-1 increased sharply(P < 0.01 vs sham group,P < 0.01 vs SAH group,P < 0.01 vs SAH+DMSO group).p38 inhibitor up-regulated autophagy level and inhibited abnormal autophagy: Compared with group sham,the levels of Atg5 and LC3-II/LC3-I in SAH group and SAH+DMSO group increased gradually(P < 0.01).After p38 inhibitor treated,the levels of Atg5 and LC3-II/LC3-I recovered to SAH group and DMSO group(P < 0.01 vs sham group,P < 0.01 vs SAH group,P < 0.01 vs SAH+DMSO group);The expression of p62 decreased after SAH(P < 0.01 vs sham group);The expression in SAH+DMSO group was up to sham group(P = 0.129 vs sham group,P < 0.01 vs SAH group).However,the expression of p62 was restored to SAH level after treatment with p38 inhibitor,(P < 0.01 vs sham group,P = 0.650 vs SAH group,P < 0.01 vs SAH+DMSO group).4.p38 inhibitor protected the neurologic function of rats after SAH: Garcia neurological function score decreased after SAH(P < 0.01 vs sham group).After treatment with p38 inhibitor,neurological function did not reach sham level,but it increased significantly compared with SAH group(P < 0.01),but not significantly compared with sham group(P = 0.063).ConclusionIn the EBI stage after SAH,p38 inhibitor down-regulats p-p38 activity,and then increases the expression of DJ-1,inhibites abnormal autophagy and maintains mitochondrial function,and exerts neuroprotective function finally.