| Objectives:Epilepsy is a common neurological disorder,which is characterized by the spontaneous recurrent neuronal discharges.Status epilepticus(SE)is the severest formation in Epilepsy for high disability rate and mortality rate.SE can lead to produce a large number of autophagosomes in the sensitive area of hippocampal neurons,demonstrating that autophagy in post-SE hippocampal neurons plays a role in the process of neuronal repair.Previous studies have shown that in a pilocarpine-induced chronic epilepsy model of SD rats,cannabinoids can effectively alleviate the epileptic behavior and play a role of antioxidant defense through neuronal autophagy.Ligands are bound to work through receptors,cannabinoid receptor 2(CB2R)is one of the endogenous cannabinoid receptors,CB2 R can alleviate the pathological process of autoimmune encephalitis by activating autophagy.Therefore,we hypothesized that CB2 R was activated in the neuronal repair process after SE in rats,and regulated neuronal death by autophagy.The purpose of this study is intended to study the dynamic expression of CB2 R in neuronal repair process in pilocarpine-induced status epilepticus rats,and explore the relationship between CB2 R and autophagy in neuronal repair process in pilocarpine-induced status epilepticus rats,and the mechanism.Methods: This study is divided into two parts: animal experiment and cellular experiment.Animal experiment:(1)A total of 180 health male Sprague-Dawley rats,weighing 43g-68.5g and aging 3 weeks,were used for this studies.Rats were divided into control group(n=16)and epilepsy group,and the epilepsy group was divided into six subgroup(according to the time of post-SE).Each experimental group was given lithium chloride-pilocarpine hydrochloride to induce status epilepticus(SE),and control animals received a comparable volume of normal saline.The animals were sacrificed at different time points after SE onset 2h,1day,3days,7days,14 days,21days,and the control animals were sacrificed at the point after SE onset 2h.The apoptosis of neurons in hippocampal tissue was observed by HE staining and TUNEL staining.The expression of CB2 R,LC3 and m TOR were observed by double-immunoinfluorescence staining,RT-PCR and Western blot.(2)A total of 80 health male Sprague-Dawley rats,weighing 43g-68.5g and aging 3 weeks,were used for this studies.Rats were divided into Vehicle group?Epilepsy group?JWH133 1ug/ul group?JWH133 3ug/ul group and JWH133+AM630 group.JWH133?JWH133+AM630 or Vehicle were treated by intracerebroventricular injection at 60 min before modeling.At 24 h after SE,the apoptosis of neurons in hippocampal tissue was observed by HE staining and TUNEL staining.The expression of LC3 and m TOR were observed by double-immunoinfluorescence staining.The expression of m TOR?p-m TOR?ULK1?p-ULK1?Beclin1?LC3?p62 were observed by western blot.Cellular experiment:(1)PC12 cells were implanted in six-well plant,and devided into four groups after trans-differentiation.Vehicle group?Epilepsy group?JWH133 3ug/ul group?JWH133+AM630 3ug/ul group.JWH133?JWH133+AM630 or Vehicle were treated at 60 min before modeling.At 24 h after modeling,the apoptosis of neurons was observed by CCK-8 method.The expression of LC3 was observed by immune--influorescence staining.The expression of m TOR?p-m TOR?ULK1?p-ULK1?Beclin1?LC3?p62 were observed by western blot.(2)PC12 cells were implanted in six-well plant,and devided into seven groups after trans-differentiation.Vehicle group,Epilepsy group,JWH133 group,Rapamycin group,Rapamycin+JWH133 group?MHY1485 group,MHY1485+JWH133 group.Rapamycin,MHY1485 or Vehicle were treated at 120 min before modeling.JWH133 or Vehicle were treated at 60 min before modeling.At 24 h after modeling,the apoptosis of neurons was observed by CCK-8 method.The expression of LC3 was observed by immuneinfluorescence staining.The expression of ULK1?p-ULK1?Beclin1?LC3?p62 were observed by western blot.Results:1.In physical condition,expression of CB2 R in hippocampal neurons was low;CB2R started to elevate in neurons of CA1?CA3?DG zones from 2 hours post-SE(p<0.05),and came to the highest at 1 day and 3 days post-SE(p<0.05),and then to decline from 7 days post-SE(p<0.05).2.In physical condition,expression of LC3 in hippocampal neurons was low;LC3 started to elevate in neurons of CA1?CA3?DG zones from 2 hours post-SE(p<0.05),and came to the highest at 1 day post-SE(p<0.05),and then to decline from 3 days post-SE(p<0.05);In physical condition,expression of m TOR in hippocampal neurons was expressive moderately;m TOR started to descend in neurons of CA1?CA3?DG zones from 2 hours post-SE(p<0.05),and came to the lowest at 1 day post-SE(p<0.05),and then to ascend from 3 days post-SE(p<0.05).3.Compared with rats in the Vehicle group,the expression of p-m TOR/m TOR?Beclin1?LC3?/LC3? were increased in the JWH133 group at 1 day post-SE(p<0.05),while p-ULK1/ ULK1?p62 ? Caspase-3 were decreased in the JWH133 group at 1 day post-SE(p<0.05),the effect was weaken when was added with AM630 and JWH133 simultaneously at 1 day post-SE(p<0.05).4.In vitro experiment,the expression of p-m TOR/m TOR?Beclin1?LC3?/LC3? were increased in the JWH133 group at 1 day post-modeling(p<0.05),while p-ULK1/ ULK1?p62 ?Caspase-3 were decreased in the JWH133 group at 1 day post-modeling(p<0.05),the effect was weaken when was added with AM630 and JWH133 simultaneously at 1 day post-modeling(p<0.05).5.The increasing-autophagy effect of JWH133 was waken by the treatment of Rapamycin which is CB2 R agonist(p<0.05),the apoptosis effect was enhanced(p<0.05);The increasing-autophagy effect of JWH133 was enhanced by the treatment of MHY1485 which is CB2 R inverse agonist(p<0.05),the apoptosis effect was wakened(p<0.05).Conclusions:1.The early stage of neutonal repair after SE,CB2 R and autophagy-related protein LC3 is highly expressed in neurons,while m TOR is lowly expressed in neurons.It's opposite.2.The autophagy after status epilepticus was inforced by activation of CB2 R,and the apoptosis was wakened accordingly.3.Effects of CB2 R on hippocampal neuronal autophagy come to ture through m TOR signaling pathway.4.CB2 R is a potent target in the early stage of neutonal repair after SE. |
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