An Antagonistic Effect And Mechanism Study Of Galanin Receptor-2 Agonist AR-M1896 On Epilepsy Patients And Rats

Objective: This study is aimed to investigate the relationship between Galanin,inflammatory cytokines,and the acute epileptic seizures and to study the effects of Galanin receptor-2(GalR2)agonist on epileptic seizure in the aspects of ethology,hippocampal CA3 region histopathology and the inflammatory cytokines levels in the brain.Therefore,this study wants to clarify the antagonistic effect of GalR2 agonist on epilepsy and its possible mechanism.Research objects and methods:Clinical investigation: 24 epilepsy patients and 16 healthy subjects were randomly enrolled.The incidences of epileptic seizures occurred in 24 hours when blood samples collected.The epilepsy patients were divided into the non-status epilepticus(NSE)group or the status epilepticus(SE)group according to whether they were status epilepticus or not.Meanwhile,16 healthy subjects worked as the control group.Age,sex,height and weight were collected from the clinical investigation.Besides,serum levels of Galanin,inflammatory cytokines like TNF-?,IL-1?,IL-6 were measured by enzyme-linked immunoassay(ELISA).Animal experiments: 60 healthy male Wistar rats(220~250g)were randomly assigned to five groups(each group n=12): the control group: normal saline(2?l)was injected in hippocampus,30 minutes later,normal saline(6ml/kg)was injected intraperitoneally;the PTZ model group: normal saline(2?l)was injected in hippocampus,30 minutes later,pentylenetetrazol(PTZ,6ml/kg)was injected intraperitoneally;the low dose of AR-M1896 group: AR-M1896(1nmol/2?l)was injected in hippocampus,30 minutes later,pentylenetetrazol(6ml/kg)was injected intraperitoneally;the medium dose of AR-M1896 group: AR-M1896(2nmol/2?l)was injected in hippocampus,30 minutes later,pentylenetetrazol(6ml/kg)was injected intraperitoneally;the high dose of AR-M1896 group: AR-M1896(5nmol/2?l)was injected in hippocampus,30 minutes later,pentylenetetrazol(6ml/kg)was injected intraperitoneally.Intrahippocampal cannulas were placed ahead of the experiments.Acute epileptic model was established by intraperitoneal injection of pentylenetetrazol,30 minutes after intrahippocampal injection.The severity and latency of seizures were observed and recorded.Nissl staining was conducted and neurons in hippocampus CA3 region were observed microscopically.The whole brain levels of inflammatory cytokines like TNF-?,IL-1? and IL-6 were measured by ELISA.Result:1.Patients with acute epileptic seizures had significantly higher serum levels of GAL,TNF-?,IL-1?,and IL-6 compared with the control group(P<0.01).However,there's no difference between the non-status epilepticus group and the status epilepticus group(P(29)0.05).2.Compared with the PTZ model group,the three different doses of AR-M1986 did not affect the latency of myoclonic seizure of the PTZ-induced epilepsy rats(P(29)0.05).3.Compared with the PTZ model group,the medium dose of AR-M1986 reduced the severity of the PTZ-induced epilepsy(P<0.05);the high dose of AR-M1986 significantly reduced the severity of PTZ-induced epilepsy(P<0.01)and significantly reduced the rate of generalized tonic-clonic seizure(P<0.01).The rates of generalized tonic-clonic seizure in the low dose and medium dose of AR-M1986 groups were lower than that in the PTZ model group,but there's no statistical difference(P(29)0.05).4.Compared with the control group,the brain levels of TNF-?,IL-1? and IL-6 were significantly increased in the PTZ model group(P<0.01).5.Compared with the PTZ model group,the low dose and medium dose of ARM1986 had not affected the levels of TNF-? and IL-1? in the brain of epileptic seizure rats(P>0.05),however,the high dose of AR-M1986 significantly inhibited the levels of TNF-? and IL-1?(P<0.01).The three different doses of AR-M1986 significantly reduced the level of IL-6 in the brain of epileptic seizure rats(P<0.01),and were shown dose dependence.6.Nissl staining: In the control group,the neurons in the hippocampus CA3 region were neatly arranged and layered tightly.The neurons were round,the cytoplasm was abundant with Nissl bodies.The nuclei were round or oval and clearly visible.In the PTZ model group,dark neurons were formed.The neurons were atrophic and scattered.The cell morphology was changed: the neurons were conical or polygon,the cytoplasm was deeply stained,and the nuclei were pyknotic;the number of neurons in CA3 region had no difference(P > 0.05),while the proportion of dark neurons increased significantly(P < 0.01).Compared with the PTZ model group,the severity of morphological abnormality in the hippocampal CA3 region in the three different doses of AR-M1986 groups was significantly alleviated and the proportion of dark neurons was significantly decreased(P < 0.01).The dose of the AR-M1986 was negatively correlated with the proportion of dark neurons(P < 0.01),but there was no significant effect on the number of neurons in the hippocampal CA3 region(P > 0.05).Conclusions:1.Epilepsy induced inflammatory response and epileptic seizure was related to galanin.2.Pentylenetetrazol(PTZ)was able to induce epileptic seizures effectively,and GalR2 agonist AR-M1896 had an antiepileptic effect and had a neuroprotective effect on the neurons in the hippocampus CA3 region.3.GalR2 agonist AR-M1896 was able to suppress brain inflammatory response on PTZ-induced epileptic seizures in rats.4.The antagonistic Effect of GalR2 agonist AR-M1896 had on epilepsy may associated with suppressing brain inflammatory response.