| Status epilepticus(SE)is a medical and neurological emergency.Intellectual disability,permanent neurological dysfunction and epilepsy are common sequelae of SE.The exact pathogenesis of SE is unclear.Changes in the function and expression of ion channels may be the cause of epilepsy.Recently studies have shown that the hyperpolarized activated cyclic nucleotidegated cation(HCN)channels are a potential ion channel associated with epilepsy generation.To date,the role of the HCN channels in status epilepticus(SE)is not fully understood.This study aims to explore three questions: 1.Expression patterns of HCN channels and mGluR1 in the hippocampus of SE rats.2.Clarify the regulatory relationship between mGluR1 and HCN1.3.The effect of mGluR1 inhibited HCN1 on the excitability of pentatetrazone-induced SE rats.4.Activate the intracellular signaling mechanism of mGluR1 inhibition of HCN1.Part Ⅰ: Expression of HCN channels and mGluR1 in hippocampus of SE ratObjective To verify the expression changes of HCN channels subtype and mGluR1 after SE,and explore whether there was a correlation between HCN channels and mGluR1.Methods Healthy adult male SD rats were randomly divided into control group and SE group.The SE group was divided into 5 subgroups: SE 30 min,SE 60 min,SE 1d,SE 2d and SE 7d.There were 16 rats in each group.The SE rat model was established by intraperitoneal injection of PTZ.To observe the behavior of epileptic seizures in rats.After SE,the rats were deeply anesthetized at different time points.Nissl staining,Neu N immunohistochemical staining,Neu N and Bax protein were used to evaluate the damage of hippocampal neurons after SE.The expression of HCN channels protein was detected by immunofluorescence staining and WB.The expression of EAAT2 and mGluR1 proteins were detected as well.The colocation of mGluR1 and HCN channels protein was detected by immunofluorescence double staining.Results1.Behavioral results: No SE occurred in the control group.After inducing SE by PTZ,all the rats in the 5 subgroups of SE group had Racine grade 4 or more,the SE duration was about within 30-40 minutes,and the mortality rate was 13.75%(11/80).2.HCN channels expression: total HCN1 channel protein expression was down-regulated at SE 1d(P < 0.001).The down-regulation of HCN1 surface protein earlier than that of total protein,and it was down-regulated at SE 60 min(P < 0.01),lowest at SE 1d.The HCN2 protein did not change after SE(P >0.001).3.Evaluation of hippocampal neuron injury in rats :The number of Neu N positive neurons in hippocampal CA1 region of SE group did not change(P >0.05),and there was no difference in Neu N and apoptotic protein Bax protein expression among all groups(P > 0.05).Nissl staining: Dark neurons appeared at SE 60 min(P < 0.05),and the proportion of dark neurons was largest at SE 1d(P < 0.05).4.EAAT2 and mGluR1 protein expressions: EAAT2 and mGluR1 protein were upregulated at 60 min after SE(P < 0.05),and the upregulation was most obvious at day 1 of SE(P < 0.05).6.Study on the relationship between mGluR1 and HCN1 :Immunofluorescence double stainning showed co-localization of mGluR1 and HCN1 in hippocampal pyramidal neurons.Conclusion HCN1 protein down-regulation after SE is not caused by neuronal death,which may be related to the up-regulation of mGluR1 protein activated by glutamate system.The down-regulation of HCN1 protein after SE was not caused by the loss of neuron;the changes of EAAT2 and mGluR1 were basically consistent,and mGluR1 and HCN1 co-located in hippocampal pyramidal neurons.The downregulation of HCN1 protein may be related to the upregulation of mGluR1 protein.Part Ⅱ: Effect and mechanism of mGluR1 on HCN1 channel protein expressionObjective Agonists and antagonists of HCN channel and mGluR1 were injected into the lateral ventricle to clarify the regulatory relationship between HCN channel and mGluR1.The experiment was divided into two sections.Ⅰ: Clarify the regulatory relationship between mGluR1 and HCN1Method A total of 80 rats were assigned into 3 groups:1)sham,i.c.v.injection of NS(10ul,n=16),2)DHPG(n=16),3)LY 367385(n=16),4)zd72885(n=16),5)8Br-c AMP(n=16).DHPG(a single dose of 500 nmol/10 ul),LY367385(a single dose of 320 nmol/10 ul),zd72885(a single dose of 5 μg/10ul)and 8Br-c AMP(a single dose of 100 μg /10 ul)were dissolved in NS.About5 days after recovery,the occurrence of spontaneous seizures was observed.Thereafter,rats were deeply anesthetized to collect brain samples.Results1.Behavioral observation: There were no spontaneous SE occurred in all rats after i.c.v injection.2.The regulatory relationship between mGluR1 and HCN1: Western blot results showed that activation of mGluR1(DHPH group)inhibited HCN1expression(P < 0.05),but inhibition of mGluR1(LY367385 group)had no effect on HCN1 expression(P > 0.05).Activation of HCN1(8Br-c AMP group)and inhibition of HCN1(ZD7288 group)had no effect on mGluR1 expression(P > 0.05).The immunofluorescence results of HCN1 and mGluR1 were consistent with western blot results: the mean fluorescence intensity of mGluR1 in DHPH group was increased(P < 0.05),while the mean fluorescence intensity of HCN1 was decreased(P < 0.05).The mean fluorescence intensity of mGluR1 in LY367385 group decreased(P < 0.05),but mean fluorescence intensity of HCN1 did not change significantly(P > 0.05).The mean fluorescence intensity of HCN1 increased(P < 0.05)or decreased(P < 0.05)when HCN1 was activated or inhibited,but the mean fluorescence intensity of mGluR1 remained unchanged(P > 0.05).Conclusion of section I: There was a unidirectional regulatory relationship between mGluR1 and HCN1,that was,mGluR1 activation leaded to HCN1 inhibition,but not HCN1 inhibition,and the expression of mGluR1 protein was upregulated.Ⅱ: Effects of mGluR1-induced HCN1 inhibition on excitability of PTZinduced epileptic state ratsMethod 68 rats were divided into 4 groups: Negative control group(i.c.v.injection of NS followed by i.p.injection of NS,n=8),NS+PTZ group(i.c.v.injection of NS followed by i.p.injection of PTZ,n=20),DHPG+PTZ group(i.c.v.injection of DHPG followed by i.p.injection of PTZ,n=20),LY367385+PTZ group(i.c.v.injection of LY367385 followed by i.p.injection of PTZ,n=20).All rats were i.p.injection of PTZ to induce SE 5 days after the i.c.v.injection.24 hours after SE,brain tissue was collected under deep anesthesia.Results1.Behavioral observation: Behavioral observation: Compared with NS+PTZ group,epilepsy score and mortality were increased in DHPG+PTZ group(P < 0.05),the mortality was 40%,and SE appeared in all rats.In LY367385+PTZ group,the jerks latency of epileptic seizure was significantly prolonged(P < 0.001),the epilepsy score was significantly decreased(P < 0.01),no animal death occurred,and the number of animals with SE was 9 animals(9/20).2.HCN1 and mGluR1 protein: Compared with NS+PTZ group,mGluR1 protein in DHPG+PTZ group was upregulated(P < 0.05),while total HCN1 protein was downregulated(P < 0.01).In LY367385+PTZ group,mGluR1 protein was downregulated(P < 0.05),while total HCN1 protein was significantly upregulated(P < 0.01).HCN1 surface protein was further detected.Compared with NS+PTZ group,HCN1 surface protein was downregulated in DHPY+PTZ group(P < 0.05),while HCN1 surface protein was significantly upregulated in LY367385+PTZ group(p < 0.001;Compared with NS group,P<0.001).There was no difference in S/T ratio between NS+PTZ group and DHPG+PTZ group(P > 0.05),but the S/T ratio of LY367385+PTZ group was significantly increased(compared with NS+PTZ group,P <0.001;Compared with NS group,P < 0.01)3.c AMP concentration: Compared with NS group,c AMP concentration in NS+PTZ group was significantly increased(P <0.001).Compared with NS+PTZ group,c AMP concentration in DHPG+PTZ group was increased(P < 0.001)but decreased in LY367385+PTZ group(P < 0.001).However,c AMP concentration in LY367385+PTZ group was still higher than NS group(P < 0.01).4.PKA protein: Compared with NS group,PKA protein in NS+PTZ group was significantly upregulated(P <0.001).Compared with NS+PTZ group,PKA protein in DHPG+PTZ group was upregulated(P < 0.001)but downregulated in LY367385+PTZ group(P < 0.001).However,Compared with NS group,PKA protein in LY367385+PTZ group return to normal control level(P > 0.05).Conclusions of part II: 1.Activation of mGluR1 inhibited the expression of HCN1 and increased the severity and mortality of PTZ-induced SE.2.Activation of mGluR1 inhibited of HCN1 protein after SE may be carried out through the c AMP-PKA signaling pathway.Part Ⅲ: Intracellular signaling mechanism of HCN1 inhibition induced by mGluR1 activation.Objective To investigate the mechanism of c AMP-PKA pathway and related TRIP8 b auxiliary protein in mGluR1 regulation of HCN1 channel in SE.Methods: 64 rats were randomly divided into 4 groups: The rats in negative control group(NS group,n=16),H89 group,NS+PTZ group(n=16),H89+PTZ group(n=16).NS+PTZ group or H89+PTZ group were i.p.injection with NS(the same volume as H89)or H89(0.2mg/100g)30 minutes before PTZ treatment.24 hours later,brain tissue was collected under deep anesthesia.Results1.Behavioral observation: Compared with NS+PTZ group,SE rats in H89+PTZ group had longer jerks latency(P < 0.001),and racine grade decreased significantly(P < 0.05).The number of SE rats was 9(9/16),and no rats died.2.HCN1、mGluR1 and PKA protein: Compared with NS+PTZ group,mGluR1 expression was downregulated(P<0.05),HCN1 total protein(P<0.01)and HCN1 surface protein(P<0.001)were upregulated in H89+PTZ group,and PKA protein was downregulated(P<0.05).Compared with NS group,the total protein of HCN1,mGluR1 and PKA did not change in H89 group(P>0.05),but the surface protein of HCN1 was upregulated in H89 group(P<0.001).3.TRIP8b(1a-4)and TRIP8b(1b-2)protein: Compared with NS group,TRIP8b(1a-4)protein in NS+PTZ group was significantly downregulated(P <0.01),and TRIP8b(1b-2)protein was significantly upregulated(P < 0.001).In addition,compared with NS+PTZ group,TRIP8b(1a-4)in H89+PTZ group was significantly upregulated(P <0.001),while TRIP8b(1b-2)was significantly downregulated(P <0.001),but both were still higher than that in NS group(P<0.001).Compared with NS group,TRIP8b(1a-4)protein was significantly upregulated by intraperitoneal injection of H89 alone(P <0.05),while TRIP8b(1b-2)protein unchanged(P >0.05).Conclusion: SE activated mGluR1,upregulated mGluR1 expression,activated c AMP-PKA signaling pathway,leaded to downregulation of TRIP8b(1a-4)expression and up-regulation of TRIP8b(1b-2)expression,and ultimately leaded to downregulation of HCN1 surface expression and increased neuronal excitability.Conclusion of all textThe downregulation of HCN1 induced by SE was related to the upregulation of mGluR1 protein.The relationship between mGluR1 and HCN1 was unidirectional,and activation of mGluR1 leaded to inhibition of HCN1.In SE rats,increased glutamate level leding to activation of mGluR1 and upregulation of mGluR1 expression,which activated the c AMP-PKA signaling pathway,leading to downregulation of TRIP8b(1a-4)expression and upregulation of TRIP8b(1b-2)expression,and ultimately to downregulation of HCN1 surface expression and increased neuronal excitability. |
