Mechanistic Study Of SPARCL1 And Integrin?1 In Epileptogenesis

ObjectiveEpilepsy,characterized by recurrent seizures,is a clinical manifestation of abnormal electrical discharge in the brain.The objective of this study was to explore whether SPARCL1 and Integrin?1 were related to refractory temporal lobe epilepsy in epileptic patients,PTZ-rats and Li Cl-PILOratsMethods1.Clinical specimen study:the temporal lobe cortex of 10 patients with refractory temporal lobe and 2 patients with the same batch of specimens were extracted from the specimen bank.Then,protein expression of SPARCL1 and Integrin?1 was analyzed in the temporal lobe of patients by Immunohistochemistry and immunoblotting methods.2.Animal experimental research:(1)A total of forty 21 d juvenile male SD rats were randomly divided into control group(n=5)and model group(n=35)The model group was further divided into seven subgroups including 3 h group,6 h group,12 h group,1d group,2 d group,3d group and 7 d group.The model group was treated with Li Cl-PILO intraperitoneally,and the control group was administered by the same dose of physiological saline,instead.(2)Twenty male SD rats born at 21 days were randomly divided into control group(n=5)and model group(n=15).The model group was treated with PTZ intraperitoneal injection,and the control group was replaced with the same dose of normal saline.Rats with a seizure level of IV or a spontaneous episode were selected as the epilepsy group,while rats that failed to reach the IV grade were classified as a control group.The m RNA and protein expression levels of SPARCL1 and Integrin?1were detected in the hippocampus and temporal lobe tissues of epileptic rats by Western blot(Wb)and RT-q PCR.ResultsCompared with the control group,the m RNA and protein expression levels of SPARCL1 and Integrin?1 were significantly increased in the brain tissue of patients with refractory epilepsy(P<0.05).The m RNA and protein expression levels of SPARCL1 and Integrin?1 were significantly higher in PTZ chronic epilepsy rats,compared with the control group(P<0.05).In the hippocampus of Li Cl-PILO-induced acute epileptic rats,expression of SPARCL1 and Integrin?1 was decreased at 3 h,6 h,12 h,compared withthe control group,.Especially,significantly decrease was found at 6 h after status epilepticus(SE)in the model group.P<0.05).Moreover,and there was significant correlation between the protein expression of SPARCL1 and Integrin?1 at this time point(r=0.993,P?).In the acute phase(1 d,2 d,3 d),SPARCL1 protein was significantly decreased at 1 d and 2 d after SE(P<0.05),however,there was no significant difference between 3 d and 7 d group(P>0.05).These results suggested that the protein expression of SPARCL1 decreased firstly and then increased within 7 days after SE.In the acute phase,Integrin?1 was not significantly different from the normal group at 1d,2d and 3d after SE(P>0.05),and the expression was significantly increased on the 7^thday(P<0.05).In summary,the expression levels of SPARCL1 and Integrin?1 were firstly decreased and then increased in the hippocampus.Overall,the overall trend was similar within 1 week after SE.In the cortical tissue,compared with the control group,there was no difference among control group,3 h group,and 6 h group on the protein expression of SPARCL1(P>0.05).However,significant decrease was found in 12 h group and 1week group(P<0.05),and the downward trend was more and more obvious.Integrin?1 began to show a significant decrease in expression at 6 h after SE,and it also showed a decreasing trend.Overall,the overall trend is very similar within 1week after SE.In the Li Cl-PILO epileptic rats,RT-q PCR results showed that the expression of SPARCL1 was significantly decreased in hippocampus within 1 d after SE compared with the control group,(P<0.05),and decreased at 3 h and 6 h.Obvious(P<0.01).The expression of SE was close to normal level from 2d to 7d after SE(P>0.05).The expression of Integrin?1 was significantly decreased at 3h and 6h after SE(P<0.05),and there was no significant difference in these two groups(P>0.05)at12 h,and significantly increased at 1d and 2d(P<0.05)at 3d and 7d.There was a decrease in near normal(P>0.05).Pearson correlation analysis showed that the correlation coefficients were 0.996,0.97 and 0.968 at 6 h,1 d and 3 d groupin hippocampus,and SPARCL1 and Integrin?1 were significantly correlated(P<0.05).ConclusionThe overall trends of SPARCL1 and Integrin?1 expression in refractory patients and Li Cl-PILO epilepsy rats are basically consistent,while significant correlation was found between SPARCL1 and Integrin?1,which suggested that SPARCL1 and Integrin?1involves in the occurrence and development of epilepsy.Objective:To explore whether SPARCL1 and Integrin?1 were the targets of the traditional and novel antiepileptic drugs(LEV,TPM,and VPA)via Li Cl-PILO-induced epileptic model.Methods:1.Fifty male Sprague-Dawley rats aged 21 days were randomly divided into NS control group(n=10),LEV control group(n=10),PILO group(n=15),and PILO+LEV group(n=15).The model group was injected intraperitoneally at 27mg/kg.After about 20 hours,atropine 1mg/kg,and PILO 50mg/kg intraperitoneal injection induced seizures.If the seizure does not reach the standard,continue to add PILO15mg/kg intraperitoneal injection 10 min until it is completely ignited.Patients with seizures of grade V were given intraperitoneal injection of diazepam 10 mg/kg10 minutes later to control seizures and reduce mortality.For the rats in the treatment group,the rats were treated with 300 mg/kg of levetiracetam daily for 3 weeks.After continuous administration for 3 weeks,the rats in each group were sacrificed,and the hippocampus and the hippocampus were detected by Wb and RT-q PCR.Changes in the expression of SPARCL1 and Integrin?1 in temporal cortex tissue.2.sixty-two male SD rats(21-days old)were divided into NS group(n = 10),TPM control group(n = 5)and VPA control group(n = 5).Other 32 rats were intraperitoneally injected by PTZ(35 mg/kg)at 9 am every day for 35 days.Rats with grade IV or above for 5 consecutive days were determined to have successful chronic ignition.Rats with seizures of grade V were treated with intraperitoneal injection of diazepam 10 mg/kg within 10 minutes to control seizures and reduce mortality.Thirty rats with a successful model of chronic ignited epilepsy were randomly divided into three groups,including PTZ,PTZ+VPA,and PTZ+TPM group.The control group was given an equal dose of NS every day.The treatment group was given TPM 50mg/kg and VPA 300 mg/kg daily for 3 weeks,and the SPARCL1 and Integrin?1 mRNAs in hippocampus and temporal cortex were detected.The level of protein expression.Results:1.The expression of SPARCL1 and Integrin?1 was increased in the PILO group compared with the NS control group(P<0.05).However,it was significantly increased in the LEV-treated group compared with the PILO group(P<0.05).2.Compared with the NS control group,the expression of SPARCL1 and Integrin?1 was decreased in PTZ-induced seizure rats(P<0.05).There was no significant difference in the expression of SPARCL1 and Integrin?1 between the VPA and TPM treatment groups compared with the PILO group(P>0.05).Conclusion:The expression of SPARCL1 and Integrin?1 was significantly decreased in the model group after seizures,suggesting that SPARCL1 and Integrin?1 are closely related to epilepsy.SPARCL1 and Integrin?1 were the targets of LEV but not VPA and TPM.