Molecular Mechanism Of Leptin Neuroprotective Effect In Neonatal Flurothyl-induced Seizure Combined With Penicillin-induced Secondary Attack Model

Objective:The effects of leptin on the protein level(or content)in the acute phase and neurobehavior,convulsion threshold and aberrant mossy fiber sprouting in the long term were investigated by using the new-stage flurothyl status epilepticus combined with penicillin-induced secondary attack model.Methods:60 Sprague-Dawley(SD)rats,6 days old(P6),were randomly divided into four groups according to the random number table method:the first group was the simple control group(Control group,,n=15),the second group was simple leptin group(Leptin group,n=15),the third group was simple status epilepticus group(SE group,n=15),and the last group was status epilepticus with leptin group(SE+Leptin group,n=15).And then one day after adaptive feeding,Rats in the SE group and the SE+Leptin group were induced with flurothyl for 30 min once daily for 7 days(P7-P13)to establish a model of status epilepticus(SE)in neonatal rats.The same operation was performed on the Control group and the Leptin group without inhaling flurothyl.Rats in the Leptin group and the SE+Leptin group were intraperitoneally injected with leptin(dissolved in PBS buffer)at a dose of 4 mg/kg 1 hour after daily seizures.The control group and SE group were intraperitoneally injected with the same does of PBS buffer.In the acute phase(24 hours after the last seizure),5 rats in each group were randomly took out.After anesthesia,the blood was collected from the heart,then the cortex and hippocampus were taken.The contents of clusterin and VEGF in the serum were measured by enzyme-linked immunosorbent assay(Elisa).Western blot analysis was used to detect the relative level of proteins in cortex and hippocampus:autophagy pathway related proteins Beclin-1、LC3II/LC3I,endoplasmic reticulum stress related proteins GRP78、ATF4,anti-apoptotic protein Bcl-2 and NLRP3 inflammatory.The remaining 40 rats were monitored for body weight changes every 3 days,and neurobehavioral tests(negative reflex,open field experiment and object recognition experiment)were performed at P23 and P33;Morris water maze were performed from P28 to P33.At P34,after intraperitoneal injection of penicillin,we closely observed for about 1 h and then recorded the seizure latency and survival rate.Then randomly took out 5 rats from the surviving rats in each group,and the brain was taken after perfusion.The aberrant mossy fiber sprouting in the dentate gyrus(DG subfield)and CA3 subfield of the hippocampus were observed by Neo-Timm staining.Results:1.Western bolt results:The levels of protein in the cerebral cortex and hippocampus in the four groups had significant differences[Beclin-1:F皮层(3,16)=9.81,P<0.05,F 海马(3,16)=9.87,P<0.05;LC3 II/LC3 I:F皮层(3,16)=96.62,P<0.05,F海马(3,16)=161.30,P<0.05;GRP78:F 皮层(3,16)=8.16,P<0.05,F海马(3,16)=7.60,P<0.05;ATF-4:F 皮层(3,16)=14.20,P<0.05,F海马(3,16)=19.54,P<0.05;Bcl-2:F皮层(3,16)=13.96,P<0.05,F海马(3,16)=20.43,P<0.05;NLRP3:F 皮层(3,16)=21.08,P<0.05,F海马(3,16)=16.99,P<0.05].Compared with the control group,the protein levels of Beclin-1、LC3 II/LC3 I、GRP78、ATF-4、NLRP3 in the SE group were significantly increased(P<0.05),and the protein level of Bcl-2 was significantly decreased(P<0.05)in the cerebral cortex and hippocampus;Compared with the SE group,the protein levels of Beclin-1、LC3 II/LC3 I、GRP78、ATF-NLRP3 in the SE+leptin group were significantly decreased(P<0.05),and the protein level of Bcl-2 was significantly increased in the cerebral cortex and hippocampus(P<0.05).2.ELISA results:The contents of clusterin and VEGF in the serum in the four groups had significant differences[clusterin:F(3,16)=14.24,P<0.05;VEGF:F(3,16)=18.69,P<0.05],The contents of clusterin and VEGF in the serum were significantly increased in the SE group in comparison to the control group(P<0.05),Leptin intervention decreased the content of clusterin in the serum after SE(P<0.05),while leptin intervention further increased the content of VEGF in the serum after SE(P<0.05).3.Weight change:There were significant differences among groups[F(3,27)=85.79,P<0.05]、days[F(7,63)=1160.48,P<0.05]and interaction[F(21,189)=9.05,P<0.05]。P11-P34,The body weight was significantly lower in the SE group and SE+Leptin group in comparison to the control group(P<0.05),Leptin intervention had no effect on body weight in the control group and SE group.4.Negative geotaxis test:The time of negative geotaxis in the four groups had significant differences at P23 and P33[F P23(3,36)=21.25,P<0.05;FP33(3,36)=33.68,P<0.05].The time of negative geotaxis is significantly longer in the SE group in comparison to the control group(P<0.05),Leptin intervention shorten the time of negative geotaxis after SE(P<0.05).5.Open field test:(1)The delay time in the four groups had significant differences at P23 and P33[F P23(3,36)=66.74,P<0.05;FP33(3,36)=106.40,P<0.05].The delay time was significantly prolonged in the SE group in comparison to the control group(P<0.05),Leptin intervention shorten the delay time after SE(P<0.05).(2)Locomotor scores:The locomotor scores in the four groups had significant differences at P23 and P33[F P23(3,36)=25.50,P<0.05;FP33(3,36)=32.60,P<0.05].The locomotor scores were significantly decreased in the SE group in comparison to the control group(P<0.05),Leptin intervention increased the locomotor scores after SE(P<0.05).6.Novel object recognition test:The recognition index in the four groups had significant differences at P23 and P33[F P23(3,36)=10.33,P<0.05;FP33(3,36)=23.99,P<0.05],The recognition index was significantly lower in the SE group in comparison to the control group(P<0.05),Leptin intervention enhanced the recognition index after SE(P<0.05).7.Morris water maze test:(1)Place navigation test:There were significant differences among groups[F(3,27)=47.02,P<0.05]、days[F(4,36)=567.311,P<0.05]and interaction[F(12,108)=3.432,P<0.05].From P29-P32,the escape latency was significantly longer in the SE group in comparison to the control group(P<0.05),Leptin intervention reduced the escape latency after SE(P<0.05).(2)The probe test:The frequency of passing through the platform in the four groups had significant differences at P33[F(3,36)=5.42,P<0.05].The frequency of passing through the platform was significantly was decreased in the SE group in comparison to the control group(P<0.05),Leptin intervention increased the frequency of passing through the platform after SE(P<0.05).8.Seizure threshold and survival rate after the second strike:(1)The seizure threshold in the four groups had significant differences at P34[F(3,36)=15.76,P<0.05].The seizure threshold was significantly lower in the SE group in comparison to the control group(P<0.05),Leptin intervention increased the seizure threshold after SE(P<0.05).(2)The survival rates of control group,Leptin group,SE group and SE+Leptin group were 90%,90%,50%and 70%,respectively.9.Neo-Timm’s staining:The aberrant mossy fiber sprouting score in the four groups had significant differences at P34[FDG(3,16)=25.73,P<0.05;FCA3(3,16)=20.33,P<0.05],The aberrant mossy fiber sprouting score in the dentate gyrus(DG subfield)and CA3 subfield of the hippocampus was significantly increased in the SE group in comparison to the control group(P<0.05),Leptin intervention decreased the aberrant mossy fiber sprouting score in the dentate gyrus(DG subfield)and CA3 subfield of the hippocampus after SE(P<0.05).Conclusion:1.Acute leptin intervention in neonatal rats after status epilepticus enhances cognitive ability,improves neurobehavioral parameters such as reactivity and balance,and is more tolerant to the second strike caused by penicillin,showing an increase in survival rate and a prolonged seizure threshold,and also inhibit aberrant hippocampal mossy fiber sprouting in the long term.The neuroprotective effect of leptin may be related to reduce seizure-induced increase in the protein level of autophagy pathway-related proteins Beclin-1、LC3II/LC3I,endoplasmic reticulum stress-related proteins GRP78、ATF4、NLRP3 inflammatory bodies,and up-regulate the protein level of the antiapoptotic molecule Bcl-2 in the cortex and hippocampus.2.In the acute phase of neonatal status epilepticus,the serum clusterin and VEGF increased.Acute leptin intervention can prevent the compensatory increase of clusterin in the serum,while further increase the content of VEGF in the serum.