| ObjectiveTo investigate the disease-modifying effect and the underlying mechanism of Pinellia total alkaloids(PTA)on the pilocarpine-induced epileptic rats,and to evaluate PTA' s inhibitory effect on motor and cognitive functions of the nervous system in healthy mice.Methods1.Ninety-two male Sprague-Dawley(SD)rats were firstly tested in a battery of behavioral tests,including forced alternation test(FAT),open field locomotion(OFL),and spontaneous alternation test(SAT).Rats were randomly assigned to a model group(n=74)and a control group(n=18).Status epilepticus(SE)was induced in the model group with lithium chloride and pilocarpine,and SE was controlled to last for 90 min.The model group was divided into an epilepsy(EP)group(n=13),a topiramate(TPM)group(n=12),a PTA-400 group(n=12),and a PTA-800 group(n=13).The control group was renamed normal control group(NC group;n=17).The TPM group was treated with TPM(60mg/kg),the PTA-400 group was treated with PTA(400mg/kg),and the PTA-800 group was treated with PTA(800mg/kg).The EP group and the NC group received normal saline at the same volume.Treatments were given intragastrically once daily for 14 days.The second session of behavioral tests was taken after the 14-day treatment.Then,spontaneous recurrent seizures(SRSs)were monitored 8 hours per day for 7 days.Brain tissues were collected after the SRS monitoring.Fresh tissues of the hippocampal formation were used in enzyme-linked immunosorbent assay to measure the abundance of gamma-aminobutyric acid(GABA),in Western-blotting(WB)analysis and quantitative polymerase chain reaction to measure the protein and mRNA expression levels of glutamate decarboxylase 65(GAD65),GABA transporter-1(GAT-1),GABA transaminase(GABA-T),and GABAA receptor(GABAAR)?4,?5,?2 and ? subunits,and in WB analysis to measure the protein expression levels of brain-derived neurotrophic factor(BDNF)and tyrosine kinase receptor B(TrkB).In-situ paraformaldehyde fixed brain tissues were used in Nissl staining to quantify neuron loss in the hippocampal formation,and in Timm staining to quantify mossy fiber sprouting(MFS)in the dentate gyrus(DG).2.Thirty male Kunming(KM)mice were firstly tested in the rotarod test(RT)and SAT at Day 0(DO).Then the mice were assigned to the PTA group(n=15)and the control group(n=15)and were intragastrically dosed once with PTA(2000mg/kg)and purified water respectively at D0.RT and SAT were reexamined within 8-h of dosing(8H),and at D7 and D14 after dosing.Rats were perfused in situ and sections of the brain and spinal cord were stained with hematoxylin and eosin(HE)and examined.3.Sixty male KM mice were firstly tested in RT and SAT at D0.Then the mice were assigned to the PTA low-dose group(n=15;81.63mg/kg),the PTA median-dose group(n=15:285.71mg/kg),the PTA high-dose group(n=15;1000mg/kg),and the control group(n=15;purified water)and were intragastrically dosed once daily for 28 d.RT and SAT were reexamined at D14,D28,and D35 after the first dose.At D28 and D35,rats from each group were randomonly selected to be perfused in situ and sections of the brain and spinal cord were stained with HE and examined.Results1.In the hippocampal formation,compared with the NC group,the EP group had lower level of GABA(P<0.001),lower expression levels of GAD65(protein P<0.01),GABAA.receptor(GABAAR)a5(mRNA P<0.001),8(mRNA P<0.001),and y2(mRNA P<0.01)subunits,and higher expression levels of GAT-1(protein P<0.05;mRNA P<0.01),GABA-T(protein P<0.01;mRNA P<0.001),and GABAAR ?(proteinP<0.001),y2(protein P<0.01),and a4(protein P<0.05)subunits.Compared with the EP group,PTA increased the level of GABA(both doses P<0.001)and the expression levels of GAD65(mRNA,PTA-800 group,P<0.01),GABAAR a5(mRNA,PTA-400 group,P<0.01),?(mRNA,PTA-800 group,P<0.05),and ?2(mRNA,PTA-400 group,P<0.05)subunits;PTA decreased the expression levels of GAT-1(mRNA,PTA-400 group P<0.05,PTA-800 group P<0.01),GABA-T(mRNA,PTA-400 group P<0.01,PTA-800 group P<0.001),and GABAAR 8(protein,PTA-800 group,P<0.05)and y2(protein,both doses,P<0.01)subunits.In the hippocampal formation,compared with the NC group,the protein expression levels of mBDNF and TrkB were decreased and the expression level of proBDNF was increased in the EP group(all P>0.05).Compared to the NC group,PTA lowered the expression levels of mBDNF(both doses P<0.01).Compared to the EP group,the incidence of SRS was lower in the PTA-400 group,and the frequencies of SRS were lower in the PTA-400 group and the PTA-800 group(all P>0.05).After the 14-d treatment,the FAT-forced alternation behavior%(FAB%)(P<0.05)and the SAT-spontaneous alternation behavior%(SAB%)(P<0.05)were lower and the OFL-count of square entries(P<0.001)was higher in the EP group comparing to the NC group.Unlike the other 3 model groups,the SAB%in the PTA-400 group was similar to the NC group and higher than the EP group(P>0.05).Compared to the first test results,the duration of time spent in the novel arm was shorter in the EP group(P<0.01).Unlike the other 3 model groups,the durations of time spent in the novel arm were not statistically different between the first and second FAT in the PTA-800 group.Compared with the NC group,the neuron counts in the CA1 area,the CA3 area,and the dentate hilus(DH)were lower(vs.EP group,all P>0.05),and MFS was detected in the inner molecular layer of the DG(vs.EP group,all P>0.05).The PTA-800 group showed higher neuron counts in the CA3 area and the DH and lesser severity of MFS(vs.NC group,all P>0.05).2,No signs of toxicity regarding the general appearance were observed in both groups and the numbers of morbidity and mortality were both 0.Compared with the control,the PTA group had higher body weight at D7 and D14(both P<0.05),lower latency to fall(LTF)at 8H,D7,and D14(all P>0.05),and higher SAB%at 8H(P<0.05).The numbers of arm entries recorded in SATs showed no significant differences between the 2 groups.No significant histopathological changes were observed in the frontal secondary motor cortex,caudate putamen,temporal cortex,hippocampal formation,substantia nigra,pons,cerebellum cortex,and ventral horns of the cervical,thoracic,and lumbar sections of the spinal cord.3.No signs of toxicity regarding the general appearance were observed in all groups,and the numbers of morbidity and mortality were both 0.Compared with the control,the PTA low-dose group and the high-dose group showed lower body weight at D7 and D14(all P<0.05).However,no significant differences concerning body weight were detected between the 4 groups at D28 and D35.Compared with the control,the 3 PTA groups had lower LTF at D14 and D28(all P>0.05).The SAB%showed no significant differences between the 4 groups.The numbers of arm entries of SATs showed no significant differences between the 4 groups at D28 and D35.Tissues collected after D28 from the control and the PTA high-dose group were examined,and no significant histopathological changes were observed in the frontal secondary motor cortex,caudate putamen,temporal cortex,hippocampal formation,substantia nigra,pons,cerebellum cortex,and ventral horns of the cervical,thoracic,and lumbar sections of the spinal cord.Conclusions1.In the hippocampal formation of pilocarpine-induced epileptic rats,PTA significantly enhanced the activity of the GABAergic system by up-regulating the level of GABA and the expression level of GAD65,and down-regulating the expression levels of GAT-1 and GABA-T.PTA down-regulated the protein expression levels of GABAARs ?5 and ?2 subunits and mBDNF,and up-regulated the mRNA expression levels of GABAARs ?5,?,and y2 subunits.PTA modified cognitive impairments and could potentially suppressed the incidence and frequency of SRSs and reduce the severities of hippocampal sclerosis and MFS.2.PTA did not exhibit significant inhibition effect on the motor and cognitive functions of the nerves system in healthy mice. |
PDF Full Text Request