| Background and objectiveEpilepsy is a common disease in the nervous system,70-80% of epilepsy patients with the treatment of antiepileptic drugs can obtain satisfactory results,but there are 20-30% of the patients still experience seizures after reasonable and standard treatment with antiepileptic drugs,and develop to be intractable epilepsy.Temporal lobe epilepsy is the most common syndrome of intractable epilepsy,and it has always been the difficulty in the cure of epilepsy.Many studies show that repeated seizures of temporal lobe epilepsy can cause the loss of hippocampus neurons and the clerosis in hippocampus,which not only have a great influence on the patient’s life and work,but also bring a great burden to the patient’s family and the society.The main act of antiepileptic drugs at present is on ion channels,which restrains the excitability of neurons,and it is just symptomatic treatment.Further study of the etiology and pathogenesis of epilepsy should be made to find new drugs and seek new treatment,and it is the direction for the future development of epilepsy treatment.Recently,more and more studies show that inflammation and epilepsy are closely connected,high mobility group protein 1 is a kind of important inflammatory factor,under normal circumstance,it mainly exists in the nucleus,and participate inmaintaining nucleosome structure and gene transcription regulation,when the tissue damagement occurs,it is rapidly released into the extracellular,cerebrospinal fluid and serum from injury of neurons,microglia and astrocytesin,the release of it is a dangerous signal to trigger the immune response,and it plays an important role in many diseases such as arthritis,sepsis.New study has found that it also has a close connection with the occurrence of epilepsy.Toll like receptor 4 is a kind of pathogen associated molecular patterns receptor,it can identify the damage or stress caused by the "danger signal" such as: HMGB1,activate nuclear factor-k-gene binding to phosphorylate.Phosphorylated NF-kB enter into the nucleus,inducing proinflammatory cytokine gene expression,and inflammation in brain lead to recurrent seizures.Since liquorice drugs have better effect on anti-inflammatory,anti-allergic and cell membrane protection,and they are of higher safety and tolerability,it has been widely used to treat hepatitis,skin diseases and other clinical diseases.Recent studies whether demostic or abroad have shown that Glycyrrhizin(Glycyrrhizin,GL),as a small molecule inhibitors of HMGB1,has a certain effect on inflammatory inhibition,and it is of good safety and tolerability,also part of the drug can pass the blood-brain barrier,so it is expected to become a candidate of antiepileptic drugs.However,the neural protective effect of licorice drugs and mechanism of antiepileptic effect is not clear.This study pretreated the SD rats on postnatal 21 st with Glycyrrhizin(Glycyrrhizin,GL),and made the model of temporal lobe epilepsy induced by Kainic acid(KA).Brain electrical and behavior performance were observed to disscuss the effect of the Glycyrrhizin acid on sensitivity and gravity with seizures induced by KA.Using the technology of Western blot and Q-rt-pcr respectively to examine the expression of HMGB1 / TLR4 / nf-kappa B on hippocampus at the level of protein and mRNA.Using HE staining and immunohistochemical to observe the loss of hippocampal neuron at the level of apoptosis.It is better to explore possible antiepileptic and neuroprotective mechanism of Glycyrrhizin,also,its relationship with the immune inflammatory reaction in the brain is dissussed.Conventional drugs are expected to be used in new,and it may find a new direction as well as provide theoretical basis for it in the treatment of epilepsy.Materials and methods21 days old SD rats,were randomly divided into control group,model groupⅠ,model groupⅡ.Model group I was induced by Kainic acid(KA),and the model group II was pretreated with GL by intraperitoneal injection 30 min before KA injection.According to the different observation time points,each group were divided into four subgroups:3h,12 h,24h and 7d.Model groupⅡ were divided into three subgroups: 10mg/kg,50mg/kg,100mg/kg,according to different dose of GL.There are at least 3 animals in each subgroup.Scored according to the Racine score,RT-PCR and Western blot were applied to detect the mRNA and protein expression of HMGB1 in the acute phase.Elisa was applied to measure the expression of HMGB1 in blood;Immunohistochemical was applied to measure the expression of Neuronal nuclear(Neu-N)in hippocampus in the chronic phase(7 d).Results1.Behavior results: model groupⅠ,4 were dead,the incidence of SE was 75%,the rate was 67.3%,SOT: 24.08±1.98min;model group II,6 were dead in total,the incidence rate of SE was respectively: 64.3%,the survival rate was 85.7%,SOT:33.39±2.66min;No animal was dead control group,the survival rate was 100%.GL pretreatment model II group,compared with the model group,the incidence of SE was decreased,the survival rate increased,the time of SOT Prolonged.2.The results of HMGB1/TLR-4 mRNA expression in acute stage of epilepsy in hippocampus: compared the model group Ⅰwith the control,with the observation time(3h,12 h,24h)extension,HMGB1,TLR-4 gene expression increased,which reached the peak at the time of 12h(with statistical significance,P < 0.05),at the 12 h time point,the expression of HMGB1/ TLR-4 gene in group II was significantly decreased,compared with the model group Ⅰ(with statistical significance,P < 0.05),there was no significant difference among the different dose intervention in model group II(P > 0.05).3.The results of HMGB1/TLR-4/p-NF-kB protein expression in acute stage of epilepsy in hippocampus: compared the model group Ⅰwith the control,with theobservation time(3h,12 h,24h)extension,the expression of TLR-4 and p-NF-kB protein increased,which reached the peak of 12h(with statistical significance,P <0.05),the expression of HMGB1 protein had no obvious change(P > 0.05).In 12 hours,the TLR-4 and p-NF-kB protein expression the in model of group II,decreased significantly compared with the model group Ⅰ(statistically significant,P< 0.05),there was no significant difference among the different dose intervention in model group II(P > 0.05).4.The results of HMGB1 expression in acute stage of epilepsy in serum :compared the model group Ⅰwith the control,the HMGB1 level in serum increased significantly at 12 h,(statistical significance,P < 0.05),at the time of 12 h,compared with the model group Ⅰ,HMGB1 concentration in serum significantly decreased in model II group(statistically significant,P < 0.05),different dose intervention among the model group II had no difference(P > 0.05).5.The result of Neu-N expression in hippocampus in chronic epilepsy phase :compared the model group Ⅱwith the control group,neurons in hippocampal were lost significantly(with statistical significance,P < 0.05),model II group compared with the model group,the loss of hippocampal neurons was significantly reduced(with statistical significance,P < 0.05),different dose intervention among the model group II had no difference(P > 0.05).Conclusion1.GL pretreatment can prolong the latency of seizures rats,reduce seizure susceptibility,reduce the onset of epilepsy in rats.2.The expression of HMGB1/TLR-4 gene in hippocampus was increased and the expression of TLR-4 protein was increased and the phosphorylation of NF-kappa B was induced in infant epilepsy rats.3.GL pretreatment can reduce the synthesis and release of HMGB1,decrease the concentration of extracellular HMGB1,reduce the expression of TLR-4 gene and protein synthesis,and inhibit the phosphorylation of NF-kappa B.4.GL pretreatment can reduce the pathological damage caused by epilepsy inhippocampus,prevent the loss of neurons,and play the role of neuroprotection. |
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