The Interfering Effect And Mechanism Of Saikosaponin A On Neuronal PC12 Cells Induced By IL-1β

Objective and significanceEpilepsy,a functional disturbance of the CNS and induced by abnormal electrical discharge,manifestes by recurrent seizure,and its prominent clinical characteristic has the unpredictability which repeatedly paroxysmic and each time manifests suddenly,which affected epilepsy sickness patient's life,the work and social interaction ability enormously,reduced patient's quality of life.According to the WHO statistics,at present the number of epilepsy patients in whole world is approximately 50,000,000,80%people in developing country.At the present time,the population of epilepsy patients in our country has climbed to more than 9,000,000,6,000,000 patients still had the outbreak every year,and every year will have nearly 400,000 new morbidity people.At present we controlled epilepsy still by chemistry medicine primarily, commonly used antiepileptic like benzene proper UK sodium,the third propyl-acetic acid sodium,the card Mussey equality which could control 70%～80%epilepsy patient to manifest suddenly epileptic seizure.But the pharmacokinetics effects and the side effect for example fetal malformationas,cognitive effects and bone density effecta limited their application.Regarding some difficult epilepsy,although may choose the surgical operation to treat,because its indication is limited,which has great risk,while the expense is expensive and recrudescence possibility,therefore the difficult epilepsy still by the pharmacological treatment primarily,so it needs to research and develop the new ant-iepileptic drugs.The traditional Chinese medicine has the good anti-epilepsy function,but its mechanism is not often explicit,obtaining from the single taste traditional Chinese medicine active constituent to study not can only be clear about the anti-epilepsy mechanism,but is also advantageous to promote modernization of traditional Chinese medicine.The cytokine is the important information carrier of nerve-internal secretion-immunity network.On the one hand,the each kind of tissue cells in central nervous system may have many kinds of cytokines,which takes the immunity nerve medium immediate influence brain cell function,and participates in nervous system's growth,the secretion and the repair process.On the other hand,disease of central nervous system may affect the expression of each kind of cytokines through different mechanism.Epilepsy,one kind of immunity relevant neurous system disease,it has close connection with cytokines.The massive experiments was confirmed that IL-1β,as one kind of significant cytokines,play an important role in epileptic pathogenesis.IL-1βparticipates in the process of epilepsy and only binds to its corresponding high-affinity receptor IL-1Râ… to implement its function.Studies have shown IL-1βplays an important role in the neuronal damage due to epileptic seizure. IL-1βdisplay affect through IL-1Râ… activates its excited signal transduction.Long-term effects mainly including the high expression of NF-κB,the MAPK-dependent genes duplication involved in structural and functional changes in glial and neuronal networks,IL-1R1 by IL-1βcan trigger rapid effects on neuronal excitability,leading to rapid ion channel changes mediated by activation of kinases, such as Src and phosphoinositoI 3 kinase(PI3K),These actions are likely to be responsible for the proconvulsive actions of IL-1βand may contribute to seizure-associated neuronal death.SSa was a effective monomer in saikosaponin which was a main pharmaco-ingredient of traditional Chinese drug-Bupleurum Chinese.In the previous studies,we have found that saikosaponins and saikosaponin a could inhibit experimental epilepsy;saikosaponins can inhibit expression of glial fibrillary acidic protein in chronic ignition big mouse.In addition,the further in vitro experiment proved that SSa not only has the good inhibitory action in the activation of hippocampal astrocyte induced by glutanic acid,but can also inhibit the realse of TNF-αand the high expression of TNFR1 in hippocampal astrocyte induced by PTZ.Method and content1.PC12 cells culture and inductionPC12 cells were purchased from cell bank of Zhongshan University.The cells were grown in complete medium DMEM/F12 containing 5%FBS,10%HS,100 ug/ml streptomycin,and 100IU/ml penicillin at 37℃in a humidified atmosphere of 95%air and 5%CO₂.Cultured cells were dissociated by using 0.125%trypsin and plated onto poly-L-lysine-coated petri dish at certain density.Afren 24 hours Cells were grown in DMEM/F12 containing NGF which can induce PC12 cells into neuronal cells about for 5 days,Then cells could be used for experiments.2.The effect of SSa on the survival of neuronal PC12 cells Induced by IL-1βPC12 cells were transferred of culture into 96-well by the density of 1×10⁴/ml 200ul per well.Afren 24 hours PC12 cells were induced to differentiate into the neuron-like PC12 cells with NGF about for 5 days,then neuron-like PC12 cells were randomly divided into six groups as follows:group A(control group),group B (10ng/ml IL-1β-induced group),group C(IL-1β10ng/ml + SSa 0.25mg/L),group D (IL-1β10ng/ml+ SSa 0.5mg/L),group E(IL-1β10ng/ml+ SSa lmg/L) and group F (IL-1β10ng/ml+ SSa 2 mg/L).The different groups treated with different drugs which were disposed by basic medium DMEM/F12 non-seroculture with 200ul per well, Being incubated for 24 hours,the cell were detected the optical density which reacts the survival rate of cells using the colorimetric MTT assay,repeated trial 3 times.3.The effect of SSa on the expression of IL-1Râ… and NF-κB p65 in neuron-like PC12 induced by IL-1βThe expression of IL-1Râ… and NF-κB p65 was detected by way of western blotting.Cells were transferred of culture into 6-well by the density of 2×10⁵/ml in the complete medium.Afren 24 hours PC12 cells were induced to differentiate into the neuron-like PC12 cells with NGF about for 5 days,then neuron-like PC12 cells were randomly divided into five groups as follows:group A(control group),group B (10ng/ml IL-1β-induced group),group C(IL-1β10ng/ml + SSa 0.25mg/L),group D (IL-1β10ng/ml+ SSa 0.5mg/L) and group E(IL-1β10ng/ml+ SSa 1mg/L).The different groups treated with different drugs which were disposed by basic medium DMEM/F 12 non-seroculture.The cells were treated with drugs for 24 hours,and then proteins in the cells were extracted by Total Protein Extraction Kit.The protein concentration was determined by coomassie brilliant blue.The expression of IL-1Râ… and NF-κB p65 was detected by way of western blotting.We evaluated the expression of protein by analyzing the gray scale,and calculated the ratio of the value of gray scale of the protein with the value of gray scale ofβ-actin.4.Statistical methodThe data analysis and treatment was used the SPSS13.0 statistics software.The measurement data indicated by the mean value gentleman standard deviation,and the result comparison of each group by MTT and wester blotting test uses the single factor variance analysis(One-way ANOVA),the multiple comparisons with the LSD law,P＜0.05 expressed that the difference has statistics significance.Result1.The effect of SSa on the survival of neuronal PC12 cells induced by IL-1βCompared with group A,the cell survival of group B was significantly lower(P＜0.01);Compared with group B,the cell survival of different doses of SSa groups(SSa 2mg/L group,SSa 1 mg/L group,SSa 0.5 mg/L)was significantly higher(P＜0.01),only the cell survival of SSa0.25 mg/L group has no significant different(P=0.131);The cell survival of SSa 2mg/L group was significantly lower while the SSa 1 mg/L group,0.5 mg/L group was significantly higher(P＜0.01) than that of group A.2.The effect of SSa on the the expression of IL-1Râ… in neuron-like PC12 cells induced by IL-1βWestern blot analysis noted that the expression of IL-1Râ… of group B was significantly higher than that of group A(P＜0.01),Compared with group B,the expression of IL-1Râ… of SSa 1mg/L group,0.5 mg/L group was significantly lower (P＜0.01),only that of SSa 0.25 mg/L group has no significant different(P=0.103); there was no significant difference between the group A and SSa1 group(P=0.700).3.The effect of SSa on the the expression of NF-κB p65 in neuron-like PC12 cells induced by IL-1βWestern blot analysis noted that the expression of IL-1Râ… of group B was significantly higher than that of group A(P＜0.01),Compared with group B,the expression of IL-1Râ… of SSa 1mg/L group,0.5 mg/L group was significantly lower (P＜0.01),only that of SSa0.25 mg/L group has no significant different(P=0.120); there was no significant difference between the group A and SSa1 group(P=0.421).Conclusions1.SSa could improve the cell viability of the neuronal PC12 cell induced by IL-1β,while SSa could inhibit the higher expression of IL-1Râ… and NF-κB p65 on neuronal PC12 cell induced by IL-1β.2.SSa could protect neuronal PC12 cells from damage induced by IL-1β.The mechanism is related to inhibit the higher expression of IL-1Râ… and NF-κB p65.