The Interfering Effect And Mechanism Of Saikosaponin A On Hippocampal Astrocytes Of Rats Activated By IL-1β

Objective and significanceEpilepsy,a functional disturbance of the CNS and induced by abnormal electrical discharges,manifests by recurrent seizures.The result of epidemiological investigation demonstrated that the morbidity rate of epilepsy in China was 4.6‰, and there are about 4.55 to 6.30 millions people who suffered epilepsy.General anti-epileptic drugs(AEDs) such as phenytoin,valproate, carbamazepine,and ethosuximide have been the main chemicals to anti-epilepsy.But the pharmacokinetics effects and the side effects for example fetal malformations, cognitive effects and bone density effects limited their application.Approximately 30 %of patients were still refractory to all types of treatments such as medication, epilepsy surgery,and vagus nerve stimulation.Tradional Chinese medicine may act anti-eipileptic effects by regulating neural transmitter,ion channel,suppressing reactive gliosis,clearing free radical.The study on the active component of single traditional Chinese drugs could not only help us understand the mechanism of antiepileptic,but also make for promoting modernization of traditional Chinese medicine.Cytokines could modulate neuronal function and metabolism.IL-1βcould increase neuronal excitability likely consists of its ability to enhance glutamate-mediated actions on neurons.This effect could be accomplished at least by two routes:enhancement of extracellular glutamate concentrations;increased function of the N-methyl-d-aspartate(NMDA) receptors.Long-term effects also may be triggered by IL-1βvia transcriptional activation of NF-κB and MAPK-dependent genes involved in structural and functional changes in glial and neuronal networks.Astrocytes were considered as part of synapse elements involved in neural activity control and synaptic transmission.Astrocytes could be actived by seizures. "Reactive astrogliosis" was the term used to describe the morphological transformation of astrocytes in response to a wide variety of neurotoxic stimuli.These changes included the proliferation and hypertrophy of astrocytes,as well as the upregulation of cytoskeletal proteins such as GFAP and vimentin.Numerous studies have demonstrated that MAPK medicates the activation of astrocytes induced by cytokines.The signal path of JNK/SAPK can be actived by cytokines(IL-1,TNFa), stress stimulates(ultraviolet ray,heat shook),EGF and G-protein-coupled receptors.SSa was a effective monomer in saikosaponin which was a main pharmaco -ingredient of traditional Chinese drug—Bupleurum chinense.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 and modulate expression of Glutamate in hippocampus.And we demonstrated that saikosaponin a may inhibit experimental epilepsy by block the effect of Glu on nervous system and reactive gliosis.In this study,we observed the effects of SSa on hippocampal astrocytes actived by IL-1βin vitro.Method and content1.Cell culture and identificationThe primary culture method of astrocytes was improved on the basis of McCarthy's method.Hippocamp parts were obtained from neonatal Spague-Dawley rats(1-3 days),which was snipped,digested and centrifuged.After adherencing for 40 minutes,the cells were implanted into culture flask by the density of 10⁶/ml.Cells were cultured at 37℃in a humidified 5%(v/v) CO₂ atmosphere for 7-9 days.When coalesce in the percentage of 70-80,the cells were put on the rocking bed which kept shaking for 18 hours(37.0℃,240 r/min).Transferred of culture,and we could obtain pure cells which were identified by way of immunocytochemistry.2.The effect of SSa on proliferation of astrocytes induced by IL-1βin vitro2.1 Test of linearity of the number of living cells and optical density.The pure astrocytes were stained by trypanblau,then transferred of culture into the 96-well by the density of 8×10⁴/ml 10μl,25μl,50μl,and 75μl,150μl per well. After incubated for 72 hours,the quantity of living cells was evaluated by technology of MTF assay.2.2 Cells were transferred of culture into the 96-well by the density of 7×10⁴/ml 200μl per well.After cells are cultivated in the complete medium 48 hours,the different groups treated with different drugs which were disposed by complete medium containing 5%FBS:control group,IL-1β(15 ng/ml) group,IL-1β+SSa(15 ng/ml,2 mg/L) group,IL-1β+SSa(15 ng/ml,1 mg/L) group,and IL-1β+SSa(15 ng/ml,0.5 mg/L) group.Each group included 6 wells,and 200μl medium containing different drugs was added into each well.Being incubated for 72 hours,the cells were detected the optical density which reacts the quantity of living cells by technology of MTT.3.The effect of SSa on cell division of hippocampal astrocytes induced by IL-1βThe cell division of rats' hippocampal astrocytes was detected by flow cytometry. Cells were transferred of culture by the density of 10⁵/ml.The cells were cultured in the complete medium for 48 hours,then were incubated with DMEM/F₁₂ medium only for 24 hours.The different groups treated with different drugs were disposed by DMEM/F₁₂ medium:control group,IL-1β(15 ng/ml) group,IL-1β+SSa(15 ng/ml,1 mg/L) group,and IL-1β+SSa(15 ng/ml,0.5 mg/L) group.24 hours later,the cells were stained by PI,and fixed by cold 70%ethanol.The cells division was detected by flow cytometry,and we evaluated the condition of proliferation by proliferation index.4.The effect of SSa on GFAP,vimentin,p-JNK and NF-κB in hippocampal astrocytes induced by IL-1βThe expression of GFAP,vimentin,p-JNK and NF-κB was detected by way of western bolt.The cells were cultured in the complete medium for 48 hours,then were incubated with DMEM/F₁₂ medium only for 24 hours.The different groups treated with different drugs were disposed by DMEM/F₁₂ medium:control group,IL-1β(15 ng/ml) group,IL-1β+SSa(15 ng/ml,1 mg/L) group,and IL-1β+SSa(15 ng/ml,0.5 mg/L) group.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 GFAP,vimentin,p-JNK,NF-κB was detected by way of western bolt.We evaluated the expression of proteins by analyzing the gray scale,and calculated the ratio of the value of gray scale of the proteins with the value of gray scale ofβ-actin.Result1.Cell culture and identificationWe observed the rats' hippocampal astrocytes in vitro primary culture through the microscope.From the glass we could see that cells were growing and adherenced well.The cells were identified by way of immunocytochemistry,showed over 95% positive staining.2.The effect of SSa on proliferation of astrocytes induced by IL-1βin vitro Analyzed by Curve Estimation,the number of the living cells was linear with the optical density detected by MTT.R Square=0.997,P＜0.001.Compared with IL-1βgroup,the optical densities of control group and SSa groups were significantly higher(P＜0.01).The results showed that IL-1βcould obviously active astrocytes in vitro culture,and SSa could inhibit the cell proliferation.The optical density of the SSa 2 mg/L group was significantly lower (P＜0.01) while the SSa 0.5 mg/L group was obviously higher(P＜0.01) than that of control group.The optical density of the astrocytes treated with SSa 1 mg/L remained normal level(P=0.058).3.The effect of SSa on cell division of hippocampal astrocytes induced by IL-1βThe result of flow cytometry showed that,the proliferation index of each group was significantly different(P＜0.01).The proliferation index of IL-1βgroup was higher than that of control group,and the proliferation index of different doses of SSa was lower than that of the IL-1βgroup.It showed that IL-1βcould promote the cell division of hippocampal astrocytes in vitro,and SSa could block the cell division of IL-1β-actived cells.4.The effect of SSa on GFAP in hippocampal astrocytes induced by IL-1βWestern blot analysis noted that the expression of GFAP was increased 1.5-fold in the astrocytes treated with IL-1β15 ng/ml(P＜0.01).SSa treatment with 1 and 0.5 mg/L could reduce increased expression of GFAP by 34.7%and 53.2%(P＜0.01). There was no significant difference between the control group and SSa 1 mg/L group (P=0.126).5.The effect of SSa on vimentin in hippocampal astrocytes induced by IL-1βWestern blot analysis noted that the expression of vimentin was increased 1.2-fold in the astrocytes treated with IL-1β15 ng/ml(P＜0.01).SSa treatment with 1 and 0.5 mg/L could reduce increased expression of vimentin by 17.6%and 23.8% (P＜0.01).There was no significant difference between the control group and SSa 1 mg/L group(P=0.375).6.The effect of SSa on p-JNK in hippocampal astrocytes induced by IL-1βWestern blot analysis noted that the expression of phospho-JNK was increased 1.1-fold in the astrocytes treated with IL-1β15 ng/ml(P＜0.01).SSa treatment with 1 and 0.5 mg/L could reduce increased expression of p-JNK by 7.7%and 8.6% (P＜0.01).There was no significant difference between the control group and SSa 1 mg/L group(P=0.184).7.The effect of SSa on NF-κB in hippocampal astrocytes induced by IL-1βWestern blot analysis noted that the expression of NF-κB was increased 1.5-fold in the astrocytes treated with IL-1β15 ng/ml(P＜0.01).SSa treatment with 1 and 0.5 mg/L could reduce increased expression of NF-κB by 32.6%and 34.3%(P＜0.01). There was no significant difference between the control group and SSa 1 mg/L group (P=0.463).Conclusions1.SSa could inhabit the reactive astrogliosis activated by IL-1β—inhabit the proliferation of hippocampal astrocytes activated by IL-1β,block the cell division actived by IL-1β,inhibit over-expression of GFAP and vimentin.2.SSa could inhibit the IL-1βactivated astrocytes via the inhibition of over-expression of p-JNK and NF-κB.