The Effects Of Saikosaponin A On Glu-actived Astrocyte Of Rats' Hippocampus In Vitro

Objective and significanceEpilepsy is a kind of neurological disease that is harmful to the health of human being. There is about 4.6‰of the population in our country affected by this neurological disease. Epileptic and their family have possibly to hide or to deny the heath condition because of orthodox prejudice of society,and epileptic beared mental stress from the world. It cant't be ignored that the problems of medical treament,family and social problem induced by epilepsy.In now, epileptic treatment still dependent on chemicals. General anti-epileptic drug(AEDs) can control epileptic attack,but security is a problem in treatment of long-term, and the method to treat refractoriness epilepsy by surgery may be confined by indication, high risk, expensive, and possible to recur. So epileptic treatment still dependent on chemicals, but it must know clearly that is pathogenesy of epilepsy before develop new chemicals.For the past few years, research of epileptic pathogenesy has been advanced. Increase in neuronal excitability and synchronize firing are two important characteristic of epileptic, which had be generally accepted. Many scholars try them best to study mechanism of form, and introduced many view. Recognition of relation of astrocyte with synaptic signal transmission have been gradually clear. Synaptically associated astrocytes should be viewed as integral modulatory elements of tripartite synapses. Glu, one of main excitatory neurotransmitters in central nervous system, is a etiological factor of epilepsy. It can not cause epilepsy but also may play a important part in the brain damage caused by epilepsy. In normal, glu which is released by neuron into synaptic cleft is intaked by astrocyte and then turned into glutamine. This cycle of glutamate- glutamine between neuron and astrocyte plays a important role in normal nervous system. Once the cycle is blocked, glu will cumulate in synaptic cleft that would excite neuron and then lead to convulsion. Astrocytes can become reactive gliosis when nervous system have been damaged. Histologic characteristics of reactive gliosis is greaten and hyperplasia of astrocyte, biochemical marker is increase in content of glial fibrillary acidic protein(GFAP)of astrocyte. The reactive gliosis have effect on synaptic transmission that excits neuron by many kinds of pathway, and is epileptogenous.SSa is a effective monomer in saikosaponin which is a main pharmaco -ingredient of traditional Chinese drug-- Bupleurum chinense. In the past research, we have found that saikosaponins and saikosaponin a can inhibit experimental epilepsy; saikosaponins can inhibit expression of glial fibrillary acidic protein and modulate expression of Glu in hippocampus. So. We suppose that saikosaponin a may inhibit experimental epilepsy by block the effect of Glu on nervous system and reactive gliosis.Up to now,the mechanism why SSa can inhibit experimental epilepsy is not clear.So our experiment invest the effects of SSa on glu actived-astrocyte in vitro to discuss the mechanism that the role of SSa in epilepsy. This expriment is supported by the State Administration of Traditional Chinese Medicine Of GuangDong Province (NO.1060133) . Method and content1 Cultivation and identification of rat' hippocampal astrocyte in vitroThe primary culture method of astrocytes was improved on the basis of McCarthy's way. Hippocamp part was obtained from neonatal Spague-Dawley rats(l-3day),which was snipped, digested and centrifuged. After adherencing 40min, the cells were implanted into culture flask by the density of 10⁶ /cm². Cells were cultured at 37℃in a humidified 5% (v/v) CO₂ atmosphere for 7-9 days. When the cells coalesced in the percentage of 70-80,put the cultures on the rocking bed which kept shaking 18 h (37.0℃, 240 r/min) .And then transferred of culture,so we could obtain pure cells which were identified by way of immunohistochemistry.2 The effect of glu on activity of rats' hippocampal astrocyte in vitro culture and the role of SSa.The quantity of alive cells were detected by technology of MTT assay. Cells were transferred of culture into 96-well by the density of 8×10⁴ /cm² 200μl per well. After cells were cultivated in the complete medium 48h,the different groups treated with drugs which were disposed by complete medium containing 5% FBS:a control,b L-Glu(1.5 mmol/L),c L-Glu + SSa(1.5 mmol/L,10 mg/L), d L-Glu + SSa(1.5mmol/L,5mg/L), e L-Glu+SSa( 1.5 mmol/L ,2.5 mg/L), f L-Glu+SSa(1.5 mmol/L,1.25 mg/L). Six wells per group,200μl per 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 glu on cell division of rats' hippocampal astrocyte in vitro culture and the role of SSa.The cell division of rats' hippocampal astrocyte was detected by flow cytometer. Cells were transferred of culture by the density of 10⁵/cm² .After cells were cultured in the complete medium 48h, the different groups treated with drugs were disposed by DMEM/F₁₂ medium: a(control);b L-Glu(1 mmol/L);c L-Glu+SSa(1 mmol/L,5 mg/L);d L-Glu+SSa(1 mmol/L.2.5 mg/L);e L-Glu+SSa(1 mmol/L,1.25 mg/L). 24 hours later, the cells were stained by PI, and transferred the cells density to 10⁶/cm² .Detected the cells division by flow cytometer 10⁴ cells per group, and we evaluated the condition of proliferation by proliferation index.4 The effect of glu on express of GFAP in rats' hippocampal astrocyte in vitro culture and the role of SSa.The express of GFAP was detected by way of western bolt. Cells were transferred of culture by the density of 10⁵/cm² .After cells were cultured in the complete medium 48h, the different groups treated with drugs were disposed by DMEM/F12 medium: a(control);b L-Glu(1.5 mmol/L);c L-Glu+SSa(1.5 mmol/L,5 mg/L);d L-Glu+SSa(1.5 mmol/L,2.5 mg/L);e L-Glu+SSa(1.5 mmol/L,1.25 mg/L). 24h later, proteins in the cells were extracted from the cells. The express of GFAP was detected by way of western bolt. The result of western-blot was analyzed, demonstrated in value of gray scale, and calculated the ratio of the value of gray scale of GFAP with the value of gray scale, ofβ-actin.Result1 Cultivation and identification of rats' hippocampal astrocyte in vitroWe observe the rats" hippocampal astrocyte in vitro primary culture through the microscope. From the glass we could see that cells were growing and adherencing well. The cells were identified by way of imrnunocytochemistry, showed over 98% positive staining.2 The effect of glu on activity of rats' hippocampal astrocyte in vitro culture and the role of SSa.Rats' hippocampal astrocyte in vitro culture were dealt with different factors. Compared with group a,the optical density of group b.c,d,f were obviously higher than that of group a(P<0.05 );Compared with group b, optical density of group a,c,d,e were obviously lower (P<0.05) ,and group f was obviously higher than group b (P<0.05) .The results showed that glu can obviously active astrocyte in vitro culture,and SSa can inhibit the cell proliferation.3 The effect of glu on cell division of rats' hippocampal astrocyte in vitro culture and the role of SSa.The results in different groups of flow cytometer: the proliferation index of each groups were obviously different (P<0.001) .The proliferation index of other groups were obviously higher than that of group a;compared with group b,the other groups' index were obviously lower. It showed that glu can promote the cell division of rats' hippocampal astrocyte invitro culture,different doses of SSa can block the cell division of glu-actived cells in some way.4 The effect of glu on express of GFAP in rats' hippocampal astrocyte in vitro culture and the role of SSa.The results of western blot between different groups: compared with a,the quatity of GFAP expressed in cells were obviously different in b,c,e groups (P<0.05) ;the quatity of group a,c,d was obviously lower than group b (P<0.05) ;the quatity of group e was obviously higher than group b(P<0.05) .It showed that glu can obviously enhance the express of GFAP in cells and make them active, SSa can inhibit the excess express.Conclusions1 The activity of rats' hippocampal astrocyte in vitro culture was encouraged by glu,and this proliferation of glu-actived cell was inhabited by SSa. It also could maintain the cell quantity in normal.2 The cell division of rats' hippocampal astrocyte in vitro culture cycle was promoted by glu,and the cell division of glu-actived cell was blocked by SSa.It also could maintain the cell division in normal.3 Glu could enhance the express of GFAP in rats' hippocampal astrocyte invirto culture,and the excess express of GFAP could be controlled by SSa. It also could maintain the quantity of GFAP in normal.