Effects Of Saikosaponin A On TNF-α Release And Its Receptor Expression In Rats' Hippocampal Astrocytes Activated By PTZ In Vivo And In Vitro

Objective and significanceEpilepsy is one of common chronic repeated relapsing diseases in nervous system,which takes the transient cerebral function disorder as characteristic,caused by abnormally epileptic discharge of neuron in brain.Epilepsy treatment has been a problem on how to effectively control the seizures,as patients often accompany the individual,domestic and social problems caused by epilepsy.Currently the most commonly used and important method of controlling epilepsy is to use chemicals. However the mainly used anti-epileptic drugs(AEDs) block seizures without influencing the underlying tendency to generate seizures,and are effective in over 70%of individuals.While the safety of the AEDs is not satisfactory because of the side effects accompanied with long term administration.So the new anti-epileptic drug should be used.So far,the mechanism of epilepsy has not been fully uncovered.Nevertheless, many experts of epilepsy have generally accepted that there are two important characters of epilepsy:the increase of neuronal excitability and the firing of highly synchronization.Relation of astrocyte with synaptic signal transmission have been gradually clear,recently.Astrocytes mainly in the CNS,play the role of supporting, separating and insulation.They can adjust internal and external regulation of ion concentration and pH value,and play an important role for the stability of the environment.During the process of epilepsy,the astrocytes also have some important abnormality of structure and function,which can affect the function of neuron. Astrocytes can become reactive gliosis.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 exciting neuronal by many kinds of pathway,and is epileptogenous.So astrocytes may be target cells of epilepsy treatment.Astrocytes can secrete lots of cytokines which are involved in the occurrence,development and prognosis of epilepsy.Cytokine is a group of polypeptides,which have a wide range of biological performance,such as regulation in development of human organs and cell growth.Also classically mediate and regulate immune response and inflammatory response.It is discovered that the expression of cytokines increased in the epileptic patients and rats model.The cytokines have participated in the activation of astrocytes and their Signal Transduction Pathway, which can keep recurrent seizures.TNF-αis a various functional cell factor,excreted by monocyte and Macrophage.Various evidence have shown that the level of TNF-αand its mRNA were increased significantly in the brain tissue and cerebrospinal fluid of epileptic patients and rats model.,while treated with mTNF-αantagonist,the sensitivity of convulsions in mice was reduced.Bupleurum chinense is one of a widely used Chinese herb.Saikosaponin a(SSa) is 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 have anti-epileptic effects and SSa is the main active component.which can inhibit expression of GFAP in hippocampal astrocytes.SSa could prolong the incubation period of tetanic convulsion,and could reduce the rate of tetanic convulsion.In order to investigate the anti-epileptic effect of SSa and to see if the anti-epileptic effect of SSa is obtained by the mechanism of cytokines,in this study we observed the effects of SSa on the release of TNF-αand the expression of TNF receptor 1(TNFR1) and GFAP in in rats hippocampal astrocytes activated by pentylenetetrazol(PTZ) in vivo and in vitro.Method and content1.Effects of saikosaponin a on TNF-αrelease and its receptor expression in rats hippocampal astroeytes activated by PTZ in vitro1.1 Cell culture and identificationThe primary culture method of astrocytes was improved on the basis of McCarthy's method.Hippocamp parts were obtained from neonatal Sprague-Dawley rats(1-3 days),which were 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.1.2 Measurement of cell viability by the MTT in vitroPrimary cells from passages were diluted with 15%complete culture medium to the seeding density(7×10⁴ cells/well),seeded in 96 well cell culture cluster(200 ml/ well).After being incubated at 37℃for another 48h,the cells were randomly divided into two big groups:â‘ 3gpoups:control group(group A),SSa group(group B,C, SSa1.25mg/L,SSa0.625mg/L),the cells were interfered in SSa for 6h,then the bsorbance at 490 nm was measured with a microplate reader.Each condition was detected for six times.â‘¡4gpoups:control group(group A),PTZ model group(group B),SSa group(group C,D,PTZ+SSa 1.25 mg/L/SSa 0.625 mg/L).In group C,D after pretreated with SSa for 6h,the cells were induced by PTZ for 2h,then the bsorbance at 490 nm was measured with a microplate reader.1.3 the effect of SSa on TNF-αin culture supernatant of astrocytes and on TNFR1/GFAP in hippocampal astrocytes induced by PTZ in vitroPrimary cells from passages were diluted with complete culture medium to the seeding density(5×10⁵ cells/well),seeded in 6 well cell culture cluster.After being cultured with culture medium without serum for 24h,the cells were randomly divided into four groups:control group(group A),PTZ model group(group B),SSa group(group C,D,PTZ+SSa 1.25 mg/L/SSa 0.625 mg/L).After pretreated with SSa for 6h,the cells were induced by PTZ for 2h,then collected the culture supematant of astrocytes induced by PTZ in vitro,and detected the cytokine TNF-α.While the cell disruption was used to detected the TNFR1/GFAP of astrocytes induced by PTZ in vitro.2.The effect of saikosaponin a on expression of TNFR1 in the hippocampus of PTZ induced epileptic rats in vivo96 health grown-up rats were randomly divided into four groups:normal control group(group A,6 rats),model group(group B,30 rats),SSa group(group SSa,30 rats),VPA group(group D,30 rats).The normal control group was induced by intraperitoneal injecting isotonic sodium chloride solution,while the other groups were induced by intraperitoneal injecting 60mg/kg PTZ and treated by different factors as above.The group B,group C and group D were then divided into five groups,TNFR1 was detected by using immunocytochemical stains at different time(1/2 h,2 h,4h,8h,12h).Result1.Effects of saikosaponin a on TNF-αrelease and its receptor expression in rats hippocampal astrocytes activated by PTZ in vitro1.1 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.Primary cultured hippocampal astrocytes of rats was identified by way of immunocytochemistry,positive rate of glial fibrillary acidic protein of the cells was more than 95 percent.1.2 Measurement of cell viability by the MTT in vitroResult of MTT is demonstratedâ‘ In the group B,the OD was increased by PTZ, and the group A,C and D have significant difference compare with the group B (P＜0.05).Compared with the group A,the group D(SSa0.625 mg/L) have no significant difference(P＞0.05).â‘¡the group B and C(SSal.25 mg/L,0.625 mg/L) both have no significant difference compared with Group A,which showed that dose of SSa has no significant effect on viability of astrocytes activated by PTZ in vitro at the certain time.1.3 the effect of SSa on GFAP in hippoeampal astroeytes induced by PTZ in vitroResult of Western blot is demonstrated:In the group B,the TNFR1 of culture supernatant of astrocytes was increased by PTZ,and the group A,C and D have significant difference compared with the group B(P＜0.01),compared with the group A,the group D(SSa0.625mg/L) have no significant difference(P=0.120)1.4 the effect of SSa on TNF-αof culture supernatant of astroeytes induced by PTZ in vitroResult of ELISA is demonstrated:In the group B,the TNF-αof culture supernatant of astrocytes was increased by PTZ,and the group A,C and D have significant difference compared with the group B(P＜0.01),compared with the group A,the group D(SSa0.625mg/L) have no significant difference(P=0.744)1.5 the effect of SSa on TNFR1 in hippocampal astrocytes induced by PTZ in vitroResult of Western blot is demonstrated:In the group B,the TNFR1 of culture supernatant of astrocytes was increased by PTZ,and the group A,C and D have significant difference compared with the group B(P＜0.01),compared with the group A,the group D(SSa0.625mg/L) have no significant difference(P=0.386)2.the effect of saikosaponin a on expression of TNFR1 in the hippoeampus of PTZ induced epileptic rats in vivoAfter induced by PTZ,the expression of TNFR1 in the Hippocampus was changed in a dynamic state:increased rapidly(P＜0.05),and then went down to a lower level, and then slowly recovered back to normal.SSa and VPA could inhibit the rapidly increasing of TNFR1 after induced by PTZ.Conclusion1.SSa could inhibit the raising of GFAP in hippocampal astrocytes induced by PTZ in vitro.The anti-epileptic effect of SSa that may be inhibit the astrocyte activated by PTZ.2.SSa could inhibit the raising of TNF-αof culture supematant of astrocytes induced by PTZ in vitro3.SSa could inhibit the raising of TNFR1 in hippocampal astrocytes induced by PTZ in vitro.The anti-epileptic effect of SSa that may be inhibit the astrocyte activated by PTZ.4.The expression of TNFR1 changed in dynamic state in epileptic rats.The expression of TNFR1 raised rapidly after induced by PTZ and decreased to normal then.And both SSa and VPA could inhibit the raising of TNFR1 after treated by PTZ.