The Regulation Effect And Mechanism Of Liver-regulating Compound Recipe On Hippocampus Nerve Cytogenesis With High Concentration Corticosterone Condition

ObjectiveThe most of neuron in central nervous system is undifferentiation and impossible regeneration after damage.Recent years,a cluster of especial neurocyte was found both in embryo nervous tissue and adult brain by researcher.This kind of cell not only possesses self-renewal,but also multi-directional differentiation potentiality.Accordingly,it named stem cell or precursor cell.The findings of nerve stem cell in brain is surmount the traditional standpoint that central nervous system neuron lack of regeneration capacity.The hippocampus is dividing into two parts,including hippocampal gyrum and gyrus dentatus, which have close correlation with senior cognition function,such as nervous system structure plasticity of human,learning,remembrance and affection,etc.The hippocampus gyrus dentatus of adult mammal possesses neurogenesis all its life.The process of neurogenesis is as following.The infragranular layer of hippocampus gyrus dentatus to generate the precursor cell of neogenesis neuron,then to migrate to granular cell layer and differentiate to granular cell,after that cytodendrite and axis cylinder is forming and to shape synapse connection which conform into the nerve circuit loop of hippocampus's function.Although the specific function of these granular cells is not clear,it's more plasticity than ripe nerve cell.Great quantity researches indicate that stress could affect the neurogenesis of hippocampus and induce the reduction and atrophies of gyrus dentatus granular cell regeneration,and ITP inhibition.The disorder of hippocampus neurogenesis is to become the important cause of depression onset,and antidepressant drug probably to produce and maintain curative effect through regulate neurogenesis.The major research aspects of our laboratory in the pass including regulating the HPA axis function change and center monoamine neurotransmitter disorder,which induce by stress,through use liver-regulating compound recipe.Moreover,the role and mechanism of this recipe to restrain the apoptosis of hippocampus neuron which induce by stress is one of the major research aspects.However,we haven't referred to the hippocampus neurogenesis domain. The research is through applied ex vivo experiment method to establish in vitro culture hippocampus nerve precursor cell model.This cell model is in high endogenous glucocorticoid(GC)condition which imitates stress.Then add the contain medicine serum to the cell model.This serum is from the rat after take the liver-regulating compound recipe, xiao yao san(XYS)and jia wei si ni san(JWSNS).The research aim is to observe the regulation effect and action route of liver-regulating compound recipe to the disequilibrium between proliferation and apoptosis of chronicity psychological stress hippocampus nerve precursor cell.Then further study the cell and molecular pharmacology targeting and its mechanism of liver-regulating compound recipe anti-chronicity irritability hippocampus impaired from hippocampus neurogenesis angle.Accordingly,to provide scientific evidence for prevention and cure mental disorder which caused by stress through apply traditional Chinese medicine.Moreover,to provide scientific evidence for the manufacture and development of new traditional Chinese drugs those apply to anti-irritability impairment as well.MethodsMake use of cell culture technology to serum-free culture rat hippocampus precursor cell in vitro,and apply traditional Chinese drug serum pharmacology method to approach the regulate effect and mechanism of liver-regulating compound recipe to the hippocampus neurogenetic.Step one.To take the hippocampus tissue of embryo rat and adopt in vitro serum-free culture method to culture hippocampus nerve precursor cell.Step two.Apply the MTT method to detect the effect of different concentration contain medicine serum to the reproductive activity of hippocampus nerve precursor cell in high concentration corticosterone condition.Step three.Apply immunofluorescence cytochemistry technology to detect the effect of contain medicine serum to the proliferation,apoptosis and differentiation of hippocampus nerve precursor cell in high concentration corticosterone condition.Step four.Adopt western blotting method to detect the effect of contain medicine serum to the proteinum expression of BDNF and CREB in high concentration corticosterone condition.Step five.Adopt real time fluorescent quantitation PCR method to detect the effect of contain medicine serum to the BDNF mRNA and NMDAR-1mRNA expression in high concentration corticosterone condition.ResultsPart one.After 5 to 7 days culture in stem cell growth medium,the hippocampus nerve precursor cell that isolating culture to form bulk of suspension vegetative nerve cluster.To process digest and transfer of culture,after 6 to 8 days could form a new generation nerve cluster.The never cluster could differentiate into ripe neuron and colloid cell in differential medium.The immunocytochemistry method confirms that cultured cell could express never precursor cell specificness nidogen and could intake BrdU which is Thymidine analog.This indicated that cultured cell is cleavage primitive nerve cell.And immunofluorescence ambi-mark demonstrates that cultured cell could differentiate into ripe neuron and colloid cell,and possess multi-directional differentiation.Part two.The results of MTT are as following.There is insignificant(P＞0.05)effect to hippocampus nerve precursor cell proliferation when apply 30μmol/L CORT,but 60μmol/L and 120μmol/L CORT could degrade the proliferation significantly(P＜0.05 or P＜0.01). However,the contain medicine serum,which contain 5%and 10%XYS and 10%JWSNS respective,could promote the proliferation significantly(P＜0.05 or P＜0.01).Moreover, all of groups show significant deviation between CORT group(P＜0.01 or P＜0.001), except for 120μmol/L CORT add 5%blank serum group.The results indicate that serum in medium could boost the reproductive activity of nerve precursor cell obviously. Furthermore,the higher concentration of serum could lead to the better reproductive activity.Compare with blank serum,nerve precursor cell in 10%contain medicine serum shows more better reproductive activity,and this difference possess statistical significance (P＜0.01 or P＜0.001).Part three. The results of immunofluorescence cytochemistry are as following.The high concentration corticosterone could restrain the proliferation of hippocampus nerve precursor cell significant(P＜0.01),and to increase this cell apoptosis(P＜0.001).However, the XYS and JWSNS contain medicine serum could promote hippocampus nerve precursor cell differentiation toward neuron,and could lower the apoptosis rate of nerve cell which origin from differentiation as well.Part four.The results of western blotting are as following.The high concentration corticosterone could lower the expression of pro-BDNF and BDNF proteinum,and compare with normal group BDNF only display degrade tendency but insignificant deviation.However,the XYS contain medicine serum could promote the expression of pro-BDNF and BDNF proteinum significant(P＜0.05 or P＜0.01).So it is JWSNS contain medicine serum,but compare with blank serum group there are insignificant deviation.Moreover,the CREB proteinum expression among all groups shows insignificant deviation.Part five.The results of real time fluorescent quantitation PCR are as following.The high concentration corticosterone could lower the expression of BDNF mRNA and NMDAR-1mRNA,but compare with normal group there only display degrade tendency but insignificant deviation.However,the antagon and all serum groups could raise the expression of BDNF mRNA and NMDAR-1mRNA significant(P＜0.01),and compare with blank serum XYS contain medicine serum group possess significant deviation.To compare with blank serum JWSNS contain medicine serum group shows insignificant deviation. ConclusionsPart one.The liver-regulating compound recipe XYS and JWSNS contain medicine serum could promote the proliferation of hippocampus nerve precursor cell and inhibit its apoptosis in high concentration corticosterone condition.Part two.The liver-regulating compound recipe XYS and JWSNS contain medicine serum could promote in vitro culture hippocampus nerve precursor cell's differentiation in high concentration corticosterone condition.The major differentiate direction is toward neuron. And the contain medicine serum could lower the apoptosis of nerve cell which come from differentiation. Part three.The liver-regulating compound recipe XYS and JWSNS contain medicine serum could promote the expression of BDNF mRNA,pro-BDNF and BDNF which from in vitro culture hippocampus nerve precursor cell.Accordingly,to fulfill it anti-irritability hippocampus impaired role.Part four.The liver-regulating compound recipe XYS and JWSNS contain medicine serum could promote the expression of NMDAR-1mRNA which from in vitro culture hippocampus nerve precursor cell.The result indicates that it might be another anti-irritability hippocampus impaired route of XYS and JWSNS.Moreover,further research is necessary.Part five.The mechanism of liver-regulating compound recipe XYS and JWSNS regulate hippocampus cell neurogenesis correlate with the upgrade of hippocampus cell's BDNF proteinum expression.However,NMDAR influence the BDNF proteinum expression of hippocampus cell in blood plasma high concentration corticosterone condition might be an intermedial link.Meanwhile,as an important transcription factor,the phosphorylation level of CREB could direct influence the transcription of BDNF gene.The role of liver-regulating compound recipe XYS and JWSNS to NMDAR and p-CREB in high concentration is still need further study.