The Study Of The Mechanisms And Effects Of Ginkgolide B On The Differentiation Of Neuron Stem Cells

Repair treatment of central nervous system is popular and difficult in the physical medicine and rehabilitation. Among the central nervous system diseases and injuries, cerebral vascular diseases have the highest proportion, followed by the nervous system degenerative diseases, brain and spinal cord injury. Though the central nervous system diseases and injuries have different etiology, but all have various degrees of neuronal loss and structure damage and dysfunction on pathology. Neural stem cells (NSCs) exist widely in the mammalian central nervous system and posses the capability of self-renewing, self-duplication, and the potentiality of differentiating into various neural cells. Neural stem cell transplantation in the treatment of central nervous system diseases and injuries in recent years attracts much attention, can be achieved to repair nerve pathways, and make nerve function recover, brings hope in the clinical practice.It is well know that the mechanism of proliferation and differentiation of NSCs is very complicated which is regulated by positive and negative regulator. Various signaling molecules which controling the fate of NSCs is synergy and mutual dependence, the inherent differentiation process of NSCs, cytokines, extracellular matrix, adhesion molecules in the local environment, the interaction between the cells all may be closely related to proliferation, differentiation of NSCs. The effect of cytokines also is regulated by many factors, including SOCS (signaling Suppressors of cytokines) family.Id (Inhibitor of DNA Binding) works as a negative regulator of bHLH (Helix-loop-helix) transcription factor. As a member of Id family, Id2 broadly expresses in the brain and spinal cord in situ hybridization and has an important effect in the development of central nervous system. Id2 can block the function of bHLH transcription factor, inhibit NSCs differentiate into neurons. SOCS (Suppressors of cytokine signaling) proteins discovered recently can be induced by a wide range of cytokines including growth factors and hormone. They work as negative feedback regulators of cytokine signal pathways. As a member of SOCS family, researchers show that SOCS2 mRNA is expressed in many tissues of embryonic and adult animals, especially in neuron. Recently studies showed that SOCS2 was a potent regulator of neural differentiation, and played an important role in the control of neural differentiation and cell fate. It can promote NSCs to differentiate into neurons, promote neurite growth. Therefore, SOCS2 plays an important role in both structure and function of neuron.Currently the study of proliferation and differentiation on NSCs concentrated on cytokines in the domestic and international. Chinese medicine is our national essence, has tremendous potential in the experimental research and clinical application. Therefore application of Chinese medicine on proliferation and differentiation of NSCs to promote nerve regeneration and restore nerve function, can opened up a new therapeutic approaches on the treatment of central nervous system diseases and injuries. But the studies of Chinese medicine on neural stem cell proliferation and differentiation limit on the phenomenon observation, and lack of in-depth study on the mechanism. Ginkgo is an ancient, magical plant, famous as "living fossil". Ginkgolides is the main active ingredient of Ginkgo biloba extract (EGb), and Ginkgolide B (GKB) is the largest effective component, and effect of anti-human platelet-activating factor (PAF) is the most powerful. Researchers found that Ginkgo biloba extract can promote regeneration of peripheral nerve, GKB can promote NSCs differentiation into neuron, but its mechanism is not yet clear. Therefore, the objective of this study was to observe the effect of GKB of various consistency on the differentiation of NSCs, explore initially the mechanisms of its action and provide a basic research about GKB in neurological experimental studies and clinical rehabilitation.To answer these questions, firstly NSCs were dissociated from SVZa of lateral cerebral ventricle of fetal SD rats. Secondly, cultured in differentiation medium containing 5% fetal bovine serum (FBS) and GKB for 6h,12h/ 24h,3d and 7d, the NSCs were induced to differentiation by different consistency of GKB to observe the effects of GKB on neurite outgrowth and the differentiation of NSCs. the neurite number, length and cell body area were photographed and measured by inverted phase-contrast micrograph, then Microtubule-associated protein(β-Tubulin),Glial fibrillary acidic protein (GFAP),Oligodendrocyte-specific protein (CC-1) expression were detected and counted by fluorescence microscope. Thirdly, cytokine factory-SOCS2,transcription factory -Id2 also were immunostained. The percentage of SOCS2,Id2 positive cells and the mean optical densities of immunoreactive products were obtained respectively.The main results are as follows:1. Successful isolated NSCs from anterior subventricular zone of lateral cerebral ventricle (SVZa) of fetal SD rats, cultured SVZa NSCs in vitro and identificated by Nestin-positive expression.2. Ginkgolide B increases neurites number and length and cell body area . At the same time (24h or 3d,7d), the neurite number,length and cell body area in the 20 mg / L,40 mg / L,60 mg / L GKB group were significantly increased than that in the control group (P <0.01); the neurite number,length and cell body area in the 40 mg / L,60 mg / L GKB group were significantly increased than that in 20mg/L GKB group (P <0.01), but there was not significantly different between 40 mg / L and 60 mg / L group (P> 0.05). At the different time (24h and 3d), the neurite length on 3d in the GKB groups and the control group were markerly increasd than that on 24h (P <0.01); the neurite number in the control group was not significantly different between 24h and 3d (P> 0.05), while the neurite number in GKB groups was significantly increased between 24h and 3d (P <0.01).3. Ginkgolide B increases the percentage ofβ-Tubulin positive neuron-like cells and GFAP positive astrocyte-like cells, but has no effect on CC-1 positive oligodendrocyte-like cells. The percentage ofβ-Tubulin positive neuron-like cells in GKB groups was significantly higher than that in the control group. The percentage ofβ-Tubulin positive neuron-like cells in the 40 mg / L,60 mg / L GKB group were significantly increased than that in 20mg/L GKB group (P <0.01), but there was not significantly different between 40 mg / L and 60 mg / L group (P> 0.05). The percentage of GFAP positive astrocyte-like cells in GKB groups was significantly higher than that in the control group, and there were significantly different in the GKB groups (P <0.05-0.01 ), The percentage of GFAP positive astrocyte-like ells increase in a dose-dependent manner with various consistency of GKB. But there were no significantly differences of CC-1 positive oligodendrocyte-like cells among GKB groups and the control group.4. Ginkgolide B decreases the percentage of Id2 positive cells and the mean optical densities of Id2 immunoreactive products. The percentage of Id2 positive cells in the control group,20mg/L,40 mg/L,60 mg/ L GKB group was 66.24%,57.46%,51.52%,52.03%, and the mean optical densities of Id2 immunoreactive products was 5.272,4.457,3.820,3.702. The percentage of Id2 positive cells and the mean optical densities of Id2 immunoreactive products in GKB groups were significantly lower than that in the control group (P <0.01); The percentage of Id2 positive cells and the mean optical densities of Id2 immunoreactive products in 40 mg / L,60 mg / L GKB group significantly decreased compared with 20 mg / L GKB group (P <0.01), but there was not significantly different between 40 mg / L and 60 mg GKB group (P> 0.05).5. Ginkgolide B increases the percentage of SOCS2 positive cells and the mean optical densities of SOCS2 immunoreactive products. The percentage of SOCS2 positive cells in the control group,20mg/L,40mg/L,60 mg/L GKB group was 22.17%,30.24%,35.46%,34.72%, and the mean optical densities of SOCS2 immunoreactive products was 1.134,1.382,1.578,1.527. The percentage of SOCS2 positive cells and the mean optical densities of SOCS2 immunoreactive products in GKB groups were significantly higher than that in the control group (P <0.01); The percentage of SOCS2 positive cells and the mean optical densities of SOCS2 immunoreactive products in 40 mg / L,60 mg / L GKB group significantly increased compared with 20 mg / L GKB group (P <0.01), but there was not significantly different between 40 mg / L and 60 mg GKB group (P> 0.05).In summary, Ginkgolide B promotes neurite growth, enhances the neurites number and length and cell body area, promotes NSCs to differentiation into neuron and astrocyte, but has no effect on oligodendrocyte differentiation, simultaneously upregulates SOCS2 expression and downregulates Id2 expression. Socs2 and Id2 might have an important role in regulating neuronal differentiation and maturing neuronal morph and function. Ginkgolide B promotes NSCs to differentiate into neuron and mature neural structure and function by upregulating SOCS2 expression and downregulating Id2 expression. We hope our study can provide some basic data about GKB for further illustrating the mechanism of differentiation of NSCs.