Effects And Mechanism Of α-mangostin Derivatives In The Differentiation Of Neural Stem Cells

Background: It is considered that the central nervous system of adult mammals cannot be regenerated.If the neuron is injured or lost, it is difficult to repair and the reconstruction of neural function is regarded as almost impossible. However, in the 1990 s, the discovery of neural stem cells altered the conditional concept that the nerve was irreparable after brain damage. Recently, all different kinds of brain damage, such as cerebral ischemia, epilepsy, can generate kinds of soluble cell damage signals to activate NSCs. It will bring hope to cure this kind of disease if we selectively regenerate the loss of neurons and neuroglia in the cortex by neurogenesis.α-Mangostin is a kind of ketone compounds separated from mangosteen peel, which has a wide range of biological and pharmacological activity. It exerts certain effect on differentiation of neural stem cell because of the similar structure with tacrine. Previous experiments have shown that this kind of derivatives can promote differentiation of neural stem cell.If it can promote the differentiation of NSCs into neurons, it is of great significance for clinical medicine. Objective: The aims of this study are to explore the optimal way of cultivating NSCs by primary culture. After adding α-mangostin derivatives, we detect the proportion of neurons with the help of flow cytometry. And explore the mechanism of differentiation via western blot.Methods: this study can be divided into three parts:(1)Neural stem cells were cultured with 1×105 /ml,1×106 /ml,1×107/ml respectively in order to find the optimal seeding density. The optimal way of culturing neurospheres was tested by either passaging cells via mechanical isolation or enzymatic digestion. Afterwards immumofluorescence staining is used to detect the markers of differentiation of neurospheres.(2) The experiments was divided into three groups, negative control group, no drug group(contain 1% FBS), drug group(100ng/ml α-Mangostin derivatives). The neurons proportion was investigated by flow cytometry by adding NSE(1:100) antibody after 7 days.(3) By means of extracting total protein from cells in A-GM2 treatment, the expression of Notch-1, Wnt-1, Axin, β-catenin, Cyclin D1 were investigated by Western-Blot. The expression of Notch-1, Wnt-1, Axin, β-catenin, Cyclin D1 at diffrent time point were investigated to explain the mechanism of the differentiation of neural stem cell.Results:(1) At the seeding density of 1×105 /ml, NSCs proliferated into different clones and only a few NSCs formed into neurosphere. while at the density of 1×106 /ml, NSCs formed into appropriate neurosphere. At the density of 1×107 /ml, NSCs developed into bigger neurosphere and the central cells were easy to die. The optimal seeding density was 1×106 /ml. In addition, the optimal culturing way was passaging cells via enzymatic digestion which was harmless to cells.(2) Compared with no drug group, the neurons differentiation ratio of several drug groups went higher. The differentiation ratio of neurons was increased from 22% to 37% with 100ng/ml treatment of A-GM2 compared with no drug treatment differentiated cells.(3) Western-Blot data showed that Notch-1 decreased gradually in the whole process of differentiation. It expressed highly in undifferentiated neurosphere and with little or almost no express at later stage of differentiation. It suggested that Notch-1 played a negative regulatory role in the differentiation of NSCs. However, β-catenin increased gradually in the whole process of differentiation because of the decrease of Axin. After that Cyclin D1 increased too.Interestingly, the amount of Wnt-1 increased at the early stage but decreased at the later stage.Conclusion:(1) The optimal seeding density was 1×106 /ml and the optimal culturing way was passaging cells via enzymatic digestion.(2) α-Mangostin derivatives could regulate the differentiation of NSCs, via promoting the differentiation of NSCs into neurons in different degrees.(3) The effect of the differentiation of NSCs into neurons may be related to the activation of Wnt-1.which then leads to the decreased expression of Axin. The phosphorylation of β-catenin was inhibited by repressing the formation of “destruction complex” with Axin, GSK3 and APC. All these contributed to the rapid accumulation of β-catenin. The increase expression of β-catenin leads to the activation of its downstream targets, such as Cyclin D1 and while inhibited the expression of Notch 1. In conclusion, Wnt/β-catenin played a positive role in the differentiation process of NSCs while Notch acted as a negative role in this process.