To Investigate The Mechanism Of The Proliferation And Differentiation Of Neural Stem Cells By Icariin From The PTEN Signaling Pathway

Objectives:To investigate the effects of the main monomer component of Chinese kidney-tonifying drug herba epimedii (icariin) on the proliferation and differentiation of NSCs based on the guidance of the theory of brain and kidneythe, separation and culture in vitro of hippocampus neural stem cells (NSCs), to preliminarily discuss the mechanism of icariin promoting NSCs proliferation and differentiation through detecting the expressions of PTEN, PS6R and other associated proteins, which could provide the experimental basis for icariin inducing proliferation and differentiation in vitro of NSCs, also could provide a novel direction for treating central nervous system disease using Chinese medicine combined with stem cell technique.Methods:1.The neonatal SD rats were killed by cervical vertebra dislocation. The bilateral hippocampi were separated under sterile condition so as to obtain the neural tissue. The neural tissue was mechanically separated. The primary culture and subculture in vitro were performed using the specific culture medium. The expression of nidogen (Nestin) in neural spheres was detected using immunofluorescence method. The result proved that they were neural stem cells.2.The second-generation neural stem cells were randomly divided into five groups after passage, namely 1000 ng/ml icariin group,100 ng/ml icariin group,10 ng/ml icariin group, DMSO group and blank control group, respectively. The effects of icariin on the proliferation of neural stem cells in five groups were detected using CKK-8 method; The effect of ICA on the expression of PTEN signal pathway was observed using Western blot method. The expressions of phosphorylated ribosome protein (P-S6R) reflecting rapamycin target protein (mTOR) activation and tubulin protein were detected.Results:(1) Nestin detection result by immunofluorescence showed that Nestin was positively expressed, proving that the cultured cells were neural stem cells with self-renewal capacity. (2)The cell proliferation experiment result by CKK-8 method showed that the absorbance value of ICA group was significantly higher than that of DMSO group and blank control group, and the highest value was 1000ng/ml (P<0.05). (3)WB detection results showed that compared with DMSO group and blank control group, the expression of PTEN protein in hippocampus of neonatal SD rats was significantly inhibited in icariin group, the expressions of PS6R protein and tubulin protein were significantly up-regulated in icariin group. Moreover, when the same drug acted on the different cells, the effect was most significant in 1000ng/ml icariin group (P<0.05).Conclusions:Icariin could promote the proliferation and differentiation of exogenous neural stem cells into neurons, which was achieved by inhibiting the expression of PTEN and increasing the expression of P-S6R.