Online Submissions: http://www.journaltcm.com J Tradit Chin Med 2017 June 15; 37(3): 308-313 [email protected] ISSN 0255-2922 © 2017 JTCM. This is an open access article under the CC BY-NC-ND license. RESEARCH ARTICLETOPIC Velvet antler polypeptide is able to induce differentiation of neural stem cells towards neurons in vitro

Zhang Lihong, Zhuang Zhihong, Sun Yanan, Ma Shuhua, Yang Weifeng, Lei Hongtao, Zuo Junling, Ouyang Jing- feng, Wang Yi aa Zhang Lihong, Zhuang Zhihong, Graduate School, Guang- zyme-linked immunosorbent assay, and detected zhou University of Chinese Medicine, Guangzhou 510006, the special neural molecules by immuno uores- China cence staining. NSCs were cultured on thefl cell Sun Yanan, Ma Shuhua, Yang Weifeng, Lei Hongtao, Ouy- climbing film. After neuronal differentiation, differ- ang Jingfeng, Wang Yi, Morphological Laboratory, Expei- entiated NSCs were mounted for immunocyto- mental Research Center, Research and Development Service Base, China Academy of Chinese Medical Sciences, Beijing chemistry with immunofluorescence technique. 100700, China RESULTS: The differentiating cells look like neuron, Zuo Junling, First Affiliated Hospital of Guangzhou Universi- some special factors, such as Glial cell line-derived ty of Traditional Chinese Medicine, Guangzhou 510405, China neurotrophic factor, nerve growth factor, in the me- Supported by the National Natural Sciences Foundation of dia can be detected while differentiated neuron -like China (No. 81470171) and Basic Science Research Fund from cells can express the special neural molecules. Ministry of Finance of China (No. ZZ2012008 and ZZ2015014) CONCLUSION: Differentiation of NSCs towards neu- Correspondence to: Prof. Ouyang Jingfeng, Morphologi- rons can be induced by velvet antler polypeptide. cal Laboratory, Experimental Research Center, Research and Development Service Base, China Academy of Chinese Med- © 2017 JTCM. This is an open access article under the ical Sciences, Beijing 100700, China. [email protected]; CC BY-NC-ND license. Prof. Wang Yi, Morphological Laboratory, Expeimental Re- search Center, Research and Development Service Base, Chi- Keywords: Cornu cervi pantotrichum; Glial cell na Academy of Chinese Medical Sciences, Beijing 100700, line-derived neurotrophic factors, Nerve growth China. [email protected] factor; Neural stem cells Telephone: +86-18311228848 Accepted: April 3, 2017 INTRODUCTION Human cerebral ischemia and neurodegenrative diseas- es such as Parkinson's disease (PD) and Alzheimer's dis- Abstract ease (AD) are caused by a loss of neurons and glia in the brain or spinal cord.1,2 Though neurons and glial OBJECTIVE: To investigate the neural differentia- cells have successfully been generated from several tion capacity of water extraction of velvet antler. kinds of stem cells such as embryonic stem cells (ESCs),3 induced pluripotent stem cell,4 mesenchymal stem METHODS: Velvet antler (Cervus Nippon Tem- cells5 and neural stem cells (NSCs),6 it seems that proto- minck) polypeptide (VAP) was used to differentiate cols of neural differentiation or co-culture system re- neural stem cells (NSCs) towards neurons in the ported were difficult, complicated and not suitable for study. Firstly, we obtain the polypeptides of VAP by routine use in clinical practice. Therefore, stem water extraction. Secondly, we observed the mor- cells-based cell therapies, transplantation included, phology, assayed the factors in the media by en- should be developed.

JTCM | www. journaltcm. com 308 June 15, 2017 |Volume 37 | Issue 3 | Zhang LH et al. / Research Article

NSCs have the special capacities of self-renew and mul- Extraction of velvet antler polypeptide tilineage differentiation, which make them possible to Extraction of velvet antler polypeptide (VAP) was per- compensate the lost neurocytes and cure neurodegener- formed as follows.8,9 Fresh velvet antler (1 kg), pur- ative diseases such as Parkinson's disease (PD) and Al- chased from Jilin Yunlu Group, were chop into pieces, zheimer's disease (AD). So it is necessary to develop rinsed several times with precooled distilled water until the convenient protocol which can differentiate NSCs the small pieces are completely bloodless, minced into towards neurons. paste, added 5000 mL pre-cooled homogenate (acetic Deer velvet antler, containing numerous regulatory sub- acid solution, pH 3.5) and repeatedly homogenized stance, chemicals and components, such as polysaccha- with a colloid mill. Homogenate was centrifuged at for ride, phosphatide, hormone, mineral composition, 20 min 8500 rpm and 4 , and supernate was ob- polypeptide and protein, is the young antler which is tained, added 95 % ethanol℃ to the supernate (final still not ossified and full of velvet of the male deer Cer- conc., 60 %), placed at 4 and continued stirring. 4 h vus Nippon Temminck.7 later, supernate was centrifuged℃ at 8500 rpm for 20 min The water extract of velvet antler has been widely used again, and after, and new supernate was obtained. Su- in the treatment of some diseases because several axon pernatant was isolated, concentrated by rotary evapora- growth promoters have been contained in the antler or tion under vacuum until a volume of 500 mL re- its extract, which could promote the organ regenera- mained, and then lyophilized the concentrate to obtain tion. Some papers have reported that the extract of vel- crude extract of velvet antler polypeptide, and stored at vet antler could differentiate NSCs, derived from neo- －20 . natal rats or fetal rats' brain, towards neurons. Howev- ℃ er, NSCs derived from brain of neonatal rats or fetal Cells culture rats are different from ones from adult rats. In this Neural stem cells were cultured in DMEM) / F12 sup- study, we aimed to investigate how the polypeptide plemented with 15% FBS, 2 mM L-Glutamine, 1 mL from velvet antler influences differentiation of NSCs NEAA (100 ×), 100 units/mL penicillin and 100 mg/ towards neurons from the brain of adult rats. mL streptomycin, named normal medium (NM), at

37 , 5% CO2 and 95% humidity. Cell culture media were℃ renewed every three days, up to the confluence of METHODS the monolayer. Cells were washed upon formation of confluent cultures, and then detached from the culture Materials flasks or dishes with trypsin-EDTA. Neural stem cells (NSCs, NE4C cell line) were pur- Cell viability assay chased from Cells Resource Center of Shanghai Insti- NSCs were seeded into 96-well micro-culture plates at tutes for Biological Sciences, Chinese Academy of Sci- a concentration of 1 × 104 ences (Shanghai, China). Dulbecco's modified Eagle cells per well. After 12 h in- medium (DMEM) /F12 were purchased from Thermo cubation, cells were exposed to extraction of VAP at Scientific, Waltham, MA, USA. The primary antibody concentrations from 100 to 10 μg/mL, positive control (goat anti neuron-specific enolase, NSE) was pur- treated with 20 ng/mL bFGF & 20 ng/mL NGF and chased from Aves Labs, Inc., China, while the other negative control (medium control). After 48 h, 10 mL primary antibody (rabbit anti-proliferating cell nuclear of MTT solution (5 mg/mL in phosphate buffered so- antigen, PCNA) was purchased from Cell Signaling lution) was added to the culture medium and incubat- Technology, Inc., China. The secondary antibodies, rat ed at 37 for a further 4 h. After removing unconvert- ℃ anti-goat IgG conjugated to phycoerythrin (PE), rat an- ed MTT, 150 mL of DMSO was added to each well ti-rabbit IgG conjugated to fluorescein Isothiocyanate and the plates shaken to dissolve the reduced MTT (FITC) were purchased from Beyond, China. Non-es- crystals (formazan); the optical density was measured sential amino acids (NEAA, 100 ×) and Fetal bovine se- on a microplate reader at a wavelength of 490 nm. The rum (FBS) were purchased from Life Technologies Cor- average of cell viability was evaluated by MTT tetrazoli- poration, Carlsbad, California, USA. Enzyme linked um dye assay. Each experiment was performed three immunosorbent assay (ELISA) quantitation kit (NGF times. and GDNF) were purchased from Abnova, Carlsbad, Neuronal differentiation of NSCs CA, USA. Dishes and 24-well plates were purchased NSCs were seeded into polylysine-coated 24-well from Corning corporation, NY 14831, USA. Basic plates and divided into three groups: VAP group, posi- broblast growth factor (bFGF) and nerve growth factorfi tive control group and negative control group. The (NGF) were purchased from PeproTech Inc., Rocky VAP group was treated with 50 μg/mL VAP, the posi- Hill, NJ, USA. Dimethyl Sulphoxide (DMSO) was tive control group was treated with 20 ng/mL bFGF purchased f from Fisher Chemical. 3-(4,5-dimethylhio- and 20 ng mL NGF together, negative control group azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was treated with normal medium. The protocol of was purchased from Sigma-Aldrich (St. Louis., MO, neuronal differentiation of NSCs was as follows USA). (Figure 1).

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