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Oleanolic acid enhances neural stem cell migration, proliferation, and differentiation in vitro by inhibiting GSK3β activity

Oleanolic acid enhances neural stem cell migration, proliferation, and differentiation in vitro... Oleanolic acid (OA), one of the bioactive ingredients in ginseng, has been reported to have neuroprotective activities. However, the effects and its mechanism on neural stem cell (NSC) induction are not entirely clear. In the present study, we investigated the effects of OA on promoting the migration, proliferation, and differentiation of neural stem cells (NSCs). Migration and proliferation were investigated by using neural-specific markers, neurosphere assay, and Cell Counting Kit-8, respectively. We found OA remarkably promoted neural migration and proliferation of NSCs in a time- and dose-dependent manner. Differentiation was analyzed by western blotting and immunofluorescence staining, which found MAP2 expression was remarkably increased, whereas Nestin was dramatically decreased. In addition, OA increased phosphorylation of GSK3β at Ser9 and expression of active forms of β-catenin. Furthermore, NSCs with constitutively active GSK3β (S9A) significantly suppressed the OA-induced proliferation and neural differentiation. These results showed that OA could stimulate NSC proliferation and neural differentiation in vitro via suppressing the activity of GSK3β. Our findings may have significant implications for the treatment of neurodegenerative diseases. Introduction differentiation but the potential mechanism of OA- Oleanolic acid (OA), one of the key bioactive ingre- mediated NSC induction is unknown. dients of ginseng, is a triterpenoid compound that possess The rapidly aging population makes neurodegenerative pharmacological properties including neuroprotective , diseases such as Alzheimer’s disease and Parkinson’s anti-cancer, and anti-inflammatory activities . Recently, disease a primary healthcare concern. The main patho- OA was suggested to be a promising neuroprotective genesis of neurodegenerative disease is the progressive 3 5 agent but its effects are far from clear. In addition, it loss of function or death of neurons . One promising was reported to improve neural stem cell (NSC) therapeutic approach for treating neurodegenerative dis- eases is neuron transplantation or inducing the neuro- genic differentiation of NSCs in situ . However, NSCs have limited ability to self-renew and differentiate into Correspondence: Stephen Cho Wing Sze (stephensze@gmail.com)or neurons, astrocytes, and oligodendrocytes under normal Ken Kin Lam Yung (kklyung@hkbu.edu.hk) conditions , and require induction by growth factors Faculty of Science, Department of Biology, Hong Kong Baptist University (HKBU), Hong Kong, China (GFs). It was reported that residual GFs may increase the HKBU Shenzhen Research Institute and Continuing Education, Shenzhen, 8 risk of developing a tumor after transplantation .As a China consequence, greater attention has been paid to the Guangzhou Institute of Cardiovascular Disease, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, China development of active ingredients extracted from natural Center for Cancer and Inflammation Research, School of Chinese Medicine, medicines that can promote NSC proliferation and neural HKBU, Hong Kong, China These two authors contributed equally: Shi Qing Zhang and Kai Li Lin. Edited by R Killick © 2018 The Author(s). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Zhang et al. Cell Death Discovery (2019) 5:48 Page 2 of 9 Fig. 1 OA increases NSC migration in the neurosphere assay. a Neurospheres seeded onto a 24-well PDL-coated plate were treated with different concentrations of OA for 24 h. Blue arrows indicate the migration distance from neurospheres. b OA-induced migration distance increased aa in a dose-dependent manner. Data are presented as means ± SD, n = 5. **p < 0.01, compared with the Ctr group; p< 0.01, compared with 10 μM bb OA group; p< 0.01, compared with 20 μM OA group. c Migrating cells expressed Nestin and MAP2 neural markers. Scale bars: 100 μm differentiation, especially active small molecules that can observed to emerge from the neurosphere and migrate effectively cross the blood–brain barrier (BBB). along a radial axis (Fig. 1). The OA treatment GSK3β/β-catenin signaling is important in the regula- significantly increased the migration distance in a tion of NSC proliferation and differentiation . Inactivation dose-dependent manner (Fig. 1a, b). The immuno- of GSK3β by phosphorylation at Ser9 accelerated the fluorescence staining showed the cells derived from the nuclear translocation of unphosphorylated active β-cate- neurospheres were MAP2-positive and mostly Nestin- nin, promoting NSC proliferation and differentiation . positive cells (Fig. 1c). These findings indicate that inactivation of GSK3β might be a promising target to induce NSC proliferation and OA promoted proliferation of NSCs differentiation. Proliferation of NSCs induced by OA was investigated In the present study, we investigated the effects of OA by CCK-8 assay and neurosphere assay. The CCK-8 on NSC proliferation and neural differentiation. Con- assay showed a slight absorbance increase on day 2 after sidering the importance of the activated state of GSK3β in culture, but this was not significant. On day 3 after controlling NSC induction, we used genome-editing treatment, the absorbance increased steeply and cell techniques to investigate the role of GSK3β activation in viability in the 20 and 40 μMOAgroupswas sig- regulating NSC proliferation and differentiation. To the nificantly higher than in the 10 μMOAgroup (Fig. 2a). best of our knowledge, this is the first study to confirm The cell proliferation and cell viability increased sig- that OA mediates NSC proliferation and neural differ- nificantly with OA treatment in a dose-dependent entiation through suppressing the activation of GSK3β. manner. The neurosphere assay showed the number and diameter of neurospheres generated from NSCs Results were significantly increased with the OA treatment in a OA increased NSC migration dose-dependent manner (Fig. 2b–d). These results Migration of NSCs was analyzed after treatment indicate that OA can significantly promote NSC pro- with OA (10, 20, and 40 μM). The neural cells were liferation and self-renewal in vitro. Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death Discovery (2019) 5:48 Page 3 of 9 Fig. 2 OA promotes the proliferation of NSCs. NSCs were treated with different concentrations of OA (10, 20, and 40 μM). OA-mediated NSC proliferation was analyzed by CCK-8 assay after OA treatment for 1, 2, or 3 days (a) and neurosphere assay was performed after OA treatment for 3 days (b–d). a CCK-8 assay showed cell viability was significantly enhanced in a dose- and time-dependent manner. Data are presented as means ± SD, n = 5. b An image of neurospheres after treatment with OA. Scale bars: 100 μm. The number (c) and the diameter (d) of neurospheres (diameter >30 μm) increased with OA treatment in a dose-dependent manner. Data are presented as mean ± SD, n = 5. *p < 0.05 and **p < 0.01, compared with the Ctr group; p< 0.05, compared with 10 μM OA group OA promoted the neural differentiation of NSCs Interestingly, we observed many MAP2 and Nestin NSCs have the potential to differentiate into multiple double-stained cells in the immunofluorescence staining, neural lineages, mostly neurons and astrocytes. Differ- indicating some of the NSC-derived neurons were entiation of NSCs after treatment with OA was deter- approaching maturation . mined by measuring the levels of three neural markers: Nestin, MAP2, and GFAP. The western blot analyses OA directly targeted GSK3β showed OA significantly and dose-dependently reduced The GSK3β/β-catenin signaling is considered to be a the expression of Nestin and upregulated the expression critical regulator for NSC proliferation and differentia- of MAP2, which is an exclusive dendritic protein in tion . Inhibition of GSK3β activity by phosphorylation at neurons. Furthermore, there was a decreasing trend in the Ser9 causes unphosphorylated active β-catenin to expression of GFAP after treatment with 20 and 40 μM accumulate in the cytoplasm and then translocate into the OA (Fig. 3a, b). These results demonstrate that OA can nucleus, promoting NSC proliferation and differentia- markedly promote neural differentiation of NSCs and tion . To explore the interaction between OA and suppress astrocyte differentiation in a dose-dependent GSK3β, we performed a molecular docking analysis using manner. These findings were also confirmed by immu- the Autodock Vina molecular docking program. The nofluorescence staining. As shown in Fig. 3c, d, the per- binding energy of the GSK3β-OA complex was − centage of MAP2-positive cells significantly increased 8.871 kcal/mol, which suggested good binding ability. The with increasing OA concentration, whereas the percen- three-dimensional binding conformation of the GSK3β- tage of GFAP-positive cells showed a downward trend. OA complex showed that OA interacted with GSK3β at Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death Discovery (2019) 5:48 Page 4 of 9 Fig. 3 OA selectively induces neural differentiation of NSCs. Treatment with OA for 7 days dose-dependently promoted the neural differentiation of NSCs, but suppressed astrocyte differentiation, as determined by western blotting (a, b) and immunofluorescence staining (c, d). a, b OA significantly and dose-dependently decreased the expression of Nestin and increased the expression of MAP2, but expression of GFAP was not significantly affected. c, d OA significantly and dose-dependently decreased the proportion of Nestin-positive cells and increased the proportion of MAP2-positive cells. Scale bars: 100 μm. All data are presented as means ± SD from three independent experiments. *p < 0.05 and **p < 0.01, a aa bb compared with the Ctr group; p< 0.05 and p< 0.01, compared with 10 μM OA group; p< 0.01, compared with 20 μM OA group LEU-188, THR-138, ASN-186, GLN-185, CYS-199, ALA- of β-catenin (active) to β-catenin compared with 83, VAL-70, ILE-62, GLY-63, PHE-67, and ASN-64 via the control group (Fig. 5a, b). The results showed that van der Waals force (Fig. 4a). To further verify the OA-mediated NSC proliferation and differentiation molecular docking results, the best conformation of might depend on the activation of GSK3β/β-catenin GSK3β-OA was taken as the starting conformation for the pathway. MD simulation by YASARA. As shown in Fig. 4b, the To further confirm the involvement of the activation of heavy atoms root-mean-square deviation (RMSD) track of GSK3β in OA-mediated NSC proliferation and differ- the GSK3β (Fig. 4b, red line) mildly fluctuated around 2 Å entiation, we activate GSK3β by transfection of con- over 0–100 ns and the RMSD track of OA (Fig. 4b, blue stitutively active GSK3β mutant at S9A using the GSK3β line) fluctuated around 0.2 Å during the MD simulation. (S9A) adenovirus. The transfection was confirmed by The surface visualization models of the GSK3β–OA western blot analysis (Fig. 5c, d). The expression ratio of complex at 0 and 100 ns are shown in Fig. 4c. We found p-GSK3β (Ser9) to GSK3β was significantly decreased to OA was stably presented at the center of the GSK3β about 30% in GSK3β (S9A) small interfering RNA binding site throughout the MD simulation. These results (siRNA) group compared with the control siRNA group, indicate that the binding between GSK3β and OA is leading to significant downregulation of the ratio of stable, and that OA likely targets GSK3β directly. β-catenin (active) to β-catenin. As expected, cell viability GSK3β (S9A) of NSCs induced by OA was significantly OA-mediated NSC induction through suppressing the attenuated (Fig. 5e). Furthermore, OA-mediated NSCs activation of GSK3β mutated with GSK3β (S9A) had significantly increased According to the results of the western blot Nestin expression, but decreased MAP2 expression analysis, NSCs treated with OA for 7 days showed (Fig. 5f, g). Overexpression of constitutively active GSK3β significant and concentration-dependent increases in (S9A) significantly attenuated OA-mediated NSC pro- the expression ratios of p-GSK3β (Ser9) to GSK3β and liferation and differentiation. All these results suggest that Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death Discovery (2019) 5:48 Page 5 of 9 Fig. 4 Molecular docking and molecular dynamics simulation. a Three-dimensional crystal structure of the complex of OA (ZINC95098891) with GSK3β (PDB ID: 4ACC). b Plots of root-mean-square deviation (RMSD) of heavy atoms of GSK3β (red) and OA (blue). c Surface presentation of the GSK3β-OA complex crystal structure at 0 and 100 ns the activated state of GSK3β is critical in OA-mediated Recently, several NSC replacement therapies have been proliferation and differentiation of NSCs. rapidly developed that may be promising treatments for neurodegenerative diseases, but they require in vitro Discussion induction of NSCs into functional neurons. NSCs have As neurodegenerative diseases are typically char- limited ability to proliferate and differentiate in natural acterized by the gradual loss of structure and/or func- conditions and require induction by GFs such as EGF tion of neurons in the brain, neuron replacement has and bFGF. However, in cell replacement therapy, GFs may become the most promising method to treat neurode- lead to a higher risk for carcinogenicity in vivo after 13 17,18 generative diseases . In the present study, we investi- transplantation . As a natural active component, OA gated OA, a bioactive molecule, as a potential treatment might be an ideal alternative for GFs to induced NSC for neurodegenerative diseases through the induction of proliferation and differentiation efficiently and safely. NSCs. We confirmed that OA could dose-dependently GSK3β has a critical role in regulating the behavior and promote the migration and proliferation of NSCs. In fate of NSCs. Inhibiting the activity of GSK3β can sig- addition, OA could dose-dependently induce NSC nificantly promote NSC proliferation and neural differ- neural differentiation, while suppressing astrocyte dif- entiation . We investigated the ability of OA to target ferentiation. Many macromolecular drugs cannot pass GSK3β directly by performing molecular docking and MD the BBB to target pathological changes in the brain . simulations to assess the mechanism by which OA and Meanwhile, a study demonstrated that OA could be GSK3β interact. The binding energy of the OA-GSK3β effectively delivered across the BBB , further support- complex indicated strong binding interactions. Moreover, ing the development of OA as a promising drug for OA binding to GSK3β was through LEU-188, THR-138, treating neurodegenerative diseases that can be admi- ASN-186, GLN-185, CYS-199, ALA-83, VAL-70, ILE-62, nistered orally. GLY-63, PHE-67, and ASN-64 via van der Waals force. Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death Discovery (2019) 5:48 Page 6 of 9 Fig. 5 OA regulates the activation of GSK3β in NSC induction. Proliferation and differentiation of NSCs mediated by OA depends on the suppressing of the activation of GSK3β. a Western blot analyses and b relative optical density of diverse pathway markers including β-catenin (active), β-catenin, p-GSK3β (Ser9), and GSK3β in NSCs after treatment with OA for 7 days. *p < 0.05 and **p < 0.01, compared with the Ctr group; p< 0.05, compared with 10 μM OA group. c Western blot analyses and d relative optical density of p-GSK3β (Ser9), GSK3β, p-GSK3β (Ser9), and GSK3β in NSCs after transfection with GSK3β (S9A) adenovirus for 2 days. **p < 0.01, compared with the Ad-Ctr group. e NSCs with GSK3β (S9A) significantly suppressed cell viability, as examined by CCK-8 assay. **p < 0.01, compared with Ad-Ctr+ 20 μM OA group. f Western blot analyses and g relative optical density of diverse differentiation markers including Nestin, MAP2, and GFAP in NSCs transfected with GSK3β (S9A) treated with OA (20 μM). NSCs with GSK3β (S9A) significantly suppressed NSC neural differentiation. **p < 0.01, compared with Ad-Ctr+ 20 μM OA group. All data are presented as means ± SD from three independent experiments These residues are key residues involved in inhibiting OA directly targets GSK3β and can potentially act as a the binding domain of GSK3β . Furthermore, the MD GSK3β inhibitor. This was consistent with the Western simulations demonstrated that the binding conformation blotting results that showed OA significantly and dose- of OA-GSK3β was stable. These findings indicated that dependently suppressed the activation of GSK3β.To Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death Discovery (2019) 5:48 Page 7 of 9 further confirm that the effect of OA on NSC induction method when the diameter of the neurospheres reached was mediated through GSK3β, we transfected NSCs with 150–200 μm. the constitutively active GSK3β (S9A) adenovirus. Wes- tern blotting showed successful knockdown of p-GSK3β Migration analysis (Ser9), leading to downregulation of the expression of β- OA (purity assayed by high-performance liquid chro- catenin (active). The proliferation and differentiation matography: 98.33%) was purchased from Must Bio- mediated by OA were significantly restrained in NSCs Technology Co., Ltd (Chengdu, China). Neurospheres transfected with GSK3β (S9A) adenovirus. These results (around 200 μm in diameter) were seeded on coverslips, suggest the activity of GSK3β might be critical in OA- which had been coated with poly-D-lysine (PDL; Sigma, mediated NSC proliferation and differentiation. In this St. Louis, MO, USA) for 30 min to enable neurospheres study, the change of activated state of GSK3β made cor- to attach. The neurospheres were exposed to a series responding change of β-catenin (active). Both GSK3β and of OA concentrations (10, 20, and 40 μM). After incuba- β-catenin (active) are regulated by multiple pathways, one tion for 24 h, photos of the neurospheres were taken of which is Wnt signaling pathway which plays a crucial under an inverted microscope. The migration distance role in NSC proliferation and differentiation . Although of neural cells from the edge of the neurospheres further research is needed to fully explore the role of Wnt was measured by Image-J software. Immunostaining signaling pathway involved in OA-mediated NSC was performed to visualize Nestin (NSCs marker) induction. and microtubule-associated protein-2 (MAP2, neuron NSCs can be an essential source of neural cells for marker) as described below. the treatment of many incurable diseases including neurodegenerative diseases. One of the major challenges Neurosphere assay is to induce neural differentiation of NSCs over Single cells were dissociated from the neurospheres and other lineages and to ensure the treatment is safe. In adjusted to a density of 2.5 × 10 cells/mL. The cells were this study, we demonstrated that OA promoted the seeded onto a 96-well plate (200 μL/well) and treated with differentiation of NSCs into neurons but suppressed various concentrations of OA (10, 20, and 40 μM) without the formation of astrocytes. OA is derived from GFs. After 3 days of incubation, the number and diameter ginseng, which has a long history in many clinical of neurospheres (diameter > 30 μm ) were analyzed by applications, and may provide a safe and efficient Image-J software. way for NSC induction. Our significant findings hold much promise for improving NSC replacement CCK-8 assay therapy in neurodegenerative diseases and other related Single cells were dissociated from the neurospheres and diseases. adjusted to a density of 2.5 × 10 cells/mL. The cells were seeded onto a 96-well plate (200 μL/well) and treated with Methods and materials various concentrations of OA (10, 20, and 40 μM) for NSC isolation and primary cell culture different experimental periods (1, 2, and 3 days). At the The study was approved by the Department of Health, end of each experimental period, CCK-8 (DOJINDO, the Government of the HKSAR. The modified experi- Rockville, MD, USA) reagent was added to measure cell mental protocol was carried out in accordance with proliferation. After further incubation at 37 °C for 4 h, the relevant guidelines and regulations of the Animal Ethics absorbance of each well was measured at a wavelength of Committee at HKBU. Rats (Sprague–Dawley) at postnatal 450 nm using an automatic microplate reader (BioTek). day 1 to 2 were purchased from the Chinese University of Hong Kong. The subventricular zone of the brain was Cell differentiation study dissected out and manually cut into pieces. After digestion Single cells were seeded at 2 × 10 cells/well onto a six- in trypsin at 37 °C for 15 min, the tissue suspension was well PDL-coated plate and then treated with different filtered through a Cell Strainer with a 40 µm mesh. The concentrations of OA (10, 20, and 40 μM). After incuba- filtrate was centrifuged for 5 min at 200 × g and the tion for 7 days, NSC differentiation was detected by supernatant was discarded. The pellet was resuspended in measuring the levels of differentiation markers, including neurobasal medium which contains 20 ng/mL epidermal Nestin, MAP2, and glial fibrillary acidic protein (GFAP, GF (EGF, PeproTech, Rocky Hill, NJ, USA), 20 ng/mL astrocytes marker) , by western blotting and immuno- basic fibroblastic GF (bFGF, PeproTech), 1% PSN fluorescence staining. (Thermo, Waltham, MA USA), and 2% B27 supplement (Thermo), and then cultured at 37 °C in a 5% CO Immunofluorescence staining humidified incubator. Half of the medium was changed Cells were fixed with 4% paraformaldehyde (Sigma) every 2–3 days. The cells were passaged by mechanical for 30 min at room temperature. The cells were then Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death Discovery (2019) 5:48 Page 8 of 9 incubated with the primary antibodies including anti- between the box and solute. The simulated annealing Nestin antibody, anti-MAP2 antibody, and anti-GFAP minimizations were initially set at 298 K and velocities antibody (1:1000, Millipore, Temecula, CA, USA) in were scaled down by 0.9 with every ten steps lasting for phosphate-buffered saline (PBS), with 0.1% Triton X-100 5 ps. After the energy was minimized, the temperature of (Sigma-Aldrich) and 2% normal goat serum (Vector the system was adjusted using a Berendsen thermostat to Laboratories, Burlingame, CA, USA) overnight at 4 °C. minimize temperature control influences. In addition, velocities were rescaled only every 100 simulation steps, After rinsing with PBS three times, the cells were incu- bated with the secondary antibodies at room temperature whenever the mean of last 100 detected temperatures for 3 h. The cells were then incubated with 1 μg/mL 4′,6- converged. Finally, 100 ns MD simulations were con- diamidino-2-phenylindole (Roche, Switzerland) for ducted every 2 fs and the coordinates of the complexes 15 min to stain the nuclei. After rinsing with PBS, fluor- were saved every 10 ps. escence mounting medium (Dako, Aglient, Santa Clara, CA, USA) was added, and immunoreactivity images were Adenovirus-mediated expression of constitutively active obtained and processed under a confocal microscope GSK3β (FluoView FV1000, Olympus, Tokyo, Japan). The pAdM-FH-GFP-GSK3β (S9A) adenoviral vector [Ad-GSK3β (S9A)] expressing the constitutively active Western blotting GSK3β with the serine residue at position 9 mutated to Cellular proteins were extracted using protein extrac- alanine [GSK3β (S9A)] and the empty adenoviral vector tion reagent (Novagen, Madison, WI, USA) supplemented were generated by Vigene Bioscience (Rockville, MD, with a protease inhibitor cocktail (Calbiochem, San Diego, USA). Single cells dissociated from neurospheres were CA, USA) on ice. Protein concentration was determined seeded at 2 × 10 cells/well onto a six-well PDL-coated with a protein assay kit (Bio-Rad, Hercules, CA, USA). plate. The NSCs were transfected with Ad-GSK3β (S9A) Next, proteins (30 μg) were separated on a 10% SDS- or empty adenoviral vector (Ad-Ctr) as the control at a polyacrylamide gel and transferred to a polyvinylidene multiplicity of infection of 40 in the presence of 6 μg/mL difluoride membrane (Bio-Rad). The membrane was polybrene (Sigma). The medium containing the virus was incubated with specific primary antibodies overnight at then exchanged for fresh medium after 12 h of transfec- 4 °C. Primary antibodies included anti-Nestin antibody tion. Cells were cultured for at least 48 h after transfection (1:1000, Millipore), anti-MAP2 antibody (1:1000, Milli- before further experiments were performed. The trans- pore), anti-GFAP antibody (1:1000, Millipore), anti-p- gene expressions were confirmed in the infected cells by GSK3β (Ser9) antibody (1:1000, Cell Signaling, Beverly, analyzing protein expressions. MA, USA), anti-GSK3β antibody (1:1000, Cell Signaling), anti-Non-p (active) β-catenin antibody (1:1000, Cell Sig- Statistical analysis naling), and anti-β-catenin antibody (1:1000, Cell Signal- All data were expressed as means ± SD. Statistical ana- ing). The membrane was then incubated with the lysis was performed by one-way analysis of variance. secondary antibody for 1 h at room temperature. β-Actin Relative risk was expressed as odds ratios with 95% con- antibodies (1:5000, Sigma) were used as the reference. fidence interval and statistical significance was defined as The bands were visualized and imaged using the Chemi- p < 0.05. The statistical analyses were conducted using Doc Touch imaging system (Bio-Rad). GraphPad Prism. Molecular simulation of the interaction of OA and GSK3β Acknowledgements This work was financially supported by National Natural Science Foundation of Autodock Vina molecular docking software (Scripps China (NSFC) grants (No. 81703728 and 81774100). Research Institute, La Jolla, CA, USA) was used to analyze the binding mechanism between GSK3β (PDB ID: Author contributions 4ACC) and OA (ZINC95098891). YASARA was used to K.K.L.Y., S.C.W.S., and S.Q.Z. designed the experiment. S.Q.Z., K.L.L., S.C.W.S., C.Y. perform the MD simulation and the energy minimiza- L., W.S.T., and S.S.M.W. generated cell culture data. S.Q.Z., K.L.L., and S.C.W.S. performed statistical analysis and drew illustrative figures. B.L. and X.Q.F. tion of the ligands, and AMBER 03 forcefield was used to performed bioinformatics analyses. S.Q.Z., B.L., S.C.W.S., and K.K.L.Y. wrote the run all simulations. The star conformation was considered manuscript. the best conformation for the molecular dynamics (MD) simulation. In this study, the inhibitor-binding domain of Conflict of interest GSK3β corresponded to the protein-ligand binding site of The authors declare that they have no conflict of interest. GSK3β. The protein structure of GSK3β was prepared by removing water molecules and bound ligands. 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Oleanolic acid enhances neural stem cell migration, proliferation, and differentiation in vitro by inhibiting GSK3β activity

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Springer Journals
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Copyright © 2018 by The Author(s)
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Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Stem Cells; Apoptosis; Cell Cycle Analysis
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2058-7716
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10.1038/s41420-018-0111-0
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Abstract

Oleanolic acid (OA), one of the bioactive ingredients in ginseng, has been reported to have neuroprotective activities. However, the effects and its mechanism on neural stem cell (NSC) induction are not entirely clear. In the present study, we investigated the effects of OA on promoting the migration, proliferation, and differentiation of neural stem cells (NSCs). Migration and proliferation were investigated by using neural-specific markers, neurosphere assay, and Cell Counting Kit-8, respectively. We found OA remarkably promoted neural migration and proliferation of NSCs in a time- and dose-dependent manner. Differentiation was analyzed by western blotting and immunofluorescence staining, which found MAP2 expression was remarkably increased, whereas Nestin was dramatically decreased. In addition, OA increased phosphorylation of GSK3β at Ser9 and expression of active forms of β-catenin. Furthermore, NSCs with constitutively active GSK3β (S9A) significantly suppressed the OA-induced proliferation and neural differentiation. These results showed that OA could stimulate NSC proliferation and neural differentiation in vitro via suppressing the activity of GSK3β. Our findings may have significant implications for the treatment of neurodegenerative diseases. Introduction differentiation but the potential mechanism of OA- Oleanolic acid (OA), one of the key bioactive ingre- mediated NSC induction is unknown. dients of ginseng, is a triterpenoid compound that possess The rapidly aging population makes neurodegenerative pharmacological properties including neuroprotective , diseases such as Alzheimer’s disease and Parkinson’s anti-cancer, and anti-inflammatory activities . Recently, disease a primary healthcare concern. The main patho- OA was suggested to be a promising neuroprotective genesis of neurodegenerative disease is the progressive 3 5 agent but its effects are far from clear. In addition, it loss of function or death of neurons . One promising was reported to improve neural stem cell (NSC) therapeutic approach for treating neurodegenerative dis- eases is neuron transplantation or inducing the neuro- genic differentiation of NSCs in situ . However, NSCs have limited ability to self-renew and differentiate into Correspondence: Stephen Cho Wing Sze (stephensze@gmail.com)or neurons, astrocytes, and oligodendrocytes under normal Ken Kin Lam Yung (kklyung@hkbu.edu.hk) conditions , and require induction by growth factors Faculty of Science, Department of Biology, Hong Kong Baptist University (HKBU), Hong Kong, China (GFs). It was reported that residual GFs may increase the HKBU Shenzhen Research Institute and Continuing Education, Shenzhen, 8 risk of developing a tumor after transplantation .As a China consequence, greater attention has been paid to the Guangzhou Institute of Cardiovascular Disease, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, China development of active ingredients extracted from natural Center for Cancer and Inflammation Research, School of Chinese Medicine, medicines that can promote NSC proliferation and neural HKBU, Hong Kong, China These two authors contributed equally: Shi Qing Zhang and Kai Li Lin. Edited by R Killick © 2018 The Author(s). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Zhang et al. Cell Death Discovery (2019) 5:48 Page 2 of 9 Fig. 1 OA increases NSC migration in the neurosphere assay. a Neurospheres seeded onto a 24-well PDL-coated plate were treated with different concentrations of OA for 24 h. Blue arrows indicate the migration distance from neurospheres. b OA-induced migration distance increased aa in a dose-dependent manner. Data are presented as means ± SD, n = 5. **p < 0.01, compared with the Ctr group; p< 0.01, compared with 10 μM bb OA group; p< 0.01, compared with 20 μM OA group. c Migrating cells expressed Nestin and MAP2 neural markers. Scale bars: 100 μm differentiation, especially active small molecules that can observed to emerge from the neurosphere and migrate effectively cross the blood–brain barrier (BBB). along a radial axis (Fig. 1). The OA treatment GSK3β/β-catenin signaling is important in the regula- significantly increased the migration distance in a tion of NSC proliferation and differentiation . Inactivation dose-dependent manner (Fig. 1a, b). The immuno- of GSK3β by phosphorylation at Ser9 accelerated the fluorescence staining showed the cells derived from the nuclear translocation of unphosphorylated active β-cate- neurospheres were MAP2-positive and mostly Nestin- nin, promoting NSC proliferation and differentiation . positive cells (Fig. 1c). These findings indicate that inactivation of GSK3β might be a promising target to induce NSC proliferation and OA promoted proliferation of NSCs differentiation. Proliferation of NSCs induced by OA was investigated In the present study, we investigated the effects of OA by CCK-8 assay and neurosphere assay. The CCK-8 on NSC proliferation and neural differentiation. Con- assay showed a slight absorbance increase on day 2 after sidering the importance of the activated state of GSK3β in culture, but this was not significant. On day 3 after controlling NSC induction, we used genome-editing treatment, the absorbance increased steeply and cell techniques to investigate the role of GSK3β activation in viability in the 20 and 40 μMOAgroupswas sig- regulating NSC proliferation and differentiation. To the nificantly higher than in the 10 μMOAgroup (Fig. 2a). best of our knowledge, this is the first study to confirm The cell proliferation and cell viability increased sig- that OA mediates NSC proliferation and neural differ- nificantly with OA treatment in a dose-dependent entiation through suppressing the activation of GSK3β. manner. The neurosphere assay showed the number and diameter of neurospheres generated from NSCs Results were significantly increased with the OA treatment in a OA increased NSC migration dose-dependent manner (Fig. 2b–d). These results Migration of NSCs was analyzed after treatment indicate that OA can significantly promote NSC pro- with OA (10, 20, and 40 μM). The neural cells were liferation and self-renewal in vitro. Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death Discovery (2019) 5:48 Page 3 of 9 Fig. 2 OA promotes the proliferation of NSCs. NSCs were treated with different concentrations of OA (10, 20, and 40 μM). OA-mediated NSC proliferation was analyzed by CCK-8 assay after OA treatment for 1, 2, or 3 days (a) and neurosphere assay was performed after OA treatment for 3 days (b–d). a CCK-8 assay showed cell viability was significantly enhanced in a dose- and time-dependent manner. Data are presented as means ± SD, n = 5. b An image of neurospheres after treatment with OA. Scale bars: 100 μm. The number (c) and the diameter (d) of neurospheres (diameter >30 μm) increased with OA treatment in a dose-dependent manner. Data are presented as mean ± SD, n = 5. *p < 0.05 and **p < 0.01, compared with the Ctr group; p< 0.05, compared with 10 μM OA group OA promoted the neural differentiation of NSCs Interestingly, we observed many MAP2 and Nestin NSCs have the potential to differentiate into multiple double-stained cells in the immunofluorescence staining, neural lineages, mostly neurons and astrocytes. Differ- indicating some of the NSC-derived neurons were entiation of NSCs after treatment with OA was deter- approaching maturation . mined by measuring the levels of three neural markers: Nestin, MAP2, and GFAP. The western blot analyses OA directly targeted GSK3β showed OA significantly and dose-dependently reduced The GSK3β/β-catenin signaling is considered to be a the expression of Nestin and upregulated the expression critical regulator for NSC proliferation and differentia- of MAP2, which is an exclusive dendritic protein in tion . Inhibition of GSK3β activity by phosphorylation at neurons. Furthermore, there was a decreasing trend in the Ser9 causes unphosphorylated active β-catenin to expression of GFAP after treatment with 20 and 40 μM accumulate in the cytoplasm and then translocate into the OA (Fig. 3a, b). These results demonstrate that OA can nucleus, promoting NSC proliferation and differentia- markedly promote neural differentiation of NSCs and tion . To explore the interaction between OA and suppress astrocyte differentiation in a dose-dependent GSK3β, we performed a molecular docking analysis using manner. These findings were also confirmed by immu- the Autodock Vina molecular docking program. The nofluorescence staining. As shown in Fig. 3c, d, the per- binding energy of the GSK3β-OA complex was − centage of MAP2-positive cells significantly increased 8.871 kcal/mol, which suggested good binding ability. The with increasing OA concentration, whereas the percen- three-dimensional binding conformation of the GSK3β- tage of GFAP-positive cells showed a downward trend. OA complex showed that OA interacted with GSK3β at Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death Discovery (2019) 5:48 Page 4 of 9 Fig. 3 OA selectively induces neural differentiation of NSCs. Treatment with OA for 7 days dose-dependently promoted the neural differentiation of NSCs, but suppressed astrocyte differentiation, as determined by western blotting (a, b) and immunofluorescence staining (c, d). a, b OA significantly and dose-dependently decreased the expression of Nestin and increased the expression of MAP2, but expression of GFAP was not significantly affected. c, d OA significantly and dose-dependently decreased the proportion of Nestin-positive cells and increased the proportion of MAP2-positive cells. Scale bars: 100 μm. All data are presented as means ± SD from three independent experiments. *p < 0.05 and **p < 0.01, a aa bb compared with the Ctr group; p< 0.05 and p< 0.01, compared with 10 μM OA group; p< 0.01, compared with 20 μM OA group LEU-188, THR-138, ASN-186, GLN-185, CYS-199, ALA- of β-catenin (active) to β-catenin compared with 83, VAL-70, ILE-62, GLY-63, PHE-67, and ASN-64 via the control group (Fig. 5a, b). The results showed that van der Waals force (Fig. 4a). To further verify the OA-mediated NSC proliferation and differentiation molecular docking results, the best conformation of might depend on the activation of GSK3β/β-catenin GSK3β-OA was taken as the starting conformation for the pathway. MD simulation by YASARA. As shown in Fig. 4b, the To further confirm the involvement of the activation of heavy atoms root-mean-square deviation (RMSD) track of GSK3β in OA-mediated NSC proliferation and differ- the GSK3β (Fig. 4b, red line) mildly fluctuated around 2 Å entiation, we activate GSK3β by transfection of con- over 0–100 ns and the RMSD track of OA (Fig. 4b, blue stitutively active GSK3β mutant at S9A using the GSK3β line) fluctuated around 0.2 Å during the MD simulation. (S9A) adenovirus. The transfection was confirmed by The surface visualization models of the GSK3β–OA western blot analysis (Fig. 5c, d). The expression ratio of complex at 0 and 100 ns are shown in Fig. 4c. We found p-GSK3β (Ser9) to GSK3β was significantly decreased to OA was stably presented at the center of the GSK3β about 30% in GSK3β (S9A) small interfering RNA binding site throughout the MD simulation. These results (siRNA) group compared with the control siRNA group, indicate that the binding between GSK3β and OA is leading to significant downregulation of the ratio of stable, and that OA likely targets GSK3β directly. β-catenin (active) to β-catenin. As expected, cell viability GSK3β (S9A) of NSCs induced by OA was significantly OA-mediated NSC induction through suppressing the attenuated (Fig. 5e). Furthermore, OA-mediated NSCs activation of GSK3β mutated with GSK3β (S9A) had significantly increased According to the results of the western blot Nestin expression, but decreased MAP2 expression analysis, NSCs treated with OA for 7 days showed (Fig. 5f, g). Overexpression of constitutively active GSK3β significant and concentration-dependent increases in (S9A) significantly attenuated OA-mediated NSC pro- the expression ratios of p-GSK3β (Ser9) to GSK3β and liferation and differentiation. All these results suggest that Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death Discovery (2019) 5:48 Page 5 of 9 Fig. 4 Molecular docking and molecular dynamics simulation. a Three-dimensional crystal structure of the complex of OA (ZINC95098891) with GSK3β (PDB ID: 4ACC). b Plots of root-mean-square deviation (RMSD) of heavy atoms of GSK3β (red) and OA (blue). c Surface presentation of the GSK3β-OA complex crystal structure at 0 and 100 ns the activated state of GSK3β is critical in OA-mediated Recently, several NSC replacement therapies have been proliferation and differentiation of NSCs. rapidly developed that may be promising treatments for neurodegenerative diseases, but they require in vitro Discussion induction of NSCs into functional neurons. NSCs have As neurodegenerative diseases are typically char- limited ability to proliferate and differentiate in natural acterized by the gradual loss of structure and/or func- conditions and require induction by GFs such as EGF tion of neurons in the brain, neuron replacement has and bFGF. However, in cell replacement therapy, GFs may become the most promising method to treat neurode- lead to a higher risk for carcinogenicity in vivo after 13 17,18 generative diseases . In the present study, we investi- transplantation . As a natural active component, OA gated OA, a bioactive molecule, as a potential treatment might be an ideal alternative for GFs to induced NSC for neurodegenerative diseases through the induction of proliferation and differentiation efficiently and safely. NSCs. We confirmed that OA could dose-dependently GSK3β has a critical role in regulating the behavior and promote the migration and proliferation of NSCs. In fate of NSCs. Inhibiting the activity of GSK3β can sig- addition, OA could dose-dependently induce NSC nificantly promote NSC proliferation and neural differ- neural differentiation, while suppressing astrocyte dif- entiation . We investigated the ability of OA to target ferentiation. Many macromolecular drugs cannot pass GSK3β directly by performing molecular docking and MD the BBB to target pathological changes in the brain . simulations to assess the mechanism by which OA and Meanwhile, a study demonstrated that OA could be GSK3β interact. The binding energy of the OA-GSK3β effectively delivered across the BBB , further support- complex indicated strong binding interactions. Moreover, ing the development of OA as a promising drug for OA binding to GSK3β was through LEU-188, THR-138, treating neurodegenerative diseases that can be admi- ASN-186, GLN-185, CYS-199, ALA-83, VAL-70, ILE-62, nistered orally. GLY-63, PHE-67, and ASN-64 via van der Waals force. Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death Discovery (2019) 5:48 Page 6 of 9 Fig. 5 OA regulates the activation of GSK3β in NSC induction. Proliferation and differentiation of NSCs mediated by OA depends on the suppressing of the activation of GSK3β. a Western blot analyses and b relative optical density of diverse pathway markers including β-catenin (active), β-catenin, p-GSK3β (Ser9), and GSK3β in NSCs after treatment with OA for 7 days. *p < 0.05 and **p < 0.01, compared with the Ctr group; p< 0.05, compared with 10 μM OA group. c Western blot analyses and d relative optical density of p-GSK3β (Ser9), GSK3β, p-GSK3β (Ser9), and GSK3β in NSCs after transfection with GSK3β (S9A) adenovirus for 2 days. **p < 0.01, compared with the Ad-Ctr group. e NSCs with GSK3β (S9A) significantly suppressed cell viability, as examined by CCK-8 assay. **p < 0.01, compared with Ad-Ctr+ 20 μM OA group. f Western blot analyses and g relative optical density of diverse differentiation markers including Nestin, MAP2, and GFAP in NSCs transfected with GSK3β (S9A) treated with OA (20 μM). NSCs with GSK3β (S9A) significantly suppressed NSC neural differentiation. **p < 0.01, compared with Ad-Ctr+ 20 μM OA group. All data are presented as means ± SD from three independent experiments These residues are key residues involved in inhibiting OA directly targets GSK3β and can potentially act as a the binding domain of GSK3β . Furthermore, the MD GSK3β inhibitor. This was consistent with the Western simulations demonstrated that the binding conformation blotting results that showed OA significantly and dose- of OA-GSK3β was stable. These findings indicated that dependently suppressed the activation of GSK3β.To Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death Discovery (2019) 5:48 Page 7 of 9 further confirm that the effect of OA on NSC induction method when the diameter of the neurospheres reached was mediated through GSK3β, we transfected NSCs with 150–200 μm. the constitutively active GSK3β (S9A) adenovirus. Wes- tern blotting showed successful knockdown of p-GSK3β Migration analysis (Ser9), leading to downregulation of the expression of β- OA (purity assayed by high-performance liquid chro- catenin (active). The proliferation and differentiation matography: 98.33%) was purchased from Must Bio- mediated by OA were significantly restrained in NSCs Technology Co., Ltd (Chengdu, China). Neurospheres transfected with GSK3β (S9A) adenovirus. These results (around 200 μm in diameter) were seeded on coverslips, suggest the activity of GSK3β might be critical in OA- which had been coated with poly-D-lysine (PDL; Sigma, mediated NSC proliferation and differentiation. In this St. Louis, MO, USA) for 30 min to enable neurospheres study, the change of activated state of GSK3β made cor- to attach. The neurospheres were exposed to a series responding change of β-catenin (active). Both GSK3β and of OA concentrations (10, 20, and 40 μM). After incuba- β-catenin (active) are regulated by multiple pathways, one tion for 24 h, photos of the neurospheres were taken of which is Wnt signaling pathway which plays a crucial under an inverted microscope. The migration distance role in NSC proliferation and differentiation . Although of neural cells from the edge of the neurospheres further research is needed to fully explore the role of Wnt was measured by Image-J software. Immunostaining signaling pathway involved in OA-mediated NSC was performed to visualize Nestin (NSCs marker) induction. and microtubule-associated protein-2 (MAP2, neuron NSCs can be an essential source of neural cells for marker) as described below. the treatment of many incurable diseases including neurodegenerative diseases. One of the major challenges Neurosphere assay is to induce neural differentiation of NSCs over Single cells were dissociated from the neurospheres and other lineages and to ensure the treatment is safe. In adjusted to a density of 2.5 × 10 cells/mL. The cells were this study, we demonstrated that OA promoted the seeded onto a 96-well plate (200 μL/well) and treated with differentiation of NSCs into neurons but suppressed various concentrations of OA (10, 20, and 40 μM) without the formation of astrocytes. OA is derived from GFs. After 3 days of incubation, the number and diameter ginseng, which has a long history in many clinical of neurospheres (diameter > 30 μm ) were analyzed by applications, and may provide a safe and efficient Image-J software. way for NSC induction. Our significant findings hold much promise for improving NSC replacement CCK-8 assay therapy in neurodegenerative diseases and other related Single cells were dissociated from the neurospheres and diseases. adjusted to a density of 2.5 × 10 cells/mL. The cells were seeded onto a 96-well plate (200 μL/well) and treated with Methods and materials various concentrations of OA (10, 20, and 40 μM) for NSC isolation and primary cell culture different experimental periods (1, 2, and 3 days). At the The study was approved by the Department of Health, end of each experimental period, CCK-8 (DOJINDO, the Government of the HKSAR. The modified experi- Rockville, MD, USA) reagent was added to measure cell mental protocol was carried out in accordance with proliferation. After further incubation at 37 °C for 4 h, the relevant guidelines and regulations of the Animal Ethics absorbance of each well was measured at a wavelength of Committee at HKBU. Rats (Sprague–Dawley) at postnatal 450 nm using an automatic microplate reader (BioTek). day 1 to 2 were purchased from the Chinese University of Hong Kong. The subventricular zone of the brain was Cell differentiation study dissected out and manually cut into pieces. After digestion Single cells were seeded at 2 × 10 cells/well onto a six- in trypsin at 37 °C for 15 min, the tissue suspension was well PDL-coated plate and then treated with different filtered through a Cell Strainer with a 40 µm mesh. The concentrations of OA (10, 20, and 40 μM). After incuba- filtrate was centrifuged for 5 min at 200 × g and the tion for 7 days, NSC differentiation was detected by supernatant was discarded. The pellet was resuspended in measuring the levels of differentiation markers, including neurobasal medium which contains 20 ng/mL epidermal Nestin, MAP2, and glial fibrillary acidic protein (GFAP, GF (EGF, PeproTech, Rocky Hill, NJ, USA), 20 ng/mL astrocytes marker) , by western blotting and immuno- basic fibroblastic GF (bFGF, PeproTech), 1% PSN fluorescence staining. (Thermo, Waltham, MA USA), and 2% B27 supplement (Thermo), and then cultured at 37 °C in a 5% CO Immunofluorescence staining humidified incubator. Half of the medium was changed Cells were fixed with 4% paraformaldehyde (Sigma) every 2–3 days. The cells were passaged by mechanical for 30 min at room temperature. The cells were then Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death Discovery (2019) 5:48 Page 8 of 9 incubated with the primary antibodies including anti- between the box and solute. The simulated annealing Nestin antibody, anti-MAP2 antibody, and anti-GFAP minimizations were initially set at 298 K and velocities antibody (1:1000, Millipore, Temecula, CA, USA) in were scaled down by 0.9 with every ten steps lasting for phosphate-buffered saline (PBS), with 0.1% Triton X-100 5 ps. After the energy was minimized, the temperature of (Sigma-Aldrich) and 2% normal goat serum (Vector the system was adjusted using a Berendsen thermostat to Laboratories, Burlingame, CA, USA) overnight at 4 °C. minimize temperature control influences. In addition, velocities were rescaled only every 100 simulation steps, After rinsing with PBS three times, the cells were incu- bated with the secondary antibodies at room temperature whenever the mean of last 100 detected temperatures for 3 h. The cells were then incubated with 1 μg/mL 4′,6- converged. Finally, 100 ns MD simulations were con- diamidino-2-phenylindole (Roche, Switzerland) for ducted every 2 fs and the coordinates of the complexes 15 min to stain the nuclei. After rinsing with PBS, fluor- were saved every 10 ps. escence mounting medium (Dako, Aglient, Santa Clara, CA, USA) was added, and immunoreactivity images were Adenovirus-mediated expression of constitutively active obtained and processed under a confocal microscope GSK3β (FluoView FV1000, Olympus, Tokyo, Japan). The pAdM-FH-GFP-GSK3β (S9A) adenoviral vector [Ad-GSK3β (S9A)] expressing the constitutively active Western blotting GSK3β with the serine residue at position 9 mutated to Cellular proteins were extracted using protein extrac- alanine [GSK3β (S9A)] and the empty adenoviral vector tion reagent (Novagen, Madison, WI, USA) supplemented were generated by Vigene Bioscience (Rockville, MD, with a protease inhibitor cocktail (Calbiochem, San Diego, USA). Single cells dissociated from neurospheres were CA, USA) on ice. Protein concentration was determined seeded at 2 × 10 cells/well onto a six-well PDL-coated with a protein assay kit (Bio-Rad, Hercules, CA, USA). plate. The NSCs were transfected with Ad-GSK3β (S9A) Next, proteins (30 μg) were separated on a 10% SDS- or empty adenoviral vector (Ad-Ctr) as the control at a polyacrylamide gel and transferred to a polyvinylidene multiplicity of infection of 40 in the presence of 6 μg/mL difluoride membrane (Bio-Rad). The membrane was polybrene (Sigma). The medium containing the virus was incubated with specific primary antibodies overnight at then exchanged for fresh medium after 12 h of transfec- 4 °C. Primary antibodies included anti-Nestin antibody tion. Cells were cultured for at least 48 h after transfection (1:1000, Millipore), anti-MAP2 antibody (1:1000, Milli- before further experiments were performed. The trans- pore), anti-GFAP antibody (1:1000, Millipore), anti-p- gene expressions were confirmed in the infected cells by GSK3β (Ser9) antibody (1:1000, Cell Signaling, Beverly, analyzing protein expressions. MA, USA), anti-GSK3β antibody (1:1000, Cell Signaling), anti-Non-p (active) β-catenin antibody (1:1000, Cell Sig- Statistical analysis naling), and anti-β-catenin antibody (1:1000, Cell Signal- All data were expressed as means ± SD. Statistical ana- ing). The membrane was then incubated with the lysis was performed by one-way analysis of variance. secondary antibody for 1 h at room temperature. β-Actin Relative risk was expressed as odds ratios with 95% con- antibodies (1:5000, Sigma) were used as the reference. fidence interval and statistical significance was defined as The bands were visualized and imaged using the Chemi- p < 0.05. The statistical analyses were conducted using Doc Touch imaging system (Bio-Rad). GraphPad Prism. Molecular simulation of the interaction of OA and GSK3β Acknowledgements This work was financially supported by National Natural Science Foundation of Autodock Vina molecular docking software (Scripps China (NSFC) grants (No. 81703728 and 81774100). Research Institute, La Jolla, CA, USA) was used to analyze the binding mechanism between GSK3β (PDB ID: Author contributions 4ACC) and OA (ZINC95098891). YASARA was used to K.K.L.Y., S.C.W.S., and S.Q.Z. designed the experiment. S.Q.Z., K.L.L., S.C.W.S., C.Y. perform the MD simulation and the energy minimiza- L., W.S.T., and S.S.M.W. generated cell culture data. S.Q.Z., K.L.L., and S.C.W.S. performed statistical analysis and drew illustrative figures. B.L. and X.Q.F. tion of the ligands, and AMBER 03 forcefield was used to performed bioinformatics analyses. S.Q.Z., B.L., S.C.W.S., and K.K.L.Y. wrote the run all simulations. The star conformation was considered manuscript. the best conformation for the molecular dynamics (MD) simulation. In this study, the inhibitor-binding domain of Conflict of interest GSK3β corresponded to the protein-ligand binding site of The authors declare that they have no conflict of interest. GSK3β. The protein structure of GSK3β was prepared by removing water molecules and bound ligands. 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