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Differentiation of human glioblastoma U87 cells into cholinergic neuron
Neuroscience Letters 704 (2019) 1–7
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Research article
Differentiation of human glioblastoma U87 cells into cholinergic neuron Honghui Liu, Jinye Xia, Tiansheng Wang, Wei Li, Yexun Song, Guolin Tan
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T
Department of Otorhinolaryngology – Head Neck Surgery, Third Xiangya Hospital, Central South University, Changsha, Hunan, 410013, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Differentiation Cholinergic neurons In vitro model U87 Akt
To facilitate research methodologies for investigating the role of cholinergic nerves in many diseases, establishing an in vitro cholinergic neuron model is necessary. In this study, we investigated whether human glioblastoma U87 cells could be differentiated into cholinergic neurons in vitro. Sodium butyrate was used as the differentiation agent. The differentiated cells established by inducing U87 cells with sodium butyrate were named D-U87 cells. Immunofluorescence was used to label the neuronal markers MAP2, NF-M, and ChAT and the glial marker GFAP in D-U87 cells. Flow cytometry was used to measure cell cycle distribution in D-U87 cells. PCR, protein chip, and western blot assays were used to measure the expression levels of muscarinic cholinergic receptor 1 (M1), M4, ChAT, SYP and Akt. ELISA was used to measure neurotransmitter levels. As a result, we found that sodium butyrate induced U87 cell differentiation into cells with neuronal characteristics and increased not only the expression levels of the cholinergic neuron-related proteins M1, M4, ChAT and SYP in DU87 cells but also the acetylcholine neurotransmitters in D-U87 cells. Moreover, the Akt protein expression in DU87 cells was increased compared with that in U87 cells. Finally, we found that M1, M4, ChAT and SYP protein expression and acetylcholine secretion levels were significantly decreased in D-U87 cells after treatment with the Akt inhibitor MK-2206. These results demonstrate that D-U87 cells exhibit cholinergic neuron characteristics and that sodium butyrate induced U87 cell differentiation into cholinergic neuron partially through Akt signaling.
1. Introduction Increasing evidence indicates that cholinergic nerves play multifunctional roles in many conditions, including central nervous system disease, autoimmune disease, digestive system disease and airway allergic disease [1–3]. For example, parasympathetic nerve dysfunction has been found in depression, epilepsy, ischemic stroke, multiple sclerosis, Parkinson’s disease and Alzheimer’s disease [1]; cholinergic nerve stimulation alleviates symptoms of rheumatoid arthritis by activating nicotinic receptors [3]; and cholinergic nerve hyperactivity accelerates the severity of rhinitis [4]. Additionally, cholinergic hypersensitivity inhibits tumor cell proliferation and contributes to cell differentiation [5]. However, many studies in the past were performed with animal models, in which the procedures are complex; subsequently, the progress of researching the mechanisms involved in various diseases is limited to some extent. Since simplifying research methodologies could promote the mechanistic studies of many diseases, it has become necessary to build an in vitro cholinergic neuron model. Several in vitro cholinergic neuron models have been studied. Most
models were derived from primary stem cells, including neural stem cells, mesenchymal stem cells, and embryonic stem cells [6–9]. Only a few studies have used cell lines, such as the mouse-derived embryonal carcinoma cell line P19, the human neuroblastoma cell line SH-SY5Y, and the rat adrenal pheochromocytoma cell line PC12 [10–12], as cholinergic neuron models. In fact, the procedure for differentiating cells into cells with cholinergic neuron characteristics via primary cell culture is more sophisticated than that via cell lines. Thus, the development of cell lines serving as in vitro cell models should be a priority. The human glioblastoma cell line U87 is derived from brain cancer cells. We have not found any studies showing that U87 cells have the potential to differentiate into cholinergic neuron. Therefore, our study aimed to explore whether U87 cells could be differentiated into cells with cholinergic neuron characteristics and the possible mechanism.
Abbreviations: M1, muscarinic cholinergic receptor 1; M4, muscarinic cholinergic receptor 4; M5, muscarinic cholinergic receptor 5; ChAT, choline acetyltransferase; SYP, synaptophysin; Akt, protein kinase B; MAP2, microtubule-associated protein-2; NF-M, neurofilament-M; GFAP, glial fibrillary acidic protein ⁎ Corresponding author. E-mail address: [email protected] (G. Tan). https://doi.org/10.1016/j.neulet.2019.03.049 Received 28 November 2018; Received in revised form 20 March 2019; Accepted 26 March 2019 Available online 28 March 2019 0304-3940/ © 2019 Elsevier B.V. All rights reserved.
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2. Materials and methods
were incubated with an IRDye®680RD goat anti-mouse IgG (H + L) or IRDye®800CW goat anti-rabbit IgG (H + L) secondary antibody (LICOR Odyssey, Germany) for 30 min in the dark. Images were obtained using an Odyssey Imaging system (LI-COR, Germany). The dilutions of the rabbit anti-M1, M4, M5 and ChAT antibodies (Abcam, USA) were 1:1000, the dilution of the rabbit anti-SYP antibody (Santa Cruz, USA) was 1:200, the dilution the of rabbit anti-Akt antibody (CST, USA) was 1:2000, and the dilution of the mouse anti-GAPDH antibody (Proteintech, USA) was 1:5000.
2.1. Cell culture The human glioblastoma cell line U87 was obtained from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The cells were cultured in Minimum Essential Medium (MEM; Gibco, Invitrogen, USA) containing 10% fetal bovine serum (FBS) (Gemini, USA) and were incubated in a humidified incubator at 37℃ with an atmosphere of 5% CO2.
2.7. Protein chip analysis 2.2. Cell differentiation and Akt inhibitor treatment The stress and apoptosis signaling molecules were screened using the protein chip PathScan® Stress and Apoptosis Signaling Antibody Array Kit (#12923, CST, USA) according to the manufacturer’s instructions. Image detection and analysis were performed using an Odyssey imaging system (LI-COR, Germany)
First, the U87 cells were seeded into six-well plates at 5 × 105 cells per well and grown in proliferation medium (MEM containing 10% FBS) for 24 h. Then, we removed the proliferation medium and differentiated the cells with differentiation medium (MEM containing 2 mM sodium butyrate (Sigma, USA) without FBS) for 48 h. In the Akt inhibition group, we added 50 nM Akt inhibitor MK-2206 (Selleck, USA) into the differentiation medium. Sodium butyrate and the Akt inhibitor MK-2206 were dissolved in MEM. The differentiated cells established by inducing U87 cells with sodium butyrate were named D-U87 cells.
2.8. ELISA The concentrations of human neurotransmitters were evaluated in cell supernatants using commercial kits: ACH (Acetylcholine) (Elabscience, China), NA/NE (Noradrenaline/Norepinephrine) (Elabscience, China), and ү-GABA (Gamma-Aminobutyric Acid) (Wuhan USCN Business Co., Ltd, China), according to the manufacturer’s instructions. The detection limits were as follows: 9.38 pg/mL for ACH; 0.19 ng/mL for NA/NE and 9.39 pg/mL for ү-GABA.
2.3. Immunofluorescence The cells were fixed with 4% paraformaldehyde for 30 min, washed thrice with PBS, and permeabilized in 0.5% TritonX-100 for 20 min. After washing with PBS, the cells were blocked in normal goat serum (ZSGB-BIO, China) for 30 min and were then incubated with primary antibodies (rabbit anti-choline acetyltransferase (ChAT, 1:1000, Abcam, USA), microtubule-associated protein-2 (MAP2, 1:200, Proteintech, USA), neurofilament-M (NF-M, 1:200, Proteintech, USA), or glial fibrillary acidic protein (GFAP, 1:200, Proteintech, USA)) at 4 °C overnight. After washing thrice with PBS, the cells were incubated with a TRITC-conjugated goat anti-rabbit secondary antibody IgG (1:100, Proteintech, USA) for 1 h at room temperature. Images were captured using a fluorescence microscope (Olympus, Japan).
2.9. Statistical analysis The statistical analysis was performed using SPSS version 22 software (SPSS Inc., USA). The data are presented as the mean ± SD. The statistical analysis of the independent samples was performed using ttests. P < 0.05 was considered statistically significant. 3. Results 3.1. Neuron characteristics of human glioblastoma U87 cells after differentiation
2.4. Flow cytometry Cells were collected and stained with 1 mL of DNA staining solution and 10 mL of permeabilization solution, according to the instructions from the Cell Cycle Staining Kit (MULTI SCIENCES, China). Cells were detected on a FACSCalibur flow cytometer (BD Biosciences, USA), and the results of cell cycle distribution (in the G0/G1, S and G2/M phase) were analyzed using FlowJo 10 software (BD Biosciences, USA).
Sodium butyrate, which is an inducer agent, was used to differentiate the U87 cells. Compared with U87 cells, D-U87 cells presented elongated nerve fibers, and the lengths of some fibers were 2 times longer than those of the neurite bodies (Fig. 1A–D). Immunofluorescence staining showed higher intensities of ChAT, MAP2 and NFM in D-U87 cells than in U87 cells (Fig. 1A–D), while the GFAP staining between U87 and D-U87 had no significant difference, indicating that D-U87 cells may present cholinergic nerve characteristics. Moreover, flow cytometry was used to measure cell cycle distribution, as shown in Fig. 1E and F, compared with U87 group, the percentage of cells in S phase in the D-U87 group was significantly lower, whilst the percentage in G0/G1 phase and G2/M phase was significantly higher. These results demonstrated that D-U87 cells presented not only neuron characteristics, including increased intensities of neuron markers of MAP2 and NF-M, and proliferation inhibition, but also cholinergic neuron characteristics, like higher intensity of cholinergic neuron marker ChAT.
2.5. Neurotransmitter receptor PCR chip (RT2 RNA QC PCR array protocol) The total RNA was extracted from the U87 and D-U87 cells using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The RNA samples were sent to the Kangcheng Company (Shanghai, China) for assistance in performing the following protocols, including RNA detection and data analysis. 2.6. Western blot
3.2. Cholinergic neuron-related molecular changes in U87 cells after differentiation
The cells were washed with ice-cold PBS and lysed using RIPA Lysis Buffer (KeyGEN BioTECH, China) containing 1 mM PMSF. The proteins (30 μg) were separated by electrophoresis via 10% SDS-polyacrylamide gels, electrophoretically transferred to PVDF (Millipore, USA) membranes via a wet transfer apparatus (Bio-Rad, USA), blocked in TBS blocking buffer (LI-COR Odyssey, Germany) and incubated with specific primary antibodies (rabbit anti-M1, -M4, -M5, -ChAT, -SYP, and -Akt and mouse anti-GAPDH) at 4 °C overnight. Next, the membranes
To further investigate whether D-U87 cells present cholinergic neuron characteristics, we screened the cholinergic receptors using a neurotransmitter mRNA chip analysis. Compared with the U87 cells that did not receive sodium butyrate treatment, the D-U87 cells exhibited increased M1, M4, N3 and CHRNE mRNA expression levels (Fig. 2A). Then, we measured the protein expression levels of the 2
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Fig. 1. Neuron characteristics of U87 cells after differentiation. (A–D) Immunofluorescence staining of ChAT, MAP2, NF-M, and GFAP between U87 and D-U87 cells, respectively. In each image, the upper panel were pictures of U87 and D-U87 observed under an inverted microscope. The lower panel were merged pictures from DAPI (Blue) and the cholinergic neuronal marker ChAT (Red), or the neuronal markers MAP2 (Red), NF-M (Red), or the glial marker GFAP (Red) respectively. Scale bar, 100 μm. (E) Cell cycle distribution between U87 and D-U87 cells was analyzed by flow cytometry using propidium iodine staining. (F) Percentages of U87 and DU87 cells in G0/G1, S and G2/M phases. Data are expressed as the mean ± SD, and at least three independent experiments were performed. *p < 0.05, vs U87 group.
butyrate exhibited increased M1, M4, ChAT and SYP levels, indicating that D-U87 cells exhibited cholinergic neuron characteristics. To clarify the potential mechanism involved in the process of sodium butyrateinduced U87 cell differentiation, we screened related proteins using a stress and apoptosis protein chip. The results showed that several proteins, including Akt, Bad, p53, JNK, and TAK1, were increased and that HSP27 was decreased in U87 cells after differentiation. Among the changed proteins, Akt was the most significantly increased protein in DU87 cells compared to U87 cells without differentiation (Fig. 3A and B). Furthermore, we tested Akt expression using western blotting, and the results showed a significant increase in Akt in D-U87 cells compared with its expression in U87 cells (Fig. 3C and D). These results demonstrate that the increased M1, M4, ChAT and SYP levels in U87 cells after
muscarinic receptors M1, M4 and M5 by western blotting. The results showed that the M1 and M4 levels were significantly increased after differentiation, and no increase in the M5 level was observed (Fig. 2B and C), consistent with the PCR screening chip results. In addition, we assessed the protein levels of ChAT, which is a specific cholinergic neuron marker, and SYP, which is a specific marker of neuron synapses, both of which are involved in neurotransmitter synthesis and release. The results showed that the ChAT and SYP levels were increased in the D-U87 cells compared with those in the U87 cells (Fig. 2B and C).
3.3. Akt protein changes in U87 cells after differentiation The above results show that U87 cells differentiated by sodium 3
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Fig. 2. Cholinergic neuron-related gene expression in D-U87 cells. (A) Cholinergic gene mRNA expression in D-U87 cells was analyzed by a neurotransmitter mRNA PCR chip. The expression levels of the muscarinic cholinergic receptor 1 (M1), M4 and the nicotinic cholinergic receptor 3 (N3) in DU87 cells were higher than those in U87 cells. CHRM, muscarinic cholinergic receptor. CHRNA, nicotinic cholinergic receptor. CHRNE, cholinergic receptor nicotinic epsilon subunit. (B) The expression levels of the cholinergic neuron-related proteins M1, M4, M5, ChAT and SYP were determined by western blot after differentiation. (C) The quantifications of M1, M4, M5, ChAT and SYP proteins were analyzed after normalization to GAPDH. M1, muscarinic receptor 1. M4, muscarinic receptor 4. M5, muscarinic receptor 5. ChAT, choline acetyltransferase. SYP, synaptophysin. Data are expressed as the mean ± SD, and at least three independent experiments were performed. *p < 0.05, vs U87 group.
cell line U87 could be differentiated into cells with cholinergic neuron characteristics. We found that sodium butyrate induced U87 cells into cholinergic neuron with increased cholinergic neuron-related protein expression levels of M1, M4, ChAT and SYP and increased secretion of acetylcholine. Furthermore, we found that Akt plays a role in the sodium butyrate-induced U87 differentiation process. Although many studies have explored in vitro cholinergic neuron models, most models have been restricted to primary cell cultures. Among the primary cell culture system, stem cells are commonly used. Neural stem cells, mesenchymal stem cells, and embryonic stem cells can differentiate toward a cholinergic phenotype in a specific medium containing nerve growth factor (NGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), neurotrophin-3 (NT3), and brain-derived neurotrophic factor (BDNF) [6–9,20–22]. In addition to various stem cells, other types of cells, such as fetal lung fibroblasts, glial cells and even sympathetic neurons, exhibit the capacity to differentiate into cells showing cholinergic neuron characteristics under special treatment conditions [23–25]. The differentiation times vary from ten to fourteen days. Considering the lengthy differentiation time and sophisticated processes involved in primary cell cultures, several researchers have established neuron models using cell lines to avoid complex separation management and culturing procedures that may lead to contamination. The mouse-derived embryonal carcinoma cell line P19 and the human neuroblastoma cell line SH-SY5Y can differentiate into cells with cholinergic characteristics with retinoic acid [10,11,26]. Nerve growth factor (NGF) can induce the rat adrenal pheochromocytoma cell line PC12 into exhibiting cholinergic nerve features [12,27]. Other agents, such as dibutyryl cAMP and IL-4, have also been used as differentiation inducers [28,29]. Generally, compared with primary cell differentiation, cell line differentiation often requires less time; only four to ten days of differentiation time is often enough, and the differentiation induction conditions are so simple that only one type of inducing agent is needed.
differentiation may be related to enhanced Akt expression. 3.4. Effect of Akt inhibition on cholinergic neuron-related molecule changes in D-U87 cells To determine whether Akt plays a role in the U87 cell differentiation process, we used western blotting to measure M1, M4, M5, ChAT and SYP expression levels following treatment with 50 nM of the Akt inhibitor MK-2206 in D-U87 cells. As shown in Fig. 4, the Akt inhibitor significantly decreased the M1, M4, ChAT and SYP expression levels but did not have an effect on M5 expression in D-U87 cells. 3.5. Neurotransmitters in U87 cells after differentiation and the effect of Akt inhibition To determine what types of neurotransmitters exist in D-U87 cells, we measured the acetylcholine (ACH), noradrenaline/norepinephrine (NA/NE) and gamma-aminobutyric acid (ү-GABA) levels in the supernatants of U87 and D-U87 cells. We found that ү-GABA level was below the detection range (9.39 pg/mL) (data not shown). The ACH level in DU87 cells was significantly increased compared with that in U87 cells, and Akt inhibitor reduced the ACH level (Fig. 5A). However, the NA/NE level in U87 cells was significantly decreased after differentiation, and the Akt inhibitor had no effect on NA/NE secretion (Fig. 5B). 4. Discussion Increasing evidence suggests that cholinergic nerve dysfunction is closely related to various conditions involving the central nervous system, the respiratory system, and autoimmune diseases [1,2,13–19]. While most studies have focused on animal models, it is necessary to build an in vitro model of cholinergic neurons to facilitate research methodologies. In this study, we investigated whether the glioblastoma 4
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Fig. 3. Akt protein expression in U87 cells after differentiation. (A) Representative images and (B) statistical analysis of the stress and apoptosis related protein expression levels, as determined by protein chip after differentiation. The red rectangular box represents the Akt protein spot. (C) Akt expression was determined by western blot after differentiation. (D) The quantifications of Akt protein were analyzed after normalization to GAPDH. Data are expressed as the mean ± SD, and at least three independent experiments were performed. *p < 0.05, vs U87 group.
Fig. 4. Effects of Akt inhibition on M1, M4, M5, ChAT and SYP protein expression levels in D-U87 cells. (A) The expression levels of the cholinergic neuron-related proteins M1, M4, M5, ChAT and SYP were determined by western blot after D-U87 cells were treated with the Akt inhibitor MK-2206. MK, MK-2206. (B) The quantifications of M1, M4, M5, ChAT and SYP proteins were analyzed after normalization to GAPDH. M1, muscarinic receptor 1. M4, muscarinic receptor 4. M5, muscarinic receptor 5. ChAT, choline acetyltransferase. SYP, synaptophysin. Data are expressed as the mean ± SD, and at least three independent experiments were performed. *p < 0.05, vs D-U87 group.
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Fig. 5. Acetylcholine and noradrenaline/norepinephrine levels in differentiated U87 cells and the effects of Akt inhibition on the neurotransmitter levels. (A) The acetylcholine (ACH) level in the supernatant from differentiated U87 (D-U87) cells after treated with Akt inhibitor MK-2206. ACH, acetylcholine. MK, MK-2206. (B) The noradrenaline/norepinephrine (NA/NE) level in the supernatant from D-U87 cells after treated with the Akt inhibitor. NA/NE, noradrenaline/norepinephrine. Data are expressed as the mean ± SD, and at least three independent experiments were performed. *p < 0.05, vs U87 group, #p < 0.05, vs D-U87 group.
time, differentiation reagents and culture processes. Therefore, additional in vitro cholinergic neuron models using cell lines and the differentiation mechanisms involved in different induction reagents will require further exploration.
Sodium butyrate, a histone deacetylase (HDAC) inhibitor, inhibits cell proliferation, contributes to cell apoptosis, induces cell cycle arrest and promotes cell differentiation [30–32,37]. Although no studies have reported that U87 cells could differentiate into cholinergic neurons, in our study, we found that sodium butyrate could induce U87 cells to exhibit neuronal fiber morphology. The entire differentiation time was only two days of culture, which significantly reduced the research period. D-U87 cells presented higher intensities of the neuronal markers MAP2 and NF-M and the cholinergic neuronal marker ChAT, and a decreased S phase whilst increased G1/G0 phase and G2/M phase than U87 cells, which demonstrated that D-U87 cells had neuron characteristics. Furthermore, the expression levels of cholinergic neuronrelated molecules, such as M1, M4, ChAT and SYP, and the neurotransmitter secretion of acetylcholine were significantly increased in U87 cells after differentiation. The cholinergic differentiation of U87 cells by sodium butyrate may occur through histone deacetylase (HDAC) inhibition. Several studies have shown that HDAC inhibition could contribute to ChAT expression [33–36]. HDAC inhibitor could significantly increase ChAT mRNA expression [33]. HDAC9 has been shown to repress ChAT expression, and HDAC inhibitor could not only restore stress-induced AChE impairment but also increase muscarinic receptor expression levels [34,34,35,36]. All of the evidence indicates that sodium butyrate is positively related to cholinergic function. Moreover, we found that sodium butyrate enhanced the expression of Akt in D-U87 cells and that the inhibition of Akt reduced the expression levels of M1, M4, ChAT and SYP in D-U87 cells. Studies have shown that Akt positively regulated sodium butyrate-mediated cell differentiation [38,39,42]. For example, sodium butyrate induces microglial process elongation through the activation of Akt [38]. Akt signaling also participated in the differentiation of the SH-SY5Y cell line [39]. In addition, butyrate increased adipogenic differentiation via activating Akt [42]. On the contrary, others have demonstrated that sodium butyrate inhibited cancer cell motility through inhibiting Akt/ ERK expression [40] and that butyrate decreased human colorectal cancer cell line CaCo-2 and SNU-C4 proliferation through inhibiting Akt [41]. The discrepancy of the effects of sodium butyrate on Akt expression in different studies may be due to the different cell lines used in the research and the inconsistent cellular functions observed under different conditions. On the other hand, several studies have shown a positive relationship between cholinergic receptors and Akt expression [44,45]. Akt could positively regulate CXCL12-induced cholinergic gene expression [43]. Akt also activates cell chemotaxis through activating G protein-coupled receptors [46]. Furthermore, activation of muscarinic receptors could increase Akt expression [47] and combine to trigger NGF-induced Akt expression [12]. In our study, sodium butyrate induced U87 cell differentiation toward cholinergic neuron, accompanied by an increase in Akt, and cholinergic neuron-related protein levels and acetylcholine secretion level were decreased via Akt inhibition. This evidence suggests that Akt partially participates in the process of U87 cell cholinergic differentiation. Differentiation using cell lines facilitates research procedures more than the use of primary cells by affecting various factors, such as culture
5. Conclusions In conclusion, our study provides a new in vitro cholinergic neuron cell model derived from U87 cell line, and sodium butyrate mediated this differentiation process at least partially through Akt signaling. We hope that our findings may provide a new possible method or cholinergic neuron model for further research on the roles played by cholinergic nerves in various diseases. Declarations of interest None. Acknowledgements This work was supported by the National Natural Science Foundation [8187078, 81702706, 81502358], the Fundamental Research Funds for the Central Universities of Central South University [2017zzts237], and the Funds of the Hunan Scientific Plan in China [2017SK2043, 12JJ4079]. References [1] B. Han, X. Li, J. Hao, The cholinergic anti-inflammatory pathway: an innovative treatment strategy for neurological diseases, Neurosci. Biobehav. Rev. 77 (2017) 358–368, https://doi.org/10.1016/j.neubiorev.2017.04.002. [2] B.J. Undem, T. Taylor-Clark, Mechanisms underlying the neuronal-based symptoms of allergy, J. Allergy Clin. Immunol. 133 (2014) 1521–1534, https://doi.org/10. 1016/j.jaci.2013.11.027. [3] D.B. Hoover, Cholinergic modulation of the immune system presents new approaches for treating inflammation, Pharmacol. Ther. 179 (2017) 1–16, https://doi. org/10.1016/j.pharmthera.2017.05.002. [4] S. Ssarin, B. Undem, A. Sanico, A. Togias, The role of the nervous system in rhinitis, J. Allergy Clin. Immunol. 118 (2006) 999–1016, https://doi.org/10.1016/j.jaci. 2006.09.013. [5] R.R. Resende, A. Adhikari, Cholinergic receptor pathways involved in apoptosis, cell proliferation and neuronal differentiation, Cell Commun. Signal 7 (2009) 20, https://doi.org/10.1186/1478-811X-7-20. [6] L. Wang, F. He, Z. Zhong, R. Lv, S. Xiao, Z. Liu, Overexpression of NTRK1 promotes differentiation of neural stem cells into cholinergic neurons, Biomed Res. Int. (2015) 857202, , https://doi.org/10.1155/2015/857202 2015. [7] T.T. Wang, A.H. Jing, X.Y. Luo, M. Li, Y. Kang, X.L. Zou, H. Chen, J. Dong, S. Liu, Neural stem cells: isolation and differentiation into cholinergic neurons, Neuroreport 17 (2006) 1433–1436, https://doi.org/10.1097/01.wnr.0000227980. 06013.31. [8] J. Liang, S. Wu, H. Zhao, S.L. Li, Z.X. Liu, J. Wu, L. Zhou, Human umbilical cord mesenchymal stem cells derived from Wharton’s jelly differentiate into cholinergiclike neurons in vitro, Neurosci. Lett. 532 (2013) 59–63, https://doi.org/10.1016/j. neulet.2012.11.014. [9] M. Nilbratt, O. Porras, A. Marutle, O. Hovatta, A. Nordberg, Neutrophic factors promote cholinergic differentiation in human embryonic stem cell-derived neurons, J. Cell. Mol. Med. 14 (2010) 1476–1484, https://doi.org/10.1111/j.1582-4934. 2009.00916.x. [10] D. Parnas, M. Linial, Cholinergic properties of neurons differentiated from an
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Research article
Differentiation of human glioblastoma U87 cells into cholinergic neuron Honghui Liu, Jinye Xia, Tiansheng Wang, Wei Li, Yexun Song, Guolin Tan
⁎
T
Department of Otorhinolaryngology – Head Neck Surgery, Third Xiangya Hospital, Central South University, Changsha, Hunan, 410013, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Differentiation Cholinergic neurons In vitro model U87 Akt
To facilitate research methodologies for investigating the role of cholinergic nerves in many diseases, establishing an in vitro cholinergic neuron model is necessary. In this study, we investigated whether human glioblastoma U87 cells could be differentiated into cholinergic neurons in vitro. Sodium butyrate was used as the differentiation agent. The differentiated cells established by inducing U87 cells with sodium butyrate were named D-U87 cells. Immunofluorescence was used to label the neuronal markers MAP2, NF-M, and ChAT and the glial marker GFAP in D-U87 cells. Flow cytometry was used to measure cell cycle distribution in D-U87 cells. PCR, protein chip, and western blot assays were used to measure the expression levels of muscarinic cholinergic receptor 1 (M1), M4, ChAT, SYP and Akt. ELISA was used to measure neurotransmitter levels. As a result, we found that sodium butyrate induced U87 cell differentiation into cells with neuronal characteristics and increased not only the expression levels of the cholinergic neuron-related proteins M1, M4, ChAT and SYP in DU87 cells but also the acetylcholine neurotransmitters in D-U87 cells. Moreover, the Akt protein expression in DU87 cells was increased compared with that in U87 cells. Finally, we found that M1, M4, ChAT and SYP protein expression and acetylcholine secretion levels were significantly decreased in D-U87 cells after treatment with the Akt inhibitor MK-2206. These results demonstrate that D-U87 cells exhibit cholinergic neuron characteristics and that sodium butyrate induced U87 cell differentiation into cholinergic neuron partially through Akt signaling.
1. Introduction Increasing evidence indicates that cholinergic nerves play multifunctional roles in many conditions, including central nervous system disease, autoimmune disease, digestive system disease and airway allergic disease [1–3]. For example, parasympathetic nerve dysfunction has been found in depression, epilepsy, ischemic stroke, multiple sclerosis, Parkinson’s disease and Alzheimer’s disease [1]; cholinergic nerve stimulation alleviates symptoms of rheumatoid arthritis by activating nicotinic receptors [3]; and cholinergic nerve hyperactivity accelerates the severity of rhinitis [4]. Additionally, cholinergic hypersensitivity inhibits tumor cell proliferation and contributes to cell differentiation [5]. However, many studies in the past were performed with animal models, in which the procedures are complex; subsequently, the progress of researching the mechanisms involved in various diseases is limited to some extent. Since simplifying research methodologies could promote the mechanistic studies of many diseases, it has become necessary to build an in vitro cholinergic neuron model. Several in vitro cholinergic neuron models have been studied. Most
models were derived from primary stem cells, including neural stem cells, mesenchymal stem cells, and embryonic stem cells [6–9]. Only a few studies have used cell lines, such as the mouse-derived embryonal carcinoma cell line P19, the human neuroblastoma cell line SH-SY5Y, and the rat adrenal pheochromocytoma cell line PC12 [10–12], as cholinergic neuron models. In fact, the procedure for differentiating cells into cells with cholinergic neuron characteristics via primary cell culture is more sophisticated than that via cell lines. Thus, the development of cell lines serving as in vitro cell models should be a priority. The human glioblastoma cell line U87 is derived from brain cancer cells. We have not found any studies showing that U87 cells have the potential to differentiate into cholinergic neuron. Therefore, our study aimed to explore whether U87 cells could be differentiated into cells with cholinergic neuron characteristics and the possible mechanism.
Abbreviations: M1, muscarinic cholinergic receptor 1; M4, muscarinic cholinergic receptor 4; M5, muscarinic cholinergic receptor 5; ChAT, choline acetyltransferase; SYP, synaptophysin; Akt, protein kinase B; MAP2, microtubule-associated protein-2; NF-M, neurofilament-M; GFAP, glial fibrillary acidic protein ⁎ Corresponding author. E-mail address: [email protected] (G. Tan). https://doi.org/10.1016/j.neulet.2019.03.049 Received 28 November 2018; Received in revised form 20 March 2019; Accepted 26 March 2019 Available online 28 March 2019 0304-3940/ © 2019 Elsevier B.V. All rights reserved.
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2. Materials and methods
were incubated with an IRDye®680RD goat anti-mouse IgG (H + L) or IRDye®800CW goat anti-rabbit IgG (H + L) secondary antibody (LICOR Odyssey, Germany) for 30 min in the dark. Images were obtained using an Odyssey Imaging system (LI-COR, Germany). The dilutions of the rabbit anti-M1, M4, M5 and ChAT antibodies (Abcam, USA) were 1:1000, the dilution of the rabbit anti-SYP antibody (Santa Cruz, USA) was 1:200, the dilution the of rabbit anti-Akt antibody (CST, USA) was 1:2000, and the dilution of the mouse anti-GAPDH antibody (Proteintech, USA) was 1:5000.
2.1. Cell culture The human glioblastoma cell line U87 was obtained from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The cells were cultured in Minimum Essential Medium (MEM; Gibco, Invitrogen, USA) containing 10% fetal bovine serum (FBS) (Gemini, USA) and were incubated in a humidified incubator at 37℃ with an atmosphere of 5% CO2.
2.7. Protein chip analysis 2.2. Cell differentiation and Akt inhibitor treatment The stress and apoptosis signaling molecules were screened using the protein chip PathScan® Stress and Apoptosis Signaling Antibody Array Kit (#12923, CST, USA) according to the manufacturer’s instructions. Image detection and analysis were performed using an Odyssey imaging system (LI-COR, Germany)
First, the U87 cells were seeded into six-well plates at 5 × 105 cells per well and grown in proliferation medium (MEM containing 10% FBS) for 24 h. Then, we removed the proliferation medium and differentiated the cells with differentiation medium (MEM containing 2 mM sodium butyrate (Sigma, USA) without FBS) for 48 h. In the Akt inhibition group, we added 50 nM Akt inhibitor MK-2206 (Selleck, USA) into the differentiation medium. Sodium butyrate and the Akt inhibitor MK-2206 were dissolved in MEM. The differentiated cells established by inducing U87 cells with sodium butyrate were named D-U87 cells.
2.8. ELISA The concentrations of human neurotransmitters were evaluated in cell supernatants using commercial kits: ACH (Acetylcholine) (Elabscience, China), NA/NE (Noradrenaline/Norepinephrine) (Elabscience, China), and ү-GABA (Gamma-Aminobutyric Acid) (Wuhan USCN Business Co., Ltd, China), according to the manufacturer’s instructions. The detection limits were as follows: 9.38 pg/mL for ACH; 0.19 ng/mL for NA/NE and 9.39 pg/mL for ү-GABA.
2.3. Immunofluorescence The cells were fixed with 4% paraformaldehyde for 30 min, washed thrice with PBS, and permeabilized in 0.5% TritonX-100 for 20 min. After washing with PBS, the cells were blocked in normal goat serum (ZSGB-BIO, China) for 30 min and were then incubated with primary antibodies (rabbit anti-choline acetyltransferase (ChAT, 1:1000, Abcam, USA), microtubule-associated protein-2 (MAP2, 1:200, Proteintech, USA), neurofilament-M (NF-M, 1:200, Proteintech, USA), or glial fibrillary acidic protein (GFAP, 1:200, Proteintech, USA)) at 4 °C overnight. After washing thrice with PBS, the cells were incubated with a TRITC-conjugated goat anti-rabbit secondary antibody IgG (1:100, Proteintech, USA) for 1 h at room temperature. Images were captured using a fluorescence microscope (Olympus, Japan).
2.9. Statistical analysis The statistical analysis was performed using SPSS version 22 software (SPSS Inc., USA). The data are presented as the mean ± SD. The statistical analysis of the independent samples was performed using ttests. P < 0.05 was considered statistically significant. 3. Results 3.1. Neuron characteristics of human glioblastoma U87 cells after differentiation
2.4. Flow cytometry Cells were collected and stained with 1 mL of DNA staining solution and 10 mL of permeabilization solution, according to the instructions from the Cell Cycle Staining Kit (MULTI SCIENCES, China). Cells were detected on a FACSCalibur flow cytometer (BD Biosciences, USA), and the results of cell cycle distribution (in the G0/G1, S and G2/M phase) were analyzed using FlowJo 10 software (BD Biosciences, USA).
Sodium butyrate, which is an inducer agent, was used to differentiate the U87 cells. Compared with U87 cells, D-U87 cells presented elongated nerve fibers, and the lengths of some fibers were 2 times longer than those of the neurite bodies (Fig. 1A–D). Immunofluorescence staining showed higher intensities of ChAT, MAP2 and NFM in D-U87 cells than in U87 cells (Fig. 1A–D), while the GFAP staining between U87 and D-U87 had no significant difference, indicating that D-U87 cells may present cholinergic nerve characteristics. Moreover, flow cytometry was used to measure cell cycle distribution, as shown in Fig. 1E and F, compared with U87 group, the percentage of cells in S phase in the D-U87 group was significantly lower, whilst the percentage in G0/G1 phase and G2/M phase was significantly higher. These results demonstrated that D-U87 cells presented not only neuron characteristics, including increased intensities of neuron markers of MAP2 and NF-M, and proliferation inhibition, but also cholinergic neuron characteristics, like higher intensity of cholinergic neuron marker ChAT.
2.5. Neurotransmitter receptor PCR chip (RT2 RNA QC PCR array protocol) The total RNA was extracted from the U87 and D-U87 cells using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The RNA samples were sent to the Kangcheng Company (Shanghai, China) for assistance in performing the following protocols, including RNA detection and data analysis. 2.6. Western blot
3.2. Cholinergic neuron-related molecular changes in U87 cells after differentiation
The cells were washed with ice-cold PBS and lysed using RIPA Lysis Buffer (KeyGEN BioTECH, China) containing 1 mM PMSF. The proteins (30 μg) were separated by electrophoresis via 10% SDS-polyacrylamide gels, electrophoretically transferred to PVDF (Millipore, USA) membranes via a wet transfer apparatus (Bio-Rad, USA), blocked in TBS blocking buffer (LI-COR Odyssey, Germany) and incubated with specific primary antibodies (rabbit anti-M1, -M4, -M5, -ChAT, -SYP, and -Akt and mouse anti-GAPDH) at 4 °C overnight. Next, the membranes
To further investigate whether D-U87 cells present cholinergic neuron characteristics, we screened the cholinergic receptors using a neurotransmitter mRNA chip analysis. Compared with the U87 cells that did not receive sodium butyrate treatment, the D-U87 cells exhibited increased M1, M4, N3 and CHRNE mRNA expression levels (Fig. 2A). Then, we measured the protein expression levels of the 2
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Fig. 1. Neuron characteristics of U87 cells after differentiation. (A–D) Immunofluorescence staining of ChAT, MAP2, NF-M, and GFAP between U87 and D-U87 cells, respectively. In each image, the upper panel were pictures of U87 and D-U87 observed under an inverted microscope. The lower panel were merged pictures from DAPI (Blue) and the cholinergic neuronal marker ChAT (Red), or the neuronal markers MAP2 (Red), NF-M (Red), or the glial marker GFAP (Red) respectively. Scale bar, 100 μm. (E) Cell cycle distribution between U87 and D-U87 cells was analyzed by flow cytometry using propidium iodine staining. (F) Percentages of U87 and DU87 cells in G0/G1, S and G2/M phases. Data are expressed as the mean ± SD, and at least three independent experiments were performed. *p < 0.05, vs U87 group.
butyrate exhibited increased M1, M4, ChAT and SYP levels, indicating that D-U87 cells exhibited cholinergic neuron characteristics. To clarify the potential mechanism involved in the process of sodium butyrateinduced U87 cell differentiation, we screened related proteins using a stress and apoptosis protein chip. The results showed that several proteins, including Akt, Bad, p53, JNK, and TAK1, were increased and that HSP27 was decreased in U87 cells after differentiation. Among the changed proteins, Akt was the most significantly increased protein in DU87 cells compared to U87 cells without differentiation (Fig. 3A and B). Furthermore, we tested Akt expression using western blotting, and the results showed a significant increase in Akt in D-U87 cells compared with its expression in U87 cells (Fig. 3C and D). These results demonstrate that the increased M1, M4, ChAT and SYP levels in U87 cells after
muscarinic receptors M1, M4 and M5 by western blotting. The results showed that the M1 and M4 levels were significantly increased after differentiation, and no increase in the M5 level was observed (Fig. 2B and C), consistent with the PCR screening chip results. In addition, we assessed the protein levels of ChAT, which is a specific cholinergic neuron marker, and SYP, which is a specific marker of neuron synapses, both of which are involved in neurotransmitter synthesis and release. The results showed that the ChAT and SYP levels were increased in the D-U87 cells compared with those in the U87 cells (Fig. 2B and C).
3.3. Akt protein changes in U87 cells after differentiation The above results show that U87 cells differentiated by sodium 3
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Fig. 2. Cholinergic neuron-related gene expression in D-U87 cells. (A) Cholinergic gene mRNA expression in D-U87 cells was analyzed by a neurotransmitter mRNA PCR chip. The expression levels of the muscarinic cholinergic receptor 1 (M1), M4 and the nicotinic cholinergic receptor 3 (N3) in DU87 cells were higher than those in U87 cells. CHRM, muscarinic cholinergic receptor. CHRNA, nicotinic cholinergic receptor. CHRNE, cholinergic receptor nicotinic epsilon subunit. (B) The expression levels of the cholinergic neuron-related proteins M1, M4, M5, ChAT and SYP were determined by western blot after differentiation. (C) The quantifications of M1, M4, M5, ChAT and SYP proteins were analyzed after normalization to GAPDH. M1, muscarinic receptor 1. M4, muscarinic receptor 4. M5, muscarinic receptor 5. ChAT, choline acetyltransferase. SYP, synaptophysin. Data are expressed as the mean ± SD, and at least three independent experiments were performed. *p < 0.05, vs U87 group.
cell line U87 could be differentiated into cells with cholinergic neuron characteristics. We found that sodium butyrate induced U87 cells into cholinergic neuron with increased cholinergic neuron-related protein expression levels of M1, M4, ChAT and SYP and increased secretion of acetylcholine. Furthermore, we found that Akt plays a role in the sodium butyrate-induced U87 differentiation process. Although many studies have explored in vitro cholinergic neuron models, most models have been restricted to primary cell cultures. Among the primary cell culture system, stem cells are commonly used. Neural stem cells, mesenchymal stem cells, and embryonic stem cells can differentiate toward a cholinergic phenotype in a specific medium containing nerve growth factor (NGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), neurotrophin-3 (NT3), and brain-derived neurotrophic factor (BDNF) [6–9,20–22]. In addition to various stem cells, other types of cells, such as fetal lung fibroblasts, glial cells and even sympathetic neurons, exhibit the capacity to differentiate into cells showing cholinergic neuron characteristics under special treatment conditions [23–25]. The differentiation times vary from ten to fourteen days. Considering the lengthy differentiation time and sophisticated processes involved in primary cell cultures, several researchers have established neuron models using cell lines to avoid complex separation management and culturing procedures that may lead to contamination. The mouse-derived embryonal carcinoma cell line P19 and the human neuroblastoma cell line SH-SY5Y can differentiate into cells with cholinergic characteristics with retinoic acid [10,11,26]. Nerve growth factor (NGF) can induce the rat adrenal pheochromocytoma cell line PC12 into exhibiting cholinergic nerve features [12,27]. Other agents, such as dibutyryl cAMP and IL-4, have also been used as differentiation inducers [28,29]. Generally, compared with primary cell differentiation, cell line differentiation often requires less time; only four to ten days of differentiation time is often enough, and the differentiation induction conditions are so simple that only one type of inducing agent is needed.
differentiation may be related to enhanced Akt expression. 3.4. Effect of Akt inhibition on cholinergic neuron-related molecule changes in D-U87 cells To determine whether Akt plays a role in the U87 cell differentiation process, we used western blotting to measure M1, M4, M5, ChAT and SYP expression levels following treatment with 50 nM of the Akt inhibitor MK-2206 in D-U87 cells. As shown in Fig. 4, the Akt inhibitor significantly decreased the M1, M4, ChAT and SYP expression levels but did not have an effect on M5 expression in D-U87 cells. 3.5. Neurotransmitters in U87 cells after differentiation and the effect of Akt inhibition To determine what types of neurotransmitters exist in D-U87 cells, we measured the acetylcholine (ACH), noradrenaline/norepinephrine (NA/NE) and gamma-aminobutyric acid (ү-GABA) levels in the supernatants of U87 and D-U87 cells. We found that ү-GABA level was below the detection range (9.39 pg/mL) (data not shown). The ACH level in DU87 cells was significantly increased compared with that in U87 cells, and Akt inhibitor reduced the ACH level (Fig. 5A). However, the NA/NE level in U87 cells was significantly decreased after differentiation, and the Akt inhibitor had no effect on NA/NE secretion (Fig. 5B). 4. Discussion Increasing evidence suggests that cholinergic nerve dysfunction is closely related to various conditions involving the central nervous system, the respiratory system, and autoimmune diseases [1,2,13–19]. While most studies have focused on animal models, it is necessary to build an in vitro model of cholinergic neurons to facilitate research methodologies. In this study, we investigated whether the glioblastoma 4
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Fig. 3. Akt protein expression in U87 cells after differentiation. (A) Representative images and (B) statistical analysis of the stress and apoptosis related protein expression levels, as determined by protein chip after differentiation. The red rectangular box represents the Akt protein spot. (C) Akt expression was determined by western blot after differentiation. (D) The quantifications of Akt protein were analyzed after normalization to GAPDH. Data are expressed as the mean ± SD, and at least three independent experiments were performed. *p < 0.05, vs U87 group.
Fig. 4. Effects of Akt inhibition on M1, M4, M5, ChAT and SYP protein expression levels in D-U87 cells. (A) The expression levels of the cholinergic neuron-related proteins M1, M4, M5, ChAT and SYP were determined by western blot after D-U87 cells were treated with the Akt inhibitor MK-2206. MK, MK-2206. (B) The quantifications of M1, M4, M5, ChAT and SYP proteins were analyzed after normalization to GAPDH. M1, muscarinic receptor 1. M4, muscarinic receptor 4. M5, muscarinic receptor 5. ChAT, choline acetyltransferase. SYP, synaptophysin. Data are expressed as the mean ± SD, and at least three independent experiments were performed. *p < 0.05, vs D-U87 group.
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Fig. 5. Acetylcholine and noradrenaline/norepinephrine levels in differentiated U87 cells and the effects of Akt inhibition on the neurotransmitter levels. (A) The acetylcholine (ACH) level in the supernatant from differentiated U87 (D-U87) cells after treated with Akt inhibitor MK-2206. ACH, acetylcholine. MK, MK-2206. (B) The noradrenaline/norepinephrine (NA/NE) level in the supernatant from D-U87 cells after treated with the Akt inhibitor. NA/NE, noradrenaline/norepinephrine. Data are expressed as the mean ± SD, and at least three independent experiments were performed. *p < 0.05, vs U87 group, #p < 0.05, vs D-U87 group.
time, differentiation reagents and culture processes. Therefore, additional in vitro cholinergic neuron models using cell lines and the differentiation mechanisms involved in different induction reagents will require further exploration.
Sodium butyrate, a histone deacetylase (HDAC) inhibitor, inhibits cell proliferation, contributes to cell apoptosis, induces cell cycle arrest and promotes cell differentiation [30–32,37]. Although no studies have reported that U87 cells could differentiate into cholinergic neurons, in our study, we found that sodium butyrate could induce U87 cells to exhibit neuronal fiber morphology. The entire differentiation time was only two days of culture, which significantly reduced the research period. D-U87 cells presented higher intensities of the neuronal markers MAP2 and NF-M and the cholinergic neuronal marker ChAT, and a decreased S phase whilst increased G1/G0 phase and G2/M phase than U87 cells, which demonstrated that D-U87 cells had neuron characteristics. Furthermore, the expression levels of cholinergic neuronrelated molecules, such as M1, M4, ChAT and SYP, and the neurotransmitter secretion of acetylcholine were significantly increased in U87 cells after differentiation. The cholinergic differentiation of U87 cells by sodium butyrate may occur through histone deacetylase (HDAC) inhibition. Several studies have shown that HDAC inhibition could contribute to ChAT expression [33–36]. HDAC inhibitor could significantly increase ChAT mRNA expression [33]. HDAC9 has been shown to repress ChAT expression, and HDAC inhibitor could not only restore stress-induced AChE impairment but also increase muscarinic receptor expression levels [34,34,35,36]. All of the evidence indicates that sodium butyrate is positively related to cholinergic function. Moreover, we found that sodium butyrate enhanced the expression of Akt in D-U87 cells and that the inhibition of Akt reduced the expression levels of M1, M4, ChAT and SYP in D-U87 cells. Studies have shown that Akt positively regulated sodium butyrate-mediated cell differentiation [38,39,42]. For example, sodium butyrate induces microglial process elongation through the activation of Akt [38]. Akt signaling also participated in the differentiation of the SH-SY5Y cell line [39]. In addition, butyrate increased adipogenic differentiation via activating Akt [42]. On the contrary, others have demonstrated that sodium butyrate inhibited cancer cell motility through inhibiting Akt/ ERK expression [40] and that butyrate decreased human colorectal cancer cell line CaCo-2 and SNU-C4 proliferation through inhibiting Akt [41]. The discrepancy of the effects of sodium butyrate on Akt expression in different studies may be due to the different cell lines used in the research and the inconsistent cellular functions observed under different conditions. On the other hand, several studies have shown a positive relationship between cholinergic receptors and Akt expression [44,45]. Akt could positively regulate CXCL12-induced cholinergic gene expression [43]. Akt also activates cell chemotaxis through activating G protein-coupled receptors [46]. Furthermore, activation of muscarinic receptors could increase Akt expression [47] and combine to trigger NGF-induced Akt expression [12]. In our study, sodium butyrate induced U87 cell differentiation toward cholinergic neuron, accompanied by an increase in Akt, and cholinergic neuron-related protein levels and acetylcholine secretion level were decreased via Akt inhibition. This evidence suggests that Akt partially participates in the process of U87 cell cholinergic differentiation. Differentiation using cell lines facilitates research procedures more than the use of primary cells by affecting various factors, such as culture
5. Conclusions In conclusion, our study provides a new in vitro cholinergic neuron cell model derived from U87 cell line, and sodium butyrate mediated this differentiation process at least partially through Akt signaling. We hope that our findings may provide a new possible method or cholinergic neuron model for further research on the roles played by cholinergic nerves in various diseases. Declarations of interest None. Acknowledgements This work was supported by the National Natural Science Foundation [8187078, 81702706, 81502358], the Fundamental Research Funds for the Central Universities of Central South University [2017zzts237], and the Funds of the Hunan Scientific Plan in China [2017SK2043, 12JJ4079]. References [1] B. Han, X. Li, J. Hao, The cholinergic anti-inflammatory pathway: an innovative treatment strategy for neurological diseases, Neurosci. Biobehav. Rev. 77 (2017) 358–368, https://doi.org/10.1016/j.neubiorev.2017.04.002. [2] B.J. Undem, T. Taylor-Clark, Mechanisms underlying the neuronal-based symptoms of allergy, J. Allergy Clin. Immunol. 133 (2014) 1521–1534, https://doi.org/10. 1016/j.jaci.2013.11.027. [3] D.B. Hoover, Cholinergic modulation of the immune system presents new approaches for treating inflammation, Pharmacol. Ther. 179 (2017) 1–16, https://doi. org/10.1016/j.pharmthera.2017.05.002. [4] S. Ssarin, B. Undem, A. Sanico, A. Togias, The role of the nervous system in rhinitis, J. Allergy Clin. Immunol. 118 (2006) 999–1016, https://doi.org/10.1016/j.jaci. 2006.09.013. [5] R.R. Resende, A. Adhikari, Cholinergic receptor pathways involved in apoptosis, cell proliferation and neuronal differentiation, Cell Commun. Signal 7 (2009) 20, https://doi.org/10.1186/1478-811X-7-20. [6] L. Wang, F. He, Z. Zhong, R. Lv, S. Xiao, Z. Liu, Overexpression of NTRK1 promotes differentiation of neural stem cells into cholinergic neurons, Biomed Res. Int. (2015) 857202, , https://doi.org/10.1155/2015/857202 2015. [7] T.T. Wang, A.H. Jing, X.Y. Luo, M. Li, Y. Kang, X.L. Zou, H. Chen, J. Dong, S. Liu, Neural stem cells: isolation and differentiation into cholinergic neurons, Neuroreport 17 (2006) 1433–1436, https://doi.org/10.1097/01.wnr.0000227980. 06013.31. [8] J. Liang, S. Wu, H. Zhao, S.L. Li, Z.X. Liu, J. Wu, L. Zhou, Human umbilical cord mesenchymal stem cells derived from Wharton’s jelly differentiate into cholinergiclike neurons in vitro, Neurosci. Lett. 532 (2013) 59–63, https://doi.org/10.1016/j. neulet.2012.11.014. [9] M. Nilbratt, O. Porras, A. Marutle, O. Hovatta, A. Nordberg, Neutrophic factors promote cholinergic differentiation in human embryonic stem cell-derived neurons, J. Cell. Mol. Med. 14 (2010) 1476–1484, https://doi.org/10.1111/j.1582-4934. 2009.00916.x. [10] D. Parnas, M. Linial, Cholinergic properties of neurons differentiated from an
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