ETM Experimental and Therapeutic Medicine 1792-0981 1792-1015 D.A. Spandidos ETM-0-0-09143 10.3892/etm.2020.9143 Articles Melatonin promotes Schwann cell dedifferentiation and proliferation through the Ras/Raf/ERK and MAPK pathways, and glial cell-derived neurotrophic factor expression TiongYee Lian 1 NgKhuen Yen 2 KohRhun Yian 3 PonnuduraiGnanajothy 4 ChyeSoi Moi 3 School of Postgraduate, International Medical University, Kuala Lumpur 57000, Malaysia School of Pharmacy, Monash University Malaysia, Subang Jaya, Selangor 47500, Malaysia School of Health Science, International Medical University, Kuala Lumpur 57000, Malaysia School of Medicine, International Medical University, Kuala Lumpur 57000, Malaysia Correspondence to: Dr Soi Moi Chye, School of Health Science, International Medical University, 126 Jalan Jalil Perkasa, 19 Bukit Jalil, Kuala Lumpur 57000, Malaysia chye_soimoi@imu.edu.my 11 2020 26 08 2020 20 5 16 06 09 2018 22 11 2019 Copyright: © Tiong et al. 2020 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Upon peripheral nerve injury (PNI), continuous proliferation of Schwann cells is critical for axon regeneration and tubular reconstruction for nerve regeneration. Melatonin is a hormone that is able to induce proliferation in various cell types. In the present study, the effects of melatonin on promoting Schwann cell proliferation and the molecular mechanism involved were investigated. The present results showed that melatonin enhanced the melatonin receptors (MT1 and MT2) expression in Schwann cells. Melatonin induced Schwann cell dedifferentiation into progenitor-like Schwann cells, as observed by immunofluorescence staining, which showed Sox2 marker expression. In addition, melatonin enhanced Schwann cell proliferation, mediated by the upregulation of glial cell-derived neurotropic factor (GNDF) and protein kinase C (PKC). Furthermore, the Ras/Raf/ERK and MAPK signaling pathways were also involved in Schwann cell dedifferentiation and proliferation. In conclusion, melatonin induced Schwann cell dedifferentiation and proliferation via the Ras/Raf/ERK, MAPK and GDNF/PKC pathways. The present results suggested that melatonin could be used to enhance the recovery of PNI.

 Schwann cells melatonin peripheral nerve injury glial cell-derived neurotrophic factor Ras/Raf/ERK MAPK Introduction

Peripheral nerve injury (PNI) is a common injury that can impact the quality of life of individuals (1). After an injury, peripheral axons activate the intrinsic growth capacity in the neuronal cell body and promote axonal regeneration (2). Previous studies have suggested that Schwann cells are important in the regeneration of peripheral axons and functional recovery (3,4). After the events of a PNI, mature Schwann cells dedifferentiate into progenitor-like Schwann cells, known as Schwann cell precursors, promote myelin debris clearance and tubular reconstruction, and also form a cellular bridge spanning the gap between ends of damaged nerves (5,6). Subsequently, Schwann cell precursors secrete neurotrophic factors to enhance Schwann cell proliferation and can lead to the formation of Bunger bands (6-8).

The Ras/Raf/ERK signaling pathway plays an important role in Schwann cell dedifferentiation (9). Previous studies have demonstrated that Ras/Raf/ERK activation results in cell cycle arrest of primary neurons and promotes differentiation (9,10). Additionally, ERK has a role in regulating Wallerian degeneration and regeneration; sustained ERK activation is known to initiate cell proliferation (7). Glial cell-derived neurotropic factor (GDNF) is a survival factor that stimulates both peripheral regeneration and the functional recovery of several types of neurons (11-13). Previous studies have shown that GDNF transcription can be increased via the activation of the Schwann cell purinergic receptor, followed by the activation of protein kinase C (PKC) and protein kinase D (PKD) (14,15). PKD, originally characterized as an isoform of PKC, regulates proliferation, migration and differentiation of numerous cell types, and promotes neuron regeneration (14,15).

Although microsurgical techniques have been introduced to repair PNI, in the past two decades only a small number of patients have regained full functional recovery (16). Therefore, it is important to search for new approaches that could promote nerve regeneration after PNI.

Melatonin is a hormone secreted by the pineal gland. Previous studies have demonstrated that melatonin stimulates the proliferation of avian astrocytes, dentate neurons and hippocampal neurons (17,18). The action of melatonin is mediated through a receptor-dependent signaling pathway (19). Additionally, melatonin has free radical scavenging and antioxidant properties, which can decrease malondialdehyde levels and may reverse the ischemia-reperfusion injury of the sciatic nerve (12). In the present study, the effects of melatonin and the molecular mechanism involved in promoting the proliferation of Schwann cells were investigated. A better understanding of the molecular mechanism of melatonin-induced Schwann cell proliferation may provide important insight regarding the potential use of melatonin in improving nerve regeneration.

 Materials and methods <sec> <title>Materials

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, EDTA were purchased from Gibco; Thermo Fisher Scientific, Inc. MTT, dimethyl sulfoxide, Triton X-100, Tergitol NP-40, Tris-HCl, PBS, dithiothreitol, SDS, ammonium acetate, Tris-borate-EDTA buffer, Bradford reagent and phenylmethyl sulfonyl fluoride were purchased from Sigma-Aldrich; Merck KGaA. Primary antibodies against SAPK-JNK (cat. no. 9926T), p38 (cat. no. 9926T), ERK (cat. no. 9926T), p-SAPK-JNK (cat. no. 9910T), p-p38 (cat. no. 9910T), p-ERK (cat. no. 9910T), Ras (cat. no. 18070T), c-Raf (cat. no. 9422T), p-c-Raf (cat. no. 9427S), b-Raf (cat. no. 9433S) and p-b-Raf (cat. no. 2696S), in addition to horseradish peroxidase-conjugated goat anti-rabbit (cat. no. 7074S) and anti-mouse (cat. no. 7076S) secondary antibodies were purchased from Cell Signaling Technology, Inc. Primary antibodies against MT1 (cat. no. sc-13177), MT2 (cat. no. sc-13186), PKC (cat. no. sc-10800), GDNF (cat. no. sc328) and α-tubulin (cat. no. sc-8035) were purchased from Santa Cruz Biotechnology, Inc. Primary antibody against Sox2 (cat. no. ab79351) and Alexa-Fluor® 488-conjugated goat anti-mouse secondary antibody (cat. no. ab150117) were purchased from Abcam. Amersham ECL-Plus Western Blotting Reagents and PVDF membranes were obtained from GE Healthcare Life Sciences. RNeasy Mini kit and QuantiNova SYBR Green PCR kit were purchased from Qiagen, GmbH.

 Cell culture and treatment

RT4-D6P2T (RT4), a rat neural Schwann cell line, was purchased from The American Type Culture Collection and cultured in DMEM supplemented with 10% heat-inactivated FBS. The cells were maintained in incubator at 37˚C with 5% CO2. Melatonin stock concentration of 50 mg/ml (215.26 µM) was freshly prepared with ethanol.

 Cell morphology

Schwann cells (3x105) were seeded into 35-mm tissue culture dishes overnight. The cells were then treated with various concentrations of melatonin (0.5-10 µM) dissolved in serum-free medium at 37˚C. After 24 h treatment, the morphological changes in the Schwann cells were observed using light microscopy (magnification, x200).

 MTT assay

Cell proliferation was determined by an MTT assay. In total, 1x105 Schwann cells were seeded in a 96-well plate overnight. The cells were treated with melatonin concentrations of 0.5-10 µM in serum-free medium. After 24 h of treatment, Schwann cells were added with MTT to a final concentration of 1 mg/ml. After 3 h incubation, formazan crystals were dissolved using 100 µl of DMSO and absorbance was measured using a microplate reader (Infinite 200 PRO; Tecan Group, Ltd.) at a wavelength of 570 nm.

 Immunofluorescence staining

Melatonin-induced Schwann cell dedifferentiation was demonstrated by Sox2 immunofluorescence staining. Schwann cells were treated with melatonin (1 and 10 µM) in serum-free medium. After 24 h treatment, Schwann cells were fixed with 4% paraformaldehyde in PBS at 37˚C for 30 min and blocked with 1% BSA (Sigma-Aldrich; Merck KGaA) for 1 h at 37˚C. The cells were then incubated with anti-Sox2 antibody (1:50) overnight at 4˚C followed by incubation with the AlexaFluor® 488-conjugated antibodies (1:200 dilution) for 1 h at 37˚C. Primary and secondary antibodies were diluted in PBS. Cells were counterstained with 0.5 µg/ml DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) diluted in PBS (1:500) for 15 min at 37˚C and observed under a fluorescent microscope (magnification, x200).

 Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA of melatonin-treated cells was extracted using the RNeasy Mini kit. The cells were homogenised with the RNeasy lysis buffer provided in the kit. RNA was collected with the RNeasy Mini spin column, according to the manufacturer's instruction. RNA (1 µg) was quantified at absorbance of 260/280 nm with a plate reader (Infinite® 200 PRO NanoQuant; Tecan Group, Ltd.).

cDNA was synthesized using the Maxima First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to manufacturer's protocol. The temperature protocol for reverse transcription was as follows: 2 min at 37˚C, 10 min at 25˚C, 15 min at 50˚C and 85˚C for 5 min. RT-qPCR was performed using QuantiNova SYBR Green PCR kit. The procedures followed the manufacturer's recommended amplification conditions with iQ5 real-time PCR detection system. The sequences of Rattus norvegicus primers used for the PCR were as follows: GAPDH-forward, 5'-TGCACCACCAACTGCTTAG-3' and reverse, 5'-GGATGCAGGGATGATGTTC-3'; MT1-forward, 5'-TCATCTTTACTATCGTGGTGG-3' and reverse, 5'ACCACAAATATATTCCCTGCG-3'; MT2-forward, 5'-ACCTGTTACTGAATGTTGCC-3' and reverse, 5'-AACGAAGTCTCACTTCAACAC-3'; GDNF-forward, 5'-TGACCAGTGACTCCAATATG-3' and reverse, 5'-TACCTTGTCACTTGTTAGCC-3'; and PKC-forward, 5'-CCAAAAGCTAGAGACAAGCG-3' and reverse, 5'-TGGAATCTGCATTCACCTAC-3'. The thermocycling conditions were as follows: Initial denaturation 95˚C for 2 min, followed by 40 cycles of 95˚C for 5 sec and 60˚C for 10 sec. Relative mRNA expression of each gene to GAPDH was calculated based on the 2-ΔΔCq method (20).

 Western blotting assay

Schwann cells treated with melatonin were lysed with 10X SDS sample buffer [62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol and 100 mM DTT]. Protein concentration was determined using the Quick Start™ Bradford Protein Assay kit (Bio-Rad Laboratories, Inc.). Protein samples (25 µg) were applied to 10% gels and separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% BSA for 1 h at room temperature and then incubated with primary antibodies against MT1, MT2, GDNF, PKC, Ras, B-raf, p-B-Raf, C-raf, p-C-Raf, ERK1/2, p-ERK1/2, p39, p-p38, SAPK-JNK, p-SAPK-JNK and α-tubulin at 4˚C overnight. All primary antibodies were prepared in 1:500 dilutions in blocking buffer. The blots were then incubated with horseradish peroxidase-conjugated anti-rabbit/mouse secondary antibodies at 1:10,000 dilution for 1 h at room temperature. The blots were subsequently developed with SuperSignal™ West Femto Maximum Sensitivity (Thermo Fisher Scientific, Inc.) substrate and visualised using ChemiDoc XRS+ system (Bio-Rad Laboratories, Inc.). Quantitative blot data were analysed and calculated using Image Lab Software (version 4.0; Bio-Rad Laboratories, Inc.).

 Statistical analysis

All experiments were repeated three times and data were presented as the mean ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by Dunnett's test (SPSS 21.0; IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>Melatonin enhances Schwann cell proliferation

An MTT assay was performed to determine the melatonin-induced proliferation of Schwann cells. The results showed that melatonin increases Schwann cell proliferation, after treatment with 0.5, 1, 5 and 10 µM melatonin (Fig. 1). The cell viability was significantly increased from 3.8 to 17.2% across the different treatment groups, suggesting that melatonin promoted Schwann cell proliferation in a dose-dependent manner.

 Melatonin increases the gene expression of MT1 and MT2 receptors in Schwann cells

To determine the presence of melatonin receptors on Schwann cells, western blotting (Fig. 2A) and RT-qPCR (Fig. 2B) were performed. The present study identified the presence of both MT1 and MT2 receptors in Schwann cells. Following treatment with 1 and 5 µM melatonin, the expression of MT1 and MT2 receptor mRNA were significantly increased compared with control. However, after treatment with 10 µM melatonin, the expression of MT1 and MT2 receptor mRNA was significantly decreased compared with 0.5, 1 and 5 µM melatonin treatment (Fig. 2B). MT1 gene expression was higher compared with MT2 following treatment with 1 and 5 µM melatonin, suggesting that MT1 was more responsive to the melatonin treatment and may serve a role in inducing proliferation in Schwann cells.

 Melatonin-induced Schwann cell dedifferentiation

To determine if dedifferentiation of Schwann cells was induced by melatonin, immunofluorescence staining and a western blotting assay were conducted. Under light microscope observation, Schwann cells have oval, spindle-shape or bipolar-like cell morphology. The cell morphology between control cells and melatonin-treated cells did not differ much (Fig. 3). Under fluorescent microscope observation, the cell nucleus was stained with DAPI (blue fluorescence). However, only melatonin-treated cells expressed the Sox2 marker and showed green fluorescence (Fig. 3), which indicated a positive cell dedifferentiation process. Moreover, Sox2 protein expression increased in a dose-dependent manner as shown by the western blotting assay (Fig. 4).

 Melatonin increases GDNF and PKC protein expressions in Schwann cells

To determine GDNF and PKC gene and protein expressions in Schwann cells, RT-qPCR and western blotting assays were conducted. The GDNF protein expression was significantly upregulated when the cells were treated with 1, 5 and 10 µM melatonin as compared with the control, whilst PKC protein expression was significantly upregulated following treatment with 1 and 5 µM melatonin compared with control (Fig. 5A and B). RT-qPCR analysis showed that GDNF and PKC gene expressions were significantly upregulated in cells treated with 1 and 5 µM melatonin as compared with the control (Fig. 5C).

 Melatonin increases Ras and p-Raf protein expression in Schwann cells

The Ras, B-Raf, p-B-Raf C-Raf and p-C-Raf protein expressions in Schwann cells were measured by western blotting. In Fig. 6A, after melatonin treatment, the Ras, p-B-Raf and p-C-Raf protein expressions increased in a dose-dependent manner. However, there were no protein expressions changes for B-Raf and C-Raf. In Fig. 6B, the normalized phosphorylated protein was compared with the respective total protein, which showed that p-B-Raf/B-Raf and p-C-Raf/C-Raf protein expressions increased in a dose-dependent manner, especially at higher concentrations of melatonin (5 and 10 µM).

 Melatonin regulates ERK, SAPK-JNK and p38 protein expressions in Schwann cells

ERK, p-ERK, SAPK-JNK, p-SAPK-JNK, p38 and p-p38 protein expressions were measured in Schwann cells using a western blotting assay. The present study showed no significant difference in the expression of total protein levels of ERK, SAPK/JNK and p38 between the control and the melatonin treated groups (Fig. 7A). However, while normalized phosphorylated proteins with the respective total proteins, the results showed that in the melatonin treated groups, p-ERK(p44)/ERK(p44) and p-ERK(p42)/ERK(p42) increased in a dose-dependent manner (Fig. 7B), especially at higher concentrations of melatonin (5 and 10 µM). Furthermore, melatonin downregulated the expressions of p-SAPK-JNK(p54)/SAPK-JNK(p54), p-SAPK-JNK(p46)/SAPK-JNK(p46) (Fig. 7D) and p-p38/p38 (Fig. 7C) proteins as compared with the control.

 Discussion

Melatonin has been shown to promote glial cell survival, axonal regeneration and neuronal stem cell proliferation (21,22). Turgut et al (23) demonstrated that melatonin inhibited both collagen production and neuroma formation, resulting in axonal regeneration of the transected sciatic nerve in rats. In pinealectomized rats, melatonin suppressed transforming growth factor-β and basic fibroblast growth factor protein expressions (24). The findings from a previous study suggested that melatonin could suppress collagen scar formation after PNI (23). Melatonin also increased the number of axons and the thickness of the myelin sheath after PNI (4). Stavisky et al (25) demonstrated that melatonin enhanced the repair of sciatic nerve crush injuries in rats. However, the exact molecular mechanism involved is not yet known.

Schwann cells play an important role in the regeneration of damaged nerves. Upon PNI, Schwann cells lose their connection to axons. This phenomenon triggers the continuous proliferation of Schwann cells and the formation of Bunger bands that provide guidance for axonal regeneration (5). The present study indicated that melatonin induced RT4 Schwann cell proliferation at concentrations of 0.5, 1, 5 and 10 µM, while Chang et al (26) identified that the optimal melatonin concentrations to enhance RSC 96 Schwann cell proliferation were 0.1, 1 and 10 nM. This difference may be due to different cell lines being used in the previous study and melatonin having different efficacies in various cell lines. Moreover, Chang et al (26) also demonstrated that melatonin enhanced RSC 96 Schwann cell proliferation mainly via the MT1 receptor as well as phosphorylation of ERK1/2 protein expression. However, the present study not only suggested that melatonin is able to dedifferentiate RT4 Schwann cells, the present study also demonstrated that melatonin may enhance cell proliferation through three different pathways, which are the Ras/Raf/ERK, MAPK and GDNF/PKC pathways. The identification of these proliferation pathways could have important implications for future development of therapeutic approaches. The present results demonstrated that melatonin induces Schwann cell proliferation via MT1 and MT2 receptors, and the MT1 receptor expression was higher than MT2. Previous studies demonstrated that MT1 activation is involved in proliferation, differentiation and firing in neuronal cells (27,28). Chern et al (29) observed that MT2 activation enhanced endogenous neurogenesis in rats. MT2 also played an important role in axonogenesis in in vivo and in vitro models (30). However, a previous study demonstrated that melatonin can induce cell proliferation in rat pancreatic stellate cells, independent of MT1/MT2 receptor activations via the MAPK pathway (31). In the present study, following treatment with 1 and 5 µM melatonin, the expression of MT1 and MT2 receptor mRNA were significantly increased compared with control. However, after treatment with 10 µM melatonin, the expression of MT1 and MT2 receptor mRNA was reduced compared with 0.5, 1 and 5 µM melatonin treatment. This means high concentrations of melatonin may not result in a high responses due to negative feedback effects that altered melatonin receptor or the saturation of melatonin receptors (32).

After PNI, Schwann cells upregulate inflammatory cytokines [tumour necrosis factor-α, interleukin (IL)-1α, IL-6, IL-1β, leukaemia inhibitory factor and monocyte chemotactic protein 1] production in the distal stump (33,34). IL-6 and leukaemia inhibitory factor attract macrophages to the injured nerve and act on neurons to promote axonal regeneration (35,36). Macrophages co-operate with the Schwann cells to degrade myelin debris that potentially inhibit axon growth (37). Macrophages also produce cytokines to promote vascularisation of the distal nerve (38,39). Specifically, upregulation of neurotrophic factors (GDNF, artemin, brain-derived neurotrophic factor, neurotrophin-3, nerve growth factor and vascular endothelial growth factor) promote axonal elongation and the survival of injured neurons (40,41). The present study identified the involvement of GDNF and PKC in the melatonin-mediated Schwann cell proliferation. Loss of GNDF signaling is one of the features of PNI, especially at the distal stump of injured sciatic nerves (42). Previous studies demonstrated that GDNF is a survival factor for several types of neurons (43-45). The addition of exogenous GDNF has been shown to improve peripheral nerve regeneration and functional recovery (46). The present study showed that GDNF was highly expressed in the melatonin-treated Schwann cells. A previous study has reported that Schwann cells secrete a range of neurotrophic factors, including GDNF, which have been shown to be involved in promoting the development and maintenance of a subset of dorsal root ganglion sensory neurons (11). Increased PKC protein expression was also observed in the present study. PKC phosphorylation is involved in the formation of the growth cone after neuronal injury, which is an important step for neuronal regeneration (47,48). Therefore, the present data supported the hypothesis that melatonin promotes Schwann cell proliferation through the activation of the GDNF/PKC pathway.

Activation of the Ras/Raf/ERK signaling pathway is an important process in the development of Schwann cell-derived tumours (9,49). Harrisingh et al (9) identified that the continuous activation of the Ras/Raf/ERK signaling pathway is able to induce Schwann cell dedifferentiation and proliferation. The present results demonstrated that melatonin induced increases in Ras, p-C-Raf/C-Raf, p-B-Raf/B-Raf, p-ERK (p44)/ERK(p44) and p-ERK(p42)/ERK(p42) protein expressions in a dose-dependent manner. There are three different Raf isoforms (Raf-1/C-Raf, B-Raf and A-Raf), which consist of three conserved regions (CR; CR1, CR2 and CR3) (50). CR1 consists of a Ras binding domain and a cysteine rich domain (51). CR2 consists of activating phosphorylation and inhibitory sites, which regulate Ras binding and Raf activation (52). CR3 consists of a kinase activation domain (53). The major differences between the three Raf isoforms are dependent on the number and location of activating phosphorylation, inhibitory and autophosphorylation sites (54,55). All Raf proteins share MEK1/2 kinases as substrates (56). MEK1/2 in turn activates ERK1/2, and this pathway regulates cell proliferation and differentiation (50,57). p-ERK (p44) and p-ERK (p42) proteins are rapidly phosphorylated in response to all mitogens and are ubiquitously expressed; there are no obvious regulatory differences inferred from their protein sequences, their regulation or their sub-cellular localization (49). With Schwann cells being dedifferentiated to Schwann cell precursors, the proliferation rate was enhanced in the presence of cAMP (10). Other previous studies have demonstrated that Ras/Raf/ERK signaling activation also drives the demyelination of peripheral nerves, and the Schwann cells remain in dedifferentiated states, which ultimately leads to axon regeneration and tubular reconstruction (9,58).

MAPKs, consisting of ERK, SAPK-JNK, and p38 MAPKs, are all activated in Schwann cells after PNI (50). Previous studies suggested that the ERK pathway seems to play a more distinct role in Schwann cell dedifferentiation than the SAPK-JNK and p38 MAPK pathways (59,60). The present results demonstrated that after melatonin treatment, p-ERK(44)/ERK(44) and p-ERK(42)/ERK(42) protein expressions increased in a dose-dependent manner. However, p-SAPK-JNK(p54)/SAPK-JNK(p54), p-SAPK-JNK(p46)/SAPK-JNK(p46), p-p38/p38 ratio decreased in manner that is negatively associated with the dose of melatonin concentration used. In conclusion, the ERK pathway plays a more important role than the SAPK-JNK and p38 MAP kinase pathways in Schwann cell dedifferentiation. In summary, the present study provided evidence that melatonin promotes Schwann cell dedifferentiation and proliferation through the GDNF/PKC, Ras/Raf/ERK and MAPK pathways. Schwann cell proliferation plays a crucial role in PNI; the present study suggested the use of melatonin as a therapeutic agent to enhance recovery from PNI. In the future, the present results (melatonin concentrations and mechanisms involved) could be applied to treat sciatic nerve injury and pinealectomized rats to determine optimal melatonin concentrations and parameters for progress in functional recovery.

 Acknowledgements

Not applicable.

 Funding

The present study was funded by The Malaysia Toray Science Foundation (grant no. 14/G72).

 Availability of data and materials

All data generated or analysed during the present study are included in this published article.

 Authors' contributions

YLT performed the experiments, data analysis and interpretation. GP performed the RT-qPCR, western blotting, data analysis and critical revision of the manuscript. RYK and KYN conceived and designed the study. SMC designed the study, wrote the original draft, edited and critically revised the manuscript. All authors read and approved the final manuscript.

 Ethics approval and consent to participate

Not applicable.

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

 References CiaramitaroPMondelliMLogulloFGrimaldiSBattistonBSardAScarinziCMigliarettiGFaccaniGCocitoDItalian Network for Traumatic NeuropathiesTraumatic peripheral nerve injuries: Epidemiological findings, neuropathic pain and quality of life in 158 patientsJ Peripher Nerv Syst1512012720102062677510.1111/j.1529-8027.2010.00260.x RichardsonPMIssaVMPeripheral injury enhances central regeneration of primary sensory neuronesNature3097917931984620420510.1038/309791a0 Fex SvennigsenADahlinLBRepair of the peripheral nerve-remyelination that worksBrain Sci31182119720132496152410.3390/brainsci3031182 AktasATurgutMKaplanSUlkayBOdacıEAkyüzOÇolakoğluSYazıcıACİnceOThe effect of intrauterine acute ethanol exposure on developing sciatic nerves and their myelination: A stereological studyJ Exp Clin Med2635412009 MirskyRJessenKRBrennanAParkinsonDDongZMeierCParmantierELawsonDSchwann cells as regulators of nerve developmentJ Physiol Paris96172420021175577910.1016/s0928-4257(01)00076-6 NaiduMDavidPMajor cellular events in peripheral nerve regeneration. A brief overviewInt Med J869722009 AgthongSKaewsemaATanomsridejchaiNChentanezVActivation of MAPK ERK in peripheral nerve after injuryBMC Neurosci74520061676205810.1186/1471-2202-7-45 BarrasFMPaschePBoucheNAebischerPZurnADGlial cell line-derived neurotrophic factor released by synthetic guidance channels promotes facial nerve regeneration in the ratJ Neurosci Res7074675520021244459610.1002/jnr.10434 HarrisinghMCPerez-NadalesEParkinsonDBMalcolmDSMudgeAWLloydACThe Ras/Raf/ERK signalling pathway drives Schwann cell dedifferentiationEMBO J233061307120041524147810.1038/sj.emboj.7600309 RidleyAJPatersonHFNobleMLandHRas-mediated cell cycle arrest is altered by nuclear oncogenes to induce Schwann cell transformationEMBO J71635164519883049071 HὃkeAHoTCrawfordTOLeBelCHiltDGriffinJWGlial cell line-derived neurotrophic factor alters axon Schwann cell units and promotes myelination in unmyelinated nerve fibersJ Neurosci2356156720031253361610.1523/JNEUROSCI.23-02-00561.2003 SayanHOzacmakVHOzenOACoskunOArslanSOSezenSCAktasRGBeneficial effects of melatonin on reperfusion injury in rat sciatic nerveJ Pineal Res3714314820041535765710.1111/j.1600-079X.2004.00145.x PiirsooMKaljasATammKTimmuskTExpression of NGF and GDNF family members and their receptors during peripheral nerve development and differentiation of Schwann cells in vitroNeurosci Lett46913514020101994474310.1016/j.neulet.2009.11.060 XuPRosenKMHedstromKReyOGuhaSHartCCorfasGNerve injury induces glial cell line-derived neurotrophic factor (GDNF) expression in Schwann cells through purinergic signaling and the PKC-PKD pathwayGlia611029104020132355360310.1002/glia.22491 SvenssonKZeidmanRTrollérUSchultzALarssonCProtein Kinase C beta1 is implicated in the regulation of neuroblastoma cell growth and proliferationCell Growth Differ11641648200011149599 MartinsRSBastosDSiqueiraMGHeiseCOTeixeiraMJTraumatic injuries of peripheral nerves: A review with emphasis on surgical indicationArq Neuropsiquiatr7181181420132421252110.1590/0004-282X20130127 Ramirez-RodriguezGKlempinFBabuHBenítez-KingGKempermannGMelatonin modulates cell survival of new neurons in the hippocampus of adult miceNeuropsychopharmacology342180219120091942116610.1038/npp.2009.46 RennieKDe ButteMPappasBAMelatonin promotes neurogenesis in dentate gyrus in the pinealectomized ratJ Pineal Res4731331720091979604510.1111/j.1600-079X.2009.00716.x FredrichMChristEKorfHWDifferential regulation of cell proliferation and apoptosis by melatonin receptor subtype-signaling in the adult murine brainNeuroendocrinology10715816620182994980210.1159/000491577 LivakKJSchmittgenTDAnalysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) methodMethods2540240820011184660910.1006/meth.2001.1262 BorlonganCVYamamotoMTakeiNKumazakiMUngsuparkornCHidaHSanbergPRNishinoHGlial cell survival is enhanced during melatonin-induced neuroprotection against cerebral ischemiaFASEB J41307131720001087782310.1096/fj.14.10.1307 KongPJByunJSLimSYLeeJJHongSJKwonKJKimSSMelatonin induces AKt phosphorylation through melatonin receptor- and PI3K-dependent pathways in primary astrocytesKorean J Physiol Pharmacol12374120082015739210.4196/kjpp.2008.12.2.37 TurgutMUyanikgilYBakaMTuncATYavapodluAYurtsevenMEKaplanSPinealectomy exaggerates and melatonin treatment suppresses neuroma formation of transected sciatic nerve in rats: Gross morphological, histological and stereoligical analysisJ Pineal Res3828429120051581390610.1111/j.1600-079X.2004.00205.x TurgutMOktemGUysalAYurtsevenMEImmunohistochemical profile of transforming growth factor-beta1 and basic fibroblast growth factor in sciatic nerve anastomosis following pinealectomy and exogenous melatonin administration in ratsJ Clin Neurosci1375375820061683155310.1016/j.jocn.2005.07.019 StaviskyRCBrittJMZuzekATruongEBittnerGDMelatonin enhances the in vitro and in vivo repair of severed rat sciatic axonsNeurosci Lett3769810120051569892810.1016/j.neulet.2004.11.033 ChangHMLiuCHHsuWMChenLYWangHPWuTHChenKYHoWHLiaoWCProliferative effects of melatonin on Schwann cells: Implication for nerve regeneration following peripheral nerve injuryJ Pineal Res5632233220142449929610.1111/jpi.12125 DubocovichMLMarkowskaMFunctional MT1 and MT2 melatonin receptors in mammalsEndocrine2710111020051621712310.1385/ENDO:27:2:101 KanekoYHayashiTYuSTajiriNBaeECSolomitaMAChhedaSHWeinbrenNLParoliniOBorlonganCVHuman amniotic epithelial cells express melatonin receptor MT1, but not melatonin receptor MT2: A new perspective to neuroprotectionJ Pineal Res5027228020112126932710.1111/j.1600-079X.2010.00837.x ChernCMLiaoJFWangYHShenYCMelatonin ameliorates neural function by promoting endogenous neurogenesis through the MT2 melatonin receptor in ischemic-stroke miceFree Radic Biol Med521634164720122233006410.1016/j.freeradbiomed.2012.01.030 LiuDWeiNManHYLuYZhuLQWangJZThe MT2 receptor stimulates axonogenesis and enhances synaptic transmission by activating Akt signallingCell Death Differ2258359620152550160110.1038/cdd.2014.195 Santofimia-CastañoPGarcia-SanchezLRuyDCSanchez-CorreaBFernandez-BermejoMTarazonaRSalidoGMGonzalezAMelatonin induces calcium mobilization and influences cell proliferation independently of MT1/MT2 receptor activation in rat pancreatic stellate cellsCell Biol Toxicol319511020152576437110.1007/s10565-015-9297-6 YuGDRusakBPigginsHDRegulation of melatonin-sensitivity and firing-rate rhythms of hamster suprachiasmatic nucleus neurons: Constant light effectsBrain Res6021911991993844866510.1016/0006-8993(93)90682-d MartiniRFischerSLópez-ValesRDavidSInteractions between Schwann cells and macrophages in injury and inherited demyelinating diseaseGlia56566157720081880332410.1002/glia.20766 RotshenkerSWallerian degeneration: The innate-immune response to traumatic nerve injuryJ Neuroinflammation810920112187812510.1186/1742-2094-8-109 HirotaHKiyamaHKishimotoTTagaTAccelerated nerve regeneration in mice by upregulated expression of interleukin (IL) 6 and IL-6 receptor after traumaJ ExpMed183262726341996867608310.1084/jem.183.6.2627 CaffertyWBGardinerNJGavazziIPowellJMcMahonSBHeathJKMunsonJCohenJThompsonSWLeukemia inhibitory factor determines the growth status of injured adult sensory neuronsJ Neurosci217161717020011154972710.1523/JNEUROSCI.21-18-07161.2001 HirataKKawabuchiMMyelin phagocytosis by macrophages and nonmacrophages during Wallerian degenerationMicrosc Res Tech5754154720021211243710.1002/jemt.10108 BarretteBHébertMAFilaliMLafortuneKValli'eresNGowingGJulienJPLacroixSRequirement of myeloid cells for axon regenerationJ Neurosci289363937620081879967010.1523/JNEUROSCI.1447-08.2008 CattinALBurdenJJVan EmmenisLMackenzieFEHovingJJGarcia CalaviaNGuoYMcLaughlinMRosenbergLHQueredaVMacrophage-induced blood vessels guide Schwann cell-mediated regeneration of peripheral nervesCell1621127113920152627919010.1016/j.cell.2015.07.021 WoodMDMackinnonSEPathways regulating modality-specific axonal regeneration in peripheral nerveExp Neurol26517117520152568157210.1016/j.expneurol.2015.02.001 BoydJGGordonTNeurotrophic factors and their receptors in axonal regeneration and functional recovery after peripheral nerve injuryMol Neurobiol2727732420031284515210.1385/MN:27:3:277 ShiJYLiuGSLiuLFKuoSMTonCHWenZHTeeRChenCHHuangHTChenCLGlial cell line-derived neurotrophic factor gene transfer exerts protective effect on axons in sciatic nerve following constriction-induced peripheral nerve injuryHum Gene Ther2272173120112160499410.1089/hum.2010.036 PascualAHidalgo-FigueroaMPiruatJIPintadoCOGómez-DíazRLópez-BarneoJAbsolute requirement of GDNF for adult catecholaminergic neuron survivalNat Neurosci1175576120081853670910.1038/nn.2136 Ortiz-OrtizMAMoránJMRuiz-MesaLMBonmattyRGFuentesJMProtective effect of the glial cell line-derived neurotrophic factor (GDNF) on human mesencephalic neuron-derived cells against neurotoxicity induced by paraquatEnviron Toxicol Pharmacol3112913620112178767710.1016/j.etap.2010.09.013 MekaDPMüller-RischartAKNidadavoluPMohammadiBMotoriEPonnaSKAboutalebiHBassalMAnnamneediAFinckhBParkin cooperates with GDNF/RET signaling to prevent dopaminergic neuron degenerationJ Clin Invest1251873188520152582202010.1172/JCI79300 ChenZYCaoLLuCLHeCBaoXProtective effect of exogenous glial cell line derived neurotrophic factor on neurons after sciatic nerve injury in ratsSheng Li Xue Bao52295300200011951110(In Chinese) KawakamiTKawakamiYKitauraJProtein kinase C beta (PKC beta): Normal functions and diseasesJ Biochem13267768220021241701510.1093/oxfordjournals.jbchem.a003273 SpinsantiPDe VitaTCarusoAMelchiorriDMisasiRCaricasoleANicolettiFDifferential activation of the calcium/protein kinase C and the canonical beta-catenin pathway by Wnt1 and Wnt7a produces opposite effects on cell proliferation in PC12 cellsJ Neurochem1041588159820081798823810.1111/j.1471-4159.2007.05111.x BuscàRPouysségurJLenormandPERK1 and ERK2 map kinases: Specific roles or functional redundancy?Front Cell Dev Biol45320162737606210.3389/fcell.2016.00053 LeichtDTBalanVKaplunASingh-GuptaVKaplunLDobsonMTzivionGRaf kinases: Function, regulation and role in human cancerBiochim Biophys Acta17731196121220071755582910.1016/j.bbamcr.2007.05.001 TranNHWuXFrostJAB-Raf and Raf-1 are regulated by distinct autoregulatory mechanismsJ Biol Chem280162441625320051571060510.1074/jbc.M501185200 DhillonASMeikleSYaziciZEulitzMKolchWRegulation of Raf-1 activation and signaling by dephosphorylationEMBO J21647120021178242610.1093/emboj/21.1.64 ChongHLeeJGuanKLPositive and negative regulation of Raf kinase activity and function by phosphorylationEMBO J203716372720011144711310.1093/emboj/20.14.3716 MorrisonDKHeideckerGRappURCopelandTDIdentification of the major phosphorylation sites of the Raf-1 kinaseJ Biol Chem268173091731619938349614 StephensRMSithanandamGCopelandTDKaplanDRRappURMorrisonDK95-kilodalton B-Raf serine/threonine kinase: Identification of the protein and its major autophosphorylation siteMol Cell Biol12373337421992150817910.1128/mcb.12.9.3733 MatallanasDBirtwistleMRomanoDZebischARauchJvon KriegsheimAKolchWRaf family kinases: Old dogs have learned new tricksGenes Cancer223226020112177949610.1177/1947601911407323 WellbrockCKarasaridesMMaraisRThe RAF proteins take centre stageNat Rev Mol Cell Biol587588520041552080710.1038/nrm1498 NapoliINoonLARibeiroSKeraiAPParrinelloSRosenbergLHCollinsMJHarrisinghMCWhiteIJWoodhooALloydACA central role for the ERK-signaling pathway in controlling Schwann cell plasticity and peripheral nerve regeneration in vivoNeuron7372974220122236554710.1016/j.neuron.2011.11.031 LeeHJShinYKParkHTMitogen activated protein kinase family proteins and c-jun signaling in injury-induced Schwann cell plasticityExp Neurobiol2313013720142496327710.5607/en.2014.23.2.130 ShinYKJangSYParkJYParkSYLeeHJSuhDJParkHTThe Neuregulin-Rac-MKK7 pathway regulates antagonistic c-jun/Krox20 expression in Schwann cell dedifferentiationGlia6189290420132350503910.1002/glia.22482

Melatonin increases proliferation of Schwann cells. Schwann cells were exposed to different concentrations of melatonin (0.5, 1, 5 and 10 µM) for 24 h and then examined for cell viability by an MTT assay. All data are presented as the mean ± standard deviation. *P<0.05 vs. the control.

Protein and gene expressions of MT1 and MT2 in RT4 Schwann cells. (A) Both MT1 and MT2 proteins were expressed in RT4 Schwann cells. (B) MT1 and MT2 were significantly upregulated at lower concentrations (0.5, 1 and 5 µM) of melatonin but not at a higher concentration (10 µM). MT1 gene expression was higher than MT2 in RT4 Schwann cells. All data are presented as the mean ± standard deviation. *P<0.05 vs. respective control and #P<0.05 vs. 0.5, 1 and 5 µM. RT4, RT4-D6P2T.

Melatonin induces Schwann cell dedifferentiation, as demonstrated by Sox2 immunofluorescence staining. (A) Schwann cells were exposed to 0 (B) 1 and (C) 10 µM melatonin for 24 h and then observed under a light microscope. (D) Schwann cells were exposed to 0 (E) 1 and (F) 10 µM melatonin for 24 h, following which immunofluorescence staining was performed for observation under a fluorescent microscope. Magnification, x200.

Protein expression of Sox2 in RT4-D6P2T Schwann cells after melatonin treatment. (A) Western blot analysis shows that melatonin upregulated Sox2 protein expression. (B) Relative protein fold compared with the control was quantified using Image Lab software and α-tubulin was used as the housekeeping gene. All data are presented as the mean ± standard deviation. *P<0.05 vs. the control.

Protein and gene expressions of GDNF and PKC in RT4-D6P2T Schwann cells after melatonin treatment. (A) Western blot analysis shows that melatonin affected GDNF and PKC protein expressions. (B) Relative protein fold compared with the control was quantified using Image Lab software and α-tubulin was used as the housekeeping gene. (C) Reverse transcription-quantitative PCR was performed to analyse gene expressions of GNDF and PKC. Both GDNF and PKC were upregulated after treatment with melatonin. All data are presented as the mean ± standard deviation. *P<0.05 vs. respective control. GDNF, glial cell-derived neurotropic factor; PKC, protein kinase C.

Protein expressions of the Ras/Raf/ERK pathway in RT4-D6P2T Schwann cells after melatonin treatment. (A) Western blot analysis shows that melatonin affected Ras, p-B-Raf and p-C-Raf protein expressions. (B) Relative protein fold compared with the control was quantified using Image Lab software and α-tubulin was used as the housekeeping gene. All data are presented as the mean ± standard deviation. *P<0.05 vs. respective control. p, phosphorylated.

Protein expressions of the MAPK pathway in RT4-D6P2T Schwann cells after melatonin treatment. (A) Representative western blotting images showing the effects of melatonin treatment on ERK, p38 and SAPK-JNK phosphorylation in Schwann cells. (B) Quantitative comparison of relative ratios of p-ERK (p44)/ERK (p44) and p-ERK (42)/ERK (p42) in melatonin treated groups compared with control. (C) Quantitative comparison of relative ratios of p-p38/p38 in melatonin treated groups with control. (D) Quantitative comparison of relative ratios of p-SAPK-JNK (p54)/SAPK-JNK (p54) and p-SAPK-JNK (p46)/SAPK-JNK (p46) in melatonin treated groups with control. All data are presented as the mean ± standard deviation. *P<0.05 vs. respective control. p, phosphorylated.