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Dendropanax morbiferus leaf extract facilitates oligodendrocyte development

Treatment of multiple sclerosis is effective when anti-inflammatory, neuroprotective and regenerative strategies are combined. Dendropanax morbiferus (DM) has anti-inflammatory, anti-oxidative properties, which may be beneficial for multiple sclerosis. However, there have been no reports on the effects of DM on myelination, which is critical for regenerative processes. To know whether DM benefits myelination, we checked differentiation and myelination of oligodendrocytes (OLs) in various primary culture systems treated with DM leaf EtOH extracts or control. DM extracts increased the OL membrane size in the mixed glial and pure OL precursor cell (OPC) cultures and changed OL-lineage gene expression patterns in the OPC cultures. Western blot analysis of DM-treated OPC cultures showed upregulation of MBP and phosphorylation of ERK1/2. In myelinating cocultures, DM extracts enhanced OL differentiation, followed by increased axonal contacts and myelin gene upregulations such as Myrf, CNP and PLP. Phytochemical analysis by LC-MS/MS identified multiple components from DM extracts, containing bioactive molecules such as quercetin, cannabidiol, etc. Our results suggest DM extracts enhance OL differentiation, followed by an increase in membrane size and axonal contacts, thereby indicating enhanced myelination. In addition, we found that DM extracts contain multiple bioactive components, warranting further studies in relation to finding effective components for enhancing myelination.

Treatment of multiple sclerosis is effective when antiinflammatory, neuroprotective and regenerative strategies are combined. Dendropanax morbiferus (DM) has antiinflammatory, anti-oxidative properties, which may be beneficial for multiple sclerosis. However, there have been no reports on the effects of DM on myelination, which is critical for regenerative processes. To know whether DM benefits myelination, we checked differentiation and myelination of oligodendrocytes (OLs) in various primary culture systems treated with DM leaf EtOH extracts or control. DM extracts increased the OL membrane size in the mixed glial and pure OL precursor cell (OPC) cultures and changed OL-lineage gene expression patterns in the OPC cultures. Western blot analysis of DM-treated OPC cultures showed upregulation of MBP and phosphorylation of ERK1/2. In myelinating cocultures, DM extracts enhanced OL differentiation, followed by increased axonal contacts and myelin gene upregulations such as Myrf, CNP and PLP. Phytochemical analysis by LC-MS/MS identified multiple components from DM extracts, containing bioactive molecules such as quercetin, cannabidiol, etc. Our results suggest DM extracts enhance OL differentiation, followed by an increase in membrane size and axonal contacts, thereby indicating enhanced myelination. In addition, we found that DM
Multiple sclerosis is a major demyelinating disease of the central nervous system leading to focal plaque of primary demyelination and diffuse neurodegeneration in the grey and white matter of the brain and spinal cord [10].
Patients with multiple sclerosis show increased oxidative stress markers and inflammation [11]. Recent studies on DM suggest that DM might be effective on multiple sclerosis symptoms because of its antioxidant [2,3], cell-protective [4] and anti-inflammatory properties [8,9].
Remyelination fails in many chronically demyelinated multiple sclerosis plaques [12,13]. The remyelination failure is mainly attributable to reduced oligodendrocyte precursor cell (OPC) recruitment and differentiation [14]. Among these events, it is thought that disrupted differentiation is the major time-limiting factor because OPC mitogen and recruitment factor PDGF overexpression do not increase remyelination [15]. Currently, it is understood that oligodendrocyte (OL) differentiation failure is the biggest cause of remyelination failure [16]. Study of various developmental markers of OLs has shown that OPC is not fully differentiated in multiple sclerosis lesions [12,13,17,18]. Therefore, enhancement of OL differentiation and myelination is an important problem to solve in relation to multiple sclerosis treatment.
Although recent research has reported that DM has anti-oxidative, cell-protective and antiinflammatory properties, which can be useful in multiple sclerosis treatment, the link between DM's benefits and myelin-forming OLs has not been investigated yet. In the present study, we aimed to reveal the effects of DM on OL differentiation, and myelination using multiple primary culture systems, including in vitro myelination cultures.

Mice
Timed pregnant females (day 13.5 of pregnancy) or pups (postnatal day 0-2) of CrljOri:CD1(ICR) mouse line were purchased from ORIENT BIO Inc. (Seongnam, Korea). All experiments were performed in compliance with the relevant laws and institutional guidelines and were approved by the University of Brain Education's Animal Care and Use Committee (approval no. 2017-AE-01).

Cell cultures
Mixed glial cultures were prepared from cortices of mice at postnatal days 0-2 on poly-D-lysine (PDL)-coated flasks or coverslips and were maintained in DMEM/F-12 medium containing 10% fetal bovine serum, 5% horse serum and 1Â penicillin-streptomycin (Gibco). For OPC pure cultures, OPCs were isolated by shaking from the flasks containing mixed glial cultures on days in vitro (DIV) 10. After removing the astrocytes by dish-panning, the OPCs were seeded on PDL-coated coverslips and were maintained in Sato medium (DMEM containing 1Â B-27 supplement, 1Â Glutamax, 1Â penicillin-streptomycin, 1% horse serum, 1Â sodium pyruvate, 0.34 mg ml 21 T3 and 0.4 mg ml 21 T4). For cocultures, mouse dorsal root ganglia (DRG) neuronal cultures and mouse glial mixed cultures were separately prepared in advance. DRG neurons were prepared as described previously [21]. The OPCs were isolated by shaking and were seeded on DRG neuronal cultures and were maintained in coculture medium (DMEM containing B-27 supplement, N-2 supplement, 5 mg ml 21 N-Acetylcysteine, 5 mM forskolin and penicillin-streptomycin). For plant extract treatment, the above prepared plant extracts were diluted with the medium into 1 : 1000 (high, if not indicated) or 1 : 100 000 (low) and filtered through a 0.22 mm syringe filter. Cells were incubated with the plant extract-diluting medium or control medium for DIV 8 -12 in the mixed glial cultures, for DIV 1 -3 in the pure OPC cultures and for DIV 1-7 or 1 -12 for DRG/OPC cocultures. Control or plant extract-containing medium was changed with fresh medium every two days. If not indicated, the leaf part of six-yearold DM was used in the treatment.

Image analysis
Images were obtained using a Leica TCS SPE microscope and Axio Imager Z1 equipped with Apotome (Carl Zeiss). Image analysis was performed using Image J. The images were given to the investigators without the sample names but only with numbers for analysis. Thus, the images were scored blinded to the plant names. For MBP þ or Caspr þ area per cell, the images were manually analysed in a random manner to detect single cells under the same threshold throughout all images. If distinguishing a single cell was difficult because of the density, the cells were excluded from the analysis. For Olig2 þ or Dapi þ cell number, the cells were automatically detected and counted under the same threshold throughout all images in Image J. For O4 þ or MBP þ area per field, marker-positive areas were automatically detected and measured under the same threshold throughout all images in Image J.

Statistical analyses
All graph data are presented as the mean + s.e.m. Statistical analyses were performed using unpaired Student's t-test with two tails, unequal variance. Sample size was based on similar studies in the field.

Non-targeted metabolome analysis
DM leaf ethanol extracts were stored at 2808C until analyses and phytochemicals were analysed by liquid chromatography (LC)-mass spectrometry (MS)/MS. For non-targeted analysis, MS (Q-Exactive focus, Thermo Fisher Scientific, San Jose, CA, USA), which enables us to perform highly selective and sensitive metabolite quantification owing to the Fourier transfer MS principle, was connected to a highperformance liquid chromatography (Ultimate3000 system, Thermo Fisher Scientific). The samples were resolved on Acculaim C18 (2.1 mm ID Â 150 mm, 3 mm particle, Thermo Fisher Scientific), using a step gradient with mobile phase A (60 : 40 (v/v) of water: acetonitrile in 10 mM ammonium formate and 0.1% formic acid) and mobile phase B (90 : 10 (v/v) of isopropyl alcohol: acetonitrile in 10 mM ammonium formate and 0.1% formic acid) at ratios of 68 : 32 (0-4 min), 55 : 45 (4-5 min), 48 : 52 (5-8 min), 34 : 66 (8-11 min), 30 : 70 (11-14 min) and 25 : 75 (14-18 min), at a flow rate of 0.2 ml min 21 and a column temperature of 458C. The Q-Exactive focus mass spectrometer was operated under an ESI-positive mode for all detections. Full mass scan (m/z 50-900), followed by three rapid datadependent MS/MS, was operated at a resolution of 70 000. The automatic gain control target was set at 3 Â 10 6 ions, and the maximum ion injection time was 100 ms. Source ionization parameters were optimized with the spray voltage at 3 kV and the other parameters were as follows: transfer temperature at 3208C, S-Lens level at 50, heater temperature at 3008C, sheath gas at 36 and auxilliary gas at 10.
Compound Discoverer 2.0 (Thermo Fisher Scientific) was used for the non-targeted metabolomics workflow as described in Zhou [23]. Briefly, this software first aligned the total ion chromatograms of different samples along the retention time. Then the detected features were extracted and merged into components. The resulting compounds were identified by both (i) formula prediction based on accurate m/z value and isotope peak patterns, and (ii) MS/MS structural validation querying to m/z cloud database (https://www.mzcloud.org/). Moreover, formula predicted signals were assigned into candidate compounds by database search (ChemSpider database; http://www.chemspider.com/).

Dendropanax morbifera extracts increased the oligodendrocyte membrane size in the mixed glial cultures
To check whether DM extract conferred any effects on the formation of the OL membrane sheath, we performed mixed glial cultures from the cortices of the postnatal days 0-2 ICR pups on PDL-coated royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 190266 coverslips. The cultures were initially incubated in the regular glial medium, and after eight days the medium was changed to Sato medium containing either the ethanol control (figure 1a,c) or the following medicinal plant extracts, which have been appeared as their neurological recovery function in traditional literature [24]: DM ( figure 1b,d), EU, AcA, XS, AM, ArP, AP, RG, LS and AA (figure 1a-e; electronic supplementary material, figure S1). On DIV 12, the cultures were fixed and stained with MBP to determine the size of the OL membrane sheath. MBP is the major marker of mature myelinating OLs [21]; therefore, we used MBP-positive percentage area per field on DIV 12 as the indication of membrane formation. Using Image J, MBP-positive percentage area per field was measured. The pictures were taken in low magnification to include as many mature OLs as possible.
Many of the investigated medicinal plant extracts in the study including DM leaf extract induced increases in MBP-positive membrane size compared with EtOH control (figure 1e). ArP and DM showed the highest effects on MBP-positive percentage area per field in the glial mixed culture system. The glial mixed cultures contain three major glial cell types: OLs, astrocytes and microglia. Therefore, the effects of DM on OL membrane sheath formation may derive not only from the OLs but also from the combined effects of the three cell types, because astrocytes and microglia also affect myelin synthesis [25,26].

Dendropanax morbiferus extracts increased the size of oligodendrocyte membrane sheath in the isolated oligodendrocyte precursor cell cultures
To clarify whether the changes in membrane size by DM extracts derive directly from OL or indirectly through astrocytes or microglia, we isolated OPCs from the mixed glial culture on DIV 10 by shaking. Isolated OPCs were seeded on PDL-coated coverslips and incubated in Sato medium containing differentiating hormones T3 and T4 [21]. On DIV 1, the medium was changed to fresh medium containing either the control EtOH     synthesis and real-time PCR. Gene expressions related to OL differentiation, such as Olig1, ID2, Ascl1 and MBP, were investigated. GAPDH was used to normalize the expression of the indicated genes. The relative expression of Olig1 and ID2 were significantly decreased ( figure 2a,b), while that of MBP showed a tendency of increase (figure 2d) compared with EtOH control. The relative expression of Ascl1 was not changed by the treatment (figure 2c). During OPC differentiation, the expressions of Olig1 [29], ID2 [30] and Ascl1 [31] are downregulated, whereas MBP expression is upregulated [32]. Therefore, real-time PCR results suggest that the DM leaf extract contains biologically effective components for OL differentiation, supporting the immunocytochemistry data, which showed the positive effects of DM leaf on OL membrane synthesis. Next, to see whether we can find changes in protein levels, we performed western blot analysis in the same system on DIV 3 using OPCs treated with either DM leaf or control during DIV 1-3. Consistent with the real-time PCR results, MBP expression normalized by b-actin was upregulated in DM leaf extract-treated OPC cultures compared with the control-treated OPC cultures (figure 2e,f; electronic supplementary material, figure S3). The extracellular signal-regulated kinase (ERK) signalling pathway plays a specific role in the timing of OL differentiation [33]. Therefore, we also checked the phosphorylation status of ERK1/2 to see whether the altered differentiation by DM leaf extract treatment affects ERK signalling. The results showed that the DM leaf extract treatment increased the phosphorylation of ERK1/2 compared with the control treatment ( figure 2e,g), suggesting that the enhanced OL differentiation by DM leaf extract is mediated by ERK signalling to a certain extent.

Dendropanax morbiferus leaf extract increased the axonal contacts of oligodendrocytes in the myelinating cultures
We found that DM leaf extract enhanced OL differentiation on DIV 7 in DRG/OPC cocultures ( figure 3). Next, we asked whether the facilitated differentiation enhances axonal contacts of OLs to initiate myelination. To test this, we immunostained cocultures on DIV 12 with antibodies against Caspr, which clusters upon OL contact and ensheathment [34,35]. Caspr-positive area per cell was measured and compared between EtOH-(figure 4a) and DM leaf extract-treated cultures ( figure 4b). The result showed that DM leaf extract significantly facilitated Caspr clustering in DRG/OPC coculture system on DIV 12 (*p , 0.05, Student's t-test, n ¼ 3 experiments, figure 4d). This suggests that DM leaf extract is effective not only for OL differentiation but also in boosting axonal contacts.
To confirm the immunocytochemical analysis in the myelinating culture system, we performed quantitative real-time PCR on myelin genes by using the same culture system on DIV12. Myelin genes such as Olig1, Myrf, CNP, MBP and PLP were investigated. Myrf is a transcription factor activating the expression of myelin genes [36]. CNP is a non-compact myelin protein [37]; MBP and PLP are important structural components of compact myelin [38]. We found that all of the investigated myelin royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 190266 genes increased its expression by DM leaf treatment and significant increases were observed in Myrf, CNP and PLP (*p , 0.05, **p , 0.01, Student's t-test, n ¼ 3 experiments, figure 4e). The increase in myelin gene expression is consistent with the immunocytochemical analysis ( figure 4a-d ), supporting the facilitating effects of DM leaf on myelination.

Discussion
In this paper, we report the function of DM on the development of OL-lineage cells: from OPC proliferation till myelination. OPCs proliferate to supply OLs producing enough myelin for the nervous system. By DM treatment, the number of OLs was not significantly changed (figure 3c), suggesting DM does not affect the proliferation of OPCs. In the OPC stage, they express differentiation inhibitory transcription factors such as ID2 [57]. DM treatment reduced ID2 expression (figure 2b), suggesting its facilitation of OL differentiation. Indeed, the expression of MYRF, a transcription factor of myelinating OL [57], was significantly increased by DM treatment in myelinating cultures on DIV12 (figure 4e), supporting this notion. Consistently, myelin genes such as CNP and PLP showed a significant increase in their expression in DM-treated myelinating cultures (figure 4e). MBP protein expression was also increased in all of the investigated culture systems by DM treatment (figures 1, 2e and 3). This suggests that DM facilitates the OL differentiation process from OPC to mature OLs forming myelin. The OPC differentiation is mediated by multiple combinatory mechanisms [58]. Among them, ERK signalling is one of the critical pathways in OL differentiation [33] as well as myelin growth [59]. Our results indicate that DM leaf extract enhances OL differentiation at least partially by activating ERK signalling pathway (figure 2e,g). For myelination, the processes of OL should contact axons, and then they initiate ensheathment to form myelin by increasing their membrane size. To analyse the effects of DM on myelination steps, we checked the axonal contacts of OLs by using myelinating cultures with Caspr staining (figure 4), and investigated OL membrane synthesis by using glia mixed cultures, OPC cultures and myelinating cultures with MBP staining or expression analysis (figures 1-4). For immunocytochemical membrane synthesis analysis, we used mixed glial cultures and OPC cultures because they present membrane sheath in a flat round form without neurons, which makes analysis easier. DM treatment increased OL axonal contacts as well as OL membrane size compared with control treatment. This is consistent with the result of facilitated OL differentiation by DM.   Previous reports about DM are about antioxidant effects [2,3], cell-protective effects [4], selective cell death [5,6], anti-thrombotic [7] and anti-inflammatory effects [8,9]. Our observation of DM functions in enhancement in OL development is novel, raising possibilities of its potential use in multiple sclerosis treatment. Non-targeted metabolome analysis by LC-high-resolution MS assigned multiple compounds in DM ethanol extracts, showing DM is a complex of mixture of phytochemicals (table 1). They contain molecules having bioactivities, such as quercetin, chlorogenic acid, rutin, carnosol, dextromethorphan, cannabidiol, bremazocine, doxapram, resolvin D2, procyclidine, 2-arachidonoylglycerol and eplerenone. Interestingly, quercetin, dextromethorphan and cannabidiol protect OL-lineage cells from the stress environments [41,44,45], while quercetin also improves myelination in the context of perinatal cerebral hypoxia ischaemia-induced brain injury by promoting the proliferation of OPCs [41]. Other components have been reported for various functions. Quercetin, chlorogenic acid and rutin mediate the anti-oxidation process [39,42]. Carnosol and resolvin D2 are related to anti-inflammatory functions [43,48]. Bremazocine is a k-opioid agonist [60] and Doxapram is a respiratory stimulant [61]. Procyclidine is a synthetic anticholinergic agent [49] and 2-Arachidonoylglycerol is an endocannabinoid that activates the cannabinoid receptors CB1 and CB2 [62][63][64]. Eplerenone has been reported to reduce both the risk of death and the risk of hospitalization among patients with systolic heart failure and mild symptoms [65]. As other components (except quercetin) have not been reported in relation with myelination, they warrant further studies to screen the effective components on myelination.
Accumulative findings suggest that treatment of progressive multiple sclerosis should be a combinatory strategy of anti-inflammation, neuroprotection and regeneration [66]. The reported functions of phytochemicals of DM leaf on inflammation and cell protection as well as our current finding of DM leaf function in myelination indicate that DM leaf extract can be considered as a candidate for relieving the symptoms of progressive multiple sclerosis, even as a complex. Medicinal plants used in multiple sclerosis care report their benefits in relieving spasticity, pain, tremor and depression [67] and enhancing fatty acid metabolism and lymphocyte function [68,69]. However, to our knowledge, no study has explored the function of medicinal plants on OL differentiation/ myelination, which is a critical step for remyelination in multiple sclerosis, and our result that DM leaf extracts affect OL development is the first observation as far as we know.
In this research, we have shown that DM enhances endogenous OL differentiation, followed by membrane synthesis, using various types of primary cultures such as glial mixed culture, pure OPC culture and myelinating cultures. However, in vivo is a much more complicated environment than in vitro. Therefore, confirmation of the DM effects in in vivo demyelination models such as cuprizone assay, lysolecithin mode, or experimental allergic encephalomyelitis may be necessary in the future. In phytochemical analysis, we identified bioactive components whose functions in myelination have not been reported yet, warranting further studies on the isolation of active components for OL differentiation and myelination from the current isolated candidates.

Conclusion
DM leaf EtOH extract enhanced the differentiation of OL and membrane formation, at least partially, by ERK signalling pathway. Moreover, DM leaf extract increased the axonal contacts of mature OLs, which is an initial step for myelination, as well as myelin gene expression. It also contained multiple bioactive molecules, warranting further studies on searching for active components on myelination. Our findings suggest that DM leaf extract may contain a novel therapeutic target in the treatment of progressive multiple sclerosis.
Ethics. Permission to provide samples that were used in the study was granted to Mago-Yeongnong-Johap and Jirisan Cheongjung Yakcho by Gyungsangnamdo Sancheongguncheong ( permit no. 2012-Gyungnamsancheong-00005). The study was reviewed and approved by the Institutional Animal Care and Use Committee (approval no. 2017-AE-01) of the University of Brain Education.
Data accessibility. All data have been submitted as figures in the main text or as electronic supplementary material.