Micronized sacchachitin promotes satellite cell proliferation through TAK1-JNK-AP-1 signaling pathway by TLR2/4 activation

a therapeutic the underlying mechanism remains unclear. This study investigated the effect and underlying molecular mechanism of micronized sacchachitin (mSC) on satellite cells (SCs), which are known as the muscle stem cells.


Methods
The myogenic cells, included SCs (Pax7 + ) were isolated from tibialis anterior muscles of a healthy rat and were cultured in growth media with different mSC concentrations. For the evaluation of SC proliferation, these cultivated cells were immunostained with Pax7 and bromodeoxyuridine assessed simultaneously. The molecular signal pathway was further investigated by using Western blotting and signal pathway inhibitors.

Results
Our results revealed that 200 µg/mL mSC had an optimal capability to signi cantly enhance the SC proliferation. Furthermore, this enhancement of SC proliferation was veri ed to be involved with activation of TAK1-JNK-AP-1 signaling pathway through TLR2, whose expression on SC surface was con rmed for the rst time here.

Conclusion
Micronized sacchachitin extracted from GT was capable of promoting the proliferation of SC under a correct concentration.

Background
Ganoderma tsugae (GT) is a representative of Ganoderma sp., the traditional medicinal mushrooms that comprise of over 80 species. The history of Ganoderma sp. could be traced back to more than 2000 years [1], and it has been used for promoting health and extending life in China, Japan, and other East Asian countries. Investigation of this traditional medicinal mushroom by using modern scienti c technology revealed that GT contains more than 400 bioactive substances, including sterols, steroids, fatty acids, polysaccharides, triterpenoids, nucleotides, proteins or peptides, and trace elements. Of these, the polysaccharides (PSs) were identi ed as the major source for biological activity and therapeutic use [2][3][4]. PSs possess antitumor [5][6][7], immune-modulatory [6,8,9], antioxidant [10][11][12], and hypoglycemi [13] effects. Ganoderma-based drugs have been developed using these pharmaceutical properties. Moreover, many of these products are authorized by State Food and Drug Administration of China for clinical use.
In addition to the therapeutic effects mentioned above, enhancement of muscular strength is another well-known effect of Ganoderma-based drugs, which have recently drawn attentions. In fact, Ganodermabased drug has already been in clinical use for treating myopathy in China, since 1973 [14]. According to previous studies, this Ganoderma-based drug has exhibited effectiveness in improving muscle strength in neuromuscular diseases, including Duchenne muscular dystrophy [15,16]. Although this Ganodermabased drug has already been clinically applied for more than 30 years, complete understanding and precise mechanism of this drug in improving muscular regeneration and thereby increasing the muscular mass and strength remains unclear.
The skeletal muscle is a large organ, accounting for approximately 40% of the body weight [17].
Homeostasis of the skeletal muscle is mediated by skeletal stem cells, also known as satellite cells (SCs). SCs reside under basal lamina of myo ber [18] and express Pax7, a paired box (PAX) family of transcription factors [19], as its cell marker. SCs are normally under a quiescent state; however, they are activated on muscle damage to proliferate, differentiate, and fuse to form new myo bers, thereby replacing the damaged tissue and restoring its motor function [20,21]. The processes of muscle regeneration are complex and regulated by multiple signaling pathways. Mitogen-activated protein kinase (MAPK) signaling pathway is closely associated with muscle damage. At least three distinct MAPK pathway targets modulate the muscle regeneration. Activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway increases the number of quiescent SCs [22] and c-Jun N-terminal kinase (JNK) signaling promotes the expansion of the activated SC during regenerative myogenesis [23]. By contrast, p38 MAPK activation inhibits self-renewal and promotes differentiation of SC into myoblasts [24][25][26]. In addition to MAPK signaling pathway, a recent study revealed that the transforming growth factor-β-activated kinase 1 (TAK1), located upstream of MAPK signaling pathway, plays a key role during muscle regeneration. Activation of TAK1 enhances the expression of JNKs and nuclear factor kappa-lightchain-enhancer of activated B cells (NK-κB), which are essential for SC survival and proliferation [27]. Hence, the therapeutic effect of Ganoderma sp. in skeletal muscle atrophy possibly involves TAK1 and MAPK signaling pathway activation in SCs.
In this study, the SCs were isolated from the tibialis anterior (TA) muscle of a healthy rat. These isolated SCs were then cultured in growth media containing different concentrations of micronized sacchachitin (mSC), a novel chitin-polysaccharide extracted from the residue of the Ganoderma fruiting body, which can induce the proliferation of the corneal epithelium according to our previous reports [2,28,29]. In this study, we successfully identi ed the optimal concentration of mSC for promoting the proliferation of SCs through coimmunostaining of Pax7 and bromodeoxyuridine (BrdU) assay. Secondly, Western blotting and activator protein-1 (AP-1) assay revealed that the enhanced SC proliferation triggered by mSC was mediated by Toll-like receptor 2 (TLR2)-TAK1-JNK signaling pathway, but not through ERK1/2 or p38 MAPK signaling pathways. This study elucidated the bene cial effect of Ganoderma sp. on proliferation of SCs and explained the possible therapeutic mechanism of Ganoderma sp. for atrophy for the rst time. Furthermore, these data revealed the potential of mSC in clinical application for the treatment of skeletal muscle atrophy.

Material And Methods
All animal handling procedures were approved by the Institutional Animal Care Committee of Taipei  Isolation of myogenic cells from TA muscle A procedure described in a previous study [30] was used for isolation of myogenic cells. Brie y, Sprague Dawley rats aged 6-8 weeks and weighing 190-210 g were used in this experiment. After scari cation, the TA muscle was excised and homogenized with scissors. The muscle tissue was digested for 30 min at 37℃ by using 0.5% trypsin-EDTA (Thermo Fisher Scienti c, Waltham, MA, USA). The sample was collected through centrifugation at 1500 × g for 5 min and titrated through a ne pipette. An enriched population of myogenic cells was recovered by differential centrifugation (500 × g for 1 min followed by 1500 × g for 5 min). The pellet was suspended in Dulbecco's Modi ed Eagle Medium (DMEM) containing 5% horse serum (Sigma-Aldrich, St. Louis, MO, USA). The suspension containing myogenic cells was then ltrated though a nylon membrane (200 µm) and plated onto 0.1% gelatin coated petri dish. The isolated cells were cultured in growth media that comprised DMEM with 5% HS and 10% Fetal Bovine Serum and 2 mg/mL, 200 µg/mL and 20 µg/mL of mSC, under 5% CO 2 at 37℃. The cell number was counted after 2 days and 8 days of plating for proliferation evaluation until it was con uent for myo ber quantity evaluation.

GT extraction
Please see supporting information for details.

Physical analysis and rheological measurement of mSC
Please see supporting information for details.

Western blotting
The extracts of primary cultures taken at 36 h or 8 days after plating were subjected to standard procedures. The protein amount was determined using BioRad protein assay. Equal amounts of protein (30 µg) were electrophoresed on 10% acrylamide gel and electrotransferred to polyvinylidene uoride membrane, followed by immunoblotting with antibodies for myosin heavy chain (MyHC; 1:1000, GTX73432, GeneTex, Taiwan AP-1/c-Jun assay AP-1 ELISA assay was performed using AP-1 Activity Assay Kit (GeneCopoeia Inc. MD. USA) according to the protocol provided by the manufacturer [32]. Nuclear fraction (50 mg) was mixed with the transcription factor-binding buffer supplied by the manufacturer and was then added to each well of the 96-well plate coated with oligo-DNA fragment containing consensus AP-1 binding sequence. After incubation for 1 h at room temperature (RT), the wells were washed with 200 mL of washing buffer supplied by manufacturer for 1 min. After the nal wash, 100 mL of diluted anti-AP-1 antibody (1:1000) solution was added to each well except the blank wells, and the plate was incubated for 1 h at RT with gentle rocking. Then, each well was washed two more times with washing buffer and incubated with 100 mL of diluted peroxidaseconjugated secondary antibody (1:1000) at RT for 1 h. After another two washes, each well was treated with chemiluminescence developing solution followed by 30 min incubation at RT with gentle agitation and light shielding. After incubation, 100 mL of the stop solution was added to each well and absorbance was measured at 450 nm wavelength using a spectrophotometric plate reader. Nuclear extract of MCF-7 cells was used as a positive control for this assay.

Statistical analysis
Data are presented as mean ± standard deviation (SD). Statistical analyses regarding different mSC concentration were conducted using one-way analysis of variance (ANOVA) with Dunnett's multiple comparisons comparing the mean of each group to the mean of control. One-way ANOVA with Tukey's multiple comparisons comparing mean of each groups were conducted for evaluation of SC proliferation with inhibitors. The rest of data were analyzed by unpaired t-test. The signi cance was set at p ≤ 0.05. Data were analyzed using GraphPad Prism (version 6.0; GraphPad Software, San Diego, CA, USA).

Physical and rheological characterizations of mSC
Physical characteristics of mSC have been reported by Chen et al [33]. It was summarized as the following. mSC was measured by particle size analyzer to show an average effective diameter of 2.06 µm with a mid-range polydispersity index (0.459). But when it was observed under SEM, it shows no sign of particles but appearing to be more like the gel matrix (Fig. 1). This indicated that those short bers of mSC was so small in both length and diameter that they could be hydrated to become nanogel when mSC was dispersed in aqueous medium. Rheological characteristics of mSC nanogel have also been reported by Chen et al [33]. Please also see supporting information for details.
Proliferation of Pax7 + SCs isolated from the TA muscle was signi cantly enhanced by 200 µg/mL mSC The concentration of mSC could possibly vary its effect [29]; therefore, we determined its optimal concentration, which could effectively promote SC proliferation. The primary cultured myogenic cells, which were isolated from the TA muscle, were cocultured in growth media that contained different concentrations of mSC, such as 2 mg/mL, 200 µg/mL, and 20 µg/mL. Coimmunostaining using anti-Pax7 antibody and BrdU assay was performed 2 days after cell plating. Pax7 is an SC marker and BrdU assay is commonly used in the detection of proliferating cells; therefore, the number of Pax7 + BrdU + cells in each assessed mSC concentration could be referred to as actively proliferating SC (Fig. 1A). The results revealed that 200 µg/mL mSC possessed the optimal effectiveness among the three concentrations for promoting Pax7 + cell proliferation (200 µg/mL mSC relative to control, 1.160 ± 0.96 and 1.000 ± 0.062 respectively, p < 0.01, n = 6, Fig. 1B). This was con rmed by evaluation of MyHC protein levels in SCs, maintained in different concentrations of mSCs, after 8 days of cell plating through Western blotting. As expected, medium with 200 µg/mL mSC had the highest MyHC protein expression (0.459 ± 0.068 and 0.314 ± 0.032 respectively, p < 0.01, n = 3, Fig. 1C); this result was compatible with the Pax7 + BrdU + coimmunostaining result. Therefore, 200 µg/mL mSC was used for later experiments.
TLR2 and TLR4 were expressed on SC and the signals of its downstream, TAK1-JNK but not p38, ERK was activated by mSC Here, 200 µg/mL mSC was the optimal condition to promote SC proliferation, the signaling pathway involved was investigated with this enhancement. According to the previous studies, TLR2 and TLR4 (TLR2/4) express on the surface of myocytes, and their downstream protein kinase MAPK is closely associated with the proliferation of SC [33]. However, the presence of TLR2/4 on SC was not yet con rmed. Therefore, immunostainings were performed to investigate the presence of TLR2/4 on SCs (Pax7 + ) and the results were as expected ( Fig. 2A). After con rming the presence of TLR2/4 on SC, the phosphorylation status of TAK1 (p-TAK1) and its downstream molecules, namely p38/MAPK (p-P38), JNK/MAPK (p-JNK), and ERK/MAPK (p-ERK), which form the MAPK signaling pathway, were examined for medium containing 200 µg/mL mSC. As expected, a signi cant elevation was observed for p-TAK1 (0.716 ± 0.057, p < 0.05, n = 3, Fig. 2B). The signi cant elevation was observed for only p-JNK in MAPK signaling pathway, but not for p-p38 or p-ERK (0.858 ± 0.036, p < 0.05; 0.576 ± 0.061, p = 0.258; and 0.765 ± 0.038, p = 0.384, respectively; n = 3; Fig. 2C).
The proliferation of SC induced by mSC was effectively suppressed by JNK inhibitor but not by p38 or

ERK inhibitor
The capability of mSC to activate a speci c MAPK signaling pathway was further con rmed by experiments using inhibitors of JNK, p38, and ERK. The effective blockage of the enhanced SC proliferation under the effect of 200 µg/mL mSC was observed with JNK inhibitor (SP600125) only and not with p38 inhibitor (SB203580) nor ERK inhibitor (U0126) (mSC + IH relative to IH, Fig. 3A, JNK inhibitor, 0.330 ± 0.057 and 0.463 ± 0.054, p = 0.0570; Fig. 3B, p38 inhibitor, 1.131 ± 0.051 and 1.131 ± 0.051 and 0.389 ± 0.046, p < 0.0001; Fig. 3C, ERK inhibitor, 0.994 ± 0.044 and 0.500 ± 0.068, p < 0.0001, respectively; n = 3). These results were compatible to the previous results of the phosphorylation status of MAPK signaling pathway molecules, which were induced by mSC. Both results indicated that the JNK/MAPK signaling pathway was the sole pathway to be activated by mSC.

mSC-induced JNK phosphorylation was predominantly initiated by the activation of TLR2 and mediated by TAK1
Immunostaining of TLR2 and TLR4 with Pax7 antibody revealed that the TLR2 and/or TLR4 were expressed on SC. Therefore, it was rational to hypothesize that this mSC-induced enhanced SC proliferation was through the activation of TLR2 and/or TLR4. To con rm this hypothesis, the protein levels of p-TAK1 and p-JNK, which were known to be located downstream of TLR2/4, were evaluated under the conditions of mSC-containing medium added with either TLR2/4 inhibitor (TLR2/4 IH) or TLR4 inhibitor (TLR4 IH). The protein levels of p-TAK1 and p-JNK were signi cantly suppressed by TLR2/4inhibitor (1.10 ± 0.05, p = 0.07 and 0.55 ± 0.06, p = 0.13, respectively, n = 3, Fig. 4) and not by TLR4inhibitior (1.903 ± 0.016 and 1.106 ± 0.052, respectively, p < 0.05; n = 3; Fig. 4), although TLR4-inhibitor also exhibited a trend of SC proliferation suppression. These results suggested that the signal transduction induced by mSC was predominantly through the activation of TLR2.
AP-1 transcriptional activity was signi cantly enhanced by mSC Lastly, the changes in AP-1 transcriptional activity of the myogenic cells isolated from TA muscle, included Pax7 + population which were cultivated with mSC (200 µg/mL) contained medium was evaluated. The AP-1 transcriptional activity of the myogenic cells in mSC-containing medium was signi cantly enhanced in relation to that of the control (1.713 ± 0.095, p < 0.05, n = 3, Fig. 5A). On the other hand, there was no remarkable increase in phosphorylation of NF-κB (p-NF-kB, 0.786 ± 0.045, p = 0.481, n = 3, Fig. 5B).

Discussion
Skeletal muscular atrophy is an unsolved issue despite of the remarkable progression of modern medication. Although novel treatments, such as photobiomodulation therapy [30], electromyostimualtion [34], and amino acid supplementation [35,36] with or without a combination of traditional rehabilitation, have been proposed to ease the aggravation of atrophy or even to increase the muscle mass, unfortunately the effectiveness of above mentioned therapies are still limited. On the other hand, Ganoderma sp., the traditional Chinese medicinal mushrooms, which were popularly used to improve pathological conditions, such as hepatic functional impairment and cancer, have recently drawn attention due to their core component, "sacchachitin" that was clinically found to be capable of improving muscular atrophy. Although this Ganoderma-based drug has been applied clinically [14], its pharmacological mechanism remains unclear. As our previous reports revealed [29] that mSC, an original product of our team, can enhance the proliferation of skin cell. However, its effect on myogenic cells, SCs in particular, has not yet been examined. This study investigated the effect of mSC on SC, which play a critical role during postnatal muscle regeneration, and simultaneously unveil the molecular mechanisms underlying the phenomenon.
In this study, different concentrations of mSC, such as 2 mg/mL, 200 µg/mL, and 20 µg/mL, were added to the SC growth medium. These mSC-containing media were used for culturing of myogenic cells, which were isolated from TA muscle of a healthy rat, revealed presence of Pax7 + SCs. Through the coimmunostaining with anti-Pax7 antibody and BrdU cell proliferation assay, we con rmed that 200 µg/mL mSC was the optimal condition to effectively promote the proliferation of SCs (Pax7 + BrdU + ). Micronized sacchachitin could boost the proliferation of SC up to 16.1% higher than that of control.
Notably, the effect of mSC on SC proliferation appeared as an U-shaped or "biphasic" response; therefore, a risk of adverse effect of mSC on SC proliferation depending on the concentration was speculated. Therefore, the effect of mSC could be a double-edged sword for SC proliferation based on the applied concentration.
Molecular signaling pathways involved in the enhancement of mSC-induced SC proliferation were investigated. TLR2 and TLR4 are expressed on myocytes [37,38]; however, their presence on SC was not identi ed. In this study, we con rmed the presence of TLR2 and TLR4 on SC for the rst time. Although both TLR2 and TLR4 were expressed on SC, results of our experiments revealed that the mSC-induced enhanced SC proliferation was predominantly dependent on the activation of TLR2. This result was compatible with the previous reports, which revealed a signi cant role of TLR2 during muscle regeneration, because TLR2 signal could be upregulated by lipopolysaccharides and this upregulation was considered critical for muscle regeneration as this process is important for SC proliferation [39,40]. Notably, the biphasic effect of mSC on SCs resembled the role of TLR2 signaling pathway, which could be dependent on the property of in ammatory condition that triggered the muscle regeneration, such as acute or chronic muscle injury. The upregulated TLR2 induced proin ammatory environment, which was favorable for muscle regeneration in acute muscle injury; by contrast, a reduced in ammatory environment, facilitated by suppressing TLR2 signaling pathway, is bene cial for chronic muscle injury [40]. Taken together, the optimal mSC concentration can differ based on the characteristics of the muscular atrophy. Thus, a careful evaluation of appropriate mSC concentration, favorable for distinct muscular atrophy type, is important.
TAK1 is located downstream of TLR2 signaling pathway. A recent report revealed that it has an essential role in SC proliferation during muscle regeneration because the inactivation of TAK1 caused precocious differentiation of SC, which eventually led to an inhibition of muscle regeneration [27]. Furthermore, the SC proliferation was also known to be closely involved with MAPK signaling pathway, which is further downstream of TAK1. In this study, the capability of mSC to activate both TAK1 and MAPK signaling pathway was con rmed. However, among the MAPK signaling pathways, JNK signaling pathway was solely activated. This result was also compatible to the previous report, which revealed that the activation of JNK signaling pathway promotes the SC expansion during muscle regeneration [23]. Although either ERK1/2 or p38 MAPK signaling pathways were not activated by mSC, according to our data, the results could not simply be explained as the incapability of mSC for increasing the number of quiescent SC or to boost SC differentiation into myoblast by activating ERK1/2 and p38 MAPK signaling, respectively, because we investigated the optimal mSC concentration by only evaluating the increase in Pax7 + BrdU + cell number but not the cell counts of quiescent SC (Pax7 + MyoD − ) and myoblast (Pax7 + MyoD + ).
The activation of AP-1, a transcription factor downstream of TAK1-JNK signaling pathway, was veri ed to be compatible with previous reports: AP-1 positively regulated the cell proliferation via c-Jun signaling [41]. Since, c-Jun is a downstream transcription factor of JNK, it is reasonable to speculate the effect of mSC, which enhanced the SC proliferation in the present study was through the activation of TAK1-JNK-AP-1 signaling pathway. AP-1 is closely involved with the regulation of matrix metalloproteinase-9 (MMP-9) [42,43]; however, the changes in MMP-9 were not evaluated in this study. Therefore, it was di cult to determine the correlation between MMP-9 and mSC. In addition, MMP-9 activity could be promoted by mSC because an elevation of AP-1 activity was observed. These results might contradict the results of our previous study, which revealed that mSC could inhibit the MMP-9 activity, whereas the inhibition of MMP-9 led to an increase in SC proliferation and Notch expression, which is also bene cial for SC proliferation. We suspect that the inconsistency between our current and previous studies may be due to the distinct in ammatory conditions, namely chronic and acute. The myogenic cells used in the present study were collected from the TA muscle of a healthy rat and the evaluation was performed within 36 h after the collection.
Therefore, the experiment model of present study tended to be in an acute, rather than chronic, phase of in ammation. By contrast, the corneal damage model or the muscular dystrophy model in our previous study resembled the model of chronic in ammation. The discrimination between the in ammatory phases of the two models might explain the different responses of SC.

Conclusion
In this study, we demonstrated the capability of mSC to promote SC proliferation for the rst time. The enhancement was predominant through TLR2, which were expressed on SC, leading to the activation of TAK1-JNK-AP-1 signaling pathway. Notably, JNK was solely activated among the MAPK signaling pathways (Fig. 5C). However, the effect of mSC on SC could be altered based on mSC concentration. Furthermore, the in ammatory phase of the skeletal muscle could also be an involved factor. Thus, applying a "correct" concentration of mSC to the "appropriate" in ammatory environment of injured skeletal muscle could be critical in determining the effectiveness of the medication. This study used SC from healthy rats; therefore, an experiment using human SCs will be our next project. The current results can provide a better understanding of mSC's effects on muscle regeneration.    Toll-like receptor 2 and TLR4 were expressed upon SC and the signals of its downstream, TAK1-JNK but not p38, ERK was activated by mSC. (A) The presence of TLR2/4 upon Pax7+ve SCs were con rmed through immunostaining. Scale bar is 30um. (B) Relative to control, a signi cant elevation of pTAK-1 was admitted under mSC 200ug/ml (0.716±0.057, *p<0.05, n=3, unpaired t-test). (C) Signi cant elevation was only observed with p-JNK but p-p38 nor p-ERK (0.858±0.036, p<0.05, 0.576±0.061, p=0.258, 0.765±0.038, p=0.384, n=3, unpaired t-test).