The Role of Moracin M in Promoting Hair Growth: Insights into Mechanisms of WNT/β- Catenin Pathway Activation and Angiogenesis Enhancement in Human Dermal Papilla Cells

doi:10.21203/rs.3.rs-5200734/v1

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The Role of Moracin M in Promoting Hair Growth: Insights into Mechanisms of WNT/β- Catenin Pathway Activation and Angiogenesis Enhancement in Human Dermal Papilla Cells

https://doi.org/10.21203/rs.3.rs-5200734/v1

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Hair follicle growth depends on the intricate interaction of cells within the follicle and its vascular supply. Current FDA-approved treatments like minoxidil have limitations, including side effects and the need for continuous use. Moracin M, a compound from Moraceae family, was investigated for its effects on hair growth and vascular regeneration. In our study, Moracin M significantly increased cell proliferation in human dermal papilla cells (hDPCs) during both the anagen and catagen phases and promoted cell migration in human umbilical vein endothelial cells (HUVECs) without cytotoxicity at concentrations up to 50 µM. Mechanistic analysis revealed that moracin M enhanced GSK-3β phosphorylation and increased non-phospho β-catenin levels, activating Wnt signaling and upregulating transcription factors LEF, TCF, and AXIN2. This resulted in elevated levels of growth factors VEGF, FGF2, KGF, HGF and MYC in hDPCs, effects comparable to those of minoxidil. Additionally, moracin M significantly increased protein and mRNA levels of VEGF, FGF2, and KGF in hDPCs under IFN-γ-induced inflammatory conditions. Moracin M treatments also resulted in notable wound width reductions in a dose-dependent manner. Further investigation showed that moracin M stimulated MMP-2 and MMP-9 expression while having no effect on MMP-7 levels.

These findings indicate that moracin M significantly enhances hair growth through the promotion of cell proliferation and angiogenesis, particularly via the activation of the Wnt signaling pathway in dermal papilla cells, presenting it as a promising therapeutic alternative to current treatments.

Moracin M

Wnt signaling pathway

angiogenesis

Hair growth

Hair follicle growth is a complex process governed by the intricate interaction of various cells within the follicular tissues located at the skin's periphery. This process includes dynamic phases such as growth, regression, and resting stages, where maintaining a delicate balance is crucial to prevent hair loss [1]. The growth of hair is closely tied to vascular health [2]. The dermal papilla, a critical structure within the hair follicle, receives essential nutrients and oxygen from the scalp's microvasculature. These nutrients and signals play a vital role in nourishing the matrix cells and stem cells located in the bulge area of the follicle, crucial for initiating and supporting new hair growth.

In the field of pharmacological treatments for hair loss, the FDA has approved two primary medications: minoxidil and finasteride [3]. Initially developed as vasodilators to reduce blood pressure, minoxidil unexpectedly showed an additional benefit of stimulating hair growth. It has since been repurposed as a prominent treatment for enhancing hair growth. Minoxidil functions by stimulating cell proliferation, enhancing the expression of vascular endothelial growth factor (VEGF), and promoting prostaglandin synthesis. Simultaneously, it inhibits collagen synthesis in various skin and follicular cell types [2, 4]. However, a notable effect of minoxidil is its tendency to shorten prematurely the resting phase (telogen phase) of existing hair fcllicles, often leading to early shedding known as telogen effluvium. Therefore, long-term and consistent application of minoxidil is typically necessary to sustain its effects [5].

According to case reports by Ponomareva et al.[6] and Dubrey et al. [7], young male patients experienced dizziness, blurred vision, general malaise, fatigue, and pre-syncope symptoms after the application of topical minoxidil solution. These symptoms rapidly resolved upon discontinuation of the minoxidil. Such cases underscore the potential for systemic.

Morus alba L. has traditionally been utilized as a medicinal herb and dietary supplement to treat various diseases and symptoms through its roots, branches, leaves, and fruits. Morus alba L. root extract demonstrated its potential to enhance vascular regeneration and proliferation of dermal fibroblasts [8]. The same research group also validated the safety and efficacy of Morus alba L. root extract when formulated into a shampoo. Furthermore, enzymatic treatment of Morus alba L. leaves (Enz_MoriL) was shown to enhance proliferation of dermal papilla cells [9]. Notably, Morus alba L. leaves are known for their effectiveness in preventing abnormal vascular responses [10]. Furthermore, according to studies utilizing animal models, Morus alba L. leaves are considered a promising alternative therapy for cardiovascular diseases (CVDs), encompassing coronary artery disease, hypertension, and the prevention or treatment of myocardial infarction and stroke [11–13].

The bark of roots, stem bark, and leaves are major sources of active compound and aryl benzofuran derivatives, including moracin derivatives [14–17]. We evaluated the proliferative effects on dermal papilla cells of chlorogenic acid, quercetin, moracin M, O, and P compounds abundantly found in Morus alba L. leaves, identifying moracin M as significantly enhancing cell proliferation compared to the control group. Subsequently, we conducted further investigations into the mechanisms of hair growth and vascular-related growth factors influenced by moracin M.

2.1 Chemicals, Animals, and Biological Kits

Moracin M, O, P were purchased from MedChem Express (Monmouth Junction, Middlesex County, NJ, USA). Quercetin, chlorogenic acid, matrigel were purchased from sigma-aldrich (Cell Signaling, CA, USA). Fetal bovine serum (FBS) and trypsin were from Gibco (Grand Island, NY, USA).

2.2 Cell culture of hDPCs and HUVEC

Human dermal papilla cells (hDPCs) were obtained from ScienCell (San Diego, CA, USA) and were primarily sourced from normal human scalp hair follicles. These cells were cultured in mesenchymal stem cell medium-basal (MSCM‐b) provided by ScienCell, which was supplemented with 10% fetal bovine serum (FBS) and 1× antibiotic solution. Human umbilical vein endothelial cells (HUVECs; PromoCell, Heidelberg, Germany) were cultured in full growth medium (endothelial cell growth medium 2; PromoCell) supplemented with exosome‐depleted FBS up to the third passage. Culturing was performed at 37°C in a 5% CO₂ environment using 100 mm cell culture dishes from Corning Inc. (Corning, New York, NY, USA) until they reached a confluence of 70–80%. The culture medium was refreshed every 3 days. Only cells within 5 passages were utilized for the experiments.

2.3 MTT Assay

To determine cell viability, the cells were added to 200 µL of 1 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma, St. Louis, MO, USA) solution/well and then incubated for 2 h in a humidified atmosphere (37°C in 5% CO₂). The medium was replaced with 200 µL dimethyl sulfoxide (DMSO) and absorbance was measured at 540 nm using a microplate reader (Molecular Devices Inc., Sunnyvale, CA, USA). Cell proliferation was expressed as percentage values in comparison with the negative PBS control, which was considered to represent 100% cell proliferation.

2.4 Wound healing

The cell migration capacity was evaluated using a scratch wound-healing assay. A synthetic wound was generated in an 80–90% confluent monolayer of HUVECs cultured in a 24-well plate, employing a 200 µl pipette tip. The impact of co-culturing HUVECs with moracin M at concentrations of 6.25, 12.5, and 25 µg/ml on cell migration was observed using microscopy at the 0 and 24-hour marks. Images were captured with an inverted microscope and analyzed using ImageJ software, with the number of cells that migrated into the wound area counted within a predetermined frame

2.5 Protein Expression Analysis

Cells were harvested and pelleted at 200× g for 3 min, washed with Tris-buffered saline (TBS; 20 mM Tris, pH 7.5, 130 mM NaCl) containing protease inhibitor and phosphatase inhibitor cocktails, and placed on ice as quickly as possible. The cells were then lysed by direct application of a lysis buffer to the dish. A nuclear/cytosol fractionation kit (Bio Vision Technology Inc., New Minas, NS, Canada) was used to separate nuclear and cytoplasmic proteins according to the manufacturer’s protocol. After isolation of the proteins, the sample concentrations were determined using a micro BCA assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). A total of 20 µg of sample per lane was electrophoresed on a 7.5–10% reducing SDS–PAGE gel and transferred onto a nitrocellulose membrane, which was blocked with 5% skim milk and sequentially incubated with one of the following antibodies: anti- phospho GSK3β, anti- GSK3β, anti- non-phospho (Active) β-Catenin (Ser33/37/Thr41), anti- β-catenin, anti-VEGF, anti- FGF2, anti-β-actin or lamin B antibodies at 4°C. Immunoreactive bands were visualized using horseradish peroxidase-conjugated secondary antibodies (1:1000 dilution; Cell Signaling, CA, USA), followed by ECL detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Protein images were captured using a FluorChem E imaging system (ProteinSimple; Santa Clara, CA, USA).

2.6 Gene Expression Analysis

Total RNA was extracted from harvested cells for reverse transcription polymerase chain reaction (RT-PCR) using Easy Blue. The RNA isolation protocol included DNase I treatment. RNA samples were quantified using the standard absorbance OD260 method. Each reaction mix and reaction contained 50–100 ng of RNA and was performed in a total reaction volume of 25 µL. The primer and probe concentrations were 300 and 200 nM, respectively. The conditions for real-time quantitative RT-PCR were as follows: 30 min at 48°C (RT), 10 min at 95°C (RT inactivation and initial activation), followed by 40 cycles of amplification for 15 s at 95°C (denaturation) and 1 min at 60°C (annealing and extension). The primers for LEF, TCF, AXIN2, VEGF, FGF2, KGF, HGF, MYC, MMP-2, MMP-7, MMP-9 quantitative amplification were validated using the TaqMan Gene Expression Assay (Applied Biosystems). Data analysis was performed using Step one software v2.3. To normalize the β-actin housekeeping gene expression, the mathematical model of the relative expression ratio, including PCR efficiency, was chosen and applied for sample quantification.

2.7 Statistical Analysis

Data are expressed as means ± SDs. Significant differences were compared using student’s t-test. Statistical significance was defined as p < 0.05. All statistical analyses were performed using GraphPad Prism v.5.0 (Chicago, IL, USA).

3.1 Effects of candidates on proliferation of hDPCs or HUVECs.

To select candidate materials based on cell proliferation in hDPCs and HUVECs, chlorogenic Acid (CA), quercetin (QC), and moracins M, O, and P were each administered at concentrations of 12.5, 25, and 50 µM. For hDPCs, the test substances were applied to groups treated with IFN-γ and groups not treated with IFN-γ to compare proliferation rates in the anagen and catagen phases. In the anagen phase, chlorogenic acid significantly increased cell proliferation at 25 µM (Fig. 1A). In the catagen phase, control (CON; 75.1 ± 2.1%) showed a significant 24.9% decrease compared to normal (NOR; 100.0 ± 2.6%). However, compared to CON, moracin M at 25 and 50 µM significantly increased cell proliferation. Moracin P showed an increase at 25 µM, but there was no significant difference at 50 µM (Fig. 1B).

In HUVEC, CA, QC, and moracin O at concentrations of 12.5, 25, and 50 µM did not show a significant increase compared to NOR. Moracin M showed a significant increase of 10.0% and 21.2% at 12.5 and 25 µM, respectively, compared to NOR, but exhibited cytotoxicity at 50 µM. Moracin P demonstrated a significant increase of 10% at 50 µM compared to NOR (Fig. 1C).

3.2 Effect of moracin M on activating Wnt/β-Catenin signaling pathways in hDPCs

To evaluate the cytotoxicity of moracin M on hDPCs, cells were exposed to concentrations of 0, 12.5, 25, 50, 100, and 200 µM. Cytotoxicity was defined as a cell viability reduction to less than 80% compared to the untreated group. The results showed no cytotoxic effects at concentrations up to 50 µM (data not shown). Therefore, all subsequent experiments with hDPCs were conducted using moracin M at concentrations of 12.5, 25, and 50 µM.

Treatment with IFN-γ resulted in decreased phosphorylation of glycogen synthase kinase-3β (GSK-3β) at Ser9 while simultaneously increasing the phosphorylation of β-catenin at Ser33/37/Thr41. These changes promoted proteasomal degradation and inhibited the nuclear translocation of β-catenin. Conversely, in the group co-treated with moracin M and IFN-γ, there was a notable increase in GSK-3β phosphorylation, increase in non-phospho (Active) β-Catenin (Ser33/37/Thr41) levels, diminished proteasomal degradation, and a significantly increased in nuclear β-catenin levels (Fig. 2A, B). The transcription factors LEF, TCF and AXIN2, which are key players in the Wnt pathway, There were notable decreases in LEF, TCF and AXIN2compared to NOR in the CON group during the catagen stage. In contrast, moracin M treatment resulted in a statistically significant increase in LEF, TCF and AXIN2expression when compared to those in the CON group (Fig. 2C). Moracin M at 50 µM exhibited hair growth efficacy similar to that of 1 µM minoxidil, which was used as a positive control.

3.3 Effect of moracin M on growth factor in hDPCs

To evaluate the impact of moracin M treatment on cytokine levels during the catagen phase, we assessed the protein and mRNA levels of growth factors in hDPCs. VEGF and FGF2 showed significant protein reductions following IFN-γ treatment. However, when co-treated with moracin M, there was a significant increase in these proteins compared to the CON group (Fig. 3A, B). IFN-γ treatment led to significant mRNA reductions in VEGF, FGF7, KGF, HGF, and MYC by 40%, 60%, 10%, 20%, and 60%, respectively, compared to the NOR group. After 24 hours of exposure, while moracin M did not affect HGF levels in hDPCs, it significantly increased the levels of other growth factors in a dose-dependent manner (Fig. 3C). The increase rate of moracin M at a 50 µM concentration was similar to that of minoxidil, used as a positive control. VEGF, FGF2, KGF, and MYC levels increased by 83.3%, 200%, 86.6%, and 200%, respectively, compared to the CON group.

3.4 Effects of moracin M on angionenesis in HUVECs.

To assess the extent of growth factor promotion by moracin M, we evaluated cell migration in HUVECs. The concentrations of moracin M used in the experiment were 6.25, 12.5, and 25 µM, which were determined to be non-cytotoxic (Fig. 4A). Minoxidil (1 µM) served as the positive control for wound width reduction in HUVECs. Under normal conditions, the wound width naturally decreased by 20.67%, whereas treatment with minoxidil resulted in a 78.6% reduction. Treatment with moracin M at concentrations of 6.25, 12.5, and 25 µM resulted in wound width reductions of 56.7%, 60.7%, and 72.3%, respectively. Moracin M enhanced HUVEC tube formation in a dosedependent manner (Fig. 4D, E). We investigated the mechanism underlying moracin M-induced HUVEC tube formation and migration further. We assessed the production of the three MMPs involved in angiogenesis. The quantitative real-time PCR results showed that moracin M dose-dependently stimulated MMP-2 and MMP-9 expression in HUVECs, while there was no change in MMP-7 (Fig. 4F).

Moracins are a group of naturally occurring benzofuran compounds primarily derived from the morus genus of the Moraceae family [15]. These benzofuran derivatives are major constituents found in various natural products within the Moraceae family. Recently, benzofuran derivatives have garnered significant interest among natural product researchers due to their valuable biological activities, including anticancer, antibacterial, immunomodulatory, antioxidant, antihyperglycemic, and anti-inflammatory properties [18]. Moracins, with benzofuran heterocycles as their fundamental scaffold, enable the generation of diverse functionalities, thereby optimizing modulation for the treatment of various diseases. To date, 26 members of this group (designated moracin A to Z) have been identified [19, 20]. Lee et al. study found that moracins M, O, and R, isolated from M. alba root bark, inhibit IL-6 production and lung inflammation through the JNK/c-Jun pathway, suggesting their potential as treatments for lung inflammatory diseases [21]. Similar to resveratrol, moracin M is a natural phosphodiesterase 4 (PDE4) inhibitor [22], exhibiting pharmacological efficacy in the treatment of idiopathic pulmonary fibrosis (IPF) and asthma [23]. Moracin M, isolated from the root bark of M. alba, demonstrated notable antibacterial and anticancer activities [24].

Our research aimed to identify compounds with hair growth-promoting activity in hDPCs and HUVEC cells, inspired by the cardiovascular benefits [25, 26], cognitive function improvements [27], and anti-hair loss [9, 28] effects associated with various morus species. Although moracin M did not promote HDPCs proliferation under conditions without IFN-γ, significant cell proliferation was observed when IFN-γ was present. The hair follicle life cycle can be divided into the anagen (growth), catagen (regression), and telogen (resting) phases, which repeat throughout the follicle's lifespan. During the anagen phase, the hair shaft grows continuously, while growth decreases during the catagen and telogen phases, preparing for the next hair cycle through structural changes. Enhanced or inhibited transition to the catagen phase increases the likelihood of hair loss. IFN-γ is one of several cytokines involved in regulating the hair follicle cycle [29]. In hDPCs during the anagen phase, IFN-γ acts as a regression inducer, terminating the anagen phase and creating conditions similar to the catagen phase. Effective hair loss treatments should prolong the anagen phase or rapidly revert the catagen phase to the anagen phase. In this context, moracin M shows potential as a candidate compound for inducing growth under catagen-like conditions.

The research conducted by Wikramanayake et al. [30] revealed that subcutaneous injections of quercetin could induce hair regrowth in established alopecia areata (AA) lesions in C3H/HeJ mice. However, this hair regrowth effect was not observed in the proliferation of dermal papilla cells during the anagen or catagen phases under non-AA conditions. Chlorogenic acid is widely used as an indicator substance for the Moraceae family. In our current experiment, similar to the findings of Tan et al. [31], proliferation of dermal papilla cells was observed during the anagen phase, but no cell proliferation was noted during the catagen phase. Additionally, in Tan's previous study, no significant results were obtained regarding the activity of 5α-reductase, an important marker in androgenic alopecia.

Blood vessels play a crucial role in hair growth by supplying nutrients to hDPCs. Angiogenesis, a hallmark of the anagen phase, is facilitated by hDPCs, which produce angiogenic factors to support vascular growth. Angiogenesis is significantly induced during the anagen phase, while it decreases during the catagen and telogen phases, and is essential for maintaining normal hair growth. Our findings indicate that moracin M enhances the proliferation rate of HUVEC cells up to a concentration of 25µM. Similarly, moracin P shows a significant increase in cell proliferation at a concentration of 50µM, suggesting the necessity for further investigation at higher concentrations.

Human dermal papilla cells (hDPCs) are a type of mesenchymal cell connected to the capillaries beneath hair follicles. They have been identified as possessing stem cell functions and are anticipated to play a crucial role in preventing hair loss and promoting hair growth. The mechanisms known to activate the proliferation of hDPCs, depending on the hair phase, include Wnt/β-catenin, JAK/STAT, PI3K/AKT, MAPK, and 5α-reductase. Currently, FDA-approved drugs for hair loss treatment include minoxidil, which activates vascular endothelial growth factor (VEGF) and Wnt/β-catenin pathways, and finasteride, which targets 5α-reductase. The recently approved baricitinib functions by acting on the JAK/STAT pathway in cases of alopecia areata.

Among various signaling pathways, the Wnt/β-catenin pathway plays a crucial role in hair follicle development, hair cycle regulation, and regeneration. In addition to M. alba, several natural extracts have been studied for their potential to activate the Wnt/β-catenin pathway [32], including Aconitie ciliare tuber [33], Centipeda minima (L.) A [34]. Ginkgo biloba [35], Red ginseng oil [36], and Salvia plebeia R. Brown (Labiatae). [37], According to shin et al. [38], various natural compounds such as baicalin, 3-Deoxysappan chalcone (3-DSC), Epigallocatechin-3-gallate (EGCG), Fisetin, Quercitrin, and Honokiol have been demonstrated to promote hair growth by stimulating Wnt/β-catenin signaling. Additionally, research on activating the Wnt/β-catenin pathway using lasers is also actively being pursued.

In the Wnt-off state, GSK-3β phosphorylates β-catenin, leading to its proteasomal degradation. However, in the Wnt-on state, β-catenin is not phosphorylated by GSK-3β, allowing it to stabilize in the cytoplasm and subsequently translocate to the nucleus [38]. Once stabilized, β-catenin translocates to the nucleus and binds to T-cell factor (TCF)/lymphoid enhancer factor (LEF). TCF/LEF transcription factors are the major endpoints mediators of Wnt/Wingless signaling across all metazoans. Because TCF/LEF were expressed in distinctly different but extensively overlapping patterns, it is common for more than one TCF/LEF to be co-expressed within a single cell. When AXIN2 is increased in dermal papilla cells, the Wnt/β-catenin signaling pathway is regulated, promoting the nuclear translocation of β-catenin. As a result, genes essential for hair growth are activated. These genes contribute to growth-promoting activities mediated through β-catenin signaling. The study by Lim et al. [39] investigated the expression of various Wnt target genes, demonstrating that Wnt/β-catenin signaling is active in the bulge during the telogen phase. Their findings establish that Axin2 expression is a reliable indicator of Wnt signaling activity in hair follicles.

This complex then activates downstream target genes responsible for cell proliferation and migration. Our results indicate that moracin M induces β-catenin accumulation by promoting the phosphorylation of GSK-3β at Ser9. This activation leads to the transcription of Wnt signaling-mediated genes, including TCF, LEF, and AXIN2, thereby promoting hair outgrowth [9, 38]. These findings suggest that moracin M may serve as a potent activator of the Wnt/β-catenin pathway, offering a novel mechanism for promoting hair growth and regeneration. Minoxidil at 1µM was used as a positive control, and moracin M exhibited Wnt/β-catenin activity equal to or greater than that of the positive control.

Cytokines within hair follicles play a crucial role in inducing and regulating hair growth, as well as maintaining the growth phase [40, 41]. Keratinocyte growth factor (KGF) is essential for hair growth, promoting proliferation and differentiation of normal hair follicles, with its mRNA levels peaking during the anagen phase. Additionally, topical application of fibroblast growth factor (FGF) in mice has demonstrated significant hair growth compared to controls. Secreted by DP, vascular endothelial growth factor (VEGF) stimulates the expression of VEGFR-2 in human epidermal cells, hence promoting their proliferation, differentiation, and migration. In terestingly, VEGF can directly act on DP and promote human HF growth by stimulating local blood vessels during anagen, with bFGF promoting VEGF angiogenesis. When catagen phase was induced with IFN-γ, a significant decrease in VEGF and FGF2 protein levels was observed, consistent with the findings of Yano et al [42]. However, moracin M showed a significantly to restore the levels of these proteins. In our study, we observed that the mRNA expression levels of these growth factors (KGF, VEGF, FGF2, and MYC) in hDPCs significantly increased in a dose-dependent manner when exposed to Moracin M. These growth factors are regulated by the Wnt/β-catenin pathway.

When it comes to hair loss research, the application of HUVECs is particularly advantageous. HUVECs can enhance angiogenesis, thereby improving blood flow to hair follicles and potentially stimulating hair growth[43]. Furthermore, their ability to detect and respond to pharmacological and disease-causing agents is invaluable in assessing the efficacy and safety of new hair loss treatments. HUVECs also interact with pericytes and other skin cells to support the regeneration and maintenance of healthy hair follicles. Thus, utilizing HUVECs in hair loss research holds promise for the development of more effective and holistic treatment approaches. Blood vessels play a crucial role in hair growth by supplying nutrients to hDPCs. Angiogenesis, a hallmark of the anagen phase, is facilitated by hDPCs, which produce angiogenic factors to support vascular growth. Angiogenesis is significantly induced during the anagen phase, while it decreases during the catagen and telogen phases, and is essential for maintaining normal hair growth. Our findings indicate that moracin M enhances the proliferation rate of HUVEC cells up to a concentration of 25µM. Similarly, Moracin P shows a significant increase in cell proliferation at a concentration of 50µM, suggesting the necessity for further investigation at higher concentrations.

In the study conducted by Yoon et al., wound healing was performed on hDPCs [44]. However, to evaluate the vascular differentiation potential of moracin M, we cultured HUVEC cells and performed a scratch assay, observing the healing effects over a 24-hour period. The results showed a significant difference in wound healing, which is an indicator of angiogenesis. Yano et al., studies, transgenic overexpression of VEGF in outer root sheath keratinocytes resulted in enhanced perifollicular vascularization, accelerated hair regrowth after depilation, and increased size of vibrissa follicles, hair follicles, and hair shafts [42]. These findings suggest that moracin M may play a crucial role in promoting vascular cell differentiation and angiogenesis.

Moracin M has been shown to enhance tube formation in HUVECs and to elevate the expression and activity of MMP-2 and MMP-9, while not affecting MMP-7 levels. The various effects of moracin M identified in this study align with findings reported by Sun et al. [45] Additional research is necessary to comprehensively and thoroughly investigate the impact of moracin M on the altered endothelial cell. MMPs facilitate endothelial cell migration and tube formation by breaking down the basement membrane. Specifically, MMP-2 and MMP-9 are highlighted due to their type IV collagenase activity, which is crucial in the early stages of angiogenesis [46]. Moracin M significantly enhances angiogenesis and endothelial cell differentiation, highlighting its potential as a therapeutic agent for hair growth and vascular health.

Moracin M has been identified as a promoter of angiogenesis and a facilitator of hair cycle regulation and growth through the Wnt/β-catenin signaling pathway. Notably, moracin M has been observed to significantly enhance cell proliferation during the catagen (regression) phase of the hair growth cycle. This study represents the first comprehensive elucidation of moracin M is effects on angiogenesis and hair growth. Despite these promising findings, further investigation is required to validate these effects within actual microenvironments or in vivo conditions. Nevertheless, our research advances the understanding of hair growth-promoting agents and serves as a critical reference for subsequent studies on the vascular benefits of moracin M. This work establishes a foundation for developing novel therapeutic strategies utilizing moracin M for dermatological and vascular health applications.

FDA

The Food and Drug Administration

hDPCs

human dermal papilla cells ()

HUVECs

human umbilical vein endothelial cells

LEF

lymphoid enhancer factor

TCF

T-cell factor

AXIN2

axis inhibition protein 2

GSK-3β

Glycogen synthase kinase-3β

Wnt

Wingless-related integration site

VEGF

vascular endothelial growth factor

FGF2

fibroblast growth factor 2

KGF

Keratinocyte growth factor

HGF

Hepatocyte Growth Factor

MYC

MYC Proto-Oncogene, BHLH Transcription Factor

IFN-γ

Interferon gamma

MMP

Matrix Metalloproteinase

CVDs

cardiovascular diseases

ECL

Enhanced chemiluminescence

Acknowledgments: This paper was supported by Wonkwang Uiversity in 2022

Competing Interests: The authors declare no conflicts of interest.

Authors' contributions: BoYoon Chang; Yuri Hwang: Conceptualization, Methodology, Validation, Investigation, Data Curation, Writing - Original Draft, Visualization. SungYeon Kim: Conceptualization, Project administration, Writing - Review & Editing. Yonghwan Kim, In Kim, Hyungmin Park: Visualization, Resources

Funding: Not applicable

Data Availability: The data used to support the findings of this study are available from the corresponding author upon request.

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The Role of Moracin M in Promoting Hair Growth: Insights into Mechanisms of WNT/β- Catenin Pathway Activation and Angiogenesis Enhancement in Human Dermal Papilla Cells

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Abstract

Figures

1. Introduction

2. Material and Method

2.1 Chemicals, Animals, and Biological Kits

2.2 Cell culture of hDPCs and HUVEC

2.3 MTT Assay

2.4 Wound healing

2.5 Protein Expression Analysis

2.6 Gene Expression Analysis

2.7 Statistical Analysis

3. Results

3.1 Effects of candidates on proliferation of hDPCs or HUVECs.

3.2 Effect of moracin M on activating Wnt/β-Catenin signaling pathways in hDPCs

3.3 Effect of moracin M on growth factor in hDPCs

3.4 Effects of moracin M on angionenesis in HUVECs.

4. Discussion

Abbreviations

Declarations

References

Additional Declarations

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Version 1