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Therapeutic Effect of Nepenthes kampotiana Lecomte Ethanol Extract (Nk-EE) on Androgenic Alopecia through the Inhibition of Apoptosis and 5a-Reductase Activity

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09 August 2023

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10 August 2023

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Abstract
Alopecia refers to the continuous shedding of hair from the scalp or other areas. Genetic factors are the most common of the variety of causes of alopecia. Male pattern baldness, also known as androgenic alopecia, is prevalent. Recently, the interest in hair loss treatment has increased due to the growing number of people experiencing anxiety and stress related to hair loss. Nepenthes kampotiana Lecomte is known for its anticancer effects, but its potential for preventing hair loss has not been researched. Therefore, this study focused on the hair loss prevention effects of N. kampotiana Lecomte ethanol extract (Nk-EE). The results showed that Nk-EE has a proliferative effect on human follicle dermal papilla cells and inhibited cell death. In vivo experiments using androgenic areata models showed that Nk-EE had a positive effect on a variety of biomarkers such as hair-to-skin ratio, hair type frequency, and hair thickness. The results of this study suggest that Nk-EE has the potential to be an effective treatment for androgenic alopecia.
Keywords: 
Alopecia areata
;  
Nepenthes Kampotiana Lecomte
;  
5a-reductase
;  
testosterone
Subject: 
Medicine and Pharmacology  -   Pharmacology and Toxicology

1. Introduction

Alopecia, also known as hair loss, refers to the gradual thinning or complete loss of hair[1]. Hair loss can occur for a variety of reasons, including genetic factors, hormonal changes, nutritional deficiencies, stress, irregular lifestyle habits, and medication side effects [2,3,4]. The most common form of hair loss is androgenic alopecia (AGA), which occurs in over 50% of men but can also occur in women [5,6]. AGA causes hair to become thinner and shorter, and can affect all hair on the body, not just the scalp [7,8,9]. AGA is caused by the role of androgen hormones. AGA starts with the hair gradually thinning and shortening, ultimately leading to complete hair loss, and typically begins around the temples and progresses to the top of the scalp [10,11,12]. While the exact cause of AGA is not fully understood, it is believed that inhibition of hair growth through the androgen receptor (AR) signaling system is the main cause [12,13,14].
Androgens are hormones that regulate reproductive development and function in both males and females, and in men they are primarily produced in the testes [15,16]. Androgens are composed of several chemical substances, including testosterone, androstenedione, dehydroepiandrosterone (DHEA), and dihydrotestosterone (DHT). DHT is the active metabolite of testosterone [15,17]. Testosterone is one of the major hormones that influences sex differentiation and maintenance, reproductive function, and muscle development [18]. DHT is produced when testosterone is converted by an enzyme called 5α-reductase [19]. This enzyme is expressed mainly in the scalp and prostate, and in some other tissues that promote DHT synthesis [20,21]. Therefore, DHT is mainly produced in the scalp and prostate, and plays an important role in male development and maintenance. DHT stimulates prostate growth and development, and inhibits the action of the female hormone estrogen, thereby affecting male reproductive function and development [22]. In addition, DHT is one of the causes of hair loss. Male pattern baldness is thought to occur due to genetic factors and excessive DHT production in the scalp [23]. If a specific genetic mutation causes excessive production of DHT in the scalp, the DHT can act on the hair follicles, causing the diameter of the hair to become smaller and thinner, ultimately leading to hair loss [11,24,25]. Androgens also have an impact on hair growth, as the cells in the hair follicle in the scalp have ARs. When androgens bind to these receptors, they trigger signals that inhibit hair growth, leading to gradual thinning and shortening of androgen-sensitive hair until they no longer grow [25]. This is the main mechanism behind AGA, and changes in the AR signaling system are one of the contributing factors.
AGA is hair loss caused by the influence of androgen hormones on genetically healthy hair. The most commonly used treatments for AGA are flutamide and finasteride, which inhibit ARs to prevent hair loss [26]. Flutamide is an AR antagonist that promotes hair growth by acting on ARs [27,28]. This drug can be applied topically to the scalp or administered by injection. Finasteride inhibits 5α-reductase and prevents the conversion of testosterone to DHT, which helps to suppress AGA, particularly in the treatment of male pattern baldness [26,29]. However, both finasteride and flutamide can have potential side effects. Finasteride can cause sexual dysfunction, breast enlargement, and other side effects, while flutamide may have an impact on the fetus of pregnant women when taken orally, making it restricted for use by pregnant women [30,31,32]. Hair follicles are complex structures that undergo continuous cycles of growth and shedding, which are regulated by a collection of genetic, hormonal, and environmental factors. These cycles are divided into four phases: anagen, catagen, telogen, and exogen [33]. During the anagen phase, the hair follicle actively grows, with new hair cells being produced at the base of the follicle that push older cells up and out of the skin surface. This phase can last anywhere from a few months to several years, depending on the location of the hair on the body. The catagen phase is a short transitional phase that marks the end of the anagen phase. During this phase, the hair follicle stops growing and begins to shrink, as the hair matrix, human dermal papilla (HDP) cells, inner root sheath keratinocyte, and outer root sheath keratinocytes undergo programmed cell death (apoptosis) [34,35]. The telogen phase is a resting phase that follows the catagen phase. During this phase, the hair follicle is dormant and the hair shaft is fully formed, but not actively growing. This phase typically lasts for several months. Finally, during the exogen phase, the old hair shaft is shed from the skin surface, allowing a new hair cycle to begin. This phase is also known as the shedding phase, and it can last several weeks [36].
This study investigated the significant changes that occur during the telogen phase of the hair growth cycle. Telogen is regulated by a variety of genetic, hormonal, and environmental factors, and compared to the anagen phase, it shows a dramatic decrease in the Bcl2/Bax ratio, leading to cell apoptosis. As a result, old hair falls out, and through the resting and growth phases, new hair is generated.

2. Results

2.1. Analysis of Components Using Gas Chromatography-Mass Spectrometry (GC-MS)

GC-MS analysis of Nk-EE revealed that the most abundant compound was silver butanoate, followed by tricarbonyl(trimethylphosphine)nickel, 4-o-(β-d-glucopyranosyl)-d-glucopyranose, and others. All compounds have been listed in Table 1.
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Figure 1. Phytochemical analysis of Nk-EE using GC-MS.
Figure 1. Phytochemical analysis of Nk-EE using GC-MS.
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2.2. Promotion of Human Dermal Papilla (HDP) Cell Growth and Recovery from Testosterone-Induced Cytotoxicity by Nk-EE

To investigate the effect of Nk-EE on AGA, we performed a 5α-reductase assay. The results showed that Nk-EE inhibited the activity of 5α-reductase in a concentration-dependent manner (Figure 2A). To further confirm the inhibitory effect and hair-growth-promoting effect of Nk-EE on 5α-reductase, we analyzed the survival and growth rates of HDP cells. The analysis showed that Nk-EE significantly increased the growth of HDP cells in a concentration-dependent and statistically significant manner (Figure 2B). Additionally, when HDP cells were treated with testosterone and Nk-EE, the survival rate decreased due to the testosterone, but it increased in a concentration-dependent manner due to the Nk-EE (Figure 2C). Finally, to check whether Nk-EE can directly bind to 5a-reductase, we evaluated binding affinity of individual components identified in Nk-EE. As Table 2 shows, finasteride showed -10.1, while 1,3,4-trimethyl-6-cyclohexylbenzene, n-hexadecanoic acid, and β-santalol exhibited affinity value with -8.4, -6.3, and -7.3, respectively.

2.3. Prevention or Inhibition of Hair Loss by Nk-EE in an AGA Mouse Model

To further analyze the preventive and inhibitory effects of alopecia, we analyzed an AGA mouse model. We injected testosterone once each week and applied Nk-EE to the skin at a dosage of 5 or 10 mg daily. We then took pictures each week to analyze the amount and growth of hair. Our analysis revealed that Nk-EE increased hair growth, which was evident during the first, second, and third weeks (Figure 3A). Next, we sacrificed the mice at week 3, extracted their skin, and performed histological analysis by hematoxylin and eosin (H&E) staining. The results showed a significant decrease in the number of hair follicles, including those containing hair, in the group treated with testosterone, while Nk-EE treatment increased the number of hair follicles (Figure 3B and C). For a more detailed analysis, we counted the number of hair follicles with or without hair in all photographs and presented them graphically. As expected, Nk-EE remarkably enhanced hair follicle numbers (Figure 3D). Meanwhile, finasteride also showed remarkable induction of hair growth at day 4, while testosterone-treated control group did not show clear hair growth (Figure 3E).

2.4. Inhibition of AR Activity by Nk-EE and its Mechanism

To analyze the levels of testosterone, we collected blood from the AGA mouse models and extracted serum for testosterone ELISA analysis. The analysis showed high concentrations of testosterone in all groups except for the control group (Figure 4A). Additionally, to indirectly analyze the activity of testosterone, we performed western blotting analysis using mouse skin tissue to analyze the activity of AR. The results showed that the activity of AR decreased with Nk-EE treatment (Figure 4B). Furthermore, we analyzed the gene expression of TGF-1B and DKK-1 in the AR signaling pathway of skin tissue using real-time PCR analysis. The results showed that Nk-EE treatment suppressed the expression of the TGF-1B and DKK-1 genes (Figure 4C, D).

2.5. Inhibition of Cell Apoptosis by Nk-EE and the Underlying Mechanism

We analyzed the effects of Nk-EE on inhibiting cell death and promoting the growth of hair follicle cells. Using skin tissue from the AGA mouse model, we used real-time PCR analysis to confirm the expression of genes related to cell death, including Bcl2 and Caspase-3, as well as growth-regulating genes HGF and IGF-1. The results indicated that Nk-EE treatment decreased the expression of the Caspase-3 gene related to cell death (Figure 5A). Furthermore, it was confirmed that the expression of the Bcl2 gene, which inhibits cell death, was increased by Nk-EE treatment (Figure 5B). The expression of HGF and IGF-1 genes related to hair follicle cell growth [37,38,39] was upregulated by Nk-EE treatment. In addition, proteins involved in cell death were analyzed with western blotting analysis. Nk-EE treatment decreased the expression of proteins such as p53, BAX, and caspase-9, which are involved in cell death [37,40], while increasing the expression of the Bcl-2 protein (Figure 5E).

3. Discussion

The purpose of our study was to investigate the potential benefits of Nk-EE in preventing and inhibiting hair loss in an AGA mouse model. AGA is a common type of hair loss that affects both men and women, and is caused by the hormone DHT, which is derived from testosterone. To test the effects of Nk-EE in alopecia, we conducted in vitro and in vivo experiments. In our in vitro experiments, we used cultured HDP cells. In our in vivo experiments, we used a mouse model of AGA.
Testosterone can prevent the normalization of the Bcl2/Bax ratio or lengthen the telogen phase through dysregulation, which disrupts the balance of hair growth stages and induces alopecia [41]. To prevent or inhibit such alopecia, we analyzed several biomarkers and confirmed that Nk-EE is effective in suppressing hair loss or promoting hair growth. We confirmed that Nk-EE stimulates growth by significantly increasing the expression of the growth factor HGF and IGF genes in HDP cells and by promoting cell growth through the downregulation of Caspase-9 protein expression induced by BAX protein while inhibiting cell death. In contrast, we found that Nk-EE decreases the survival of prostate cancer cells that use the hormone testosterone as a growth factor. Furthermore, the results showed that by inhibiting the activation of the AR signaling pathway induced by testosterone, Nk-EE can not only promote cell growth and inhibit cell death in hair follicle cells but also inhibit the effects induced by testosterone. These effects suggested that Nk-EE has potential value as a material for treating or preventing alopecia.
Based on our study, we have found that Nk-EE has the potential to offer several benefits in terms of preventing hair loss and promoting hair growth. Our results suggest that Nk-EE can inhibit the activity of 5α-reductase, an enzyme responsible for converting testosterone to DHT. Moreover, we have observed that Nk-EE can stimulate the growth of HDP cells. These findings indicate that Nk-EE has the potential to promote the overall health and growth of hair follicles, thereby positively impacting hair growth and preventing alopecia. Furthermore, our experiments demonstrated that Nk-EE may increase hair growth and the number of hair follicles, while also decreasing the expression of genes related to cell death and increasing the expression of genes related to hair follicle cell growth. These findings suggest that Nk-EE may have a positive effect on the regulation of hair follicle growth and may help to prevent alopecia.
Our research suggests that Nk-EE's mechanism of action involves inhibiting and regulating a variety of genes and proteins involved in the AR signaling pathway as well as cell death and growth. We observed that Nk-EE inhibited AR activity by decreasing the expression of two key genes involved in the AR signaling pathway, TGF-1B and DKK-1 [42]. By reducing the expression of these genes, Nk-EE may reduce the activation of the AR signaling pathway, ultimately leading to decreased cell proliferation and growth. In addition, our findings suggest that Nk-EE may also inhibit cell apoptosis (cell death) by modulating the expression of certain genes. Specifically, Nk-EE decreased the expression of the Caspase-3 gene, which is involved in promoting cell apoptosis, while increasing the expression of the Bcl2 gene, which is involved in inhibiting cell apoptosis [43]. This dual effect may lead to an overall reduction in cell death.
Moreover, Nk-EE regulated the expression of other proteins involved in cell death and growth. For example, Nk-EE increased the expression of the HGF and IGF-1 genes, which are involved in promoting cell growth and survival, while decreasing the expression of the p53 and BAX genes, which are involved in promoting cell death [44]. Additionally, Nk-EE regulated the expression of the caspase-9 gene, which is involved in promoting apoptosis. Overall, these changes in protein expression contribute to Nk-EE's effects on cell growth and death.
Our study provides compelling evidence that Nk-EE may hold significant promise as a potential treatment for hair loss. The mechanisms of action identified in our research suggest that Nk-EE could be effective in inhibiting the AR signaling pathway and promoting hair growth by regulating cell death and growth pathways [45]. However, further studies are necessary to confirm the safety and efficacy of Nk-EE in human clinical trials. In particular, the optimal dosage and duration of treatment need to be investigated to ensure that the benefits of the treatment outweigh any potential risks or side effects. In addition to efficacy and safety considerations, future research should also aim to gain a deeper understanding of the underlying mechanisms of Nk-EE in the context of hair growth. This could involve further investigation into the specific genes and proteins that are targeted by Nk-EE as well as the interactions between these molecular pathways. Overall, while our study provides promising initial evidence of the potential of Nk-EE as a hair loss treatment, further research is needed to fully realize its therapeutic potential and to advance our understanding of the underlying mechanisms of hair growth and loss.

4. Materials and Methods

4.1. Materials and Reagents

The following reagents were purchased from the mentioned suppliers: testosterone, ethanol, of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), finasteride, and dimethyl sulfoxide (DMSO) from Sigma-Aldrich in St. St. Louis, MO, USA; trypsin (0.25%) from HyClone Laboratories in Logan, UT, USA; HDP cells, which are a human hair follicle dermal papilla cell line, and CEFOgro™ HDP Growth Medium were obtained from CEFO Co. in Seoul, Korea; and phosphate-buffered saline (PBS) from Samchun Pure Chemical Co. in Gyeonggi-do, Korea. The antibodies for Bcl-2, Bax, caspase 3, and cleaved caspase 3 were obtained from Cell Signaling Technology in Beverly, MA, USA, and the antibodies for b-actin and AR were purchased from Santa Cruz Biotechnology, Inc. in Dallas, TX, USA. TRIzol reagent was bought from Molecular Research Center, Inc. in Cincinnati, OH, USA.

4.2. Preparation of Nepenthes Kampotiana Lecomte Ethanol Extract (Nk-EE) and GC-MS

We obtained Nk-EE from the National Institute for Biological Resources in Incheon, Korea. To prepare the sample, the whole plant was pulverized and granulated for 24 h at 20–22 °C using 70% ethanol. The ethanol was completely removed using a rotary flash evaporator (Büchi Labortechnik AG, Flawil, Switzerland) for filtering and concentrating under a vacuum of 10 hPa at 40 °C. The resulting aqueous solution was further evaporated at 5 mTorr and -85 °C, and the extract was subsequently lyophilized. We conducted GC-MS analysis at the Cooperative Center for Research Facilities of SKKU located in Gyeoggido, Korea.

4.3. Cell Culture and Cell Viability Assay

The HDP cells used in this study were cultured in HDP growth medium at 37 °C. They were incubated with 5% CO2. HDP cells were seeded in 96-well plates at a density of 1 × 104 cells/mL. Then, Nk-EE (0–200 μg/mL) was added to each well. After incubation for 24 h, 10 μL of MTT solution was added to the cells, which were then further cultured for 3 h. Next, 100 μL of 10% sodium dodecyl sulfate (SDS) in 0.01 M HCl was added to each well, and the cells were incubated for 24 h to stop the reaction and dissolve the formazan. Finally, the absorbance of the MTT formazan was measured at 540 nm.

4.4. Binding affinity calculations with Autodock Vina

3D structure of 5α-reductase 2 was obtained from PDB database, and 2D structure of each compound identified in Nk-EE was acquired from PubChem. 2D structure of each compound was modified with PyMOL. The binding affinity was calculated by Autodock Vina.

4.5. Animals and Testosterone-Induced Mouse Models (AGA Mouse Models)

We obtained 5-week-old male C57BL/6 mice and 10-week-old male Sprague Dawley rats from Orient Bio (Iksan, Korea). They were housed in plastic cages with plenty of water and food. Our study followed the guidelines set forth by the Institutional Animal Care and Use Committee at Sungkyunkwan University (SKKUIACUC-2021-07-18-1). We divided the male C57BL/6 mice (n = 6) into four groups: the Normal group, the Vehicle (PBS: DMSO = 1:1) control group, the 1 mg/kg/week testosterone + Nk-EE 5 mg/day group, and the 1 mg/kg/week testosterone + Nk-EE 10 mg/day group. To confirm the hair-growth-promoting effect of Nk-EE, we applied hair removal cream and shaved the dorsal part of each mouse seven and three days before the first testosterone treatment. We subcutaneously injected 1 mg/kg of testosterone dissolved in sesame oil once per week for 3 weeks. During the three weeks of testosterone injection, we applied Nk-EE (5 and 10 mg/kg) diluted with PBS and DMSO (ratio of 1:1) to the skin on the back of each mouse every day. One day after the last testosterone injection, we euthanized all groups of C57BL/6 mice and isolated the dorsal skin for further analysis.

4.6. Hematoxylin and Eosin (H&E) Staining

The mouse skin tissue was fixed in 3.7 % formaldehyde solution at 4 °C for 3 days, and then cut and embedded in 4-μm-thick paraffin. The cut samples were deparaffinized with xylene and rehydrated with ethanol at different concentrations. Subsequently, they were stained with H&E solution. The stained tissue sections were mounted on clean slides and examined under a microscope to observe the result.

4.7. Quantitative Real-Time PCR

TRIzol reagent was used to extract RNA from mouse skin tissue in the alopecia model. To synthesize cDNA from total RNA, a cDNA synthesis kit from Thermo Fisher Scientific was utilized, following established protocols. Real-time PCR with SYBR premix Ex Taq was performed to measure the mRNA levels of Bcl-2, Bax, caspase 3, TGF-B1, DKK-1, HGF, and IGF-1. The expression levels were determined relative to GAPDH. The primer sequences are listed in Table 3.

4.8. Western Blotting

C57BL/6 mouse skin tissues were pulverized in liquid nitrogen and stored at −70 °C. To prepare for western blotting analysis, the tissues were lysed using the previously described lysis buffer (50 mM Tris-HCl, pH 7.4; 120 mM NaCl; 25 mM β-glycerol phosphate, pH 7.5; 20 mM NaF; 2% Nonidet P-40; and protease inhibitors). Following sonication, the lysates were pelleted by centrifugation at 12,000g for 3 min at 4 °C, and the resulting supernatants were used for western blotting analysis. Protein samples were subjected to 10–15% SDS-polyacrylamide gel electrophoresis, and the levels of AR, Bcl-2, caspase 9, cleaved caspase 9, and β-actin were measured using the corresponding antibodies.

4.9. 5α-Reductase Activity Assay

The entire liver of a 10-week-old male Sprague Dawley was extracted and lysed with lysis buffer containing protease inhibitors and additional components (7.5 mM K2HPO4, 3.25 mM KH2PO4, 1 mM DTT, 32 mM sucrose, and 0.2 mM PMSF) to obtain 5α-reductase. To initiate the reaction, reduced nicotinamide adenine dinucleotide phosphate (NADPH, 34 mM), testosterone (0.4 mM), and McIlvaine buffer (pH 5.0) were added to 4 μL of the enzyme extract that had been treated with different concentrations of Nk-EE. After incubation, the reaction was terminated by heating for 40 min at 37 °C. The oxidation of NADPH was detected by measuring the absorbance at 340 nm.

4.10. Statistical Analysis

The data presented in this paper represent the means ± standard errors of at least two independent experiments. Kruskal-Wallis and Mann-Whitney U tests were performed to analyze the data. Statistical significance was defined as p-values < 0.05. SigmaPlot version 14.0 (Systat Software Inc., San Jose, CA, USA), and SPSS version 25.0 (SPSS Inc., Chicago, IL, USA) were used for data analysis.

5. Conclusions

AGA is a disease that causes hair loss, and its major cause is excessive response to androgenic hormones. Here, we conducted a 5α-reductase assay with Nk-EE. 5α-Reductase converted testosterone to its active form, DHT, and Nk-EE successfully downregulated enzymatic activity. The results showed that Nk-EE promoted hair growth in an AGA mouse model, and upregulated the proliferation of HFDPC cells. These effects were exerted through the anti-apoptotic effect of Nk-EE.
Our result suggests the possibility of a new alopecia drug using Nk-EE. Also, recently there have been a few papers reporting on plant extracts that use mass spectrometry data to show a protective effect against hair loss. In combination with these previous reports, we can make new research finding a common chemical which might be a main factor eliciting an anti-alopecia effect.
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Figure 6. Schematic representation showing the anti-androgenic and anti-apoptotic effects of Nk-EE.
Figure 6. Schematic representation showing the anti-androgenic and anti-apoptotic effects of Nk-EE.
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Author Contributions

Conceptualization, Dong Seon Kim, Won Young Jang, Jong-Hoon Kim, Jongsung Lee, and Jae Youl Cho; Data curation, Jong-Hoon Kim, Jongsung Lee, and Jae Youl Cho, Jongsung Lee and Jae Youl Cho; Formal analysis, Dong Seon Kim and Jae Youl Cho; Funding acquisition, Jae Youl Cho; Investigation, Dong Seon Kim, Won Young Jang, Sang Hee Park, Ji Hye Yoon, Chae Yun Shin, Lei Huang, Kry Masphal, Chhang Phourin, Hye-Woo Byun, Jino Son, Ga Ryun Kim; Project administration, Jae Youl Cho; Writing – original draft, Dong Seon Kim, Won Young Jang; Writing – review & editing, Jong-Hoon Kim, Jongsung Lee, and Jae Youl Cho.

Funding

This research was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR202207203).

Data Availability Statement

Data is contained within the article

Conflicts of Interest

The authors declare no conflict of interest.

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  26. Bachelot, A.; Chabbert-Buffet, N.; Salenave, S.; Kerlan, V.; Galand-Portier, M.B. Anti-androgen treatments. Ann Endocrinol (Paris) 2010, 71, 19-24. https://doi.org/10.1016/j.ando.2009.12.001. [CrossRef]
  27. Zhao, J.; Harada, N.; Okajima, K. Dihydrotestosterone inhibits hair growth in mice by inhibiting insulin-like growth factor-I production in dermal papillae. Growth Horm IGF Res 2011, 21, 260-267. https://doi.org/10.1016/j.ghir.2011.07.003. [CrossRef]
  28. Yazdabadi, A.; Sinclair, R. Treatment of female pattern hair loss with the androgen receptor antagonist flutamide. Australas J Dermatol 2011, 52, 132-134. https://doi.org/10.1111/j.1440-0960.2010.00735.x. [CrossRef]
  29. York, K.; Meah, N.; Bhoyrul, B.; Sinclair, R. A review of the treatment of male pattern hair loss. Expert Opin Pharmacother 2020, 21, 603-612. https://doi.org/10.1080/14656566.2020.1721463. [CrossRef]
  30. Brufsky, A.; Fontaine-Rothe, P.; Berlane, K.; Rieker, P.; Jiroutek, M.; Kaplan, I.; Kaufman, D.; Kantoff, P. Finasteride and flutamide as potency-sparing androgen-ablative therapy for advanced adenocarcinoma of the prostate. Urology 1997, 49, 913-920. https://doi.org/10.1016/s0090-4295(97)00091-5. [CrossRef]
  31. Diviccaro, S.; Melcangi, R.C.; Giatti, S. Post-finasteride syndrome: An emerging clinical problem. Neurobiol Stress 2020, 12, 100209. https://doi.org/10.1016/j.ynstr.2019.100209. [CrossRef]
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  33. Lin, X.; Zhu, L.; He, J. Morphogenesis, Growth Cycle and Molecular Regulation of Hair Follicles. Front Cell Dev Biol 2022, 10, 899095. https://doi.org/10.3389/fcell.2022.899095. [CrossRef]
  34. Oh, J.W.; Kloepper, J.; Langan, E.A.; Kim, Y.; Yeo, J.; Kim, M.J.; Hsi, T.C.; Rose, C.; Yoon, G.S.; Lee, S.J.; Seykora, J.; Kim, J.C.; Sung, Y.K.; Kim, M.; Paus, R.; Plikus, M.V. A Guide to Studying Human Hair Follicle Cycling In Vivo. J Invest Dermatol 2016, 136, 34-44. https://doi.org/10.1038/JID.2015.354. [CrossRef]
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Figure 2. Inhibition of 5α-reductase activity of Nk-EE promotes HDP cell growth. (A) 5α-Reductase assay with Nk-EE. (B) Cell proliferation assay on HDP cells with Nk-EE. (C) Analysis of cell viability in HDP cells treated with Nk-EE and testosterone.
Figure 2. Inhibition of 5α-reductase activity of Nk-EE promotes HDP cell growth. (A) 5α-Reductase assay with Nk-EE. (B) Cell proliferation assay on HDP cells with Nk-EE. (C) Analysis of cell viability in HDP cells treated with Nk-EE and testosterone.
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Figure 3. In vivo analysis of the hair growth promoting and hair loss prevention effects of Nk-EE. (A) Photographs showing weekly monitoring of hair growth of mice treated with testosterone (1 mg/kg) once per week and Nk-EE (5 and 10 mg/kg/100 μL) daily. (B) Hair-to-skin ratio histological data were obtained by H&E staining from the mice shown in (A). (C) Analysis of the number of hair follicles including hair in the histological analysis results with H&E staining. (D) Total hair follicle count using H&E staining. (E) Effect of finasteride (Fina, 1 mg/kg) was also examined with testosterone (1 mg/kg)-treated mice.
Figure 3. In vivo analysis of the hair growth promoting and hair loss prevention effects of Nk-EE. (A) Photographs showing weekly monitoring of hair growth of mice treated with testosterone (1 mg/kg) once per week and Nk-EE (5 and 10 mg/kg/100 μL) daily. (B) Hair-to-skin ratio histological data were obtained by H&E staining from the mice shown in (A). (C) Analysis of the number of hair follicles including hair in the histological analysis results with H&E staining. (D) Total hair follicle count using H&E staining. (E) Effect of finasteride (Fina, 1 mg/kg) was also examined with testosterone (1 mg/kg)-treated mice.
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Figure 4. Analysis of Nk-EE’s regulation of the AR signaling pathway using skin tissue. (A) Analysis of Nk-EE’s regulation of testosterone levels with ELISA. (B) Analysis of Nk-EE’s regulation of AR protein levels with western blotting. (C, D) Analysis of Nk-EE’s regulation of the AR signaling pathway-related genes, TGF-b1 and DKK-1, through real-time PCR. Data are presented in mean + s.d. #,## represents p < 0.05, p < 0.01 compared with the normal control and *,** represents p < 0.05, p < 0.01 compared with the testosterone treated group.
Figure 4. Analysis of Nk-EE’s regulation of the AR signaling pathway using skin tissue. (A) Analysis of Nk-EE’s regulation of testosterone levels with ELISA. (B) Analysis of Nk-EE’s regulation of AR protein levels with western blotting. (C, D) Analysis of Nk-EE’s regulation of the AR signaling pathway-related genes, TGF-b1 and DKK-1, through real-time PCR. Data are presented in mean + s.d. #,## represents p < 0.05, p < 0.01 compared with the normal control and *,** represents p < 0.05, p < 0.01 compared with the testosterone treated group.
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Figure 5. Analysis of Nk-EE's anti-apoptotic effect in vivo using skin tissue. (A, B) Analysis of caspase 3 and Bcl-2 gene regulation related to the apoptosis signaling pathway by Nk-EE using real-time PCR analysis. (C, D) Analysis of HGF and IGF-1 gene regulation related to growth promotion by Nk-EE using real-time PCR analysis. (E) Analysis of protein regulation of the apoptosis signaling pathway by Nk-EE using western blotting analysis. # represents p < 0.05 compared with the normal control and *,** represents p < 0.05 and p < 0.01 compared with the testosterone treated group.
Figure 5. Analysis of Nk-EE's anti-apoptotic effect in vivo using skin tissue. (A, B) Analysis of caspase 3 and Bcl-2 gene regulation related to the apoptosis signaling pathway by Nk-EE using real-time PCR analysis. (C, D) Analysis of HGF and IGF-1 gene regulation related to growth promotion by Nk-EE using real-time PCR analysis. (E) Analysis of protein regulation of the apoptosis signaling pathway by Nk-EE using western blotting analysis. # represents p < 0.05 compared with the normal control and *,** represents p < 0.05 and p < 0.01 compared with the testosterone treated group.
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Table 1. List of phytochemicals in Nk-EE analyzed by GC-MS.
Table 1. List of phytochemicals in Nk-EE analyzed by GC-MS.
Peak# RT Compound Corrected area Area (%)
1 1.467 Hydroxylamine 57022153 3.064
2 1.691 Tricarbonyl(trimethylphosphine)nickel 121766953 6.542
3 3.257 Glyceraldehyde 6573734 0.353
4 3.616 2-Furanmethanol 7382840 0.397
5 3.987 2-(Methylthio)ethylamine 15486459 0.832
6 4.494 Methylenecyclopropanecarboxylic acid 30449212 1.636
7 5.032 Glycerin 13883619 0.746
8 5.438 2H-Pyran-2,6(3H)-dione 15745744 0.846
9 6.347 2-Furancarboxylic acid 9522821 0.512
10 6.567 4,5-Diamino-2-hydroxypyrimidine 10469278 0.562
11 6.786 N-Butylarachidamide 9562344 0.514
12 7.477 N-Nitrosodiethylamine 11101059 0.596
13 7.617 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one 19365108 1.040
14 8.375 Catechol 7437001 0.400
15 8.583 Dihydro-5-propyl-2(3H)-furanone 9178831 0.493
16 8.779 5-Hydroxymethylfurfural 24210497 1.301
17 8.974 Glycerol 1-acetate 12032863 0.646
18 9.475 2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one 23091763 1.241
19 10.049 2-Methoxy-4-vinylphenol 7925320 0.426
20 10.296 Isovaleric acid, 8-chlorooctyl ester 16915750 0.909
21 10.808 1-bromo-pentaborane(9) 9024608 0.485
22 11.168 2-bromo-pentaborane(9) 7963850 0.428
23 11.396 Propanedioic acid, 3-thienyl- 10538919 0.566
24 11.674 1,7-Dihydro-6H-purin-6-one, 35302111 1.897
25 12.318 (1S,5S,6R)-6-Methyl-2-methylene-6-(4-methylpent-3-en-1-yl)bicyclo[3.1.1]heptane 5075342 0.273
26 12.742 1,5,5,6-Tetramethyl-1,3-cyclohexadiene, 12622932 0.678
27 13.187 (1S)-;2,2-Dimethyl-3-methylene-bicyclo[2.2.1]heptane 33499401 1.80
28 13.473 3-((3R)-2,3-Dimethyltricyclo[2.2.1.02,6]heptan-3-yl)propanal 8724900 0.469
29 13.889 Silver butanoate 313129982 16.824
30 14.251 4-o-(β-d-Glucopyranosyl)-d-glucopyranose 67520633 3.628
31 14.987 β-Santalol 53417474 2.870
32 15.223 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol 50422487 2.709
33 15.457 Bicyclo[4.3.0]nonane, 2-methylene-, cis- 22777451 1.224
34 15.601 7-Oxabicyclo[4.1.0]heptane, 1-methyl-4-(2-methyloxiranyl)- 33239934 1.786
35 15.999 (3-Nitro-2-oxo-2H-pyridin-1-yl)-acetic acid 42554276 2.286
36 17.403 n-Hexadecanoic acid 47635172 2.559
37 17.739 Hexadecanoic acid, ethyl ester 42916698 2.306
38 18.130 Hexyl(trimethyl)silane 7164569 0.385
39 18.886 Phytol 11829108 0.636
40 19.056 Linoelaidic acid 47916166 2.574
41 19.585 Octadecanoic acid, ethyl ester 2675125 0.144
42 20.231 Isolongifolol 7865272 0.423
43 28.571 Stigmasterol 33849418 1.819
44 29.362 3H-3a,7-Methanoazulene, 2,4,5,6,7,8-hexahydro-1,4,9,9-tetramethyl-, 33499388 1.800
45 29.654 4-Methyl-2-trimethylsilyloxy-acetophenone 19417777 1.043
46 29.800 1,4-Dimethyl-8-isopropylidenetricyclo[5.3.0.0(4,10)]decane 49452870 2.657
47 30.112 4-Dehydroxy-N-(4,5-methylenedioxy-2-nitrobenzylidene)tyramine 19238008 1.034
48 30.633 2-(Acetoxymethyl)-3-(methoxycarbonyl)biphenylene 19840482 1.066
49 30.829 2,4-dimethylbenzo[h]quinoline 18185091 0.977
50 31.614 Benzo[g]pteridin-4(3H)-one, 6,7,8,9-tetrahydro- 30888689 1.660
51 32.028 2-Nitro-4-(trifluoromethyl)phenol 41702434 2.241
52 32.324 1,3,4-Trimethyl-6-cyclohexylbenzene 60248563 3.237
53 32.494 Taraxasterol 41985363 2.256
54 33.080 Ursolic aldehyde 32160264 1.728
55 33.378 Hexamethylcyclotrisiloxane 11199989 0.602
56 33.511 2,4-Dimethylbenzo[h]quinoline 11932089 0.641
57 33.627 N-Methyl-1-adamantaneacetamidet 16886466 0.907
58 33.971 1H-Indole, 1-methyl-2-phenyl- 13943280 0.749
59 34.150 Uvaol 38698032 2.079
60 35.927 Uvaol 30791918 1.654
61 36.530 2'-Hydroxy-5'-methylacetophenone, TMS derivative 20006348 1.075
62 37.457 N-Methyl-1-adamantaneacetamide 14381084 0.773
Table 2. Binding affinity of each compound in Nk-EE to 5α- reductase 2.
Table 2. Binding affinity of each compound in Nk-EE to 5α- reductase 2.
Compound Max binding affinity
b-D-Glucopyranose, 4-O-b-D-galactopyranosyl- -8.5
1,3.4-Trimethyl-6-cyclohexylbenzen -8.3
b-Santalol -7.2
Linoelaidic acid -7.2
2-Nitro-4-(trifluoromethyl)phenol -7.1
(3-Nitro-2-oxo-2H-pyridin-1-yl)-acetic acid -7
4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol -6.9
n-Hexadecanoic acid -6.5
Hexadecanoic acid, ethyl ester -6.2
hydroxylamine -3.2
Finasteride -10.1
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Copyright: This open access article is published under a Creative Commons CC BY 4.0 license, which permit the free download, distribution, and reuse, provided that the author and preprint are cited in any reuse.
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