1. INTRODUCTION:

Hair undergoes the process of growth and loss under the influences of environmental and lifestyle factors, climate change, psychological or physical stress, diseases, and hormones. Hair protects the skin from external stimuli and protects the head from cold or hot temperatures, physical shock, and ultraviolet rays.

Unhygienic scalp conditions and exposure to chemicals can accelerate hair thinning that can lead to alopecia, where the growth phase of hair becomes shorter.¹

Alopecia refers to hair loss at areas where hair should normally grow.² The general causes of alopecia include genetics, poor circulation, malnutrition, disease, fever, dandruff, environment, stress, anemia, and hormones. Androgenic alopecia or male pattern alopecia, the most common type of alopecia that occurs in men following puberty, is reported to occur due to hormonal and genetic influences.³

Androgenic alopecia causes local thinning, shortening, and eventually diffuse hair loss of hair in the middle of the scalp (frontal and parietal regions). As people who are genetically predisposed to androgenic alopecia age, testosterone in the scalp gets converted to dihydrotestosterone (DHT) by 5α-reductases to delay protein synthesis by hair follicle cells, and as a result, growing hair follicles enter the resting phase much earlier, leading to hair thinning and visible hair loss.^4-5

5α-reductases inhibitor and finasteride are commonly used to treat androgenic alopecia. 5α-reductasesexists in two forms in males. Type I 5α-reductases is distributed more in the sweat glands, sebaceous glands, keratinocytes, and fibrocytes, while the type II isoenzyme exists in the inner root sheaths of the urinary prostate, hair follicles, seminal vesicles, and epididymismainly.^6-7Finasteride selectively inhibits type II 5α-reductases to reduce the conversion of testosterone to DHT, and thereby induces hair growth. However, for patients who respond little or do not respond to 5α-reductases inhibitors at all, it is necessary to consider that the side-effects of finasteride include reduced sexual function and high risk of prostate cancer according to the FDA.⁸Research on safer and more effective natural medications of androgenic alopecia are currently needed.

In Korean medicine, androgenic alopecia has been mentioned as doklak, jeondok, and baljutalbal, and hyeolhur, shineumhur, pungyeol, ohyol, food, and psychological factors are considered as causes of androgenic alopecia.⁹Numerous studies have reported that the therapeutic effects of Korean medicine treatments for androgenic alopecia have been excellent.^10-12

The SM-215 used in this experiment consists of Bockboonja, Omija, baekjain, jimo, maekmundong, hyunsam, sanggisaeng, baekbugeun and pagoji. Since it has been previously reported that these natural products have significantly improve hair growth through hair follicle stimulation¹³, we hypothesized that they would also be effective for androgenic alopecia.

The effects of SM-215 on external morphology, alleviation of androgen-induced hair follicle damage, regulation of androgen-induced dermal inflammation, and the activation of resting hair follicles were investigated using immunohistochemical staining and a TUNEL assay.

2. MATERIALS AND METHODS:

2.1. Experimental animals:

Male C3H/HeNSic (10 weeks old) mice were used for this study(Orient Bio, Korea) and stable in a standard laboratory environment. They consisted of a control group and an aging-elicited group (AE group), no treatment was received in the control group. Testosterone was injected (Catalent France) at 30 mg/kg into the mice after shaving them. In the SM-215 treatment group (ST group), the samples were treated with SM-215 (30 mg/kg/day) for 5 weeks after shaving after 2 weeks.

The study was approved by the Semyung University Institutional Animal Care and Use Committee (smecac 16-05-02). Laboratory animals were managed and used in accordance with NIH guidelines.

2.2. Shaving:

The dorsal hair of all groups were removed using hair removal cream and an electric shaver.

2.3. Preparation of SM-215 extract:

To prepare SM-215 extract, 750 g of SM-215 (table 1) mixed 2 L of distilled water was put into a round flask and boiled at 100°C for 2 hours. After that, it was concentrated using a rotator evaporator (Eyela, Japan) and was freeze-dried to obtain 40.8 g of a SM-215 powder.

Table 1.The amount and composition of SM-215.

Name of Herbs	Pharmacognostic Name	Weight(g)
Pagoji	Psoraleacorylifolia L	100
Baekbugeun	Stemona japonica (Bl.) Miq.	100
Sanggiseang	Loranthusparasiticus(L) Merr.	100
Macmoondong	Liriopeplatyphylla Wang et Tang	75
Hyunsam	ScrophulariabuergerianaMiquel	75
Jimo	AnemarrhenaasphodeloidesBunge	75
Baekjain	ThujaorientalisLinne	75
Omija	SchizandrachinensisBaillon	75
Bockboonja	RubuscoreanusMiquel	75
Total		750

2.4. Measurement of the effects of SM-215 on androgenetic alopecia:

2.4.1. Preparation of femur tissue samples:

The mice were anesthetized with ether and all skin removed, which was then placed in 10% NBF at 36.5°C for 24h. The tissue samples were treated with decalcification solution for 12 h and were then embedded in paraffin and sliced into 5-µm-thick films.

2.4.2. Immunohistochemistry:

Skin fragments were proteolytically digested for 5 minutes in proteinase K (20 µg/ml) and then blocked by blocking 10% normal goat serum for 2 h. The samples were then mixed with one of the following primary antibodies at 4°C for 72 h in a humidified chamber: mouse anti-SREBP-1 (1:500, Santa Cruz Biotech), mouse anti-SCAP (Santa Cruz Biotech,1:50), mouse anti-p-JNK (Santa Cruz Biotech, 1:200), mouse anti-p-IkB (Santa Cruz Biotech, 1:200), mouse anti-CD34 (Santa Cruz Biotech, 1:50), mouse anti-SHH (Santa Cruz Biotech, 1:100), and mouse anti-IGF-1 (Santa Cruz Biotech, 1:100). The secondary antibody, biotinylated goat anti-mouse IgG (DAKO, 1:100), was applied at 37°C for 24 h and reacted with the avidin-biotin complex kit (VectorLabs) at room temperature for 1 hour. Finally, after staining with 0.05M tris-HCl buffer (pH 7.4) containing 0.05% 3,3'-diaminobenzidine and 0.01% HCl, the cells were stained with hematoxylin.

2.4.3. TUNEL assay:

Terminal deoxynucleotidetransferase-mediated dUTP-biotin nick-end labeling (TUNEL) was performed using the ApopTag® Peroxidase In Situ Apoptosis Detection Kit(Apoptag, Intergen, USA). First, tissue slices were subjected to proteolysis by proteinase K for 5 min and treated in an equilibration buffer for 20 s. Next, they were treated with strength TdT enzyme (36 µl TdT enzyme:72 µl reaction buffer) in a humidified chamber for 1 hat 37°C, and treated in a strength stop/wash buffer for 10 minutes. Then the tissue slices were reacted with anti-digoxigenin-peroxidase for 1 h, followed by DAB. Eosin was used for contrast. The tissue slices were observed using an optical microscope.

2.5. Image analysis and statistical analysis:

Immunohistochemistry and TUNEL assay results were expressed as numbers (means ± standard error) following image analysis using Image-Pro Plus (Media cybernetics, USA). After obtaining images of randomly selected skin tissues from each sample at 200X magnification, image analysis was performed to quantify the positive pixels/20 000 000 pixels. Statistical analysis was performed by using SPSS 20.0 software (SPSS Inc., USA), and statistical significance was tested by one-way ANOVA (P<0.05).

3. RESULTS:

3.1. External morphology:

In the ST group, A larger amount of hair was seen in the back in comparison with the AE group using the naked eye in figure 1.

3.2. Alleviation of androgen-induced hair follicle damage by SM-215:

Immunohistochemical staining was performed to determine the effects on alleviation of androgen-induced hair follicle damage of SM-215. The SPREBP-1-positive reaction significantly reduced by 33% in the ST group relative to the AE group. In addition, the SCAP-positive reaction significantly reduced by 76% in the ST group relative to the AE group.

The TUNEL assay, apoptosis significantly reduced by 66% in the ST group relative to the AE groupin figure 2.

Figure 1.Differences in external morphology

3.3. Regulation of androgen-induced dermal inflammation by SM-215

Immunohistochemical staining was performed to carry out the effects of SM-215 on the regulation of androgen-induced dermal inflammation. The p-JNK-positive reaction significantly reduced by 30% in the ST group relative to the AE group, and significantly reduced by 36% in the ST group relative to the AE group. The SPTLC-2-positive reaction significantly reduced by 66% in the ST group relative to the AE groupin figure 3.

3.4. Activation of resting hair follicles by SM-215

Immunohistochemical staining was performed to investigate the effects of SM-215 on the activation of resting hair follicles. The CD34-positive, SHH-positive, and IGF-1-positive reaction significantly increased by 356%, 315%, and 289% in the ST group relative to the AE group, respectively in figure 4.

4. DISCUSSION:

A normal hair growth cycle consists of the growth, lasting 3–5 years, cessation, lasting3 weeks and rest phaseslasting3 months respectively. New hair forms after the resting phase. In the case of normal hair, hair papilla cells undergo cell division during the formation of new hair from resting hair, or new hair papilla cells are produced from hair root sheaths. Inhibition of the production of hair papilla cells by androgens can lead to the thickening of newly formed hair, shortening of the normal hair growth cycle, and eventually androgenic alopecia.^14-17

Androgenic alopecia is gradual hair loss at the frontal head caused by androgens that is particularly common in men who are genetically predisposed. Hair loss does not occur before puberty; it begins as men reach their 20s after puberty, during which androgens are actively released.¹⁸

In general, when blood testosterone, a type of androgen, enters hair papilla cells through capillaries, it gets converted to the even more potent androgen DHT by 5α-reductase present within the hair papilla cells. DHT binds to androgen receptors and moves to the nucleus, where it regulates the expression of genes that respond to androgens. Androgen activity is locally amplified after 5α-reductase converts testosterone into DHT, and the effects of androgens are even more evident in hair follicles located in regions where hair loss is taking place. This is because 5α-reductase exists in large amounts in these regions, where they produce a profuse amount of DHT, contrary to aromatase that converts testosterone to estrogen, a female hormone.^19-20Androgens have a paradoxical role in hair growth. While hair in other regions of the body, including the beard, grow upon stimulation from androgens, hair in the frontal head and the crown of the head become thin and turn into soft hair in men genetically predisposed to androgenic alopecia.^21-22Therefore, when the levels of DHT and other androgens increase in the hair follicles, hair begins to thin, resulting in hair loss.

Medications for androgenic alopecia include finasteride and dutasteride. They act on 5α-reductase to inhibit DHT production.^23-24 However, they cannot be used in patients who respond little or do not respond at all to 5α-reductase. They can also give rise to side-effects such as reduced sexual function and high-risk prostate cancer, according to the FDA⁸. Safer and more effective natural medications need to be developed.

The SM-215 used in this study is a prescription consisting entirely of medicinal herbs. Park et al. have previously reported that SM-215 has a significant effect on hair growth through hair follicle stimulation.¹³Therefore, we hypothesized that it would also be effective against androgenic alopecia.

Male C3H/HeNSic (10 weeks of age) mice were separated into the untreated control group, the AE group treated with DHT, and the ST group treated with SM-215. The effects of SM-215 on external morphology, alleviation of androgen-induced hair follicle’s damage, regulation of androgen-induced dermal inflammation, and the activation of resting hair follicles were assessed.

First, a larger amount of hair was observed in the ST group than in the AE group in our external morphological analysis, indicating that SM-215 is effective against androgenic alopecia.

SREBP-1- and SCAP-positive reactions were reduced in the ST group in comparison with the AE group. In the TUNEL assay, apoptosis was decreased in the ST group in comparison with the AE group.

When DHT that is converted from testosterone by 5α-reductase binds to AR, the expression of SCAP increases, and SCAP binds SREBP inside the nuclear membrane and endoplasmic reticulum to move to the Golgi apparatus. This induces ER stress, which affects the expression of caspase-3 and caspase-12, and induces apoptosis.^25-26 Therefore, reduced SREBP-1- and SCAP-positive reactions at hair follicles indicate that apoptosis induced by ER stress was inhibited. This was confirmed by the TUNEL assay results that revealed that apoptosis was reduced. The results demonstrate that SM-215 reduces apoptosis at hair follicles damaged by androgens to inhibit androgenic alopecia.

In immunohistochemical staining performed to investigate the ability of SM-215 to regulate androgen-induced dermal inflammation, p-JNK-, p-IkB-, and SPTLC-2-positive reactions were decreased in the ST group relative to the AE group. The scalp is skin tissue that protects the head. It has well-developed hair roots, and large and small sweat glands, and it protects the brain from external shock or pressure. It is also associated with the excretion of heavy metals from the body and hair production. Physiological causes of scalp damage include contamination around pores and the scalp by secretions such as sebum, sweat, and dead skin cells. These secretions not only prevent the scalp from breathing, but also block pores to inhibit absorption of nutrients from external sources, inhibit new hair growth, and contribute to the propagation of germs, leading to increased dandruff, red spots, inflammation, and hair loss.²⁷

SPTLC-2 is an enzyme that catalyzes the first stage of sphingolipid biosynthesis.^28-29 Increased levels of SPTLC-2 indicate increased lipids in the scalp and are also closely associated with inflammation. Immunohistochemical staining was performed to determine the effects of SM-215 on the regulation of dermal inflammation, revealing that SM-215 reduced p-JNK-, p-IkB-, and SPTLC-2-positive reactions. This indicates that SM-215 inhibits androgenic alopecia through its involvement in mechanisms that reduce scalp inflammation.

Lastly, in the analysis of the SM-215 effects on the activation of resting hair follicle cells, SM-215 increased CD-34, SHH-, and IGF-1-positive reactions in the ST group relative to the AE group.

CD34 is a glycosylated cell surface protein expressed in endothelial and immature hematopoietic cells. In mice hair follicles, CD34 was found to be a subset of bulge keratinocytes with the characteristics of stem cells³⁰. In addition, SHH is a protein that constitutes the HH signaling pathway and is involved in cell differentiation and proliferation.^31-32 Therefore, increased CD-34- and SHH-positive reactions at resting hair follicles indicate that SM-215 activated resting hair follicles, indicating that SM-215 can inhibit androgenic alopecia through this mechanism.

In general, cell proliferation and differentiation are mediated by various cytokines. Cytokines associated with hair growth include IGF, VEGF, PRL, and PL. These factorsinduce the proliferation of vascular system, extracellular matrix, and dermal papillaand increase the amount of the extracellular matrix within dermal papilla in the maintenance of growing hair follicles. IGF-1, which is produced from dermal papilla, promotes the growth of hair follicles and epithelial cells, and prevents apoptosis of hair follicles to accelerate hair growth.³³ Based on the increased levels of IGF-positive reactions following SM-215 treatment in the present study, it is reasonable to assume that SM-215 activates IGF-1, thereby promoting the growth of hair follicles and epithelial cells, and preventing hair follicle apoptosis, in the inhibition of androgenic alopecia.

Based on the abovementioned results, SM-215 reduces androgen-induced apoptosis at hair follicles and scalp inflammation, activates resting hair follicle cells, and promotes the growth of epithelial cells around hair follicles. Therefore, it may be suggested to be used as a natural medication that has no side-effects and is effective against androgenic alopecia.

5. CONCLUSION:

To examine the effects of SM-215 on androgenic alopecia, which was previously reported to effectively induce hair growth by hair follicle stimulation, experiments were performed that assess external morphology, hair follicle damage, dermal inflammation, and resting hair follicles. The following results were observed:

1. Growth of a larger number of dorsal hairs was visible following SM-215 treatment.

2. SM-215 significantly reduced SREPB-1- and SCAP-positive reactions in hair follicles. In a TUNEL assay, SM-215 significantly reduced apoptosis.

3. SM-215 significantly reduced p-JNK-, p-IkB-, and SPTLC-positive reactions.

4. SM-215 significantly increased CD34-, SHH-, and IGF-1-positive reactions.

Based on these results, SM-215 alleviates hair follicle damage caused by androgens, reduces scalp inflammation, activates resting hair follicles, and induces the growth of epithelial cells around hair follicle cells; therefore, it can be considered effective against androgenic alopecia.

6. ACKNOWLEDGMENT:

This study was conducted with School Research Funds (2016) granted by Semyung University.

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Received on 12.12.2017 Modified on 15.01.2018

Research J. Pharm. and Tech 2018; 11(5): 1745-1751.

DOI: 10.5958/0974-360X.2018.00324.4