Ann Dermatol.  2019 Oct;31(5):530-537. 10.5021/ad.2019.31.5.530.

RE-ORGA, a Korean Herb Extract, Can Prevent Hair Loss Induced by Dihydrotestosterone in Human Dermal Papilla Cells

• Kang MG
• Park D
• Han HY
• Shim H
• Hong Y
• Moon J
• Yoon S
• Kwon B

• KMID: 2456230
• DOI: http://doi.org/10.5021/ad.2019.31.5.530

Abstract

BACKGROUND
Androgenic alopecia (AGA) is the most common type of hair loss. It is likely inherited genetically and is promoted by dihydrotestosterone. 5α-reductase has been proven a good target through finasteride use. However, the pathogenesis of AGA cannot be fully explained based only on dihydrotestosterone levels.
OBJECTIVE
To identify similar hairloss inhibition activity of RE-ORGA with mode of action other than finasteride.
METHODS
We prepared RE-ORGA from Korean herb mixtures. We performed MTT assays for cytotoxicity, Cell Counting Kit-8 assays for cell proliferation, and western blot to identify expression levels of 5α-reductase and Bax. RNA-sequencing was performed for the expression patterns of genes in dihydrotestosterone-activated pathways. Anti-inflammatory activity was also assessed by the expression levels of tumor necrosis factor-alpha (TNF-α) and interleukin 6.
RESULTS
REORGA could promote the proliferation of human dermal papilla cells and showed low cytotoxicity. It also inhibited the expression of 5α-reductases and Bax in the cells. RNA-sequencing results verified that the mRNA expressions of SRD5A1, Bax, transforming growth factor-beta 1 (TGF-β1), and TGF-β1 induced transcript 1 (TGFβ1I1) were decreased, whereas expression of protein tyrosine kinase 2 beta (PTK2β) was more elevated. REORGA also showed anti-inflammatory activity through decreased mRNA levels of TNF-α.
CONCLUSION
Transcriptionally, up-regulation of PTK2β and concomitant down-regulation of TGFβ1I1 imply that RE-ORGA can modulate androgen receptor sensitivity, decreasing the expression of 5α-reductase type II and Bax together with TGF-β1 transcripts; RE-ORGA also showed partial anti-inflammatory activity. Overall, RE-ORGA is expected to alleviate hair loss by regulating 5α-reductase activity and the receptor's androgen sensitivity.

Keyword

Androgenic alopecia; Dihydrotestosterone; Finasteride; Herbal extract; 5 alpha-reductase

MeSH Terms

Alopecia
Blotting, Western
Cell Count
Cell Proliferation
Cholestenone 5 alpha-Reductase
Dihydrotestosterone*
Down-Regulation
Finasteride
Hair*
Humans*
Interleukin-6
Protein-Tyrosine Kinases
Receptors, Androgen
RNA, Messenger
Tumor Necrosis Factor-alpha
Up-Regulation
Cholestenone 5 alpha-Reductase
Dihydrotestosterone
Finasteride
Interleukin-6
Protein-Tyrosine Kinases
RNA, Messenger
Receptors, Androgen
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Viabilities of dermal papilla cells after treatment with RE-ORGA at various concentrations, as measured using an 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Optical densities were measured at a wavelength of 570 nm using a microplate reader and averaged from six replicates of cells seeded in 96-well plates at a cell density of 10,000 cells/well. MTT assays showed survival rates close to 100% for both cell types after treatment with RE-ORGA at a concentration below 20%.

• 

  Fig. 2 Cell proliferation in human dermal papilla (DP) cells after treatment with RE-ORGA at various concentrations. The highest number of proliferated DP cells was measured on day 5. Human DP cells proliferated most efficiently in groups treated with RE-ORGA at 1% and 5% on day 1 and in groups treated with RE-ORGA at 1%, 5%, and 10% on day 5. Growth rates of human DP cells after treatment with 10% RE-ORGA were similar to those after treatment with 20 ng/ml epidermal growth factor (EGF) on all days.

• 

  Fig. 3 (A, B) Western blot analysis of 5-α-reductase type II and Bax in human dermal papilla cells. Expression levels of 5-α-reductase type II decreased after addition of RE-ORGA. After treatment with 20% RE-ORGA, the expression level of 5-α-reductase type II dropped to 21.3% of that treated with 100 nM dihydrotestosterone (DHT) and 34.0% of that treated with 200 nM finasteride. Reduction in expression level of Bax was not higher than that of 5-α-reductase type II. However, treatment with 20% RE-ORGA resulted in decreased expression level of the Bax gene (86%) compared to treatment with 200 nM finasteride (78%).

• 

  Fig. 4 RNA-seq analysis of dihydrotestosterone (DHT)-stimulated dermal papilla (DP) cells treated with or without RE-ORGA. In DHT-stimulated cells, transforming growth factor-beta 1 (TGF-β1), glycogen synthatase kinase 3 beta (GSK3β), TGF-β1 induced transcript 1 (TGFβ1I1), and Bax genes were highly expressed. However, these genes are downregulated in DHT-stimulated DP cells treated with RE-ORGA. On the other hand, protein tyrosine kinase 2 beta (PTK2β), a negative regulator of androgen receptor, was upregulated, whereas 5-α-reductase type I and Bax were downregulated in DHT-stimulated DP cells treated with RE-ORGA. IL6: interleukin 6, DKK1: Dickkopf-1, CTNNB1: catenin beta-1.

• 

  Fig. 5 Anti-inflammatory activity of RE-ORGA in Mast cell-1. Tumor necrosis factor-alpha (TNF-α) (A) and interleukin 6 (IL6) (B) were chosen as biomarkers for inflammation. Levels of TNF-α (0.28±0.015) that were high after treatment with 40 nM phorbol-12-myristate 13-acetate plus calcium ionophore A23187 (PMACI) showed RE-ORGA-concentration dependent reduction. They decreased to 0.22±0.004 (79.2%) in the 10% RE-ORGA treatment group and 0.20±0.008 (72.4%) in the 20% RE-ORGA treatment group, showing a statistically significant difference compared to their levels in the 0% RE-ORGA treatment group. However, the maximum inhibition of IL6 level was observed in the 1% RE-ORGA treatment group (p-value=0.053). **p-value<0.01 compared with 0% Re-ORGA.

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