ABSTRACT
Dihydrotestosterone (DHT) is the most potent male hormone that causes androgenetic alopecia. The type II 5alpha-reductase is an enzyme that catalyzes the conversion of testosterone (T) to DHT, therefore it can be expected that specific inhibitors for type II 5alpha-reductase may improve the pathophysiologic status of androgenetic alopecia. In this study, we attempted to establish the reliable and convenient screening model for type II 5alpha-reductase inhibitors. After transfection of human cDNA for type II 5alpha-reductase into HEK293 cells, the type II 5alpha-reductase over-expressing stable cells were selected by G418 treatment. RT-PCR and Western blot analyses confirmed that type II 5alpha-reductase gene was expressed in the stable cells. In in vitro enzymatic assay, 10 microg of stable cell extract completely converted 1 microCi (approximately 0.015 nmol) of T into DHT. The type II 5alpha-reductase activity was inhibited by finasteride in a dose-dependent manner, confirming the reliability of screening system. In cell culture condition, 2 x 10(5) of stable cells completely converted all the input T (approximately 0.03 nmol) into DHT by 4h incubation, demonstrating that the stable cell line can be used as a cell-based assay system. Using this system, we selected the extracts of Curcumae longae rhizoma and Mori ramulus as the potential inhibitors for type II 5alpha-reductase. These results demonstrate that the type II 5alpha-reductase over-expressing stable cell line is a convenient and reliable model for screening and evaluation of inhibitors.
Subject(s)
Cholestenone 5 alpha-Reductase/metabolism , Enzyme Inhibitors/pharmacology , Base Sequence , Blotting, Western , Cell Line , Cholestenone 5 alpha-Reductase/antagonists & inhibitors , DNA Primers , Humans , Models, Theoretical , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: Hair loss is a distressing condition for an increasing number of men and women. It is of great importance; therefore, to develop new therapies for the treatment of hair loss. OBJECTIVE: We examined the effects of 45 plant extracts that have been traditionally used for treating hair loss in oriental medicine in order to identify potential stimulants of hair growth. METHODS: Six-week-old female C57BL/6 and C3H mice were used for evaluating the hair growth-promoting effects of the plant extracts. Topical application onto the backs of the C57BL/6 and C3H mice was performed daily for 30 days and 45 days, respectively. Protein synthesis was measured by the cysteine uptake assay, using cultured murine vibrissae follicles. Proliferation of the immortalized human keratinocyte cell line (HaCaT) and human dermal papilla (DP) cells was evaluated by the MTT and thymidine incorporation assays. The mRNA levels of several growth factors that have been implicated in hair growth control were measured by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Among the tested plant extracts, the extract of Asiasari radix showed the most potent hair growth stimulation in C57BL/6 and C3H mice experiments. In addition, this extract markedly increased the protein synthesis in vibrissae follicle cultures and the proliferation of both HaCaT and human DP cells in vitro. Moreover, the A. radix extract induced the expression of VEGF in human DP cells that were cultured in vitro. CONCLUSION: These results suggest that the A. radix extract has hair growth-promoting potential, and that this effect may be due to its regulatory effects on both cell growth and growth factor gene expression.
Subject(s)
Alopecia/drug therapy , Hair Follicle/drug effects , Hair/drug effects , Plant Extracts/pharmacology , Vibrissae/drug effects , Animals , Cell Proliferation , Cell Survival , Cholestenone 5 alpha-Reductase/metabolism , Cysteine/metabolism , DNA Primers/chemistry , Female , Humans , Keratinocytes/metabolism , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Plant Preparations/pharmacology , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Skin/cytology , Skin/drug effects , Tetrazolium Salts/pharmacology , Thiazoles/pharmacology , Thymidine/chemistry , Time FactorsABSTRACT
In search of natural extracts for hair growth, we found that the extract of dried root of Sophora flavescens has outstanding hair growth promoting effect. After topical application of Sophora flavescens extract onto the back of C57BL/6 mice, the earlier conversion of telogen-to-anagen was induced. The growth of dermal papilla cells cultured in vitro, however, was not affected by Sophora flavescens extract treatment. RT-PCR analysis showed that Sophora flavescens extract induced mRNA levels of growth factors such as IGF-1 and KGF in dermal papilla cells, suggesting that the effects of Sophora flavescens extract on hair growth may be mediated through the regulation of growth factors in dermal papilla cells. In addition, the Sophora flavescens extract revealed to possess potent inhibitory effect on the type II 5alpha-reductase activity. Taken together, these results suggest that Sophora flavescens extract has hair growth promoting potential and can be used for hair growing products.
