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The hair growth promoting effect and action mechanism of 4-O-methylhonokiol from Magnolia officinalis

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Alternative Title
후박에서 분리한 4-O-methylhonokiol의 육모 효능 및 작용기전
loss, regardless of its type, is a common and distressing phenomenon. Recently, there has been increasing number of people suffering from hair loss or thinning. It is very important to develop new therapeutic materials. To prevent hair loss and to enhance hair growth, therefore, we screened the extracts of plants that have traditionally been used in oriental medicine and discovered that Magnolia officinalis (M. officinalis) potentially promoted the hair growth among them. M. officinalis has been used as a traditional remedy in China and Japan for treatment of gastrointestinal disorders, anxiety and allergic diseases including bronchial asthma. There have been many pharmacological reports of the activities of extracts or constituents from the bark of M. officinalis such as anti-inflammatory, antiallergic, antibacterial, and neurite spouting activities.
In the present study, we investigated the promotion effect of M. officinalis as well as 4-O-methylhonokiol, a neolignan compound from M. officinalis, on the growth of hair. When rat vibrissa follicles were treated with a EtOH extract of M. officinalis, the hair-fiber lengths of the vibrissa follicles increased significantly. In particular, 4-O-methylhonokiol, a principal of the extract, also increased the hair-fiber lengths of vibrissa follicles. In addition, after daily topical application of 4-O-methylhonokiol onto the back of C57BL/6 mice, anagen progression of the hair shaft was induced. Moreover, 4-O-methylhonokiol increased both the expression of proliferating cell nuclear antigen (PCNA) in the bulb region and proliferation of immortalized vibrissa dermal papilla cells. In order to determine the mechanism by which 4-O-methylhonokiol promotes hair growth, we examined the expression levels of transforming growth factor-β1 (TGF-β1) and TGF-β2 which have been known to play an important role in anagen to catagen transition via the induction of keratinocyte apoptosis. When the vibrissa follicles in the anagen phase were treated with 4-O-methylhonokiol for 7 days, the expressions of TGF-β1 in cells of outer root sheath (ORS) and epithelial strand, i.e., in the epithelial residue of the regressing hair bulb during catagen were found to be lower than those of the control follicles that were expected to be in the anagen-catagen transition phase. Also, 4-O-methylhonokiol decreased the expression of TGF-β2 in the bulb matrix region of the 7-day cultured follicles. These results suggest that 4-O-Methylhonokiol has the potential to promote hair growth via down regulation of TGF-β1 and TGF-β2, as well as the proliferation of dermal papilla.
To investigate how the 4-O-methylhonokiol could inhibit the TGF-β1-induced keratinocyte growth arrest, human keratinocyte HaCaT cell was used. When HaCaT cells were pretreated with 4-O-methylhonokiol, the TGF-β1-induced p21 expression was decreased. Moreover, 4-O-methylhonokiol inhibited nuclear translocation of smad2/3 and smad4 and Sp1activation by TGF-β1. We observed that ERK activation by TGF-β1 was significantly attenuated by treatment with 4-O-methylhonokiol. On the other hand, TGF-β has been reported to increase reactive oxygen species (ROS) intracellular content in different cell types as well as the effects of TGF-β on the cell growth arrest and apoptosis have been kwon to be mediated by oxidative stress. 4-O-Methylhonokiol inhibited TGF-β1-induced ROS production and suppressed mRNA expression of NOX4. These results indicate that 4-O-methylhonokiol could inhibit TGF-β1-induced cell growth arrest through down regulation of smad2/3, smad4, and NOX4 in Human keratinocyte HaCaT cell.
탈모인구는 해마다 계속 증가하고 있으며, 최근에는 중년 남성뿐만 아니라 젊은 층과 여성층에서도 탈모가 보편적으로 나타나고 있다. 특히 유전적인 요인, 호르몬의 과다, 정신적 스트레스, 자가면역질환 및 화학요법 등이 탈모에 관여하는 것으로 보고되고 있다. 그런 까닭에 모발 소실 방지 및 모발 성장 증진을 위한 새로운 치료물질을 개발하는 것은 무엇보다 중요하다. 새로운 탈모치료제를 찾기 위한 일한으로 천연물에서 유래하는 물질을 이용하여 육모효능을 검색 한 결과 후박 추출물이 탁월한 육모 효능을 확인 할 수 있었다. 후박은 중국 및 일본에서 전통적으로 위장질환, 불안장애 및 천식을 포함한 알레르기 질환 치료에 이용 되고 있다. 후박추출물 및 그 성분은 antiinflammatory, antiallergic, antibacterial 및 neurite spouting activity등 많은 약리활성이 보고 되고 있다.
본 연구에서 후박추출물 및 후박추출물의 성분인 4-O-metylhonokiol의 육모 효능에 대해 조사하였다. Rat vibrissa 모낭에 후박 추출물을 처리하였을 때, vibrissa 모낭의 hair fiber 길이는 유의성 있게 증가 하였다. 특히 후박추출물의 성분인 4-O-methtylhonokiol 역시 vibrissa 모낭의 hair fiber 길이를 증가 시켰다. 게다가 C57BL/6 마우스에 4-O-methtylhonokiol을 도포한 경우, 성장기 모낭을 유도하였다. 그리고 4-O-methtylhonokiol은 bulb region에서 PCNA 발현과 immortalized vibrissa dermal papilla 세포 증식을 증가 시켰다. 4-O-methtylhonokiol의 육모 효능작용기전을 알아보기 위해, keratinocyte apoptosis 유도에 의한 anagen에서 catagen 전이시 중요한 역할을 하는 TGF-β1과 TGF-β2의 발현을 조사하였다. 성장기 모낭에 4-O-methtylhonokiol를 처리하여 7일간 배양 후 TGF-β1과 TGF-β2의 발현을 조사 한 결과, TGF-β1은 대조군 보다 outer root sheat (ORS)와 epithelial strand에 약하게 발현되는 것을 관찰되었다. 또한 4-O-methylhonokiol을 처리한 7일째 모낭의 bulb matrix region에서는 TGF-β2 발현이 감소하였다. 이와 같은 결과는 TGF-β1과 TGF-β2의 down-regulation과 dermal papilla 세포 증식을 통하여 4-O-methtylhonokiol이 모낭 성장을 촉진한다고 생각됩니다.
다음은 TGF-β1에 의해 유도된 keratinocyte growth arrest을 4-Omethtylhonokiol의해 어떻게 억제 할 수 있는지 조사 하였습니다. HaCaT cell에 4-O-methtylhonokiol을 전처리 하였을 때, TGF-β1에 의해 유도되는 cell cycle arrest가 감소되는 것을 확인하였다. Cell cycle arrest와 밀접한 관계가 있는 p21 발현에 4-O-methylhonokiol의 효과를 알아본 결과, 4-O-methylhonokiol처리에 의해 TGF-β1에 의해서 유도된 p21 발현은 감소하였다. TGF-β1에 의한 p21 발현은 Smad pathway와 non-Smad pathway를 통하여 일어난다. TGF-β1에 의해서 유도되는 Smad2/3, Smad4의 nuclear translocation와 Sp1 활성에 4-O-methylhonokiol의 효과를 알아본 결과, 4-O-methylhonokiol은 TGF-β1에 의해서 유도된 Smad2/3, Smad4의 nuclear translocation와 Sp1 활성을 억제하는 것을 확인하였다. 또한 TGF-β1에 의해 유도되는 ERK 활성에 4-O-methylhonokiol이 효과를 알아본 결과, 4-Omethylhonokiol를 처리하였을 때 ERK 활성이 감소되는 것을 확인 할 수 있었다. 한편, TGF-β는 다양한 세포에서 ROS 생성을 증가시키고, 증가된 ROS는 MAPKs를 활성화시켜 growth arrest 및 apoptosis에 관여한다고 알려져 있습니다. TGF-β1에 의해 유도되는 ROS 증가 및 NOX4 mRNA 발현을 4-O-methylhonokiol 처리에 의해 감소하는 것을 확인 할 수 있었다. 따라서 4-O-methylhonokiol은 Smad2/3, Smad4 및 NOX4의 down regulation함으로써 TGF-β1에 의해 유도되는 cell cycle arrest를 막을 수 있을 것으로 사료됩니다.
Issued Date
Awarded Date
2010. 2
Alternative Author(s)
Kim, Sang Cheol
제주대학교 대학원
대학원 의학과
Table Of Contents
Ⅰ. Introduction 1
Ⅱ. Materials and Methods 18
1. Reagents 18
2. Isolation of 4-O-methylhonokiol from Magnolia officinalis 18
3. Animals 20
4. Cell cultures 20
5. Isolation and culture of rat vibrissa follicles 21
6. Hair growth activity in vivo 22
7. Hematoxylin and eosin staining 23
8. Immunohistochemistry 23
9. MTT Assay 24
10. RNA preparation and RT-PCR 25
11. Cell cycle analysis 26
12. Measurement of intracellular reactive oxygen species (ROS) 27
13. Superoxide Dismutase (SOD) Activity 28
14. Catalase (CAT) Activity 29
15. Assay of rat prostatic 5α-reductase 29
16. Western blot analyses 31
17. Statistical analyses 32
Ⅲ. Results 36
1. The Hair Growth Promoting Effect of 4-O-methylhonokiol from Magnolia officinalis 36
1.1 The effect of M. officinalis extract on rat vibrissa follicle elongation 36
1.2. The effect of 4-O-methylhonokiol from M. officinalis on the promotion of hair-growth 39
1.3. The effect of 4-O-methylhonokiol on anagen induction in C57BL/6 mice 42
1.4. The effect of 4-O-methylhonokiol on cell proliferation of hair follicles 45
1.5. Inhibitory effect of 4-O-methylhonokiol on the activity of prostatic 5α-reductase 49
1.6. Proliferation effect of 4-O-methylhonokiol on cultured NIH3T3 fibroblast cells 51
1.7. The effect of 4-O-methylhonokiol on TGF-β1 and TGF-β2 expression in rat vibrissa follicles 53
2. Inhibitory Effect of 4-O-methylhonokiol from Magnolia Officinalis on TGF-β1-induced Cell Cycle Arrest in a Human Keratinocyte Cell Line (HaCaT) 57
2.1. 4-O-methylhonokiol blocked TGF-β1-induced G1 arrest in HaCaT cell 57
2.2. 4-O-methylhonokiol inhibited TGF-β1-induced p21 expression in HaCaT cell 59
2.3. 4-O-methylhonokiol inhibited nuclear translocation of Smads by TGF-β 1 61
2.4. 4-O-methylhonokiol inhibited TGF-β1-induced Sp1 activation in HaCaT cell. 63
2.5. Effect of MAPK inhibitors on TGF-β1-induced p21 expression in HaCaT cells 65
2.6. Effect of 4-O-methylhonokiol on TGF-β1-induced MAPKs activation in HaCaT cells 67
2.7. Effect of 4-O-methylhonokiol on the TGF-β1-induced ROS production in HaCaT cells 70
2.8. Effect of 4-O-methylhonokiol on enzymatic and non-enzymatic antioxidant systems 73
2.9. Effect of 4-O-methylhonokiol on the TGF-β1-induced NADPH oxidase NOX4 in HaCaT cells 74
Ⅳ. Discussion 76
Ⅴ. Reference 86
Ⅵ. Abstract in Korean 104
제주대학교 대학원
김상철. (2010). The hair growth promoting effect and action mechanism of 4-O-methylhonokiol from Magnolia officinalis
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