Effects of Topical application of Astragalus membranaceus in Spontaneous Alopecia Mice Model

Article information

J Korean Med. 2018;39(1):1-12

Publication date (electronic) : 2018 March 31

doi : https://doi.org/10.13048/jkm.18001

Hyeok Je Kwon

, Mi Hye Kim

, Woong Mo Yang

Department of Convergence Korean Medical Science, College of Korean Medicine, Kyung Hee University

Correspondence to: 양웅모 (Woong Mo Yang), 서울시 동대문구 회기동 1번지 경희대학교 한의과대학, Tel:+82-2-961-2209, E-mail:wmyang@khu.ac.kr

Received 2017 November 21; Revised 2017 December 14; Accepted 2018 January 17.

Abstract

Objectives

Astragalus membranaceus has been reported to inhibit immune responses, but its effect on hair loss is not clear. In this study, the effect of A. membranaceus extract (AM) on hair regrowth in C57BL/6 mice with natural hair loss in the telogen phase was investigated.

Methods

Mice with natural hair loss were topically treated with 1% AM on the dorsal skin for 2 weeks. Dorsal skin samples were stained with hematoxylin and eosin and probed with an anti-mouse CD8a IgG. The mRNA expression levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-4 were measured by reverse transcription polymerase chain reaction and quantitative real-time polymerase chain reaction.

Results

AM treatment induced hair regrowth in hair loss mice, while control mice suffered continued hair loss. Tapering hair shafts and broken hair follicles were decreased as well as CD8+ T lymphocyte infiltration. In addition, the expressions of TNF-α, IFN-γ and IL-4 were reduced by AM treatment. Also, AM treatment significantly increased the KGF expressions in Hs68 fibroblast cells.

Conclusion

These results suggest that topical application of A. membranaceus may be an alternative therapy for hair loss.

Keywords: Astragalus membranaceus; hair loss; hair follicle; T lymphocyte; cytokine

Fig. 1

Quality evaluation of Astragalus membranaceus. HPLC chromatograms of standard formononetin (A) and A. membranaceus extract (B). It takes 31.7 min for the retention times of formononetin itself and formononetin in A. membranaceus extract.

Fig. 2

A. membranaceus induces hair regrowth in hair loss mice. Morphological fingdings on the back of the C57BL/6J mice. The mice of A. membranaceus group were treated with 100 μ L of 1% (w/v) A. membranaceus for 14 days. Following challenge for 15 days, photographs were taken and mice were sacrificed. Normal, non-treated normal control mice; Control, vehicle-treated alopecia mice; AM, AM-treated alopecia mice.

Fig. 3

A. membranaceus recovers the dystrophy of hair follicles. Histological findings by hematoxylin and eosin (H&E) staining of dorsal skin sections (n = 7). The magnifications were × 100 (upper panel) and × 200 (lower panel). Arrow indicates ‘swarm of bees’. Arrowhead indicates tapering hair shaft. Normal, non-treated normal control mice; Control, vehicle-treated alopecia mice; AM, AM-treated alopecia mice.

Fig. 4

A. membranaceus reduces the infiltration of CD8+ cells in skin tissues. Immunohistochemical CD8⁺ cell staining. Arrow indicates brown-stained CD8⁺ cells. Normal, non-treated normal control mice; Control, vehicle-treated alopecia mice; AM, AM-treated alopecia mice.

Fig. 5

A. membranaceus decreases the mRNA expressions of cytokines in skin tissues. Expression of TNF-α, IL-4 and IFN-γ mRNA levels by RT-PCR (A) and qPCR (B). Results are presented as mean ± S.E.M. ^## and ^### indicates the mean differs significantly between Normal group and Control group (p < 0.01 and p < 0.001, respectively). ^*** indicates that the mean differs significantly between Control and AM group (p < 0.001).

Fig. 6

A. membranaceus increases the KGF expression in Hs68 fibroblast cells. Expression of KGF fluorescence by immunofluorescence staining (A) and protein level by Western blotting (B) in Hs68 fibroblast cells. Green, KGF. Blue, DAPI. Results are presented as mean ± S.E.M. ^*** indicates that the mean differs significantly between non-treated cells and AM-treated cells (p < 0.001).

References

1. Stenn KS, Paus R. Controls of Hair Follicle Cycling. Physiological Reviews 2001;81(1):449–94.

2. Min BG. Diagnosis and treatment of hair & scalp disorders. Hanmi Book 2005;:21–30.

3. Zoe DD. Cosmetics in dermatology 2ndth ed. London: Churchill Livingstone; 1995. p. 179–91.

4. Ahn SG, Ji HG, Hwang SM, Jung J, Jang KH. Common Skin Disease Pacific publisher; 2003. p. 437.

5. Korean Dermatology Association. Textbook of Dermatology 6Thth ed. Seoul: Med book; 2014. p. 551–7.

6. Lee SH, Lee JR. Association of Diffuse Hair loss of Adult Male on Stress, Self-confidence, and Depression. Journal of the Society of Cosmetic Scientists of Korea 2010;16(4):1171–9.

7. Fitzpatrick TB, Wolff K. Fitzpatrick’s dermatology in general medicine 7Thth ed. NewYork: McGraw-Hill; 2008. p. 766–9.

8. Moustafa AET, Hassan I, Essam AN, Mai SAD. Platelets rich plasma versus minoxidil 5% in treatment of alopecia areata: A trichoscopic evaluation. Dermatologic Therapy 2017;30(1):e12437.

9. Amin SS, Sachdeva S. Alopecia areata: A review. Saudi Society of Dermatology and Dermatologic Surgery 2013;17:37–45.

10. Blumeyer A, Tosti A, Messenger A, Reygagne P, Del Marmol V, Spuls PI, et al. Evidence-based (S3) guideline for the treatment of androgenetic alopecia in women and in men. Journal der Deutschen Dermatologischen Gesellschaft 2011;9(6):51–7.

11. Varothai S, Bergfeld WF. Androgenetic Alopecia: An Evidence-Based Treatment Update. American Journal of Clinical Dermatology 2014;15(3):217–30.

12. Levy LL, Emer JJ. Female pattern alopecia: current perspectives. International Journal of Women’s Health 2013;5:541–56.

13. Yamana K, Labrie F, Luu-The V. Human type 3 5α-reductase is expressed in peripheral tissues at higher levels than types 1 and 2 and its activity is potently inhibited by finasteride and Dutasteride. Hormone Molecular Biology and Clinical Investigation 2010;2:293–9.

14. Lee SY, Chun SW, Kim JB, No BI. A Case of Combination Therapy with Finasteride and Low Dose Dutasteride in the Treatment of Androgenetic Alopecia. Korean Journal of Dermatology 2017;55(2):147–8.

15. Mounsey AL, Reed SW. Diagnosing and treating hair loss. American Family Physician 2009;80(4):356–62.

16. Eun HC, Kwon OS, Yeon JH, Skin HS, Kim BY, Ro BI, et al. Efficacy, safety, and tolerability of dutasteride 0.5 mg once daily in male patients with male pattern hair loss: A randomized, double-blind, placebo-controlled, phase III study. Journal of the American Academy of Dermatology 2010;63:252–8.

17. Choi GS, Kim JH. Safety and Tolerability of the Dual 5-Alpha Reductase Inhibitor Dutasteride in the Treatment of Androgenetic Alopecia. Annals of Dermatology 2016;28(4):444–50.

18. Lee WS, Ahn HJ, Kim YH. The Effect of Coapplication of Capsaicin and Minoxidil on the Murine Hair growth. Korean Journal of Dermatology 2003;41(4):451–60.

19. Zappacosta AR. Reversal of baldness in patient receiving minoxidil for hypertension. The New England Journal of Medicine 1980;303:1480–1.

20. Fiedler-Weiss VC. Potential mechanisms of minoxidil-induced hair growth in alopecia areata. Journal of the American Academy of Dermatology 1987;16:653–6.

21. Messenger AG, Rundegren J. Minoxidil: mechanisms of action on hair growth. British Journal of Dermatology 2004;150:186–94.

22. Evelyn YC. Androgenetic alopecia (male pattern hair loss) in the United States: What treatments should primary care providers recommend? Journal of the American Association of Nurse Practitioners 2013;25(8):395–401.

23. Alkhalifah A, Alsantali A, Wang E, McElwee KJ, Shapiro J. Alopecia areata update. Part II: treatment. Journal of the American Academy of Dermatology 2010;62:191–202.

24. Olsen EA, Carson SC, Turney EA. Systemic steroids with or without 2% topical minoxidil in the tretment of alopecia areata. Archives of Dermatology 1992;128:1467–73.

25. Chiang KS, Mesinkovska NA, Piliang MP, Bergfeld WF. Clinical efficacy of diphenylcyclopropenone in alopecia areata: retrospective data analysis of 50 patients. Journal of Investigative Dermatology Symposium Proceedings 2015;17:50–5.

26. Ohtsuki A, Hasegawa T, Komiyama E, Takagi A, Kawasaki J, Ikeda S. 308-nm Excimer Lamp for the Treatment of Alopecia Areata: Clinical Trial on 16 Cases. Indian Journal of Dermatology 2013;58(4):326.

27. Munck A, Gavazzoni MF, Trüeb RM. Use of low-level laser therapy as monotherapy or concomitant therapy for male and female androgenetic alopecia. Canadian Medical Association Journal 2013;185(18):1579–85.

28. Fukuoka H, Suga H. Hair Regeneration Treatment Using Adipose-Derived Stem Cell Conditioned Medium: Follow-up With Trichograms. Eplasty 2015;15:e10.

29. Zawar VP, Karad GM. Liquid nitrogen cryotherapy in recalcitrant alopecia areata: a study of 11 patients. International Journal of Trichology 2016;8:15–20.

30. Khatu SS, More YE, Gokhale NR, Chavhan DC, Bendsure N. Platelet-rich plasma in androgenic alopecia: myth or an effective tool. Journal of Cutaneous and Aesthetic Surgery 2014;7(2):107–10.

31. Lee WG. Sinpyeonsomunjipju Seoul: Daeseongmunhwasa; 1994. p. 894.

32. Kwak AC. Hwangjenaegyeong yeongchugyojueoseok Busan: Iljungsa; 1993. 123p. 213.

33. Gal H. Galhongjuhubigeupbang Beijing: Inminwi-saengchulpansa; 1996. p. 196.

34. So WB. Sossijebyeongwonhuchongnon Shanghai: Soinchulpansa; 1980. p. 695–6.

35. Son SM. Bigeupcheongeumyobang Seoul: Deseongmunhwasa; 1984. p. 434–5.

36. Jin SG. Oegwajeongjong Beijing: Inminwisaengchulpansa; 1983. p. 256.

37. Oh G. Uijonggeumgam Seoul: Daeseongmunhwasa; 1983. p. 97.

38. Gao J, Liu H, Xie Y, Zhao H, Xie Z, Zhong Z. Clinical observation of the therapy of catgut embedding and moxibustion on androgenetic alopecia. Modern Journal of Integrated Traditional Chinese and Western Medicine 2013;22(1):8–15.

39. Lin KR, Jiang YF, Yu AS. Clinical observation of Chinese comprehensive therapy on androgenetic alopecia of damp and heat in the spleen and the stomach pattern. Shanghai Journal of Traditional Chinese Medicine 2014;48(8):53–6.

40. Yang SS. Junguiimsangdaejeon Seoul: Daeseongchulpansa; 1988. p. 912.

41. Chen GT. Siryongjungseouigyeolhapjindan-chiryohak Busan: Iljungsa; 1992. p. 1508.

42. Gu BK. Junguioegwahak Shanghai: Sanghaegwahakg-isulchulpansa; 1985. p. 309–12.

43. All oriental medicine university bonchohak teacher. Bonchohak Seoul: Yunglimsa; 2007. p. 576–8.

44. Kim SH, Jun YM, Lim JJ, Kim SH, Chung IM, Kim EH. Variation of Astragalosides Contents in Cultivated Astragalus membranaceus. Korean Journal of Medicinal Crop Science 2012;20(5):372–80.

45. Bai F, Makino T, Kono K, Nagatsu A, Ono T, Mizukami H. Calycosin and formononetin from astragalus root enhance dimethylarginine dimethylaminohydrolase 2 and nitric oxide synthase expressions in Madin Darby Canine Kidney II cells. Journal of Natural Medicines 2013;67(4):782–9.

46. Park CI. Study on Effects of Anti-oxidant and Viscoelastic on Emulsion by the Extract of Astragalus membranaceus. The Korea Journal of Herbology 2012;27(2):93–7.

47. Baek NI, Kim YS, Kyung JS, Park KH. Isolation of anti-hepatotoxic agent from the root of Astragalus membranacus. Korean Journal of Pharmacognosy 1996;27:111–6.

48. Hikino H, Funayama S, Endo K. Hypotensive principles of Astragalus and Hedysarum roots. Planta Medica 1976;30:297–302.

49. Zang YD, Wang YL, Shen JP, Li DX. Hypotensive and anti-inflammatory effects of Astragalus saponin 1. Acta Pharmacologica Sinica 1984;19:333–7.

50. Kim JH, Kim MH, Yang GS, Huh YB, Kim SH, Yang WM. Effects of topical application of Astragalus membranaceus on allergic dermatitis. Immunopharmacology and Immunotoxicology 2012;35(1):151–6.

51. Kim MH, Kim SH, Yang WM. Beneficial Effects of Astragaloside IV for Hair Loss via Inhibition of Fas/Fas L-Mediated Apoptotic Signaling. PLoS One 2014;9(3):e92984.

52. Paus R, Ito N, Takigawa M, Ito T. The hair follicle and immune privilege. Journal of Investigative Dermatology Symposium Proceedings 2003;8(2):188–94.

53. Ansel J, Perry P, Brown J. Cytokine modulation of keratinocyte cytokines. Journal of Investigative Dermatology 1990;94(6):1015–75.

54. Whicher JT, Evans SW. Cytokines in disease. Clinical Chemistry 1990;36:1269–81.

55. Philpott MP, Sanders DA, Bowen J, Kealey T. Effects of interleukins, colony-stimulating factor and tumour necrosis factor on human hair follicle growth in vitro: a possible role for interleukin-1 and tumour necrosis factor-α in alopecia areata. British Journal of Dermatology 1996;135(6):942–8.

56. Hoffmann R, Eicheler W, Huth A, Wenzel E, Happle R. Cytokines and growth factors influence hair growth in vitro: Possible implications for the pathogenesis and treatment of alopecia areata. Archives of Dermatological Research 1996;288:153–6.

57. Gilhar A, Etzioni A, Assy A, Eidelman S. Response of grafts from patients with alopecia areata transplanted onto nude mice, to administration of interferon-gamma. Clinical Immunology and Immunopathology 1993;66:120–6.

58. Sato-Kawamura M, Aiba S, Tagami H. Strong expression of CD40, CD54 and HLA-DR antigen and lack of evidence for direct cellular cytotoxicity are unique immunohistopathological features in alopecia areata. Archives of Dermatological Research 2003;294(12):536–43.

59. Freyschmidt-Paul P, McElwee KJ, Hoffmann R, Sundberg JP, Vitacolonna M, Kissling S, et al. Interferon-gamma-deficient mice are resistant to the development of alopecia areata. British Journal of Dermatology 2006;155(3):515–21.

60. Teraki Y, Imanishi K, Shiohara T. Cytokines in alopecia areata: contrasting cytokine profiles in localized form and extensive form (alopecia universalis). Acta Dermato-Venereologica 1996;76(6):421–3.

61. Attia EA, El Shennawy D, Sefin A. Serum Interleukin-4 and Total Immunoglobulin E in Nonatopic Alopecia Areata Patients and HLA-DRB1 Typing. Dermatology Research and Practice 2010;2010:1–6.

62. Hom DB. Growth factors in wound healing. Otolaryngologic Clinics of North America 1995;28(5):933–53.

63. Wemer S, Smola H, Liao X, Longaker MT, Krieg T, Hofschneider PH, et al. The function of KGF in morphogenesis of eithelium and reepithelialization of wounds. Science 1994;266:819.

64. Pierce GF, Yanagihara D, Klopchin K, Danilenko DM, Hsu E, Kenney WC, et al. Stimulation of all epithelial elements during skin regeneration by keratinocyte growth factor. The Journal of Experimental Medicine 1994;179:831–40.