The Androgenic Alopecia Protective Effects Of Saw Palmetto Extracts And The Molecular Regulation In A Mouse Model

Androgenetic alopecia(AGA),also known as Seborrhoeica alopecia or male pattern alopecia,is the most common type of hair loss caused by androgen,which is characterized by progressive reduction of hair density,diminished hair follicles,shortened growing period,decreased ratio of growing phase/resting phase of hair follicles,and substitution of vellus hair for terminal hair.The pathogenesis remains unclear and it is generally believed that androgen,androgen receptor(AR)and 5 α-reductase type 2 play a pivotal role in its development.The dihydrotestosterone(DHT),isoform of testosterone,is an androgen derivate and one of the main materials which are the cause of AGA.DHT affects the growth of hair follicles by promoting premature entry into catagen,thereby causing hair loss.DHT promotes an alopecia mechanism relates to transforming growth factor(TGF)-β2,which inhibits hair growth by shortening the anagen phase and promoting a more rapid entry into the catagen phase.TGF-β2 not only inhibits hair growth by reducing anagen and promoting early entry into catagen but also increase the expression of caspase-9 and 3.TGF-F-β2 also influences the Bcl-2/Bax ration related to mitochondrial apoptosis.Finasteride is widely used agent that has obtained State Food and Drug Administration approval for promoting hair growth and preventing hair loss.However,side effects have recently reported because it uses.For this reason,natural materials are being studies as substitutes for synthetic products in order to avoid side effects and to allow sustainable use.The extract of mature dry fruit of Saw palmetto(SPE)is composed of multiple free fatty acids,which has been widely used to treat dysfunction of the urinary system caused by benign prostatic hyperplasia.Its efficacy as a noncompetitive inhibition of 5a-reductase(type 1 and type 2)and inhibition of DHT binding to the cytosolic androgen receptor,anti-inflammatory,anti-apoptosis,have recently been reported.However,the effectiveness of this compound as a hair loss treatment has not yet been investigated.In this study,we have examined SPE for its potential in preventing hair loss both in vitro and in vivo using hair cell cultures and the male C57BL/6 mouse model that has characterized androgenic alopecia,respectively.Morphological and histological findings verified that retardation of entry into catagen occurred in response to SPE administration.We also verified the effects of SPE treatment on protein expression related to androgenic alopecia in the back skin of the mice by western blot examination.Methods1.Culture of human keratinocytes and human hair dermal papilla cells.Different treatment was performed when cells grew adherently.Grouping and treatment of HaCaT cells:blank group:no treatment;DHT group:0.03μg/mL DHT culture solution was added;DHT + finasteride group:culture solution containing 0.03μg/mL DHT and 0.08μg/mL finasteride;DHT+ SPE group:culture solution containing 0.03μg/mL DHT and SPE(1μg/mL,5μg/mL,25μg/mL,100μg/mL or 200μg/mL)was added respectively.Grouping and treatment of HHDPC cells:blank group:no treatment;DHT group:0.03μg/mL DHT culture solution was added;DHT+ finasteride group:culture solution containing 0.03μg/mL DHT and 0.08μg/mL finasteride;DHT +SPE group:culture solution containing 0.03μg/mL DHT and SPE(1μg/mL,5μg/mL,25μg/mL or 100μg/mL)was added respectively.The MTT assay was used to determine the cell proliferation rate.2.Experiment animals2.1 Establishment of androgenic alopecia mouse models and sampling.The mice were divided into four groups randomly with eight for each group:blank group(A),DHT group(B),DHT+ finasteride group(C)and DHT+SPE group(D).Androgenic alopecia protective effect was assessed by measuring changes in mouse back skin color.We took a picture of C57BL/6 mice back skin area once a week.2.2 Analysis of histological changes.Back skin was harvested for histological analysis on day 35.All hair was shaven off and weighed.2.3 HE staining.Harvested dorsal skins were observed and analyzed through an optical microscope.The number of follicles in anagen phase(A)and those in telogen phase(T)were counted.2.4 Western blot.TGF-β2,cleaved caspase3,Bcl-2 and BAX were detected.Results1.The proliferation rate of human keratinocytes(HaCaTs):The cell survival rates at the three time points(24h,48h and 72h)of each group generally showed a consistent trend.As the culture period prolonged,the survival rate of HaCaTs in DHT group was decreased;as shown in the DHT+ finasteride group,finasteride could improve the living status of HaCaTs treated by DHT,and the trend became evident as the culture period prolonged(to 72h);within a range froml to 100ug/ml,SPE exerted increased positive effects on the survival of HaCaTs,which reached the peak at 100ug/ml and the enhancement became even more evident with time compared with finasteride;however,when SPE reached 200ug/ml,the trend was reversed possibly.2.The proliferation rate of human dermal papilla cells(HDPPC):The cell survival rates at the three time points(24h,48h and 72h)of each group generally showed a consistent trend with a nuance in SPE lug/ml group.As the culture period prolonged,the survival rate of HHDPCs in DHT group was lowered;as shown in the DHT+ finasteride group,finasteride can improve the living status of HHDPCs treated by DHT,and the trend became slightly weakened with time(peaked at 24h relatively);SPE can improve the survival of HHDPCs and this effect showed a remarkably increased trend within a range from 1 to 5ug/ml,and the enhancement became greater than finasteride with time(peaked at 48h and 5ug/ml relatively);however,when SPE reached 25ug/ml and 100ug/ml,the trend was reversed possibly.3.Establishment of androgenic alopecia mouse models:The androgenic alopecia mouse model was built based on the protocol and back skin of all mice showed pink color.4.Morphological changes induced by SPE administration:The result showed that no hair developed in the shaven regions of group B within 35 days,which indicated an evident inhibitory effect of DHT;blank group(A),DHT+ finasteride group(C)and DHT+SPE group(D)had consistent grades at the 28th and 35th day,which indicated that SPE can promote hair growth to a certain extent;however,on the 14th day and 21 st day,A group had the highest grade followed by C group while D group had relatively low score,which indicated that the effect of SPE was lagged compared to finasteride.The hair weighing result showed that A and C group were close,D group weighed only a half of the former,while B group weighed 0,indicating successful establishment;SPE can indeed promote hair growth but there was a weaker effect compared with finasteride in this study.5.Histological change analysis by SPE administration:The development of hair follicles was closed in SPE group and finasteride group,and they were more apparent than in the DHT and blank groups,indicating a protective effect of SPE against DHT-induced androgenic alopecia.However,a larger number of hair follicles in the anagen was seen in SPE-treated group than in the finasteride group,indicating that the anagen phase was maintained in the SPE-treated group and entry into catagen was delayed compared to the murine hair cycle pattern seen in response to DHT.6.Protein expression during androgenic alopecia in mouse back skin in response to SPE:The result showed that B group had a significantly higher content of apoptosis factor cleaved caspase3 and BAX than other groups with group C and D close to each other(especially BAX),follow by group A,and the same trend was also seen in TGF-P2;group B had a remarkably lower content of apoptosis inhibition factor Bcl-2X with group C and D close to each other(C was slightly higher),and A had the highest content,which indicated that SPE can reduce the expression of cleaved caspase3,BAX and TGF-β2 via increasing the expression of Bcl-2X of skin in the animals treated with DHT to improve the survival of HHDPC cells and promote hair growth.Conclusion:The administration of SPE can promote hair growth and prevent apoptosis by similar mechanism with finasteride,but the effect is poorer and slower than finasteride.The mechanism of SPE possibly involves decreases in the expression of TGF-β2,cleaved caspase-3 and Bax and increases in Bcl-2.