The Screening Of 5?-reductase Inhibitor For Seborrheic Alopecia From Traditional Chinese Herbs And The Pharmacological Evaluation Of Cacumen Platycladi

ObjectiveSeborrheic alopecia(SA)is the most common hair loss disease in clinical.It is an androgen-dependent polygenic autosomal dominant hereditary baldness,the excessive androgens or increased sensitivity to androgens are the major factors in the occurrence of the disease,the increased levels of Testosterone(T),its metabolite dihydrotestosterone(DHT)and androgen receptor(AR)are directly related,and the 5?-reductase(5a-R)plays an important role in it.In addition,long-term mental stress,eating disorders,mental imbalance and virus infection are important factors to induce or aggravate the disease.Epidemiological investigations show that the incidence rates of SA in 50-year-old male are more than 50%in European countries,and approximately 21.3%in our country.Currently,the market is flooded with many synthetic steroidal and non-steroidal 5a-R inhibitors,but these synthesis inhibitors often have many adverse reactions,and it is difficult to meet the needs of the majority of patients.Traditional Chinese medical theory believes poliosis and lapsus pilorum are related to the weakness of liver,kidney and blood vigor.Therefore,the cure must rely on the reinforcement of the factors above.In China,many traditional Chinese medicines have been used for nourishing the liver and kidney,promoting blood generation,cooling blood and blackening hair for hundreds of years,For example,polygoni multiflori and angelica sinensis nourishing blood and essence,Cacumen Platycladi and Eclipta prostrate cooling blood and blacking hair.Modern medical theory proves that many of them could promote hair follicle growth.Traditional Chinese medicine treatment has the advantage of mild and few side effects.But its therapeutic target is not clear,and the mechanism also needs further study.The study was conducted according to TCM understanding of hair loss,with the help of modern medicine's understanding of seborrheic alopecia that 5?-R is an effective target for treatment SA and combined with our previous findings.we established the in vitro screening platform,and screened traditional Chinese medicine that could inhibit 5?-R activity.On the other hand,we evaluated the impact of traditional Chinese medicine 5a-R inhibitor on hair growth of the C57 mice through in vivo experiments and explore the mechanism,Clarify the relationship of traditional Chinese medicine between the treatment effect of SA and 5?-R.This study will elucidate the mechanism of herbal treatment of seborrheic alopecia and lay the foundation for the development of a new generation of product to treat seborrheic alopecia,but also to bring the Gospel to patients at home and abroad.Methods1.The 5a-reductase inhibitors' model in vitro was established which were composed of Type ? 5?-reductase extracted from prostates of male rats,testosterone(as a substrate)and NADPH(as a hydrogen supplier).The concentration of T which measured by High Performance Liquid Chromatography could show the activity of enzyme.The specificity of the model was judged with finasteride as a positive control.2.On the basis of the in vitro screening platform which has been established,the inhibitory activity of different concentrations of Chinese herb extracts were examined.3.Rosin/paraffin mixture(1:1)was used for hair removal of C57BL6 mice,inducing anagen.The next day 0.05%testosterone solution was smeared,30min later corresponding medicaments was smeared,continuous for 28d.Changes in skin color and hair growth was observed daily and hair growth scored every other day.HE staining was used to observe the hair follicle morphology and the count of hair follicles.Hair follicle staging was scored with reference to Muller-Rover' follicles morphological stage.4.Enzyme-linked immunosorbent assay(ELISA)was used to detect levels of T,DHT,and E2 in serum and skin tissue and immunofluorescence was used to detect the expression of type ? 5?-R.Image J software was used for measuring fluorescence intensity and semi-quantitative analysis.Pearson correlation analysis the relationship between the expression of 5?-R and the DHT levels in skin tissue.Results1.Establishing the HPLC method in vitro screening model,Chromatographic conditions Col?mn:Beijing Dumas DiamonsilTM C18(250mm×4.6mm,5?m)stainless steel column;mobile phase:methanol/water 70/30(V/V),column temperature:room temperature;detecting wavelength:242 nm;flow rate:1.0mL/min;injection volume:20?L;enzymatic reaction system conditions the concentration of substrate T 1000?mol/L,NADPH concentration of 1mmol/L,crude enzyme concentration 3 mg/mL,the reaction temperature is 37?,reaction time 30min.Finasteride showed 5a-R inhibition activity in a dose-dependent manner,and the inhibition rate was up to 83.67%,IC50 was 212nmol/L.2.The extract of Ganoderma Lucidum,Polygonum Multiflori,Cacumen Platycladi and Cynomorium songaricum could significantly inhibit 5?-R activity(P<0.01)in a dose-dependent manner.3.Compared with the model group,the high and low dose of Cac?men Platycladi could significantly shorten the time mouse skin got black and the hair grew(P<0.01),and could significantly promote hair growth in mice;Compared with the model group,the high and low dose of Cacumen Platycladi could significantly reduce the staging score of hair follicle morphology in mice(P<0.01)and increase the number of follicles per visual field(P<0.01).4.Compared with the model group,there was no significant difference(P>0.05)of the T,DHT,and E2 levels in serum of the high and low dose of Cacumen Platycladi,while DHT levels were significantly decreased in skin(P<0.01);Compared with the model group,the mean fluorescence intensity was significantly lower in the high and low dose of Cacumen Platycladi(P<0.01).The expression 5?-R was positively correlated with DHT levels in skin(Pearson correlation coefficient r = 0.837,P<0.01)Conclusion1.This study established a stable,reproducible 5?-R inhibitors in vitro screening platform for the screening of TCM 5a-R inhibitor.2.The extract of Ganoderma Lucidum,Polygonum Multiflori,Cacumen Platycladi and Cynomorium songaricum could significantly inhibit 5?-R activity in a dose-dependent manner.3.The extract of Cacumen Platycladi could induce the hair enters growth period from telogen and inhibit hair follicles enter the telogen and promote hair growth.4.After applying testosterone,DHT levels and 5?-R expression of skin tissue increased and was positively correlated,after effects of Cacumen Platycladi,DHT levels and 5?-R expression was down-regulated.