The Function And Mechanisms Of Bioactive Molecules In Regulating The Growth And Cyclle Of Hair Follicle

Background:About 70%of people in China suffer from hair loss.Androgenic alopecia(AGA),also known as seborrheic alopecia,is one of the most common types of hair loss,with a high prevalence of about 21.3%in males and 6.0%in females.AGA is characterized by the involvement of genetic factors and the action of androgen,and is autosomal dominant inheritance.At present,most research believe that an important factor of AGA is that abnormal elevation of 5 ?-reductase leads to excessive androgen conversion to dihydrotestosterone(DHT),which affects the function of hair follicle(HF)cells,and eventually leads to hair loss.DHT can shrink HFs until they disappear,shorten hair occurrence for a long time,decrease the number/density of hair,decrease the terminal hair and increase the wolly hair.The classical symptoms of AGA are as follows:in men,thinning hair,progressive retraction of forehead hairline and top hair;in women,hair thinning,normal forehead hairline,and thinning of the whole top and forehead hair.Finasteride and minoxidil,the most widely used FDA-approved drugs in the treatments of AGA,are available.However,the effect of minoxidil is transient and may cause hypertrichosis or contact dermatitis,and the usage of finasteride is contraindicated in women.These results in poor patient compliance.Therefore,it is urgent to investigate more efficient and safe treatment methods.Epithelial-mesenchymal interaction(EMI)plays an important role in HF growth and hair cycle regulation.It is critical for cell differentiation,embryogenesis and biological activity of postnatal HF cells.HF outer root sheath cell(ORSC),derived from HF epithelium,is one of the most important paracrine target cells.ORSC not only regulates hair stem cells(SC)growth,but also participates in EMI.ORSC maintains HFSC and its microenvironment,and participates the hair cycle.Wnt/?-catenin signaling is one of the most important pathways for HF growth,development and hair cycle regulation.?-catenin,the critical transducer of Wnt/?-catenin signaling,accumulates in the cytoplasm and translocates into the nucleus after activated in adult tissues.Then it dimerizes with lymphoid enhancer factor/T-cell factor(LEF/TCF)family members of transcription factors.This complex regulates the transcription of downstream target genes and then regulates cells function,such as differentiation,migration and proliferation.Previous studies have shown that Wnt/?-catenin signaling pathway is critical for hair growth,development and tissue regeneration.Wnt signaling is a key regulator of hair cell(such as ORSC and derma papilla cell(DPC))migration,proliferation and differentiation.What's more,Wnt signaling pathway is a key player in induction of anagen onset and maintenance of cycling transition during regeneration of HFs.In addition,Wnt/?-catenin signaling pathway stimulates HF angiogenesis and shows an essential role in the HF's morphogenesis.So,Wnt/?-catenin signaling pathway is crucial for the regulation of HFs and could be a potential target of hair loss preventation and treatment.Bioactive molecules play an important role in cell biology,organogenesis and tissue regeneration.Their endogenous members include lipids,proteins and so on,while the exogenous members include plant extracts and synthetic compounds.Bioactive molecules take an important part in the communication between cells and extracellular matrix(ECM)and the maintenance of homeostasis,morphology and function in tissues.They are closely related to Wnt signals.This study aims to investigate the endogenous polyglycoprotein decorin and exogenous morroniside.Decorin is a member of small leucine-rich proteoglycan family,which participates in various biological processes,such as regulating the structure and formation of collagen,cellular proliferation,differentiation and migration.Decorin has been reported to play a potential role in HF biology.It is expressed in DPC ECM and regulates some important HF growth-related receptors,which may be involved in EMI.Our group has previously reported that decorin was highly expressed in the anagen of mouse HFs,but relatively reduced in the catagen and telogen.Also we found that Wnt signaling pathway in decorin treatment group was upregulated,suggesting that decorin plays a role as "anagen inducer" in HFs and may participate in the HF growth,cycle and morphogenesis regulation.However,the function and specific mechanisms of decorin in regulating the HF cell behavior and cycle transition remains unknown.Recent years,traditional Chinese medicine monomers or extracts have been shown to have positive effects in stimulating hair cell function,improving HF miniaturization,preventing alopecia and promoting hair regrowth significantly.Morroniside is the most abundant iridoid glycosides extracted from Cornus officinalis,which is regarded as one of the most widely used vegetable drugs in China.Previous studies have reported that morroniside has many bioactivities,including promoting cell proliferation,protecting cells against apoptosis and facilitating tissue regeneration.Hu et al.demonstrated that morroniside stimulated bone marrow mesenchymal SC proliferation through the secreted factors,cellular adhesion molecules and extracellular matrices.Li et al.reported that morroniside inhibited cell apoptosis,stimulated MC3T3-E1 cell proliferation and reduced bone resorption directly.Xu et al.demonstrated that morroniside had protective effects on umbilical vein endothelial cells for diabetic angiopathies,promoted endothelial progenitor cell proliferation and microvascular function after ischemia.However,the function and mechanisms of morroniside on HF growth and cycling regulation hasn not been investigated.Therefore,in this study,we aim to further explore the function and mechanisms of decorin and morroniside on the phenotypes of HF cells and the hair cycle regulation.PART I:Decorin promotes proliferation and migration of ORSCs and maintains hair anagen in miceObjective:Explore the effects of decorin on the proliferation,cell cycle and migration ability of human HF ORSCs in vitro.Clarify the mechanisms of decorin affecting the function of human HF ORSCs.Clarify the role of decorin in mouse hair cycle transition and its related mechanism using decorin knockout(KO)transgenic mice model.Methods:Normal human HF ORSCs were isolated and cultured in vitro.Decorin was overexpressed by adenovirus transfection,the mRNA and protein expression of decorin was verified by qRT-PCR and Western Blot(WB)assay.MTS assay was used to detect the effect of decorin on the proliferation of ORSCs.Transwell assay was used to detect the migration rate of ORSCs.The gene and protein expression of Wnt/?-catenin signaling pathway in decorin overexpression group and control group were compared by qRT-PCR and WB assay;The expression and localization of the key molecule(3-catenin in the Wnt/?-catenin signaling pathway were detected by immunofluorescence(IF),and the effect of decorin on cell function was verified by Dickkopf-related protein 1(DKK1)inhibiting the Wnt signaling.Then,the decorin KO transgenic mice were constructed and the new hair cycle was induced by depilation.Hematoxylin-eosin(HE)staining was used to detect the histomorphology of the samples.The thickness of the skin,the diameter of hair bulb,the proportion of HFs and the percentage of HFs in each stage of hair cycle were compared.The expression and localization of ?-catenin in the two groups were compared by immunohistochemistry and WB assay.Results:1.Decroin overexpression promoted the proliferation of human HF ORSCs.2.Decorin overexpression promoted the migration ability of human HF ORSCs.3.Decorin overexpression upregulated the expression of Wnt/?-catenin signaling pathway in human HF ORSCs.4.The upregulated proliferation and migration of human HF ORSCs were partly inhibited by DKK1(a Wnt/p-catenin signaling pathway inhibitor).5.The telogen of HFs in DCN-/-mice was delayed into anagen compared with wild type(WT)mice.6.The anagen of HFs in DCN-/-mice was accelerated into catagen compared with WT mice.7.DCN-/-mice mice showed down-regulated expression of(3-catenin both in telogen-anagen transition and anagen-catagen transition compared with WT mice.Conclusion:The experiment further explored the role and mechanisms of decorin on HF growth and hair cycle regulation.In vitro,decorin promoted the proliferation and migration of human HF ORSCs.In vivo,decorin maintained the anagen of mouse HFs.These phenomena were partially mediated by Wnt/?-catenin signaling pathway.In conclusion,our study shows that decorin plays an active role in HF growth and plays a role as"anagen inducer" in hair cycle transition,which may provide some new possibilities for the treatment of hair loss.PART II:Morroniside regulates hair growth and cycle transition via activation of the Wnt/?-catenin signaling pathwayObjective:Clarify the effects of morroniside on the activity(such as proliferation and migration)of normal human HF ORSCs cultured in vitro.Clarify the mechanisms of morronside on the function of human HF ORSCs cultured in vitro.Elucidate the role of morroniside in the hair cycle transition of C57BL/6 mice through the study of hair regeneration cycle model induced by depilation.Based on the above fundings,we aim to provide a potential drug for the treatment of hair loss.Methods:Primary HF ORSCs were isolated and obtained from normal healthy people scalp skin,then treated with morroniside.The effect of morroniside on the proliferation of human HF ORSCs in vitro was detected by MTS assay,cell cycle changes were detected by flow cytometry,cell proliferation was inhibited by mitomycin C treatment,cell migration was detected by Transwell assay.The gene and protein expressions of Wnt/?-catenin signaling pathway in morroniside and control group were investigated by WB assay.The expression and localization of key molecule of Wnt/?-catenin signaling pathway was detected by IF assay;the activation of Wnt pathway was detected by TOP/FOP Flash assay;and DKK1 was used to inhibit Wnt/?-catenin signaling pathway to verify that the effect of morroniside.The HF cycle model of C57BL/6 mice was induced by depilation.Morroniside was injected intradermally.The samples were examined histomorphologically by HE staining.The thickness of skin,the diameter of hair bulb,the proportion of HFs in each stage of hair cycle and the percentage of hair cycle were analyzed.The expression and location of Ki67 were tested by IF assay.The expression of ?-catenin protein was compared using WB assay.Results:1.Morroniside promoted the proliferation of human HF ORSCs and increase the S,G2 and S/G1 proportion of ORSCs.2.Morroniside promoted migration of human HF ORSCs.3.Morroniside activated Wnt/?-catenin signaling pathway in human HF ORSCs.4.The upregulated proliferation and migration of human HF ORSCs stimulated by morroniside were partially down-regulated by DKK1.5.Intradermal injection of morroniside accelerated telogen-anagen transition and delayed anagen-catagen transition.6.?-catenin,the critical signal molecule of Wnt/?-catenin signaling pathway,was upregulated both at telogen-anagen and anagen-catagen transition in mice treated with morroniside compared with control group.Conclusion:Morroniside promoted the proliferation and migration of HF ORSCs in vitro.In addition,morroniside induced the anagen,delayed the catagen and produced larger bulges and longer hair shafts in morroniside-treated mice compared with controls.These in vitro and in vivo effects were mediated by Wnt/?-catenin signaling.These results highlight a positive role of morroniside in the regulation of HF development and growth and provide a potential method for the treatment of alopecia.