| Backgrounds and ObjetivesHair follicle is an important skin accessory organ of mammals.Its morphological formation is divided into three stages:induction period,organ formation period,and cell differentiation period.In the inducing phase,the Wnt/β-catenin signals from the mesenchymal cells induces the formation of a placode of the epithelial cells,which in turn induces the formation of a dermal condensate underlying epithelial cells.Hair follicles in the organ formation phase and cell differentiation phase,a variety of complex signal interactions between epithelial and mesenchymal cells prompted the gradual formation of hair follicle morphological structure.Under the regulation of the cell differentiation signal network,a large number of dermal and epithelial cells differentiate and proliferate to complete the main structure of the hair.Subsequently,the cycle of hair growth starts:anagen,catagen and telogen.The normal development and periodic regeneration of hair follicles depends on the interaction between epithelial cells and mesenchymal cells.It is currently believed that although the signal transmission between the two types of cells is mutual,dermal cells are the inducers of epithelial cell responses,and the activity of hair follicle dermal papilla cells is a key factor in inducing and maintaining the growth state of hair follicles.Studies confirmed that alkaline phosphatase(ALP),Versican,Corin,CD 133 and a-smooth muscle actin(a-SMA)are molecular markers of hair follicle papilla cells.The immune privilege of the hair follicle is a periodic,relative privilege state.Studies have shown that it exists only during the growth phase of hair follicles.One of the mechanisms is the local potent immunosuppressants secretions of hair bulbs in human and mouse growing stages including α-MSH,TGF-β1,TGF-β2,ACTH,IGF-1,and IL-10.Additionally,these autocrine cytokines and hormone expression levels are highest in the skin of the growing hair follicle.This is crucial for restoring and maintaining the immune privilege of the hair follicle.Natural medicines show great advantages and ideal application prospects because of their low toxic and side effects,low price,and easy availability.Finding and screening safe and effective natural medicines is the preferred direction and task.According to reports in the literatures,there are a few types of natural drugs used for hair growth,and most of the studies have not clarified the material basis of natural drugs.Scutellaria baicalensisis one of the common prescriptions for the treatment of alopecia(inside and outside treatment)by traditional Chinese medicine.Baicalin is the main active ingredient of scutellaria baicalensis.Previous studies have found that baicalin can improve immune function,antioxidant,anti-aging,anti-tumor,anti-virus,and promoting hair follicle growth.However,its mechanism of regulating hair growth still lacks in-depth research.This study was divided into two parts:In the first,the reconstituted hair follicle growth models were established on the back of BALB/c-nu nude mice,simulating the morphogenesis of hair follicles and its periodic growth process.The baicalin was dropped onto the hair follicles in anagen.The effects of baicalin on Wnt/p-catenin signaling pathway and alkaline phosphatase(ALP)activity of dermal papilla cells of hair follicles were tested.In the second,the models of hair follicles in anagen were established on the back of C57/BL6 mice by depilating hairs,and topical applicating baicalin to examine its effects on the immune privilege of hair follicles.Methods and Results1.The intact skins taking from 0 to 1 day old C57BL/6 mice were trypsinizedOvernight(for 12 to 16 hours),and then the dermis and the epidermis were separated using aseptic scissors and forceps.The single dermis and epidermal cells were prepared by collagenase digestion and blow.The dermal and epidermal cells isolated from skin of C57BL/6 mice were mixed at a ratio of 1:1 and transplanted to the back of an immune-deficient nude mouse,in which a silicon chamber had been implanted.After one week,the dome of the silicon chamber was taken out,exposing the wound surface.Two weeks later,black hair outgrew at the transplant site.The baicalin-treated group and the minoxidil-positive control group received 50 μmol,100μmol baicalin,and 100 μmol minoxidil solution(100 μl,respectively)application topically.The blank control group was treated with 100 μl of vehicle,and theWnt/β-catenin pathway blocker IWR-1 group was treated with 100 μmol of baicalin plus 1 μmol of IWR-1.The growth status of the hair was observed by photographs.After 14 days of treatment,mice were sacrificed to obtain skin specimens,and hair follicle growth status were detected by counting hair follicles,using HE staining,histochemistry,real-time quantitative polymerase chain reaction(qRT-PCR)and Western-blotting methods.The expressions of Wntβ-catenin pathway-associated proteins Wnt5a,Wnt3a,frizzled7,disheveled2,GSK3β,β-catenin,Lefl and dermal papilla alkaline phosphatase ALP were determined.In the baicalin-treated groups and the positive control group,the hair densities were thicker than the control group and the Wnt/p-catenin pathway blocker IWR-1 group,and the Wnt/β-catenin pathway blocker IWR-1 group had no hair growth almost;The expressions of Wnt3a,Wnt5a、frizzled7,disheveled2,β-catenin、Lefl,and alkaline phosphatase ALP were significantly increased in the skin tissue of the hair follicle,and the expression of GSK3β was significantly decreased compared with control group and Wnt/β-catenin pathway blocker IWR-1 group.The difference was significant,whereas there was no significant difference between the treatment group and the positive control group.However,the blank control group was significantly different from the Wnt/β-catenin pathway blocker IWR-1 group.The outcomes of hair follicle counting and HE staining showed that the number of hair follicles or hair shafts increased in the treatment groups and the positive control group compared to control group and Wnt/β-catenin pathway blocker IWR-1 group.2.After be melted,the mixture of rosin and wax were topically applicated on the back of C57/BL6 mice by a scoop to depilate hair and establish models of hair follicle in anagen.On the second day after depilating,baicalin-treated group,positive control group and blank control group were treated with 100 μl baicalin(100 μmol),100 μl minoxidil(100 μmol),and 100 μl of vehicle for 17 days respectively.Photographs of hair growth were recorded daily.On the 19th day,the mice were sacrificed,and specimens were obtained,while hair shafts randomly extracted(10 mice/mouse)at the site of the hair regeneration,measuring the lengths of shafts.Immunohistochemistry,enzyme-linked immunosorbent assay(ELISA),real-time quantitative polymerase chain reaction(RT-qPCR),and Western-blotting test were used to detect the expressions hair follicle immunosuppressive factors such as α-MSH,TGF-β,and IGF-1 in local skin tissues.The direct observation results showed that the treatment group and the positive control group had more denser hair and deeper coat color,and the hair shaft measurement length showed of that the treatment group and the positive control group were longer than the control group.The difference was statistically significant.HE staining showed that the volumes of hair follicles in the treatment group and the positive control group were greater than that in the control group.The expressions of α-MSH,TGF-β1 and IGF-1 in local tissues of the treatment group and positive control group were increased compared to the control group,the difference was significant,whereas there was no significant difference between the treatment group and the positive control group.Conclusions1.Baicalin promotes hair growth by activating the canonical Wnt/β-catenin pathway of hair follicles and increasing the activity of hair follicle dermal papilla cells.Baicalin activates Wnt/β-catenin signaling pathways in hair follicles through acting on the upstream molecules of Axin/CKla/APC/GSK3β complex in cytoplasm.2.Baicalin stimulates the secretion of immunosuppressive factors including α-MSH,TGF-β1,and IGF-1 of the hair follicles in the local tissue,and then enhances the restoration of hair follicle immune privilege during anagen.Therefore,the hair follicles return into the growth phase earlier and generate longer hair shafts. |
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