Effect Of 8DSS On Hair Growth And Its Possible Mechanisms

Background:Hair play a role in protecting,promoting social interaction and maintaining beautiful. Hair follicle is a complex structure of the skin appendages which undergoes a cycle of growth(anagen phase),regression(catagen phase) and resting(telogen phase). When the hair follicle cycle is in disorder, hair loss will occurred. In China, alopecia areata and androgenic alopecia are the most common type of hair loss. With the social pressure, environmental problems increasingly become prominent for the people,the incidence of hair loss increased year by year, and the trend was younger. Hair loss not only affects the appearance and mental on patients and their families, but also resulting in decreased quality of life and communication problems for patients.However, the therapeutic effect of hair loss are still very scarce.Currently,the only FDA-approved oral medication finasteride and externally applied agent minoxidil have definite effect. The use of these agents is associated with adverse effects, including dryness, itchiness, and loss of libido or erectile dysfunction.Therefore,to promote hair follicle regeneration, new drugs research which can promote hair regrowth is becoming increasingly important.Hair follicles, composed of multiple layers of epithelial and mesenchymal cells, named dermal papilla, undergo regeneration through cycles ranging from anagen, catagen, to telogen.The dermal papilla cells play an essential role in the induction of new hair follicles, the regulation of morphogenesis, and the control of the hair growth cycle. Several molecules and signal pathways have been implicated in proliferation and the regulation of hair cycling.Signaling through the β-Catenin pathway regulates morphogenesis and regeneration of hair. The Wnt/β-catenin signaling pathway is essential in maintaining the anagen-phase characteristics and hair-inducing activity of the dermal papilla. Glycogen synthase kinase 3(GSK-3β), a serine/threonine kinase, suppresses cell proliferation and survival.β-catenin can be degraded by phosphorylation at its N-terminal domain by GSK-3β.The Akt pathway is involved in regulating the cell survival of hDPCs and have crosstalk with the Wnt/β-catenin signaling.Aspartate-serine-serine(DSS) repeats are abundant in naturally occurring proteins that are criticalfor tooth formation.And is amino acid repeat sequence in dentin phosphoprotein(DPP). Octuplet repeats of aspartate-serine-serine(8DSS) peptides is a synthetic peptides sequences. 8DSS has a strong ability to bind calcium ions, and play an important role in dental matrix deposition and mineralization. After external use of 8DSS on the alopecia of Balb/c mice(Pub.No.US2010 / 0239503 A1),the hair follicle regrowth rapidly. Accordingly, we speculate 8DSS may regulate the hair follicle cycle through PI3K/Akt/GSK-3β pathway.To confirm this, we take the hair follicle cycle as the starting point, focusing on the Wnt/β-catenin and PI3K/Akt signaling pathway, using Real-time PCR, Western blot and other methods to investigate inducing function of the cellular and molecular by 8DSS in dermal papilla cells, to investigate its mechanism and the effects on the Wnt/β-catenin and PI3K/Akt signaling pathway in hDPCs. We demonstrated the promoting effect on hair regrowth in the mouse model of alopecia by 8DSS, and to reveal the role and its mechanism in hair regrowth. This study may provide new therapeutic targets and strategies for the prevention of hair loss diseases.Objective:1. To demonstrate 8DSS can promote hair regrowth of C57 BL / 6 mice and regulate the hair follicle cycle.2. To investigate whether 8DSS regulate the proliferation, apoptosis and cytokine secretion in hair papilla cells.3. To investigate if 8DSS regulate inducibility of DPCs through PI3 K / Akt and Wnt / β-catenin signaling pathway.Methods:1. Using 1% fluorescence 8DSS on the shaved area of mice to observe its dermal penetration pathway.The backs of the mice were depilated at seven weeks of age, at which time all of the hair follicles were synchronized in the telogen stage. The mice were topically treated with 50 μl of 1% 8DSS or sham daily for 31 days. After 7 days treatment,5 mices in each group were euthanized.Use HE staining and immunohistochemistry to detect the morphological changes of hair follicle and the expression of β-catenin.2. Use improved "two-step enzyme digestion" to extract the dermal papilla, an culturedermal papilla cells.Use immunofluorescence to identify the DPCs.3. Use CCK8 assay to detect the proliferation of DPCs.Use qRT-PCR to detect effect on expression of molecules.4. Use Western blot to detect expression of p-GSK-3β, p-Akt,Bcl-2 and the nuclear β-catenin.5. Use Western blot to detect expression of p-GSK-3β, p-Akt after inhibit the PI3K/Akt signaling pathway.Results:1.8DSS penetrates to hair follicle along the ORS way.After topical use of fluorescence 8DSS on shaved area of mice,green fluorescence was observed mainly in the epidermis in one hour, two hours after,the 8DSS gradually penetrate along the outer root sheath to the the bulb of follicle, indicating 8DSS can act on the hair follicle site.2.8DSS induces regeneration of hair follicleThe skin color became pink to black after topical use of 8DSS on shaved area of mice was observed,indicating an earlier telogen-to-anagen transition than mock treated mice.HE staining showed follicles growing in V-phase by 8DSS however, a phase II in control group.Immunohistochemistry showed significantly expressed β-catenin.by 8DSS.3. 8DSS induces signaling molecules in dermal papilla cellsCCK8 showed no effect on proliferation with low concentration, as the concentration of trans dermal papilla cells inhibited;RT-PCR showed upregulated expression of Wnt signaling-related genes and transcription factors such as Wnt10 b, Lef-1 and c-myc,suggesting 8DSS can promote the capacity of hair papilla cells that induce epithelial cell proliferation and differentiation..4. 8DSS induces the nuclear β-catenin to activate Wnt/β-catenin signalingAfter treatment dermal papilla cells with 8DSS,Western blot showed increased expression of nuclear β-catenin in time-dependent at 1h and 24 h, suggesting that 8DSS could activite Wnt/β-catenin signaling pathway in dermal papilla cells.5. 8DSS can activate PI3K/Akt signaling pathway in the dermal papilla cellsAfter 8DSS treatment at 5min, 15 min, 30 min and 1h,Western blot showed increased expression of Bcl-2, p-GSK-3β p-Akt in time-dependent, suggesting that 8DSS can activate PI3K/Akt signaling pathway in dermal papilla cells and increase the expression of downstream target genes Bcl-2 and p-GSK-3β.6. 8DSS induces hair follicles regeneration via PI3K/Akt/GSK-3β signaling pathwayAfter blocking the PI3K/Akt signaling pathway by LY294002,8DSS can not enhance the phosphorylation of GSK-3β and Akt,suggesting that 8DSS increase the activity of Wnt/β-catenin signaling pathway in dermal papilla cells may be through activation of PI3K/Akt/GSK-3β signaingl pathway.Conclusions: 8DSS promoted hair regrowth through the PI3K/Akt/GSK-3β signaling pathway. These findings suggest that 8DSS may be a potentially useful agent for treatment of hair loss.