OXTR, a seven-transmembrane G protein-coupled receptor, activates several signaling cascades, including the mitogen-activated protein kinase, protein kinase C, or phosphoinositide-specific phospholipase C pathways17. These signaling pathways maintain homeostasis in various organs and tissues, and activation of OXTR may represent a therapeutic strategy for certain diseases. Previous studies have shown that OXTR agonists improve social interactions in a mouse model of autism,14 reduce alcohol intake in male mice,18 and improve social and cognitive function in a rat model of schizophrenia. has been reported19. In the skin, dermal fibroblasts and keratinocytes express OXTR, and oxytocin signaling plays a role in regulating cell proliferation, inflammation, aging, and oxidative stress responses. However, to our knowledge, there are no studies on the effects of OXTR on hair follicle cells. In this study, we demonstrated that LIT001 and WAY267464, which are non-peptide-specific agonists of OXTR, have a hair growth-promoting effect and hold great promise as potential therapeutic agents for alopecia. Therefore, these insights will accelerate the search for new OXTR-targeted drugs.
To address the issue of oxytocin stability and skin permeability, this study investigated OXTR agonists. Molecules with MW > 500 have been reported to have difficulty penetrating healthy skin21. The molecular weights of LIT001 and WAY267464 are 531 and 655, respectively, which are smaller than oxytocin (molecular weight 1007), but still exceed this value. However, since the target of OXTR agonists is hair follicles, specifically DP cells, these molecules may reach DP cells through the hair follicle pores. This is partially supported by a previous report that bioactive peptides with relatively large molecular weights (molecular weights 509 and 774) were delivered to the DP through the follicular pore and promoted hair growth in the skin. They used nanoliposomes to load the peptides and deliver them to the DP. The permeation kinetics and effects of LIT001 and WAY267464 on human skin samples will be the next subject. Furthermore, combinations with other OXTR agonists with smaller molecular weights and higher permeation rates and drug delivery systems through the follicular pore 23 may provide a better approach.
Bioinformatics analysis showed that OXTR expression levels were low in DP cells of alopecia patients (Fig. 2a). This result may be due to a variety of factors, including genetic factors common to alopecia patients. Psychological environmental factors such as happiness and stress. Growth factors produced by hair removal. This finding may be both a cause and a consequence of alopecia. In this study, OXTR knockdown in DP cells suppressed the expression of hair growth-related genes (Fig. 2f). This indicates that decreased OXTR expression levels are mainly associated with hair loss. However, the sample size for bioinformatics analysis was small. Further studies incorporating diverse samples across different ages and genders are needed to elucidate the relationship between OXTR expression and androgenetic alopecia (AGA).
OXTR activation can be achieved using oxytocin itself or OXTR agonists. Oxytocin is produced in both men and women in response to skin stimulation (such as hugs and massages), music therapy, and interaction with pets5,24,25. OXTR activation is promoted by lifestyle changes, and oxytocin’s ease of use is another advantage of oxytocin. However, due to low receptor binding specificity and activation of other receptors, continuous administration of oxytocin may reduce efficacy and cause side effects. OXTR agonists with high receptor binding specificity may reduce these risks.
The top 10 KEGG terms related to WAY267464 or LIT001 treatment included neuroactive ligand-receptor interaction, oxytocin signaling pathway, and cytokine-cytokine receptor interaction. Of note, these pathways were upregulated by oxytocin treatment in our previous study8. Therefore, although WAY267464 and LIT001 are potential hair growth promoters that can replace oxytocin, several pathways were upregulated differently by LIT001 and WAY267464. These agonists may have different mechanisms and provide additional hair growth effects when used in combination with LIT001 and WAY267464. Future research will need to elucidate the mechanism and verify the effects of the combination. Additionally, more than 10 oxytocin agonists are available, and future studies will classify them based on the genes activated in DP cells and test their effectiveness in inducing hair growth using follicularoids. It’s planned.
Although we used OXTR agonists to activate oxytocin signaling, approaches to increasing OXTR using RNA-based therapies are also possible. Target genes can be selectively regulated using RNA-based therapies26,27. In hair research, RNA interference can selectively suppress expression of the androgen receptor, which mediates a series of biomolecular changes that lead to hair loss. Clinical studies of siRNA applied to the scalp have confirmed its hair growth promoting effects29. Approaches that use mRNA therapy to increase OXTR expression levels may also be effective in hair loss treatment and will be investigated in future studies.
In our previous studies, we developed a technique to prepare folliculoids that efficiently regenerate hair follicles in vitro16,30,31. Hair follicles are generated through epithelial-mesenchymal interactions,32 and hair follicle generation in vitro is possible by reconstituting the interactions between epithelial and mesenchymal cell populations (including DP cells). It has become. In the hair follicle, DP cells produce several growth factors that stimulate the proliferation of epithelial cells and elongate the hair shaft. Hair growth-promoting drugs increase hair elongation through activation of growth factor secretion by DP cells. Therefore, hair follicleoids can be used to evaluate the hair growth promoting effect of drugs using the elongation of rod-like structures after addition of drug candidates as an indicator. Treatment with existing hair growth-promoting drugs, such as minoxidil, promotes the elongation of rod-like structures16. In this study, hair follicles were cultured in medium containing WAY267464 or LIT001. Although these drug candidates elongated rod-like structures, the germination length with WAY267464 or LIT001 treatment was shorter than that with minoxidil treatment. To enhance the hair growth effect of WAY267464 or LIT001 treatment, culture conditions such as drug concentration, timing, duration, and drug combinations may need to be optimized. Minoxidil was originally developed as an antihypertensive agent, and its ability to increase blood flow was suggested as a mechanism for promoting hair growth. However, subsequently, several other mechanisms such as opening of potassium channels, activation of β-catenin signaling and extracellular signal-regulated kinase and Akt signaling, and increased release of growth factors such as FGF733, 34, 35, 36 is proposed. . Combination with minoxidil, which has a different mechanism of action than OXTR agonists, may be an effective treatment for alopecia.
OXTR agonist increased the expression of hair growth-related genes in DP cells and promoted the elongation of hair follicles sprouting from folliculoids. These findings were further supported by results from human hair follicle organ culture, a standard assay for investigating hair growth promoters. However, testing many drugs using this approach is hampered by the limited number of hair follicles available and the unique nature of individual hair follicles, such as in vivo anagen/telogen and isolation processes. It is difficult because the results vary based on the condition. To address this limitation, the present study often used approaches that reconstitute hair follicle organoids from cells dispersed from multiple hair follicles. A significant problem with using follicularoids for drug testing is that there is no way to expand human follicular keratinocytes without rapidly losing their hair-producing ability. In this study, follicularoids were prepared using human hair follicle keratinocytes without subculture to maintain functionality. As a result, the number of experiments using cells from the same human sample was limited, and control experiments using the same cell vial were required to normalize all hair growth data. To alleviate this limitation in hair follicle-based drug testing, new methods are needed to expand hair follicle keratinocytes while preserving function.
Our findings represent a first step toward demonstrating the hair growth-promoting effects of OXTR agonists. Future studies should use organ culture and animal models to obtain further evidence of its effects. Specifically, organ culture studies should investigate four categories: (i) Prolongation of the anagen phase or inhibition of the catagen phase. (ii) promoting proliferation or inhibiting apoptosis in hair matrix cells; (iii) regulation of key hair growth mediators at the protein expression level, particularly in DP cells, hair matrix cells, and/or outer root sheaths; (iv) Observation of OTXR-specific effects of WAY267464 and LIT001, where OTXR silencing demonstrated significant antagonism. In animal experiments, we will investigate effective conditions by optimizing the concentration, administration time, and administration frequency of WAY267464 and LIT001. Although the cells used in this study were from healthy donors, the efficacy of WAY267464 and LIT001 needs to be evaluated in the future using donor cells isolated from alopecia patients. We will continue to conduct these analyses, and move forward with the development of new hair loss treatments.
In conclusion, OXTR expression levels in DP cells of alopecia patients were low. OXTR knockdown in DP cells suppressed the expression of hair growth-related genes. WAY267464 and LIT001 increased the expression of hair growth-related genes in DP cells and promoted the elongation of hair follicles sprouting from follicularoids. Therefore, our findings showed that WAY267464 and LIT001 are potential hair growth promoting agents targeting OXTR, facilitating their application as therapeutics. Our findings will accelerate the development of OXTR-targeted therapies.
