Possible role of ß-hydroxybutyrate in inducing inflammation in alopecia areata.

Alopecia areata (AA) is an autoimmune inflammatory disease characterized by non-scarring hair loss due to an immune response that targets hair follicles. The current treatment approach for AA involves the use of immunosuppressants and immunomodulators to reduce cytokine levels around affected hair follicles. Sodium-glucose cotransporter 2 (SGLT2) inhibitors have emerged as potential anti-inflammatory agents with diverse beneficial effects in various medical conditions. This study investigates the role of beta-hydroxybutyrate (BHB), a ketone body produced during SGLT2 inhibition, in the pathogenesis of AA. Serum BHB levels were found to be significantly elevated in patients with AA compared with healthy controls, with higher levels correlating with severity of hair loss. BHB treatment increased inflammatory cytokine production in outer root sheath (ORS) cells, mimicking the inflammatory conditions seen in AA. The results suggest that elevated BHB levels may exacerbate the inflammatory immune response in AA patients and may be associated with chronic hair loss and resistance to treatment. Serum BHB levels may serve as a potential marker of poor prognosis in patients with severe AA. Further research is needed to elucidate the precise role of BHB in the pathogenesis of AA and its implications for disease management.

The crosstalk between PTEN-induced kinase 1-mediated mitophagy and the inflammasome in the pathogenesis of alopecia areata.

Alopecia areata (AA) is a T-cell-mediated autoimmune disease that causes chronic, relapsing hair loss; however, its precise pathogenesis remains to be elucidated. Recent studies have provided compelling evidence of crosstalk between inflammasomes and mitophagy-a process that contributes to the removal of damaged mitochondria. Our previous studies showed that the NLR family pyrin domain containing 3 (NLRP3) inflammasome is important for eliciting and progressing inflammation in AA. In this study, we detected mitochondrial DNA damage in AA-affected scalp tissues and IFNγ and poly(I:C) treated outer root sheath (ORS) cells. In addition, IFNγ and poly(I:C) treatment increased mitochondrial reactive oxygen species (ROS) levels in ORS cells. Moreover, we showed that mitophagy induction alleviates IFNγ and poly(I:C)-induced NLRP3 inflammasome activation in ORS cells. Lastly, PTEN-induced kinase 1 (PINK1) knockdown increased NLRP3 inflammasome activation, indicating that PINK1-mediated mitophagy plays a critical role in NLRP3 inflammasome activation in ORS cells. This study supports previous studies showing that oxidative stress disrupts immune privilege status and promotes autoimmunity in AA. The results emphasize the significance of crosstalk between mitophagy and inflammasomes in the pathogenesis of AA. Finally, mitophagy factors regulating mitochondrial dysfunction and inhibiting inflammasome activation could be novel therapeutic targets for AA.

SIRT1 downregulation provokes immune-inflammatory responses in hair follicle outer root sheath cells and may contribute to development of alopecia areata.

BACKGROUND: Silent information regulator 1 (SIRT1), a type III histone deacetylase, is involved in various cutaneous and systemic autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, and psoriasis. However, little is known about the role of SIRT1 in the development of alopecia areata (AA). OBJECTIVES: This study investigated whether SIRT1 regulates the hair follicle immune system and is involved in AA pathogenesis. METHODS: SIRT1 expression in human scalp tissue was analyzed using immunohistochemical staining, qPCR, and western blotting. The regulatory effect of SIRT1 was evaluated after stimulation with the double-stranded RNA mimic polyinosinic:polycytidylic acid (poly I:C) in hair follicle outer root sheath (ORS) cells and C3H/HeJ mice. RESULTS: SIRT1 expression was significantly reduced in the AA scalp compared to the normal scalp. SIRT1 inhibition upregulated MHC class I polypeptide-related sequence A and UL16 binding protein 3 in hair follicle ORS cells. SIRT1 inhibition also promoted the production of Th1 cytokines (IFN-γ and TNF-α), IFN-inducible chemokines (CXCL9 and CXCL10), and T cell migration in ORS cells. Conversely, SIRT1 activation suppressed the autoreactive inflammatory responses. The counteractive effect of the immune response by SIRT1 was mediated through the deacetylation of NF-κB and phosphorylation of STAT3. CONCLUSION: SIRT1 downregulation induces immune-inflammatory responses in hair follicle ORS cells and may contribute to AA development.

Pitavastatin Induces Apoptosis of Cutaneous Squamous Cell Carcinoma Cells through Geranylgeranyl Pyrophosphate-Dependent c-Jun N-Terminal Kinase Activation.

BACKGROUND: Pitavastatin is a cholesterol-lowering drug and is widely used clinically. In addition to this effect, pitavastatin has shown the potential to induce apoptosis in cutaneous squamous cell carcinoma (SCC) cells. OBJECTIVE: The purpose of this study is to investigate the effects and possible action mechanisms of pitavastatin. METHODS: SCC cells (SCC12 and SCC13 cells) were treated with pitavastatin, and induction of apoptosis was confirmed by Western blot. To examine whether pitavastatin-induced apoptosis is related to a decrease in the amount of intermediate mediators in the cholesterol synthesis pathway, the changes in pitavastatin-induced apoptosis after supplementation with mevalonate, squalene, geranylgeranyl pyrophosphate (GGPP) and dolichol were investigated. RESULTS: Pitavastatin dose-dependently induced apoptosis of cutaneous SCC cells, but the viability of normal keratinocytes was not affected by pitavastatin at the same concentrations. In supplementation experiments, pitavastatin-induced apoptosis was inhibited by the addition of mevalonate or downstream metabolite GGPP. As a result of examining the effect on intracellular signaling, pitavastatin decreased Yes1 associated transcriptional regulator and Ras homolog family member A and increased Rac family small GTPase 1 and c-Jun N-terminal kinase (JNK) activity. All these effects of pitavastatin on signaling molecules were restored when supplemented with either mevalonate or GGPP. Furthermore, pitavastatin-induced apoptosis of cutaneous SCC cells was inhibited by a JNK inhibitor. CONCLUSION: These results suggest that pitavastatin induces apoptosis of cutaneous SCC cells through GGPP-dependent JNK activation.

Mechanistic Investigation of WWOX Function in NF-kB-Induced Skin Inflammation in Psoriasis.

Psoriasis is a chronic inflammatory skin disease characterized by epidermal hyperproliferation, aberrant differentiation of keratinocytes, and dysregulated immune responses. WW domain-containing oxidoreductase (WWOX) is a non-classical tumor suppressor gene that regulates multiple cellular processes, including proliferation, apoptosis, and migration. This study aimed to explore the possible role of WWOX in the pathogenesis of psoriasis. Immunohistochemical analysis showed that the expression of WWOX was increased in epidermal keratinocytes of both human psoriatic lesions and imiquimod-induced mice psoriatic model. Immortalized human epidermal keratinocytes were transduced with a recombinant adenovirus expressing microRNA specific for WWOX to downregulate its expression. Inflammatory responses were detected using Western blotting, real-time quantitative reverse transcription polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay. In human epidermal keratinocytes, WWOX knockdown reduced nuclear factor-kappa B signaling and levels of proinflammatory cytokines induced by polyinosinic: polycytidylic acid [(poly(I:C)] in vitro. Furthermore, calcium chelator and protein kinase C (PKC) inhibitors significantly reduced poly(I:C)-induced inflammatory reactions. WWOX plays a role in the inflammatory reaction of epidermal keratinocytes by regulating calcium and PKC signaling. Targeting WWOX could be a novel therapeutic approach for psoriasis in the future.

Screening of Plant-Derived Natural Extracts to Identify a Candidate Extract Capable of Enhancing Lipid Synthesis in Keratinocytes.

BACKGROUND: Reduced lipid content in the stratum corneum is a major cause of skin-barrier dysfunction in various pathological conditions. Promoting lipid production is a potential strategy to improve skin-barrier function. Recent evidence supports the beneficial effects of adiponectin on lipid metabolism and senescence in keratinocytes. OBJECTIVE: This study aimed to investigate whether plant extracts can enhance skin-barrier function. METHODS: We screened fruit and herb extracts that enhance the lipid synthesis of keratinocytes via AMP-activated protein kinase (AMPK) activation and SIRT1 signaling in the adiponectin pathway. The levels of major lipid synthesis enzymes and transcription factors as well as epidermal barrier lipids involved in adiponectin-associated epidermal barrier formation were evaluated in the herbal extracts- or adiponectin-treated human epidermal keratinocyte and equivalent models. The mRNA expression of major lipid synthesis enzymes increased following treatment with Lycii Fructus , Prunus tomentosa , and Melia toosendan extracts. RESULTS: The expression of transcription factors SIRT1, liver X receptor α, peroxisome proliferator-activated receptors (PPARs), and sterol regulatory element-binding proteins (SREBPs) were upregulated. Levels of free fatty acids, cholesterol, and ceramides were elevated. The expression of keratinocyte differentiation markers increased. In particular, among fruit extracts with a detectable effect, Melia toosendan induced the highest expression of lipid synthase. CONCLUSION: These results indicate that Melia toosendan is a promising candidate for improving skin-barrier function.

Augmentation of NAD+ by Dunnione Ameliorates Imiquimod-Induced Psoriasis-Like Dermatitis in Mice.

Background: Dunnione has anti-inflammatory properties arising from its ability to alter the ratio of NAD+/NADH through NAD(P)H quinone oxidoreductase 1 (NQO1) enzymatic action, followed by subsequent inhibition of NF-κB and inflammatory cytokines. Psoriasis is a chronic, inflammatory skin disorder in which the IL-23/Th17 axis plays an important role in inflammation. However, it is unclear whether modulation of NAD+ levels affects psoriasis, such as skin inflammation. Therefore, in this study, we investigated the effect of NAD+/NADH ratio modulation on imiquimod (IMQ)-induced, psoriasis-like skin inflammation in mice. Methods: Psoriasis-like skin inflammation was generated by daily topical application of IMQ cream. The severity of dermatitis was assessed using the Psoriasis Area Severity Index (PASI) and histochemistry. Expression of inflammatory cytokines was detected by enzyme-linked immunosorbent assay and quantitative PCR. Acetylation of NF-κB p65 and STAT3 was determined by Western blotting. Results: Dunnione improved IMQ-induced epidermal hyperplasia and inflammation, consistent with decreased levels of inflammatory cytokines (IL-17, IL-22, and IL-23) in skin lesions. Moreover, we found that an increase in the NAD+/NADH ratio by dunnione restored SIRT1 activity, thereby reduced imiquimod-induced STAT3 acetylation, which modulates the expression of psoriasis-promoting inflammatory cytokines, such as IL-17, IL-22, and IL-23. Conclusion: Pharmacological modulation of cellular NAD+ levels could be a promising therapeutic approach for psoriasis-like skin disease.

Role of Substance P in Regulating Micro-Milieu of Inflammation in Alopecia Areata.

BACKGROUND: Alopecia areata (AA) is an autoimmune disease characterized by chronic inflammation, the pathogenesis of which is unknown. Stress is believed to play a role; however, evidence remains insufficient. A recent study showed that substance P (SP) damaged hair follicles by causing neurogenic inflammation, activating perifollicular mast cells, and inducing keratinocyte apoptosis. OBJECTIVE: We aimed at studying the role of SP in AA pathogenesis. We investigated the SP levels in the lesional scalp tissues and serum. We also studied the effect of SP on the inflammatory response and hair growth in the outer root sheath (ORS) cells. METHODS: We compared the serum levels of SP in 58 AA patients and 28 healthy subjects. Then, we checked the expression of SP and SP receptor, neurokinin-1 receptor (NK-1R) in the scalps of AA patients and healthy controls using immunohistochemical staining. Finally, we analyzed the mRNA expression of inflammatory cytokines and hair growth-related factors in ORS cells. RESULTS: SP and NK-1R expression were markedly higher in the hair follicles and interfollicular epidermis of the scalp lesions of AA patients. However, there was no statistically significant difference in serum SP levels between controls and patients, regardless of the type of alopecia. SP significantly increased the mRNA expression of inflammatory cytokines and decreased hair growth-related growth factors in ORS cells, but the results were not dramatic. CONCLUSION: SP triggered a localized micro-inflammation in lesional hair follicles, provoked an inflammatory response, and inhibited hair growth, thereby confirming the pathogenic role of SP in AA.

Inhibitory Effect of Mitoxantrone on Collagen Synthesis in Dermal Fibroblasts.

Background: Fibroblasts produce collagen molecules that support the structure of the skin. The decrease and hypersynthesis of collagen causes skin problems such as skin atrophy, wrinkles and scars. Objective: The purpose of this study is to investigate the mechanism of mitoxantrone on collagen synthesis in fibroblasts. Methods: Cultured fibroblasts were treated with mitoxantrone, and then collagen synthesis was confirmed by reverse transcription-polymerase chain reaction and Western blot. Results: Mitoxantrone inhibited the expression of type I collagen in fibroblasts at both the mRNA and protein levels. In the collagen gel contraction assay, mitoxantrone significantly inhibited gel contraction compared to the control group. Mitoxantrone inhibited transforming growth factor (TGF)-ß-induced phosphorylation of SMAD3. Finally, mitoxantrone inhibited the expression of LARP6, an RNA-binding protein that regulates collagen mRNA stability. Conclusion: These results suggest that mitoxantrone reduces collagen synthesis by inhibiting TGF-ß/SMAD signaling and LARP6 expression in fibroblasts, which can be developed as a therapeutic agent for diseases caused by collagen hypersynthesis.

The potential role of fibroblast-derived multi-peptide factors in activation of growth factors and ß-Catenin in hair follicle cells.

BACKGROUND: Dermal fibroblasts play a pivotal role in hair follicle regeneration during wound repair. Recently, dermal fibroblast-conditioned medium (DFCM), which contains multi-peptide factors (MPFs), has been used to promote wound repair. AIM: This study aimed to investigate the stimulatory effects of MPF-containing DFCM on hair growth. METHODS: MPF-containing DFCM was prepared using human neonatal dermal fibroblasts. Outer root sheath (ORS) and dermal papilla (DP) cells were cultured in MPF-containing DFCM. We examined the expression and secretion of growth factors and cytokines using quantitative polymerase chain reaction and a growth factor array. In addition, the effect of MPFs on ß-catenin activity was determined using the TOPflash assay. All experiments were repeated at least three times with separate batches of cells. RESULTS: MPF-containing DFCM increased keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) mRNA expression in ORS cells and KGF and VEGF mRNA expression in DP cells. When ORS cells were treated with MPF-containing DFCM, the secretion of several growth factors, including EGF, VEGF, insulin-like growth factor-binding protein (IGFBP)-4, IGFBP-6, and fibroblast growth factor-7, was increased in the cell-cultured medium compared with that in control. Additionally, MPF-containing DFCM increased the transcriptional activation of ß-catenin in DP cells. CONCLUSIONS: These results suggest that MPF-containing DFCM might stimulate hair growth by inducing growth factors in ORS and DP cells and regulating ß-catenin in DP cells.

Sorafenib induces pigmentation via the regulation of ß-catenin signalling pathway in melanoma cells.

We conducted large-scale screening test on drugs that were already approved for other diseases to find pigmentation-modulating agents. Among drugs with potential for pigmentation control, we selected sorafenib and further investigated the effect on pigmentation using HM3KO melanoma cells. As a result of treating melanoma cells with sorafenib, pigmentation was promoted in terms of melanin content and tyrosinase activity. Sorafenib increased mRNA and protein levels of pigmentation-related genes such as MITF, tyrosinase and TRP1. To uncover the action mechanism, we investigated the effect of sorafenib on the intracellular signalling pathways. Sorafenib reduced phosphorylation of AKT and ERK, suggesting that sorafenib induces pigmentation through inhibition of the AKT and ERK pathways. In addition, sorafenib significantly increased the level of active ß-catenin, together with activation of ß-catenin signalling. Mechanistic study revealed that sorafenib decreased phosphorylation of serine 9 (S9) of GSK3ß, while it increased phosphorylation of tyrosine 216 (Y216) of GSK3ß. These results suggest that sorafenib activates the ß-catenin signalling through the regulation of GSK3ß phosphorylation, thereby affecting the pigmentation process.

Activation of NLRP3 Inflammasome by Palmitic Acid in Human Sebocytes.

BACKGROUND: Sebocytes are the main cells involved in the pathogenesis of acne by producing lipids and inflammatory cytokines. Although palmitic acid (PA) has been suggested to induce an inflammatory reaction, its effect on sebocytes remains to be elucidated. OBJECTIVE: In the present study, we investigated whether PA promotes inflammasome-mediated inflammation of sebocytes both in vivo and in vitro. METHODS: We intradermally injected PA into the mice ears. And, we treated cultured human sebocytes with PA. Inflammasome-mediated inflammation was verified by immunohistochemistry, Western blot and ELISA. RESULTS: PA-treated mice developed an inflammatory response associated with increased interleukin (IL)-1ß expression in the sebaceous glands. When PA was added to cultured human sebocytes, caspase-1 activation and IL-1ß secretion were significantly enhanced. In addition, NLRP3 knockdown attenuated IL-1ß production by sebocytes stimulated with PA. PA-mediated inflammasome activation required reactive oxygen species. CONCLUSION: These findings indicate that PA activates the NLRP3 inflammasome before induction of an inflammatory response in sebocytes. Thus, PA may play a role in the inflammation of acne.

Azidothymidine Downregulates Insulin-Like Growth Factor-1 Induced Lipogenesis by Suppressing Mitochondrial Biogenesis and Mitophagy in Immortalized Human Sebocytes.

BACKGROUND: Increased sebum secretion is considered the main causative factor in the pathogenesis of acne. There is an unmet pharmacological need for a novel drug that can control sebum production with a favorable adverse effect profile. OBJECTIVE: To investigate the effect of azidothymidine on lipid synthesis in sebocytes and to identify the underlying mechanism of the inhibitory effect of azidothymidine on insulinlike growth factor (IGF)-1-induced lipid synthesis in sebocytes. METHODS: Immortalized human sebocytes were used for the analysis. Thin-layer chromatography (TLC) and Oil Red O staining were performed to evaluate lipid synthesis in the sebocytes. The differentiation, lipid synthesis, mitochondrial biogenesis, and mitophagy in sebocytes were investigated. RESULTS: TLC and Oil Red O staining revealed that azidothymidine reduced IGF-1 induced lipid synthesis in the immortalized human sebocytes. Azidothymidine also reduced IGF-1-induced expression of transcriptional factors and enzymes involved in sebocyte differentiation and lipid synthesis, respectively. Moreover, we found that IGF-1 upregulated the levels of peroxisome proliferator-activated receptorgamma coactivator-1α, LC-3B, p62, and Parkin, major regulators of mitochondrial biogenesis and mitophagy in immortalized human sebocytes. In contrast, azidothymidine inhibited IGF-1 induced mitochondrial biogenesis and mitophagy in the sebocytes. CONCLUSION: These results suggest that azidothymidine downregulates IGF-1-induced lipogenesis by dysregulating the quality of mitochondria through suppression of mitochondrial biogenesis and mitophagy in immortalized human sebocytes. Our study provides early evidence that azidothymidine may be an effective candidate for a new pharmacological agent for controlling lipogenesis in sebocytes.

Potential Role of Cytosolic RNA Sensor MDA5 as an Inhibitor for Keratinocyte Differentiation in the Pathogenesis of Psoriasis.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease. The etiology of psoriasis is not fully understood, but the genetic background is considered to be the most important factor. To date, many psoriasis-related genes have been discovered, but the role of many important genes has not been well understood. OBJECTIVE: The purpose of this study is to uncover possible roles of MDA5 in psoriasis. METHODS: Expression of MDA5 was investigated using immunohistochemistry. Then, MDA5 was overexpressed in keratinocytes using a recombinant adenovirus. RESULTS: As a result of immunohistochemical staining, the expression of MDA5 was significantly increased in the epidermis of psoriasis compared to normal skin. Similarly, the expression of MDA5 was increased in imiquimod-induced psoriasiform dermatitis model. In cultured keratinocytes, toll-like receptor 3 agonist poly(I:C) induced expression of MDA5 at both mRNA and protein levels. When MDA5 was overexpressed using a recombinant adenovirus, poly(I:C)-induced cytokine expression was significantly increased. Finally, MDA5 overexpression significantly inhibited calcium-induced differentiation of keratinocytes. CONCLUSION: These results suggest that MDA5 increases in psoriasis and negatively regulates keratinocyte differentiation.

Adenosine and Cordycepin Accelerate Tissue Remodeling Process through Adenosine Receptor Mediated Wnt/ß-Catenin Pathway Stimulation by Regulating GSK3b Activity.

Adenosine is a cellular metabolite with diverse derivatives that possesses a wide range of physiological roles. We investigated the molecular mechanisms of adenosine and cordycepin for their promoting effects in wound-healing process. The mitochondrial energy metabolism and cell proliferation markers, cAMP responsive element binding protein 1 (CREB1) and Ki67, were enhanced by adenosine and cordycepin in cultured dermal fibroblasts. Adenosine and cordycepin stimulated adenosine receptor signaling via elevated cAMP. The phosphorylation of mitogen-activated protein kinase kinase (MEK) 1/2, mammalian target of rapamycin (mTOR) and glycogen synthase kinase 3 beta (Gsk3b) and Wnt target genes such as bone morphogenetic protein (BMP) 2/4 and lymphoid enhancer binding factor (Lef) 1 were activated. The enhanced gene expression by adenosine and cordycepin was abrogated by adenosine A2A and A2B receptor inhibitors, ZM241385 and PSH603, and protein kinase A (PKA) inhibitor H89, indicating the involvement of adenosine receptor A2A, A2B and PKA. As a result of Wnt/ß-catenin pathway activation, the secretion of growth factors such as insulin-like growth factor (IGF)-1 and transforming growth factor beta (TGFß) 3 was increased, previously reported to facilitate the wound healing process. In addition, in vitro fibroblast migration was also increased, demonstrating their possible roles in facilitating the wound healing process. In conclusion, our data strongly demonstrate that adenosine and cordycepin stimulate the Wnt/ß-catenin signaling through the activation of adenosine receptor, possibly promoting the tissue remodeling process and suggest their therapeutic potential for treating skin wounds.