Anti-bacterial and anti-inflammatory properties of capric acid against Propionibacterium acnes: A comparative study with lauric acid

BackgroundPropionibacterium acnes (P. acnes) is a commensal bacterium which is possibly involved in acne inflammation. The saturated fatty acid, lauric acid (C12:0) has been shown to possess antibacterial and anti-inflammatory properties against P. acnes. Little is known concerning the potential effects of its decanoic counterpart, capric acid (C10:0).ObjectiveTo examine the antibacterial and anti-inflammatory activities of capric acid against P. acnes and to investigate the mechanism of the anti-inflammatory action.MethodsThe antimicrobial activity of fatty acids was detected using the broth dilution method. An evaluation of P. acnes-induced ear edema in mice was conducted to evaluate the in vivo anti-inflammatory effect. To elucidate the in vitro anti-inflammatory effect, human SZ95 sebocytes and monocytic THP-1 cells were treated with P. acnes alone or in the presence of a fatty acid. The mRNA levels and secretion of pro-inflammatory cytokines were measured by qRT-PCR and enzyme immunoassay, respectively. NF-κB activation and MAPK expression were analyzed by ELISA and Western blot, respectively.ResultsLauric acid had stronger antimicrobial activity against P. acnes than capric acid in vitro and in vivo. However, both fatty acids attenuated P. acnes-induced ear swelling in mice along with microabscess and significantly reduced interleukin (IL)-6 and CXCL8 (also known as IL-8) production in P. acnes-stimulated SZ95 sebocytes. P. acnes-induced mRNA levels and secretion of IL-8 and TNF-α in THP-1 cells were suppressed by both fatty acids, which inhibited NF-κB activation and the phosphorylation of MAP kinases.ConclusionOur data demonstrate that both capric acid and lauric acid exert bactericidal and anti-inflammatory activities against P. acnes. The anti-inflammatory effect may partially occur through the inhibition of NF-κB activation and the phosphorylation of MAP kinases.     

Development of In Vitro Co-Culture Model to Mimic the Cell to Cell Communication in Response to Urban PM2.5

BackgroundAirborne particulate matter (PM), a widespread air contaminant, is a complex mixture of solids and aerosols composed of particles suspended in the air. PM is associated with inflammatory responses and may worsen inflammatory skin diseases. However, the mechanisms through which PM affects atopic dermatitis (AD) remain unclear.ObjectiveTo establish an in vitro model that more accurately mimics AD using human keratinocyte (HaCaT), dermal fibroblast (HDF), and mast cell (HMC-1) and using this model to investigate the mechanism through which PMs affect AD.MethodsAn AD-like in vitro model was established by seeding HaCaT, HDF, and HMC-1 cells with recombinant human interleukin (IL)-1α and polyinosinic:polycytidylic acid. We confirmed the effect of PM on the inflammatory cytokine expression of a triple-cell culture model. SRM 1649b Urban Dust, which is mainly composed of polycyclic aromatic hydrocarbons, was used as the reference PM. The effects of PM on the expression levels of proinflammatory cytokines and skin barrier markers were assessed using quantitative real-time polymerase chain reaction and western blotting. Inflammatory cytokine levels were measured using an enzyme-linked immunosorbent assay.ResultsInteractions between various skin cell types were evaluated using a co-culture system. PM treatment increased mRNA and protein levels of the inflammatory cytokines IL-6, IL-1α, tumor necrosis factor-α, IL-4, and IL-1β and decreased the expression of the skin barrier markers filaggrin and loricrin.ConclusionOur results suggest that an in vitro triple-cell culture model using HaCaT, HDF, and HMC-1 cells may be reliable for obtaining more physiological, functional, and reproducible data on AD and skin barriers.     

Mechanism of Macrophage-Derived Chemokine/CCL22 Production by HaCaT Keratinocytes

BackgroundCC chemokine ligand 17 (CCL17) and CCL22 are the functional ligands for CCR4. We previously reported that inhibitors of nuclear factor-kappa B and p38 mitogen-activated protein kinase (p38 MAPK), but not of extracellular signal-related kinase (ERK), inhibited tumor necrosis factor (TNF)-α- and interferon (IFN)-γ-induced production of CCL17 by the human keratinocyte cell line, HaCaT. Further, an inhibitor of epidermal growth factor receptor (EGFR) enhanced the CCL17 production by these keratinocytes.ObjectiveTo identify the mechanism underlying CCL22 production by HaCaT cells.MethodsWe investigated the signal transduction pathways by which TNF-α and IFN-γ stimulate HaCaT cells to produce CCL22 by adding various inhibitors.ResultsTNF-α- and IFN-γ-induced CCL22 production was inhibited by PD98059, PD153035, Bay 11-7085, SB202190, c-Jun N-terminal kinase (JNK) inhibitor II, and Janus kinase (JAK) inhibitor 1.ConclusionOur results indicate that CCL22 production in HaCaT cells is dependent on ERK, EGFR, p38 MAPK, JNK, and JAK and is mediated by different signal pathways from those regulating CCL17 production. Altogether, our previous and present results suggest that EGFR activation represses CCL17 but enhances CCL22 production by these cells.     

Negative Air Ions Alleviate Particulate Matter-Induced Inflammation and Oxidative Stress in the Human Keratinocyte Cell Line HaCaT

BackgroundRecent studies have revealed that particulate matter induces inflammation, oxidative stress, and several skin diseases. Experimental results have also shown that negative air ions are highly effective in removing particulate matter-induced inflammation.ObjectiveThe present study aimed to investigate whether negative air ions can inhibit inflammatory responses and reduce oxidative stress in HaCaT cells exposed to particulate matters.MethodsHaCaT cells were treated with particulate matter in the presence or absence of negative air ions and the viability was evaluated by the MTT assay. Reactive oxygen species (ROS) generation was quantified by the dichlorodihydrofluorescein diacetate assay. The expression of genes and proteins was analyzed by real-time polymerase chain reaction and Western blot. Levels of inflammatory cytokines were quantified by enzyme-linked immunosorbent assay.ResultsNegative air ions were observed to downregulate the mRNA and protein levels of particulate matter-induced pro-inflammatory cytokines in HaCaT cells. In addition, negative air ion treatment suppressed particulate matter-induced intracellular ROS generation, p38 mitogen-activated protein kinase activation, and activator protein 1 (c-Fos and c-Jun) activation.ConclusionOur findings indicate that negative air ions exert anti-inflammatory and antioxidant effects in HaCaT cells exposed to particulate matter. Therefore, negative air ions can be used for the prevention and treatment of particulate matter-related inflammatory skin diseases.     

Botulinum toxin blocks mast cells and prevents rosacea like inflammation

Background

Rosacea is a chronic inflammatory skin condition whose etiology has been linked to mast cells and the antimicrobial peptide cathelicidin LL-37. Individuals with refractory disease have demonstrated clinical benefit with periodic injections of onabotulinum toxin, but the mechanism of action is unknown.

佛波酯对HaCaT细胞环氧合酶-2表达水平的影响

目的 研究佛波酯对培养的人永生化角质形成细胞株HaCaT细胞环氧合酶-2（COX-2）表达水平的影响，探讨佛波酯的皮肤细胞毒性机制。方法 以体外培养的HaCaT细胞为对象，采用RT－PCR和Western印迹方法，检测HaCaT 细胞经0.1，1.0，10 mg/L佛波酯处理24 h后COX-2 mRNA和蛋白表达情况。结果 对照组HaCaT 细胞COX-2 mRNA和蛋白微弱表达或不表达，1.0，10.0 mg/L佛波酯组COX-2 mRNA和蛋白表达水平均显著高于对照组和0.1 mg/L佛波酯组（P值均 < 0.01），而且10.0 mg/L佛波酯组均高于1.0 mg/L佛波酯组，差异均有统计学意义（P < 0.01）。结论 佛波酯可能通过上调HaCaT细胞表达COX-2，促进前列腺素的合成释放，参与皮肤角质形成细胞恶性肿瘤的发生。     

Accelerated human epidermal turnover driven by increased hyaluronan production

BackgroundHyaluronan (HA) is an essential component of extracellular matrix in the skin, but its functions in the epidermis remain elusive.ObjectiveWe examined the interaction of increased HA production mediated by 1-ethyl-β-N-acetylglucosaminide (β-NAG2), a newly developed highly selective inducer of HA production which is intracellularly converted to UDP-N-acetylglucosamine, a substrate of HA, with epidermal proliferation and differentiation.MethodsThe amount, molecular size and epidermal tissue distribution of HA and expression of CD44, a cell surface receptor for HA, were analyzed in β-NAG2-treated organ cultured human skin, reconstructed human skin equivalents or cultured human skin keratinocytes. The relationship between HA and epidermal proliferation or differentiation was examined.Resultsβ-NAG2 significantly increased HA production in the epidermis of skin explants or skin equivalents without affecting molecular size of HA (>2000 kDa) or CD44 mRNA expression. Histochemical experiments revealed that β-NAG2 enhances HA signals in the basal to granular layers of the epidermis of skin equivalents, accompanying increased epidermal stratification. Immunohistochemical experiments demonstrated that signals of Ki67, transglutaminase 1 and filaggrin are increased in β-NAG2-treated skin equivalents, and these observations were confirmed by the data showing that mRNA expression of PCNA, transglutaminase 1 (TGM1) and filaggrin (FLG) is significantly up-regulated by β-NAG2 in skin equivalents. Importantly, blockade of HA production by inhibiting conversion of β-NAG2 to UDP-NAG abolished β-NAG2-mediated up-regulation of PCNA, TGM1 and FLG mRNA expression in cultured keratinocytes.ConclusionThese results suggest that increased epidermal HA production plays a key role in epidermal morphogenesis and homeostasis by accelerating keratinocyte proliferation and differentiation.     

Dieckol Inhibits the Effects of Particulate Matter 10 on Sebocytes,Outer Root Sheath Cells,and Cutibacterium Acnes-Pretreated Mice

BackgroundParticulate matter (PM) is an air pollutant that can impair the human skin. Antioxidants have been tested to improve PM-induced skin inflammation.ObjectiveIn this study, we investigated the effects of dieckol on PM-induced inflammation on cultured human sebocytes, outer root sheath (ORS) cells, and mice pretreated with Cutibacterium acnes.MethodsWe cultured and treated the sebocytes and ORS cells with 5 µM of dieckol and 100 µg/ml of PM10 for 24 h. The C. acnes-pretreated mice received 5 µM of dieckol and 100 µg/ml of PM10. We measured cell viability using MTT assay. Real-time PCR and measurement of reactive oxygen species (ROS) and sebum production analyzed the effects.ResultsDieckol inhibited the upregulation of the gene expression of the inflammatory cytokines, matrix metalloproteinase (MMP), aryl hydrocarbon receptor, and nuclear factor kappa-light-chain-enhancer of activated B cells by PM10 in the cultured sebocytes and ORS cells and inhibited an increase in ROS production by PM10 in the cultured sebocytes. In addition, dieckol decreased the inflammatory cytokines, MMP, and sebum production in C. acnes-pretreated mice.ConclusionDieckol effectively reduced the expression of inflammatory biomarkers and the production of sebum in cultured sebocytes, ORS cells, and C. acnes-pretreated mice.     

Therapeutic Effects and Immunomodulation of Suanbo Mineral Water Therapy in a Murine Model of Atopic Dermatitis

Background

Balneotherapy is widely used as an alternative treatment modality for AD. Although the clinical benefit of some mineral waters has been established, their mechanisms of action in alleviating AD are only partly understood.

Objective

The clinical modification and immunomodulatory or anti-inflammatory effects of mineral water from the Suanbo hot springs on the differentiation and cytokine production of Th1, Th2, and regulatory T cells (T_reg) were investigated using spleen, skin tissue, and serum from NC/Nga mice.

Methods

The therapeutic effects of bathing in mineral water in a Dermatophagoides farinae body extract ointment (Dfb ointment)-induced AD mouse model were assessed by measuring the modified Scoring atopic dermatitis (SCORAD) index scores, transepidermal water loss (TEWL), histological and immunohistochemical changes of the skin lesion, serum levels of interferon (IFN)-γ, interleukin (IL)-4, IL-5 and immunoglobulin E, mRNA expression of IFN-γ, IL-4 and IL-5 of dorsal skin, and helper T cell differentiation in the spleen.

Results

Bathing in mineral water significantly reduced the modified SCORAD index scores, TEWL, epidermal hyperplasia, and inflammatory cell infiltration. IL-4 production and Th2 cell differentiation showed a decreasing tendency with mineral water bathing, but the Th1 cells did not. On the contrary, differentiation to T_reg cells was promoted with mineral water bathing.

Conclusion

Balneotherapy not only has anti-inflammatory activity, but also shows positive effects on cutaneous barrier homeostasis. These results suggest that the favorable effects of balneotherapy may be mediated by modifying the Th2 response, and possibly in part by inducing T_reg cell differentiation.     

MiR-23a regulates DNA damage repair and apoptosis in UVB-irradiated HaCaT cells

BackgroundMiRNAs remain at a constant level under physiological conditions. However, how the expression of miRNAs is regulated and what are the roles of miRNAs in response to UVB damage to skin cells is still not fully understood. In our preliminary study, we observed that miR-23a was upregulated following a treatment with a DNA repair agent and UVB exposure.ObjectiveTo investigate the regulation and function of miR-23a in response to UVB-induced injury in human keratinocyte cell line (HaCaT) cells.MethodsThe changes in expression of miR-23a after UVB irradiation of HaCaT cells were measured by qRT-PCR. The level of miR-23a expression was also modulated by transfecting with a miR-23a mimic or an inhibitor. Cell viability was assessed by the CCK-8 assay. Immunofluorescence staining and Southwestern dot blotting were used to detect the levels of cyclobutane pyrimidine dimers (CPDs). Flow cytometry, Hoechst staining, and measurements of caspase-3 activity were employed to measure the incidence of apoptosis. The mRNA and protein expression levels of genes related to DNA reparation and apoptosis, such as topoisomerase-1, caspase-7, and STK4, were analyzed by qRT-PCR and Western blotting, respectively.ResultsMiR-23a expression was remarkably up-regulated at 4 h and 24 h after the UVB irradiation of HaCaT cells. UVB-induced apoptosis was increased by down-regulation of miR-23a. UVB-induced removal of CPDs was accelerated by miR-23a up-regulation and delayed by miR-23a down-regulation. Forced over-expression of miR-23a decreased the expression of UVB-induced topoisomerase-1\caspase7\STK4 at both the mRNA and protein levels, and these effects were reversed by down-regulation of miR-23a.ConclusionThe protection of HaCaT cells against UVB damage is afforded by miR-23a through regulation of topoisomerase-1\caspase7\STK4, and this miRNA may be a novel therapeutic target in skin diseases related to UVB radiation.     

Propionibacterium acnes-induced iNOS and COX-2 protein expression via ROS-dependent NF-κB and AP-1 activation in macrophages

BackgroundPropionibacterium acnes (P. acnes), a gram-positive anaerobic bacterium, plays a critical role in the development of inflammatory lesion as a result of cytokines production by keratinocytes and macrophages activation. However, effect of P. acnes on iNOS/NO and COX-2/PGE₂ production in macrophages is still uninvestigated.ObjectiveThis study aimed at determining the reactive oxygen species (ROS), inducible nitric oxide (NO) synthase (iNOS)/nitric oxide (NO), and cyclooxygenase (COX)-2/prostaglandin (PG)E₂ produced by macrophages upon P. acnes infection, and dissecting the mechanism of P. acnes-stimulated multiplicity of infection (MOI)-dependent increases in iNOS and COX-2 protein expressions in accordance with the elevation of NO and PGE₂ production by RAW264.7 macrophages.MethodsUsing an in vitro cell culture system, the effects of P. acnes on iNOS/NO, COX-2/PGE₂, ROS production, ERK/JNK, and AP-1/NF-κB activation were examined via Western blotting, a flow cytometric analysis, and luciferase assay. In pharmacological studies, the ROS scavenger, N-acetyl cysteine (NAC), the NADPH oxidase inhibitor, diphenylene iodide (DPI), and mitogen-activated protein kinase (MAPK) inhibitors (U0126 and SP600125) were applied to investigate the mechanism.ResultsWe found that P. acnes exposures increased iNOS/NO and COX-2/PGE₂ expression in RAW264.7, J774A.1, and peritoneal macrophages via a MOI-dependent manner. Increased ROS production, ERK/JNK protein phosphorylation, and elevated AP-1/NF-κB luciferase activity are identified in P. acnes-induced iNOS/NO and COX-2/PGE₂ production. Additionally, hispolon but not its analogs, hispolon methylether or dehydroxyhispolon, showed significant inhibition of P. acnes-induced iNOS/NO and COX-2/PGE₂ production, indicating an important role of OH at C5 for hispolon's inhibition of P. acnes-induced inflammatory events in macrophages.ConclusionROS-dependent stimulation of ERK, JNK, NF-κB, and AP-1 activation contributes to P. acnes-induced iNOS/NO and COX-2/PGE₂ in macrophages, and chemicals such as hispolon possessing ability to block iNOS/NO and COX-2/PGE₂ production reserve potential to be further developed for treatment of the early phase of inflammation elicited by P. acnes.     

Involvement of EGF receptor activation in the induction of cyclooxygenase-2 in HaCaT keratinocytes after UVB

Because selective inhibition of cyclooxygenase-2 (COX-2) suppressed the induction of skin tumors in mice by UV and as UV has been shown to induce expression of COX-2 in skin and cells, COX-2 may be crucial for photocarcinogenesis of the skin. We studied the mechanism of UVB-induced expression of COX-2 focusing on the signal transduction pathway involved. Hydrogen peroxide (H2O2) treatment of HaCaT cells induced expression of COX-2 and pretreatment with the antioxidant N-acetylcysteine (NAC) partly inhibited the UVB-induced expression of COX-2 protein in HaCaT cells, suggesting that oxidative stress contributes to COX-2 induction. To examine the signaling pathways involved in the UVB-induced expression of COX-2 in HaCaT cells, we analysed the expression of COX-2 protein after treatment with various inhibitors of signaling molecules. Inhibition of EGFR by a specific inhibitor and by a neutralizing antibody suppressed the induction of COX-2 expression by UV. Although a neutralizing antibody to transforming growth factor-alpha (TGF-alpha) suppressed COX-2 expression induced by TGF-alpha, it did not suppress COX-2 expression by UV, indicating that a direct activation of EGFR is involved. Treatment of cells at low temperature (4 degrees C) inhibited UVB-induced JNK activation, but it did not inhibit COX-2 expression by UV. Inhibitors of MEK, p38 MAP kinase and PI3-kinase, suppressed the induction of COX-2 expression by UV. In contrast, an erbB-2 inhibitor augmented the UVB-induced increase of COX-2 protein. These data indicate that oxidative stress in association with activation of EGFR, ERK, p38 MAP kinase, and PI3-kinase plays crucial roles in the UVB induction of expression of COX-2.     

Validity of skin blot examination for albumin and nerve growth factor β to detect itching of the skin in Indonesian older adults

AimItching, a common skin disorder, impacts the quality of life of individuals. Itchy skin occurs more with increasing age and the prediction of itchy skin prognosis is necessary to provide good skincare. This study validated biomarkers in skin blotting to identify and measure itching sensation as well as conventional methods to measure skin barrier function.Materials and methodsFrom a cross-sectional study conducted in Long-term Care (LTC) facilities in Indonesia itching symptoms were obtained through a questionnaire. Skin conditions were assessed using photographs, stratum corneum (SC) hydration, skin pH, and skin blotting for biomarkers: albumin, interleukin 2 (IL2), nerve growth factor β (NGFβ), and thymic stromal lymphopoietin (TSLP). Association of skin measurements with the presence of skin blotting and trends analysis were conducted.ResultsAltogether, 564 LTC residents (average age, 70 years) participated. The SC hydration, skin pH, albumin, and NGFβ were associated with the presence of itch (p value= <0.001, <0.001, <0.001, and <0.001, respectively). The signal levels of skin blotting biomarkers were higher in itch group than in the non-itch group. Additionally, the higher quantile of SC hydration was significantly associated with a lower intensity level of NGFβ and TSLP (p value = 0.005, 0.003, respectively). The lower quantile of skin pH (better skin condition) was significantly associated with lower albumin, NGFβ, and TSLP (p value = 0.048, 0.035, and <0.001, respectively).ConclusionThe albumin, NGFβ, and TSLP could be a candidate for measurement of itchy skin among older adult with disrupted skin barrier function and local skin inflammation.     

Negative feedback regulation of phosphatidylinositol 3-kinase/Akt pathway by over-expressed cyclooxygenase-2 in human epidermal cancer cells

While enhanced expression of cyclooxygenase (COX)-2 has been observed in human skin epidermal cancer, the mechanisms underlying COX-2 expression have not been completely elucidated. Recently, a role for the phosphatidylinositol-3 (PI3) kinase pathway in COX-2 expression has attracted attention. We investigated COX-2 expression, PI3 kinase activity, and the phosphorylation level of Akt, a downstream effector of PI3 kinase, in the human skin cancer cell line HSC-5. Compared to the nontumorigenic keratinocyte HaCaT, in HSC-5 cells, COX-2 protein expression and PI3 kinase activity were increased. The PI3 kinase inhibitor LY294002 reduced COX-2 expression in HSC-5 cells and, contrary to our expectation, the phosphorylation of Akt was significantly decreased. The expression of Bcl-2, which is regulated by Akt, was reduced, and apoptosis was induced in HSC-5 cells compared to HaCaT cells. COX-2 inhibitor NS398 up-regulated Akt phosphorylation. These results imply that constitutively over-expressed COX-2 down-regulates the Akt phosphorylation through a negative feedback mechanism.     

Ampelopsis japonica Makino Extract Inhibits the Inflammatory Reaction Induced by Pathogen-Associated Molecular Patterns in Epidermal Keratinocytes

BackgroundKeratinocytes are the major cells in epidermis, providing barrier components such as cornified cells through the sophisticated differentiation process. In addition, keratinocytes exerts their role as the defense cells via activation of innate immunity. It has been known that pathogen-associated molecular patterns (PAMPs) including double-strand RNA and nucleotides can provoke inflammatory reaction in keratinocytes.ObjectiveThe aim of this study is to evaluate the effect of Ampelopsis japonica Makino extract (AE) on PAMPs-induced inflammatory reaction of keratinocytes.MethodsThe effects of AE were determined using poly (I:C)-induced inflammation and imiquimod-induced psoriasiform dermatitis models.ResultsIn cultured keratinocytes, AE significantly inhibited poly(I:C)-induced expression of inflammatory cytokines, such as interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor-α. AE significantly inhibited poly(I:C)-induced release of caspase-1 active form (p20), and down-regulated nuclear factor-κB signaling pathway. In imiquimod-induced psoriasiform dermatitis model, topical application of AE resulted in significant reduction of epidermal hyperplasia.ConclusionThese results suggest that AE may be a potential candidate for the treatment of skin inflammation.     

Keratinocyte-Like Cells Trans-Differentiated from Human Adipose-Derived Stem Cells,Facilitate Skin Wound Healing in Mice

BackgroundMesenchymal stem cells (MSCs) have been reported to promote wound healing in both animal models and human studies. Among MSCs, adipose-derived stem cells (ADSCs) can be easily harvested in large quantities.ObjectiveWe investigated whether skin wound healing in mice can be facilitated by keratinocyte-like cells differentiated from ADSCs (KC-ADSCs).MethodsFor the wound contraction and epithelialization model, a 20 mm×20 mm fullthickness skin wound was made on the dorsum. For the wound epithelialization model, a 6 mm×6 mm full-thickness skin wound was made on the dorsum. A nitrile rubber stent with an inner diameter of 8 mm was sutured around the wounds to minimize wound contraction. Undifferentiated ADSCs (uADSCs) or KC-ADSCs was injected around the wound base in both models. To evaluate whether the injected ADSCs could enhance wound contraction in a skin wound, the contractile activity of ADSCs was assessed by an in vitro type I collagen gel contraction assay. Alpha-smooth muscle actin (αSMA) expressions in uADSCs and KC-ADSCs were also evaluated by flow cytometry and real-time polymerase chain reaction.ResultsIn a wound contraction and epithelialization model, KC-ADSCs further facilitated wound healing compared with uADSCs. In a wound epithelialization model, KC-ADSCs also further facilitated wound epithelialization compared with uADSCs. The contractile activity of KC-ADSCs was lower than that of uADSCs. The uADSCs expressed high levels of αSMA, which decreased after the differentiation into keratinocyte-like cells.ConclusionOur results suggest that the wound healing effect of KC-ADSCs depends primarily on re-epithelialization rather than wound contraction.