Ursodeoxycholic acid alleviates atopic dermatitis-associated inflammatory responses in HaCaT and RBL-2H3 cells and DNCB/DFE-treated mice.

AIMS: Ursodeoxycholic acid (UDCA) is a hydrophilic dihydroxy bile acid used for cholestatic liver disease and exhibits antioxidant, antitumor, and anti-inflammatory effects. However, its potential effects on atopic dermatitis (AD) have not been elucidated. This study aimed to evaluate the efficacy of UDCA in inhibiting the inflammatory response and alleviating lesions in AD-like mice. MAIN METHODS: To investigate the efficacy of UDCA in AD-like inflammatory responses, tumor necrosis factor-alpha (TNF-α)- and interferon-gamma (IFN-γ)-stimulated HaCaT cells and anti-dinitrophenyl immunoglobulin E (DNP-IgE)- and human serum albumin (HSA)-stimulated RBL-2H3 cells were used to investigate the levels of inflammatory factors and their mechanisms. AD-like lesions were induced by applying DNCB/DFE to mice. The effect of UDCA administration in AD-like mice was analyzed by assessing organ weight, serum IgE and inflammatory cytokine levels, and histopathological changes using immunohistochemical and immunofluorescent staining. KEY FINDINGS: In HaCaT cells, UDCA significantly diminished TARC, MDC, MCP-1, and IL-6 expression by inhibiting the phosphorylation of nuclear NF-κB and cytoplasmic IκB, and also increased the levels of skin barrier protein. In RBL-2H3 cells, UDCA reduced ß-hexosaminidase and IL-4 levels. In AD-like mice, UDCA suppressed organ hypertrophy, ear edema, SCORAD index, DFE-specific IgE levels, inflammatory cytokine levels, skin hypertrophy, mast cell invasion, skin barrier loss, and thymic stromal lymphopoietin-positive areas. SIGNIFICANCE: UDCA suppressed the expression of pro-inflammatory cytokines by keratinocytes and mast cells. It also alleviated atopy by suppressing symptoms without organ toxicity in AD-like mice. UDCA may be an effective and safe treatment for AD.

Effect of Bacillus subtilis and Bacillus coagulans spores on induced allergic contact dermatitis in dogs.

BACKGROUND: Probiotic strains have the potential to modulate immune responses, reduce intestinal inflammation, normalize intestinal mucosal function and decrease allergic reactions. OBJECTIVE: This study aimed to investigate the effect of oral probiotic supplements containing Bacillus subtilis and Bacillus coagulans spores on clinical symptoms, haematological factors and immune responses to allergic contact dermatitis in dogs induced by dinitrochlorobenzene (DNCB). METHODS: DNCB was injected subcutaneously into the scapular region of 20 healthy adult dogs of both sexes, divided into four groups, to induce experimental allergic contact dermatitis. Dogs in Group 1 received food without probiotics or medication. Oral prednisolone was administered to Group 2 for 30 days at a dosage of 0.25 mg/kg every other day. The dogs in Group 3 were treated with a combination of oral prednisolone and probiotics. The dogs in Group 4 were fed daily with a mixture of 109 B. subtilis and B. coagulans bacteria for 30 days. The immune system responses and related gene expression were analysed in the treated animals. RESULTS: The administration of probiotics for 30 days resulted in a reduction in clinical symptoms and duration of wound repair. The probiotics treatment also significantly increased the serum bactericidal effects against Staphylococcus aureus and Escherichia coli. It enhanced both the classic and alternative activity of the complement, as well as lysozyme activity. Additionally, the probiotics led to higher total immunoglobulin levels and significant reductions in anti-trypsin and C-reactive protein levels. Furthermore, the expression of IgE, induction of interferon-gamma and IL-4 genes were also reduced. CONCLUSIONS: According to the results, B. subtilis and B. coagulans can be further investigated as a viable alternative to corticosteroids in treating allergic contact dermatitis in dogs.

Identification of Dihydrobenzofuran Neolignans as Novel PDE4 Inhibitors and Evaluation of Antiatopic Dermatitis Efficacy in DNCB-Induced Mice Model.

Atopic dermatitis is a chronic relapsing skin disease characterized by recurrent, pruritic, localized eczema, while PDE4 inhibitors have been reported to be effective as antiatopic dermatitis agents. 3',4-O-dimethylcedrusin (DCN) is a natural dihydrobenzofuran neolignan isolated from Magnolia biondii with moderate potency against PDE4 (IC50 = 3.26 ± 0.28 µM) and a binding mode similar to that of apremilast, an approved PDE4 inhibitor for the treatment of psoriasis. The structure-based optimization of DCN led to the identification of 7b-1 that showed high inhibitory potency on PDE4 (IC50 = 0.17 ± 0.02 µM), good anti-TNF-α activity (EC50 = 0.19 ± 0.10 µM), remarkable selectivity profile, and good skin permeability. The topical treatment of 7b-1 resulted in the significant benefits of pharmacological intervention in a DNCB-induced atopic dermatitis-like mice model, demonstrating its potential for the development of novel antiatopic dermatitis agents.

N-benzyl-N-methyldecan-1-amine, derived from garlic, and its derivative alleviate 2,4-dinitrochlorobenzene-induced atopic dermatitis-like skin lesions in mice.

Given the intricate etiology and pathogenesis of atopic dermatitis (AD), the complete cure of AD remains challenging. This study aimed to investigate if topically applying N-benzyl-N-methyldecan-1-amine (BMDA), derived from garlic, and its derivative [decyl-(4-methoxy-benzyl)-methyl-1-amine] (DMMA) could effectively alleviate AD-like skin lesions in 2,4-dinitrochlorobenzene (DNCB)-treated mice. Administering these compounds to the irritated skin of DNCB-treated mice significantly reduced swelling, rash, and excoriation severity, alongside a corresponding decrease in inflamed epidermis and dermis. Moreover, they inhibited spleen and lymph node enlargement and showed fewer infiltrated mast cells in the epidermis and dermis through toluidine-blue staining. Additionally, they led to a lower IgE titer in mouse sera as determined by ELISA, compared to vehicle treatment. Analyzing skin tissue from the mice revealed decreased transcript levels of inflammatory cytokines (TNF-α, IL-1ß, and IL-6), IL-4, iNOS, and COX-2, compared to control mice. Simultaneously, the compounds impeded the activation of inflammation-related signaling molecules such as JNK, p38 MAPK, and NF-κB in the mouse skin. In summary, these findings suggest that BMDA and DMMA hold the potential to be developed as a novel treatment for healing inflammatory AD.

Immunomodulatory and anti-angiogenesis effects of excavatolide B and its derivatives in alleviating atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin condition primarily driven by T helper 2 (Th2) cytokines, resulting in skin barrier defects, angiogenesis, and inflammatory responses. The marine natural product excavatolide B (EXCB), isolated from the Formosan Gorgonian coral Briareum stechei, exhibits anti-inflammatory and analgesic properties. To enhance solubility, EXCB is chemically modified into the derivatives EXCB-61 salt and EXCB-79. The study aims to investigate the therapeutic effects of these compounds on dinitrochlorbenzene (DNCB)-induced skin damage and to elucidate the underlying anti-inflammatory and anti-angiogenesis mechanism. In vitro, using lipopolysaccharide (LPS)-induced RAW 264.7 cells, all compounds at 10 µM significantly inhibited expression of inflammatory proteins (inducible nitric oxide synthase and cyclooxygenase-2), vascular endothelial growth factor (VEGF), and cytokines (interleukin (IL)-1ß, IL-6, and IL-17A). In vivo, topical application of these compounds on DNCB-induced AD mice alleviated skin symptoms, reduced serum levels of IgE, IL-4, IL-13, IL-17, and interferon-γ, and moderated histological phenomena such as hyperplasia, inflammatory cell infiltration, and angiogenesis. The three compounds restored the expression of skin barrier-related proteins (loricrin, filaggrin, and claudin-1) and reduced the expression of angiogenesis-related proteins (VEGF and platelet endothelial cell adhesion molecule-CD31) in the tissues. This is the first study to indicate that EXCB, EXCB-61 salt, and EXCB-79 can treat AD disease by reducing inflammation and angiogenesis. Hence, they may be considered potential candidates for the development of new drugs for AD.

Proof of concept testing of a positive reference material for in vivo and in vitro sensitization testing of medical devices.

In vivo skin sensitization tests are required to evaluate the biological safety of medical devices in contact with living organisms to provide safe medical care to patients. Negative and positive reference materials have been developed for biological tests of cytotoxicity, implantation, hemolysis, and in vitro skin irritation. However, skin sensitization tests are lacking. In this study, polyurethane sheets containing 1 wt/wt % 2,4-dinitrochlorobenzene (DNCB-PU) were developed and evaluated as a positive reference material for skin sensitization tests. DNCB-PU sheet extracts prepared with sesame oil elicited positive sensitization responses for in vivo sensitization potential in the guinea pig maximization test and the local lymph node assay. Furthermore, DNCB-PU sheet extracts prepared with water and acetonitrile, 10% fetal bovine serum-containing medium, or sesame oil elicited positive sensitization responses as alternatives to animal testing based on the amino acid derivative reactivity assay, human cell line activation test, and epidermal sensitization assay, respectively. These data suggest that the DNCB-PU sheet is an effective extractable positive reference material for in vivo and in vitro skin sensitization testing in medical devices. The formulation of this reference material will lead to the development of safer medical devices that contribute to patient safety.

Erigeron annuus Extract Improves DNCB-Induced Atopic Dermatitis in a Mouse Model via the Nrf2/HO-1 Pathway.

Atopic dermatitis (AD) is a persistent inflammatory skin condition resulting from an intricate interplay among genetic, immunological, and environmental factors. Erigeron annuus (EA), an annual winter plant belonging to the family Asteraceae, possesses anti-inflammatory, cytoprotective, and antioxidant activities. In this study, we hypothesized that Erigeron annuus extract (EAE) could be an effective agent for ameliorating AD-like symptoms. To confirm this hypothesis in vitro, we used H2O2-stimulated human keratinocytes (HaCaT cells) to demonstrate that pre-treatment with EAE protected against oxidative stress. HaCaT cells pretreated with EAE and stimulated with H2O2 showed decreased intracellular malondialdehyde content, increased superoxide dismutase activity, and reduced intracellular reactive oxygen species accumulation. To verify the in vivo hypothesis based on the intracellular results, an AD disease mouse model was induced with 1-chloro-2,4-dinitrobenzene (DNCB), and EAE was orally administered at a non-toxic concentration according to the toxicity evaluation results. The results showed that AD disease models in BALB/c mice exhibited reduced ear epidermal thickness, scratching behavior, and mast cell infiltration. In conclusion, our results indicate that EAE has the potential to improve AD by upregulating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway.

Supercritical CO2 fluid extract from Stellariae Radix ameliorates 2,4-dinitrochlorobenzene-induced atopic dermatitis by inhibit M1 macrophages polarization via AMPK activation.

Yin chai hu (Radix Stellariae) is a root medicine that is frequently used in Chinese traditional medicine to treat fever and malnutrition. In modern medicine, it has been discovered to have anti-inflammatory, anti-allergic, and anticancer properties. In a previous study, we were able to extract lipids from Stellariae Radix using supercritical CO2 extraction (SRE), and these sterol lipids accounted for up to 88.29% of the extract. However, the impact of SRE on the development of atopic dermatitis (AD) has not yet been investigated. This study investigates the inhibitory effects of SRE on AD development using a 2,4-dinitrochlorobenzene (DNCB)-induced AD mouse model. Treatment with SRE significantly reduced the dermatitis score and histopathological changes compared with the DNCB group. The study found that treatment with SRE resulted in a decrease of pro-inflammatory cytokines TNF-α, CXC-10, IL-12, and IL-1ß in skin lesions. Additionally, immunohistochemical analysis revealed that SRE effectively suppressed M1 macrophage infiltration into the AD lesion. Furthermore, the anti-inflammatory effect of SRE was evaluated in LPS + INF-γ induced bone marrow-derived macrophages (BMDMs) M1 polarization, SRE inhibited the production of TNF-α, CXC-10, IL-12, and IL-1ß and decreased the expression of NLRP3. Additionally, SRE was found to increase p-AMPKT172, but had no effect on total AMPK expression, after administration of the AMPK inhibitor Compound C, the inhibitory effect of SRE on M1 macrophages was partially reversed. The results indicate that SRE has an inhibitory effect on AD, making it a potential therapeutic agent for this atopic disorder.

Antioxidant, Antibacterial Properties of Novel Peptide CP by Enzymatic Hydrolysis of Chromis notata By-Products and Its Efficacy on Atopic Dermatitis.

This study investigated the antioxidant, antimicrobial, and anti-atopic dermatitis (AD) effects of a novel peptide (CP) derived from a Chromis notata by-product hydrolysate. Alcalase, Flavourzyme, Neutrase, and Protamex enzymes were used to hydrolyze the C. notata by-product protein, and the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical-scavenging activity was measured. Alcalase hydrolysate exhibited the highest ABTS radical-scavenging activity, leading to the selection of Alcalase for further purification. The CHAO-1-I fraction, with the highest ABTS activity, was isolated and further purified, resulting in the identification of the peptide CP with the amino acid sequence Ala-Gln-Val-Met-Lys-Leu-Pro-His-Arg-Met-Gln-His-Ser-Gln-Ser. CP demonstrated antimicrobial activity against Staphylococcus aureus, inhibiting its growth. In a 2,4-dinitrochlorobenzene (DNCB)-induced AD-like skin model in mice, CP significantly alleviated skin lesions, reduced epidermal and dermal thickness, and inhibited mast cell infiltration. Moreover, CP suppressed the elevated levels of interleukin-6 (IL-6) in the plasma of DNCB-induced mice. These findings highlight the potential of CP as a therapeutic agent for AD and suggest a novel application of this C. notata by-product in the fish processing industry.

[MOR106 alleviates inflammation in mice with atopic dermatitis by blocking the JAK2/STAT3 signaling pathway and inhibiting IL-17C-mediated Tfh cell differentiation].

Objective To explore the significance of interleukin-17C(IL-17C)-mediated follicular helper T cell (Tfh) differentiation in atopic dermatitis (AD) model. Methods BALB/c mice were divided into control group, AD model group, low-dose MOR106 (anti-IL-17C huIgG1)(MDR106-L)treatment group and high-dose MOR106 (MOR106-H) treatment group, 8 mice in each group. Except for the control group, all the other groups were treated with 2, 4- dinitrochlorobenzene (DNCB) to establish AD models. The low-dose and high-dose MOR106 groups were treated with 5 mg/kg or 10 mg/kg MOR106 respectively. The differentiation of Tfh cell subsets in peripheral blood of mice was analyzed by flow cytometry, and the expression of Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3) signal pathway protein in skin tissue was detected by Western blot analysis. Results Compared with the control group, the dermatitis severity score, mass difference between two ears, spleen mass and spleen index of DNCB group increased significantly, while those of MOR106-L group and MOR106-H group decreased significantly. Compared with the control group, the Tfh subgroup of AD mice showed deregulated differentiation, resulting in a significant increase in the percentage of CD4+CXCR5+IFN-γ+Tfh1 cells, CD4+CXCR5+IL-17A+Tfh17 and CD4+CXCR5+IL-21+Tfh21 cells, and a significant decrease in the percentage of CD4+CXCR5+IL-10+Tfh10 cells and CD4+CXCR5+FOXP3+Tfr cells in peripheral blood. The protein levels of phosphorylated JAK2(p-JAK2) and p-STAT3 were significantly increased. MOR106 effectively reversed these changes of Tfh1, Tfh10, Tfh17, Tfh21 and Tfr cells in peripheral blood of AD mice. Compared with AD group, the levels of p-JAK2 and p-STAT3 protein in low-dose and high-dose MOR106 treatment groups decreased significantly. Conclusion MOR106 can reduce the inflammatory response of AD mice by blocking JAK2/STAT3 signaling pathway and inhibiting the differentiation of Tfh cells mediated by IL-17C.

Qing-Re-Chu-shi decoction ameliorates 2,4-dinitrochlorobenzene-induced atopic dermatitis in NC/Nga mice through anti-inflammation and immunoregulatory mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE: Qing-Re-Chu-Shi Decoction (QRCSD), a traditional Chinese herbal formula, has been employed as a complementary and alternative therapy for inflammatory skin diseases. However, its active constituents and the mechanistic basis of its action on atopic dermatitis remain in adequately understood. AIM OF THE STUDY: Atopic dermatitis (AD) is an allergic dermatitis marked by eczematous lesions and pruritus. The study aimed to elucidate the underlying effects of QRCSD on AD and to identify the components responsible for its therapeutic efficacy in a mouse model. MATERIALS AND METHODS: Network pharmacology and UPLC-mass analysis were used to anticipate the pharmacological mechanisms and to identify active components of QRCSD, respectively. A DNCB-induced AD-like model was established in NC/Nga mice. QRCSD or prednisolone (as a positive control) was administered via gavage every other day from day14 to day 21. Dermatitis severity score, scratching behavior, skin barrier function, spleen index, Th1/Th2 lymphocyte ratio, and serum IgE levels were evaluated. Protein arrays, including 40 inflammatory cytokines, were performed on skin lesions, followed by confirmation experiments of Western blotting in dorsal skin lesions. RESULTS: The construction of a QRCSD-AD-Network and topological analysis firstly proposed potential targets of QRCSD acting on AD. Animal experiments demonstrated that oral administration of QRCSD ameliorated AD-like lesions, reduced epidermal thickness and mast cell count, decreased serum IgE levels, augmented tight junction protein (Claudin 1, Occludin) levels, and regulated the Th1/Th2 balance in the spleen, as well as spleen index. Elevated levels of interleukin (IL)-4, IL-5, IL-6, IL-17, and Eotaxin were revealed in AD-like skin lesions by protein arrays. Western blotting confirmed that the phosphorylation levels of ERK, P38, JNK, STAT3 and P65 were downregulated, and IL-6 expression was also reduced following QRCSD treatment. CONCLUSIONS: The study enhances the understanding of the anti-inflammatory and immunomodulatory effects of QRCSD, showcasing its significant protective role against atopic dermatitis. Treatment with QRCSD may be considered as a viable candidate for complementary and alternative therapy in managing atopic dermatitis.

Exosomes derived from human dermal fibroblasts (HDFn-Ex) alleviate DNCB-induced atopic dermatitis (AD) via PPARα.

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Skin barrier dysfunction is the initial step in the development of AD. Recently, exosomes have been considered as potential cell-free medicine for skin defects such as aging, psoriasis and wounds. The aim of this study was to investigate the effects of human dermal fibroblast-neonatal-derived exosome (HDFn-Ex) on AD. HDFn-Ex increased the expression of peroxisome proliferator activated receptor α (PPARα) and alleviated the 1-chloro-2,4-dinitrobenzene (DNCB)-mediated downregulation of filaggrin, involucrin, loricrin, hyaluronic acid synthase 1 (HAS1) and HAS2 in human keratinocyte HaCaT cells. However, these effects were inhibited by the PPARα antagonist GW6471. In the artificial skin model, HDFn-Ex significantly inhibited DNCB-induced epidermal hyperplasia and the decrease in filaggrin and HAS1 levels via a PPARα. In the DNCB-induced AD-like mouse model, HDFn-Ex administration reduced epidermis thickening and mast cell infiltration into the dermis compared to DNCB treatment. Moreover, the decreases in PPARα, filaggrin and HAS1 expression, as well as the increases in IgE and IL4 levels induced by DNCB treatment were reversed by HDFn-Ex. These effects were blocked by pre-treatment with GW6471. Furthermore, HDFn-Ex exhibited an anti-inflammatory effect by inhibiting the DNCB-induced increases in IκBα phosphorylation and TNF-α expression. Collectively, HDFn-Ex exhibited a protective effect on AD. Notably, these effects were regulated by PPARα. Based on our results, we suggest that HDFn-Ex is a potential candidate for treating AD by recovering skin barrier dysfunction and exhibiting anti-inflammatory activity.

Evaluation the effect of dietary vitamin E, sesamin and thymoquinone bioactive compounds on immunological response, intestinal traits and MUC-2 gene expression in broiler Japanese quails (Coturnix japonica).

The current study was performed to determine the effect of dietary vitamin E, sesamin and thymoquinone bioactive lignans derived from sesame and black seed on immunological response, intestinal traits and Mucin2 gene expression in broiler quails. Three hundred and fifty (one days-old) quails were allotted to seven dietary treatments with five replicates as an experimental randomized design study. Treatments were basal diet as a control, control +100 and +200 mg of vitamin E, sesamin and thymoquinone per each kg of diet respectively. At 35 d of age, two quails from each pen were chosen, weighted, slaughtered, eviscerated and lymphoid organ relative weights were measured. Anti-body titers against Newcastle disease (ND), Sheep red blood cell (SRBC), and infectious bronchitis virus (IBV) and Avian influenza (AI) vaccination were determined. The serum activities of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and serum antioxidant activates such as superoxide dismutase (SOD),glutathione peroxidase(GPX), catalase (CAT) and total antioxidant capacity (TAC) were examined. The cell mediated immunity by dinitrochlorobenzene (DNCB) and phytohemagglutinin (PHA) challenges were assessed. The microflora populations of ileum, morphological traits of jejunum and mucin2 gene expression were analyzed. Data showed that the lymphoid organ (thymus, spleen and Bursa) relative weights and antibody titer against HI, AI, SRBC and IB vaccination were increased compared to the control (p ≤ 0.05). Serum activities of ALP, ALT and AST were decreased under influences of dietary treatments (p ≤ 0.05). The serum antioxidant activates of GPX,SOD,CAT and TAC were increased and Increasing in mean skin thickness after DNCB challenge and decrease wing web swelling response to PHA mitojen injection were observed (p ≤ 0.05). Salmonella enterica, E-coli and Coliforms colonies were decrease and Lactobacillus colonies increased instead (p ≤ 0.05). The villus height and surface, crypt depth and goblet cells density were increased compared to the control (p ≤ 0.05). The expression of MUC2 gene increased under influnces of vitamin E, sesamin and thymoquinone supplemented diets (p ≤ 0.05).

Systemic Type 2 Inflammation-Associated Atopic Dermatitis Exacerbates Periodontitis.

INTRODUCTION: Atopic dermatitis (AD) is a prevalent and chronic inflammatory skin disease characterized by Th2 cell-mediated type 2 inflammation. Emerging evidence indicated that AD patients exhibit an increased incidence of oral disorders. In the present study, we sought mechanistic insights into how AD affects periodontitis. METHODS: Onset of AD was induced by 2,4-dinitrochlorobenzene (DNCB). Furthermore, we induced periodontitis (P) in AD mice. The effect of AD in promoting inflammation and bone resorption in gingiva was evaluated. Hematoxylin and eosin staining, tartrate-resistant acid phosphatase staining, immunofluorescence assay, and flow cytometry were used to investigate histomorphology and cytology analysis, respectively. RNA sequencing of oral mucosa is used tissues to further understand the dynamic transcriptome changes. 16S rRNA microbial analysis is used to profile oral microbial composition. RESULTS: Compared to control group, mice in AD group showed inflammatory signatures and infiltration of a proallergic Th2 (Th2A)-like subset in oral mucosa but not periodontitis, as identified by not substantial changes in mucosa swelling, alveolar bone loss, and TRAP+ osteoclasts infiltration. Similarly, more Th2A-like cell infiltration and interleukin-4 levels were significantly elevated in the oral mucosa of DNCB-P mice compared to P mice. More importantly, AD exacerbates periodontitis when periodontitis has occurred and the severity of periodontitis increased with aggravation of dermatitis. Transcriptional analysis revealed that aggravated periodontitis was positively correlated with more macrophage infiltration and abundant CCL3 secreted. AD also altered oral microbiota, indicating the re-organization of extracellular matrix. CONCLUSIONS: These data provide solid evidence about exacerbation of periodontitis caused by type 2 dermatitis, advancing our understanding in cellular and microbial changes during AD-periodontitis progression.

Inhibitory effects of catalpol on DNCB-induced atopic dermatitis and IgE-mediated mast cells reaction.

Atopic dermatitis (AD) is a chronic, inflammatory cutaneous disease driven by immune dysregulation. Catalpol is an iridoids, possessing anti-inflammatory, antioxidant, and neuroprotective activities. It can be added to food as a dietary supplement. To evaluate the effects and mechanisms of catalpol on AD, both in vitro and in vivo studies were conducted. It was found that catalpol downregulated the phosphorylation of Lyn and Syk to inhibit various downstream pathways, including intracellular Ca2+ elevation, cytokines generation, and histamine release, which ultimately controlled mast cell (MCs) degranulation. The results showed that catalpol alleviated AD-like skin lesions and MC infiltration via regulation of pro-Th2 and Th2 cytokines in vivo. Furthermore, this compound reduced the levels of IgE in AD mice and improved allergic reactions in PCA mice. The results provided that catalpol was potentially developed as a dietary supplement to improve AD and other atopic diseases.

Histone deacetylase 3 inhibition alleviates 2,4-dinitrochlorobenzene-induced atopic dermatitis via epigenetically upregulating Nrf2/HO-1 signaling pathway.

Atopic dermatitis (AD) is a frequent skin disorder that is associated with immune dysfunction and skin inflammation. Histone deacetylase 3 (HDAC3) possesses strong immune and inflammatory modulatory properties in multiple diseases. However, the role and mechanism of HDAC3 in AD remain unknown. Here, we reported that HDAC3 expression was aberrantly upregulated in 2,4-dinitrochlorobenzene (DNCB)-induced lesional AD skin in mice. Inhibition of HDAC3 by RGFP966 protected against DNCB-induced AD, indicated by improved histological damages, relieved inflammatory and immune dysfunction. Nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) signaling pathway activity in lesional AD skin was significantly decreased and RGFP966 attenuated the decrease. Inhibition of Nrf2/HO-1 signaling pathway via Nrf2 inhibitor ML385 blunted anti-AD effect of RGFP966 in DNCB-treated mice. Mechanistically, RGFP966 promoted Nrf2 expression and upregulated H3K27ac deposition on the promoter region of Nrf2. Collectively, HDAC3 inhibition protects against AD via epigenetically activating Nrf2 transcription to upregulate Nrf2/HO-1 signaling pathway activity. HDAC3 may act as a promising therapeutic target for the treatment of AD.

Characterization of Different Inflammatory Skin Conditions in a Mouse Model of DNCB-Induced Atopic Dermatitis.

The mouse model of 2,4-dinitrochlorbenzene (DNCB)-induced human-like atopic dermatitis (hlAD) has been widely used to test novel treatment strategies and compounds. However, the study designs and methods are highly diverse, presenting different hlAD disease patterns that occur after sensitization and repeated challenge with DNCB on dorsal skin. In addition, there is a lack of information about the progression of the disease during the experiment and the achieved pheno- and endotypes, especially at the timepoint when therapeutic treatment is initiated. We here examine hlAD in a DNCB-induced BALB/cJRj model at different timepoints: (i) before starting treatment with dexamethasone, representing a standard drug control (day 12) and (ii) at the end of the experiment (day 22). Both timepoints display typical AD-associated characteristics: skin thickening, spongiosis, hyper- and parakeratosis, altered cytokine and gene expression, increased lipid mediator formation, barrier protein and antimicrobial peptide abnormalities, as well as lymphoid organ hypertrophy. Increased mast cell infiltration into the skin and elevated immunoglobulin E plasma concentrations indicate a type I allergy response. The DNCB-treated skin showed an extrinsic moderate sub-acute hlAD lesion at day 12 and an extrinsic mild sub-acute to chronic pheno- and endotype at day 22 with a dominating Th2 response. A dependency of the filaggrin formation and expression in correlation to the disease severity in the DNCB-treated skin was found. In conclusion, our study reveals a detailed classification of a hlAD at two timepoints with different inflammatory skin conditions and pheno- and endotypes, thereby providing a better understanding of the DNCB-induced hlAD model in BALB/cJRj mice.

Epidermal keratinocyte-specific STAT3 deficiency aggravated atopic dermatitis-like skin inflammation in mice through TSLP upregulation.

Atopic dermatitis (AD) is one of the most common inflammatory skin diseases with complex pathogenesis involving epidermal barrier dysfunction, skin microbiome abnormalities and type-2-skewed immune dysregulation. Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that plays critical roles in various biological processes. However, the role of STAT3 in epidermal keratinocytes in AD remains unclear. In this study, we generated an epidermal keratinocyte-specific Stat3-deficient mouse strain (termed Stat3 cKO mice). After topical 2,4-dinitrochlorobenzene (DNCB) treatment, Stat3 cKO mice developed worsened AD-like skin inflammation with increased Ki67+ cells, decreased filaggrin and loricrin expression, and downregulated S100A9 and LL37. The dominant microbial population in Stat3 cKO mice changed from Ralstonia to Staphylococcus. DNCB-treated Stat3 cKO mice displayed more infiltrating type-2 inflammatory cells, including mast cells, eosinophils, and CD4+T cells, accompanied by increased skin IL-4 and serum IgE levels. Moreover, thymic stromal lymphopoietin (TSLP), mainly produced by keratinocytes, was highly expressed in the ear skin of Stat3 cKO mice and chemoattracted more TSLPR+ cells. TSLP blockade significantly alleviated DNCB-induced AD-like skin inflammation in Stat3 cKO mice. Thus, epidermal keratinocyte-specific STAT3 deficiency can aggravate AD-like skin inflammation in mice, possibly through TSLP dysregulation.

Anti-Atopic Dermatitis Effect of TPS240, a Novel Therapeutic Peptide, via Suppression of NF-κB and STAT3 Activation.

Atopic dermatitis (AD) is a relapsing skin disease with persistent inflammation as a causal factor for symptoms and disease progression. Current therapies provide only temporary relief and require long-term usage accompanied by side effects due to persistent relapses. A short peptide, TPS240, has been tested for its potential to subside AD. In this study, we confirmed the anti-atopic effect of TPS240 in vivo and in vitro using a DNCB-induced AD mouse model and TNF-α/IFN-γ-stimulated HaCaT cells. In the AD mouse model, topical treatment with TPS240 diminished AD-like skin lesions and symptoms such as epidermal thickening and mast cell infiltration induced by DNCB, similar to the existing treatment, dexamethasone (Dex). Furthermore, skin atrophy, weight loss, and abnormal organ weight changes observed in the Dex-treated group were not detected in the TPS240-treated group. In TNF-α/IFN-γ-stimulated HaCaT cells, TPS240 reduced the expression of the inflammatory chemokines CCL17 and CCL22 and the pruritic cytokines TSLP and IL-31 by inhibiting NF-κB and STAT3 activation. These results suggest that TPS240 has an anti-atopic effect through immunomodulation of AD-specific cytokines and chemokines and can be used as a candidate drug for the prevention and treatment of AD that can solve the safety problems of existing treatments.

A human 3D immune competent full-thickness skin model mimicking dermal dendritic cell activation.

We have integrated dermal dendritic cell surrogates originally generated from the cell line THP-1 as central mediators of the immune reaction in a human full-thickness skin model. Accordingly, sensitizer treatment of THP-1-derived CD14-, CD11c+ immature dendritic cells (iDCs) resulted in the phosphorylation of p38 MAPK in the presence of 1-chloro-2,4-dinitrobenzene (DNCB) (2.6-fold) as well as in degradation of the inhibitor protein kappa B alpha (IκBα) upon incubation with NiSO4 (1.6-fold). Furthermore, NiSO4 led to an increase in mRNA levels of IL-6 (2.4-fold), TNF-α (2-fold) and of IL-8 (15-fold). These results were confirmed on the protein level, with even stronger effects on cytokine release in the presence of NiSO4: Cytokine secretion was significantly increased for IL-8 (147-fold), IL-6 (11.8-fold) and IL-1ß (28.8-fold). Notably, DNCB treatment revealed an increase for IL-8 (28.6-fold) and IL-1ß (5.6-fold). Importantly, NiSO4 treatment of isolated iDCs as well as of iDCs integrated as dermal dendritic cell surrogates into our full-thickness skin model (SM) induced the upregulation of the adhesion molecule clusters of differentiation (CD)54 (iDCs: 1.2-fold; SM: 1.3-fold) and the co-stimulatory molecule and DC maturation marker CD86 (iDCs ~1.4-fold; SM:~1.5-fold) surface marker expression. Noteworthy, the expression of CD54 and CD86 could be suppressed by dexamethasone treatment on isolated iDCs (CD54: 1.3-fold; CD86: 2.1-fold) as well as on the tissue-integrated iDCs (CD54: 1.4-fold; CD86: 1.6-fold). In conclusion, we were able to integrate THP-1-derived iDCs as functional dermal dendritic cell surrogates allowing the qualitative identification of potential sensitizers on the one hand, and drug candidates that potentially suppress sensitization on the other hand in a 3D human skin model corresponding to the 3R principles ("replace", "reduce" and "refine").