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.

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.

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.

PYR-41, an inhibitor of ubiquitin-activating enzyme E1, attenuates 2,4-dinitrochlorobenzene-induced atopic dermatitis-like skin lesions in mice.

PYR-41 is an irreversible and cell permeable inhibitor of ubiquitin-activating enzyme E1, and has been reported to inhibit the degradation of IκB protein. Previous studies have shown that PYR-41 has effects on anti-inflammatory, but whether it has therapeutic effects on allergic dermatitis is unclear. The aim of this research was to explore the therapeutic effects of PYR-41 on atopic dermatitis. The effects of PYR-41 on the activation of NF-κB signaling pathway and the expression of inflammatory genes in HaCat cells were tested by western blot and qPCR. A mouse model was built, and the AD-like skin lesions were induced by 2,4-dinitrochlorobenzene (DNCB). Then, the treatment effects of PYR-41 were examined by skin severity score, ear swelling, ELISA, and qPCR. The results showed that PYR-41 can significantly reduce the K63-linked ubiquitination level of nuclear factor-κB essential modulator (NEMO) and tumor necrosis factor receptor associated factor 6 (TRAF6), inhibit the proteasomal degradation of IκBα, thereby activate TNF-α-induced NF-κB signaling pathway in HaCat cells. In addition, DNCB-treated mice have significant reduction in symptoms after treated by PYR-41, including reduced ear thickening and reduced skin damage. Serum tests showed that PYR-41 significantly reduced the expression of IgE, IFN-γ, and TNF-α. In conclusion, the current results suggest that PYR-41 has potential to reduce the symptoms of atopic dermatitis.

Ozonated oil alleviates dinitrochlorobenzene-induced allergic contact dermatitis via inhibiting the FcεRI/Syk signaling pathway. / 医用臭氧油通过抑制FcεRI/Syk信号通路缓解DNCB诱导的变应性接触性皮炎（英文）.

OBJECTIVES: Ozone is widely applied to treat allergic skin diseases such as eczema, atopic dermatitis, and contact dermatitis. However, the specific mechanism remains unclear. This study aims to investigate the effects of ozonated oil on treating 2,4-dinitrochlorobenzene (DNCB)-induced allergic contact dermatitis (ACD) and the underling mechanisms. METHODS: Besides the blank control (Ctrl) group, all other mice were treated with DNCB to establish an ACD-like mouse model and were randomized into following groups: a model group, a basal oil group, an ozonated oil group, a FcεRI-overexpressed plasmid (FcεRI-OE) group, and a FcεRI empty plasmid (FcεRI-NC) group. The basal oil group and the ozonated oil group were treated with basal oil and ozonated oil, respectively. The FcεRI-OE group and the FcεRI-NC group were intradermally injected 25 µg FcεRI overexpression plasmid and 25 µg FcεRI empty plasmid when treating with ozonated oil, respectively. We recorded skin lesions daily and used reflectance confocal microscope (RCM) to evaluate thickness and inflammatory changes of skin lesions. Hematoxylin-eosin (HE) staining, real-time PCR, RNA-sequencing (RNA-seq), and immunohistochemistry were performed to detct and analyze the skin lesions. RESULTS: Ozonated oil significantly alleviated DNCB-induced ACD-like dermatitis and reduced the expressions of IFN-γ, IL-17A, IL-1ß, TNF-α, and other related inflammatory factors (all P<0.05). RNA-seq analysis revealed that ozonated oil significantly inhibited the activation of the DNCB-induced FcεRI/Syk signaling pathway, confirmed by real-time PCR and immunohistochemistry (all P<0.05). Compared with the ozonated oil group and the FcεRI-NC group, the mRNA expression levels of IFN-γ, IL-17A, IL-1ß, IL-6, TNF-α, and other inflammatory genes in the FcεRI-OE group were significantly increased (all P<0.05), and the mRNA and protein expression levels of FcεRI and Syk were significantly elevated in the FcεRI-OE group as well (all P<0.05). CONCLUSIONS: Ozonated oil significantly improves ACD-like dermatitis and alleviated DNCB-induced ACD-like dermatitis via inhibiting the FcεRI/Syk signaling pathway.

Expression of interleukin-1ß, interleukin-4, interferon-γ and tumour necrosis factor α in different tissue in a dinitrochlorobenzene-induced ear swelling test in mice.

OBJECTIVE: To study the expression of interleukin-1ß (IL-1ß), interleukin-4 (IL-4), interferon-γ (IFN-γ) and tumour necrosis factor α (TNF-α) in different tissue in a dinitrochlorobenzene (DNCB)-induced ear swelling test in mice and further evaluate the correlation between the cytokine expression in different tissues and the degree of ear swelling. METHODS: The mice were sensitised with a 0.50% DNCB solution on their back for 3 days. After 7 days, the thickness of their ears was measured and grouped. Different concentrations of the DNCB solution were challenged in the left ear of each group of mice, and the right ear was used as the control. The thickness of both ears was measured every 24 h, and the mice were sacrificed 72 h after the challenge. The expressions of IL-1ß, IL-4, IFN-γ and TNF-α in the mouse serum, lymph node and ear tissue were quantified by enzyme-linked immunosorbent assay, respectively. RESULTS: There was a linear positive correlation between the swelling index of the mouse lateral ear and the challenge concentration of DNCB (r = 0.96, p < 0.01). The high expression of IL-1ß and IL-4 in the lateral ear tissue of the mice was positively correlated with the ear swelling index 48 h after the challenge. The correlation coefficient was 0.78 (p < 0.01). Furthermore, IFN-γ and TNF-α had no significant correlation with the ear swelling index 48 h after the challenge. CONCLUSION: There is a correlation between the degree of ear swelling in mice and the concentration of DNCB and the expression of IL-1ß and IL-4 in the lateral ear tissue. There is a sub-clinical skin sensitivity state in contact allergy.

Oral Cadmium Intake Enhances Contact Allergen-induced Skin Reaction in Rats.

Objective: The effect of oral cadmium (Cd) intake to influence contact skin allergies was examined, since it is known that Cd is a heavy metal that affects many tissues, including the skin, in which it disturbs homeostasis, thus resulting in inflammation and injury. Methods: Male rats were evoked with experimental contact hypersensitivity reaction (CHS) to hapten dinitrochlorobenzene (DNCB), after prolonged (30 day) oral exposure to an environmentally relevant Cd dose (5 ppm). The ear cell population was analyzed with flow cytometry. Cytokine production by ear skin cells and the activity of skin-draining lymph node (DLN) cells were measured using enzyme-linked immunosorbent assay (ELISA). Results: Orally acquired Cd (5 ppm) increased CHS intensity only in Dark Agouti (DA) rats by affecting inflammatory responses in both the sensitization (an increase of IFN-γ and IL-17 cytokine production) and challenge (an increase of CD8 + and CD4 + cell number and TNF, IFN-γ and IL-17 cytokine production) phases. An increased CHS reaction was seen in Albino Oxford (AO) rats only at a high Cd dose (50 ppm), during the challenge phase (an increase of CD8 + and CD4 + cell number and TNF, IFN-γ and IL-17 cytokine production). Conclusion: These novel data indicate that oral Cd intensifies the skin response to sensitizing chemicals such as DNCB.

Evaluation of the Anti-Atopic Dermatitis Effects of α-Boswellic Acid on Tnf-α/Ifn-γ-Stimulated HaCat Cells and DNCB-Induced BALB/c Mice.

Boswellic acids, triterpenoids derived from the genus Boswellia (Burseraceae), are known for their anti-inflammatory and anti-tumor efficacy. Atopic dermatitis is a chronic, non-infectious inflammatory skin disease. However, the effects of α-boswellic acid on atopic dermatitis have not been studied. Therefore, in this study we examined the expression level of pro-inflammatory cytokines, histopathological analysis, and physiological data from BALB/c mice with atopic-like dermatitis induced by 2,4-dinitrochlorobenzene and TNF-α/IFN-γ-stimulated HaCaT cells to better understand the agent's anti-atopic dermatitis efficacy. First, we found that α-boswellic reduced the epidermal thickening, mast cell numbers, and dermal infiltration of 2,4-dinitrochlorobenzene-induced atopic-like dermatitis in BALB/c mice. Furthermore, we also found that α-boswellic acid can restore transepidermal water loss and skin reddening in mice. In human keratinocytes inflamed by TNF-α/IFN-γ, α-boswellic acid inhibited MAP kinase activation and showed a reduction in NF-κB nuclear translocation. Finally, α-boswellic acid can reduce the expression level of cytokines (IL-1ß, IL-6, and IL-8) following the stimulation of TNF-α/IFN-γ in HaCaT cells. Taken together, our study suggests that α-boswellic acids are a potential component for the development of anti-atopic dermatitis drugs.

Artemisia annua water extract attenuates DNCB-induced atopic dermatitis by restraining Th2 cell mediated inflammatory responses in BALB/c mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua L. (A. annua) is a traditional Chinese medicine that has been used since ancient times to treat malaria, eczema, dermatomycosis, jaundice, and boils. Modern pharmacological studies show that it has immunosuppressive and anti-inflammatory effects. However, the mechanism of A. annua in the treatment of atopic dermatitis (AD) remains unclear. AIM OF THE STUDY: This study was aimed to investigate the effect of A. annua water extract (AWE) on 2,4-dinitrochlorobenzene (DNCB)-induced AD mouse model and tried to explore its possible underlying mechanisms. MATERIALS AND METHODS: AD was induced in BALB/c mice by the topical repeated application of DNCB. Oral drug intervention of AWE and dexamethasone (DEX, positive control) began from the 7th day and continued for 13 consecutive days. The clinical skin score, ear thickness and the weight of ear and spleen were assessed. The ear tissue were stained with toluidine blue and hematoxylin and eosin (H&E) to detect inflammatory cell infiltration. IgE, terleukin (IL)-4 and IL-13 levels in the serum and IgE level in splenocytes were quantified by enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of IL-4, IL-6, IL-13, IL-17, tumor necrosis factor (TNF)-α and thymic stromal lymphopoietin (TSLP) were measured by quantitative real time polymerase chain reaction. The phosphorylation levels of mitogen-activated protein kinases (MAPKs)-p38 and nuclear factor (NF)-κB in ear tissue were detected by Western blot. RESULTS: Results demonstrated that AWE treatment significantly attenuated the AD-like symptoms in DNCB-induced BALB/c mice, including the skin dermatitis severity and ear edema. Further study disclosed that AWE treatment suppressed the expressions of IgE, IL-4, IL-6, IL-13, IL-17, TNF-α and TSLP at mRNA and protein levels. Moreover, AWE showed inhibitory effect on the phosphorylation of p38 MAPK and NFκB in ear tissues of AD mice. CONCLUSIONS: Collectively, our results suggested that AWE suppressed DNCB-induced AD in mice probably by restraining Th2 type inflammatory response. These findings might pave the road for the potential clinical application of AWE for AD treatment.

Oleanolic Acid Alleviates Atopic Dermatitis-like Responses In Vivo and In Vitro.

Oleanolic acid (OA) is a pentacyclic triterpenoid, abundantly found in plants of the Oleaceae family, and is well known for its beneficial pharmacological activities. Previously, we reported the inhibitory effect of OA on mast cell-mediated allergic inflammation. In this study, we investigated the effects of OA on atopic dermatitis (AD)-like skin lesions and its underlying mechanism of action. We evaluated the inhibitory effect of OA on AD-like responses and the possible mechanisms using a 1-chloro-2,4-dinitrochlorobenzene (DNCB)-induced AD animal model and tumor necrosis factor (TNF)-α/interferon (IFN)-γ-stimulated HaCaT keratinocytes. We found that OA has anti-atopic effects, including histological alterations, on DNCB-induced AD-like lesions in mice. Moreover, it suppressed the expression of Th2 type cytokines and chemokines in the AD mouse model and TNF-α/IFN-γ-induced HaCaT keratinocytes by blocking the activation of serine-threonine kinase Akt, nuclear factor-κB, and the signal transducer and activator of transcription 1. The results demonstrate that OA inhibits AD-like symptoms and regulates the inflammatory mediators; therefore, it may be used as an effective and attractive therapeutic agent for allergic disorders, such as AD. Moreover, the findings of this study provide novel insights into the potential pharmacological targets of OA for treating AD.

Effect of Neferine on DNCB-Induced Atopic Dermatitis in HaCaT Cells and BALB/c Mice.

Atopic dermatitis (AD) is a chronic and persistent inflammatory skin disease characterized by eczematous lesions and itching, and it has become a serious health problem. However, the common clinical treatments provide limited relief and are accompanied by adverse effects. Therefore, there is a need to develop novel and effective therapies to treat AD. Neferine is a small molecule compound isolated from the green embryo of the mature seeds of lotus (Nelumbo nucifera). It has a bisbenzylisoquinoline alkaloid structure. Relevant studies have shown that neferine has many pharmacological and biological activities, including anti-inflammatory, anti-thrombotic, and anti-diabetic activities. However, there are very few studies on neferine in the skin, especially the related effects on inflammatory skin diseases. In this study, we proved that it has the potential to be used in the treatment of atopic dermatitis. Through in vitro studies, we found that neferine inhibited the expression of cytokines and chemokines in TNF-α/IFN-γ-stimulated human keratinocyte (HaCaT) cells, and it reduced the phosphorylation of MAPK and the NF-κB signaling pathway. Through in vivo experiments, we used 2,4-dinitrochlorobenzene (DNCB) to induce atopic dermatitis-like skin inflammation in a mouse model. Our results show that neferine significantly decreased the skin barrier damage, scratching responses, and epidermal hyperplasia induced by DNCB. It significantly decreased transepidermal water loss (TEWL), erythema, blood flow, and ear thickness and increased surface skin hydration. Moreover, it also inhibited the expression of cytokines and the activation of signaling pathways. These results indicate that neferine has good potential as an alternative medicine for the treatment of atopic dermatitis or other skin-related inflammatory diseases.

Gomisin M2 Ameliorates Atopic Dermatitis-like Skin Lesions via Inhibition of STAT1 and NF-κB Activation in 2,4-Dinitrochlorobenzene/Dermatophagoides farinae Extract-Induced BALB/c Mice.

The extracts of Schisandra chinensis (Turcz.) Baill. (Schisandraceae) have various therapeutic effects, including inflammation and allergy. In this study, gomisin M2 (GM2) was isolated from S. chinensis and its beneficial effects were assessed against atopic dermatitis (AD). We evaluated the therapeutic effects of GM2 on 2,4-dinitrochlorobenzene (DNCB) and Dermatophagoides farinae extract (DFE)-induced AD-like skin lesions with BALB/c mice ears and within the tumor necrosis factor (TNF)-α and interferon (IFN)-γ-stimulated keratinocytes. The oral administration of GM2 resulted in reduced epidermal and dermal thickness, infiltration of tissue eosinophils, mast cells, and helper T cells in AD-like lesions. GM2 suppressed the expression of IL-1ß, IL-4, IL-5, IL-6, IL-12a, and TSLP in ear tissue and the expression of IFN-γ, IL-4, and IL-17A in auricular lymph nodes. GM2 also inhibited STAT1 and NF-κB phosphorylation in DNCB/DFE-induced AD-like lesions. The oral administration of GM2 reduced levels of IgE (DFE-specific and total) and IgG2a in the mice sera, as well as protein levels of IL-4, IL-6, and TSLP in ear tissues. In TNF-α/IFN-γ-stimulated keratinocytes, GM2 significantly inhibited IL-1ß, IL-6, CXCL8, and CCL22 through the suppression of STAT1 phosphorylation and the nuclear translocation of NF-κB. Taken together, these results indicate that GM2 is a biologically active compound that exhibits inhibitory effects on skin inflammation and suggests that GM2 might serve as a remedy in inflammatory skin diseases, specifically on AD.

Lycii radicis cortex inhibits glucocorticoid­induced bone loss by downregulating Runx2 and BMP­2 expression.

Lycii radicis cortex (LRC) has been used to regulate high blood pressure, body temperature, pain and bone disorders in East Asia. Glucocorticoids (GCs), also known as steroids, are potent immunity regulators widely used in the treatment of inflammatory diseases. However, despite their effectiveness, GC usage is strictly controlled due to severe side­effects, such as osteoporosis. However, further research is required as to date, at least to the best of our knowledge, there is no appropriate model to overcome secondary osteoporosis as a side­effect of GC use. Thus, the aim of the present study was to establish an experimental model of osteoporosis induced by GC. Furthermore, the present study aimed to establish the research methodology for medical evaluations of the effectiveness and side­effects of GCs. A secondary osteoporosis animal model was established, and the animals were divided into two groups as follows: The allergic contact dermatitis (ACD)­induced group and the non­ACD­induced group. In the ACD­induced group, a GC topical application group was compared with a GC subcutaneous injection group. The results revealed that the presence of ACD affected the induction of GC­mediated osteoporosis. Therefore, the group exhibiting induced ACD that was treated with a topical application of GC was selected for examining the side­effects of GCs. The effects of LRC on secondary osteoporosis were confirmed in vivo and in vitro. The results indicated that LRC regulated dexamethasone­induced osteoblast apoptotic markers, including caspase­6, caspase­9, X­linked inhibitor of apoptosis, apoptosis inhibitor 1 and apoptosis inhibitor 2, and increased the expression of osteoblast differentiation­related genes, such as Runt­related transcription factor 2 and bone morphogenetic protein 2 in the MC3T3E­1 cell line. LRC also significantly reduced GC­induced osteoporosis and exerted anti­inflammatory effects in vivo. In addition, LRC inhibited the reduction of calbindin­D28k in the kidney. Overall, the results of the present study suggest that the use of LRC alleviates GC­induced secondary osteoporosis.

Chrysin Inhibits TNFα-Induced TSLP Expression through Downregulation of EGR1 Expression in Keratinocytes.

Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that acts as a critical mediator in the pathogenesis of atopic dermatitis (AD). Various therapeutic agents that prevent TSLP function can efficiently relieve the clinical symptoms of AD. However, the downregulation of TSLP expression by therapeutic agents remains poorly understood. In this study, we investigated the mode of action of chrysin in TSLP suppression in an AD-like inflammatory environment. We observed that the transcription factor early growth response (EGR1) contributed to the tumor necrosis factor alpha (TNFα)-induced transcription of TSLP. Chrysin attenuated TNFα-induced TSLP expression by downregulating EGR1 expression in HaCaT keratinocytes. We also showed that the oral administration of chrysin improved AD-like skin lesions in the ear and neck of BALB/c mice challenged with 2,4-dinitrochlorobenzene. We also showed that chrysin suppressed the expression of EGR1 and TSLP by inhibiting the extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) 1/2 mitogen-activated protein kinase pathways. Collectively, the findings of this study suggest that chrysin improves AD-like skin lesions, at least in part, through the downregulation of the ERK1/2 or JNK1/2-EGR1-TSLP signaling axis in keratinocytes.

Anti-atopic dermatitis effects of dictamni cortex: Studies on in vitro and in vivo experimental models.

BACKGROUND: Dictamni Cortex (DC), a Chinese herbal medicine with wind dispelling and itchiness relieving effects, is the most popular single herb prescribed for the treatment of atopic dermatitis (AD), as it is used in up to 12.68% of all herbal prescriptions for AD. PURPOSE: The present study aimed to evaluate the anti-AD effect of Dictamni Cortex extract (DCE) and elucidate the underlying molecular mechanisms of its action using the 1-chloro-2,4-dinitrobenzene (DNCB)-induced AD-like mouse model and a relevant in vitro experimental model. METHODS: Female Balb/c mice were sensitized with 200 µl 0.5% DNCB for three days. After sensitization, mice were challenged with 200 µl 1% DNCB on the same dorsal skin and also 20 µl 1% DNCB on each ear every 3 days, and orally administrated by gavage with DCE (0.6, 1.2 and 2.4 g/kg) daily from day 14 to day 29 for 16 consecutive days. At the end of experiment, the clinical scores for AD on the mice were calculated to evaluate the therapeutic effect of DCE; and serum, ears and dorsal skin of the mice were collected for mechanistic study. The anti-allergic activity of DCE was also evaluated using antigen-induced RBL-2H3 cell line. The release of selected cytokines, chemokines and ß-hexosaminidase was measured to determine the anti-allergic activity of DCE. In addition, intracellular Ca2+ level, MAPKs and Lyn phosphorylations were further investigated to reveal its anti-allergic molecular mechanisms. RESULTS: Our results demonstrated that DCE could markedly improve the AD-like symptoms in AD-like mice by inhibiting the mast cell infiltration, suppressing the production of Th2-associated cytokine (IL-4) and pro-inflammatory cytokines (TNF-α), and enhancing the protein expression of filaggrin through inhibition of the MAPKs and NF-κB pathways. Moreover, DCE suppressed mast cell degranulation through decreasing the intracellular Ca2+ level and inactivation of Lyn, Syk and PLCγs, suggesting DCE could regulate mast-cell-mediated allergic response. CONCLUSION: Our experimental results unambiguously indicate that DCE possesses potent anti-allergic effect, and help place the application of DC for the treatment of AD on a scientific footing.

TRPA1 deficiency alleviates inflammation of atopic dermatitis by reducing macrophage infiltration.

AIMS: The aim of this study was to investigate the role of TRPA1 in the pathogenesis of AD. MAIN METHODS: The experimental atopic dermatitis (AD)-like skin lesions were established using 2,4-dinitrochlorobenzene (DNCB). Mice were divided into three groups: TRPA1-/- and WT groups were treated with DNCB dissolved in a 3:1 mixture of acetone and olive oil; the negative control group was treated with 3:1 mixture of acetone and olive oil without DNCB. The treatment lasted for 21 days, after which the animals were sacrificed and their blood, ears and dorsal skin tissue samples were collected for analysis. KEY FINDINGS: Lower dermatitis score, ear thickness, pruritus score, and epidermal hyperplasia were observed in mice in TRPA1-/- mice compared to the WT group. Besides, lower dermal mast cell infiltration, proinflammatory cytokines, Th2 cytokines and the infiltration of macrophages were observed in the TRPA1-/- mice compared to the WT group. Furthermore, we demonstrated that TRPA1 antagonist HC-030031 could alleviate AD-like symptoms and reduce the degree of epidermal hyperplasia in mice. SIGNIFICANCE: TRPA1 has a crucial role during the AD pathogenesis in mice, thus may be used as a potential new target for treating patients with chronic skin inflammatory disease.

Proteomic analysis of the cellular response to a potent sensitiser unveils the dynamics of haptenation in living cells.

Haptenation of model nucleophiles, representing the key MIE in skin sensitisation, is routinely measured in chemico to provide data for skin allergy risk assessment. Better understanding of the dynamics of haptenation in human skin could provide the metrics required to improve determination of sensitiser potency for risk assessment of chemicals. We have previously demonstrated the applicability and sensitivity of the dual stable isotope labelling approach to detect low level haptenation in complex mixtures of proteins. In the present study, we investigated haptenation in a relevant living cell model over time at a subtoxic concentration. DNCB, an extremely potent sensitiser, caused minimal changes in overall protein differential expression in HaCaT cells and haptenated approximately 0.25 % of all available nucleophiles when applied at a subtoxic concentration (10µM) for 4 h. The data shows that the maximum level of haptenation occurs at 2 h and that DNCB, whilst being a promiscuous hapten, shows a preference for Cys residues, despite the considerably higher concentration of amine-based nucleophiles. Although a proportion of highly abundant proteins were haptenated, numerous haptenated sites were also detected on low abundant proteins. Certain proteins were modified at residues buried deep inside the protein structure which are less accessible to haptenation compared with surface exposed nucleophiles. The microenvironment of the buried residues may be a result of several factors influencing the reactivity of both the target nucleophile and the hapten.

Ginsenoside Rh2 Ameliorates Atopic Dermatitis in NC/Nga Mice by Suppressing NF-kappaB-Mediated Thymic Stromal Lymphopoietin Expression and T Helper Type 2 Differentiation.

Ginsenosides are known to have various highly pharmacological activities, such as anti-cancer and anti-inflammatory effects. However, the search for the most effective ginsenosides against the pathogenesis of atopic dermatitis (AD) and the study of the effects of ginsenosides on specific cytokines involved in AD remain unclear. In this study, ginsenoside Rh2 was shown to exert the most effective anti-inflammatory action on thymic stromal lymphopoietin (TSLP) and interleukin 8 in tumor necrosis factor-alpha and polyinosinic: polycytidylic acid induced normal human keratinocytes by inhibiting proinflammatory cytokines at both protein and transcriptional levels. Concomitantly, Rh2 also efficiently alleviated 2,4-dinitrochlorobenzene-induced AD-like skin symptoms when applied topically, including suppression of immune cell infiltration, cytokine expression, and serum immunoglobulin E levels in NC/Nga mice. In line with the in vitro results, Rh2 inhibited TSLP levels in AD mice via regulation of an underlying mechanism involving the nuclear factor κB pathways. In addition, in regard to immune cells, we showed that Rh2 suppressed not only the expression of TSLP but the differentiation of naïve CD4+ T-cells into T helper type 2 cells and their effector function in vitro. Collectively, our results indicated that Rh2 might be considered as a good therapeutic candidate for the alternative treatment of AD.

dl-Malic acid as a component of α-hydroxy acids: effect on 2,4-dinitrochlorobenzene-induced inflammation in atopic dermatitis-like skin lesions in vitro and in vivo.

Background: dl-Malic acid (dl-M) is used widely in cosmetic formulations as a pH-adjuster or as a preservative. dl-M is used as an exfoliator in the form of α-hydroxy acids. However, the role of dl-M in skin diseases (including atopic dermatitis (AD)) has not been studied deeply. We wished to reveal the effect of dl-M on AD induced by 2,4-dinitrochlorobenzene (DNCB) in Balb/c mice.Methods: The thickness and immune-cell infiltration into the dermis and epidermis were evaluated. Moreover, serum levels of cytokines, as well as expression of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) in tissue were measured in AD mice. We also studied the effect of dl-M on inflammatory mediators in a human keratinocyte (HaCaT) cell line. Results: The dl-M (high) group improved skin condition compared with the DNCB-treated group. The dl-M (high) group inhibited phosphorylation of MAPK and NF-κB in skin tissue. dl-M reduced serum levels of interleukin-4 and IgE. Finally, dl-M decreased the expression of thymus and activation-regulated chemokine, monocyte chemoattractant protein-1 and intercellular cell adhesion molecule induced by interferon-gamma/tumor necrosis factor-α in HaCaT cells. Discussion: These results suggest that dl-M can improve the skin conditions of AD mice.