Stimulated HaCaT Cells Research Articles

Anti-atopic dermatitis effect of fraxinellone via inhibiting IL-31 in vivo and in vitro

Chronic recurrent itch and skin inflammation are prominent features of atopic dermatitis (AD), which is closely related to the immune response driven by T-helper type 2 (Th2) cells. The expression of interleukin 31 (IL-31) is positively correlated with the severity of dermatitis. Anti-IL-31 receptor α (IL-31RA) targeted drugs have been used to treat AD, however, they are expensive and have side effects. Fraxinellone (FRA) is one of the main limonoid components in the dried root bark of Dictamnus dasycarpus Turcz.; however, its anti-inflammatory and antipruritic effects on atopic dermatitis (AD) have not been previously reported. In this study, we investigated the anti-dermatitis effect of FRA and its potential mechanism of action using a 2,4-dinitrofluorobenzene (DNFB)-induced AD-like mouse model and lipopolysaccharide (LPS)-stimulated HaCaT cells. FRA significantly inhibited chronic pruritus, epidermal thickening, and inflammatory infiltration in AD mice. FRA not only inhibited the levels of IL-31 in the serum and lesioned skin of AD mice but also significantly downregulated the mRNA expression and protein levels of IL-31, IL-31RA, transient receptor potential (TRP) V1, and TRPA1 in the lesioned skin and dorsal root ganglion (DRG) of AD mice. In LPS-stimulated HaCaT cells, FRA inhibited the production of iNOS and COX2, as well as the protein levels of IL-31, IL-31RA, TRPV1 and TRPA1, showing significant anti-inflammatory effects. In summary, our findings suggest that FRA exerts antipruritic and anti-inflammatory effects in AD by regulating the IL-31 pathway, and may hold promise for the clinical treatment of AD.

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

AimsUrsodeoxycholic 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 methodsTo 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 findingsIn 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 β-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. SignificanceUDCA 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.

Inhibition of NLRP3 Inflammasome Activation by 3H-1,2-Dithiole-3-Thione: A Potential Therapeutic Approach for Psoriasis Treatment.

Psoriasis is a chronic autoimmune skin disease with a significant impact on quality of life and potential for severe comorbidities. Inflammation in the skin is induced by immune cells that overexpress pro-inflammatory cytokines, with the Th17 cell playing a crucial role. NLRP3 inflammasome activation is associated with inflammatory diseases and abnormal T cell differentiation. 3H-1,2-dithiole-3-thione (D3T), isolated from cruciferous vegetables, has anti-inflammatory effects and inhibits Th17 differentiation. This study aimed to investigate how D3T reduces skin inflammation and modulates Th17 cell differentiation by inhibiting NLRP3 inflammasome activation. In an imiquimod-induced psoriasis mouse model, D3T treatment demonstrated significant reductions in ear thickness, skin redness, and scaling compared to a control group. Our study also observed decreased expression of ki-67, NLRP3 inflammasome, and cleaved caspase-1 in skin samples, reduced levels of IL-6 and IL-17A in serum samples, and inhibition of Th17 differentiation after D3T application. D3T could also inhibit the expression of NLRP3, caspase-1, and IL-1β in TNF-α stimulated HaCaT cells. The mechanical study also revealed that D3T could inhibit NLRP3 inflammasome activation by inhibiting the JNK pathway in HaCaT cells. These results indicate that targeting NLRP3 inflammasome activation is a promising strategy in the treatment of psoriasis.

In-situ monitoring of signal transduction between skin cells induced by UVB irradiation using scanning electrochemical microscopy

Skin is the first barrier of the immune system, protecting the body from various damages of the external environment, the signal transduction between skin cells plays an important role in skin healing and injury. Scanning electrochemical microscopy (SECM) combined with a Transwell co-culture device has been proposed to in-situ investigate the signal transduction between skin cells with UVB irradiation, in which, keratinocyte HaCaT cells and fibroblast HFF cells were used as signaling cells and signal-receiving cells, respectively. Extracellular ROS and pH, cell membrane permeability and cell height were in-situ monitored by multi-potential step waveform in SECM (SECM-MPSW), potentiometric mode, shear force mode and the probe approach curve (PAC) combined with COMSOL simulation, respectively. The results revealed that UVB irradiation stimulated HaCaT cells to release excess H+, which diffused to HFF cells and increased the ROS release and membrane permeability with unchanged cell height. Therefore, this work provides an effective way to in-situ investigate intercellular signal transduction.

The pivotal role of Bifida Ferment Lysate on reinforcing the skin barrier function and maintaining homeostasis of skin defenses in vitro.

The semiactive or inactive probiotics or their extracts used in dermatology have interesting properties to ameliorate signs of irritated skin and enhance the skin barrier. Bifidobacterium, as the most common probiotics, which has been found to be effective in reducing acne and improving the skin barrier function of atopic dermatitis. Bifida Ferment Lysate (BFL) can be obtained from Bifidobacterium by fermentation and extraction. In this study, we investigated the effect of a topically used BFL on the skin using in vitro evaluation methods. The results showed that upregulation of skin physical barrier gene (FLG, LOR, IVL, TGM1, and AQP3) and antimicrobial peptide gene (CAMP and hBD-2) in HaCaT cells by BFL might be responsible for skin barrier resistance. In addition, BFL had strong antioxidant properties representing a dose-dependent increasing of the scavenging capacity of DPPH, ABTS, hydroxyl, and superoxide radicals. BFL treatment also fundamentally inhibited the intracellular ROS and MDA production and improved the activities of antioxidant enzymes (CAT and GSH-Px) in H2 O2 -stimulated HaCaT cells. As a good immunomodulatory factor, BFL efficiently decreased the secretion of IL-8 and TNF-α cytokines, and COX-2 mRNA expression in LPS-induced THP-1 macrophages. BFL can strengthen the skin barrier function and stimulate skin barrier resistance, to reinforce the skin against oxidative stress and inflammatory stimuli.

Use of a Silkworm (Bombyx mori) Larvae By-Product for the Treatment of Atopic Dermatitis: Inhibition of NF-κB Nuclear Translocation and MAPK Signaling.

Atopic dermatitis (AD) is a long-lasting inflammatory skin disease that contributes to the global health burden and impacts 10-20% of the world's population. In this study, we determined the anti-AD effect of a by-product of silkworm (Bombyx mori) larval powder, strain Yeonnokjam (SLPY), as a sustainable, natural source for the development of therapeutic agents for AD. HaCaT cells were used to assess the in vitro anti-inflammatory activity of SLPY, and a 1-chloro-2,4-dinitrobenzene (DNCB)-induced mouse model was used to study the in vivo anti-AD effects. SLPY treatment downregulated the expression of the inflammatory cytokines TNF-α, IL1β, IL-8, and Cox-2 in stimulated HaCaT cells. Similarly, the topical application of SLPY in DNCB-treated mice downregulated the expression of inflammatory cytokines and proteins while ameliorating the clinical features of AD. Further, SLPY treatment inhibited the nuclear translocation of NF-κb p65, thereby supporting the efficacy of SLPY in the treatment of AD.

Etanercept ameliorates psoriasis progression through regulating high mobility group box 1 pathway.

As a common skin disease, psoriasis is related to inflammation and immune response. Due to the frequent recurrence of psoriasis, the treatment of psoriasis remains a clinical challenge. As an effective tumor necrosis factor-alpha (TNF-α) inhibitor, etanercept has been used for the treatment of psoriasis. However, some patients with psoriasis have no response to etanercept or discontinue treatment. To improve the therapeutic effect of etanercept, searching the potential biomarkers and investigating the related mechanisms of etanercept in the treatment of psoriasis are vital. We stimulated HaCaT cells with lipopolysaccharide (LPS) to generate cellular psoriatic changes and established an imiquimod (IMQ)-induced psoriasis-like mouse model, and then used an etanercept to treat cell and mouse model. Etanercept alleviated IMQ-induced pathological changes and inflammation, and it also decreased the protein expression of high mobility group box 1 (HMGB1), receptor for advanced glycation end-products, and toll-like receptor 4. Moreover, the results of in vitro experiments showed that etanercept inhibited proliferation and inflammation, and promoted cell cycle arrest and apoptosis in LPS-treated HaCaT cells. Knockdown of HMGB1 further enhanced the inhibitory effects of etanercept on LPS-treated HaCaT cell viability and inflammation, while overexpression of HMGB1 notably reversed the inhibitory effects of etanercept on LPS-induced HaCaT cell viability and inflammation. Etanercept inhibited proliferation and inflammation and promoted cell cycle arrest and apoptosis in LPS-induced HaCaT cells, and etanercept ameliorated inflammation in a psoriasis-like mouse model.

Enhanced skin anti-inflammatory and moisturizing action of gold nanoparticles produced utilizing Diospyros kaki fruit extracts

Inflammatory skin diseases (ISD) cause very severe itchy skin and dryness which is now a days an important issue which has to be taken care. Nanotechnology plays a main role in manufacturing cosmetic ingredients at a nanoscale size. Among different nanoparticles, gold (Au) is one of the non-toxic materials synthesized organically or inorganically. For synthesizing nanoparticles (NPs), using inorganic methods may cause some toxicity to cells, but using organic synthesis like plant extract is less toxic and environmentally friendly. Therefore, we synthesized DK-AuNPs using Diospyros kaki fruit extract. UPLC-MS/MS was used to evaluate phytochemicals responsible for converting salt into nanoparticles. The DK-AuNPs were characterized to confirm the formation of NPs. Furthermore, we analyzed the activity of DK-AuNPs on human keratinocytes (HaCaT cells). The DK-AuNPs showed 98.2 % cell survival upto 200 µg/mL against HaCaT cells. Additionally, compared to DK treatment, DK-AuNPs therapy decreased ROS production in TNF-α/IFN-γ (T + I) stimulated HaCaT cells by 68.7 %, whereas DK treatment reduced ROS generation by 27.8 %. Moreover, the skin anti-inflammatory potential and moisturizing effect of DK-AuNPs were analyzed using HaCaT cells. Furthermore, skin inflammatory activity biomarkers were downregulated through the MAPK/NFκB signaling pathway and showed significant inhibition by DK-AuNPs. Also, the skin moisturizing biomarkers such as HAS (1–3) were upregulated and HYAL (1–2) were downregulated by PI3K/AKT/NFκB through HAS2 regulation. Therefore, skin anti-inflammatory and moisturizing activity were enhanced by treatment with DK-AuNPs. In summary, we conclude that the DK-AuNPs could be a new alternative for skin disease.

Anti-Inflammatory Effects Exerted by 14-Methoxyalternate C from Antarctic Fungal Strain Pleosporales sp. SF-7343 via the Regulation of NF-κB and JAK2/STAT3 in HaCaT Human Keratinocytes

Atopic dermatitis (AD) is a chronic inflammatory skin disease with a profound negative impact on patients' quality of life. Four known secondary fungal metabolites were found in the chemical study of the Antarctic fungus Pleosporales sp. SF-7343, including 14-methoxyalternate C (1), 5'-methoxy-6-methyl-biphenyl-3,4,3'-triol (2), 3,8,10-trihydroxy-4-methoxy-6-methylbenzocoumarin (3), and alternariol monomethyl ether (4). Additionally, we identified the skin anti-inflammatory composition from the SF-7343 strain. Interleukin-8 and -6 Screening results showed that compound 1 inhibited IL-8 and IL-6 in tumor necrosis factor-α/interferon-γ stimulated HaCaT cells. Compound 1 showed inhibitory effects on MDC and RANTES. It also downregulated the expression of intercellular adhesion molecule-1 (ICAM-1) and upregulated the expression of involucrin. The results of the mechanistic study showed that compound 1 inhibited the nuclear translocation of nuclear factor-kappa B p65 and STAT3. In conclusion, this study demonstrates the potential of the Antarctic fungal strain SF-7343 as a bioactive resource to inhibit skin inflammation, such as AD.

416 Pyroptosis of keratinocyte in Stevens-Johnson syndrome / toxic epidermal necrolysis

Stevens Johnson syndrome (SJS)/ toxic epidermal necrolysis (TEN) are characterized by the remarkable cell death of keratinocytes. Our previous studies have revealed that keratinocyte death pathway involve apoptosis and necroptosis. However, in recent years, it has been found that there is a crosstalk between apoptosis, necroptosis and pyroptosis. Pyroptosis is an inflammatory programmed cell death characterized by caspase 1-mediated cleavage of GSDMD and the release of IL-1b and IL-18. The aim of our study is to reveal whether pyroptosis is associated with the pathogenesis of SJS/TEN and develop specific therapeutic agents that can suppress these cell death programs in SJS/TEN. Expression of cleaved GSDMD and cleaved caspase1, which were key players of pyroptosis, were observed in the keratinocytes of SJS/TEN patients by immunofluorescent staining. To prove that pyroptosis can occur in keratinocytes, we stimulated HaCaT cells with pyroptosis inducers, LPS and nigertin, and evaluated pyroptosis reaction by live/dead staining assay, observation of morphological changes and cleaved GSDMD expression. HaCaT cells after LPS and nigertin stimulation formed large bubbles and a punctate positive reaction of cleaved GSDMD on the cell membrane, occurring as a result of membrane pore-forming activity of the gasdermin-N domain. Pyroptosis occurred even after stimulating HaCaT cells with SJS serum. Our study showed that pyroptosis occurred in keratinocytes of SJS/TEN patients and substances that induce pyroptosis to keratinocyte were present in the serum of the patients with SJS/TEN. We have investigated SJS/TEN-specifically elevated proteins by selected reaction monitoring. We are currently exploring the substances that induce pyroptosis. It is expected that understanding the mechanisms of keratinocyte death in SJS/TEN lead us to develop a specific therapeutic drug that can suppress cell death in SJS/TEN.

Spatholobus suberectus Dunn Water Extract Ameliorates Atopic Dermatitis–Like Symptoms by Suppressing Proinflammatory Chemokine Production In Vivo and In Vitro

S. patholobus suberectus Dunn, a traditional Chinese herbal medicine, has various pharmacological activities, such as anti-inflammatory properties. However, to the best of our knowledge, its therapeutic effect on atopic dermatitis (AD) has not been investigated. In this study, we explored the effect of S. suberectus Dunn water extract (SSWex) on AD in vivo and in vitro. In Dermatophagoides farina extract (DfE)–treated NC/Nga mice, the oral administration of SSWex alleviated AD-like symptoms, such as ear thickness, dermatitis score, epidermal thickness, immune cell infiltration, and levels of AD-related serum parameters (immunoglobulin E, histamine, and proinflammatory chemokines). In HaCaT cells, the production of proinflammatory chemokines induced by interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) was inhibited by SSWex pretreatment. SSWex treatment inhibited the phosphorylation of mitogen-activated protein kinase and activation and translocation of transcriptional factors, such as signal transducer and activator of transcription 1 and nuclear factor kappa B in IFN-γ/TNF-α–stimulated HaCaT cells. These results indicate that SSWex may be developed as an efficient therapeutic agent for AD.

D-tryptophan triggered epithelial-mesenchymal transition by activating TGF-β signaling pathway

D-tryptophan is a special kind of nonprotein amino acid showing multiple physiological functions, but the detailed mechanisms are not fully revealed, impairing its further development and applications. This work was to investigate D-tryptophan physiological function and demonstrate the underlying mechanisms. D-tryptophan suppressed HaCaT cell proliferation but increased cell migration. Specifically, D-tryptophan decreased E-cadherin and increased Snail, Twist, and Slug expression, resulting in the development of an epithelial-mesenchymal transitions (EMT) phenomenon. Moreover, D-tryptophan promoted the expression of transforming growth factor-β (TGF-β) 1, and Smad4 knockout damages D-tryptophan's ability. These results indicated that D-tryptophan stimulated HaCaT cells to produce TGF-β1 and thus activated the TGF-β/Samd pathway, resulting in the triggering of EMT. This study revealed the molecular mechanisms of D-tryptophan activity, provided D-tryptophan as a potential approach for cancer treatment, wound healing, organ development and other relevant applications.

Enhancing the anti-psoriatic activity of vitamin D3 employing nanostructured archaeolipid carriers

Psoriasis (Ps) is a multifactorial autoimmune skin disease, where oxidative stress plays a key role in promoting a vicious producing cycle between keratinocytes and immune cells. Vitamin D3 (VD3) regulates the differentiation, apoptosis, and proliferation of keratinocytes, also displaying antioxidant and anti-inflammatory activities. Previous attempts of using topical VD3 as anti-psoriatic agent, failed because of its poor solubility, high hydrophobicity, structural lability, and low bioavailability. Specifically tailored nanoparticles for topical co-delivery of VD3 and antioxidants to activated keratinocytes and macrophages could make Ps treatments more efficient. In this work, structural features, and in vitro activity of nanostructured archaeolipid carriers (NAC) containing VD3 plus the antioxidant C50 dipolar carotenoid bacterioruberin (BR) (NAC-VD3), are presented. Ultra-small (70 nm), −39 mV ζ potential, ∼5 mg VD3/ml, 0.35 BR μg/ml NAC-VD3, with good storage stability (at least 6 month) consisted of a compritol and BR core, covered by a shell of sn 2,3 ether linked archaeolipids and Tween 80 (2: 2: 1.2: 3% w/w) were obtained. Raman, DSC and SAXS analysis showed that VD3 was trapped within the disordered compritol-BR core, impairing its fast release, and protecting VD3 against thermal degradation. NAC-VD3 were extensively captured and displayed high anti-proliferative (65%), anti-inflammatory (IL-8 release) and antioxidant activities (ROS reduction) on a psoriatic model made of CaCl2 differentiated-imiquimod stimulated HaCaT cells, and on lipopolysaccharide induced THP-1 macrophages. Interestingly, the BR extract alone displayed high anti-Staphylococcus aureus activity including anti-biofilm formation. Overall, the results suggest that NAC-VD3 protect the labile structure of VD3, while enhancing its anti-psoriatic activity and deserves further in vivo exploration.

Punicalagin Alleviates Psoriasis by Inhibiting NF-κB-Mediated IL-1β Transcription and Caspase-1-Regulated IL-1β Secretion.

Psoriasis is a chronic and inflammatory skin disorder characterized by inflammation and epidermal hyperplasia. Punicalagin (PUN) is a main active ingredient of pomegranate (Punica granatum L.) peel with multiple biological activities, such as antibacterial, antioxidant and anti-tumor effects. However, the potential effect of PUN on psoriasis remains unknown. In this study, we want to investigate the pharmacological effect of PUN on psoriasis by using imiquimod (IMQ)-induced psoriatic mice model in vivo and tumor necrosis factor a (TNF-α) and interleukin-17A (IL-17A)-stimulated HaCaT cells in vitro. Our results showed that PUN can effectively alleviate the severity of psoriasis-like symptoms. Mechanistically, PUN potently suppresses the aberrant upregulation of interleukin-1β (IL-1β) and subsequent IL-1β-mediated inflammatory cascade in keratinocytes by inhibiting the nuclear factor kappa B (NF-κB) activation and cleaved caspase-1 expression in vitro and in vivo. Taken together, our findings indicate that PUN can relieve psoriasis by repressing NF-κB-mediated IL-1β transcription and caspase-1-regulated IL-1β secretion, which provide evidence that PUN might represent a novel and promising candidate for the treatment of psoriasis.

Kirenol inhibits TNF-α-induced proliferation and migration of HaCaT cells by regulating NF-κB pathway

Psoriasis is a common chronic, inflammatory skin disease possessing properties of inflammatory cell infiltration and excessive proliferation of keratinocytes, the occurrence and development of which remain fully elucidated. Therefore, the study was designed to determine the effects of kirenol (50, 100 and 200 μg/mL) on Cultured Human Keratinocytes (cells) (HaCaT) in vitro and reveal its molecular mechanism. The in vitro psoriasis model was established utilizing tumor necrosis factor-α (TNF-α)-stimulated HaCaT cells. Kirenol, a diterpenoid compound, was applied at different concentrations (50, 100 and 200 μg/mL) to HaCaT cells for 24 h. The Cell Counting Kit-8 (CCK-8) and thymidine monobromodeoxyuridine (BrdU) assays were used to assess cell viability and proliferation, followed by assessment of cell migration by Transwell assay. Subsequently, inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA), and Western blot assay was used to evaluate expressions of p65, p-p65, IκBα and p-IκBα. Activities of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and malondialdehyde (MDA) contents were measured spectrophotometrically. The results demonstrated that TNF-α induced a significant increase in cell viability and inflammatory cytokines, including expressions of Interleukin (IL)-6, IL-8, IL-22 and IL-1β in HaCaT cells, which was dose-dependently inhibited by kirenol. Similarly, TNF-α-induced cell migration was also suppressed by kirenol treatment. Furthermore, TNF-α stimuli induced the upregulation of phosphorylation levels of p65 and IκBα as well as p-p65–p65 and p-IκBα–IκBα ratios, whereas kirenol significantly suppressed the activation of cellular nuclear factor-kappa B (NF-κB) signaling pathway. In addition, kirenol significantly decreased the level of MDA but increased the levels of SOD, CAT and GSH in a dose-dependent manner. These results proposed that kirenol could inhibit the proliferation, migration, expression of inflammatory factors, and oxidative stress in HaCaT cells via suppressing NF-κB signaling pathway.

Circ-IGF1R plays a significant role in psoriasis via regulation of a miR-194-5p/CDK1 axis.

Psoriasis is a skin disorder that is classed as an autoimmune disease. It is characterized by excessive proliferation, abnormal migration and differentiation of keratinocytes, as well as inflammatory cell infiltration. Circular RNAs (circRNAs/circ) have been reported to play an important role in several aspects of psoriasis. Thus in the present study, the role of circ-insulin-like growth factor 1 receptor (circ-IGF1R) in the development of psoriasis was assessed, and the involvement of microRNA (miR)-194-5p was also investigated as its expression was downregulated in psoriasis. StarBase analysis and dual luciferase reporter assays confirmed the interaction between circ-IGF1R with miR-194-5p. The increased expression of circ-IGF1R and decreased expression of miR-194-5p were further confirmed by reverse transcription-quantitative polymerase chain reaction in interleukin (IL-22)-stimulated HaCaT cells. The increased proliferation, migration and invasion, as well as decreased apoptosis, caspase 3 activity and cleaved-caspase 3/caspase 3 ratio were observed in IL-22-stimulated HaCaT cells. Conversely, transfection of circ-IGF1R-small interfering (si)RNA resulted in significantly increased expression of miR-194-5p with or without stimulation of IL-22 in HaCaT cells, and also overcame the effects of the miR-194-5p inhibitor. Additionally, transfection of circ-IGF1R-siRNA inhibited cell proliferation, migration and invasion, which were reversed by transfection of a miR-194-5p inhibitor. Similarly, circ-IGF1R-siRNA promoted apoptosis, caspase 3 activity and the cleaved-caspase 3/caspase 3 ratio, which were reversed by miR-194-5p inhibitor. These results showed that circ-IGF1R could affect the proliferation, apoptosis, migration and invasion of IL-22-stimulated HaCaT cells by regulating the expression of miR-194-5p. Based on TargetScan prediction and dual luciferase reporter assays, it was shown that cyclin-dependent kinase (CDK)1 was targeted by miR-194-5p. Additionally, the expression of CDK1 was upregulated following stimulation with IL-22 in HaCaT cells at the mRNA and protein levels. Transfection of miR-194-5p mimic or miR-194-5p inhibitor negatively regulated CDK1 expression in the IL-22 induced HaCaT cells. In conclusion, circ-IGF1R-siRNA could inhibit the cell proliferation, migration and invasion, and induce apoptosis by regulating the miR-194-5p/CDK1 axis. circ-IGF1R may thus serve as a potential treatment target for psoriasis.

Fuzhenghefuzhiyang Formula (FZHFZY) Improves Epidermal Differentiation via Suppression of the Akt/mTORC1/S6K1 Signalling Pathway in Psoriatic Models.

Psoriasis is a chronic proliferative skin disorder characterised by abnormal epidermal differentiation. The Fuzhenghefuzhiyang (FZHFZY) formula created by Chuanjian Lu, a master of Chinese medicine in dermatology, has been external used in the Guangdong Provincial Hospital of Chinese Medicine for the treatment of psoriasis, but its mechanisms of action against psoriasis remain poorly understood. This study involved an exploration of the effects of FZHFZY on epidermal differentiation and its underlying mechanisms in interleukin (IL)-17A/IL-22/interferon (IFN)-γ/tumour necrosis factor (TNF)-α–stimulated HaCaT cells and in a mouse model of imiquimod (IMQ)-induced psoriasis. Cell viability was assessed by MTT assay. Epidermal differentiation was detected by reverse-transcription polymerase chain reaction and western blotting. Histological evaluation of the skin tissue was performed via haematoxylin and eosin staining, and the Akt/mTORC1/S6K1 pathway was analysed by western blotting. FZHFZY inhibited proliferation and improved epidermal differentiation in IL-17A/IL-22/IFN-γ/TNF-α–induced HaCaT cells. FZHFZY ameliorated symptoms of psoriasis, regulated epidermal differentiation and inhibited phosphorylation of the Akt/mTORC1/S6K1 pathway in the skin of mice with imiquimod-induced psoriasis. Our results suggest that FZHFZY may exhibit therapeutic action against psoriasis by regulating epidermal differentiation via inhibition of the Akt/mTORC1/S6K1 pathway.

Effects of topical application of a tri-herb formula on inflammatory dry-skin condition in mice with oxazolone-induced atopic dermatitis

BackgroundAtopic dermatitis (AD) is a chronic relapsing inflammatory and pruritic skin disease, affecting 10-20% of the population worldwide. Paeonia suffruticosa Andrews (Paeoniaceae) (Cortex Moutan) and Mentha haplocalyx Briq. (Labiatae) (Herba Menthae) have shown beneficial effects on AD. Calendula officinalis L. (Asteraceae) is commonly used for treating skin rashes and wounds. PurposeIn the present study, a three-herbs formula including Cortex Moutan and Herba Menthae, together with C. officinalis at 1:1:1 weight ratio was used as a topical agent and its therapeutic effects on AD was investigated. MethodsIn vitro effects of individual herbs and three-herbs formula (0.125–1 mg/ml) were examined using cytokine release assay on human mast HMC-1 cells, inflammation test on murine macrophage RAW cells and human keratinocyte (HaCaT) cells, and migration scratch assay on human umbilical vein endothelial cells (HUVEC). The contributing functional pathway of three-herbs formula in AD was explored using Western Blot assay in HMC-1 cells. Oxazolone-induced AD-like mice model was also used to investigate the in vivo therapeutic effect of the topical application of the three-herbs formula. ResultsHerba Menthae, Cortex Moutan, and three-herbs formula significantly reduced the production of IL-6 and tumor necrosis factor (TNF)-α in HMC-1 cells, inhibited the expression of IL-6, IL-8 and CCL2 in TNF-α/IFN-γ stimulated HaCaT cells, and suppressed the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cells. Moreover, Herba Menthae and three-herbs formula significantly suppressed CCL2 and TNF-α production in LPS-induced RAW 264.7 cells. C. officinalis and three-herbs formula promoted wound healing in HUVEC. For intracellular mechanisms, three-herbs formula inhibited the expressions of molecules in STAT1 and STAT3-dependent pathways. In vivo model showed that topical application of three-herbs formula on challenged ear reduced ear swelling and mice scratching frequencies. H&E and toluidine blue staining of the challenged ear tissue demonstrated that three-herbs formula reduced the epidermal thickness and mast cell infiltration, respectively. ConclusionThe three-herbs formula of Cortex Moutan, Herba Menthae and C. officinalis at 1:1:1 (w/w) exhibited anti-inflammatory effect and promotion of cell migration in vitro. It also alleviated ear redness, swelling, epidermal thickness and inflammation of the OXA-induced AD mice. These findings suggest a potential beneficial role of the topical application of the three-herbs formula for treatment of AD.

Water Extract of Rubus coreanus Prevents Inflammatory Skin Diseases In Vitro Models.

Atopic dermatitis (AD) is a chronic inflammatory skin disease caused by immune hypersensitivity reaction. The cause of AD is unclear, but its symptoms have a negative effect on quality of life; various treatment methods to alleviate these symptoms are underway. In the present study, we aimed to evaluate in vitro antioxidant and anti-inflammatory effects of Rubus coreanus water extract (RCW) on AD. Total phenolic compounds and flavonoid content of RCW were 4242.40 ± 54.84 mg GAE/g RCE and 1010.99 ± 14.75 mg CE/g RCW, respectively. RCW reduced intracellular reactive oxygen species level and increased the action of antioxidant enzymes, such as catalase, superoxide dismutase, and glutathione peroxidase in tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ)-stimulated HaCaT cells. Moreover, mRNA expression of the pro-inflammatory cytokines, including TNF-α, interleukin-1β, and interleukin-6, was downregulated by RCW in the TNF-α/IFN-γ-stimulated cells. The levels of inflammatory chemokines (thymus- and activation-regulated chemokine; eotaxin; macrophage-derived chemokine; regulated on activation, normal T-cell expressed and secreted; and granulocyte-macrophage colony-stimulating factor) and intercellular adhesion molecule-1 were decreased in the TNF-α/IFN-γ-stimulated HaCaT cells after RCW treatment. Additionally, the mRNA expression levels of filaggrin and involucrin, proteins that form the skin, were increased by RCW. Furthermore, RCW inhibited the nuclear factor kappa-light-chain-enhancer of the activated B cells pathway in the TNF-α/IFN-γ-stimulated HaCaT cells. Collectively, the present investigation indicates that RCW is a potent substance that inhibits AD.

MicroRNA-126-5p Regulates Proliferation and Apoptosis of IL-22-Stimulated Human Keratinocytes Through Regulating Caspase 1

Objective: The study was aimed to explore the roles of miR-126-5p in psoriasis and the underlying molecular mechanisms. Methods: In vitro cell model of psoriasis was established by IL-22 induction. CASP1, the target gene of miR-126-5p, was predicted by TargetScan and verified through the dual luciferase reporter gene system. qRT-PCR was used to measure the mRNA expression of miR-126-5p and CASP1 in IL-22 stimulated HaCaT cells. The protein expression of CASP1, cleaved-caspase3 and caspase3 were measured by Western blot analysis. MTT assay and flow cytometry analysis were performed to detect the cell proliferation and apoptosis. A Caspase3 Activity Assay kit was used to detect the activity of Caspase3. Results: miR-126-5p was high expressed in IL-22 stimulated HaCaT cells compared with normal HaCaT cells. We predicted and verified that CASP1 was a direct target of miR-126-5p, and the mRNA and protein expression of CASP1 were reduced in IL-22 stimulated HaCaT cells compared with the normal HaCaT cells. miR-126-5p inhibitor and CASP1-siRNA significantly decreased the expression of miR-126-5p and CASP1 in HaCaT cells respectively. miR-126-5p inhibitor up-regulated the expression of CASP1 in HaCaT cells, and the effect was reversed by the transfection with CASP1-siRNA. In comparison with the control group, miR-126-5p inhibitor decreased the cell proliferation, induced apoptosis, and improved the activity of Caspase3, enhanced cleaved-caspase3/caspase3 ratio in IL-22 stimulated HaCaT cells, and all the effects were reversed by down-regulating CASP1. Conclusion: We demonstrated that miR-126-5p inhibitor played a protective role in psoriasis by targeting CASP1, evidenced by inhibiting IL-22-induced HaCaT cell proliferation and inducing apoptosis.