IL-32γ suppressed atopic dermatitis through inhibition of miR-205 expression via inactivation of nuclear factor-kappa B.

BACKGROUND: IL-32 is a novel cytokine involved in many inflammatory diseases. However, the role of IL-32γ, an isotype of IL-32, in atopic dermatitis (AD) has not been reported. OBJECTIVE: We investigated the effects of IL-32γ on development of AD and its action mechanisms. METHODS: We used phthalic anhydride (PA) and an MC903-induced AD model using wild-type and IL-32γ transgenic mice. We conducted the therapy experiments by using recombinant IL-32γ protein in a reconstructed human skin model and PA-induced model. We conducted a receiver operating characteristic analysis of IL-32γ with new AD biomarkers, IL-31 and IL-33, in serum from patients with AD. RESULTS: Dermatitis severity and epidermal thickness were significantly reduced in PA- and MC903-induced IL-32γ transgenic mice compared with in wild-type mice. The concentration of AD-related cytokines was reduced in PA- and MC903-induced IL-32γ transgenic mice compared with in wild-type mice. Subsequent analysis showed that IL-32γ inhibits miR-205 expression in PA- and MC903-induced skin tissue samples and TNF-α/IFN-γ-treated HaCaT cells. IL-32γ reduced NF-κB activity in skin tissue samples from PA- and MC903-induced mice and TNF-α/IFN-γ-treated HaCaT cells. NF-κB inhibitor treatment with IL-32γ expression further suppressed expression of inflammatory mediators as well as miR-205 in TNF-α/IFN-γ-treated HaCaT cells. Furthermore, recombinant IL-32γ protein alleviated AD-like inflammation in in vivo and reconstructed human skin models. Spearman correlation analysis showed that serum levels of IL-32γ and miR-205 were significantly concordant in patients with AD. CONCLUSION: Our results indicate that IL-32γ reduces AD through the inhibition of miR-205 expression via inactivation of NF-κB.

Combination Effect of Titrated Extract of Centella asiatica and Astaxanthin in a Mouse Model of Phthalic Anhydride-Induced Atopic Dermatitis.

PURPOSE: In our previous study, we demonstrated that both titrated extract of Centella asiatica (TECA) and astaxanthin (AST) have anti-inflammatory effects in a 5% phthalic anhydride (PA) mouse model of atopic dermatitis (AD). The increasing prevalence of AD demands new therapeutic approaches for treating the disease. We investigated the therapeutic efficacy of the ointment form of TECA, AST and a TECA + AST combination in a mouse model of AD to see whether a combination of the reduced doses of 2 compounds could have a synergistic effect. METHODS: An AD-like lesion was induced by the topical application of 5% PA to the dorsal ear and back skin of an Hos:HR-1 mouse. After AD induction, TECA (0.5%), AST (0.5%) and the TECA (0.25%) + AST (0.25%) combination ointment (20 µg/cm²) were spread on the dorsum of the ear or back skin 3 times a week for 4 weeks. We evaluated dermatitis severity, histopathological changes and changes in protein expression by Western blotting for inducible nitric oxide synthase (iNOS), cyclocxygenase (COX)-2, and nuclear factor (NF)-κB activity. We also measured the concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and immunoglobulin E (IgE) in the blood of AD mice by enzyme-linked immunosorbent assay (ELISA). RESULTS: PA-induced skin morphological changes and ear thickness were significantly reduced by TECA, AST and TECA + AST treatments, but these inhibiting effects were more pronounced in the TECA + AST treatment. TECA, AST and the TECA+AST reatments inhibited the expression of iNOS and COX-2; NF-κB activity; and the release of TNF-α, IL-6 and IgE. However, the TECA+AST treatment showed additive or synergistic effects on AD. CONCLUSIONS: Our results demonstrate that the combination of TECA and AST could be a promising therapeutic agent for AD by inhibiting NF-κB signaling.

Inhibitory effect of Carnosol on UVB-induced inflammation via inhibition of STAT3.

Ultraviolet B (UVB) irradiation causes sunburn, inflammatory responses, dysregulation of immune function, oxidative stress, DNA damage and photocarcinogenesis on skin. Rosemary (Rosmarinus officinalis L.) has been reported to inhibit inflammation. Carnosol, a major component of Rosemary, has prominent anti-inflammatory effects. However, its protective effect on UVB-induced inflammatory skin responses has not yet been reported. Here, we investigated the effectiveness of carnosol on UVB-induced inflammation. We examined the anti-inflammation effect of topical application of carnosol (0.05 µg/cm2) on UVB (540 mJ/cm2, for 3 successive days)-induced skin inflammation in HR1 mice. Topical application of carnosol inhibited UVB-induced erythema, epidermal thickness, inflammatory responses in HR1 mice. Carnosol reduced the level of Immunoglobulin-E and IL-1ß in blood serum of UVB-induced mice. Carnosol also significantly inhibited the UVB-induced expression of inflammatory marker protein (iNOS and COX-2) in back skin of mice. In addition, carnosol treated skin decreased activation of STAT3, a transcriptional factor regulating inflammatory genes. Our study suggested that carnosol has protective effects on skin inflammatory skin damages by UVB.

Astaxanthin alleviated ethanol-induced liver injury by inhibition of oxidative stress and inflammatory responses via blocking of STAT3 activity.

Astaxanthin (AXT) is classified as a xanthophyll carotenoid compound which have broader functions including potent antioxidant, anti-inflammatory and neuroprotective properties. Considerable researches have demonstrated that AXT shows preventive and therapeutic properties against for Diabetes, Osteoarthritis and Rheumatoid Arthritis. However, the protective effect of AXT on liver disease has not yet been reported. In this study, we investigated effects of AXT on ethanol-induced liver injury in chronic plus binge alcohol feeding model. The hepatic steatosis and inflammation induced by ethanol administration were alleviated by AXT. Serum levels of aspartate transaminase and alanine transaminase were decreased in the livers of AXT administrated group. The ethanol-induced expression of cytochrome P450 2E1 (CYP2E1), pro-inflammatory proteins, cytokines, chemokines and reactive oxygen species (ROS) levels were also reduced in the livers of AXT administrated group. Moreover, ethanol-induced infiltration of neutrophils was decreased in the livers of AXT administrated group. Docking model and pull-down assay showed that AXT directly binds to the DNA binding site of STAT3. Moreover, AXT decreased STAT3 phosphorylation in the liver of AXT administration group. Therefore, these results suggest that AXT could prevent ethanol-induced hepatic injury via inhibition of oxidant and inflammatory responses via blocking of STAT3 activity.

Imidazole Antifungal Drugs Inhibit the Cell Proliferation and Invasion of Human Breast Cancer Cells.

Breast cancer is currently the most prevalent cancer in women, and its incidence increases every year. Azole antifungal drugs were recently found to have antitumor efficacy in several cancer types. They contain an imidazole (clotrimazole and ketoconazole) or a triazole (fluconazole and itraconazole) ring. Using human breast adenocarcinoma cells (MCF-7 and MDA-MB-231), we evaluated the effects of azole drugs on cell proliferation, apoptosis, cell cycle, migration, and invasion, and investigated the underlying mechanisms. Clotrimazole and ketoconazole inhibited the proliferation of both cell lines while fluconazole and itraconazole did not. In addition, clotrimazole and ketoconazole inhibited the motility of MDA-MB-231 cells and induced G1-phase arrest in MCF-7 and MDA-MB-231 cells, as determined by cell cycle analysis and immunoblot data. Moreover, Transwell invasion and gelatin zymography assays revealed that clotrimazole and ketoconazole suppressed invasiveness through the inhibition of matrix metalloproteinase 9 in MDA-MB-231 cells, although no significant changes in invasiveness were observed in MCF-7 cells. There were no significant changes in any of the observed parameters with fluconazole or itraconazole treatment in either breast cancer cell line. Taken together, imidazole antifungal drugs showed strong antitumor activity in breast cancer cells through induction of apoptosis and G1 arrest in both MCF-7 and MDA-MB-231 cells and suppression of invasiveness via matrix metalloproteinase 9 inhibition in MDA-MB-231 cells. Imidazole drugs have well-established pharmacokinetic profiles and known toxicity, which can make these generic drugs strong candidates for repositioning as antitumor therapies.

Anti-inflammatory effect of astaxanthin in phthalic anhydride-induced atopic dermatitis animal model.

In this study, we investigated anti-dermatitic effects of astaxanthin (AST) in phthalic anhydride (PA)-induced atopic dermatitis (AD) animal model as well as in vitro model. AD-like lesion was induced by the topical application of 5% PA to the dorsal skin or ear of Hos:HR-1 mouse. After AD induction, 100 µL of 1 mg/mL and 2 mg/mL of AST (10 µg or 20 µg/cm2 ) was spread on the dorsum of ear or back skin three times a week for four weeks. We evaluated dermatitis severity, histopathological changes and changes in protein expression by Western blotting for inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and nuclear factor-κB (NF-κB) activity. We also measured tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and immunoglobulin E (IgE) concentration in the blood of AD mice by enzyme-linked immunosorbent assay (ELISA). AST treatment attenuated the development of PA-induced AD. Histological analysis showed that AST inhibited hyperkeratosis, mast cells and infiltration of inflammatory cells. AST treatment inhibited expression of iNOS and COX-2, and NF-κB activity as well as release of TNF-α, IL-1ß, IL-6 and IgE. In addition, AST (5, 10 and 20 µM) potently inhibited lipopolysaccharide (LPS) (1 µg/mL)-induced nitric oxide (NO) production, expression of iNOS and COX-2 and NF-κB DNA binding activities in RAW 264.7 macrophage cells. Our data demonstrated that AST could be a promising agent for AD by inhibition of NF-κB signalling.

Peroxiredoxin 6 overexpression attenuates lipopolysaccharide-induced acute kidney injury.

Peroxiredoxin 6 (PRDX6) is a member of the PRDX family of antioxidant enzymes and correlated with inflammatory response. Therefore, we investigated the role of PRDX6 during lipopolysaccharide (LPS)-induced acute kidney injury. Both 3 months aged PRDX6-overexpressing transgenic mice (PRDX6 mice) and wild type (WT) mice had acute renal injury induced by intraperitoneal injection of LPS (10 mg/kg)., PRDX6 mice showed decreased mortality and renal injury following LPS challenge compared to WT mice. Furthermore, infiltration of macrophages, T-cells and neutrophils, and the number of apoptotic cells were more decreased by LPS treatment in PRDX6 mice than in WT mice. Because LPS induces reactive oxygen species (ROS) production which induces inflammation through c-Jun N-terminal Kinase (JNK) and p38 MAPK activation, we investigated ROS concentration and MAPK signaling pathway in the kidney of PRDX6 mice. As expected, LPS-induced oxidative stress was attenuated, and p38 MAPK and JNK activation was decreased in the kidney of PRDX6 mice. Inhibitory effect of PRDX6 on LPS-induced apoptosis and MAPK activation in the primary renal proximal tubular cells were overcome by treatment with PRDX6 inhibitor or hydrogen peroxide. These results suggest that PRDX6 overexpression inactivates p38 MAPK and JNK pathway through decrease LPS-induced ROS concentration in the kidney, resulting in inhibition of renal apoptosis and leukocyte infiltration and led to attenuation of LPS-induced acute kidney injury.

Anti-Inflammatory Effect of Titrated Extract of Centella asiatica in Phthalic Anhydride-Induced Allergic Dermatitis Animal Model.

Centella asiatica has potent antioxidant and anti-inflammatory properties. However, its anti-dermatitic effect has not yet been reported. In this study, we investigated the anti-dermatitic effects of titrated extract of Centella asiatica (TECA) in a phthalic anhydride (PA)-induced atopic dermatitis (AD) animal model as well as in vitro model. An AD-like lesion was induced by the topical application of five percent PA to the dorsal skin or ear of Hos:HR-1 mouse. After AD induction, 100 µL of 0.2% and 0.4% of TECA (40 µg or 80 µg/cm²) was spread on the dorsum of the ear or back skin three times a week for four weeks. We evaluated dermatitis severity, histopathological changes and changes in protein expression by Western blotting for inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and NF-κB activity, which were determined by electromobility shift assay (EMSA). We also measured TNF-α, IL-1ß, IL-6, and IgE concentration in the blood of AD mice by enzyme-linked immunosorbent assay (ELISA). TECA treatment attenuated the development of PA-induced atopic dermatitis. Histological analysis showed that TECA inhibited hyperkeratosis, mast cells and infiltration of inflammatory cells. TECA treatment inhibited expression of iNOS and COX-2, and NF-κB activity as well as the release of TNF-α, IL-1ß, IL-6, and IgE. In addition, TECA (1, 2, 5 µg/mL) potently inhibited Lipopolysaccharide (LPS) (1 µg/mL)-induced NO production, expression of iNOS and COX-2, and NF-κB DNA binding activities in RAW264.7 macrophage cells. Our data demonstrated that TECA could be a promising agent for AD by inhibition of NF-κB signaling.

C-C chemokine receptor type 5 deficiency exacerbates alcoholic fatty liver disease through pro-inflammatory cytokines and chemokines-induced hepatic inflammation.

BACKGROUND AND AIM: Chemokines and chemokine receptors implicated with alcoholic liver disease. Studies have shown that inflammation and oxidative stress induce fat molecules aggregation in liver. We evaluated the relationship between alcoholic fatty liver disease and C-C chemokine receptor 5 (CCR5) and impact of inflammation and oxidative stress in fat molecule deposition. METHODS: Lieber-DeCarli diet containing ethanol or isocaloric control diets were fed to wild-type and CCR5 knockout mice for 10 days and gavaged with a single dose of ethanol or isocaloric maltose dextrin at 11th day. Cytokine, chemokine, and reactive oxygen species levels were measured in liver tissues to study the role of CCR5 in alcoholic fatty liver disease. RESULTS: C-C chemokine receptor type 5 knockout mice exacerbated ethanol-induced liver injury. Serum levels of aspartate aminotransferase and alanine aminotransferase were higher in CCR5 knockout mice than wild-type mice, and CCR5 knockout mice showed more severe lipid accumulation in liver tissue than wild-type mice after ethanol feeding. Increased expressions of pro-inflammatory cytokines TNF-α and IL-6 and chemokines CCL2, CCL3, CCL4, and CCL5 result in exacerbation of hepatitis in CCR5 knockout mice after ethanol feeding. Oxidative stress induced by reactive oxygen species was more severe in CCR5 knockout mice, and increasing level of fatty acid import and decreasing level of lipid degradation resulted in lipid accumulation in ethanol-fed CCR5 knockout mice. CONCLUSION: Deficiency of CCR5 exacerbates alcoholic fatty liver disease by hepatic inflammation induced by pro-inflammatory cytokines and chemokines and oxidative stress.

Synergistic Inhibitory Effects of Cetuximab and Cisplatin on Human Colon Cancer Cell Growth via Inhibition of the ERK-Dependent EGF Receptor Signaling Pathway.

The purpose of this study was to evaluate the anticancer efficacy of cetuximab combined with cisplatin (combination treatment) on colon cancer growth, as well as its underlying action mechanism. Combination treatment synergistically potentiated the effect of cetuximab on cell growth inhibition and apoptosis induction in HCT116 and SW480 cells. Combination treatment further suppressed the expression of the activated form of epidermal growth factor receptor (EGFR) and MAP kinase (p-ERK and p-p38) and also significantly inhibited the activity of activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB). Additionally, the expression of cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) mRNA was significantly reduced by the combination treatment as compared to the expression seen for treatment with cetuximab or cisplatin alone. We found that the synergistic inhibitory effects of cetuximab and cisplatin on AP-1 and NF-κB activation, as well as on cell viability, were reversed by pretreatment with an ERK inhibitor. Results demonstrate that combined treatment with cetuximab and cisplatin exerts synergistic anticancer effects on colon cancer cells and also suggest that the ERK pathway plays a critical role in these effects via the suppression of the EGFR signaling pathway, along with the inhibition of COX-2, IL-8, and AP-1 and NF-κB.

Exacerbation of collagen antibody-induced arthritis in transgenic mice overexpressing peroxiredoxin 6.

OBJECTIVE: Peroxiredoxin 6 plays important and complex roles in the process of inflammation, but its role in the development of rheumatoid arthritis (RA) remains unclear. We undertook this study to investigate the roles and mechanisms of peroxiredoxin 6 in the development of collagen antibody-induced arthritis (CAIA) and antigen-induced arthritis (AIA) in peroxiredoxin 6-overexpressing transgenic mice, in peroxiredoxin 6-transfected RAW 264.7 cells, in macrophages isolated from peroxiredoxin 6-overexpressing transgenic mice, and in synoviocytes from arthritis patients. METHODS: CAIA and AIA were induced using standard methods. Peroxiredoxin 6-transfected RAW 264.7 cells, macrophages isolated from peroxiredoxin 6-overexpressing transgenic mice, and synoviocytes from arthritis patients were used to study proinflammatory responses and mechanisms. Clinical scores and histopathologic changes were determined in peroxiredoxin 6-overexpressing transgenic mice and wild-type (WT) mice with CAIA or AIA. Generation of nitric oxide (NO), expression of inducible NO synthase and cyclooxygenase 2, and activity of NF-κB and activator protein 1 (AP-1) were determined in cultured macrophages and synoviocytes as well as in joint tissue from mice by Western blotting, electrophoretic mobility shift assay, and immunohistochemical analysis. RESULTS: Development of CAIA and AIA and proinflammatory responses were more exacerbated in peroxiredoxin 6-overexpressing transgenic mice than in WT mice. Overexpression of peroxiredoxin 6 increased lipopolysaccharide-induced inflammatory responses in RAW 264.7 cells, in macrophages isolated from peroxiredoxin 6-overexpressing transgenic mice, and in synoviocytes from arthritis patients, and this was accompanied by up-regulation of the JNK pathway. Moreover, a JNK inhibitor completely blocked RA development and proinflammatory responses. CONCLUSION: Our findings suggest that overexpression of peroxiredoxin 6 might promote development of RA through NF-κB and AP-1 activity via the JNK pathway.

Interleukin-32ß ameliorates metabolic disorder and liver damage in mice fed high-fat diet.

OBJECTIVE: Chronic excessive food intake leads to energy imbalance, resulting in hepatic steatosis and inflammation. Interleukin-32 (IL-32) is known to be a pro-inflammatory cytokine associated with chronic inflammation and cancer. Therefore, the relationship between IL-32 and chronic excessive food intake-induced liver disease was investigated. METHODS: Male IL-32ß transgenic and wild-type mice were fed a high-fat diet (HFD) for 15 weeks. They were compared with wild-type mice on a standard chow diet. Daily food intake, body and liver weight, serum biochemistry, histopathological analysis of the liver, and hepatic immune response were determined. RESULTS: IL-32ß mice on HFD showed lower lipid accumulation, reduced infiltration of immune cells, and lower production of pro-inflammatory cytokines in the liver. The expression of the peroxisome proliferator-activated receptor γ (PPARγ) was downregulated and the adenosine 50-monophosphate (AMP)-activated protein kinase (AMPK) was activated in the liver of IL-32ß mice compared to wild-type mice. Furthermore, IL-32ß over-expression activated the AMPK pathway and IL-32ß downregulation inactivated the AMPK pathway in HepG2 cells under high-glucose conditions. CONCLUSIONS: These data suggest that IL-32ß modulates lipid accumulation through inhibition of PPARγ expression and AMPK activation.