Gypenoside A from Gynostemma pentaphyllum Attenuates Airway Inflammation and Th2 Cell Activities in a Murine Asthma Model.

Our previous study found that oral administration of Gynostemma pentaphyllum extract can attenuate airway hyperresponsiveness (AHR) and reduce eosinophil infiltration in the lungs of asthmatic mice. Gypenoside A is isolated from G. pentaphyllum. In this study, we investigated whether gypenoside A can effectively reduce asthma in mice. Asthma was induced in BALB/c mice by ovalbumin injection. Asthmatic mice were treated with gypenoside A via intraperitoneal injection to assess airway inflammation, AHR, and immunomodulatory effects. In vitro, gypenoside A reduced inflammatory and oxidative responses in inflammatory tracheal epithelial cells. Experimental results showed that gypenoside A treatment can suppress eosinophil infiltration in the lungs, reduce tracheal goblet cell hyperplasia, and attenuate AHR. Gypenoside A significantly reduced Th2 cytokine expression and also inhibited the expression of inflammatory genes and proteins in the lung and bronchoalveolar lavage fluid. In addition, gypenoside A also significantly inhibited the secretion of inflammatory cytokines and chemokines and reduced oxidative expression in inflammatory tracheal epithelial cells. The experimental results suggested that gypenoside A is a natural compound that can effectively reduce airway inflammation and AHR in asthma, mainly by reducing Th2 cell activation.

Acacetin Protects against Non-Alcoholic Fatty Liver Disease by Regulating Lipid Accumulation and Inflammation in Mice.

We previously demonstrated that acacetin reduces adipogenesis in adipocytes, and decreases lipid accumulation in visceral adipocyte tissue. Here we investigated whether acacetin regulated the mechanisms of lipogenesis and inflammation in non-alcoholic fatty liver disease (NAFLD) in obese mice. Male C57BL/6 mice were fed a high-fat diet (HFD), and then administered acacetin by intraperitoneal injection. Acacetin reduced body weight and liver weight in obese mice. Acacetin-treated obese mice exhibited decreased lipid accumulation, increased glycogen accumulation, and improved hepatocyte steatosis. Acacetin regulated triglycerides and total cholesterol in the liver and serum. Acacetin decreased low-density lipoprotein and leptin concentrations, but increased high-density lipoprotein and adiponectin levels in obese mice. Acacetin effectively weakened the gene expressions of transcription factors related to lipogenesis, and promoted the expressions of genes related to lipolysis and fatty acid ß-oxidation in liver. Acacetin also reduced expressions of inflammation-related cytokines in the serum and liver. Oleic acid induced lipid accumulation in murine FL83B hepatocytes, and the effects of acacetin treatment indicated that acacetin may regulate lipid metabolism through the AMPK pathway. Acacetin may protect against hepatic steatosis by modulating inflammation and AMPK expression.

Urolithin A Inactivation of TLR3/TRIF Signaling to Block the NF-κB/STAT1 Axis Reduces Inflammation and Enhances Antioxidant Defense in Poly(I:C)-Induced RAW264.7 Cells.

Urolithin A is an active compound of gut-microbiota-derived metabolites of polyphenol ellagic acid that has anti-aging, antioxidative, and anti-inflammatory effects. However, the effects of urolithin A on polyinosinic acid-polycytidylic acid (poly(I:C))-induced inflammation remain unclear. Poly(I:C) is a double-stranded RNA (dsRNA) similar to a virus and is recognized by Toll-like receptor-3 (TLR3), inducing an inflammatory response in immune cells, such as macrophages. Inflammation is a natural defense process of the innate immune system. Therefore, we used poly(I:C)-induced RAW264.7 cells and attenuated the inflammation induced by urolithin A. First, our data suggested that 1-30 µM urolithin A does not reduce RAW264.7 cell viability, whereas 1 µM urolithin A is sufficient for antioxidation and the decreased production of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and C-C chemokine ligand 5. The inflammation-related proteins cyclooxygenase-2 and inducible nitric oxide synthase were also downregulated by urolithin A. Next, 1 µM urolithin A inhibited the levels of interferon (INF)-α and INF-ß. Urolithin A was applied to investigate the blockade of the TLR3 signaling pathway in poly(I:C)-induced RAW264.7 cells. Moreover, the TLR3 signaling pathway, subsequent inflammatory-related pathways, and antioxidation pathways showed changes in nuclear factor-κB (NF-κB) signaling and blocked ERK/mitogen-activated protein kinase (MAPK) signaling. Urolithin A enhanced catalase (CAT) and superoxide dismutase (SOD) activities, but decreased malondialdehyde (MDA) levels in poly(I:C)-induced RAW264.7 cells. Thus, our results suggest that urolithin A inhibits TLR3-activated inflammatory and oxidative-associated pathways in macrophages, and that this inhibition is induced by poly(I:C). Therefore, urolithin A may have antiviral effects and could be used to treat viral-infection-related diseases.

Sophoraflavanone G from Sophora flavescens Ameliorates Allergic Airway Inflammation by Suppressing Th2 Response and Oxidative Stress in a Murine Asthma Model.

Sophoraflavanone G (SG), isolated from Sophora flavescens, has anti-inflammatory and anti-tumor bioactive properties. We previously showed that SG promotes apoptosis in human breast cancer cells and leukemia cells and reduces the inflammatory response in lipopolysaccharide-stimulated macrophages. We investigated whether SG attenuates airway hyper-responsiveness (AHR) and airway inflammation in asthmatic mice. We also assessed its effects on the anti-inflammatory response in human tracheal epithelial cells. Female BALB/c mice were sensitized with ovalbumin, and asthmatic mice were treated with SG by intraperitoneal injection. We also exposed human bronchial epithelial BEAS-2B cells to different concentrations of SG to evaluate its effects on inflammatory cytokine levels. SG treatment significantly reduced AHR, eosinophil infiltration, goblet cell hyperplasia, and airway inflammation in the lungs of asthmatic mice. In the lungs of ovalbumin-sensitized mice, SG significantly promoted superoxide dismutase and glutathione expression and attenuated malondialdehyde levels. SG also suppressed levels of Th2 cytokines and chemokines in lung and bronchoalveolar lavage samples. In addition, we confirmed that SG decreased pro-inflammatory cytokine, chemokine, and eotaxin expression in inflammatory BEAS-2B cells. Taken together, our data demonstrate that SG shows potential as an immunomodulator that can improve asthma symptoms by decreasing airway-inflammation-related oxidative stress.

Oleuropein attenuates inflammation and regulates immune responses in a 2,4-dinitrochlorobenzene-induced atopic dermatitis mouse model.

BACKGROUND: Olive (Olea europaea Linn) leaves contain a phenolic compound oleuropein (Ole) has antioxidant, anti-inflammatory, and immunomodulatory activities. However, whether Ole might be an effective treatment for atopic dermatitis (AD) remains unknown. OBJECTIVE: This study investigated the functional role of oleuropein in a 2,4-dinitrochlorobenzene-induced AD-like mouse model, with a focus on allergic inflammation. METHODS: We evaluated cytokine gene expression, COX-2 inflammatory protein production, and Th2 related cytokine regulation of mast cells and eosinophils that infiltrated AD-like skin lesions. RESULTS: A topical application of Ole significantly reduced Th2-related cytokine gene expression (IL-4 and IL-5) and inflammatory COX-2 protein production in AD-like skin lesions. Additionally, Ole suppressed serum IgE levels. Furthermore, Ole effectively reduced ear swelling and epidermal and dermal thickening. CONCLUSIONS: These results suggested that, mechanistically, Ole treatment improved allergic inflammation by blocking the Th2-driven inflammatory axis. In conclusion, our findings indicated that Ole showed promise in treating AD by regulating serum IgE and Th2 cytokine levels. Although the effects of Ole on AD in humans require clinical trials, our results provided insights into how AD treatments might be improved.

Tomatidine Improves Pulmonary Inflammation in Mice with Acute Lung Injury.

Tomatidine, which is isolated from green tomato, can ameliorate inflammation and oxidative stress in cells and animal experiments and has been shown to improve airway inflammation in a murine model of asthma. Here, we investigated whether tomatidine can ameliorate acute lung injury in mice. Mice were given tomatidine by intraperitoneal injection for 7 consecutive days, and then, lung injury was induced via intratracheal instillation of lipopolysaccharide (LPS). Tomatidine reduced inflammatory cytokine expressions in bronchoalveolar lavage fluid (BALF), attenuated neutrophil infiltration in the BALF and lung tissue, increased superoxide dismutase activity and glutathione levels, and alleviated myeloperoxidase expression in the lung tissue of mice with lung injury. Tomatidine also decreased inflammatory cytokine and chemokine gene expression in inflammatory lungs and attenuated the phosphorylation of mitogen-activated protein kinase and nuclear factor kappa B. Furthermore, tomatidine enhanced the production of heme oxygenase-1, decreased the secretion of inflammatory cytokines and chemokines in LPS-stimulated lung epithelial cells, and attenuated THP-1 monocyte adhesion. Our findings suggest that tomatidine attenuates oxidative stress and inflammation, improving acute lung injury in mice.

Maslinic acid protects against obesity-induced nonalcoholic fatty liver disease in mice through regulation of the Sirt1/AMPK signaling pathway.

Maslinic acid is a pentacyclic triterpenoid that is distributed in the peel of olives. Previous studies found that maslinic acid inhibited inflammatory response and antioxidant effects. We investigated whether maslinic acid ameliorates nonalcoholic fatty liver disease in mice with high-fat-diet (HFD)-induced obesity and evaluated the regulation of lipogenesis in hepatocytes. Male C57BL/6 mice fed a normal diet or HFD (60% fat, w/w) were tested for 16 wk. After the fourth week, mice were injected intraperitoneally with maslinic acid for 12 wk. In another experiment, HepG2 cells were treated with oleic acid to induce lipid accumulation or maslinic acid to evaluate lipogenesis. Maslinic acid significantly reduced body weight compared with HFD-fed mice. Maslinic acid reduced liver weight and liver lipid accumulation and improved hepatocyte steatosis. Furthermore, serum glucose, leptin, and free fatty acid concentrations significantly reduced, but the serum adiponectin concentration was higher, in the maslinic acid group than in the HFD group. In liver tissue, maslinic acid suppressed transcription factors involved in lipogenesis and increased adipose triglyceride lipase. In vitro, maslinic acid decreased lipogenesis by activating AMPK. These findings suggest that maslinic acid acts against hepatic steatosis by regulating enzyme activity involved in lipogenesis, lipolysis, and fatty acid oxidation in the liver.-Liou, C.-J., Dai, Y.-W., Wang, C.-L., Fang, L.-W., Huang, W.-C. Maslinic acid protects against obesity-induced nonalcoholic fatty liver disease in mice through regulation of the Sirt1/AMPK signaling pathway.

Luteolin Attenuates IL-1ß-Induced THP-1 Adhesion to ARPE-19 Cells via Suppression of NF-κB and MAPK Pathways.

Cytokine-induced endothelial dysfunction leads to inflammation and vascular adhesion molecule production in retinal pigment epithelium (RPE) cells. Inflammation is a critical mediator in retinal degeneration (RD) diseases, including age-related macular degeneration (AMD), and RD progression may be prevented through anti-inflammatory activity in RPE cells. The flavonoid polyphenol luteolin (LU) has anti-inflammatory and antidiabetes activities, but its effects regarding retinal protection remain unknown. Here, we examined the ability of luteolin to alleviate markers of inflammation related to RD in cytokine-primed APPE-19 cells. We found that luteolin decreased the levels of interleukin- (IL-) 6, IL-8, soluble intercellular adhesion molecule-1 (sICAM-1), and monocyte chemoattractant protein-1 (MCP-1) and attenuated adherence of the human monocytic leukemia cell line THP-1 to IL-1ß-stimulated ARPE-19 cells. Luteolin also increased anti-inflammatory protein heme oxygenase-1 (HO-1) levels. Interestingly, luteolin induced protein kinase B (AKT) phosphorylation, thus inhibiting nuclear factor- (NF-) κB transfer from cytoplasm into the nucleus and suppressing mitogen-activated protein kinase (MAPK) inflammatory pathways. Furthermore, cotreatment with MAPK inhibitors and luteolin decreased inflammatory cytokine and chemokine levels, and further suppressed THP-1 adhesion. Overall, these results provide evidence that luteolin protects ARPE-19 cells from IL-1ß-stimulated increases of IL-6, IL-8, sICAM-1, and MCP-1 production by blocking the activation of MAPK and NF-κB signaling pathways, thus ameliorating the inflammatory response.

Helminthostachys zeylanica Water Extract Ameliorates Airway Hyperresponsiveness and Eosinophil Infiltration by Reducing Oxidative Stress and Th2 Cytokine Production in a Mouse Asthma Model.

Helminthostachys zeylanica is a traditional folk herb used to improve inflammation and fever in Taiwan. Previous studies showed that H. zeylanica extract could ameliorate lipopolysaccharide-induced acute lung injury in mice. The aim of this study was to investigate whether H. zeylanica water (HZW) and ethyl acetate (HZE) extracts suppressed eosinophil infiltration and airway hyperresponsiveness (AHR) in asthmatic mice, and decreased the inflammatory response and oxidative stress in tracheal epithelial cells. Human tracheal epithelial cells (BEAS-2B cells) were pretreated with various doses of HZW or HZE (1 µg/ml-10 µg/ml), and cell inflammatory responses were induced with IL-4/TNF-α. In addition, female BALB/c mice sensitized with ovalbumin (OVA), to induce asthma, were orally administered with HZW or HZE. The result demonstrated that HZW significantly inhibited the levels of proinflammatory cytokines, chemokines, and reactive oxygen species in activated BEAS-2B cells. HZW also decreased ICAM-1 expression and blocked monocytic cells from adhering to inflammatory BEAS-2B cells in vitro. Surprisingly, HZW was more effective than HZE in suppressing the inflammatory response in BEAS-2B cells. Our results demonstrated that HZW significantly decreased AHR and eosinophil infiltration, and reduced goblet cell hyperplasia in the lungs of asthmatic mice. HZW also inhibited oxidative stress and reduced the levels of Th2 cytokines in bronchoalveolar lavage fluid. Our findings suggest that HZW attenuated the pathological changes and inflammatory response of asthma by suppressing Th2 cytokine production in OVA-sensitized asthmatic mice.

Fisetin Protects Against Hepatic Steatosis Through Regulation of the Sirt1/AMPK and Fatty Acid ß-Oxidation Signaling Pathway in High-Fat Diet-Induced Obese Mice.

BACKGROUND/AIMS: Fisetin is a naturally abundant flavonoid isolated from various fruits and vegetables that was recently identified to have potential biological functions in improving allergic airway inflammation, as well as anti-oxidative and anti-tumor properties. Fisetin has also been demonstrated to have anti-obesity properties in mice. However, the effect of fisetin on nonalcoholic fatty liver disease (NAFLD) is still elusive. Thus, the present study evaluated whether fisetin improves hepatic steatosis in high-fat diet (HFD)-induced obese mice and regulates lipid metabolism of FL83B hepatocytes in vitro. METHODS: NAFLD was induced by HFD in male C57BL/6 mice. The mice were then injected intraperitoneally with fisetin for 10 weeks. In another experiment, FL83B cells were challenged with oleic acid to induce lipid accumulation and treated with various concentrations of fisetin. RESULTS: NAFLD mice treated with fisetin had decreased body weight and epididymal adipose tissue weight compared to NAFLD mice. Fisetin treatment also reduced liver lipid droplet and hepatocyte steatosis, alleviated serum free fatty acid, and leptin concentrations, significantly decreased fatty acid synthase, and significantly increased phosphorylation of AMPKα and the production of sirt-1 and carnitine palmitoyltransferase I in the liver tissue. In vitro, fisetin decreased lipid accumulation and increased lipolysis and ß-oxidation in hepatocytes. CONCLUSION: This study suggests that fisetin is a potential novel treatment for alleviating hepatic lipid metabolism and improving NAFLD in mice via activation of the sirt1/AMPK and ß-oxidation pathway.

Fucoxanthin attenuates fatty acid-induced lipid accumulation in FL83B hepatocytes through regulated Sirt1/AMPK signaling pathway.

The fucoxanthin, isolated from brown algae, was reported to have multiple biological functions to anti-inflammation, anti-tumor, and ameliorated obesity in mice. In this study we investigated whether fucoxanthin could inhibit lipids accumulation in FL83B hepatocytes. FL83B cells were induced as fatty liver cell model by 0.5 mM oleic acid for 48 h, and treated with various concentration of fucoxanthin for 24 h. The results demonstrated that fucoxanthin significantly suppressed lipid accumulation and decreased lipid peroxidation in hepatocytes. Fucoxanthin could decrease lipogenesis-related transcription factor expression, including sterol regulatory element-binding proteins 1c and peroxisome proliferator-activated receptor γ. It also reduced fatty acid synthase expression and increased adipose triglyceride lipase and the phosphorylation of hormone-sensitive lipase production for lipolysis. Furthermore, fucoxanthin significantly increased phosphorylation of AMP-activated protein kinase (AMPK), and decreased activity of acetyl-CoA carboxylase for regulating fatty acid synthesis. The results suggest that fucoxanthin is an effective marine nature compound for increasing lipolysis and inhibiting lipogenesis in oleic acid induced fatty liver cells through promoted Sirt1/AMPK pathway.

Ginkgolide C reduced oleic acid-induced lipid accumulation in HepG2 cells.

Ginkgolide C, isolated from Ginkgo biloba, is a diterpene lactone that has multiple biological functions and can improve Alzheimer disease and platelet aggregation. Ginkgolide C also inhibits adipogenesis in 3T3-L1 adipocytes. The present study evaluated whether ginkgolide C reduced lipid accumulation and regulated the molecular mechanism of lipogenesis in oleic acid-induced HepG2 hepatocytes. HepG2 cells were treated with 0.5 mM oleic acid for 48 h to induce a fatty liver cell model. Then, the cells were exposed to various concentrations of ginkgolide C for 24 h. Staining with Oil Red O and the fluorescent dye BODIPY 493/503 revealed that ginkgolide C significantly reduced excessive lipid accumulation in HepG2 cells. Ginkgolide C decreased peroxisome proliferator-activated receptor γ and sterol regulatory element-binding protein 1c to block the expression of fatty acid synthase. Ginkgolide C treatment also promoted the expression of adipose triglyceride lipase and the phosphorylation level of hormone-sensitive lipase to enhance the decomposition of triglycerides. In addition, ginkgolide C stimulated CPT-1 to activate fatty acid ß-oxidation, significantly increased sirt1 and phosphorylation of AMP-activated protein kinase (AMPK), and decreased expression of acetyl-CoA carboxylase for suppressed fatty acid synthesis in hepatocytes. Taken together, our results suggest that ginkgolide C reduced lipid accumulation and increased lipolysis through the sirt1/AMPK pathway in oleic acid-induced fatty liver cells.

Tomatidine Attenuates Airway Hyperresponsiveness and Inflammation by Suppressing Th2 Cytokines in a Mouse Model of Asthma.

Tomatidine is isolated from the fruits of tomato plants and found to have anti-inflammatory effects in macrophages. In the present study, we investigated whether tomatidine suppresses airway hyperresponsiveness (AHR) and eosinophil infiltration in asthmatic mice. BALB/c mice were sensitized with ovalbumin and treated with tomatidine by intraperitoneal injection. Airway resistance was measured by intubation analysis as an indication of airway responsiveness, and histological studies were performed to evaluate eosinophil infiltration in lung tissue. Tomatidine reduced AHR and decreased eosinophil infiltration in the lungs of asthmatic mice. Tomatidine suppressed Th2 cytokine production in bronchoalveolar lavage fluid. Tomatidine also blocked the expression of inflammatory and Th2 cytokine genes in lung tissue. In vitro, tomatidine inhibited proinflammatory cytokines and CCL11 production in inflammatory BEAS-2B bronchial epithelial cells. These results indicate that tomatidine contributes to the amelioration of AHR and eosinophil infiltration by blocking the inflammatory response and Th2 cell activity in asthmatic mice.

Matrine Attenuates COX-2 and ICAM-1 Expressions in Human Lung Epithelial Cells and Prevents Acute Lung Injury in LPS-Induced Mice.

Matrine is isolated from Sophora flavescens and shows anti-inflammatory effects in macrophages. Here we evaluated matrine's suppressive effects on cyclooxygenase 2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1) expressions in lipopolysaccharide- (LPS-) stimulated human lung epithelial A549 cells. Additionally, BALB/c mice were given various matrine doses by intraperitoneal injection, and then lung injury was induced via intratracheal instillation of LPS. In LPS-stimulated A549 cells, matrine inhibited the productions of interleukin-8 (IL-8), monocyte chemotactic protein-1, and IL-6 and decreased COX-2 expression. Matrine treatment also decreased ICAM-1 protein expression and suppressed the adhesion of neutrophil-like cells to inflammatory A549 cells. In vitro results demonstrated that matrine significantly inhibited mitogen-activated protein kinase phosphorylation and decreased nuclear transcription factor kappa-B subunit p65 protein translocation into the nucleus. In vivo data indicated that matrine significantly inhibited neutrophil infiltration and suppressed productions of tumor necrosis factor-α and IL-6 in mouse bronchoalveolar lavage fluid and serum. Analysis of lung tissue showed that matrine decreased the gene expression of proinflammatory cytokines, chemokines, COX-2, and ICAM-1. Our findings suggest that matrine improved lung injury in mice and decreased the inflammatory response in human lung epithelial cells.

Sesamol suppresses the inflammatory response by inhibiting NF-κB/MAPK activation and upregulating AMP kinase signaling in RAW 264.7 macrophages.

OBJECTIVES AND DESIGN: Sesamol is a lignan isolated from sesame seed oil. In recent years, it was found that sesamol could decrease lung inflammation and lipopolysaccharide (LPS)-induced lung injury in rats. In this study, we investigated whether sesamol exhibited anti-inflammatory activity in LPS-stimulated macrophages. MATERIALS AND METHODS: RAW 264.7 cells were treated with sesamol, then treated with LPS to induce inflammation. The levels of proinflammatory cytokines were analyzed with ELISA. The gene and protein expression of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), and nuclear factor erythroid-2-related factor 2 (Nrf2) were evaluated with real-time PCR and Western blots, respectively. We also examined inflammatory signaling pathways, including nuclear transcription factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. RESULTS: Sesamol inhibited production of nitric oxide, prostaglandin E2 (PGE2), and proinflammatory cytokines. Sesamol markedly suppressed mRNA and protein expression of iNOS and COX-2. Sesamol enhanced the protective antioxidant pathway represented by Nrf2 and HO-1. Moreover, sesamol suppressed NF-κB transport into the nucleus and decreased MAPK activation, but it promoted adenosine monophosphate-activated protein kinase (AMPK) activation. CONCLUSIONS: These data suggested that sesamol ameliorated inflammatory and oxidative damage by upregulating AMPK activation and Nrf2 signaling and blocking the NF-κB and MAPK signaling pathways.

Monoclonal antibody against a putative myristoylated membrane protein encoded by grouper iridovirus 59L gene.

Groupers (Epinephelus spp.) are economically important fish species worldwide, and ranaviruses are major viral pathogens causing heavy economic losses in grouper aquaculture. In this study, the 59L gene of grouper iridovirus (GIV-59L) was cloned and characterized. This gene is 1521 bp and encodes a protein of 506 amino acids with a predicted molecular mass of 53.9 kDa. Interestingly, GIV-59L and its homologs are found in all genera of the family Iridoviridae. A mouse monoclonal antibody specific for the C-terminal domain (amino acid positions 254-506) of the GIV-59L protein, GIV-59L(760-1518)-MAb-21, was produced and proved to be well suited for use in a number of GIV immunoassays. RT-PCR, Western blotting, and cycloheximide and cytosine arabinoside drug inhibition analyses indicated that GIV-59L is a viral late gene in GIV-infected grouper kidney cells. Immunofluorescence analysis revealed that GIV-59L protein mainly accumulates in the cytoplasm of infected cells and is finally packed into a whole virus particle. The GIV-59L(760-1518)-MAb-21 characterized in this study could have widespread application in GIV immunodiagnostics and other research on GIV. In addition, the results presented here offer important insights into the pathogenesis of GIV.

Characterization of protein marker expression, tumorigenicity, and doxorubicin chemoresistance in two new canine mammary tumor cell lines.

BACKGROUND: Canine mammary tumors (CMTs) are the most common type of cancer found in female dogs. Establishment and evaluation of tumor cell lines can facilitate investigations of the biological mechanisms of cancer. Different cell models are used to investigate genetic, epigenetic, and cellular pathways, cancer progression, and cancer therapeutics. Establishment of new cell models will greatly facilitate research in this field. In the present study, we established and characterized two new CMT cell lines derived from a single CMT. RESULTS: We established two cell lines from a single malignant CMT specimen: DTK-E and DTK-SME. Morphologically, the DTK-E cells were large, flat, and epithelial-like, whereas DTK-SME cells were round and epithelial-like. Doubling times were 24 h for DTK-E and 18 h for DTK-SME. On western blots, both cell lines expressed cytokeratin AE1, vimentin, cytokeratin 7 (CK7), and heat shock protein 27 (HSP27). Moreover, investigation of chemoresistance revealed that DTK-SME was more resistant to doxorubicin-induced apoptosis than DTK-E was. After xenotransplantation, both DTK-E and DTK-SME tumors appeared within 14 days, but the average size of DTK-SME tumors was greater than that of DTK-E tumors after 56 days. CONCLUSION: We established two new cell lines from a single CMT, which exhibit significant diversity in cell morphology, protein marker expression, tumorigenicity, and chemoresistance. The results of this study revealed that the DTK-SME cell line was more resistant to doxorubicin-induced apoptosis and exhibited higher tumorigenicity in vivo than the DTK-E cell line. We anticipate that the two novel CMT cell lines established in this study will be useful for investigating the tumorigenesis of mammary carcinomas and for screening anticancer drugs.

Protective effects of myricetin on airway inflammation and oxidative stress in ovalbumin-induced asthma mice.

Myricetin, a flavonoid isolated from many edible vegetables and fruits, has multiple biological effects, including anti-inflammatory and anti-tumor effects. Myricetin could inhibit mast cell degranulation in vitro, and it reduced the eosinophil content in bronchoalveolar lavage fluid (BALF) of ovalbumin (OVA)-sensitized mice. However, it remains unclear whether myricetin alleviates airway hyperresponsiveness (AHR), airway inflammation, and oxidative stress in asthma. Here, we investigated whether myricetin attenuated AHR, airway inflammation, and eosinophil infiltration in lungs of asthmatic mice. Mice were sensitized with OVA, then injected intraperitoneally with myricetin to investigate anti-inflammatory and antioxidant effects of myricetin. Moreover, we examined its effects on human bronchial epithelial BEAS-2B cells stimulated with TNF-α and IL-4, in vitro. Myricetin effectively mitigated eosinophil infiltration, AHR, and goblet cell hyperplasia in lung, and it reduced Th2 cytokine expression in BALF from asthmatic mice. Myricetin effectively promoted glutathione and superoxide dismutase productions and mitigated malondialdehyde expressions in mice by promoting Nrf2/HO-1 expression. Myricetin also reduced the production of proinflammatory cytokines, eotaxins, and reactive oxygen species in BEAS-2B cells. Myricetin effectively suppressed ICAM-1 expression in inflammatory BEAS-2B cells, which suppressed monocyte cell adherence. These results suggested that myricetin could effectively improve asthma symptoms, mainly through blocking Th2-cell activation, which reduced oxidative stress, AHR, and airway inflammation.

Licochalcone D ameliorates lipid metabolism in hepatocytes by modulating lipogenesis and autophagy.

Metabolic dysfunction-associated fatty liver disease is a metabolic disease caused by abnormal lipid accumulation in the liver. Excessive lipid accumulation results in liver inflammation and fibrosis. Previous studies have demonstrated that the chalcone licochalcone D, which is isolated from Glycyrrhiza inflata Batal, has anti-tumor and anti-inflammatory effects. The present study explored whether licochalcone D can regulate lipid accumulation in fatty liver cells. FL83B hepatocytes were incubated with oleic acid to establish a fatty liver cell model, and then treated with licochalcone D to evaluate the molecular mechanisms underlying the regulation of lipid metabolism. In addition, male C57BL/6 mice were fed a methionine/choline-deficient diet to induce an animal model of metabolic dysfunction-associated steatohepatitis (MASH) and given 5 mg/kg licochalcone D by intraperitoneal injection. In cell experiments, licochalcone D significantly reduced lipid accumulation in fatty liver cells and reduced sterol regulatory element-binding protein 1c expression, blocking fatty acid synthase production. Licochalcone D increased adipose triglyceride lipase and carnitine palmitoyltransferase 1 expression, enhancing lipolysis and fatty acid ß-oxidation, respectively. Licochalcone D also significantly increased SIRT-1 and AMPK phosphorylation, reducing acetyl-CoA carboxylase phosphorylation and inhibiting fatty acid synthesis. Licochalcone D also increased the fusion of autophagosomes and lysosomes to promote autophagy, reducing oil droplet accumulation in fatty liver cells. In the animal experiments, licochalcone D effectively reduced the number of lipid vacuoles and degree of fibrosis in liver tissue and inhibited liver inflammation. Thus, licochalcone D can improve MASH by reducing lipid accumulation, inhibiting inflammation, and increasing autophagy.

Punicalagin from pomegranate ameliorates TNF-α/IFN-γ-induced inflammatory responses in HaCaT cells via regulation of SIRT1/STAT3 axis and Nrf2/HO-1 signaling pathway.

Punicalagin (PUN) was isolated from the peel of pomegranate (Punica granatum L.), is a polyphenol with anti-inflammatory, hepatoprotective, and antioxidant activities. However, it remains unclear whether PUN alleviates the inflammation and anti-inflammatory mechanisms in pro-inflammatory cytokines-induced human keratinocyte HaCaT cells. Here, we investigated that tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) mixture-stimulated HaCaT cells were treated with various concentrations of PUN, followed by analyzed the expression of inflammation-related mediators and evaluate anti-inflammatory-related pathways. Our results demonstrated that PUN ≤ 100 µM did not reduce HaCaT cell viability, and PUN ≥ 3 µM was sufficient to decrease interleukin-6 (IL-6), IL-8, monocyte chemoattractant protein-1 (MCP-1), chemokine ligand 5 (CCL5), CCL17 and CCL20 concentrations. We found that PUN ≥ 10 µM and ≥ 3 µM significantly increased sirtuin 1 (SIRT1) expression and inhibited signal transducer and activator of transcription 3 (STAT3) phosphorylation, respectively. PUN downregulated inflammation-related proteins cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), enhanced nuclear factor erythroid-2-related factor-2 (Nrf2) and heme oxygenase-1 (HO-1) expression. Moreover, PUN decreased intercellular adhesion molecule-1 (ICAM-1) expression and inhibited monocyte adhesion to inflamed HaCaT cells. PUN also suppressed inflammatory-related pathways, including mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways in TNF-α/IFN-γ- stimulated HaCat cells. Collectively, there is significant evidence that PUN has effective protective defenses against TNF-α/IFN-γ-induced skin inflammation by enhancing SIRT1 to mediate STAT3 and Nrf2/HO-1 signaling pathway.