Aromadendrin Inhibits Lipopolysaccharide-Induced Inflammation in BEAS-2B Cells and Lungs of Mice.

Aromadendrin is a phenolic compound with various biological effects such as anti-inflammatory properties. However, its protective effects against acute lung injury (ALI) remain unclear. Therefore, this study aimed to explore the ameliorative effects of aromadendrin in an experimental model of lipopolysaccharide (LPS)-induced ALI. In vitro analysis revealed a notable increase in the levels of cytokine/chemokine formation, nuclear factor kappa B (NF-κB) activation, and myeloid differentiation primary response 88 (MyD88)/toll-like receptor (TLR4) expression in LPS-stimulated BEAS-2B lung epithelial cell lines that was ameliorated by aromadendrin pretreatment. In LPS-induced ALI mice, the remarkable upregulation of immune cells (ICs) and IL-1ß/IL-6/TNF-α levels in the bronchoalveolar lavage fluid (BALF) and inducible nitric oxide synthase (iNOS)/cyclooxygenase-2 (COX-2)/CD68 expression in lung was decreased by the oral administration of aromadendrin. Histological analysis revealed the presence of cells in the lungs of acute lung injury (ALI) mice, which was alleviated by aromadendrin. In addition, aromadendrin ameliorated lung edema. This in vivo effect of aromadendrin was accompanied by its inhibitory effect on LPS-induced NF-κB activation, MyD88/TLR4 expression, and signal transducer and activator of transcription 3 (STAT3) activation. Furthermore, aromadendrin increased the expression of heme oxygenase-1 (HO-1)/ NAD(P)H quinone dehydrogenase 1 (NQO1) in the lungs of ALI mice. In summary, the in vitro and in vivo studies demonstrated that aromadendrin ameliorated endotoxin-induced pulmonary inflammation by suppressing cytokine formation and NF-κB activation, suggesting that aromadendrin could be a useful adjuvant in the treatment of ALI.

Citropten Inhibits Vascular Smooth Muscle Cell Proliferation and Migration via the TRPV1 Receptor.

Vascular smooth muscle cell (VSMC) proliferation and migration play critical roles in arterial remodeling. Citropten, a natural organic compound belonging to coumarin and its derivative classes, exhibits various biological activities. However, mechanisms by which citropten protects against vascular remodeling remain unknown. Therefore, in this study, we investigated the inhibitory effects of citropten on VSMC proliferation and migration under high-glucose (HG) stimulation. Citropten abolished the proliferation and migration of rat vascular smooth muscle cells (RVSMCs) in a concentration-dependent manner. Also, citropten inhibited the expression of proliferation-related proteins, including proliferating cell nuclear antigen (PCNA), cyclin E1, cyclin D1, and migration-related markers such as matrix metalloproteinase (MMP), MMP2 and MMP9, in a concentration-dependent manner. In addition, citropten inhibited the phosphorylation of ERK and AKT, as well as hypoxia-inducible factor-1α (HIF-1α) expression, mediated to the Krüppel-like factor 4 (KLF4) transcription factor. Using pharmacological inhibitors of ERK, AKT, and HIF-1α also strongly blocked the expression of MMP9, PCNA, and cyclin D1, as well as migration and the proliferation rate. Finally, molecular docking suggested that citropten docked onto the binding site of transient receptor potential vanilloid 1 (TRPV1), like epigallocatechin gallate (EGCG), a well-known agonist of TRPV1. These data suggest that citropten inhibits VSMC proliferation and migration by activating the TRPV1 channel.

Ancistrocladus tectorius Extract Inhibits Obesity by Promoting Thermogenesis and Mitochondrial Dynamics in High-Fat Diet-Fed Mice.

Root extracts of Ancistrocladus tectorius (AT), a shrub native to China, have been shown to have antiviral and antitumor activities, but the anti-obesity effects of AT aerial parts, mainly the leaves and stems, have not been investigated. This study is the first to investigate the anti-obesity effects and molecular mechanism of AT 70% ethanol extract in 3T3-L1 adipocytes and high-fat diet (HFD)-fed C57BL/6J mice. Treatment with AT extract inhibited lipid accumulation in 3T3-L1 cells and decreased the expression of adipogenesis-related genes. AT extract also upregulated the mRNA expression of genes related to mitochondrial dynamics in 3T3-L1 adipocytes. AT administration for 12 weeks reduced body weight and organ weights, including liver, pancreas, and white and brown adipose tissue, and improved plasma profiles such as glucose, insulin, homeostasis model assessment of insulin resistance, triglyceride (TG), and total cholesterol in HFD-fed mice. AT extract reduced HFD-induced hepatic steatosis with levels of liver TG and lipogenesis-related genes. AT extract upregulated thermogenesis-related genes such as Cidea, Pgc1α, Ucp1, Prdm16, Adrb1, and Adrb3 and mitochondrial dynamics-related genes such as Mff, Opa1, and Mfn2 in brown adipose tissue (BAT). Therefore, AT extract effectively reduced obesity by promoting thermogenesis and the mitochondrial dynamics of BAT in HFD-fed mice.

Platycodi Radix Extract Prevents Hepatic Steatosis by Enhancing Bile Acid Synthesis in a High-Fat Diet-Induced Fatty Liver Mouse Model.

We aimed to identify the mechanism underlying the preventive effects of non-alcoholic fatty liver disease (NAFLD) through Platycodi Radix consumption using liver proteomic and bioinformatic analysis. C57BL/6J mice were categorized into three groups: those receiving a standard chow diet (NCD), those on a high-fat diet (HFD), and those on an HFD supplemented with 5% Platycodi Radix extract (PRE). After a 12-week period, PRE-fed mice exhibited a noteworthy prevention of hepatic steatosis. Protein identification and quantification in liver samples were conducted using LC-MS/MS. The identified proteins were analyzed through Ingenuity Pathway Analysis software, revealing a decrease in proteins associated with FXR/RXR activation and a concurrent increase in cholesterol biosynthesis proteins in the PRE-treated mouse liver. Subsequent network analysis predicted enhanced bile acid synthesis from these proteins. Indeed, the quantity of bile acids, which was reduced in HFD conditions, increased in the PRE group, accompanied by an elevation in the expression of synthesis-related proteins. Our findings suggest that the beneficial effects of PRE in preventing hepatic steatosis may be mediated, at least in part, through the modulation of FXR/RXR activation, cholesterol biosynthesis, and bile acid synthesis pathways.

Evodiae Fructus extract suppresses inflammatory response in HaCaT cells and improves house dust mite-induced atopic dermatitis in NC/Nga mice.

This study was conducted to assess the effect of Evodiae Fructus 70% ethanol extract (EFE) on the pathology of atopic dermatitis using in vitro and in vivo models. The major compounds in EFE were identified by ultra-performance liquid chromatography with tandem mass spectrometry as rutaecarpine, evodiamine, evodol, dehydroevodiamine, limonin, synephrine, evocarpine, dihydroevocarpine, and hydroxyevodiamine. EFE significantly decreased chemokine levels in tumor necrosis factor-α/interferon-γ-stimulated HaCaT cells. In house dust mite-treated NC/Nga mice, topical application of EFE significantly decreased the dermatitis score, epidermal hyperplasia and thickening, mast cell infiltration, and plasma levels of histamine and corticosterone. Thymic stromal lymphopoietin, CD4+ T cells, interleukin-4, and intercellular adhesion molecule-1 expression in the lesioned skin was reduced in the treated mice. The mechanism of EFE was elucidated using transcriptome analysis, followed by experimental validation using Western blotting in HaCaT cells. EFE down-regulated the activation of Janus kinase (JAK)-signal transducers and activators of transcription (STAT) and mitogen-activated protein kinases (MAPK) signaling pathways in HaCaT cells. EFE improves atopic dermatitis-like symptoms by suppressing inflammatory mediators, cytokines, and chemokines by regulating the JAK-STAT and MAPK signaling pathways, suggesting its use as a potential agent for the treatment of atopic dermatitis.

Antarctic Krill Oil from Euphausia superba Ameliorates Carrageenan-Induced Thrombosis in a Mouse Model.

FJH-KO obtained from Antarctic krill, especially Euphausia superba, has been reported to contain high amounts of omega-3 polyunsaturated fatty acids (n-3 PUFA) and to exhibit anticancer and anti-inflammatory properties. However, its antithrombotic effects have not yet been reported. This study aimed to investigate the antithrombotic effects of FJH-KO in carrageenan-induced thrombosis mouse models and human endothelial cells. Thrombosis was induced by carrageenan injection, whereas the mice received FJH-KO pretreatment. FJH-KO attenuated carrageenan-induced thrombus formation in mouse tissue vessels and prolonged tail bleeding. The inhibitory effect of FJH-KO was associated with decreased plasma levels of thromboxane B2, P-selectin, endothelin-1, ß-thromboglobulin, platelet factor 4, serotonin, TNF-α, IL-1ß, and IL-6. Meanwhile, FJH-KO induced plasma levels of prostacyclin I2 and plasminogen. In vitro, FJH-KO decreased the adhesion of THP-1 monocytes to human endothelial cells stimulated by TNF-α via eNOS activation and NO production. Furthermore, FJH-KO inhibited the expression of TNF-α-induced adhesion molecules such as ICAM-1 and VCAM-1 by suppressing the NF-κB signaling pathway. Taken together, our study demonstrates that FJH-KO protects against carrageenan-induced thrombosis by regulating endothelial cell activation and has potential as an antithrombotic agent.

Development of Genetically Engineered Feeder Cells for Natural Killer Cell Expansion.

BACKGROUND/AIM: To obtain sufficient numbers of high-quality natural killer (NK) cells, we developed feeder cells using synthetic biology techniques. MATERIALS AND METHODS: K562 cells were engineered to express membrane bound interleukin-2 (mbIL2) or interleukin-13 (mbIL13). RESULTS: The incubation of human primary NK cells isolated from peripheral blood mononuclear cells (PBMCs) with these feeder cells significantly increased the number of activated NK cells compared to K562 parental cells. Fluorescence-activated cell sorting (FACS) analysis demonstrated that NKG2D activating receptors were abundant on the surface of NK cells expanded by K562-mbIL2 or mbIL13 cells. NK cells expanded on K562-mbIL2 or mbIL13 lysed cancer cells more effectively than those cultured with normal K562 cells. Using NK cells incubated with our feeder cells, we developed anti-CD19 chimeric antigen receptor (CAR)-NK cells. They showed robust cytotoxic effect against CD19 positive cancer cell line. CONCLUSION: Our newly developed feeder cells could provide useful tools for NK cell therapy.

Sesamin ameliorates lipotoxicity and lipid accumulation through the activation of the estrogen receptor alpha signaling pathway.

Nonalcoholic fatty liver disease (NAFLD) has been linked to fat accumulation in the liver and lipid metabolism imbalance. Sesamin, a lignan commonly found in sesame seed oil, possesses antioxidant, anti-inflammatory, and anticancer properties. However, the precise mechanisms by which sesamin prevents hepatic steatosis are not well understood. This study aimed to explore the molecular mechanisms by which sesamin may improve lipid metabolism dysregulation. A in vitro hepatic steatosis model was established by exposing HepG2 cells to palmitate sodium. The results showed that sesamin effectively mitigated lipotoxicity and reduced reactive oxygen species production. Additionally, sesamin suppressed lipid accumulation by regulating key factors involved in lipogenesis and lipolysis, such as fatty acid synthase (FASN), sterol regulatory element-binding protein 1c (SREBP-1c), forkhead box protein O-1, and adipose triglyceride lipase. Molecular docking results indicated that sesamin could bind to estrogen receptor α (ERα) and reduce FASN and SREBP-1c expression via the Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß)/AMP-activated protein kinase (AMPK) signaling pathway. Sesamin attenuated palmitate-induced lipotoxicity and regulated hepatic lipid metabolism in HepG2 cells by activating the ERα/CaMKKß/AMPK signaling pathway. These findings suggest that sesamin can improve lipid metabolism disorders and is a promising candidate for treating hepatic steatosis.

Dendropanoxide Alleviates Thioacetamide-induced Hepatic Fibrosis via Inhibition of ROS Production and Inflammation in BALB/C Mice.

Hepatic fibrosis results from overproduction and excessive accumulation of extracellular matrix (ECM) proteins in hepatocytes. Although the beneficial effects of dendropanoxide (DPx) isolated from Dendropanax morbifera have been studied, its role as an anti-fibrotic agent remains elucidated. We investigated the protective effect of DPx in BALB/C mice that received thioacetamide (TAA) intraperitoneally for 6 weeks. Later DPx (20 mg/kg/day) or silymarin (50 mg/kg/day) was administered daily for 6 weeks, followed by biochemical and histological analyses of each group. Hematoxylin and eosin staining of the livers showed TAA-induced hepatic fibrosis, which was significantly reduced in the DPx group. DPx treatment significantly decreased TAA-induced hyperlipidemia as evidenced by the decreased AST, ALT, ALP, γ-GTP and serum TG concentrations and reduced the activities of catalase (CAT) and superoxide dismutase (SOD) activity. ELISA revealed reduced levels of total glutathione (GSH), malondialdehyde (MDA) and Inflammatory factors (IL-6, IL-1ß, and TNF-α). Immunostaining showed reduced in collagen-1, α-SMA, and TGF-ß1 expression and western blotting showed reduced levels of the apoptotic proteins, TGF-ß1, p-Smad2/3, and Smad4. RT-qPCR and Western blotting revealed modifications in SIRT1, SIRT3 and SIRT4. Thus, DPx exerted a protective effect against TAA-induced hepatic fibrosis in the male BALB/C mouse model by inhibiting oxidative stress, inflammation, and apoptosis via TGF-ß1/Smads signaling.

Heterotypic cell-in-cell structures between cancer and NK cells are associated with enhanced anticancer drug resistance.

The heterotypic CIC structures formed of cancer and immune cells have been observed in tumor tissues. We aimed to assess the feasibility of using heterotypic CICs as a functional biomarker to predict NK susceptibility and drug resistance. The heterotypic CIC-forming cancer cells showed a lower response to NK cytotoxicity and higher proliferative ability than non-CIC cancer cells. After treatment with anticancer drugs, cancer cells that formed heterotypic CICs showed a higher resistance to anticancer drugs than non-CIC cancer cells. We also observed the formation of more CIC structures in cancer cells treated with anticancer drugs than in the non-treated group. Our results confirm the association between heterotypic CIC structures and anticancer drug resistance in CICs formed from NK and cancer cells. These results suggest a mechanism underlying immune evasion in heterotypic CIC cancer cells and provide insights into the anticancer drug resistance of cancer cells.

Effect of 3-caffeoyl, 4-dihydrocaffeoylquinic acid from Salicornia herbacea on endothelial nitric oxide synthase activation via calcium signaling pathway.

3-Caffeoyl-4-dicaffeoylquinic acid (CDCQ) is a natural chlorogenic acid isolated from Salicornia herbacea that protects against oxidative stress, inflammation, and cancer. Nitric oxide (NO) plays a physiologically beneficial role in the cardiovascular system, including vasodilation, protection of endothelial cell function, and anti-inflammation. However, the effect of CDCQ on NO production and eNOS phosphorylation in endothelial cells is unclear. We investigated the effect of CDCQ on eNOS phosphorylation and NO production in human endothelial cells, and the underlying signaling pathway. CDCQ significantly increased NO production and the phosphorylation of eNOS at Ser1177. Additionally, CDCQ induced phosphorylation of PKA, CaMKII, CaMKKß, and AMPK. Interestingly, CDCQ increased the intracellular Ca2+ level, and L-type Ca2+ channel (LTCC) blockade significantly attenuated CDCQ-induced eNOS activity and NO production by inhibiting PKA, CaMKII, CaMKKß, and AMPK phosphorylation. These results suggest that CDCQ increased eNOS phosphorylation and NO production by Ca2+-dependent phosphorylation of PKA, CaMKII, CaMKKß, and AMPK. Our findings provide evidence that CDCQ plays a pivotal role in the activity of eNOS and NO production, which is involved in the protection of endothelial dysfunction.

Upregulation of CYP1B1 by hypoxia is mediated by ERα activation in breast cancer cells.

Endocrine therapy for breast cancer often leads to drug resistance and tumor recurrence; tumor hypoxia is also associated with mortality and tumor relapse. Cytochrome P450 1B1 (CYP1B1) regulates estrogen metabolism in breast cells and is known to be overexpressed in breast cancer tissue. Although the individual association of hypoxia-induced hypoxia-inducible factor-1-alpha (HIF-1α) and CYP1B1 with tumorigenesis is well known, the association between HIF-1α and CYP1B1 leading to tumorigenesis has not been investigated. Here, we investigated the correlation between hypoxia and CYP1B1 expression in breast cancer cells for tumorigenesis-related mechanisms. Hypoxia was induced in the human breast cancer cell lines MCF-7 (Er-positive) and MDA-MB-231 (triple-negative) and the normal breast epithelial cell line MCF10A; these cell lines were then subjected to immunoblotting, transient transfection, luciferase assays, gene silencing using small interfering RNA, polymerase chain reaction analysis, chromatin immunoprecipitation, co-immunoprecipitation, and mammalian two-hybrid assays. Furthermore, immunofluorescence analysis of the tumor microarrays was performed, and the pub2015 and the Cancer Genome Atlas patient datasets were analyzed. HIF-1α expression in response to hypoxia occurred in both normal and breast cancer cells, whereas CYP1B1 was induced only in estrogen receptor α (ERα)-positive breast cancer cells under hypoxia. HIF-1α activated ERα through direct binding and in a ligand-independent manner to promote CYP1B1 expression. Therefore, we suggested the mechanism by which hypoxia and ER-positivity orchestrate breast cancer relapse.

Puerarin attenuates hepatic steatosis via G-protein-coupled estrogen receptor-mediated calcium and SIRT1 signaling pathways.

Puerarin, the major bioactive ingredient isolated from the root of Pueraria lobata (Willd.), attenuates body weight gain and reduces lipid levels in high-fat diet-induced obese mice; however, the underlying mechanism responsible for regulating lipid metabolism remains unclear. This study investigated the molecular mechanism(s) underlying the role of puerarin in regulating lipogenesis and lipolysis in human HepG2 cells. In this study, puerarin strongly inhibited the expression of fatty acid synthase (FASN) and sterol regulatory element binding protein 1c (SREBP-1c). Moreover, puerarin significantly induced the expression of adipose triglyceride lipase (ATGL), which is responsible for triacylglycerol hydrolase activity in cells. Puerarin enhanced 5' AMP-activated protein kinase (AMPK) activity, which is a central regulator of hepatic lipid metabolism. Furthermore, this AMPK activation could be mediated by sirtuin 1 (SIRT1) and calcium signaling pathways involved in G protein-coupled estrogen receptor (GPER) signaling. GPER blockage significantly reversed the effect of puerarin on lipid accumulation and the related signaling pathways. Docking studies showed that puerarin could bind in the GPER in a similar manner as GPER agonist G1. Our results suggest that puerarin can improve hepatic steatosis by activating GPER; it's signaling cascade sequentially induced calcium and SIRT1 signaling pathways. Thus, puerarin may be a potential therapeutic agent for the treatment of non-alcoholic fatty liver disease.

Effect of Porcine Whole Blood Protein Hydrolysate on Slow-Twitch Muscle Fiber Expression and Mitochondrial Biogenesis via the AMPK/SIRT1 Pathway.

Skeletal muscle is a heterogeneous tissue composed of a variety of functionally different fiber types. Slow-twitch type I muscle fibers are rich with mitochondria, and mitochondrial biogenesis promotes a shift towards more slow fibers. Leucine, a branched-chain amino acid (BCAA), regulates slow-twitch muscle fiber expression and mitochondrial function. The BCAA content is increased in porcine whole-blood protein hydrolysates (PWBPH) but the effect of PWBPH on muscle fiber type conversion is unknown. Supplementation with PWBPH (250 and 500 mg/kg for 5 weeks) increased time to exhaustion in the forced swimming test and the mass of the quadriceps femoris muscle but decreased the levels of blood markers of exercise-induced fatigue. PWBPH also promoted fast-twitch to slow-twitch muscle fiber conversion, elevated the levels of mitochondrial biogenesis markers (SIRT1, p-AMPK, PGC-1α, NRF1 and TFAM) and increased succinate dehydrogenase and malate dehydrogenase activities in ICR mice. Similarly, PWBPH induced markers of slow-twitch muscle fibers and mitochondrial biogenesis in C2C12 myotubes. Moreover, AMPK and SIRT1 inhibition blocked the PWBPH-induced muscle fiber type conversion in C2C12 myotubes. These results indicate that PWBPH enhances exercise performance by promoting slow-twitch muscle fiber expression and mitochondrial function via the AMPK/SIRT1 signaling pathway.

Immune-Enhancing Effect of Submerged Culture of Ceriporia lacerata Mycelia on Cyclophosphamide-Induced Immunosuppressed Mice and the Underlying Mechanisms in Macrophages.

The white-rot fungi Ceriporia lacerata is used in bioremediation, such as lignocellulose degradation, in nature. Submerged cultures and extracts of C. lacerata mycelia (CLM) have been reported to contain various active ingredients, including ß-glucan and extracellular polysaccharides, and to exert anti-diabetogenic properties in mice and cell lines. However, the immunostimulatory effects have not yet been reported. This study aimed to identify the immunomodulatory effects, and underlying mechanisms thereof, of submerged cultures of CLM using RAW264.7 macrophages and cyclophosphamide (CTX)-induced immunosuppression in mice. Compared to CTX-induced immunosuppressed mice, the spleen and thymus indexes in mice orally administered CLM were significantly increased; body weight loss was alleviated; and natural killer (NK) cytotoxicity, lymphocyte proliferation, and cytokine (tumor necrosis factor [TNF]-α, interferon [IFN]-γ, and interleukin [IL]-2) production were elevated in the serum. In RAW264.7 macrophages, treatment with CLM induced phagocytic activity, increased the production of nitric oxide (NO), and promoted mRNA expression of the immunomodulatory cytokines TNF-α, IFN-γ, IL-1ß, IL-6, IL-10, and IL-12. In addition, CLM increased the inducible NO synthase (iNOS) concentration in macrophages, similar to lipopolysaccharide (LPS) stimulation. Mechanistic studies showed that CLM induced the activation of the NF-κB, PI3k/Akt, ERK1/2, and JNK1/2 pathways. Moreover, the phosphorylation of NF-κB and IκB induced by CLM in RAW264.7 cells was suppressed by specific MAPKs and PI3K inhibitors. Further experiments with a TLR4 inhibitor demonstrated that the production of TNF-α, IL-1ß, and IL-6 induced by CLM was decreased after TLR4 was blocked. Overall, CLM protected against CTX-induced adverse reactions by enhancing humoral and cellular immune functions, and has potential as an immunomodulatory agent.

Rutaecarpine Increases Nitric Oxide Synthesis via eNOS Phosphorylation by TRPV1-Dependent CaMKII and CaMKKß/AMPK Signaling Pathway in Human Endothelial Cells.

Rutaecarpine (RUT) is a bioactive alkaloid isolated from the fruit of Evodia rutaecarpa that exerts a cellular protective effect. However, its protective effects on endothelial cells and its mechanism of action are still unclear. In this study, we demonstrated the effects of RUT on nitric oxide (NO) synthesis via endothelial nitric oxide synthase (eNOS) phosphorylation in endothelial cells and the underlying molecular mechanisms. RUT treatment promoted NO generation by increasing eNOS phosphorylation. Additionally, RUT induced an increase in intracellular Ca2+ concentration and phosphorylation of Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß), AMP-activated protein kinase (AMPK), and Ca2+/calmodulin-dependent kinase II (CaMKII). Inhibition of transient receptor potential vanilloid type 1 (TRPV1) attenuated RUT-induced intracellular Ca2+ concentration and phosphorylation of CaMKII, CaMKKß, AMPK, and eNOS. Treatment with KN-62 (a CaMKII inhibitor), Compound C (an AMPK inhibitor), and STO-609 (a CaMKKß inhibitor) suppressed RUT-induced eNOS phosphorylation and NO generation. Interestingly, RUT attenuated the expression of ICAM-1 and VCAM-1 induced by TNF-α and inhibited the inflammation-related NF-κB signaling pathway. Taken together, these results suggest that RUT promotes NO synthesis and eNOS phosphorylation via the Ca2+/CaMKII and CaM/CaMKKß/AMPK signaling pathways through TRPV1. These findings provide evidence that RUT prevents endothelial dysfunction and benefit cardiovascular health.

Effects of tobacco compound 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on the expression of epigenetically regulated genes in lung carcinogenesis.

Cigarette smoking is a major cause of lung cancer. Although tobacco smoking-induced genotoxicity has been well established, there is apparent lack of abundance functional epigenetic effects reported On cigarette smoke-induced lung carcinogenesis. The aim of this study was to determine effects of intratracheal administration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) utilizing target gene expression DNA methylation patterns in lung tissues of mice following twice weekly for 8 weeks treatment. An unbiased approach where genomic regions was undertaken to assess early methylation changes within mouse pulmonary tissues. A methylated-CpG island recovery assay (MIRA) was performed to map the DNA methylome in lung tissues, with the position of methylated DNA determined using a Genome Analyzer (MIRA-SEQ). Alterations in epigenetic-regulated target genes were confirmed with quantitative reverse transcription-PCR, which revealed 35 differentially hypermethylated genes including Cdkn1C, Hsf4, Hnf1a, Cdx1, and Hoxa5 and 30 differentially hypomethylated genes including Ddx4, Piwi1, Mdm2, and Pce1 in NNK-exposed lung tissue compared with controls. The main pathway of these genes for mediating biological information was analyzed using the Kyoto Encyclopedia of Genes and Genomes database. Among them, Rssf1 and Mdm2 were closely associated with NNK-induced lung carcinogenesis. Taken together, our data provide valuable resources for detecting cigarette smoke-induced lung carcinogenesis.

G protein-coupled estrogen receptor regulates the KLF2-dependent eNOS expression by activating of Ca2+ and EGFR signaling pathway in human endothelial cells.

G protein-coupled estrogen receptor (GPER) is important for maintaining normal blood vessel function by preventing endothelial cell dysfunction. It has been reported that G-1, an agonist of GPER, increases nitric oxide (NO) production through the phosphorylation of endothelial nitric oxide synthase (eNOS). However, the effect of GPER activation on eNOS expression has not been studied. Our results show that G-1 significantly increased the expression of eNOS and Kruppel-like factor 2 (KLF2) in human endothelial EA.hy926 cells. The individual silences of KLF2 and GPER attenuated G-1-induced eNOS expression. In addition, inhibition of the Gαq and Gßγ suppressed G-1-induced the expression of eNOS and KLF2 in EA.hy926 cells. Interestingly, these effects were similar in HUVECs. Furthermore, we found that GPER-mediated Ca2+ signaling increased the phosphorylation of CaMKKß, AMPK, and CaMKIIα in the cells. The phosphorylation of histone deacetylase 5 (HDAC5) by activation of AMPK and CaMKIIα increased the expression of eNOS via transcriptional activity of KLF2. We further demonstrate that GPER activation increased the phosphorylation of Src, EGFR, ERK5, and MEF2C and consequently induced the expression of eNOS and KLF2. Meanwhile, inhibition of ERK5 and HDAC5 suppressed the expression of eNOS and KLF2 induced by G-1 in the cells. These findings suggest that GPER provides a novel mechanism for understanding the regulation of eNOS expression and is an essential therapeutic target in preventing cardiovascular-related endothelial dysfunction.

Influence of o,p'-DDT on MUC5AC expression via regulation of NF-κB/AP-1 activation in human lung epithelial cells.

o,p'-Dichlorodiphenyltrichloroethane (o,p'-DDT) is a representative endocrine disruptor, and exposure to o,p'-DDT may produce immune disorders and inflammation, leading to various diseases such as cancer. Chronic airway inflammation is characterized by excessive mucus secretion resulting in chronic obstructive pulmonary disease (COPD). Mucin 5AC  (MUC5AC), one of the mucus genes, plays an important role in mucus secretion and inflammation in the airways. The aim of this study was to examine the effects of o,p'-DDT on the regulation of MUC5AC expression in human lung epithelial A549 cell line. o,p'-DDT increased mRNA levels and the promoter activity of MUC5AC. Transient transfection with mutation promoter constructs of MUC5AC demonstrated that nuclear factor kappa-b (NF-κB) and activator protein 1(AP-1) response elements were essential for the consequences of o,p'-DDT on MUC5AC expression. In addition, o,p'-DDT induced phosphorylation of ERK, JNK, p38, and Akt, which are involved in the regulation of MUC5AC expression. It is noteworthy that inhibitors of NF-κB, AP-1, Akt, and MAPKs blocked enhanced o,p'-DDT-induced MUC5AC mRNA expression. Data indicate that o,p'-DDT increase in NF-κB, and AP-1 transcriptional activation-dependent MUC5AC expression is associated with stimulation of Akt and MAPK signaling pathways in A549 cells.

Metallothionein 3 Promotes Osteoblast Differentiation in C2C12 Cells via Reduction of Oxidative Stress.

Metallothioneins (MTs) are intracellular cysteine-rich proteins, and their expressions are enhanced under stress conditions. MTs are recognized as having the ability to regulate redox balance in living organisms; however, their role in regulating osteoblast differentiation is still unclear. In this research, we found that the expression of MT3, one member of the MT protein family, was specifically upregulated in the differentiation process of C2C12 myoblasts treated with bone morphogenetic protein 4 (BMP4). Transfection with MT3-overexpressing plasmids in C2C12 cells enhanced their differentiation to osteoblasts, together with upregulating the protein expression of bone specific transcription factors runt-related gene 2 (Runx2), Osterix, and distal-less homeobox 5 (Dlx5). Additionally, MT3 knockdown performed the opposite. Further studies revealed that overexpression of MT3 decreased reactive oxygen species (ROS) production in C2C12 cells treated with BMP4, and MT3 silencing enhanced ROS production. Treating C2C12 cells with antioxidant N-acetylcysteine also promoted osteoblast differentiation, and upregulated Runx2/Osterix/Dlx5, while ROS generator antimycin A treatment performed the opposite. Finally, antimycin A treatment inhibited osteoblast differentiation and Runx2/Osterix/Dlx5 expression in MT3-overexpressing C2C12 cells. These findings identify the role of MT3 in osteoblast differentiation and indicate that MT3 may have interesting potential in the field of osteogenesis research.