iNOS Gene Expression Research Articles

Neuron-Derived Extracellular Vesicles Modulate Microglia Activation and Function.

Simple SummaryIn this study we investigated how neuron-derived extracellular vesicles (NDEVs) mediate neuroimmune regulation in primary cell culture systems. Rat cortical neurons released EVs that improved microglial survival and inhibited the expression of activation markers on microglia. Furthermore, NDEVs reduced the LPS-induced proinflammatory response and promoted an anti-inflammatory response. Thus, neurons critically regulate microglia activity and control inflammation via EV-mediated neuron–glia communication.Microglia act as the immune cells of the central nervous system (CNS). They play an important role in maintaining brain homeostasis but also in mediating neuroimmune responses to insult. The interactions between neurons and microglia represent a key process for neuroimmune regulation and subsequent effects on CNS integrity. However, the molecular mechanisms of neuron-glia communication in regulating microglia function are not fully understood. One recently described means of this intercellular communication is via nano-sized extracellular vesicles (EVs) that transfer a large diversity of molecules between neurons and microglia, such as proteins, lipids, and nucleic acids. To determine the effects of neuron-derived EVs (NDEVs) on microglia, NDEVs were isolated from the culture supernatant of rat cortical neurons. When NDEVs were added to primary cultured rat microglia, we found significantly improved microglia viability via inhibition of apoptosis. Additionally, application of NDEVs to cultured microglia also inhibited the expression of activation surface markers on microglia. Furthermore, NDEVs reduced the LPS-induced proinflammatory response in microglia according to reduced gene expression of proinflammatory cytokines (TNF-α, IL-6, MCP-1) and iNOS, but increased expression of the anti-inflammatory cytokine, IL-10. These findings support that neurons critically regulate microglia activity and control inflammation via EV-mediated neuron–glia communication. (Supported by R21AA025563 and R01AA025591).

The Inducible Nitric Oxide Synthase Pathway Promotes Osteoclastogenesis under Hypoxic Culture Conditions

Bone homeostasis depends on the balance between bone resorption by osteoclasts (OCs) and bone formation by osteoblasts. Bone resorption can become excessive under various pathologic conditions, including rheumatoid arthritis. Previous studies have shown that OC formation is promoted under hypoxia. However, the precise mechanisms behind OC formation under hypoxia have not been elucidated. The present study investigated the role of inducible nitric oxide synthase (iNOS) in OC differentiation under hypoxia. Primary bone marrow cells obtained from mice were stimulated with receptor activator of NF-κB ligand and macrophage colony-stimulating factor to induce OC differentiation. The number of OCs increased in culture under hypoxia (oxygen concentration, 5%) compared with that under normoxia (oxygen concentration, 20%). iNOS gene and protein expression increased in culture under hypoxia. Addition of an iNOS inhibitor under hypoxic conditions suppressed osteoclastogenesis. Addition of a nitric oxide donor to the normoxic culture promoted osteoclastogenesis. Furthermore, insulin-like growth factor 2 expression was significantly altered in both iNOS inhibition experiments and nitric oxide donor experiments. These data might provide clues to therapies for excessive osteoclastogenesis under several hypoxic pathologic conditions, including rheumatoid arthritis.

Intestinal inflammation-associated hypersensitivity is attenuated in a DSS model of colitis in Sigma-1 knockout C57BL/6 mice

Sigma-1 receptors (σ1R) have been implicated in several pain pathways. We assessed the implication of σ1Rs in the development of intestinal inflammation and inflammation-associated referred hypersensitivity in a model of colitis in σ1R knockout (KO) mice. Colitis was induced with dextran sulfate sodium (DSS) in wild type (WT) and σ1R KO mice. The development of referred mechanical hypersensitivity (von Frey test) was assessed. Colonic and spinal changes in expression of immune- and sensory-related markers were also investigated (RT-qPCR/Western blot). Absence of σ1Rs had little impact in colitis generation and progression, although during the chronic phase a reduction in edema and a down-regulation of iNOS gene expression was observed. In σ1R KO mice, inflammation-associated hypersensitivity was significantly attenuated (paw) or completely prevented (abdomen). During colitis, in WT mice, changes in the colonic expression of nociceptive markers were observed during the acute and chronic phases of inflammation. Although σ1R KO mice showed similar regulation in the acute phase, an attenuated response was observed during the chronic phase of colitis. These differences were especially relevant for CB2 and TRPV1 receptors, which could play an important role in σ1-mediated regulation of sensitivity. No changes were detected on ERK phosphorylation at the level of the lumbosacral spinal cord. In summary, intestinal inflammation-associated referred hyperalgesia was reduced (paw) or absent (abdomen) in σ1R KO mice, thus confirming an important role for σ1R in the development of colitis-associated hypersensitivity. These results identify σ1Rs as a possible therapeutic target for the treatment of hypersensitivity associated to intestinal inflammation.

Fipronil induced oxidative stress in neural tissue of albino rat with subsequent apoptosis and tissue reactivity

Fipronil (FIP) insecticide is extensively used in agriculture, public health and veterinary medicine. Although it is considered as a neurotoxin to insects (target organisms) and exhibits neurological signs upon vertebrates (non-target organisms) exposure, slight is known about its potential neurotoxic effects and its molecular mechanisms on vertebrates. The current study is designed to assess oxidative stress as a molecular mechanism of FIP neurotoxicity subordinated with apoptosis and neural tissue reactivity. Ten adult male albino rats received 10 mg/kg body weight fipronil technical grade by oral gavage daily for 45 days (subacute exposure). Brain neural tissue regions (hippocampus, cerebellum and caudate putamen) were processed to examine oxidative stress induced cellular macromolecular alterations as MDA, PCC and DNA fragmentation. Besides, TNF-α and Bcl-2 gene expression and immunoreactivity for caspase-3 (active form), iNOS and GFAP were evaluated. Also, histopathological assessment was conducted. We found that FIP significantly raised MDA, PCC and DNA fragmentation (p ≤ 0.05). Also, it significantly upregulated TNF-α and non-significantly down-regulated Bcl-2 gene expression (p ≤ 0.05). Further, significant increased immunoreactivity to GFAP, iNOS and caspase-3 (active form) in these brain neural tissue regions in FIP treated group was noticed (p ≤ 0.05). Histopathological findings, including alterations in the histological architecture and neuronal degeneration, were also observed in these brain regions of FIP treated group. In conclusion, we suggest the ability of FIP to induce oxidative stress mediated macromolecular alterations, leading to apoptosis and tissue reaction in these brain regions which showed variable susceptibility to FIP toxic effects.

Anti-Oxidant and Anti-Inflammatory Substance Generated Newly in Paeoniae Radix Alba Extract Fermented with Plant-Derived Lactobacillus brevis 174A.

Fermentation of medicinal herbs can be a significant technique to obtain bioactive compounds. Paeoniae Radix (PR) used in the present study is a well-known herbal medicine that exhibits anti-inflammatory and immunomodulatory activity. The aim of this study is to explore the possibility that a bioactive compound is newly generated in PR extract by fermentation with a plant-derived lactic acid bacteria Lactobacillus brevis 174A. We determined the anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The PR extract fermented with Lactobacillus brevis 174A markedly increased the total phenolic content, decreased intracellular ROS levels, inhibited the release of nitric oxide (NO). It also suppressed inflammatory cytokines IL-6, TNF-ɑ, while simultaneously downregulating the gene expressions of iNOS, IL-6, TNF-ɑ, and IL-1β compared to the unfermented PR extract. Furthermore, the bioactive compound newly generated from the fermentation was identified as pyrogallol. It inhibits the inflammatory responses in a dose-dependent manner suggesting that fermentation of the herbal extract used as a medium together with the plant-derived lactic acid bacterial strain may be a practical strategy to produce medicines and supplements for healthcare.

A Cytokine Network Balance Influences the Fate of Leishmania (Viannia) braziliensis Infection in a Cutaneous Leishmaniasis Hamster Model

The golden hamster is a suitable model for studying cutaneous leishmaniasis (CL) due to Leishmania (Viannia) braziliensis. Immunopathological mechanisms are well established in the L. (L.) major-mouse model, in which IL-4 instructs a Th2 response towards progressive infection. In the present study, we evaluated the natural history of L. braziliensis infection from its first stages up to lesion establishment, with the aim of identifying immunological parameters associated with the disease outcome and parasitism fate. To this end, hamsters infected with 104, 105, or 106 promastigotes were monitored during the first hours (4h, 24h), early (15 days, 30 days) and late (50 days) post-infection (pi) phases. Cytokines, iNOS and arginase gene expression were quantified in the established lesions by reverse transcription-quantitative PCR. Compared to the 105 or 106 groups, 104 animals presented lower lesions sizes, less tissue damage, and lower IgG levels. Basal gene expression in normal skin was high for TGF-β, and intermediary for TNF, IL-6, and IL-4. At 4hpi, no cytokine induction was observed in the 104 group, while an upregulation of IL-6, IL-10, and IL-4 was observed in the 106 group. At 15dpi, lesion appearance was accompanied by an increased expression of all assessed cytokines, markedly in the 105 and 106 groups. Upregulation of all investigated cytokines was observed in the late phase, although less expressive in the 104 group. IFN-γ was the depending variable influencing tissue damage, while IL-6 was associated to parasite load. The network correlating gene expression and clinical and laboratorial parameters indicated inoculum-independent associations at 15 and 30dpi. A strong positive network correlation was observed in the 104 group, but not in the 105 or 106 groups. In conclusion, IL-4, IL-6, IL-10, and TGF-β are linked o L. braziliensis progression. However, a balanced cytokine network is the key for an immune response able to reduce the ongoing infection and reduce pathological damage.

Functional Exploration of Desmarestia ligulate and Anti-inflammatory Activity of Functional Substance D-mannuronic acid

This study was conducted to investigate the anti-inflammatory activity of Desmarestia ligulate (D. ligulate) leaf. The effects on cell viability and anti-inflammatory activities of D. ligulata leaf extract, as well as those of D-mannuronic acid (DMA), on RAW 264.7 cells (macrophages) and peripheral blood mononuclear cells (PBMCs) during inflammation-induced stress were analyzed by determining the levels of NO, IL-17, IL-4, and TNF-α using ELISA and evaluating the expression of iNOS and MPO genes. D. ligulata leaf extract and DMA exhibited low toxicity in RAW 264.7 cells and PBMCs, which secreted lower levels of TNF-α and IL-4. The anti-inflammatory effect of DMA was higher than that of D. ligulata leaf extract as indicated by the levels of the inflammatory cytokine IL-17. Furthermore, treatment with D. ligulata leaf extract and DMA decreased the expression of iNOS and MPO genes after stress induction. These findings suggested that D. ligulata leaf extract and DMA exert anti-inflammatory effects as well as regulate cytokine release activities and inflammatory gene expression. Therefore, D. ligulata leaf extract and DMA may be used as cosmetic materials to control skin inflammation. Keywords: Anti-inflammatory; Desmarestia ligulate; D-mannuronic acid; IL-17; IL-6

Anti-Inflammatory Effects of Abeliophyllum distichum Nakai (Cultivar Okhwang 1) Callus through Inhibition of PI3K/Akt, NF-κB, and MAPK Signaling Pathways in Lipopolysaccharide-Induced Macrophages

One of the Korean endemic plants, Abeliophyllum distichum Nakai (Oleaceae), contains acteoside, which is a glycoside exhibiting neuroprotective, anti-inflammation effects and antibacterial capacities. We conducted an investigation on the effects of the callus of A. distichum (cultivar Okhwang 1, CAO) on pro-inflammatory mediators released following nuclear factor-кB (NF-кB), phosphatidylinositol 3-kinase/Akt (PI3K-Akt) and mitogen-activated protein kinase (MAPK) signal activation in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Immunoblotting was employed to find out the expression of cyclooxygenase-2 (COX-2), inducible nitric oxide (iNOS), and activation of MAPK molecules, NF-κB and Akt. Cytokines, COX-2, and iNOS gene expression were assessed using polymerase chain reaction techniques. Cytokines, COX-2, and iNOS gene expression were assessed using polymerase chain reaction techniques. High-performance liquid chromatography revealed that CAO was rich in acteoside and isoacteoside. As a result, CAO inhibited the generation of NO, cytokines, COX-2, and iNOS expression. Further, translocation to the nuclear of NF-κB p65 and degradation of the inhibitor of NF-кB (IкB) were alleviated by suppressing phosphorylation. Additionally, CAO significantly impacted MAPK pathway activation by potentially reducing phosphorylation of MAPKs. These results indicate that the anti-inflammatory effect of CAO is mediated via the inhibition of MAPK, PI3K/Akt, and NF-κB signaling pathways, probably via glycosides, phenolics, and flavonoids bioactivity derived from plants. CAO can serve as a potential anti-inflammatory agent, which alleviates inflammation factors and act through specific cell signaling pathways.

Protective effect of (+)-catechin against lipopolysaccharide-induced inflammatory response in RAW 264.7 cells through downregulation of NF-κB and p38 MAPK.

Catechin, a flavonol belonging to the flavonoid group of polyphenols is present in many plant foods. The present study was done to evaluate the effect of catechin on various inflammatory mediators using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The effect of catechin on total cyclooxygenase (COX) activity, 5-lipoxygenase (5-LOX), myeloperoxidase, nitrite and inducible nitric oxide synthase (iNOS) level, secretion of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) were assessed in LPS-stimulated RAW 264.7 cells. The expression of COX-2, iNOS, TNF-α, nuclear factor-ĸB (NF-κB) and p38 mitogen-activated protein kinase (MAPK) genes were also investigated. The effect was further analyzed using human PBMCs by assessing the level of TNF-α and IL-10. The study demonstrated that the inflammatory mediators such as COX, 5-LOX, nitrite, iNOS, and TNF-α were significantly inhibited by catechin in a concentration-dependent manner whereas IL-10 production was up-regulated in RAW 264.7 cells. Moreover, catechin down-regulated the mRNA level expression of COX-2, iNOS, TNF-α, NF-κB and p38 MAPK. The current study ratifies the beneficial effect of catechin as a dietary component in plant foods to provide protection against inflammatory diseases.

Anti-inflammatory Effects of Empagliflozin and Gemigliptin on LPS-Stimulated Macrophage via the IKK/NF-κB, MKK7/JNK, and JAK2/STAT1 Signalling Pathways.

Background Sodium-glucose cotransporter 2 (SGLT2) and dipeptidyl peptidase-4 (DPP-4) inhibitors are glucose-lowering drugs whose anti-inflammatory properties have recently become useful in tackling metabolic syndromes in chronic inflammatory diseases, including diabetes and obesity. We investigated whether empagliflozin (SGLT2 inhibitor) and gemigliptin (DPP-4 inhibitor) improve inflammatory responses in macrophages, identified signalling pathways responsible for these effects, and studied whether the effects can be augmented with dual empagliflozin and gemigliptin therapy. Methods RAW 264.7 macrophages were first stimulated with lipopolysaccharide (LPS), then cotreated with empagliflozin, gemigliptin, or empagliflozin plus gemigliptin. We conducted quantitative RT-PCR (qRT-PCR) to determine the most effective anti-inflammatory doses without cytotoxicity. We performed ELISA and qRT-PCR for inflammatory cytokines and chemokines and flow cytometry for CD80, the M1 macrophage surface marker, to evaluate the anti-inflammatory effects of empagliflozin and gemigliptin. NF-κB, MAPK, and JAK2/STAT signalling pathways were examined via Western blotting to elucidate the molecular mechanisms of anti-inflammation. Results LPS-stimulated CD80+ M1 macrophages were suppressed by coincubation with empagliflozin, gemigliptin, and empagliflozin plus gemigliptin, respectively. Empagliflozin and gemigliptin (individually and combined) inhibited prostaglandin E2 (PGE2) release and COX-2, iNOS gene expression in LPS-stimulated RAW 264.7 macrophages. These three treatments also attenuated the secretion and mRNA expression of proinflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IFN-γ, and proinflammatory chemokines, such as CCL3, CCL4, CCL5, and CXCL10. All of them blocked NF-κB, JNK, and STAT1/3 phosphorylation through IKKα/β, MKK4/7, and JAK2 signalling. Conclusions Our study demonstrated the anti-inflammatory effects of empagliflozin and gemigliptin via IKK/NF-κB, MKK7/JNK, and JAK2/STAT1 pathway downregulation in macrophages. In all cases, combined empagliflozin and gemigliptin treatment showed greater anti-inflammatory properties.

Enhancement of nutraceutical properties of licorice callus cultures using sample pre‐treatment strategy

AbstractLicorice is an herbal plant in the Leguminosae family, and its roots and rhizomes are used as sweeteners in food and confectionery products. Moreover, it has a distinct inflammatory activity. In the present study, a sample pre‐treatment method to induce the deglycosylation of active metabolites in callus cultures of Glycyrrhiza inflata (GI) and Glycyrrhiza glabra (GG) was developed. The results of the method evaluation showed the biotransformation of ononin to formononetin, a rare flavonoid found in trace amounts in licorice. The magnitude of enhancement was 3‐ and 19‐fold in the GI and GG samples, respectively. Moreover, the anti‐inflammatory activity assay showed that the potency of the sample pre‐treatment group was higher than that of the untreated group because it exerted an enhanced suppression of cyclo‐oxygenase 2 (COX‐2), interleukin‐6 (IL‐6) and inducible nitric oxide synthase (iNOS) gene expression. This is the first report on the anti‐inflammatory activity of licorice callus, which has the potential to be utilised as a functional food for health promotion. These findings support the idea of using sample preparation to impart nutraceutical properties to plant products.

Effects of Probiotic and/or Prebiotic Supplementations on Immune Response, Haematology, Oxidant-antioxidant Biomarkers, and Cytokine mRNA Expression Levels in the Caeca of Broilers Infected with Salmonella

The present study was conducted to evaluate the effects of incorporation of the probiotic mix, prebiotic, and synbiotic supplements on immune response, interleukin-6, interleukin-10, and inducible nitric oxide synthase (iNOS) gene expression, hematology, and oxidant-antioxidant biomarkers of broilers infected with Salmonella Enteritidis and Salmonella Typhimurium mixed infection. A total of 273 commercial Cobb 500 chicks were randomly allocated into seven experimental groups, including NC group: fed only a basal diet (negative control), PC group; fed a basal diet and infected with Salmonellae on 3 rd life day (positive control), ProSa group: administrated probiotic mix in drinking water and then infected with Salmonellae, Presa group: fed prebiotic with basal diet and then infected with Salmonellae, SynSa: received probiotic mix and prebiotic (synbiotic) and then infected with Salmonellae, Pro group: given probiotic mix in feed, and Pre group: basal diet supplemented with prebiotic. The probiotic mix, prebiotic, and synbiotic supplementation significantly increased total IgY levels in the sera of infected birds in comparison to those of positive control. Also, the additives significantly downregulated IL-6 and iNOS gene expressions while they significantly upregulated IL-10 gene expression in the caeca of infected birds, in comparison to those of positive control. Also, the supplementary treatments significantly improved Salmonella-induced changes in hematology and oxidant-antioxidant biomarkers of infected groups. Compared with different supplementary treatments, the synbiotic significantly robust immune response of Salmonellainfected birds when compared with single supplementation of probiotic mix or prebiotic. In conclusion, probiotics, prebiotic, and in particular synbiotic supplements improved immune response, hematological and antioxidative biomarkers of birds experimentally infected with mixed Salmonellae.

Loss of Interleukin (IL)-10 Is Associated With Increased Vascular Inflammation and Sex-Differences in Metabolic Outcomes of Normal Diet-Fed Mice

ObjectivesThe anti-inflammatory cytokine, IL-10, plays an important role in reducing the risk of many inflammatory diseases. This study investigated the time and sex effects of IL-10 gene deletion on metabolic risk factors that contribute to the development of cardiovascular disease. MethodsSix-wk-old male and female B6.129P2-Il10tm1Cgn/J (IL-10–/–) and C57BL/6 (WT) mice (n = 12–16/group) were randomly assigned to 12- or 24-wk time points and were fed growth (AIN-93G) diet up to 3 m of age and then maintenance diet (AIN-93M) for the remainder of the study. Monthly fasting glucose was assessed as well as intraperitoneal glucose tolerance test (ipGTT), body composition, and serum metabolic parameters at each study end point. Cardiac and vascular adhesion molecules, macrophage marker F4/80, and sterol metabolism genes were assessed using qPCR. Data were analyzed using t-test and 2-way ANOVA with strain and gender as factors, and α = 0.05. ResultsIL-10 deletion resulted in weight loss (p < 0.05) coinciding with reduced fat mass and % fat (P < 0.05) in both sexes of IL-10–/–. Loss of IL-10 had no effect on fasting glucose at any time point in either sex; however, a delayed response to glucose challenge and increased AUC with the ipGTT (P < 0.05) occurred in male IL-10–/– vs WT mice. No strain effect was observed on serum lipids at 12 wks, but cholesterol and high-density lipoprotein (HDL)-C were reduced (P < 0.05) in IL-10–/– vs WT mice at 24 wks. Only male IL-10–/– mice exhibited elevated (P < 0.05) non-HDL cholesterol and tended (P = 0.072) to have elevated triglycerides vs WT mice at 24 wks. In conjunction with serum lipid changes, male IL-10–/– mice increased (P < 0.05) hepatic transcription of β-hydroxy β-methylglutaryl-CoA (HMG-CoA), whereas HMGCoA transcript tended to be repressed (≥ –53.5%; P = 0.08) in female IL-10–/– vs WT mice. At 12 and 24 wks, IL-10–/– exhibited increased (P < 0.05) circulating c-reactive protein and aortic and cardiac gene expression of VCAM-1, ICAM-1, and iNOS. The only increase in the F4/80 macrophage marker occurred in male IL-10–/– mice vs WT at 24 wks. ConclusionsLoss of IL-10 was associated with different metabolic responses in male and female mice and could be detrimental to cholesterol-mediated metabolic processes in female mice on a control diet. Funding SourcesOklahoma Agriculture Experiment Station and the Jim and Lynn Williams Professorship.

Chemical profiling and in-vitro anti-inflammatory activity of bioactive fraction(s) from Trichodesma indicum (L.) R.Br. against LPS induced inflammation in RAW 264.7 murine macrophage cells

Ethnopharmacological relevanceTrichodesma indicum (L.) R. Br. (family: Boraginaceae) is a medicinal herb largely used to treat arthralgia, rheumatoid arthritis, wound healing, dysentery, etc. It's mechanism of anti-inflammatory activity has not been systematically analyzed yet. Aim of the studyThe present study was undertaken to examine the anti-inflammatory effects of successive solvent extracts (n-hexane extract (HE), ethyl acetate extract (EA), ethanol extract (EE), aqueous extract (AE) and fractions of HE) from the aerial parts of Trichodesma indicum (TI) against lipopolysaccharide (LPS) stimulated inflammatory reaction using mouse macrophage RAW 264.7 cells. Materials and methodsCytotoxic effects of the extracts and fractions of TI were assessed by MTT assay. The effect of extracts and fractions on the production of nitric oxide (NO) in RAW 264.7 macrophages were measured using the Griess reagent method. IL - 6, IL - 1β, TNF-α, iNOS and COX-2 gene expressions were examined by a qRT-PCR method. ResultsRAW 264.7 macrophages pretreated with HE, EA, EE and AE of TI showed a significant decrease in the production of proinflammatory cytokines and NO without exhibiting cytotoxicity. The potent HE was fractionated using flash chromatography into FA, FB, FC, FD and FE. Among the five fractions, FE displayed a stronger ability to reduce IL - 1β, TNF-α, iNOS, COX2 and NO importantly no cytotoxicity was observed. The phytochemical compounds present in FE were further screened by Gas chromatography - Mass spectroscopy (GC-MS). GC-MS analysis revealed that 1,2-benzenedicarboxylic acid diisooctyl ester is the major compound in FE. Molecular docking analysis showed good inhibition of 1,2-benzenedicarboxylic acid diisooctyl ester against TLR-4, NIK and TACE. ConclusionOur results suggested that 1,2-benzenedicarboxylic acid diisooctyl ester could be a potential candidate in alleviating inflammatory reactions in TI.

Helicase-like transcription factor-deletion from the tumor microenvironment in a cell line-derived xenograft model of colorectal cancer reprogrammed the human transcriptome-S-nitroso-proteome to promote inflammation and redirect metastasis.

Methylation of the HLTF gene in colorectal cancer (CRC) cells occurs more frequently in men than women. Progressive epigenetic silencing of HLTF in tumor cells is accompanied by negligible expression in the tumor microenvironment (TME). Cell line-derived xenografts (CDX) were established in control (Hltf+/+) and Hltf-deleted male Rag2-/-IL2rg-/- mice by direct orthotopic cell microinjection (OCMI) of HLTF+/+HCT116 Red-FLuc cells into the submucosa of the cecum. Combinatorial induction of IL6 and S100A8/A9 in the Hltf-deleted TME with ICAM-1 and IL8 in the primary tumor activated a positive feedback loop. The proinflammatory niche produced a major shift in CDX metastasis to peritoneal dissemination compared to controls. Inducible nitric oxide (iNOS) gene expression and transactivation of the iNOS-S100A8/A9 signaling complex in Hltf-deleted TME reprogrammed the human S-nitroso-proteome. POTEE, TRIM52 and UN45B were S-nitrosylated on the conserved I/L-X-C-X2-D/E motif indicative of iNOS-S100A8/A9-mediated S-nitrosylation. 2D-DIGE and protein identification by MALDI-TOF/TOF mass spectrometry authenticated S-nitrosylation of 53 individual cysteines in half-site motifs (I/L-X-C or C-X-X-D/E) in CDX tumors. POTEE in CDX tumors is both a general S-nitrosylation target and an iNOS-S100A8/A9 site-specific (Cys638) target in the Hltf-deleted TME. REL is an example of convergence of transcriptomic-S-nitroso-proteomic signaling. The gene is transcriptionally activated in CDX tumors with an Hltf-deleted TME, and REL-SNO (Cys143) was found in primary CDX tumors and all metastatic sites. Primary CDX tumors from Hltf-deleted TME shared 60% of their S-nitroso-proteome with all metastatic sites. Forty percent of SNO-proteins from primary CDX tumors were variably expressed at metastatic sites. Global S-nitrosylation of proteins in pathways related to cytoskeleton and motility was strongly implicated in the metastatic dissemination of CDX tumors. Hltf-deletion from the TME played a major role in the pathogenesis of inflammation and linked protein S-nitrosylation in primary CDX tumors with spatiotemporal continuity in metastatic progression when the tumor cells expressed HLTF.

Assessment of the Vanillin Anti-Inflammatory and Regenerative Potentials in Inflamed Primary Human Gingival Fibroblast.

Background Inflammatory responses have been associated with delayed oral mucosal wound healing and the pathogenesis of the periodontal disease. The invasion of microbes into the tissues and the establishment of a chronic infection may be due to impaired healing. The protracted inflammatory phase may delay wound healing and probably support tissue fibrosis and reduce tissue regeneration. Vanillin is a well-known natural compound with potential anti-inflammatory capacity. Hence, we hypothesized that Vanillin could accelerate wound healing reducing inflammation and especially cytokine production making the oral tissue repair process easier. Methods Our hypothesis was tested using primary human gingival fibroblast (HGF) cell pretreated with Vanillin and primed with IL-1β, as inductor of proinflammatory environment. After 24 hours of treatments, the gene expression and production of IL-6, TNF-α, IL-8, COX-2, iNOS, and nitric oxide (NO) generation and the wound healing rate were determined. Results In IL-1β-primed cells, preincubation with Vanillin reduced IL-6, IL-8, COX-2, and iNOS expression and NO release, compared to IL-1β-primed cells. Moreover, Vanillin determines the increased gene expression of nAChRα7, leading us to hypothesize a role of Vanillin in the activation of the cholinergic anti-inflammatory pathway. Furthermore, in presence of mechanical injury, the Vanillin preincubation, wound closure may be reducing the expression and release of IL-6 and TNF-α and upregulation of COX-2 and IL-8. Conclusion Together, the results of this study highlight the anti-inflammatory and tissue repair ability of Vanillin in IL-1β-primed HGF. Therefore, Vanillin shows a potential therapeutic interest as an inflammatory modulator molecule with novel application in periodontal regeneration and oral health.

55. PREVENTION OF LIPOPOLYSACCHARIDE-INDUCED ENDOTHELIAL DYSFUNCTION IN HUVEC CELLS: COMPARISON BETWEEN DECAFFEINATED LIGHT ROASTED GREEN COFFEE EXTRACT AND GREEN TEA EXTRACT

Background: Endothelial dysfunction contributes to hypertension complications. Coffee consumption still gave rise to conflicting data regarding its preventive action on endothelial dysfunction. Objective: This study aimed to compare the effect of decaffeinated light roasted green coffee extract (GCE) and green tea extract (GTE) on endothelial dysfunction biomarkers in lipopolysaccharide (LPS) treated HUVEC cells. Methods: The HUVEC cells were maintained in M200 medium containing 2% LSGS and 1% penicillin streptomycin. The cells were seeded in 6-well plate with 1 × 106 cells/well density. At 80% confluency, the cells were treated for 48 hours with either GCE or GTE at the dose of 125, 250, and 500 μg/mL and 1 mg/mL of Simvastatin. The cells were then treated with 1 mg/mL LPS for another 48 hours. Finally the cells were harvested and the gene expression of NOX-1, TNFα, ENOS, INOS, and MCP-1 were analysed by RT-PCR method Results: The gene expression of NOX-1, TNFα, INOS, and MCP-1 were significantly lower in all GCE, GTE, and simvastatin group (P &lt; 0.000). The ENOS gene expression were significantly higher in all GCE and GTE group (P &lt; 0.000) compared to LPS treated HUVEC. In general, the administration of GCE significantly improved the endothelial dysfunction biomarkers compared to that of GTE administration in the same doses. Administration of GCE 250 μg/mL resulted in significantly lower NOX-1, TNFα, INOS, and MCP-1 expression (P &lt; 0.000) and significantly higher ENOS expression (P &lt; 0.000) compared to those of GCE 250 μg/mL administration Conclusion: GCE administration significantly prevented endothelial dysfunction through improvement of NOX-1, TNFα, ENOS, INOS, and MCP-1 compared to that of GTE administration in LPS treated HUVEC cells.

Hesperetin, a dietary flavonoid, inhibits AGEs-induced oxidative stress and inflammation in RAW264.7 cells

• Three typical structural derivatives of Naringenin were tested in this paper. • Hesperetin could significantly inhibit both AGEs-induced ROS production and inflammation. • Gene set enrichment analysis was performed and twenty-three key genes related were screened. Advanced glycation end products (AGEs) excessive accumulation in the body either by exogenous intake or endogenous formation makes individuals prone to oxidative stress and inflammation damage. In this study, DCFH-DA fluorescence test and qPCR experiment were carried out to study the effect of some dietary flavonoids on AGEs induced toxicity. Among four tested flavonoids, it was found that hesperetin could significantly inhibit both AGEs-induced ROS production and gene expressions of TNF-α, IL-1β, IL-6, MCP-1, COX-2 and iNOS. High-throughput transcriptome analysis showed that the protective effect of hesperetin against AGEs-induced toxicity might be attributed to inhibit the activation of MAPK, JAK, NF-κB-related pathways. Furthermore, gene set enrichment analysis was performed using the MSigDB database and twenty-three key genes related were screened. This study might provide new ideas for the prevention of AGEs-related diseases and lay a theoretical foundation for the application of dietary flavonoids.

Ergostatrien-3β-ol (EK100) from Antrodia camphorata Attenuates Oxidative Stress, Inflammation, and Liver Injury In Vitro and In Vivo.

Hepatic ischemia/reperfusion (IR) injury is a complication that occurs during liver surgery, whereby hepatic tissue is injured by oxygen deficiency during ischemia, then further damaged by a cascade of inflammatory and oxidative insults when blood is resupplied during reperfusion. Antrodia camphorata is an indigenous fungus in Taiwan and an esteemed Chinese herbal medicine with various bioactivities. This study examined the effect of ergostatrien-3β-ol (EK100), an active compound found in both the fruiting body and mycelia of A. camphorata, on IR injury pathologies in rats and cell models of oxidative and inflammatory stress. Male Sprague-Dawley rats were randomly assigned to receive a vehicle or 5 mg/kg EK100 prior to hepatic IR injury induced by 1 h ischemia followed by 24 h reperfusion, or a sham operation. RAW 264.7 murine macrophages and HepG2 hepatocytes were pretreated with EK100, then inflammation was induced with lipopolysaccharides in the former and oxidative stress was induced with hydrogen peroxide in the latter. EK100 decreased IR-induced elevation in serum levels of alanine aminotransferase and aspartate aminotransferase and lowered levels of the inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-6, and IL-1β. In addition, EK100 significantly reduced hepatic mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2, as well as nitrite production and iNOS gene expression in both hepatocyte and macrophage cell lines. We demonstrated that EK100 exhibits potent protec-tion against hepatic IR injury, which may be used to design strategies to ameliorate liver damage during liver surgery.

In vivo anti-inflammatory effects of Prasiola japonica ethanol extract

Abstract The aim of this study was to investigate the efficacy of Prasiola japonica ethanol extract (Pj-EE) in various mouse inflammation models. For edema mice models, eight-week-old female ICR mice were orally administrated with Pj-EE (25 and 50 mg/kg) for one week, before ear edema induction with carrageenan or xylene. The weight, thickness, and volume of paw and ear edema were decreased in Pj-EE-treated mice after 3 h of edema induction. The mRNA expression levels of iNOS, COX-2, IL-6, IL-1β, and IFN-γ were decreased in Pj-EE-treated conditions. The phosphorylation forms of IκBα and IKKα/β were suppressed by Pj-EE at 50 mg/kg. The effect of Pj-EE on gastrointestinal disorder was examined in HCl/EtOH-induced gastritis model. Oral administration of Pj-EE ameliorated HCl/EtOH-stimulated gastric ulcers. In addition, Pj-EE reduced gene expressions of iNOS, COX-2, IL-6, and IL-1β. Alverine, one of ingredient compounds in Pj-EE, was also confirmed to show anti-edema activity, in vitro, and in vivo. Conclusively, therapeutic effects of Pj-EE were demonstrated in various inflammatory mouse models, suggesting a possibility as a promising anti-inflammatory functional food.