Biodiesel exhaust–induced cytotoxicity and proinflammatory mediator production in human airway epithelial cells

Increasing use of biodiesel has prompted research into the potential health effects of biodiesel exhaust exposure. Few studies directly compare the health consequences of mineral diesel, biodiesel, or blend exhaust exposures. Here, we exposed human epithelial cell cultures to diluted exhaust generated by the combustion of Australian ultralow‐sulfur‐diesel (ULSD), unprocessed canola oil, 100% canola biodiesel (B100), and a blend of 20% canola biodiesel mixed with 80% ULSD. The physicochemical characteristics of the exhaust were assessed and we compared cellular viability, apoptosis, and levels of interleukin (IL)‐6, IL‐8, and Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) in exposed cultured cells. Different fuel types produced significantly different amounts of exhaust gases and different particle characteristics. All exposures resulted in significant apoptosis and loss of viability when compared with control, with an increasing proportion of biodiesel being correlated with a decrease in viability. In most cases, exposure to exhaust resulted in an increase in mediator production, with the greatest increases most often in response to B100. Exposure to pure canola oil (PCO) exhaust did not increase mediator production, but resulted in a significant decrease in IL‐8 and RANTES in some cases. Our results show that canola biodiesel exhaust exposure elicits inflammation and reduces viability of human epithelial cell cultures in vitro when compared with ULSD exhaust exposure. This may be related to an increase in particle surface area and number in B100 exhaust when compared with ULSD exhaust. Exposure to PCO exhaust elicited the greatest loss of cellular viability, but virtually no inflammatory response, likely due to an overall increase in average particle size. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 44–57, 2016.     

Esculetin inhibits histamine‐induced expression of inflammatory cytokines and mucin in nasal epithelial cells

Allergic rhinitis (AR) is a type of respiratory disease closely associated with chronic inflammation. Esculetin is a natural coumarin derivative and has been reported to possess anti‐allergic and anti‐inflammatory effects. However, the roles of esculetin in AR have not been studied. In this study, we aimed to examine the effect of esculetin on AR using an in vitro model. The human nasal epithelial cells (HNEpC) were stimulated by histamine for 24 hours with or without the pretreatment of esculetin. The mRNA levels and production of inflammatory cytokines including IL‐6 and IL‐8, as well as mucin 5AC (MUC5AC) were measured using qRT‐PCR and ELISA, respectively. The results showed that esculetin suppressed histamine‐induced expression and secretion of IL‐6, IL‐8, and MUC5AC in HNEpCs. Furthermore, we examined the effect of esculetin on NF‐κB pathway by detecting the expression levels of NF‐κB p65, p‐p65 and IκBα using western blot analysis. Esculetin treatment suppressed the histamine‐induced p‐p65 expression and p‐IκBα degradation. Inhibiting NF‐κB pathway suppressed histamine‐induced production of IL‐6, IL‐8, and MUC5AC in HNEpCs. These findings suggested that esculetin suppressed histamine‐induced production of inflammatory cytokines and mucin in HNEpCs, which were partly mediated by the inhibition of NF‐κB pathway.     

Exendin-4 and GLP-1 decreases induced expression of ICAM-1, VCAM-1 and RAGE in human retinal pigment epithelial cells

BackgroundAdvanced glycation end products (AGEs) take part in the development of diabetic retinopathy. Hyperglycemia triggers an inflammatory response in the retina. These mechanisms may lead to an enhanced expression of adhesion molecules (ICAM-1 and VCAM-1) in human retinal pigment epithelium (HRPE). Glucagon-like peptide 1 (GLP-1) functions as an incretin hormone with antidiabetogenic properties. GLP-1 also possesses vasoprotective properties.MethodsThe aim of our study was to evaluate the influence of glycated albumin (GlyAlb; 100; 500 and 1000 mg/l) and proinflammatory cytokine, TNF-α (2.5 and 10  ng/ ml), on expression of RAGE, ICAM-1 and VCAM-1 and to evaluate the influence of GLP-1 (100 nM) and its analogue, exendin-4 (10 nM), on the expression of RAGE, ICAM-1 and VCAM-1 in stimulated HRPE.ResultsTNF-α increased RAGE expression in HRPE cells. The addition of GlyAlb (500 and 1000 mg/l) resulted in a decrease of RAGE expression. Both TNF-α and GlyAlb increased the secretion of both adhesion molecules. In cells co-treated with GLP-1 or exendin-4 both incretins decreased RAGE expression in TNF-α treated cells, and in GlyAlb group. The ICAM-1 expression was lowered by exendin-4 and GLP-1 in cells stimulated by TNF-α and GlyAlb. The similar results were obtained for VCAM-1. All observed alterations were statistically significant.ConclusionsThe obtained results indicate that both GLP-1 and exendin-4 by decreasing the expression of RAGE in HRPE can make these cells more resistant to circulating AGEs, and decreased expression of circulating VCAM-1 and ICAM-1, can be the result of anti-inflammatory properties of incretins and decreased expression of RAGE.     

Amantadine inhibits RANTES production by influenzavirus-infected human bronchial epithelial cells

1. Amantadine can prevent and decrease airway inflammation by inhibiting influenza virus (IV) replication; however, the effect of amantadine on RANTES production by human bronchial epithelial cells (BEC) has not been determined. In the present study, we examined the effect of amantadine on RANTES production and also analysed p38 mitogen-activated protein (MAP) kinase and c-Jun-NH2-terminal kinase (JNK) activation to clarify the mechanism in the effect of amantadine on RANTES production, since we have previously shown that p38 MAP kinase and JNK regulate RANTES production by IV-infected BEC. 2. BEC that had been preincubated with amantadine were infected with IV and then p38 MAP kinase and JNK activation in the cells and RANTES concentrations in the culture supernatants were determined. 3. Amantadine-induced inhibition of virus replication resulted in a decrease in p38 MAP kinase and JNK activity and decreased expression of RANTES in IV-infected cells. 4. Amantadine did not inhibit p38 MAP kinase and JNK activation induced by tumour necrosis factor-alpha (TNF-alpha) as a non-viral stimulus. 5. These results indicate that amantadine inhibits IV infection-induced RANTES production by human BEC and that the inhibition by amantadine of RANTES production might result from an indirect inhibitory effect of amantadine on p38 MAP kinase and JNK activation via the inhibition of virus replication, and we emphasize that amantadine may produce a beneficial effect on controlling bronchial asthma exacerbation caused by IV infection.     

Trivalent arsenic species induce changes in expression and levels of proinflammatory cytokines in intestinal epithelial cells

Chronic arsenic (As) toxicity in humans has been documented in many countries where exposure mostly occurs through drinking water. The As immunotoxic effects have been demonstrated in animal models as well as in humans. The studies of the immunotoxicity of As have centered on organs related to immune response or target organs, with few data being available at intestinal level. The present study has evaluated the changes in the expression and release of cytokines in Caco-2 cells, widely used as an intestinal epithelial model. Differentiated cells were exposed to 1 μM of As(III), 0.1 μM of monomethylarsonous acid [MMA(III)] and 1 μM of dimethylarsinous acid [DMA(III)] during 2, 4, 6 and 24 h. Additionally, the effect of As coexposure with lipopolysaccharide (LPS, 10 ng/mL) has been evaluated. The results show trivalent species to induce increases in the expression and release of the proinflammatory cytokines tumor necrosis factor alpha (TNFα), IL6, IL8 – the magnitude and time of response being different for each As species. The response of greatest magnitude corresponds to DMA(III), followed by As(III), while MMA(III) generates a limited response. Furthermore, the presence of LPS in the co-exposed cells could affect the expression and secretion of cytokines compared with individual exposure to arsenicals, especially for As(III)/LPS and DMA(III)/LPS.     

Extract from Nandina domestica inhibits lipopolysaccharide-induced cyclooxygenase-2 expression in human pulmonary epithelial A549 cells

Extract from fruits of Nandina domestica THUNBERG (NDE) has been used to improve cough and breathing difficulty in Japan for many years. To explore whether NDE may alleviate respiratory inflammation, we investigated its effect on expression of cyclooxygenase-2 (COX-2) and production of prostaglandin E? (PGE?) in human pulmonary epithelial A549 cells in culture. Treatment with lipopolysaccharide (LPS; 6?μg/mL) resulted in an increase of COX-2 expression and PGE? production in A549 cells. Both the LPS-induced COX-2 expression and PGE? production were significantly inhibited by NDE (1-10?μg/mL) in a concentration-dependent manner. NDE did not affect COX-1 expression nor COX activity. These results suggest that NDE downregulates LPS-induced COX-2 expression and inhibits PGE? production in pulmonary epithelial cells. Furthermore, higenamine and nantenine, two major constituents responsible for tracheal relaxing effect of NDE, did not mimic the inhibitory effect of NDE on LPS-induced COX-2 expression in A549 cells. To identify active constituent(s) of NDE responsible for the anti-inflammatory effect, NDE was introduced in a polyaromatic absorbent resin column and stepwise eluted to yield water fraction, 20% methanol fraction, 40% methanol fraction, 99.8% methanol fraction, and 99.5% acetone fraction. However, none of these five fractions alone inhibited LPS-induced COX-2 expression. On the other hand, exclusion of water fraction from NDE abolished the inhibitory effect of NDE on LPS-induced COX-2 expression. These results suggest that constituent(s) present in water fraction is required but not sufficient for the anti-inflammatory activity of NDE, which may result from interactions among multiple constituents.     

Induction of cyclo-oxygenase-2 by cytokines in human pulmonary epithelial cells: regulation by dexamethasone.

1. Cyclo-oxygenase metabolizes arachidonic acid to prostaglandin H2 (PGH2) and exists in at least two isoforms. Cyclo-oxygenase-1 (COX-1) is expressed constitutively whereas COX-2 is induced by lipopolysaccharide (LPS) and some cytokines in vitro and at the site of inflammation in vivo. Epithelial cells may be an important source of prostaglandins in the airways and we have, therefore, investigated the expression of COX-1 or COX-2 isoforms in primary cultures of human airway epithelial cells or in a human pulmonary epithelial cell line (A549). 2. COX-1 or COX-2 protein was measured by western blot analysis using specific antibodies to COX-2 and selective antibodies to COX-1. The activity of COX was assessed by the conversion of either endogenous or exogenous arachidonic acid to four metabolites, PGE2, PGF2 alpha, thromboxane B2 or 6-oxo PGF1 alpha measured by radioimmunoassay. Thus, COX-1 or COX-2 activity was measured under two conditions; initially the accumulation of the COX metabolites formed from endogenous arachidonic acid was measured after 24 h. In other experiments designed to measure COX activity directly, cells were treated with cytokines for 12h before fresh culture medium was added containing exogenous arachidonic acid (30 microM) for 15 min after which COX metabolites were measured. 3. Untreated primary cells or A549 cells contained low amounts of COX-1 or COX-2 protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

NaDC3抑制人肾小管上皮细胞SIRT1的表达和活性

目的 探讨高亲和力钠依赖性二羧酸转运蛋白（NaDC3）加速肾脏衰老的作用机制.方法 采用解放军总医院肾内科实验室构建的正反义NaDC3的逆转录病毒载体转染人肾小管上皮细胞系（HKC）,通过调控NaDC3的表达水平,观察其对蛋白去乙酰化酶（SIRT1）mRNA、蛋白质表达和酶活性的影响.结果 在HKC中NaDC3可通过增强能量代谢,降低尼克酰胺腺嘌呤二核苷酸（NAD）＋/烟酰胺嘌呤二核苷酸还原态（NADH）的比值,抑制SIRT1的蛋白质表达和酶活性,即在转录后水平调控SIRT1.结论 NaDC3通过能量代谢调控SIRT1的活性加速肾脏衰老.     

Pentoxifylline inhibits platelet-derived growth factor-stimulated cyclin D1 expression in mesangial cells by blocking Akt membrane translocation

Pentoxifylline (PTX) is a potent inhibitor of mesangial cell proliferation, but its underlying mechanism is poorly understood. Here, we demonstrate that in platelet-derived growth factor (PDGF)-stimulated mesangial cells, PTX causes G1 arrest by down-regulation of cyclin D1 expression, which subsequently attenuates Cdk4 activity. In vivo, PTX similarly reduces cyclin D1 expression in mesangial cells of rats with acute Thy1 glomerulonephritis. The mechanism by which PTX reduces cyclin D1 is also investigated. PTX blocks Akt but not phosphatidylinositol 3-kinase (PI3K) activation in response to PDGF and abrogates cyclin D1 induction by PI3K, suggesting an effect of PTX on Akt itself. Indeed, PTX is capable of blocking the membrane translocation of Akt, and enforced targeting of Akt to cell membrane prevents the inhibition of Akt and cyclin D1 by PTX. Because PTX is known to increase intracellular cAMP levels by inhibiting phosphodiesterase, the role of protein kinase A (PKA) in these events is investigated. The PKA antagonist N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89) abolishes cell proliferation effects of PTX and restores cyclin D1 expression as well as Akt membrane translocation and activation by PDGF, whereas dibutyryl cAMP and forskolin recapitulate the functions of PTX in mesangial cells. In conclusion, our results indicate that PTX, acting through PKA, interferes with PDGF signaling to Akt activation by blocking Akt membrane translocation, thereby inhibiting cyclin D1 expression and mesangial cell proliferation.     

Pentoxifylline inhibits tumor necrosis factor-alpha induced synthesis of complement component C3 in human endothelial cells

Vascular endothelium is a major target for the inflammatory damage that occurs with multiple organ dysfunction associated with sepsis and other trauma. The growing appreciation of endothelium as a target of inflammation has obscured the importance of these cells as a source of inflammatory mediators. In the following study we evaluated the ability of tumor necrosis factor-alpha (TNF) to induce the synthesis of complement component C3 in human umbilical vein endothelial cells (HUVEC) and whether pentoxifylline (PTX) could reduce C3 expression. Confluent monolayers of HUVEC were treated with increasing concentrations of TNF with and without two concentrations of PTX. Concentrations of C3 were determined every 48 h for 144 h in cellular supernatants and C3 mRNA was amplified using RT-PCR. TNF increased C3 release from HUVEC in a concentration dependent manner. PTX added at the same time as TNF significantly reduced C3 release at the 96 h time point. Consistent with data on C3 release PTX inhibited the increased C3 mRNA expression associated with TNF treatment. TNF increases C3 synthesis and release from endothelial cells which were inhibited by clinical concentrations of PTX. This data further supports the potential benefit of PTX in multiple organ dysfunction and other inflammatory processes involving the endothelium by inhibiting one of the major mediators of vascular damage.     

Tumour necrosis factor-alpha-induced ICAM-1 expression in human vascular endothelial and lung epithelial cells: modulation by tyrosine kinase inhibitors.

1. Tumour necrosis factor-alpha (TNF alpha) increases the expression of the adhesion molecule intercellular adhesion molecule-1 (ICAM-1) on cultured endothelial and epithelial cells and modulation of this may be important in controlling inflammation. Activation of tyrosine kinase(s) is known to be involved in the signal transduction pathways of many cytokines. In this study we have investigated the effects of the tyrosine kinase inhibitors, ST638, tyrphostin AG 1288 and genistein, on TNF alpha-induced ICAM-1 expression in human alveolar epithelial (A549) and vascular endothelial (EAhy926) cell lines and also normal human lung microvascular endothelial cells (HLMVEC). 2. ICAM-1 expression on cultured cells was determined by a sensitive enzyme-linked immunosorbant assay (ELISA). Endothelial or epithelial monolayers were exposed to increasing doses of TNF-alpha (0.01-10 ng ml-1), in the presence or absence of either ST638 (3-100 microM), AG 1288 (3-100 microM) or genistein (100 microM) and ICAM-1 expression was measured at 4 and 24 h. Control experiments examined the effect of ST638 on phorbol 12-myristate 13-acetate (PMA, 20 ng ml-1, 4 h)-stimulated ICAM-1 and compared it to that of a specific protein kinase C inhibitor, R031-8220 (10 microM). Also, functional consequences of changes in ICAM-1 expression were assessed by measuring adhesion of 111 In-labelled human neutrophils to EAhy926 endothelial and A549 epithelial monolayers treated with TNF alpha, in the presence or absence of ST638. 3. ST638 caused a concentration-dependent reduction in TNF alpha- (0.1-10 ng ml-1)-induced ICAM-1 on EAhy926 endothelial (at 4 h) and A549 epithelial monolayers (at 4 and 24 h). In contrast, ST638 caused a concentration-dependent increase in TNF alpha- (0.1-10 ng ml-1)-induced ICAM-1 on EAhy926 endothelial cells at 24 h. Similar effects were seen with AG 1288 or genistein. ST638 (100 microM) significantly (P < 0.01) inhibited ICAM-1 expression on HLMVEC endothelial cells induced by 0.01 ng ml-1 TNF alpha at 4 or 24 h or 0.1 ng ml-1 at 4 h, but increased ICAM-1 expression induced by 0.1 ng ml-1 TNF alpha at 24 h. ST638 did not significantly change the expression of PMA-stimulated ICAM-1 on either A549 epithelial, EAhy926 or HLMVEC endothelial cells. However, PMA-induced ICAM-1 expression was inhibited by Ro31-8220. Also, treatment of epithelial or endothelial monolayers with TNF alpha and ST638 altered adhesion of human neutrophils to A549 epithelial or EAhy926 endothelial cells in a manner that corresponded to the alteration in ICAM-1 expression. 4. These results show that tyrosine kinase inhibitors alter TNF alpha-induced ICAM-1 expression, but that the cell type, concentration of TNF alpha and time of exposure to this cytokine determine whether expression is decreased or increased by the inhibitor. An increased understanding of the signal transduction pathway(s) involved in TNF alpha-induced ICAM-1 expression on lung epithelial and vascular endothelial cells may be of potential therapeutic value in the treatment of inflammatory disease.     

阿托伐他汀对内皮细胞微粒诱导人脐静脉内皮细胞VCAM-1和ICAM-1表达的影响

目的:探讨阿托伐他汀对内皮细胞微粒(EMPs)诱导的人脐静脉内皮细胞(HUVECs)表达血管细胞粘附分子(VCAM)-1和细胞间粘附分子(ICAM)-1的影响。方法:取生长良好的第4,5代人脐静脉内皮细胞,将细胞分为3大组:对照组、EMPs组、EMPs+阿托伐他汀组。对照组加入培养基,EMPs组以不同浓度的EMPs(0/mL,1×102/mL,1×103/mL,1×104/mL,1×105/mL)与HUVECs共同孵育24 h,EMPs+阿托伐他汀组以不同浓度的阿托伐他汀(0.05,0.1,1.0,10μmol.L-1)与HUVECs作用1 h后,加入105/mL EMPs共同孵育24 h。分别采用实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测VCAM-1和ICAM-1 mRNA和蛋白的表达。结果:HUVECs受EMPs刺激后,VCAM-1和ICAM-1 mRNA及蛋白表达呈浓度依赖性增加,阿托伐他汀可不同程度上抑制EMPs的作用。结论:阿托伐他汀抗动脉粥样硬化作用可能部分与抑制EMPs诱导的内皮细胞VCAM-1和ICAM-1的表达有关。     

Metformin inhibits proliferation and proinflammatory cytokines of human keratinocytes in vitro via mTOR-signaling pathway

Context: The antidiabetic drug metformin exhibits antiproliferative and pro-apoptotic effects in various cells, suggesting its potential to treat a variety of malignant and non-malignant hyperplastic diseases. Clinical studies indicate that psoriasis patients with metformin treatment have a better response than those without metformin.

Objective: The present study evaluates the antiproliferative activity and anti-inflammatory responses of metformin in human keratinocytes in vitro and explores the underlying mechanisms.

Materials and methods: HaCaT cells were incubated with metformin at 0, 25, 50, and 100?mM for 48?h. Antiproliferative activity was evaluated by MTT and apoptotic response was examined by flow cytometry. ELISA was used to detect IL-6, TNF-α, and VEGF protein expression. Western blot was used to investigate the expression of the mammalian target of rapamycin (mTOR) and its downstream effectors p70 ribosomal S6 kinase (p70S6K).

Results: The survival rates of HaCaT cells treated with metformin at 50?mM were reduced to 75.6, 59.4, and 30.3% at 24, 48, and 72?h, respectively. The number of apoptotic HaCaT cells was significantly increased at 50?mM metformin after 48?h treatment. Metformin can exert an anti-inflammatory effect by direct inhibition of IL-6, TNF-α, and VEGF. Metformin at 50?mM significantly reduced the phosphorylation of mTOR and p70S6K, by 49.0 and 62.1%, respectively.

Discussion and conclusion: Metformin treatment significantly inhibited proliferation and proinflammatory responses in HaCaT cells by a mechanism associated with inhibition of the mTOR signaling pathway. The results indicate that metformin may be used as a potential therapeutic agent for psoriasis.     

Flavonoids inhibit histamine release and expression of proinflammatory cytokines in mast cells

Mast cells participate in allergy and inflammation by secreting inflammatory mediators such as histamine and proinflammatory cytokines. Flavonoids are naturally occurring molecules with antioxidant, cytoprotective, and antiinflammatory actions. However, effect of flavonoids on the release of histamine and proinflammatory mediator, and their comparative mechanism of action in mast cells were not well defined. Here, we compared the effect of six flavonoids (astragalin, fisetin, kaempferol, myricetin, quercetin, and rutin) on the mast cell-mediated allergic inflammation. Fisetin, kaempferol, myricetin, quercetin, and rutin inhibited IgE or phorbol-12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-mediated histamine release in RBL-2H3 cells. These five flavonoids also inhibited elevation of intracellular calcium. Gene expressions and secretion of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-8 were assessed in PMACI-stimulated human mast cells (HMC-1). Fisetin, quercetin, and rutin decreased gene expression and production of all the proinflammatory cytokines after PMACI stimulation. Myricetin attenuated TNF-α and IL-6 but not IL-1β and IL-8. Fisetin, myricetin, and rutin suppressed activation of NF-κB indicated by inhibition of nuclear translocation of NF-κB, NF-κB/DNA binding, and NF-κB-dependent gene reporter assay. The pharmacological actions of these flavonoids suggest their potential activity for treatment of allergic inflammatory diseases through the down-regulation of mast cell activation.