LPS priming potentiates and prolongs proinflammatory cytokine response to the trichothecene deoxynivalenol in the mouse

Simultaneous exposure to lipopolysaccharide (LPS) markedly amplifies induction of proinflammatory cytokine expression as well as IL-1-driven lymphocyte apoptosis by trichothecene deoxynivalenol (DON) in the mouse. The purpose of this research was to test the hypothesis that LPS priming will sensitize a host to DON-induced proinflammatory cytokine induction and apoptosis. In mice primed with LPS (1 mg/kg bw) ip. and treated 8 h later with DON po., the minimum DON doses for inducing IL-1alpha, IL-1beta, IL-6 and TNF-alpha serum proteins and splenic mRNAs were significantly lower than the DON doses required for vehicle-primed mice. LPS priming also decreased onset time and dramatically increased magnitude and duration of cytokine responses. LPS-primed mice maintained heightened sensitivity to DON for up to 24 h. LPS priming doses as low as 50 microg/kg bw evoked sensitization. DNA fragmentation analysis and flow cytometry also revealed that mice primed with LPS (1 mg/kg) for 8 h and exposed to DON (12.5 mg/kg) exhibited massive thymocyte loss by apoptosis 12 h later compared to mice exposed to DON or LPS alone. LPS priming decreased DON-induced p38 and ERK 1/2 phosphorylation suggesting that enhanced mitogen-activated protein kinase activation was not involved in increased cytokine responses. Taken together, exposure to LPS rendered mice highly susceptible to DON induction of cytokine expression and this correlated with increased apoptosis in the thymus.     

Comparative study of deoxynivalenol, 3-acetyldeoxynivalenol,and 15-acetyldeoxynivalenol on intestinal transport and IL-8 secretion in the human cell line Caco-2

The effects of the trichothecene mycotoxin deoxynivalenol (DON) and its acetylated derivatives, 3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON) on human intestinal cell Caco-2 were investigated by the studies of transepithelial transport, gene expression, and cytokine secretion. Permeability across a Caco-2 cell monolayer was evaluated by transport study. Transport rates were ranked as DON, 3ADON < 15ADON in apical–basolateral direction. 15ADON showed the highest permeability, induced the highest decrease in transepithelial electrical resistance (TEER), and prompted significant Lucifer Yellow permeability. These results showed that 15ADON affect paracellular barrier function extremely. In addition, gene expressions induced by toxins were screened by DNA microarray for investigating cellular effect on Caco-2 cell. The most remarkable gene induced by DON and 15ADON was inflammatory chemokine IL-8 and thus mRNA expression and secretion of IL-8 were analyzed by PCR and ELISA. Both DON and acetylated DONs could induce mRNA expression and production of IL-8. In particular, ELISA assay showed that the ability to produce IL-8 was ranked as 3ADON < DON < 15ADON. Our results indicated that 15ADON caused the highest permeability and highest IL-8 secretion among DON, 3ADON, and 15ADON in human intestinal cell.     

Influence of deoxynivalenol on NF-kappaB activation and IL-8 secretion in human intestinal Caco-2 cells

Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. In human intestinal Caco-2 cells, DON activates the mitogen-activated protein kinases (MAPKs). We hypothesized a link between DON ingestion and intestinal inflammation, and used Caco-2 cells to assess the effects of DON, at plausible intestinal concentrations (250-10,000 ng/ml), on inflammatory mediators acting downstream the MAPKs cascade i.e. activation of nuclear factor-kappaB (NF-kappaB) and interleukin-8 (IL-8) secretion. In addition, Caco-2 cells were co-exposed to pro-inflammatory stimuli in order to mimic an inflamed intestinal epithelium. Dose-dependent increases in NF-kappaB activity and IL-8 secretion were observed, reaching 1.4- and 7.6-fold, respectively using DON at 10 microg/ml. Phosphorylation of inhibitor-kappaB (IkappaB) increased (1.6-fold) at DON levels <0.5 microg/ml. Exposure of Caco-2 cells to pro-inflammatory agents, i.e. 25 ng/ml interleukin-1beta, 100 ng/ml tumor necrosis factor-alpha or 10 microg/ml lipopolysaccharides, activated NF-kappaB and increased IL-8 secretion. Synergistic interactions between these stimuli and DON were observed. These data show that DON induces NF-kappaB activation and IL-8 secretion dose-dependently in Caco-2 cells, and this effect was accentuated upon pro-inflammatory stimulation, suggesting DON exposure could cause or exacerbate intestinal inflammation.     

Activation of ERK mitogen-activated protein kinase in human cells by the mycotoxin patulin

Patulin (PAT), a mycotoxin produced by certain species of Penicillium and Aspergillus, is often detectable in moldy fruits and their derivative products. PAT led to a concentration-dependent and time-dependent increase in phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in human embryonic kidney (HEK293) cells, human peripheral blood mononuclear cells (PBMCs), and Madin-Darby canine kidney (MDCK) cells. Exposure of HEK293 cells to concentrations above 5 μM PAT for 30 min induced ERK1/2 phosphorylation; activation of ERK1/2 was also observed after 24 h incubation with 0.05 μM of PAT. Treatment of human PBMCs for 30 min with 30 μM PAT dramatically increased the phosphorylated ERK1/2 levels. Both MEK1/2 inhibitors, U0126 and PD98059, suppressed ERK1/2 activation in either HEK293 or MDCK cells. In HEK293 cells, U0126-mediated inhibition of PAT-induced ERK1/2 phosphorylation resulted in a significant decrease in levels of DNA damage, expressed as tail moment values, in the single cell gel electrophoresis assay. Conversely, U0126 did not affect cell viability, lactate dehydrogenase release, and the DNA synthesis rate in PAT-treated cultures. Exposure of HEK293 cells for 90 min to 15 μM PAT elevated the levels of early growth response gene-1 (egr-1) mRNA, but not of c-fos, fosB, and junB mRNAs. These results indicate that in human cells, PAT causes a rapid and persistent activation of ERK1/2 and this signaling pathway plays an important role in mediating PAT-induced DNA damage and egr-1 gene expression.     

Mitogen-activated protein kinase (MAPK) pathway mediates the oestrogen-like activities of ginsenoside Rg1 in human breast cancer (MCF-7) cells

Background and purpose:

The present study was designed to determine how ginsenoside Rg1, an active ingredient in ginseng root, exerts its oestrogenic effects. We hypothesize that Rg1 may exert oestrogen-like actions in MCF-7 cells by activating the mitogen-activated protein kinase (MAPK) pathway in a ligand-independent manner.

Experimental approach:

MCF-7 cells were co-incubated with the MAPK inhibitor PD98059 to determine whether the stimulant effects of Rg1 on cell proliferation, the induction of IGF-IR and pS2, the functional transactivation of oestrogen receptor-α (ERα), as well as ERα phosphorylation are dependent on MAPK. The time-dependent responses of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated protein kinase (ERK) to Rg1 in MCF-7 cells were studied. The responses of MEK phosphorylation to Rg1 in oestrogen receptor (ER)-negative HEK293 cells were also determined. The effects of Rg1 on cell proliferation and IGF-IR protein expression were studied in the presence of tyrosine kinase inhibitor genistein to elucidate the involvement of tyrosine kinase in mediating these effects.

Key results:

The oestrogenic effects of Rg1 in MCF-7 cells were abolished in the presence of PD98059. Rg1 could induce MEK protein expression and the phosphorylation level of MEK and ERK significantly in a time- and dose-dependent manner. Rg1 activated MEK phosphorylation in ER-negative HEK293 cells in a time- and dose-dependent manner. Rg1 induction of cell proliferation and IGF-IR protein expression was abolished by co-treatment with genistein.

Conclusions and implications:

Taken together, these results show that the MAPK pathway is involved in mediating the oestrogen-like actions of Rg1 in MCF-7 cells and suggest that Rg1 may activate ERα via MEK/ERK in a ligand-independent manner.     

NAC和地塞米松对脂多糖诱导大鼠肺泡巨噬细胞p38蛋白激酶的影响

目的　观察脂多糖 (LPS)诱导肺泡巨噬细胞 (AM )p38蛋白激酶活化及抗炎药物地塞米松 (DEX)和N 乙酰半胱氨酸 (NAC)对其影响。方法　分离培养大鼠肺泡巨噬细胞 ,设正常对照组、LPS刺激组、DEX或NAC干预组 ,共 4组。分别采用Western印迹和放射免疫分析法检测AM核提取物 p38蛋白激酶和细胞培养上清TNF α、IL 8含量。 结果　LPS刺激组核蛋白提取物p38蛋白激酶和细胞培养上清TNF α、IL 8含量较正常对照组升高 (P <0 0 1 )。DEX组和NAC组虽较正常对照组高 ,但均低于LPS刺激组 (P <0 0 1 )。核提取物 p38蛋白激酶和细胞培养上清TNF α、IL 8含量之间分别呈正相关 (r =0 754、0 62 5 ,P <0 0 1 )。结论　LPS诱导肺泡巨噬细胞 p38蛋白激酶活化 ,进而导致TNF α、IL 8表达增多 ;DEX和NAC可能通过抑制 p38活化而减少炎性介质TNF α、IL 8的释放     

Involvement of p38 mitogen-activated protein kinase in the induction of interleukin-12 p40 production in mouse macrophages by berberine,a benzodioxoloquinolizine alkaloid

Interleukin (IL)-12 plays a pivotal role in the development of T helper type 1 (Th1)-immune response, which may have therapeutic effects on diseases associated with pathologic Th2 responses such as allergic disorders and asthma. In this study, we investigated the effects of berberine, a benzodioxoloquinolizine alkaloid with anti-microbial and anti-tumor activities, on the production of IL-12 p40, an inducible subunit of IL-12, in mouse macrophages. Berberine-induced IL-12 p40 production and activation of p38 mitogen-activated protein kinase (MAPK) in dose-dependent manners, which were significantly inhibited by p38 MAPK inhibitors and yohimbine, indicating that p38 MAPK and alpha(2)-adrenergic receptor were involved in the induction of IL-12 p40 production in mouse macrophages by berberine. Furthermore, berberine significantly enhanced IL-12 p40 production in mouse macrophages when combined with lipopolysaccharide, a well-known inducer of IL-12 production. These findings may explain some of the known biological effects of berberine and suggests berberine as an immunotherapeutic compound for induction of IL-12, which is potentially applicable for tumors, infectious disease, and airway inflammation.     

Involvement of multiple signaling pathways in the post-bariatric induction of IL-6 and IL-8 mRNA and release in human visceral adipose tissue

The present studies were designed to determine the site of and the mechanism for the rapid increase in IL-6 and IL-8 mRNA observed in human visceral adipose tissue after removal during laparoscopic bariatric surgery. Upregulation of IL-6 and IL-8 mRNA as well as their release were seen within 3h whether one intact piece of tissue or minced pieces of adipose tissue were incubated in vitro. Most of the IL-6 and IL-8 mRNA content of visceral adipose tissue after 3h of incubation was in the non-fat cells. Actinomcyin D markedly reduced the upregulation of IL-6 and IL-8 mRNA. Incubation of adipose tissue explants with a soluble TNFalpha receptor (etanercept) plus a blocking antibody against IL-lbeta reduced by 55% the increase in IL-6 mRNA and by 42% that of IL-8 mRNA seen between 1 and 5h of incubation. The upregulation of IL-8 and IL-6 mRNA accumulation as well as their release over a 2 or 4h incubation was reduced by around 50% in the presence of an inhibitor of the p38 MAPK or an inhibitor of the NFkappaB pathway and by 85% in the presence of both inhibitors. The data suggest that the relative trauma and/or hypoxia that occurs when adipose tissue is removed results in the release of TNFalpha and IL-1beta. These cytokines, and probably other factors as well, enhance IL-6 and IL-8 mRNA accumulation in human adipose tissue explants through mechanisms involving the p38 MAPK and NFkappaB pathways.     

Inhibition of interleukin-8 (CXCL8/IL-8) responses by repertaxin, a new inhibitor of the chemokine receptors CXCR1 and CXCR2

Repertaxin is a new non-competitive allosteric blocker of interleukin-8 (CXCL8/IL-8) receptors (CXCR1/R2), which by locking CXCR1/R2 in an inactive conformation prevents receptor signaling and human polymorphonuclear leukocyte (PMN) chemotaxis. Given the unique mode of action of repertaxin it was important to examine the ability of repertaxin to inhibit a wide range of biological activities induced by CXCL8 in human leukocytes. Our results show that repertaxin potently and selectively blocked PMN adhesion to fibrinogen and CD11b up-regulation induced by CXCL8. Reduction of CXCL8-mediated PMN adhesion by repertaxin was paralleled by inhibition of PMN activation including secondary and tertiary granule release and pro-inflammatory cytokine production, whereas PMN phagocytosis of Escherichia coli bacteria was unaffected. Repertaxin also selectively blocked CXCL8-induced T lymphocyte and natural killer (NK) cell migration. These data suggest that repertaxin is a potent and specific inhibitor of a wide range of CXCL8-mediated activities related to leukocyte recruitment and functional activation in inflammatory sites.     

Role of TNF-alpha, IL-1beta and IL-6 in the local tissue damage induced by Bothrops asper snake venom: an experimental assessment in mice.

The role of the cytokines TNF-alpha, IL-1beta and IL-6 in the acute local pathological effects induced by Bothrops asper snake venom was assessed in mice. Intramuscular injection of this venom induced increments in IL-1beta and IL-6 in muscle, but no elevations of TNF-alpha were detected. Pentoxifylline (PTX), a methylxanthine derivative that inhibits the synthesis of TNF-alpha, and antibodies against these three cytokines were used to assess the role of these cytokines in venom-induced effects. As a control, PTX pretreatment was effective at abrogating lethality and serum TNF-alpha increments in mice subjected to endotoxemia induced by injection of Escherichia coli lipopolysaccharide, although it did not affect the increment in IL-1beta and IL-6 in such endotoxic model. PTX failed to reduce lethality, hemorrhage, myonecrosis, dermonecrosis and edema induced by B. asper venom. Moreover, pretreatment with anti-cytokine antibodies was also ineffective at reducing venom-induced myonecrosis and hemorrhage. It is concluded that TNF-alpha, IL-1beta and IL-6 do not have a significant role in the pathogenesis of the acute local pathological effects induced by B. asper venom in mice, although this does not exclude the possibility that these cytokines play a role in other aspects of venom-induced local pathology, as well as in the reparative and regenerative responses that take place after the onset of tissue damage.     

The role of adenosine A2A and A2B receptors in the regulation of TNF-alpha production by human monocytes

Adenosine is an endogenous nucleoside that regulates many physiological processes through the activation of its four receptors: A(1), A(2A), A(2B) and A(3). Previous studies have identified the involvement of A(2) receptors in the inhibitory activity of adenosine analogues on tumor necrosis factor-alpha (TNF-alpha) production by lipopolysaccharide (LPS) activated monocytes, but the relative contributions of A(2A) versus A(2B) receptors have not been determined in human primary monocytes. Nor has the role of A(1) and A(3) been clearly identified in the system. The lack of such information impacts on the selection of adenosine receptor agonists for disease intervention. Using LPS-stimulated human primary monocytes, we found that the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) or the A(2A) receptor agonist, 4-[2-[[6-amino-9-(N-ethyl-b-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680) produced a concentration-dependent inhibition of TNF-alpha production, with IC(50)s of 58.4nM (32.7-104.5nM, 95% confidence interval) and 49.2nM (22.7-105.9nM, 95% confidence interval), respectively. The selective A(2A) receptor blocker, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylaminso]ethyl)phenol (ZM241385, 30nM), antagonized the effects of NECA and CGS21680 (pK(B) estimates were 8.7+/-0.1 and 8.9+/-0.1, respectively), while the selective A(2B) antagonist, N-(4-cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide (MRS1754, 100nM), failed to antagonize the effects of either agonist. Furthermore, neither the A(1) receptor agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA) nor the A(3) receptor agonist, 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-b-d-ribofuranuronamide (2-Cl-IB-MECA) showed significant inhibitory activity at concentrations that effectively bind to their respective receptors. We conclude that A(2A) receptor activation is predominantly responsible for the inhibitory effects of adenosine receptor agonists on TNF-alpha production from LPS-stimulated monocytes.     

MiR-215-5p inhibits the inflammation injury in septic H9c2 by regulating ILF3 and LRRFIP1

MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) playing crucial roles in sepsis-induced diseases, including myocardial inflammation. Nevertheless, the expression pattern and role of miR-215-5p in myocardial inflammation are still un-investigated up to now. The purpose of our study is to further inquire the effect of miR-215-5p on lipopolysaccharide (LPS)-activated inflammation injury in H9c2 cells and the possibly associated mechanisms. First of all, LPS-induced H9c2 cells models were constructed and affirmed via detection of pro-inflammatory factors, the viability and apoptosis. MiR-215-5p was overtly down-regulated in LPS-treated H9c2 cells and miR-215-5p overexpression could suppress the inflammation injury. LRRFIP1 was proved to be the target gene of miR-215-5p and meanwhile, miR-215-5p also targeted ILF3 that experimented to bind to and stabilize LRRFIP1. Final rescue assays confirmed that the overexpression of LRRFIP1 or ILF3 rescued the repressive effect of miR-215-5p up-regulation on the inflammation injury in septic H9c2. Totally, miR-215-5p exerted protective function in the inflammation injury in septic H9c2 via targeting ILF3 and LRRFIP1, suggesting an additional treatment method for sepsis-activated myocardial inflammation.