Differential effects of selenium and knock-down of glutathione peroxidases on TNFα and flagellin inflammatory responses in gut epithelial cells

Selenium (Se) is essential for human health. Despite evidence that Se intake affects inflammatory responses, the mechanisms by which Se and the selenoproteins modulate inflammatory signalling, especially in the gut, are not yet defined. The aim of this work was to assess effects of altered Se supply and knock-down of individual selenoproteins on NF-κB activation in gut epithelial cells. Caco-2 cells were stably transfected with gene constructs expressing luciferase linked either to three upstream NF-κB response elements and a TATA box or only a TATA box. TNFα and flagellin activated NF-κB-dependent luciferase activity and increased IL-8 expression. Se depletion decreased expression of glutathione peroxidase1 (GPX1) and selenoproteins H and W and increased TNFα-stimulated luciferase activity, endogenous IL-8 expression and reactive oxygen species (ROS) production. These effects were not mimicked by independent knock-down of either GPX1, selenoprotein H or W; indeed, GPX1 knock-down lowered TNFα-induced NF-κB activation and did not affect ROS levels. GPX4 knock-down decreased NF-κB activation by flagellin but not by TNFα. We hypothesise that Se depletion alters the pattern of expression of multiple selenoproteins that in turn increases ROS and modulates NF-κB activation in epithelial cells, but that the effect of GPX1 knock-down is ROS-independent.     

Reactive oxygen species mediate IL-8 expression in Down syndrome candidate region-1-overexpressed cells

Reactive oxygen species (ROS) have been considered to mediate inflammation in Down syndrome (DS). The present study is purposed to examine the mechanism of increased ROS levels and inflammatory cytokine IL-8 expression in Down syndrome candidate region-1 (DSCR1)-transfected cells, by determining ROS levels, IL-8 expression, NF-κB activation, and SOD1 levels in human embryonic kidney (HEK) 293 cells. The cells were treated with an antioxidant N-acetyl cysteine (NAC) or a calcium chelator BAPTA and stimulated with or without IL-1β. As a result, basal levels of ROS, IL-8, and NF-κB-DNA binding activity were higher, and basal SOD1 levels were higher in DSCR1-transfected cells than pcDNA-transfected cells. BAPTA and NAC inhibited increase in ROS (intracellular and mitochondrial levels) in DSCR-1-transfected cells without treatment of IL-1β. DSCR1 transfection-induced changes were increased by treatment with IL-1β, which was suppressed by NAC and BAPTA. Transfection of SOD1 inhibited ROS levels in DSCR1-transfected cells. In conclusion, ROS activate NF-κB and IL-8 induction in DSCR1-transfected cells in a calcium-dependent manner, which is augmented by IL-1β since IL-1β increases calcium and ROS levels in the cells. Reducing ROS levels by treatment of antioxidants may be beneficial for preventing DS-associated inflammation by suppressing cytokine expression.     

Rg3 inhibits gemcitabine-induced lung cancer cell invasiveness through ROS-dependent,NF-κB- and HIF-1α-mediated downregulation of PTX3

PTX3, a member of the long pentraxin subfamily, associated with innate immunity is indispensable for resistance to some cancer. Gemcitabine, an analog of cytosine arabinoside, has shown restrained benefits because of profound chemoresistance. The PTX3 expression on GEM in human lung cancer cells have not yet been clarified; the present study aimed to show reactive oxygen species (ROS) mediatory PTX3 expression through distinct mechanisms. Whereas ginsenoside Rg3 is a herbal medicine with strong antitumor activity. Furthermore, we tested the hypothesis; Rg3 abrogates GEM-induced production of ROS-mediated activation of Akt and extracellular signal-regulated kinase (ERK) pathways and inhibits nuclear piling-up of nuclear factor kappa B (NF-κB) and HIF-1α. On the basis of time and dose-dependent manner, our data demonstrated that GEM-induced PTX3 expression was dependent on ROS generation as it was abrogated by pretreatment of lung cancer cells with the free radical scavenger N-acetyl-l -cysteine. Our data demonstrated that PTX3 upregulation by GEM correlated with the time-dependent escalation of NF-κB and HIF-1α in the nucleus resulted from phosphorylation-induced degradation of IκBα, whereas HIF-1α upregulation was NF-κB-dependent. Increase in ROS expression in lung cancer cells on GEM treatment preceded the nuclear accumulation of NF-κB and HIF-1α and suppression of ROS diminished these effects. ERK1/2 and Akt activation mediated the effect of ROS on NF-κB and HIF-1α and their pharmacological inhibition suppressed GEM-induced PTX3. Our study findings reinforced the role regarding PTX3 signaling in GEM-induced resistance and pointed toward an unintended and undesired effect of chemotherapy and to get an active regimen; the synergy was associated with NF-κB downregulation in lung cancer.     

Peroxisome proliferator-activated receptor gamma depletion stimulates Nox4 expression and human pulmonary artery smooth muscle cell proliferation

Hypoxia stimulates pulmonary hypertension (PH) in part by increasing the proliferation of pulmonary vascular wall cells. Recent evidence suggests that signaling events involved in hypoxia-induced cell proliferation include sustained nuclear factor-kappaB (NF-κB) activation, increased NADPH oxidase 4 (Nox4) expression, and downregulation of peroxisome proliferator-activated receptor gamma (PPARγ) levels. To further understand the role of reduced PPARγ levels associated with PH pathobiology, siRNA was employed to reduce PPARγ levels in human pulmonary artery smooth muscle cells (HPASMC) in vitro under normoxic conditions. PPARγ protein levels were reduced to levels comparable to those observed under hypoxic conditions. Depletion of PPARγ for 24–72 h activated mitogen-activated protein kinase, ERK 1/2, and NF-κB. Inhibition of ERK 1/2 prevented NF-κB activation caused by PPARγ depletion, indicating that ERK 1/2 lies upstream of NF-κB activation. Depletion of PPARγ for 72 h increased NF-κB-dependent Nox4 expression and H₂O₂ production. Inhibition of NF-κB or Nox4 attenuated PPARγ depletion-induced HPASMC proliferation. Degradation of PPARγ depletion-induced H₂O₂ by PEG-catalase prevented HPASMC proliferation and also ERK 1/2 and NF-κB activation and Nox4 expression, indicating that H₂O₂ participates in feed-forward activation of the above signaling events. Contrary to the effects of PPARγ depletion, HPASMC PPARγ overexpression reduced ERK 1/2 and NF-κB activation, Nox4 expression, and cell proliferation. Taken together these findings provide novel evidence that PPARγ plays a central role in the regulation of the ERK1/2–NF-κB–Nox4–H₂O₂ signaling axis in HPASMC. These results indicate that reductions in PPARγ caused by pathophysiological stimuli such as prolonged hypoxia exposure are sufficient to promote the proliferation of pulmonary vascular smooth muscle cells observed in PH pathobiology.     

Magnesium isoglycyrrhizinate suppresses LPS-induced inflammation and oxidative stress through inhibiting NF-κB and MAPK pathways in RAW264.7 cells

Magnesium Isoglycyrrhizinate (MgIG), a novel molecular compound extracted from licorice root, has exhibited greater anti-inflammatory activity and hepatic protection than glycyrrhizin and β-glycyrrhizic acid. In this study, we investigated the anti-inflammatory effect and the potential mechanism of MgIG on Lipopolysaccharide (LPS)-treated RAW264.7 cells. MgIG down-regulated LPS-induced pro-inflammatory mediators and enzymes in LPS-treated RAW264.7 cells, including TNF-α, IL-6, IL-1β, IL-8, NO and iNOS. The generation of reactive oxygen species (ROS) in LPS-treated RAW264.7 cells was also reduced. MgIG attenuated NF-κB translocation by inhibiting IKK phosphorylation and IκB-α degradation. Simultaneously, MgIG also inhibited LPS-induced activation of MAPKs, including p38, JNK and ERK1/2. Taken together, these results suggest that MgIG suppresses inflammation by blocking NF-κB and MAPK signaling pathways, and down-regulates ROS generation and inflammatory mediators.     

褪黑素对小鼠MFC前胃癌细胞ERK、Akt及NF-κB表达的影响

目的探讨褪黑素对小鼠MFC前胃癌细胞ERK、Akt及NF-κB表达的影响。方法建立褪黑素在不同时间点干预胃癌细胞的体外模型,免疫印迹法观察褪黑素对小鼠MFC前胃癌细胞p-ERK/ERK、p-Akt/Akt,NF-κB表达的影响,CCK-8检测PI3K抑制剂Wortmannin对MFC细胞增殖的作用。结果①2mmol/L褪黑素作用24h、48h后明显下调MFC细胞ERK1/2、Akt的磷酸化,但对ERK1/2、Akt表达无影响;②Wortmannin明显抑制MFC细胞增殖活性,与MLT作用有明显协同效应;③褪黑素对NF-κB表达无影响。结论褪黑素可通过抑制ERK1/2、Akt的磷酸化从而抑制胃癌细胞增殖。     

MicroRNA-181a regulates the activation of the NLRP3 inflammatory pathway by targeting MEK1 in THP-1 macrophages stimulated by ox-LDL

Atherosclerosis (AS) is a chronic inflammatory disease that is characterized by the deposition of lipids in the vascular wall and the formation of foam cells. Macrophages play a critical role in the development of this chronic inflammation. An increasing amount of research shows that microRNAs affect many steps of inflammation. The goal of our study was to investigate the regulatory effect of miR-181a on the NLRP3 inflammasome pathway and explore its possible mechanism. Compared with the control group, the expression of miR-181a was downregulated in the carotid tissue of AS group mice, while the expression of MEK1 and NLRP3-related proteins was upregulated significantly. In vitro, when THP-1 macrophages were stimulated with oxidized low-density lipoprotein (ox-LDL), the expression of miR-181a was decreased, the MEK/ERK/NF-κB inflammatory pathways were activated and the expression of NLRP3 inflammasome-related proteins was upregulated. Exogenous overexpression of miR-181a downregulated the activation of the MEK/ERK/NF-κB pathway and decreased the expression of NLRP3 inflammasome-related proteins (such as NLRP3, caspase-1, interleukin-18 [IL-18], IL-1β, etc). Exogenous miR-181a knockdown showed the opposite results to those of overexpression group. A luciferase reporter assay proved that miR-181a inhibited the expression of MEK1 by binding to its 3′-untranslated region. When we knocked down miR-181a and then treated cells with U0126 before ox-LDL stimulation, we found that U0126 reversed the increased activation of the MEK/ERK/NF-κB pathway and upregulation of NLRP3 inflammasome-related proteins (NLRP3, caspase-1, IL-18, IL-1β) that resulted from miR-181a knockdown. Our study suggests that miR-181a regulates the activation of the NLRP3 inflammatory pathway by altering the activity of the MEK/ERK/NF-κB pathway via targeting of MEK1.     

Shikonin, a Chinese plant-derived naphthoquinone, induces apoptosis in hepatocellular carcinoma cells through reactive oxygen species: A potential new treatment for hepatocellular carcinoma

Although shikonin, a naphthoquinone derivative, has showed anti-cancer activity, its precise molecular anti-tumor mechanism remains to be elucidated. In this study, we investigated the effects of shikonin on human hepatocellular carcinoma (HCC) in vitro and in vivo. Our results showed that shikonin induced apoptosis of Huh7 and BEL7402 but not nontumorigenic cells. ROS generation was detected, and ROS scavengers completely inhibited shikonin-induced apoptosis, indicating that ROS play an essential role. Although the JNK activity was significantly elevated after shikonin treatment, JNK was not linked to apoptosis. However, downregulation of Akt and RIP1/NF-κB activity was found to be involved in shikonin-induced apoptosis. Ectopic expression of Akt or RIP1 partly abrogated the effects of shikonin, and Akt inhibitor and RIP1 inhibitor synergistically induced apoptosis in conjunction with shikonin treatment. ROS scavengers blocked shikonin-induced inactivation of Akt and RIP1/NF-κB, but Akt or RIP1/NF-κB did not regulate ROS generation, suggesting that Akt and RIP1/NF-κB signals are downstream of ROS generation. In addition, the results of xenograft experiments in mice were consistent with in vitro studies. Taken together, our data show that shikonin, which may be a promising agent in the treatment of liver cancer, induced apoptosis in HCC cells through the ROS/Akt and RIP1/NF-κB pathways.     

Immunomodulatory beta-glucan from Lentinus edodes activates mitogen-activated protein kinases and nuclear factor-kappaB in murine RAW 264.7 macrophages

Lentinan, a cell wall β-glucan from the fruiting bodies of Lentinus edodes, is well known to be a biological defense modifier, but the signal transduction pathway(s) induced by Lentinan have not been elucidated. In this study, we extracted Lentinan (LNT-S) by ultrasonication from Lentinus edodes and report that, in murine RAW 264.7 macrophages, LNT-S glucan activated NF-κB p65 and triggered its nuclear translocation as determined by Western blotting. Moreover, LNT-S enhanced NF-κB-luciferase activity in the Dual-Luciferase gene system assay. Its upstream signaling molecules, MAPKs such as ERK1/2 and JNK1/2, were shown to be activated by assessing the level of phosphorylation in a time- and concentration-dependent manner, but its downstream proinflammatory enzyme, inducible NOS, was not observed. The data evaluated using a TNF-α ELISA kit and Griess reagent further demonstrated that no proinflammatory mediators such as TNF-α and NO were produced by LNT-S stimulation in RAW 264.7 cells. In contrast, LPS significantly induced inducible NOS expression and increased NO and TNF-α production, which are associated with activation of the NF-κB p65/p50 heterodimer complex. It is possible that LNT-S did not activate NF-κB p65/p50, and the activation of NF-κB p65 was not sufficient to stimulate cytokine production. These data demonstrate that LNT-S glucan carries out its immunomodulating activity by activating MAPK signaling pathways without secretion of TNF-α and NO.     

JAK2 is an important signal transducer in IL-33-induced NF-κB activation

IL-33, a member of the IL-1 family of cytokines, has been shown to activate NF-κB and MAP kinase family through the IL-1 receptor-related protein, ST2L. In this study, we found that IL-33 rapidly activated a tyrosine kinase, JAK2. Interestingly, we demonstrated the functional involvement of JAK2 in IL-33-induced IκBα degradation and NF-κB activation, since a JAK2 inhibitor, AG490, effectively inhibited this signaling pathway. Furthermore, IL-33 failed to induce IκBα degradation and NF-κB activation in JAK2-deficient MEFs expressing ST2L, compared with wild-type MEFs expressing ST2L. In addition, the introduction of wild-type JAK2 but not kinase dead JAK2 mutant (K882R) restored the IL-33-induced efficient activation of NF-κB in JAK2-deficient MEFs expressing ST2L, resulting in the induction of IL-6, CCL2/MCP-1 and CXCL1/KC expression. On the other hand, the activation of ERK, JNK and p38 was unaffected by JAK2 inhibition and JAK2 deficiency. Thus, these data demonstrate that JAK2 plays an important role in regulating IL-33-induced NF-κB activation.     

Protection against UVB-induced damages in human dermal fibroblasts: efficacy of tricin isolated from enzyme-treated Zizania latifolia extract

This study was undertaken to determine the effects of enzyme-treated Zizania latifolia (ETZL) and of its major compound tricin on skin photo-aging and to investigate the mechanisms involved. It was found ETZL and tricin suppressed matrix metalloproteinase (MMP) production and increased type I-procollagen production in UVB-irradiated human dermal fibroblasts (HDFs). Furthermore, ETZL and tricin significantly up-regulated the expressions of the antioxidant enzymes HO-1 and SOD1, reduced UVB-induced reactive oxygen species (ROS) generation and mitogen-activated protein kinase (MAPK) induction by ROS and thereby attenuated activator protein-1 (AP-1) expression. In addition, ETZL and tricin both reduced the phosphorylations of IκBα and IKKα/ß and κB blocked the nuclear translocation of nuclear factor-κB (NF-κB) p65. These results show that ETZL have skin protective effects against UVB and suggest tricin as major efficacious material in ETZL protecting skin photoaging.