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

Effects of selenium and thyroid hormone deficiency on peritoneal macrophages adhesion and occurrence of natural IGM antibodies in juvenile rats

Both selenium, as an effector and regulator of antioxidative enzymes activity, and thyroid hormones are potent immunomodulators. Besides, selenium incorporated into iodothyronine deiodinases is involved in the thyroid function and thus indirectly regulates the immune response. Studies of the mutual infl uence of selenium and thyroid hormones on the immune response are scarce, hence we analyzed the effects of an iodothyronine deiodinases blocker, propylthiouracil (PTU), and selenium defi ciency on the function of peritoneal macrophages, and titer of naturally occurring anti-sheep red blood cells (SRBC) IgM antibodies in juvenile rats. The experiment was carried out on 64 Wistar male rats allotted to 4 groups: controlselenium adequate PTU-group; selenium adequate, PTU+ group; selenium defi cient, PTU-group; and selenium defi cient, PTU+. The selenium adequate and selenium defi cient groups were fed a diet containing 0.334 and 0.031 mg Se/kg, respectively. PTU+ groups received PTU (150 mg/L) in drinking water. After 3 weeks, thyroxine (T-4), triiodothyronine (T-3), and thyroid stimulating hormone (TSH) were determined. The animals having "intermediate" concentrations of T-3 (1.56-1.69 nmol/L) and T 4 (41-50 nmol/L) were excluded from further analysis. Thus, PTU+ groups included hypothyroid animals (T-3 lt = 1.55 nmol/L; T-4 lt = 40 nmol/L), while PTU-groups included euthyroid rats (T-3 lt = 1.70 nmol/L; T-4 lt = 50 nmol/L). Both groups of selenium defi cient rats had, when compared to the control group, a signifi cantly lower activity of glutathione peroxidase GPx1 and GPx3. Neither selenium defi ciency nor PTU infl uenced the adherence of peritoneal macrophages. Selenium defi ciency signifi cantly decreased the peroxide synthesis in macrophages and signifi cantly increased the titer of anti-SRBC IgM. Hypotyroidism alone or in combination with selenium defi ciency had no infl uence on these parameters

Synthesis and evaluation of the anti-inflammatory properties of selenium-derivatives of celecoxib

Celecoxib is a selective cyclooxygenase (COX)-2 inhibitor used to treat inflammation, while selenium is known to down-regulate the transcription of COX-2 and other pro-inflammatory genes. To expand the anti-inflammatory property, wherein celecoxib could inhibit pro-inflammatory gene expression at extremely low doses, we incorporated selenium (Se) into two Se-derivatives of celecoxib, namely; selenocoxib-2 and selenocoxib-3. In vitro kinetic assays of the inhibition of purified human COX-2 activity by these compounds indicated that celecoxib and selenocoxib-3 had identical K(I) values of 2.3 and 2.4 μM; while selenocoxib-2 had a lower K(I) of 0.72 μM. Furthermore, selenocoxib-2 inhibited lipopolysaccharide-induced activation of NF-κB leading to the down-regulation of expression of COX-2, iNOS, and TNFα more effectively than selenocoxib-3 and celecoxib in RAW264.7 macrophages and murine bone marrow-derived macrophages. Studies with rat liver microsomes followed by UPLC-MS-MS analysis indicated the formation of selenenylsulfide conjugates of selenocoxib-2 with N-acetylcysteine. Selenocoxib-2 was found to release minor amounts of Se that was effectively inhibited by the CYP inhibitor, sulphaphenazole. While these studies suggest that selenocoxib-2, but not celecoxib and selenocoxib-3, targets upstream events in the NF-κB signaling axis, the ability to effectively suppress NF-κB activation independent of cellular selenoprotein synthesis opens possibilities for a new generation of COX-2 inhibitors with significant and broader anti-inflammatory potential

Gambogic acid covalently modifies IκB kinase-β subunit to mediate suppression of lipopolysaccharide-induced activation of NF-κB in macrophages

Gambogic acid (GA) is a polyprenylated xanthone abundant in the resin of Garcinia morella and G. hanburyi with a long history of use as a complementary and alternative medicine. The anti-tumor activity of GA has been well demonstrated and is thought to arise partly from the associated anti-inflammatory activity. Recent studies have indicated that the anti-tumor activity of GA is mediated by its ligation of the transferrin receptor TfR1. Since the cellular expression of TfR1 is down-regulated by lipopolysaccharide (LPS), we hypothesized that an alternative pathway exists in immune cells, such as macrophages, where GA could mitigate the expression of pro-inflammatory genes. Here we demonstrate that GA inhibits the LPS-dependent expression of nuclear factor-κB (NF-κB) target pro-inflammatory genes in macrophages. Western immunoblot, NF-κB luciferase reporter, and gel shift analyses revealed that GA strongly blocked the activation of NF-κB induced by LPS; while 9,10-dihydroGA that lacks the reactive α,β-unsaturated carbonyl group was ineffective. Moreover, GA was able to decrease nuclear p65 levels in RAW264.7 macrophages, where the expression of TfR1 was down-regulated by RNA interference. In-vitro kinase assays coupled with interaction studies using biotinylated GA as well as proteomic analysis demonstrated that IKKβ, a key kinase of the NF-κB signaling axis, was covalently modified by GA at Cys179 causing significant inhibition of its kinase activity. Taken together, these data demonstrate the potent anti-inflammatory activity of GA

Thioredoxin Reductase-1 Negatively Regulates HIV-1 Transactivating Protein Tat-dependent Transcription in Human Macrophages*

Epidemiological studies suggest a correlation between severity of acquired immunodeficiency syndrome (AIDS) and selenium deficiency, indicating a protective role for this anti-oxidant during HIV infection. Here we demonstrate that thioredoxin reductase-1 (TR1), a selenium-containing pyridine nucleotide-disulfide oxidoreductase that reduces protein disulfides to free thiols, negatively regulates the activity of the HIV-1 encoded transcriptional activator, Tat, in human macrophages. We used a small interfering RNA approach as well as a high affinity substrate of TR1, ebselen, to demonstrate that Tat-dependent transcription and HIV-1 replication were significantly increased in human macrophages when TR1 activity was reduced. The increase in HIV-1 replication in TR1 small interfering RNA-treated cells was independent of the redox-sensitive transcription factor, NF-κB. These studies indicate that TR-1 acts as a negative regulator of Tat-dependent transcription. Furthermore, in vitro biochemical assays with recombinant Tat protein confirmed that TR1 targets two disulfide bonds within the Cys-rich motif required for efficient HIV-1 transactivation. Increasing TR1 expression along with other selenoproteins by supplementing with selenium suggests a potential inexpensive adjuvant therapy for HIV/AIDS patients

Synthesis and evaluation of the anti-inflammatory properties of selenium-derivatives of celecoxib

Celecoxib is a selective cyclooxygenase (COX)-2 inhibitor used to treat inflammation, while selenium is known to down-regulate the transcription of COX-2 and other pro-inflammatory genes. To expand the anti-inflammatory property, wherein celecoxib could inhibit pro-inflammatory gene expression at extremely low doses, we incorporated selenium (Se) into two Se-derivatives of celecoxib, namely; selenocoxib-2 and selenocoxib-3. In vitro kinetic assays of the inhibition of purified human COX-2 activity by these compounds indicated that celecoxib and selenocoxib-3 had identical K(I) values of 2.3 and 2.4μM; while selenocoxib-2 had a lower K(I) of 0.72μM. Furthermore, selenocoxib-2 inhibited lipopolysaccharide-induced activation of NF-κB leading to the down-regulation of expression of COX-2, iNOS, and TNFα more effectively than selenocoxib-3 and celecoxib in RAW264.7 macrophages and murine bone marrow-derived macrophages. Studies with rat liver microsomes followed by UPLC-MS-MS analysis indicated the formation of selenenylsulfide conjugates of selenocoxib-2 with N-acetylcysteine. Selenocoxib-2 was found to release minor amounts of Se that was effectively inhibited by the CYP inhibitor, sulphaphenazole. While these studies suggest that selenocoxib-2, but not celecoxib and selenocoxib-3, targets upstream events in the NF-κB signaling axis, the ability to effectively suppress NF-κB activation independent of cellular selenoprotein synthesis opens possibilities for a new generation of COX-2 inhibitors with significant and broader anti-inflammatory potential