The anti-inflammatory effects of the tellurium redox modulating compound, AS101, are associated with regulation of NFkappaB signaling pathway and nitric oxide induction in macrophages

Abstract

Background: LPS-activated macrophages produce mediators which are involved in inflammation and tissue injury, and especially those associated with endotoxic shock. The non toxic tellurium compound ammonium tri-chloro(dioxoethylene-O,O'-)tellurate, AS101, has been recently shown to exert profound anti-inflammatory properties in animal models, associated with its Te(IV) redox chemistry. This study explores the anti-inflammatory properties of AS101 with respect to modulation of inflammatory cytokines production and regulation of iNOS transcription and expression in activated macrophages via targeting the NFkB complex.

Results: AS101 decreased production of IL-6 and in parallel down-regulated LPS-induced iNOS expression and NO secretion by macrophages. AS101 reduced IkB phosphorylation and degradation, and reduced NFkB nuclear translocalization, albeit these effects were exerted at different kinetics. Chromatin immunoprecipitation assays showed that AS101 treatment attenuated p50-subunit ability to bind DNA at the NFkB consensus site in the iNOS promotor following LPS induction.

Conclusions: Besides AS101, the investigation of therapeutic activities of other tellurium(IV) compounds is scarce in the literature, although tellurium is the fourth most abundant trace element in the human body. Since IKK and NFkB may be regulated by thiol modifications, we may thus envisage, inview of our integrated results, that Te(IV) compounds, may have important roles in thiol redox biological activity in the human body and represent a new class of anti-inflammatory compounds.

Figure 1

Effect of AS101 on LPS-induced iNOS protein expression (A-D) and NO(E) and IL-6 (F) secretion. (A) LPS-stimulated RAW264.7 cells (1 × 10⁶/ml) were treated with AS101(0.5 or 2 [μg/ml]) for 1 h (A) and 4 h (C). The iNOS level was analyzed by immunoblotting using anti-iNOS. Actin was used as an internal loading control. Bar graphs represent the quantitative densitometric value of the expressed protein vs actin: 1 h (B) and 4 h (D). *p < 0.05 vs LPS. Data shown are representative of three different experiments. (E-F) LPS-stimulated RAW264.7 cells (1 × 10⁶/ml) were incubated with AS101 (2 μg/ml) for 24 h. The culture supernatants were subsequently isolated and analyzed for nitrite and IL-6 levels. Data expressed as mean ± SE of four independent experiments. ** p < 0.05 vs. control, * p < 0.05 vs. LPS.

Figure 2

Effect of AS101 on degradation and phosphorylation of IKBα in RAW264.7 macrophages. (A,D) Cells were treated with LPS in the absence or in the presence of AS101 for 1 h (A) and 4 h (D). Total cellular proteins were prepared and immunoblotted using anti-pIKBα^ser32/36, anti-IKBα and anti-actin. Bar graphs represent the quantitative densitometric value of the expressed protein vs actin: pIKBα^ser32/36 1 h (B) and 4 h (E), IKBα- 1 h (C) and 4 h (F). *p < 0.05 vs LPS. Data shown are representative of three different experiments.

Figure 3

Effect of AS101 on NFkB translocalization. LPS-stimulated RAW264.7 cells (1 × 10⁶/ml) were treated with AS101 for 1 h (A) and 4 h (C). Cytosolic and nuclear extracts were immunoblotted using anti-p65 of NFκB and anti-actin. Extracts were immunoblotted using RCC1 indicating nuclear purity of the fractions (not shown). Bar graphs represent the quantitative densitometric value of the expressed protein vs actin: p65 1 h (B) and 4 h (D). *p < 0.05 vs LPS. Data shown are representative of three different experiments.

Figure 4

Inhibition of p50 DNA-binding in iNOS promoter by AS101 treatment. (A) ChIP analysis of LPS-stimulated RAW264.7 cells (1 × 10⁶/ml) treated with AS101 for 1 h. Bar graph represent the quantitative densitometric value of the p50 DNA-binding in the iNOS promoter vs input (B). *p < 0.05 vs LPS. Data are representative of three different experiments.