Inflammation induced by increased frequency of intermittent hypoxia is attenuated by tempol administration

Abstract

The levels of serum inflammatory cytokines and the activation of nuclear factor kappa B (NF-κB) and hypoxia inducible factor-1α (HIF-1α) in heart tissues in response to different frequencies of intermittent hypoxia (IH) and the antioxidant tempol were evaluated. Wistar rats (64 males, 200-220 g) were randomly divided into 6 experimental groups and 2 control groups. Four groups were exposed to IH 10, 20, 30, or 40 times/h. The other 2 experimental groups were challenged with IH (30 times/h) plus tempol, either beginning on day 0 (IH30T0) or on day 29 (IH30T29). After 6 weeks of challenge, serum levels of tumor necrosis factor (TNF)-α, intracellular adhesion molecule (ICAM)-1, and interleukin-10 were measured, and western blot analysis was used to detect NF-κB p65 and HIF-1α in myocardial tissues. Serum levels of TNF-α and ICAM-1 and myocardial expression of NF-κB p65 and HIF-1α were all significantly higher in IH rats than in controls (P<0.001). Increased IH frequency resulted in more significant changes. Administration of tempol in IH rats significantly reduced levels of TNF-α, ICAM-1, NF-κB and HIF-1α compared with the non-tempol-treated group (F=16.936, P<0.001). IH induced an inflammatory response in a frequency-dependent manner. Additionally, HIF-1α and NF-κB were increased following IH administration. Importantly, tempol treatment attenuated this effect.

Figure 1. Serum levels of circulating cytokines in groups exposed to different intermittent hypoxia (IH) frequencies. Serum levels of TNF-α (A), ICAM-1 (B), and IL-10 (C) were measured in animals from each group (n=8 each). Data are reported as means±SD. Rats received compressed air (SC) or IH at a frequency of 10 (IH10), 20 (IH20), 30 (IH30), and 40 (IH40) times/h. TNF-α: tumor necrosis factor alpha; ICAM-1, intracellular adhesion molecule-1; IL-10, interleukin-10. *P<0.05 vs SC; #P<0.05 vs IH10; ^$P<0.05 vs IH20 (one-way ANOVA and post hoc Tukey’s test).

Figure 2. Changes in myocardial expression of phospho-nuclear factor kappa B (NF-κB) and hypoxia inducible factor-1α (HIF-1α) with increasing intermittent hypoxia (IH) frequency. A, Western blot analysis of phosphorylated NF-κB p65 in rats challenged with increasing frequencies of IH. GADPH was used as a loading control. B, The intensity of phosphorylated NF-κB p65 was normalized to that of GADPH, which was then plotted as means±SD. C, Western blot analysis of HIF-1α in rats challenged with increasing frequencies of IH. GADPH was used as a loading control. D, The intensity of HIF-1α was normalized to that of GADPH, which was then plotted as means±SD. *P<0.05 vs SC; ^#P<0.05 vs IH10; ^$P<0.05 vs IH20 (one-way ANOVA and post hoc Tukey’s test).

Figure 3. Changes in serum levels of circulating cytokines with tempol treatment. Animals received intermittent hypoxia (IH) 30 times/h as in the IH30 group. In addition, 1 mL/kg of 10% tempol was administered each time before IH exposure starting from the first day of treatment (IH30T0 group) or starting from day 29 (IH30T29 group) of the procedure. Serum levels of tumor necrosis factor (TNF)-α (A), intracellular adhesion molecule (ICAM)-1 (B), and interleukin (IL)-10 (C) were measured in the animals (n=8 per group) and compared with animals from the compressed air (SC) group. Data are reported as means±SD. D, Myocardial nuclear NF-κB phosphorylation in different IH30 groups with or without tempol intervention were determined. The quantitative data of D are reported as means±SD. E, Myocardial hypoxia inducible factor-1α (HIF-1α) protein levels in the IH30 groups with or without tempol intervention were determined. F, Representative Western blot for NF-κB phosphorylation. G, Representative Western blot for HIF-1α. *P<0.05 vs IH30; ^#P<0.05 vs IH30T29; ^$P<0.05 vs IH30T0 (one-way ANOVA and post hoc Tukey’s test).