Intensive Running Enhances NF-κB Activity in the Mice Liver and the Intervention Effects of Quercetin

Abstract

Background: Emerging evidence has supported that intensive exercise induces weakened performance and immune and metabolic disorders. We systematically evaluated the effects of quercetin against hepatic inflammatory damage caused by repeated intensive exercise and explored the potential mechanism.

Methods: Male BALB/c mice were administered quercetin (100 mg/kg BW) for four weeks, and performed a treadmill running protocol of 28 m/min, 5° slope, 90 min/day concurrently for the last seven days.

Results: Quercetin administration reduced the leakage of aspartic acid and alanine aminotransferase and improved ultrastructural abnormalities such as swelling, and degeneration caused by high-intensity running in mice. Quercetin significantly decreased the hepatic and plasmatic levels of inflammatory cytokines IL-1β, IL-6, TNF-α, inducible nitric oxide synthase, cyclooxygenase-2 and intercellular adhesion molecule-1-provoked by over-exercise. Furthermore, diminished activation and nuclear translocation of NF-κB were found after quercetin treatment through inhibiting IKKα and Iκbα phosphorylation of intensive running mice.

Conclusion: Quercetin offers protection for mouse livers against intensive sports-induced inflammatory injury, and the suppression of the NF-κB signal transduction pathway may play a role in its anti-inflammatory effects. Our findings broaden our understanding of natural phytochemicals as a promising strategy to prevent excessive exercise damage.

Keywords: inflammation; intensive exercise; liver damage; quercetin.

Figure 1

Effect of intensive running with or without quercetin administration on serum aspartate transaminase (AST) (A) and alanine transaminase (ALT) levels (B). BALB/c mice were pretreated or not with quercetin for 4 weeks and subsequently exposed to intensive exercise for successive 7 days. Results presented as mean ± SD (n = 8). Ct—rested control; Ex—intensive exercise; Ex + Qu—Ex and quercetin; Qu—rested + quercetin. ** p < 0.01 vs. Ct; ## p < 0.01 vs. Ex.

Figure 2

Effect of quercetin intervention on liver morphology disturbed by intensive exercise. Liver samples processed by hematoxylin–eosin were observed by Olympus BX50 light microscope with HMIAS-2000 medical imaging system (×200) (A). The ultrastructure of the liver was shown in a transmission electron microscope (TEM) images (B). Ct—rested control; Ex—intensive exercise; Ex + Qu—Ex and quercetin; Qu—rested + quercetin.

Figure 3

Serum and hepatic inflammatory cytokines levels of intensive exercise and/or quercetin prophylaxis. (A) Serum inflammatory cytokines were quantified by ELISA; results represented as mean ± SD (n = 8); (B) liver TNF-α, IL-1β, IL-6 and IL-10 levels evaluated by qRT-PCR; values presented as multiples of control following normalization by β-actin and mean ± SD (n = 8). Ct—rested control; Ex—intensive exercise; Ex + Qu—Ex + quercetin; Qu—rested + quercetin. * p < 0.05, ** p < 0.01 vs. Ct; # p < 0.05, ## p < 0.01 vs. Ex.

Figure 4

Quercetin decreased the expression of iNOS, COX-2 and ICAM-1 in mice liver subjected to intensive exercise. (A) mRNA expression determined by real-time PCR following normalization to β-actin; data expressed as fold-change compared to the control group (mean ± SD, n = 8); (B) protein expression determined by immunohistochemistry and observed by Olympus BX50 light microscope with HMIAS-2000 medical imaging system (×200). Ct—rested control; Ex—intensive exercise; Ex + Qu—Ex + quercetin; Qu—rested + quercetin. * p < 0.05, ** p < 0.01 vs. Ct; # p < 0.05, ## p < 0.01 vs. Ex.

Figure 5

Phosphorylated protein level of IKKα (A), IκBα and P-IκBα (B) in mice liver exposed to intensive running with or without quercetin pretreatment. Figures show representative western blot (upper panel) and densitometric analysis (lower panel). Data presented as fold-change compared to the control group. Results represent mean ± SD (n = 8). Ct—rested control; Ex—intensive exercise; Ex + Qu—Ex + quercetin; Qu—rested + quercetin. * p < 0.05, ** p < 0.01 vs. Ct; # p < 0.05, ## p < 0.01 vs. Ex.

Figure 6

Effect of quercetin on the nuclear expression level and activity of NF-κB in mice liver following intensive running. (A) Liver sections were probed with NF-κB (green) and Iκbα (red) antibodies, while DAPI was used to stain the nuclei (blue); (B) activity of NF-κB-DNA binding was quantified by densitometry. Specific binding was verified by the addition of unlabeled (cold) oligonucleotide (competitor, Ct-) or labeled oligonucleotide mutate (noncompetitor, Ct+). Representative EMSA (upper panel) and densitometric analysis (lower panel). Data presented as fold-change in contrast to the control group. Values presented as mean ± SD (n = 8). Ct—rested control; Ex—intensive exercise; Ex + Qu—Ex + quercetin; Qu—rested + quercetin. ** p < 0.01 vs. Ct; ## p < 0.01 vs. Ex.