Korean J Physiol Pharmacol.  2016 May;20(3):229-236. 10.4196/kjpp.2016.20.3.229.

Resveratrol attenuates 4-hydroxy-2-hexenal-induced oxidative stress in mouse cortical collecting duct cells

• Bae EH
• Joo SY
• Ma SK
• Lee J
• Kim SW

• KMID: 2285594
• DOI: http://doi.org/10.4196/kjpp.2016.20.3.229

Abstract

Resveratrol (RSV) may provide numerous protective eff ects against chronic inflammatory diseases. Due to local hypoxia and hypertonicity, the renal medulla is subject to extreme oxidative stress, and aldehyde products formed during lipid peroxidation, such as 4-hydroxy-2-hexenal (HHE), might be responsible for tubular injury. This study aimed at investigating the eff ects of RSV on renal and its signaling mechanisms. While HHE treatment resulted in decreased expression of Sirt1, AQP2, and nuclear factor erythroid 2-related factor 2 (Nrf2), mouse cortical collecting duct cells (M1) cells treated with HHE exhibited increased activation of p38 MAPK, extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and increased expression of NOX4, p47(phox), Kelch ECH associating protein 1 (Keap1) and COX2. HHE treatment also induced NF-κB activation by promoting IκB-α degradation. Meanwhile, the observed increases in nuclear NF-κB, NOX4, p47(phox), and COX2 expression were attenuated by treatment with Bay 117082, N-acetyl-l-cysteine (NAC), or RSV. Our findings indicate that RSV inhibits the expression of inflammatory proteins and the production of reactive oxygen species in M1 cells by inhibiting NF-κB activation.

Keyword

4-hydroxy-2-hexenal; Collecting duct; Oxidative stress; Resveratrol; Sirtuin 1

MeSH Terms

Acetylcysteine
Animals
Anoxia
Bays
JNK Mitogen-Activated Protein Kinases
Lipid Peroxidation
Mice*
Oxidative Stress*
p38 Mitogen-Activated Protein Kinases
Phosphotransferases
Reactive Oxygen Species
Sirtuin 1
Acetylcysteine
JNK Mitogen-Activated Protein Kinases
Phosphotransferases
Reactive Oxygen Species
Sirtuin 1
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Fig. 1 Effects of 4-hydroxy-2-hexenal (HHE) on the protein expression of Sirt1 and aquaporin 2 (AQP2) in mouse cortical collecting duct cells (M1 cells).Cells were treated with HHE (10 µM). After 1, 3, 6, and 8 hr, the protein expression of Sirt1 and AQP2 was decreased. Results are presented as means±SEM of three individual experiments. *p<0.05 vs. control.

• 

  Fig. 2 Effects of HHE on the protein expression of NOX4, p47phox, iNOS and COX2.M1 cells were treated with HHE (10 µM). After 1, 3, 6, and 8 hr, protein expression of NOX4, p47phox, iNOS and COX2 were increased (A). Effects of N-acetyl-l-cysteine (NAC) on expression of NF-κB p65 subunit, Sirt 1, NOX4, COX2 and AQP2. The protein expression of Sirt1, NOX4, COX2 and AQP2 was attenuated by 1 hr pre-treated NAC (10 mM) (B). Results are presented as means±SEM of three individual experiments. *p<0.05 vs. control. #p<0.05 vs. HHE.

• 

  Fig. 3 Formation of ROS detected using the ROS-sensitive fluorescent dye DCF.HHE caused increase of DCF fluorescence after incubation for 8 h, which was attenuated by N-acetyl-L-cysteine (NAC, 10 mM) 1 h pre-treatment. *p<0.05 versus control. #p<0.05 vs. HHE.

• 

  Fig. 4 Expression of NF-κB p65 subunit levels in nuclear extracts of M1 cells incubated with HHE (10 µM) (A). The expression started to increase 30 min after HHE incubation. Cytoplasmic total IκBα expression began to decrease at 30 min, and kept decreased at 1 and 2 hrs. Effects of NF-κB inhibitor (Bay, 10 µM) on expression of NF-κB p65 subunit, Sirt1, NOX4 and COX2 (B). The HHE-induced increased expression of NF-κB p65 subunit in nuclear extracts of M1 cells was also attenuated by 1 hr pre-treated Bay 117082 (10 µM), NF-kB inhibitor. Results are presented as means±SEM of three individual experiments. *p<0.05 vs. control. #p<0.05 vs. HHE.

• 

  Fig. 5 Effects of resveratrol (RSV) in HHE-treated M1 cells.The protein expression of NF-κB p65 subunit was attenuated and cytoplasmic total IκBα expression was upregulated by resveratrol treatment in M1-cells (A). The protein expression of Sirt1 and aquaporin 2 (AQP2) was decreased by HHE, while NOX4 and COX2 protein expressions were increased. These changes were counteracted by RSV (25 µM) 1 h pre-treatment (B). The protein expression of the phosphorylation of extracellular signal-regulated kinase (pERK 1/2), c-Jun N-terminal kinase (pJNK) and pP38 were increased by HHE treatment, which was attenuated by RSV (25 µM) 1 h pre-treatment (C). The protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2) was increased and decreased in Kelch ECH associating protein 1 (Keap1) with HHE (10 µM) treatment, which was counter-regulated by 1 h RSV pre-treatment (D). N*p<0.05 vs. control, #p<0.05 vs. HHE.

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