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. 2013 Sep;19(9):675-81.
doi: 10.1111/cns.12131. Epub 2013 Jun 3.

Resveratrol protects vascular endothelial cells from high glucose-induced apoptosis through inhibition of NADPH oxidase activation-driven oxidative stress

Feng Chen  1 , Li-Hua Qian, Bo Deng, Zhi-Min Liu, Ying Zhao, Ying-Ying Le
Affiliations

Resveratrol protects vascular endothelial cells from high glucose-induced apoptosis through inhibition of NADPH oxidase activation-driven oxidative stress

Feng Chen et al. CNS Neurosci Ther. 2013 Sep.

Abstract

Introduction: Hyperglycemia-induced oxidative stress has been implicated in diabetic vascular complications in which NADPH oxidase is a major source of reactive oxygen species (ROS) generation. Resveratrol is a naturally occurring polyphenol, which has vasoprotective effects in diabetic animal models and inhibits high glucose (HG)-induced oxidative stress in endothelial cells.

Aims: We aimed to examine whether HG-induced NADPH oxidase activation and ROS production contribute to glucotoxicity to endothelial cells and the effect of resveratrol on glucotoxicity.

Results: Using a murine brain microvascular endothelial cell line bEnd3, we found that NADPH oxidase inhibitor (apocynin) and resveratrol both inhibited HG-induced endothelial cell apoptosis. HG-induced elevation of NADPH oxidase activity and production of ROS were inhibited by apocynin, suggesting that HG induces endothelial cell apoptosis through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunit Nox1 but not Nox2, Nox4, and p22(phox) expression through NF-κB activation, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced endothelial cell apoptosis through inhibiting HG-induced NF-κB activation, NADPH oxidase activity elevation, and ROS production.

Conclusions: HG induces endothelial cell apoptosis through NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide new potential therapeutic targets against brain vascular complications of diabetes.

Keywords: Apoptosis; Hyperglycemia; NADPH oxidase; Resveratrol; Vascular endothelial cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Resveratrol inhibits high glucose (HG)–induced vascular endothelial apoptosis. bEnd3 cells were cultured in medium containing normal glucose (NG, 5.6 mM) or HG (HG, 25 mM) in the presence or absence of different concentrations of apocynin (Apo) or resveratrol (RV) for 24 h, cell viability were examined with MTT assay (A), and apoptotic cells were observed under fluorescence microscope after staining with Hoechst 33258 (B). Results represent the mean ± SD of three independent experiments. *P < 0.05 compared with cells cultured in medium containing NG alone; # P < 0.05, ## P < 0.01 compared with cells cultured in medium containing HG alone. The representative images of Hoechst 33258 staining are shown in the left part of B.
Figure 2
Figure 2
Resveratrol inhibits high glucose (HG)–induced NADPH oxidase activation in vascular endothelial cells. bEnd3 cells were cultured in medium containing 5.6 mM glucose (NG) or 25 mM glucose (HG) for different times (A) or for 12 h in the presence or absence of 200 nM apocynin or 25 μM resveratrol (B), and NADPH oxidase activity was measured by lucigenin chemiluminescence method. Results represent the mean ± SD of three independent experiments.*P < 0.05 compared with cells cultured in NG alone. # P < 0.05 compared with cells cultured in HG alone.
Figure 3
Figure 3
Resveratrol inhibits high glucose (HG)–induced reactive oxygen species (ROS) productions. bEnd3 cells were cultured in medium containing 5.6 mM glucose (NG) or 25 mM glucose (HG) for different times (A) or for 12 h in the presence or absence of different concentrations of apocynin (B) or resveratrol (C), and ROS production was examined with H2 DCF‐DA. Results represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 compared with cells exposed to NG alone. # P < 0.05, ## P < 0.01 compared with cells exposed to HG alone.
Figure 4
Figure 4
High glucose (HG) enhances NADPH oxidase activity in endothelial cells through upregulation of Nox1 expression. (A–B) bEnd3 cells were cultured in medium containing 5.6 mM glucose (NG) or 25 mM glucose (HG) for different periods of time, the mRNA levels of Nox1, Nox2, Nox4, and p22phox were examined by RT‐PCR (A), and protein levels of Nox1 were examined by Western blot (B). (C–D) bEnd3 cells transfected with control siRNA or Nox1 siRNA for 36 h were incubated with 5.6 mM glucose (NG) or 25 mM glucose (HG) for additional 36 h and then examined for Nox1 expression by Western blot (C) and NADPH oxidase activity (D). All results represent the mean ± SD of three independent experiments. The representative images of RT‐PCR and Western blot are shown in the upper part of A, B, and C, respectively. A–B: *P < 0.05, **P < 0.01 compared with cells cultured with NG. C–D: **P < 0.01 compared with control siRNA transfected cells cultured with NG. # P < 0.05, ## P < 0.01 compared with control siRNA transfected cells cultured with HG.
Figure 5
Figure 5
Resveratrol attenuates Nox1 upregulation in endothelial cells by high glucose (HG) through inhibition of NF‐κB activation. (A) bEnd3 cells pretreated with or without 25 μM resveratrol (RV) for 2 h were cultured in medium containing 5.6 mM glucose (NG) or 25 mM glucose (HG) for 36 h, and then, Nox1 mRNA level was examined by RT‐PCR. (B) bEnd3 cells pretreated with 25 μM resveratrol (RV) or 50 μM sulfasalazine (Sul) for 1 h were incubated with medium containing 5.6 mM glucose (NG) or 25 mM glucose (HG) for 36 h and then examined for Nox1 expression by Western blot. (C) bEnd3 cells transfected with dominant‐negative IκB (DN‐IκB) or control vector flag‐zeo for 36 h were treated with 5.6 mM glucose (NG) or 25 mM glucose (HG) for another 36 h and then examined for Nox1 expression by Western blot. (D–E) bEnd3 cells were exposed to 5.6 mM glucose (NG) or 25 mM glucose (HG) in the presence or absence of 50 μM sulfasalazine, reactive oxygen species production was examined with H2DCF‐DA after 12 h (D), and cell viability was examined with MTT assay after 24 h (E). (F) bEnd3 cells pretreated with or without 25 μM resveratrol for 1 h were incubated with medium containing 5.6 mM glucose (NG) or 25 mM glucose (HG) for the indicated times and then examined for IκBα phosphorylation by Western blot. All results represent the mean ± SD of three independent experiments. The representative images of RT‐PCR and Western blot are shown in the upper part of A–C and F. A, B, D, E: **P < 0.01 compared with cells cultured with NG alone; # P < 0.05, ## P < 0.01 compared with cells cultured with HG alone. C: *P < 0.05 compared with control plasmid flag‐zeo‐transfected cells cultured with NG; ## P < 0.01 compared with flag‐zeo‐transfected cells cultured with HG. F: *P < 0.05, **P < 0.01 compared with cells cultured with NG. # P < 0.05, ## P < 0.01 compared with cells treated with HG alone for corresponding time.
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