Resveratrol protects vascular smooth muscle cells against high glucose-induced oxidative stress and cell proliferation in vitro

Abstract

Background: Resveratrol exhibits beneficial effects against numerous degenerative diseases at different stages of pathogenesis. This study investigated potential mechanisms and resveratrol effects on high glucose (HG)-induced oxidative stress (30 mM D-glucose, 30 min) and cell proliferation (30 mM D-glucose, 24 h) in vascular smooth muscle cells (VSMCs).

Material/methods: Intracellular reactive oxygen species (ROS) generation was detected by 2',7'-dichlorofluorescein diacetate (DCFH-DA). Total antioxidant capacity (TAC), malonyldialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) were measured to evaluate oxidative stress. VSMC proliferation was measured by CCK-8 assays and through propidium iodide-based cell cycle analysis. Expression of NAD(P)H oxidase, proliferation proteins, and cell signalling were assessed by immunoblot analysis.

Results: Co-treatment of primary cultures of VSMCs with 1-100 μM resveratrol decreased HG-induced ROS overproduction (P<0.05). Resveratrol also abolished HG-induced phosphorylation of oxidase subunit p47 phox and reduced HG-induced cyclin D1, cyclin E, and PCNA expression in a concentration-dependent manner. Furthermore, resveratrol (10 μM) attenuated HG-induced phosphorylation of Akt, p38 mitogen-activated protein kinase (MAPK), ERK 1/2, and JNK1/2 without affecting total levels. HG stimulation enhanced downstream IκB-α phosphorylation and NF-κB activity, and resveratrol repressed these effects.

Conclusions: Resveratrol inhibits HG-induced oxidative stress and VSMC proliferation by suppressing ROS generation, NADPH oxidase, Akt phosphorylation, p38 MAPK/JNK/ERK phosphorylation, and IκB-α and NF-κB activities.

Figure 1

Parameters of viability in the VSMCs incubated with different concentrations of resveratrol. (A) Cells were incubated for 24 h with resveratrol from 1 to 500 μM in the incubation medium, as described in the Materials and Methods section, and viability was assessed by CCK-8 assay. (B) Cytotoxic effects of resveratrol on VSMCs at various concentrations. Values are expressed as means ±S.D. of triplicate experiments. R: resveratrol. * P<0.05 indicates significant differences from the NG (5 mM) group.

Figure 2

Inhibitory effect of resveratrol on HG-induced ROS overproduction and p47 phox expression in VSMCs. Cells were pretreated with resveratrol for 24 h prior to HG exposure for 30 min. ROS were detected using a 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probe. (A) Representative images of ROS fluorescence in different groups: NG (5 mM) group, HG (30 mM) group, and groups of VSMCs treated with different concentrations of resveratrol (1, 10, 50, and 100 mM) prior to HG exposure. (B) Fluorescence intensity measured by spectrofluorometer. (C) Blotting data for phospho-p47 phox and β-actin protein expressions from the VSMCs. Values are expressed as means ±S.D. from 3 independent experiments. Bar: 200 μm. ^# P<0.05 indicates significant difference from the HG (30 mM) group. * P<0.01 indicates significant difference from the HG (30 mM) group.

Figure 3

FCM results of intracellular ROS production and effects of resveratrol on other redox parameters after HG stimulation. ROS were detected using a 2′,7′-dichlorofluorescin diacetate (DCFH-DA) probe by FCM. (A) Representative images of FCM in VSMCs treated with or without different concentrations of resveratrol (1, 10, and 100 mM) prior to HG exposure. (B) Quantitative analysis of the ratio of DCFH-DA probe positive cells. (C) SOD, (D) MDA, (E) GSH and (F) TAC were assessed as described in the Material and Methods section. R: resveratrol. * P<0.05 indicates significant difference from the HG (30 mM) group. ^# P<0.01 indicates significant difference from the HG (30 mM) group.

Figure 4

Effect of resveratrol on HG-induced VSMC proliferation and expression of proliferation-related proteins. (A) Representative histograms showing cell cycle distribution upon HG treatment (30 mM, 24 h) in the presence or absence of resveratrol (10 μM). (B) Bar graph summarizing cell cycle data. (C) Representative immunoblot detecting levels of expression of cyclin D1, PCNA, and cyclin E in HG-treated VSMCs in the presence or absence of resveratrol (10 μM). (D–F) Quantified band density for cyclin D1, PCNA, and cyclin E. Values are expressed as means ±S.D. from 3 independent experiments. * P<0.01 indicates significant difference from the HG (30 mM) group.

Figure 5

Western blot data showing the effects of 10 μM resveratrol on phosphorylation of Akt (A), p38 MAPK (B), ERK 1/2 (C), and JNK 1/2 (D) in 30 mM HG-exposed VSMCs. Total cell protein extracts were blotted with a primary antibody against phosphorylated Akt, p38 MAPK, ERK 1/2, and phosphorylated JNK 1/2. β-actin protein was used as an internal control. Values are expressed as means ±S.D. from 3 independent experiments. * P<0.01 indicates significant differences from the HG (30 mM) group.

Figure 6

Inhibition of phosphorylation of IκB-α (A) and NF-κB (B) by resveratrol in HG-exposed VSMCs. Western blot analysis was performed with a primary antibody against phosphorylated IκB-α and NF-κB. Values are expressed as means ±S.D. from 3 independent experiments. * P<0.01 indicates significant difference from the HG (30 mM) group.

Figure 7

Sketch of the signal cascade involved in HG-induced intracellular oxidative stress and VSMCs proliferation, as well as the effects of resveratrol. Elevated glucose concentration initiates the downstream signalling cascade. This subsequently promotes ROS overproduction; NAD(P)H oxidative stress; phosphorylation of Akt, p38 MAPK, ERK 1/2, and JNK 1/2; and NF-κB nuclear translocation. On the other hand, ROS also can phosphorylate Akt, p38 MAPK, ERK 1/2, and JNK 1/2 and initiate NF-κB nuclear translocation. The activated NF-κB then interferes with the gene expression of some target genes such as cyclin D1, PCNA, and cyclin E, directly or indirectly. Resveratrol, either by binding directly to the kinases or upon entering the cytoplasm, inhibits ROS generation and downstream signalling. The symbol “⊥” indicates inhibition or blockade.