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Atorvastatin attenuates homocysteine-induced apoptosis in human umbilical vein endothelial cells via inhibiting NADPH oxidase-related oxidative stress-triggered p38MAPK signaling.

Bao XM, Wu CF, Lu GP - Acta Pharmacol. Sin. (2009)

Bottom Line: Atorvastatin inhibited endothelial cell apoptosis induced by 1 mmol/L Hcy in a dose-dependent manner and the maximal inhibitory effect was reached at 100 micromol/L.Furthermore, atorvastatin inhibited Hcy-induced phosphorylation of p38 MAPK (1.7+/-0.1 vs 2.22+/-0.25, P<0.05), similar effects occurred with DPI, NAC and SB203580.Atorvastatin may inhibit Hcy-induced ROS accumulation and endothelium cell apoptosis through an NADPH oxidase and/or p38MAPK-dependent mechanisms, all of which may contribute to atorvastatin-induced beneficial effect on endothelial function.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

ABSTRACT

Aim: To examine the effect of atorvastatin on homocysteine (Hcy)-induced reactive oxygen species (ROS) production and apoptosis in human umbilical vein endothelial cells (HUVECs).

Methods: HUVECs were cultured with Hcy (0.1-5 mmol/L) in the presence or absence of atorvastatin (1-100 micromol//L) or various stress signaling inhibitors, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI, 10 micromol/L), the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 (10 micromol/L) and antioxidants N-acetyl cysteine (NAC, 1 mmol/L). Cell apoptosis was evaluated by Annexin V/propidium iodide staining and flow cytometry. ROS were detected by 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFH-DA). NADPH oxidases were evaluated with lucigenin-enhanced chemiluminescence. Hcy-induced expression of p38MAPK protein was measured by Western blotting analysis.

Results: Atorvastatin inhibited endothelial cell apoptosis induced by 1 mmol/L Hcy in a dose-dependent manner and the maximal inhibitory effect was reached at 100 micromol/L. Atorvastatin (10 micromol/L) significantly suppressed Hcy (1 mmol/L for 30 min) induced ROS accumulation (3.17+/-0.33 vs 4.34+/-0.31, P<0.05). Atorvastatin (10 micromol/L) also antagonized Hcy (1 mmol/L for 30 min) induced activation of NADPH oxidase (2.57+/-0.49 vs 3.33+/-0.6, P<0.05). Furthermore, atorvastatin inhibited Hcy-induced phosphorylation of p38 MAPK (1.7+/-0.1 vs 2.22+/-0.25, P<0.05), similar effects occurred with DPI, NAC and SB203580.

Conclusion: Atorvastatin may inhibit Hcy-induced ROS accumulation and endothelium cell apoptosis through an NADPH oxidase and/or p38MAPK-dependent mechanisms, all of which may contribute to atorvastatin-induced beneficial effect on endothelial function.

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Atorvastatin suppressed Hcy-induced activation of NADPH oxidase. (A) Hcy (1 mmol/L) increased NADPH oxidase activity in a time-dependent manner. (B) Pretreatment of cells with atorvastatin 30 min prior to 1 mmol/L Hcy abolished NAD(P)H oxidase activation at concentrations of 10 μmol/L and 100 μmol/L. NAD(P)H oxidase activity was assessed as lucigenin-enhanced chemiluminescence using NADPH as a substrate. Results are the means±SD from three independent experiments. cP<0.01 vs untreated cells. eP<0.05, fP<0.01 vs only 1 mmol/L Hcy-treated cells. Ator: atorvastatin; Hcy, homocysteine.
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fig2: Atorvastatin suppressed Hcy-induced activation of NADPH oxidase. (A) Hcy (1 mmol/L) increased NADPH oxidase activity in a time-dependent manner. (B) Pretreatment of cells with atorvastatin 30 min prior to 1 mmol/L Hcy abolished NAD(P)H oxidase activation at concentrations of 10 μmol/L and 100 μmol/L. NAD(P)H oxidase activity was assessed as lucigenin-enhanced chemiluminescence using NADPH as a substrate. Results are the means±SD from three independent experiments. cP<0.01 vs untreated cells. eP<0.05, fP<0.01 vs only 1 mmol/L Hcy-treated cells. Ator: atorvastatin; Hcy, homocysteine.

Mentions: To explore the underlying mechanism by which atorvastatin suppressed intracellular oxidative stress, we measured NADPH oxidase activity with lucigenin-enhanced chemiluminescence. Stimulation with 1 mmol/L Hcy led to a time-dependent increase of NADPH oxidase activity to 275%±47% at 20 min and 333%±60% at 30 min (Figure 2A). As demonstrated in Figure 2B, pretreatment of the cells with atorvastatin (10, 100 μmol/L) reduced Hcy-dependent NADPH oxidase activation.


Atorvastatin attenuates homocysteine-induced apoptosis in human umbilical vein endothelial cells via inhibiting NADPH oxidase-related oxidative stress-triggered p38MAPK signaling.

Bao XM, Wu CF, Lu GP - Acta Pharmacol. Sin. (2009)

Atorvastatin suppressed Hcy-induced activation of NADPH oxidase. (A) Hcy (1 mmol/L) increased NADPH oxidase activity in a time-dependent manner. (B) Pretreatment of cells with atorvastatin 30 min prior to 1 mmol/L Hcy abolished NAD(P)H oxidase activation at concentrations of 10 μmol/L and 100 μmol/L. NAD(P)H oxidase activity was assessed as lucigenin-enhanced chemiluminescence using NADPH as a substrate. Results are the means±SD from three independent experiments. cP<0.01 vs untreated cells. eP<0.05, fP<0.01 vs only 1 mmol/L Hcy-treated cells. Ator: atorvastatin; Hcy, homocysteine.
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Related In: Results  -  Collection

Show All Figures
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fig2: Atorvastatin suppressed Hcy-induced activation of NADPH oxidase. (A) Hcy (1 mmol/L) increased NADPH oxidase activity in a time-dependent manner. (B) Pretreatment of cells with atorvastatin 30 min prior to 1 mmol/L Hcy abolished NAD(P)H oxidase activation at concentrations of 10 μmol/L and 100 μmol/L. NAD(P)H oxidase activity was assessed as lucigenin-enhanced chemiluminescence using NADPH as a substrate. Results are the means±SD from three independent experiments. cP<0.01 vs untreated cells. eP<0.05, fP<0.01 vs only 1 mmol/L Hcy-treated cells. Ator: atorvastatin; Hcy, homocysteine.
Mentions: To explore the underlying mechanism by which atorvastatin suppressed intracellular oxidative stress, we measured NADPH oxidase activity with lucigenin-enhanced chemiluminescence. Stimulation with 1 mmol/L Hcy led to a time-dependent increase of NADPH oxidase activity to 275%±47% at 20 min and 333%±60% at 30 min (Figure 2A). As demonstrated in Figure 2B, pretreatment of the cells with atorvastatin (10, 100 μmol/L) reduced Hcy-dependent NADPH oxidase activation.

Bottom Line: Atorvastatin inhibited endothelial cell apoptosis induced by 1 mmol/L Hcy in a dose-dependent manner and the maximal inhibitory effect was reached at 100 micromol/L.Furthermore, atorvastatin inhibited Hcy-induced phosphorylation of p38 MAPK (1.7+/-0.1 vs 2.22+/-0.25, P<0.05), similar effects occurred with DPI, NAC and SB203580.Atorvastatin may inhibit Hcy-induced ROS accumulation and endothelium cell apoptosis through an NADPH oxidase and/or p38MAPK-dependent mechanisms, all of which may contribute to atorvastatin-induced beneficial effect on endothelial function.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

ABSTRACT

Aim: To examine the effect of atorvastatin on homocysteine (Hcy)-induced reactive oxygen species (ROS) production and apoptosis in human umbilical vein endothelial cells (HUVECs).

Methods: HUVECs were cultured with Hcy (0.1-5 mmol/L) in the presence or absence of atorvastatin (1-100 micromol//L) or various stress signaling inhibitors, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI, 10 micromol/L), the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 (10 micromol/L) and antioxidants N-acetyl cysteine (NAC, 1 mmol/L). Cell apoptosis was evaluated by Annexin V/propidium iodide staining and flow cytometry. ROS were detected by 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFH-DA). NADPH oxidases were evaluated with lucigenin-enhanced chemiluminescence. Hcy-induced expression of p38MAPK protein was measured by Western blotting analysis.

Results: Atorvastatin inhibited endothelial cell apoptosis induced by 1 mmol/L Hcy in a dose-dependent manner and the maximal inhibitory effect was reached at 100 micromol/L. Atorvastatin (10 micromol/L) significantly suppressed Hcy (1 mmol/L for 30 min) induced ROS accumulation (3.17+/-0.33 vs 4.34+/-0.31, P<0.05). Atorvastatin (10 micromol/L) also antagonized Hcy (1 mmol/L for 30 min) induced activation of NADPH oxidase (2.57+/-0.49 vs 3.33+/-0.6, P<0.05). Furthermore, atorvastatin inhibited Hcy-induced phosphorylation of p38 MAPK (1.7+/-0.1 vs 2.22+/-0.25, P<0.05), similar effects occurred with DPI, NAC and SB203580.

Conclusion: Atorvastatin may inhibit Hcy-induced ROS accumulation and endothelium cell apoptosis through an NADPH oxidase and/or p38MAPK-dependent mechanisms, all of which may contribute to atorvastatin-induced beneficial effect on endothelial function.

Show MeSH
Related in: MedlinePlus