J Lipid Atheroscler.  2020 Jan;9(1):184-194. 10.12997/jla.2020.9.1.184.

Curcumin Attenuates Acrolein-induced COX-2 Expression and Prostaglandin Production in Human Umbilical Vein Endothelial Cells

Affiliations
  • 1Department of Microbiology, College of Medicine, Kyung Hee University, Seoul, Korea. yongseek@khu.ac.kr
  • 2Department of Biomedical Science, Graduate School, Kyung Hee University, Seoul, Korea.

Abstract


OBJECTIVE
Inflammation is crucial to limiting vascular disease. Previously we reported that acrolein, a known toxin in tobacco smoke, might play an important role in the progression of atherosclerosis via an inflammatory response involving cyclooxygenase-2 (COX-2) and prostaglandin production in human umbilical vein endothelial cells (HUVECs). Curcumin has been known to improve vascular function and have anti-inflammatory properties. In this study, we investigated whether curcumin prevents the induction of inflammatory response caused by acrolein.
METHODS
Anti-inflammatory effects of curcumin were examined in acrolein-stimulated HUVECs. Induction of proteins, mRNA, prostaglandin and reactive oxygen species (ROS) were measured using immunoblot analysis, real-time reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay and flow cytometry, respectively.
RESULTS
Curcumin attenuates inflammatory response via inhibition of COX-2 expression and prostaglandin production in acrolein-induced human endothelial cells. This inhibition by curcumin results in the abolition of phosphorylation of protein kinase C, p38 mitogen-activated protein kinase, and cAMP response element-binding protein. Furthermore, curcumin suppresses the production of ROS and endoplasmic reticulum stress via phosphorylation of eukaryotic initiation factor-2α caused by acrolein.
CONCLUSION
These results suggest that curcumin might be a useful agent against endothelial dysfunction caused by acrolein-induced inflammatory response.

Keyword

Inflammation; Acrolein; Curcumin; Cyclooxygenase-2; Human umbilical vein endothelial cells

MeSH Terms

Acrolein
Atherosclerosis
Curcumin*
Cyclic AMP Response Element-Binding Protein
Cyclooxygenase 2
Endoplasmic Reticulum Stress
Endothelial Cells
Enzyme-Linked Immunosorbent Assay
Flow Cytometry
Human Umbilical Vein Endothelial Cells*
Humans*
Inflammation
Phosphorylation
Polymerase Chain Reaction
Protein Kinase C
Protein Kinases
Reactive Oxygen Species
RNA, Messenger
Smoke
Tobacco
Vascular Diseases
Acrolein
Curcumin
Cyclic AMP Response Element-Binding Protein
Cyclooxygenase 2
Protein Kinase C
Protein Kinases
RNA, Messenger
Reactive Oxygen Species
Smoke

Figure

  • Fig. 1 Curcumin suppresses acrolein-induced COX-2 and prostaglandin production in HUVECs. (A) HUVECs were preincubated with 25 µM curcumin before treatment with 10 µM acrolein and COX-2 protein levels were assessed by western blot. (B) Relative expression of COX-2 was measured by real-time quantitative PCR as described in the methods section. (C) The production of PGE2 was measured in the supernatants as described in the methods section. (D) Inhibitory effect of curcumin on acrolein-induced activation of COX-2 promoter. HUVECs were transfected with COX-2 promoter construct (−1,432 to +59) for 48 hours. After transfection, cells were preincubated with 25 µM curcumin for 30 minutes before exposure to 10 µM acrolein for 6 hours. Data represents luciferase activity that has been normalized to co-transfected β-galactosidase activity. Data were analyzed by the Student's t-test. Data represent the mean±standard deviation of results from three independent experiments. HUVEC, human umbilical vein endothelial cell; COX-2, cyclooxygenase-2; PGE2, prostaglandin E2. *p<0.05 vs control group.

  • Fig. 2 Effect of NAC on acrolein-induced COX-2 and PGE2 production in HUVECs. (A) HUVECs were preincubated with 10 mM NAC for 12 hours. The cells were treated with 10 µM acrolein for 16 hours and western blotting was performed for COX-2 expression. (B) HUVECs were preincubated with 10 mM NAC for 12 hours. The cells were then treated with 10 µM acrolein for 16 hours and then release of PGE2 was measured from supernatants as described in the methods section. The values shown for PGE2 production are the mean±standard deviation of 3 independent experiments. Data were analyzed by the Student's t-test. Results are from 3 independent experiments. NAC, N-acetyl-cysteine; COX-2, cyclooxygenase-2; HUVEC, human umbilical vein endothelial cell; PGE2, prostaglandin E2. *p<0.05 compared to untreated control cells.

  • Fig. 3 Effect of curcumin on acrolein-induced intracellular peroxide production. (A) Cells were incubated with (black area) or without (white area) 10 µM acrolein for 30 minutes, and then treated with a peroxide sensitive dye, H2DCF-DA (20 µM) during the final 30 minutes of each treatment. The relative peroxide concentrations in cells were then measured by flow cytometry. (B) After preincubation with 10 mM NAC for 4 hours, the cells were treated with (black area) or without (white area) 10 µM acrolein and flow cytometric analysis was performed. X-axis represents intensity of fluorescence. NAC, N-acetyl-cysteine.

  • Fig. 4 Effects of curcumin on acrolein-induced phosphorylation of PKCδ, p38, and CREB in HUVECs. Cells were pre-treated with curcumin (10 and 25 µM) for 30 minutes prior to acrolein treatment (10 µM). After 30 minutes of incubation, the cell lysates (20 µg) were prepared and western blot analysis was performed with Abs against phosphorylated PKCδ, p38, and CREB or total PKCδ, p38, and CREB. Quantitative data were obtained using an imaging densitometer (ImageJ version 1.52a software, NIH, Bethesda, MD, USA). Data were analyzed by the Student's t-test. Data represent the mean±standard deviation of results from 3 independent experiments. PKC, protein kinase C; CREB, cAMP response element-binding protein; HUVEC, human umbilical vein endothelial cell; Ab, antibody; p, phosphorylated. *p<0.05 vs. control group.

  • Fig. 5 Effects of curcumin on acrolein-induced phosphorylation of eIF-2α in HUVECs. HUVECs were treated with curcumin (10 and 25 µM) for 30 minutes before acrolein (10 µM) treatment. After 30 minutes, cell lysates were prepared, and 20 µg of protein was used to perform western blotting using the anti-phosphorylated eIF-2α, and total eIF-2α antibody was used as a loading control. Quantitative data were obtained using an imaging densitometer (ImageJ version 1.52a software, NIH). Data were analyzed by the Student's t-test. Data represent the mean±standard deviation of results from three independent experiments. eIF, eukaryotic initiation factor; HUVEC, human umbilical vein endothelial cell; p, phosphorylated. *p<0.05 vs. control group.


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