Curcumin Exerts its Anti-hypertensive Effect by Down-regulating the AT1 Receptor in Vascular Smooth Muscle Cells

Abstract

Curcumin exerts beneficial effects on cardiovascular diseases, including hypertension. However, its mechanisms are unknown. We propose that curcumin prevents the development of hypertension by regulating AT1 receptor (AT1R) expression in arteries. The present study examined how curcumin regulates AT1R expression in vascular smooth muscle cells and investigated the physiological significance of this regulation in angiotensin (Ang) II-induced hypertension. The results showed that curcumin decreased AT1R expression in a concentration- and time-dependent manner in vascular smooth muscle cells. Using luciferase reporters with an entire AT1 or a mutant AT1R in A10 cells, the AT1R promoter activity was inhibited by 10(-6 )M curcumin, and the proximal element (from -61 to +25 bp) of the AT1R promoter was crucial for curcumin-induced AT1R down-regulation. An electrophoretic mobility shift assay showed that curcumin decreased specificity protein 1 (SP1) binding with the AT1R promoter in A10 cells. Curcumin treatment reduced Ang II-induced hypertension in C57Bl/6J mice, which was accompanied by lower AT1R expression in the arteries and decreased Ang II-mediated vasoconstriction in the mesenteric artery. These findings indicate that curcumin down-regulates AT1R expression in A10 cells by affecting SP1/AT1R DNA binding, thus reducing AT1R-mediated vasoconstriction and subsequently prevents the development of hypertension in an Ang II-induced hypertensive model.

Figure 1. The inhibitory effect of curcumin on AT₁R protein and mRNA expression in A10 cells.

Curcumin decreased the AT₁R protein expression in a concentration- (for 24 hrs) (A) and time- (at 10⁻⁶M) (B) dependent manner. (C) AT₁R mRNA expression was also inhibited by curcumin (CUR, 10⁻⁶M/24 hrs) compared with the control (C). Cropped gels/blots were used in the figure; the AT₁R expression was normalized against β-actin. The values are expressed as the mean ± SEM (*P < 0.05 vs. control, n = 6).

Figure 2. Effect of cycloheximide and actinomycin D on curcumin-induced AT₁R downregulation.

(A) The effect of cycloheximide (CHX) on curcumin (CUR)-induced AT₁R protein down-regulation was examined by Western blot analysis; cropped blots are shown. The AT₁R expression level was normalized against β-actin (*P < 0.05 vs. control, ^#P < 0.05 vs. CHX, n = 7). (B) The effect of actinomycin D (ActD) on CUR-induced AT₁R mRNA down-regulation was examined by RT-PCR analysis. The AT₁R expression was normalized against β-actin. The values are expressed as the mean ± SEM (*P < 0.05 vs. control, n = 7).

Figure 3. Effect of curcumin on AT₁R mRNA transcription.

(A) Schematic of deletion mutants of the AT₁R promoter/luciferase fusion DNA construct. (B) The bar graphs indicate luciferase activity normalized against β-galactosidase activity derived from the corresponding deletion. The data indicate the luciferase activity of curcumin-stimulated A10 cells (white bars) relative to unstimulated (black bars) in each group. The values (mean ± SEM) are expressed as a percent (*P < 0.05 vs. unstimulated, n = 6).

Figure 4. Effect of curcumin on the binding ability of SP1 at the AT₁R gene promoter in A10 cells.

The binding activity of the AT₁R gene promoter (−40 bp to −6 bp), which contains an Sp1 site, was examined in the nuclear protein from unstimulated (lane 2) or curcumin (10⁻⁶M)-stimulated (lane 3) A10 cells by electrophoretic mobility shift assay; DNA-binding protein (arrow) was decreased compared with unstimulated A10 cells. No nuclear extracts or 50 times of unlabeled probe were added to the reaction mixture as controls (lanes 1 and 4, respectively).

Figure 5. Effect of curcumin on AT₁R expression and function in arteries from Ang II-induced hypertensive C57Bl/6J mice.

The blood pressure (A) and heart rate (B) were examined in Ang II-induced hypertensive C57Bl/6J mice with or without curcumin treatment (*P < 0.05 vs. control, n = 5). The Ang II-mediated vasoconstriction in the third-order mesenteric arteries (B) and the AT₁R expression in the aorta (D) were examined in these mice (*P < 0.05 vs. others, n = 7). Cropped blots were used in this figure; the AT₁R expression was normalized against β-actin. The values are expressed as the mean ± SEM (*P < 0.05 vs. control group, #P < 0.05 vs. Ang II group, n = 7).