In Vitro Effects of Curcumin on Transforming Growth Factor-β-mediated Non-Smad Signaling Pathway, Oxidative Stress, and Pro-inflammatory Cytokines Production with Human Vascular Smooth Muscle Cells

Transforming growth factor-beta (TGF-beta) induces pro-inflammatory cytokines expression including interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) and these cytokines are associated with the development of atherosclerosis. Curcumin has anti-atherogenic effects and anti-inflammatory properties in the vascular wall, but the relative mechanisms are almost unknown. In the present study, we investigate the effect of curcumin on modulating the pro-inflammatory action of TGF-beta in human vascular smooth muscle cells (VSMCs) and its molecular mechanisms. Cultured VSMCs were seeded into several groups: a control group (untreated group), a group treated with TGF-beta, and several groups treated with TGF-beta plus inhibitors. The cells were pre-treated with diphenyleneiodonium chloride, DPI, (20 mu M), curcumin (5, 10 and 20 mu M) and N-Acetyl-L-Cysteine, NAC, (10 mM) and then TGF-beta (5 ng/mL) was added to the culture medium. The mRNA levels of IL-6 and TNF-alpha were detected by quantitative Real-Time Polymerase Chain Reaction. For monitoring the Smad2 linker region phosphorylation (pSmad2L), the western-blotting technique was applied and reactive oxygen species (ROS) generation was measured by utilizing 2',7'-dichlorofluorescein diacetate-based assay. TGF-beta increased the mRNA expression of IL-6 (p=0.02 and p=0.001) and TNF-alpha (p=0.014 and p=0.001) in a time-dependent manner, ROS production (p=0.03) and Smad2L phosphorylation (p=0.015). Pre-treatment with curcumin, DPI and NAC inhibited TGF-beta-induced IL-6 (p=0.04) and TNF-alpha ( p=0.001) mRNA expression, Smad2L phosphorylation (p=0.02) and ROS production (0.03). Pharmacological inhibition by Curcumin blocks TGF-beta-induced ROS production, Smad2L phosphorylation, and IL-6 and TNF-alpha mRNA expression in human VSMCs.