Figure 3.

Reactive oxygen species (ROS)-mediated IL-6 is essential for NaVO₃-mediated VSMC migration and proliferation. (A) Plasma ROS levels in mice treated with vehicle (endotoxin-free water) (n = 9) or NaVO₃ (n = 8) once weekly for 12 weeks were measured by thiobarbituric acid reactive substances (TBARS) assay. The solid black line denotes the mean value. (B) Quiescent VSMCs were treated with NaVO₃ for the indicated time. Intracellular ROS levels were measured by 6-carboxy-2,7-dichlorodihydrofluorescein diacetate (DCFDA). (C–E) After pretreating VSMCs with different concentrations of n-acetyl-l-cysteine (NAC) for 30 min, cells were stimulated with vehicle or NaVO₃ (1 µg/mL) for 24 h. (C) Intracellular ROS levels and mitochondrial ROS were measured by DCFDA. (D) VSMC proliferation was measured by BrdU incorporation assay. (E) VSMC migration was then measured by the transwell assays. (F) VSMCs were treated with NaVO₃ (1 µg/mL) with or without different concentrations of NAC for 48 h. Cell lysates were immunoblotted with antibodies for vimentin, smooth muscle α-actin (SMα), SM22α, or β-actin. Densitometry analysis of SMα, SM22α, and vimentin protein expression relative to β-actin. Data represent mean ± SEM of three experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.