Fig. 4

PQS attenuates high phosphate-induced vascular smooth muscle cell calcification by activating Nrf2. A VSMCs were incubated with 3 mM P in the presence or absence of PQS for 48 h, and intracellular ROS levels were determined as the mean fluorescence intensity. ^*p < 0.05 or ^**p < 0.01 vs. blank, and ^##p < 0.01 vs. Pi control. B VSMCs were transfected with the Nrf2 transcription reporter gene ARE-luc and the internal control gene pRL-TK-luc then treated with various concentrations of PQS for 48 h. Nrf2 transcriptional activity was assessed using dual luciferase assay. ^*p < 0.05 vs. blank, and ^#p < 0.05 vs. Pi control. C and D Cells were treated with calcification medium containing 3 mM Pi for 6 days. The expression of Nrf2 and HO-1 was determined using RT-PCR and Western blot analysis. ^*p < 0.05 vs. blank, and ^#p < 0.05 or ^##p < 0.01 vs. Pi control. (E and F) VSMCs were transfected with Nrf2-siRNA or control siRNA for 48 h, and further incubated with or without PQS in calcification medium for 6 days. The expression of Runx2, SM22α and Nrf2 was determined using Western blot analysis. Calcium deposition was visualized by alizarin red staining. Scale bar: 200 μm. The values are presented as the means ± SD (n = 3). ^*p < 0.05 or ^**p < 0.01 vs. blank, ^#p < 0.05 vs. Pi control, and ^&p < 0.05 vs. Pi + PQS