Effects of HSM on the expression of P2X4R and P2X7R and ROS production in human macrophages.
Cells were pretreated with various concentrations (1 or 2%) of HSM extract for 20 h, followed by treatment with LPS (0.5 μg/ml) for 3 h and ATP (5 mM) for 1 h. (a) The mRNA expression levels of P2X4R and P2X7R were determined by RT-PCR, using β-actin as the internal control. (b) P2X4R and P2X7R mRNAs were quantified using real-time PCR. β-actin gene expression was used for normalization. The results are expressed as fold changes, considering one as the value of untreated cells. (c) Cell lysates were analyzed by Western blot analysis used specific anti-P2X4R and anti-P2X7R antibodies. (d) ROS production was measured with the total ROS detection kit, using a fluorescence microplate reader. Pyocyanin (200 μM), a ROS inducer, was used as a positive control for ROS formation. Data are presented as means ± SE of three experiments preformed in duplicate. #P < 0.01 versus untreated cells. versus HSM-untreated control (ethanol) cells. *P < 0.01 versus HSM-untreated control (ethanol) cells. †P < 0.05 versus HSM (1%) treated cells.