Abstract

Different effects of simvastatin on human airway smooth muscle cell proliferation induced by platelet activating factor

Author(s): Yi Shen*, Chunlai Feng, Sujuan Zhang

Determine whether and how simvastatin (Sim) may influence human airway smooth muscle cells (HASMCs) proliferation induced by platelet activating factor (PAF) through NADPH oxidase-ROS signaling pathway. Cell proliferation was analyzed according to the protocol of CCK-8 assay. Intracellular reactive oxygen species was measured by the fluorescence using an oxidation sensitive fluorescent probe, 2′7′-dichlorofluorescin diacetate (DCFHDA). The mRNA of Nox4, MnSOD, and Catalase were determined by real-time PCR. Protein levels of Nox4, phospho-ERK, total ERK were determined by western blotting. Inhibitor of MEK1/2was used to confirm the involved signal pathway. Our data demonstrated that PAF induced HASMCs proliferation with the maximal effect seen at 10-6 M and 24 h, the proliferation rate was (1.77 ± 0.51)as fold as the control group. Simvastatin (at the concentration of 10-5 M) was (0.67 ± 0.18), inhibited HASMCs proliferation induced by PAF (P<0.01).Compared with the negative control group (76.79 ± 6.05), PAF-treated group (98.89 ± 1.28) led to a significant increase in DCF fluorescence, PAF combined with simvastatin group (66.40 ± 2.87) decreased ROS level (P<0.01).PAF could increase Nox4 mRNA expression, reduced MnSOD, Catalase levels, simvastatin played opposite role. Treating HASMCs with simvastatin abrogated the phosphorylation of ERK induced by PAF. When added N-acetyl-cysteine (NAC) or PD98059 (ERK inhibitor) to PAF group separately, phosphorylation of ERK reduced at different degree. PAF induced the proliferation of HASMCs through NADPH oxidase-ROS signaling pathway, which was reversed by Simvastatin. Pretreatment of Antioxidant (NAC) significantly reduced ERK phosphorylation. The present study demonstrates ROS dependent activation of ERK/MAPK signaling way.


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