Objective      To determine the effect of metformin on the proliferation of rat pulmonary arterial smooth muscle cells (PASMCs) exposed to nicotine

and explore the underlying mechanisms.       Methods      Rat PASMCs were freshly isolated from SD rat pulmonary arteries and identified with immunocytochemical staining for α-actin. Then the cells at generations 3 to 8 were treated with 0.01

0.1

1.0 and 10 μmmol/L nicotine for 48 h. The proliferation of PASMCs exposed to nicotine at above determined doses was measured by CCK-8 assay in presence or absence of 2 mmol/L metformin. Expression of IL-6 and TNF-α in the culture supernatant were detected by ELISA assay. The level of reactive oxygen species (ROS) was detected by ROS fluorescent Probe-DHE and flow cytometry. The levels of total adenosine monophosphate activated protein kinase (AMPK) and phosphorylated AMPK in the PASMCs was determined by Western blot analysis.       Results      Immunocytochemical staining displayed that α-actin was highly expressed and distributed filamentously in the cytoplasm of PASMCs

indicating the isolated cells being PASMCs. Nicotine treatment at 0.1 μmmol/L for 48 h obviously induced the proliferation of PASMC compared with control PASMCs (P0.05). However

the proliferation of PASMCs induced by 0.1 μmmol/L nicotine was inhibited significantly after metformin pretreatment (P0.05). Additionally

the levels of IL-6 and TNF-α released by PASMCs were increased after nicotine treatment

and so was the ROS level (P0.05)

but metformin pretreatment reversed the levels (P0.05). Nicotine induced the transient phosphorylation of AMPK after exposure to nicotine

while metformin maintained the phosphorylation of AMPK (P0.05).       Conclusion      Nicotine induces the release of IL-6

TNF-α and ROS

and enhances the proliferation in PASMCs. However

metformin inhibits the above effects induced by nicotine

which might be dependent on activation of AMPK pathway.