Small interfering RNA against ERK1/2 attenuates cigarette smoke‑induced pulmonary vascular remodeling

Figure 1.. Effect of ERK1/2-siRNA on the expression of cyclin E1 induced by CSE. (A) Cells were treated with CSE or the control medium for 24 h, then the mRNA levels of ERK1/2 and cyclin E1 were determined by RT-qPCR. (B) Cells were transfected with ERK1/2-siRNA or NC-siRNA at a final concentration of 30 nmol/l for 24 h, then the cells were treated with CSE or the control medium for another 24 h. The mRNA levels of ERK1/2 and cyclin E1 were determined by RT-qPCR. (C) Cells were treated with CSE or the control medium for 24 h, then the protein levels of ERK1/2 and cyclin E1 were determined by western blot analysis. (D) Cells were treated as in (B), then the protein levels of ERK1/2 and cyclin E1 were determined by RT-qPCR. *P<0.05. ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; CSE, cigarette smoke extract; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NC, negative control.

Figure 2.. Dose-response curve of ERK1/2-siRNA in rat pulmonary artery smooth muscle cells. Cells were transfected with ERK1/2-siRNA at a final concentration of 0, 10, 20 and 30 nmol/l (presented left to right). The protein levels of ERK1/2 and cyclin E1 were determined by western blot analysis. (A) The optical density of each band was detected and normalized against β-actin. (B) Normalized optical density of each band. ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA.

Figure 3.. Effect of CSE and ERK1/2-siRNA on rat pulmonary artery smooth muscle cell cycle and cell proliferation. (A) Cells were treated with CSE or the control medium for 24 h, then the distribution of cell cycle stages was detected. The left red, right red and blue areas represent G0/G1, G2/M and S phase, respectively. (B) Cells were transfected with ERK1/2-siRNA or NC-siRNA at a final concentration of 30 nmol/l for 24 h, then cells were treated with CSE or control medium for a further 24 h. (C) Cells were treated with CSE or the control medium for 24 h, then the cell number of each group was counted. (D) Cells were treated as in (B), then the cell number of each group was counted. (E) Cells were treated with CSE or the control medium for 24 h, then incubated with 50 mmol BrdU for 4 h at 37°C and fixed with cold acetone. The BrdU-positive cells were counted. (F) Cells were treated as in (B), then incubated with 50 mmol BrdU for 4 h at 37°C. *P<0.05 vs. control. ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; CSE, cigarette smoke extract; NC, negative control; BrdU, 5-bromo-2-deoxyuridine.

Figure 4.. Effect of ERK1/2-siRNA on the expression of ERK1/2 and cyclin E1 in the pulmonary vessels of cigarette smoke-exposed rats. (A) The mRNA levels of ERK1/2 and cyclin E1. (B) Protein levels of ERK1/2 and cyclin E1 were evaluated by western blot analysis. Immunostaining for (C) p-ERK1/2 and (D) cyclin E1 was also performed in each group (C) p-ERK or (D) cyclin E1. Blue, indicated the nucleus; red, indicated the cytoplasm; and dark brown indicated p-ERK or cyclin E1. Magnification, ×200. *P<0.05 vs. control. ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; NC, negative control.

Figure 5.. Effect of smoking and ERK1/2-siRNA on rat pulmonary vascular remodeling. Sections were stained with hematoxylin and eosin and anti-α-smooth muscle actin antibody. (A) Representative photomicrographs of pulmonary vessel WT are shown (magnification, ×200). The WT of different groups were expressed as the percentage of total area in small pulmonary arteries. (B) Representative photomicrographs of the muscularization of small pulmonary arteries are shown (magnification, ×200). Blue, indicated the nucleus; red, indicated the cytoplasm; and dark brown, indicated α-smooth muscle actin. *P<0.05 vs. control. ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; NC, negative control; WT, wall thickness.