Cigarette smoke extract stimulates human pulmonary artery smooth muscle cell proliferation: Role of inflammation and oxidative stress

Abstract

Objectives: Cigarette smoke may play a direct role in proliferation of human pulmonary artery smooth muscle cells (HPASMCs). However, the mechanism involved and the effect of interventions remain unclear. We aimed to evaluate the effect of cigarette smoke extract (CSE) on HPASMCs, explore the role of inflammation and oxidative stress, and the effects of Tempol and PDTC in this process.

Materials and methods: HPASMCs were subjected to normal control (NC), CSE, CSE+Tempol (CSE+T), and CSE+PDTC (CSE+P) groups. Proliferation of HPASMCs was measured by CCK-8 and Western blot. TNF-α, IL-6, MDA, and SOD levels were determined by ELISA and commercial kits. Nuclear translocation of NF-κB p65 was evaluated by western blot.

Results: 1%, 2.5%, and 5% CSE all promoted proliferation of HPASMCs, and effect of 1% CSE was the most significant, however, 7.5% and 10% CSE inhibited viability of cells (all P<0.05). Compared with the NC group, TNF-α, IL-6, and MDA levels increased, SOD activity decreased (all P<0.05), and NF-κB p65 expression in nuclei increased (P=0.04) in the CSE group. Tempol and PDTC inhibited the proliferation of HPASMCs induced by CSE (all P<0.05). And compared with the CSE group, TNF-α, IL-6, and MDA levels in CSE+T and CSE+P groups decreased, while SOD activity increased (all P<0.05). Tempol reduced the expression of NF-κB p65 in nuclei but did not achieve a significant difference (P=0.08). PDTC inhibited the nuclear translocation of NF-κB p65 (P=0.03).

Conclusion: CSE stimulates HPASMCs proliferation in a certain concentration range. The CSE-induced proliferation of HPASMCs involved excessive inflammatory response and oxidative stress. Tempol and PDTC attenuate these effects of CSE on HPASMCs.

Keywords: Cigarette smoking; Inflammation; NF-κB; Oxidative stress; Pulmonary arterial - hypertension.

Figure 1

The HPASMCs were treated with increasing concentrations of CSE for 24 hr. Cell proliferation was analyzed by CCK-8 assay. The data were shown as mean±standard deviation from five duplicated experiments. ^aP<0.05 compared with NC group, ^bP<0.01 compared with 1% CSE group, ^cP<0.01 compared with 2.5% CSE group, ^dP<0.01 compared with 5% CSE group, ^eP<0.01 compared with 7.5% CSE group

Figure 2

HPASMCs were pretreated with Tempol or PDTC before exposure to 1% CSE. Cell viability was analyzed by CCK-8 assay. The data were shown as mean±standard deviation from three replicated experiments. *P<0.05 compared with NC group, #P<0.05 compared with 1% CSE group

Figure 3

CSE induced up-expression of α-SMA and nuclear translocation of NF-κB p65 in HPASMCs. a: Western blot analysis showed the expression level of α-SMA, Nuclear NF-κB p65, Cytoplasm NF-κB p65, β-actin, and H3. b: Summarized data showed the average protein level of α-SMA and NF-κB p65 in the nuclear and cytoplasm fraction. *P<0.05 compared with NC group, #P<0.05 compared with 1% CSE group

Figure 4

Effect of CSE on TNF-α (a) and IL-6 (b) levels in the supernatants of HPASMCs with or without Tempol and PDTC. The data were expressed as the mean±standard deviation of three replicated experiments.*P<0.01 compared with NC group, #P<0.01 compared with 1% CSE groupC: normal control; PDTC: pyrrolidine dithiocarbamate; HPASMCs: human pulmonary artery smooth muscle cells; CSE: cigarette smoke extract

Figure 5

Levels of MDA concentration (a) and SOD activity (b) in different exposure conditions. The data were expressed as the mean±standard deviation of three replicated experiments.*P<0.05 compared with NC group, #P<0.05 compared with 1% CSE group