Effect of NADPH oxidase on Toll-like receptor 4-mediated proilflammatory phenotype of cultured vascular smooth muscle cells in nice

Abstract

Objective To investigate the effect of the reduced nicotinamide adenine dinucleotide phosphate （NADPH） oxidase on Toll-like receptor 4 （TLR4）-mediated proinflammatory phenotype of cultured vascular smooth muscle cells （VSMCs） in mice.Methods NADPH oxidase agonist platelet-derived growth factor- BB （PDGF-BB） and inhibitor apocynin were used respectively to treat cultured VSMCs from C57BL/6J and TLR4-/- mice. The fluorescent probe 2＇,7＇-dichlorodihydrofluorescein diacetate was used to detect the reactive oxyen species （ROS） level in VSMCs. An enzyme-linked immunosorbent assay was used to detect the expressions of interletddn （IL）-6, IL-1β, and tumor necrosis factor-α（TNF-α in VSMCs. Tetrazolium blue staining and Boyden chamber assay were used to detect the proliferation and migration of VSMC. Results TheROS levels were increased in VSMCs both from C57BL/6J and TLR4-/- mice after PDGF-BB treatment, and this could be inhibited by apocynin. PDGF-BB pretreatment sigfificantly upregulated the expressions of IL-6 （52. 69±3.49 ng/ml vs. 35. 04 ±2. 74 ng/ml; P =0. 001）, IL-1β （79. 68 ±2. 33 ng/ml vs. 62. 38 ±0. 54 ng/ml; P=0.000）, and TNF-a （218.35± 5.42 ng/mlvs. 124.74±4.59 ng/ml; P= 0.000） in VSMCs from C57BL/6J mice, and the abilities of proliferation （1.69 ± 0. 53 vs. 1.04 ±0. 40; P = 0. 000） and migration （42. 11 ±4. 05 vs. 1.69 ± 0. 53; P = 0. 000） were increased significantly; apocynin pretreatment significantly inhibit the expressions of IL-6 （42. 11 ± 4. 05 ng/ml vs. 52. 69 ± 3.49 ng/ml; P = 0. 010）, IL-1β （67. 57 ± 1.36 ng/ml vs. 79. 68 ±2. 33 ng/ml; P = 0. 000） and TNF-α （156. 18 ±6. 98 ng/ml vs. 218. 35 ± 5.42 ng/ml; P =0. 000）, as well as proliferation （1.23 ±0. 42 vs. 1.69 ±0. 53; P =0. 000） and migration （42. 11 ±4. 05 vs. 52. 69±3.49; P = 0. 000）. While there were no significant changes in the expressions of IL-6, IL-1β, and TNF-α in VSMCs from TLR4 /- mice after PDGF-BB and apocynin pretreatment. Conclusions NADPH oxidase-derived ROS involved in the TLR4-mediated VSMC inflammatory phenotype as well as proliferation and migration, which may be the important mechanisms of its influencing on the occurrence and development of atherosclerosis. Key words: NADPH Oxidase; Toll-Like Receptor 4; Inflammation; Muscle, Smooth, Vascular; Atherosclerosis; Cells, Cultured; Mice