LY333531 attenuates contraction of tumor necrosis factor-α-sensitized human airway smooth muscle cells

Abstract

Objective Protein kinase C (PKC) has been implicated in the increased contraction of human airway smooth muscle cells (HASMCs) in asthma. Using the three-dimensional collagen gel contraction system, the study aimed to determine the effects of LY333531, a specific inhibitor of the PKC-β isoform, on the contraction of tumor necrosis factor (TNF)-α-sensitized HASMCs. Methods Cultured HASMCs were divided into five groups: the control group received no treatment, and the cells in the TNF-α group were sensitized with 10 ng/mL TNF-α for 48 h, while TNF-α was administered to sensitize HASMCs in the presence of 0.1, 0.2, and 0.5 μM LY333531 for 48 h in the 0.1LY, 0.2LY, and 0.5LY groups, respectively. Following this, HASMCs contraction was stimulated with 1 mM acetylcholine (ACh) for up to 24 h in each group and assessed using a three-dimensional collagen gel contraction assay. Furthermore, western blot and immunofluorescence analysis were performed. Results The collagen gel contraction assay revealed that TNF-α increased the protein expression of phosphorylated PKC-β2, CPI-17, and MLC while exacerbating ACh-induced HASMCs contraction. LY333531 significantly attenuated HASMCs contraction and downregulated the protein expression of both p-CPI-17 and p-MLC. Conclusions At least in part by regulating CPI-17 and MLC phosphorylation, LY333531 attenuates augmented contraction of TNF-α-sensitized HASMCs in a collagen gel contraction system.