Y-R Dai1, H-Y Wu, L-Q Wu, H Xu, J Yin, S-S Yan, W-X Zeng. 1. Department of Respiratory, the Second Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang, China. yuanrongdai@126.com.
Abstract
OBJECTIVES: The purpose of this study was to investigate the effect of roxithromycin on apoptosis of airway smooth muscle cells (ASMCs) from a rat model of asthma and uncover signaling pathway underlying the cytotoxicity of roxithromycin. MATERIALS AND METHODS: ASMCs were isolated from a rat model of asthma and treated with or without roxithromycin for 48 h before parameter detection. Cell viability was assessed by WST-8 assay and flow cytometry after Annexin V/PI double staining. Changes in the mitochondrial membrane potential (ΔΨm) were measured by flow cytometry using JC-1. Cytochrome C (Cyt c), cleaved Caspase-9/3 and P27 were evaluated by Western Blot. RESULTS: Incubation with roxithromycin reduced ASMCs proliferation and enhanced apoptosis in a dose-dependent manner. Flow cytometry revealed a loss of ΔΨm and Western Blot displayed Caspase-9/3 activation as well as Cyt c release from mitochondria to the the cytosol after the treatment of roxithromycin. In addition, P27 were more strongly expressed in AMSCs treated with roxithromycin compared with the control group. CONCLUSIONS: Roxithromycin induced apoptosis of ASMCs derived from a rat model of asthma in a dose-dependent manner via a caspase-3- and caspase-9-dependent mitochondrial pathway, involving the up-regulation of P27.
OBJECTIVES: The purpose of this study was to investigate the effect of roxithromycin on apoptosis of airway smooth muscle cells (ASMCs) from a rat model of asthma and uncover signaling pathway underlying the cytotoxicity of roxithromycin. MATERIALS AND METHODS:ASMCs were isolated from a rat model of asthma and treated with or without roxithromycin for 48 h before parameter detection. Cell viability was assessed by WST-8 assay and flow cytometry after Annexin V/PI double staining. Changes in the mitochondrial membrane potential (ΔΨm) were measured by flow cytometry using JC-1. Cytochrome C (Cyt c), cleaved Caspase-9/3 and P27 were evaluated by Western Blot. RESULTS: Incubation with roxithromycin reduced ASMCs proliferation and enhanced apoptosis in a dose-dependent manner. Flow cytometry revealed a loss of ΔΨm and Western Blot displayed Caspase-9/3 activation as well as Cyt c release from mitochondria to the the cytosol after the treatment of roxithromycin. In addition, P27 were more strongly expressed in AMSCs treated with roxithromycin compared with the control group. CONCLUSIONS:Roxithromycin induced apoptosis of ASMCs derived from a rat model of asthma in a dose-dependent manner via a caspase-3- and caspase-9-dependent mitochondrial pathway, involving the up-regulation of P27.