Liyi Huang1, Qing Chen1, Lehua Yu2, Dingqun Bai3. 1. Department of Rehabilitation Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, PR China. 2. Department of Rehabilitation Medicine, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, PR China. 3. Department of Rehabilitation Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, PR China. Electronic address: baidingqun2014@163.com.
Abstract
BACKGROUND: This study aimed to determine the effect of pyropheophorbide-α methyl ester (MPPa)-mediated photodynamic therapy (MPPa-PDT) on the apoptosis and inflammation of murine macrophage RAW264.7 cells. METHODS: Uptake and subcellular localization of MPPa was detected by flow cytometry and confocal fluorescence microscope. Cell viability was assessed by CCK-8; ROS levels were assessed by DCFH-DA. Cell apoptosis was measured by flow cytometry and Hoechst 33342 staining, whereas mitochondrial membrane potential was detected by JC-1 staining. Secretion of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) was determined using ELISA kits. Caspase-3, cleaved caspase-3, procaspase-9, cleaved caspase-9, PARP, cleaved PARP, Bcl-2, Bax, NF-κB p-p65, p-IKKα/β, and p-IκBα were measured by western blotting. Nuclear factor κB (NF-κB)-p65 nuclear translocation was observed by immunofluorescence. RESULTS: MPPa -PDT influenced cell viability in a light dose-dependent manner. It induced ROS formation and RAW264.7 cell apoptosis. It also increased the expression of cleaved caspase-3, cleaved caspase-9, cleaved PARP and Bax, decreased the expression of Bcl-2. While TNF-α, IL-1β, and IL-6 increased in LPS group (model of inflammation), it deceased in LPS-MPPa-PDT group. NF-κB p-p65, p-IKKα/β, and p-IκBα had higher expression in LPS group while that reduced in LPS-MPPa-PDT group. Simultaneously, MPPa-PDT inhibited nuclear translocation of NF-κB-p65 caused by LPS. CONCLUSIONS: MPPa-PDT can induce apoptosis and attenuate inflammation in mouse RAW264.7 macrophages, thereby suggesting a promising therapy for atherosclerosis.
BACKGROUND: This study aimed to determine the effect of pyropheophorbide-α methyl ester (MPPa)-mediated photodynamic therapy (MPPa-PDT) on the apoptosis and inflammation of murine macrophage RAW264.7 cells. METHODS: Uptake and subcellular localization of MPPa was detected by flow cytometry and confocal fluorescence microscope. Cell viability was assessed by CCK-8; ROS levels were assessed by DCFH-DA. Cell apoptosis was measured by flow cytometry and Hoechst 33342 staining, whereas mitochondrial membrane potential was detected by JC-1 staining. Secretion of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) was determined using ELISA kits. Caspase-3, cleaved caspase-3, procaspase-9, cleaved caspase-9, PARP, cleaved PARP, Bcl-2, Bax, NF-κB p-p65, p-IKKα/β, and p-IκBα were measured by western blotting. Nuclear factor κB (NF-κB)-p65 nuclear translocation was observed by immunofluorescence. RESULTS:MPPa -PDT influenced cell viability in a light dose-dependent manner. It induced ROS formation and RAW264.7 cell apoptosis. It also increased the expression of cleaved caspase-3, cleaved caspase-9, cleaved PARP and Bax, decreased the expression of Bcl-2. While TNF-α, IL-1β, and IL-6 increased in LPS group (model of inflammation), it deceased in LPS-MPPa-PDT group. NF-κB p-p65, p-IKKα/β, and p-IκBα had higher expression in LPS group while that reduced in LPS-MPPa-PDT group. Simultaneously, MPPa-PDT inhibited nuclear translocation of NF-κB-p65 caused by LPS. CONCLUSIONS:MPPa-PDT can induce apoptosis and attenuate inflammation in mouse RAW264.7 macrophages, thereby suggesting a promising therapy for atherosclerosis.