Department of Rehabilitation Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, PR China.
Department of Rehabilitation Medicine, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, PR China.
Photodiagnosis Photodyn Ther. 2019 Mar;25:148-156. doi: 10.1016/j.pdpdt.2018.12.002. Epub 2018 Dec 15.
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.
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.
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.
MPPa-PDT can induce apoptosis and attenuate inflammation in mouse RAW264.7 macrophages, thereby suggesting a promising therapy for atherosclerosis.
本研究旨在探讨焦脱镁叶绿酸-α 甲酯（MPPa）介导的光动力疗法（MPPa-PDT）对小鼠巨噬细胞 RAW264.7 细胞凋亡和炎症的影响。
采用流式细胞术和共聚焦荧光显微镜检测 MPPa 的摄取和亚细胞定位。通过 CCK-8 评估细胞活力；通过 DCFH-DA 评估 ROS 水平。通过流式细胞术和 Hoechst 33342 染色测量细胞凋亡，通过 JC-1 染色检测线粒体膜电位。使用 ELISA 试剂盒测定肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）和白细胞介素-6（IL-6）的分泌。通过 Western blot 测定 caspase-3、cleaved caspase-3、procaspase-9、cleaved caspase-9、PARP、cleaved PARP、Bcl-2、Bax、NF-κB p-p65、p-IKKα/β 和 p-IκBα。通过免疫荧光观察 NF-κB-p65 的核转位。
MPPa-PDT 以光剂量依赖的方式影响细胞活力。它诱导 ROS 形成和 RAW264.7 细胞凋亡。它还增加了 cleaved caspase-3、cleaved caspase-9、cleaved PARP 和 Bax 的表达，降低了 Bcl-2 的表达。而在 LPS 组（炎症模型）中，TNF-α、IL-1β 和 IL-6 增加，而在 LPS-MPPa-PDT 组中则减少。LPS 组中 NF-κB p-p65、p-IKKα/β 和 p-IκBα 的表达较高，而 LPS-MPPa-PDT 组中则减少。同时，MPPa-PDT 抑制了 LPS 引起的 NF-κB-p65 的核转位。
MPPa-PDT 可诱导小鼠 RAW264.7 巨噬细胞凋亡并减轻炎症，因此为动脉粥样硬化的治疗提供了一种有前途的方法。
