摘要
目的:磷脂酰肌醇-3-激酶-蛋白激酶B-哺乳动物雷帕霉素靶蛋白(phosphatidylinositol-3-kinase-protein kinase B-mammalian target of rapamycin,PI3K/Akt/mTOR)信号通路是自噬相关主要信号通路之一。自噬在硅沉着病纤维化形成过程中发挥关键作用。肺成纤维细胞向肌成纤维细胞表型转化是硅沉着病由炎症期进入纤维化期的标志之一。本研究旨在探讨PI3K/Akt/mTOR通路是否通过介导巨噬细胞自噬影响矽尘诱导的肺成纤维细胞向肌成纤维细胞表型转化。方法:采用100 ng/mL佛波酯诱导人单核细胞白血病细胞系THP-1细胞24 h获得巨噬细胞。以不同浓度(0、25、50、100、200、400μg/mL)二氧化硅(silicon dioxide,SiO_(2))粉尘悬浮液染毒巨噬细胞不同的时间(0、6、12、24、48 h)。采用细胞计数试剂盒-8(cell counting kit-8,CCK-8)法检测巨噬细胞存活率,酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)法检测巨噬细胞上清液中转化生长因子β1(transforming growth factor-β1,TGF-β1)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)的含量。采用Transwell技术建立巨噬细胞和HFL-1细胞共培养体系,设空白对照组、SiO_(2)组、LY294002组、SC79组、LY294002+SiO_(2)组、SC79+SiO_(2)组。LY294002+SiO_(2)组、SC79+SiO_(2)组分别用LY294002(PI3K抑制剂)、SC79(Akt激活剂)预处理巨噬细胞18 h、24 h,再用SiO_(2)(100μg/mL)粉尘悬浮液染毒巨噬细胞12 h。采用免疫荧光法检测巨噬细胞中微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)的表达情况;蛋白印迹法检测巨噬细胞PI3K、Akt、mTOR、Beclin-1、LC3的蛋白质表达水平及HFL-1细胞中Ⅲ型胶原蛋白(collagen Ⅲ,Col Ⅲ)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、纤维连接蛋白(fibronectin,FN)、基质金属蛋白酶-1(matrix metalloproteinase-1,MMP-1)、金属蛋白酶组织抑制因子-1(tissue matrix metalloproteinase inhibitor-1,TIMP-1)的蛋白质表达水平�
Objective:The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway is one of the main signaling pathways related to autophagy.Autophagy plays a key role in the formation of silicosis fibrosis.The phenotypic transformation of lung fibroblasts into myofibroblasts is a hallmark of the transition from the inflammatory phase to the fibrotic phase in silicosis.This study aims to investigate whether the PI3K/Akt/mTOR pathway affects the phenotypic transformation of silicosis-induced lung fibroblasts into myofibroblasts via mediating macrophage autophagy.Methods:The human monocytic leukemia cell line THP-1 cells were differentiated into macrophages by treating with 100 ng/mL of phorbol ester for 24 h.Macrophages were exposed to different concentrations(0,25,50,100,200,400μg/mL)and different times(0,6,12,24,48 h)of SiO_(2) dust suspension.The survival rate of macrophages was measured by cell counting kit-8(CCK-8)method.Enzyme linked immunosorbent assay(ELISA)was used to measure the contents of transforming growth factor-β1(TGF-β1)and tumor necrosis factor-α(TNF-α)in the cell supernatant.The co-culture system of macrophages and HFL-1 cells was established by transwell.A blank control group,a SiO_(2) group,a LY294002 group,a SC79 group,a LY294002+SiO_(2) group,and a SC79+SiO_(2) group were set up in this experiment.Macrophages in the LY294002+SiO_(2) group were pretreated with LY294002(PI3K inhibitor)for 18 hours,and macrophages in the SC79+SiO_(2) group were pretreated with SC79(Akt activator)for 24 hours,and then exposed to SiO_(2)(100μg/mL)dust suspension for 12 hours.The expression of microtubule-associated protein 1 light chain 3(LC3)protein in macrophages was detected by the immunofluorescence method.The protein expressions of PI3K,Akt,mTOR,Beclin-1,LC3 in macrophages,and collagen III(Col III),α-smooth muscle actin(α-SMA),fibronectin(FN),matrix metalloproteinase-1(MMP-1),tissue metalloproteinase inhibitor-1(TIMP-1)in HFL-1 cells were measured by Western blotting.Result
作者
杜悦
黄芳财
关岚
曾明
DU Yue;HUANG Fangcai;GUAN Lan;ZENG Ming(Department of Health Toxicology,Xiangya School of Public Health,Central South University,Changsha 410006,China)
出处
《中南大学学报（医学版）》
CAS
CSCD
北大核心
2023年第8期1152-1162,共11页
Journal of Central South University ：Medical Science
基金
国家自然科学基金(81673225)。
关键词
矽尘
肺成纤维细胞
肌成纤维细胞
巨噬细胞自噬
PI3K/Akt/mTOR通路
转化生长因子β1
肿瘤坏死因子α
silica dust
lung fibroblasts
myofibroblasts
macrophage autophagy
phosphatidylinositol-3-kinase-protein kinase B-mammalian target of rapamycin pathway
transforming growth factor-β1
tumor necrosis factor-α
