摘要
目的:探讨苦味受体2型味觉受体(T2R)38激活对香烟烟雾暴露诱导人气道上皮NuLi-1细胞铁死亡的影响,并阐明其可能的机制。方法:人气道上皮NuLi-1细胞分为对照组(不进行任何处理)、香烟提取物(CSE)组(5%CSE处理细胞24 h)和CSE+T2R38特异性激动剂苯基硫脲(PTC)组(CSE+PTC组)(5%CSE和1 mmo·l L-1 PTC处理细胞24 h)。采用实时荧光定量PCR(RT-qPCR)法和Western blotting法检测各组NuLi-1细胞中T2R38的mRNA及蛋白表达水平,细胞计数试剂盒8(CCK-8)法检测各组细胞活性,试剂盒检测各组细胞中诱导型一氧化氮合酶(iNOS)、内皮型NOS(eNOS)和超氧化物歧化酶(SOD)活性,DAX-J2红色荧光探针法检测各组细胞中一氧化氮(NO)水平,荧光探针试剂盒检测各组细胞中ROS水平,酶联免疫吸附测定(ELISA)法检测各组细胞中丙二醛(MDA)、Fe2+和还原型谷胱甘肽(GSH)水平,Western blotting法检测各组细胞中核因子红细胞2相关因子2(Nrf2)和谷胱甘肽过氧化物酶4(GPx4)蛋白表达水平。结果:与对照组比较,CSE组NuLi-1细胞中T2R38 mRNA和蛋白表达水平升高(P<0.05)。与对照组比较,CSE组NuLi-1细胞活性降低(P<0.05),细胞中iNOS和SOD活性升高(P<0.05),NO和ROS水平升高(P<0.05),MDA和Fe2+水平升高(P<0.05),细胞中GSH水平降低(P<0.05),Nrf2和GPx4蛋白表达水平降低(P<0.05)。与CSE组比较,CSE+PTC组NuLi-1细胞活性升高(P<0.05),细胞中SOD活性升高(P<0.05),GSH水平升高(P<0.05),细胞中iNOS活性降低(P<0.05),NO和ROS水平降低(P<0.05),MDA和Fe2+水平降低(P<0.05),细胞中Nrf2和GPx4蛋白表达水平升高(P<0.05)。对照组、CSE组和CSE+PTC组细胞中eNOS活性比较差异无统计学意义(P>0.05)。结论:激活苦味受体T2R38可抑制香烟烟雾暴露诱导人气道上皮NuLi-1细胞铁死亡,其主要机制可能与降低细胞中iNOS活性有关。
Objective:To investigate the effect of type 2 taste receptor(T2R)38 activation on ferroptosis of human airway epithelium NuLi-1 cells induced by cigarette smoke exposure,and to clarify its possible mechanism.Methods:The human airway epithelial NuLi-1 cells were divided into control group(without any treatment),cigarette smoke extract(CSE)group(treated with 5%CSE for 24 h)and CSE+T2R38 specific agonist phenylthiocarbamide(PTC)group(CSE+PTC group)(treated with 5%CSE and 1 mmol·L^(-1)PTC for 24 h).The expression levels of T2R38 mRNA and protein in NuLi-1 cells in various groups were determined by real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods.The cell viabilities in various groups were determined by cell counting kit-8(CCK-8)assay.The activities of inducible nitric oxide synthase(iNOS),endothelial nitric oxide synthase(eNOS),and superoxide dismutase(SOD)in the cells in various groups were measured by kits.DAX-J2 red fluorescence probe was used to determine the levels of nitric oxide(NO)in the cells in various groups.The reactive oxygen species(ROS)levels in the cells in various groups were detected by fluorescent probe kit.The levels of malondialdehyde(MDA),Fe^(2+),and reduced glutathione(GSH)in the cells in various groups were determined by enzyme-linked immunosorbent assay(ELISA)method.Western blotting method was used to determine the expression levels of nuclear factor erythroid 2-related factor 2(Nrf2)and glutathione peroxidases 4(GPx4)proteins in the cells in various groups.Results:Compared with control group,the expression levels of T2R38 mRNA and protein in NuLi-1 cells in CSE group were increased(P<0.05).Compared with control group,the viability of NuLi-1 cells in CSE group was decreased(P<0.05),the activities of iNOS and SOD in cells in CSE group were increased(P<0.05),the levels of NO and ROS were increased(P<0.05),the levels of MDA and Fe^(2+)were increased(P<0.05),and the GSH level and the expression levels of Nrf2 and GPx4 proteins were decreased.Compared with CSE group,the viability of NuLi-1 cells in CSE+PTC group was increased(P<0.05),the activity of SOD and the GSH level in the cells were increased(P<0.05),the activity of iNOS in cells was decreased(P<0.05),the levels of NO and ROS in cells were decreased(P<0.05),the levels of MDA and Fe^(2+)were decreased(P<0.05),and the expression levels of Nrf2 and GPx4 proteins were increased(P<0.05).There was no significant difference in eNOS activity among control group,CSE group,and CSE+PTC group(P>0.05).Conclusion:Activation of bitter taste receptor T2R38 can inhibit ferroptosis in human airway epithelium NuLi-1 cells induced by cigarette smoke exposure,and its mechanism may be related to the reduction of iNOS activity in the cells.
作者
李亮
周向东
王杰
徐超群
朱梦霞
余善君
李琪
LI Liang;ZHOU Xiangdong;WANG Jie;XU Chaoqun;ZHU Mengxia;YU Shanjun;LI Qi(Department of Respiratory and Critical Care Medicine,First Affiliated Hospital,Hainan Medical University,Hainan Provincial Clinical Medical Center for Respiratory Diseases,Haikou 570100,China)
出处
《吉林大学学报(医学版)》
北大核心
2025年第2期333-340,共8页
Journal of Jilin University:Medicine Edition
基金
国家自然科学基金项目(81860001,82160012,82260001)
海南省科技厅重点研发计划国际科技合作项目(GHYF2022011)
海南省科技厅重点研发项目(ZDYF2020223)
海南省科技厅重大科技专项(ZDKJ2021036)
海南省科技厅创新团队项目(820CXTD448)
海南省卫健委卫生健康科技创新联合项目(WSJK2024QN071)。
关键词
慢性阻塞性肺疾病
人气道上皮细胞
苦味受体
2型味觉受体38
诱导型一氧化氮合酶
铁死亡
Chronic obstructive pulmonary disease
Human airway epithelium cell
Bitter taste receptor
Type 2 taste receptor 38
Inducible nitric oxide synthase
Ferroptosis
