华支睾吸虫NOSIP对小鼠单核巨噬细胞RAW264.7氧化应激作用的研究  

Effect of Clonorchis sinensis NOSIP on oxidative stress in mouse mononuclear macrophages RAW264.7

作　　者：边萌 周翰宗 朱宇 许青霞 BIAN Meng;ZHOU Hanzong;ZHU Yu;XU Qingxia(Department of Clinical Laboratory,the Affiliated Cancer Hospital of Zhengzhou University&Henan Cancer Hospital,Zhengzhou,Henan 450008,China;Department of Hepatobiliary Surgery,Zhengzhou People's Hospital,Zhengzhou,Henan 450008,China;Department of Clinical Laboratory,National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100021,China)

机构地区：[1]郑州大学附属肿瘤医院(河南省肿瘤医院)检验科,河南郑州450008 [2]郑州人民医院肝胆外科,河南郑州450008 [3]国家癌症中心/国家肿瘤临床医学研究中心/中国医学科学院北京协和医学院肿瘤医院检验科,北京100021

出　　处：《热带医学杂志》2024年第12期1676-1680,共5页Journal of Tropical Medicine

基　　金：河南省医学科技攻关计划省部共建重点项目(SBGJ202102070)。

摘　　要：目的探讨不同浓度的重组华支睾吸虫一氧化氮合酶相互作用蛋白(rCsNOSIP)对脂多糖(LPS)诱导小鼠巨噬细胞系RAW264.7氧化应激的作用的研究,为探究华支睾吸虫一氧化氮合酶相互作用蛋白(CsNOSIP)对宿主氧化应激作用奠定基础。方法将纯化蛋白rCsNOSIP去内毒素,取对数生长期的RAW264.7细胞,1×106个/孔接种于六孔板中,分为对照组、LPS组(1μg/mL)、LPS+5μg/mL rCsNOSIP组、LPS+10μg/mL rCsNOSIP组、LPS+20μg/mL rCsNOSIP组和LPS+无关蛋白组;其中LPS+5μg/mL rCsNOSIP组、LPS+10μg/mL rCsNOSIP组、LPS+20μg/mL rCsNOSIP组分别加入不同浓度的去内毒素的rCsNOSIP,终浓度分别为5、10、20μg/mL。分别与RAW264.7细胞作用24 h,用流式细胞仪检测细胞内活性氧(ROS)水平;Griess法测定各组细胞培养上清液中一氧化氮(NO)水平;应用丙二醛(MDA)和超氧化物歧化酶(SOD)检测试剂盒检测各组细胞培养上清液中MDA及SOD水平。结果与对照组比较,LPS组NO、ROS、MDA和SOD水平显著升高,差异均有统计学意义(t=8.139、14.300、4.578、4.717,P均<0.05);与LPS组比较,LPS+5μg/mL rCsNOSIP组、LPS+10μg/mL rCsNOSIP组、LPS+20μg/mL rCsNOSIP组NO、ROS、MDA均升高,差异均有统计学意义(t=5.807、13.110、15.320,9.900、11.980、17.440,5.048、10.960、13.830,P均<0.05);与LPS组比较,LPS+5μg/mL rCsNOSIP组、LPS+10μg/mL rCsNOSIP组、rCsNOSIP组、LPS+20μg/mL rCsNOSIP组SOD水平降低,差异均有统计学意义(t=7.111、12.090、11.780,P均<0.05)。结论CsNOSIP可能通过加重巨噬细胞氧化应激损伤,促进肝脏疾病的发生。Objectives To investigate the effect of different concentrations of recombinant Clonorchis sinensis nitric oxide synthase interacting protein(rCsNOSIP)on lipopolysaccharide(LPS)⁃induced oxidative stress in the mouse macrophage cell line RAW264.7.In order to lay a foundation for exploring the effect of Clonorchis sinensis nitric oxide synthase interacting protein(CsNOSIP)on host oxidative stress.Methods The purified rCsNOSIP was purified to remove endotoxin,and RAW264.7 cells in the logarithmic growth phase were seeded into 6⁃well plates at 1×106 cells/well and divided into control group,LPS group(1μg/mL),and LPS+5μg/mL rCsNOSIP group,LPS+10μg/mL rCsNOSIP group,LPS+20μg/mL rCsNOSIP group and LPS+irrelevant protein group;LPS+5μg/mL rCsNOSIP group,LPS+10μg/mL rCsNOSIP group and LPS+20μg/mL rCsNOSIP group were treated with LPS plus 5,10 or 20μg/mL of rCsNOSIP,respectively.After treating for 24 hours,flow cytometry was used to detect intracellular reactive oxygen species(ROS)levels;the Griess method was used to determine the nitric oxide(NO)levels in the cell culture supernatants of each group;malonaldehyde(MDA)and superoxide dismutase(SOD)detection reagents were used to detect the levels of MDA and SOD in the cell culture supernatant of each group.Results Compared with the control group,the levels of NO,ROS,MDA and SOD were significantly increased in the LPS group(t=8.139,14.300,4.578,4.717;all P<0.05);compared with the LPS group,the levels of NO,ROS,and MDA were all significantly increased in the LPS+5μg/mL rCsNOSIP group,LPS+10μg/mL rCsNOSIP group,and LPS+20μg/mL rCsNOSIP group(t=5.807,13.110,15.320;9.900,11.980,17.440;5.048,10.960,13.830;all P<0.05);compared with the LPS group,the SOD levels were all decreased in the LPS+5μg/mL rCsNOSIP group,LPS+10μg/mL rCsNOSIP group,LPS+20μg/mL rCsNOSIP group,and the differences were statistically significant(t=7.111,12.090,11.780;all P<0.05).Conclusion CsNOSIP might promote the occurrence of liver diseases by aggravating oxidative stress damage in macrophage

关 键 词：华支睾吸虫 一氧化氮合酶相互作用蛋白 氧化应激 RAW264.7细胞 

分 类 号：R383.2[医药卫生—医学寄生虫学]