磷酸三苯酯对小鼠巨噬细胞Raw264.7的毒性及吞噬功能的影响  

Effects of triphenyl phosphate on toxicity and phagocytosis of mouse macrophage RAW264.7

作　　者：张梓涵 李丽 张文锋 张艳超 陈慧玲 石明 ZHANG Zihan;LI Li;ZHANG Wenfeng;ZHANG Yanchao;CHEN Huiling;SHI Ming(School of Public Health,Guangdong Medical University,Dongguan Key Laboratory of Environmental Medicine,Dongguan 523808;Liaobu Hospital,Guangdong Medical University,Dongguan 523400,Guangdong,China)

机构地区：[1]广东医科大学公共卫生学院,东莞市环境医学重点实验室,广东东莞523808 [2]广东医科大学寮步医院,广东东莞523400

出　　处：《癌变．畸变．突变》2021年第4期269-275,共7页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：广东省自然科学基金(2018A030307009,2020A1515010521);广东省高等学校优秀青年教师培养计划(YQ2015082);广东医科大学学科建设项目(4SG21003G)。

摘　　要：目的:探讨磷酸三苯酯(TPHP)对巨噬细胞的毒性和机制,为评估TPHP暴露对免疫系统的影响和毒性风险提供参考。方法:分别用不同浓度(0、12.5、25、50、100、200、400μmol/L)的TPHP作用小鼠巨噬细胞系(Raw264.7)24、48或72 h后,采用CCK-8比色法和乳酸脱氢酶(LDH)释放法测定细胞活性,采用流式细胞术检测巨噬细胞吞噬荧光微球的能力,采用Western blot法检测Toll样受体4(TLR4)、丝/苏氨酸激酶(Akt)和哺乳动物雷帕霉素靶蛋白(mTOR)表达水平的变化。结果:与对照组相比,随着TPHP浓度和作用时间的增加,细胞存活率逐渐降低,TPHP对Raw264.7细胞的毒性作用呈剂量依赖性(r=0.960,P<0.05)和时间依赖性(r=0.980,P<0.05);50~400μmol/L组细胞培养基中LDH水平均明显升高(P<0.05);当TPHP浓度为25和50μmol/L时,FITCA+细胞的比例显著增加,说明细胞吞噬荧光微球的能力增加(P<0.05)。Western blot法检测结果显示,与对照组相比,12.5~50μmol/L TPHP处理组TLR4的表达水平均升高(P<0.05或P<0.01),并可触发Akt、mTOR蛋白的磷酸化。结论:TPHP对Raw264.7细胞呈现明显的细胞毒性,导致巨噬细胞的吞噬功能增强,并促进了TLR4、Akt和mTOR蛋白的表达。OBJECTIVE:To investigate immunotoxicity of triphenyl phosphate(TPHP)on mouse RAW264.7 in vitro.METHODS:Raw264.7 cells were treated with different concentrations of TPHP(0,12.5,25,50,100,200,400μmol/L)for 24,48 or 72 h.CCK-8 colorimetry and lactate dehydrogenase(LDH)release assays were used to determine cell activities.Flow cytometry was used to detect phagocytosis of macrophages to fluorescent microspheres.Western blot was used to detect changes in expression levels of Tolllike receptor 4(TLR4),serine/threonine kinase(Akt)and mammalian target of rapamycin(mTOR).RESULTS:Compared with the control group,survival rates of RAW264.7 cells were reduced together with increase of TPHP exposure concentrations and times,therefore,significant dose-(r=0.960,P<0.05)and time-dependent(r=0.980,P<0.05)toxicity.Levels of LDH in cell culture medium increased significantly when the exposure concentrations were 50-400μmol/L(P<0.05).When exposed to 25 and 50μmol/L of TPHP,percentages of FITC-A+cells were significantly increased,indicating significant increases of phagocytosis of fluorescent microspheres(P<0.05).Western blot results show that TPHP increased expression of TLR4 protein and triggered phosphorylation of Akt and mTOR(P<0.05 or P<0.01).CONCLUSION:TPHP exhibitedsignificant cytotoxicity to Raw264.7 cells,leading to enhanced phagocytosis of macrophages,and increased expressions of TLR4,Akt and mTOR.

关 键 词：磷酸三苯酯 巨噬细胞 吞噬作用 细胞毒性 

分 类 号：R114[医药卫生—卫生毒理学]