二氧化硫体内衍生物通过RO...釉器细胞AE2蛋白表达上调_任洪玥.pdf

生态毒理学报Asian Journal of Ecotoxicology第 17 卷 第 6 期 2022 年 12 月Vol.17,No.6 Dec.2022 基金项目:国家自然科学基金资助项目(41967051,42167059)第一作者:任洪玥(1996),女,硕士研究生,研究方向为环境毒理学,E-mail: *通信作者(Corresponding author),E-mail:DOI:10.7524/AJE.1673-5897.20220110001任洪玥,杨进,刘霞,等.二氧化硫体内衍生物通过 ROS/JNK/AP-1 通路诱导大鼠切牙成釉器细胞 AE2 蛋白表达上调J.生态毒理学报,2022,17(6):256-265Ren H Y,Yang J,Liu X,et al.Internal derivatives of sulfur dioxide induced up-regulation of AE2 protein expression of rat enamel organ through ROS/JNK/AP-1 pathway J.Asian Journal of Ecotoxicology,2022,17(6):256-265(in Chinese)二氧化硫体内衍生物通过 ROS/JNK/AP-1 通路诱导大鼠切牙成釉器细胞 AE2 蛋白表达上调任洪玥1,2,杨进1,2,刘霞1,2,姚杰1,2,林昌虎1,涂成龙1,2,3,*1.贵州医科大学公共卫生与健康学院,贵阳 5500252.贵州医科大学环境污染与疾病监控教育部重点实验室,贵阳 5500253.贵州医科大学毒性检测中心,贵阳 550025收稿日期:2022-01-10 录用日期:2022-02-21摘要:探究二氧化硫体内衍生物混合液(亚硫酸钠与亚硫酸氢钠物质的量比为 31)对雄性大鼠切牙组织抗氧化防御系统及切牙成釉器细胞丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)信号转导通路关键蛋白与阴离子交换蛋白 2(ani-on exchanger 2,AE2)表达的影响。30 只雄性 Wistar 大鼠按体质量随机分为高(100 mgkg-1d-1)、中(50 mgkg-1d-1)、低(25mg kg-1 d-1)二氧化硫衍生物混合液染毒组、高剂量二氧化硫衍生物混合液+N-乙酰半胱氨酸(N-acetylcysteine,NAC)染毒组及生理盐水对照组(0.9%氯化钠注射液)。各组均以 2 mL kg-1每日行腹腔注射一次,高剂量+NAC 组每次腹腔注射前 30 min予 200 mg kg-1NAC 水溶液灌胃再施予高剂量组相同染毒操作,连续 4 周。取大鼠下颌骨切牙组织进行超氧化物歧化酶(su-peroxide dismutase,SOD)活性、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性、还原型谷胱甘肽(reduced glutathione,GSH)及丙二醛(malondialdehyde,MDA)含量、活性氧簇(reactive oxygen species,ROS)水平测定。剥离包绕切牙根部的下颌骨分离成釉器细胞,以 Western blot 法检测成釉器细胞 MAPKs 信号通路关键蛋白及 AE2 蛋白表达情况。结果显示,低剂量染毒组与对照组比较,大鼠切牙组织多类抗氧化指标及过氧化产物含量的改变均不具统计学意义。中、高剂量染毒组较对照组大鼠切牙组织 SOD 活性、GSH-Px 活性及 GSH 含量下降、MDA、ROS 水平升高,差异有统计学意义(P0.05)。Western blot 结果显示,随二氧化硫衍生物剂量升高,各组大鼠成釉器细胞 p-p38 MAPK/p38 MAPK 及 p-ERK/ERK 水平较对照组未见明显变化,而中、高剂量组 p-JNK/JNK 水平明显升高,AP-1 蛋白发生核转位分布,AE2 蛋白表达明显升高,差异均有统计学意义(P0.05);而 ROS 抑制剂 NAC 能使高剂量下 AP-1 蛋白核转位情况得到改善,并能在一定程度上逆转高剂量二氧化硫衍生物暴露下引起的 p-JNK/JNK 水平升高及 AE2 蛋白表达上调。以上结果提示,二氧化硫体内衍生物混合液可造成大鼠切牙组织氧化应激水平升高并通过 ROS/JNK/AP-1 通路诱导切牙成釉细胞 AE2 表达上调,具有干扰成釉器细胞成釉功能的潜在毒性。关键词:二氧化硫体内衍生物;成釉器细胞;氧化应激;丝裂原活化蛋白激酶;激活蛋白-1;阴离子交换蛋白 2文章编号:1673-5897(2022)6-256-10 中图分类号:X171.5 文献标识码:AInternal Derivatives of Sulfur Dioxide Induced Up-regulation of AE2 Pro-tein Expression of Rat Enamel Organ through ROS/JNK/AP-1 PathwayRen Hongyue1,2,Yang Jin1,2,Liu Xia1,2,Yao Jie1,2,Lin Changhu1,Tu Chenglong1,2,3,*1.School of Public Health,Guizhou Medical University,Guiyang 550025,China2.Key Laboratory of Environmental Pollution Monitoring and Disease Control,Ministry of Education,Guizhou Medical University,第 6 期任洪玥等:二氧化硫体内衍生物通过 ROS/JNK/AP-1 通路诱导大鼠切牙成釉器细胞 AE2 蛋白表达上调257 Guiyang 550025,China3.Toxicity Testing Center of Guizhou Medical University,Guiyang 550025,ChinaReceived 10 January 2022 accepted 21 February 2022Abstract:To investigate the effects of sulfur dioxide derivative mixture(molar ratio of sodium sulfite to sodiumbisulfite is 31)on antioxidant defense system and expression of key proteins in mitogen-activated protein kinasesignal transduction pathway and anion exchanger protein 2(AE2)in enamel organ of incisors of male rats.Thirtymale Wistar rats were randomly divided into high(100 mg kg-1d-1),medium(50 mgkg-1d-1),low(25 mgkg-1d-1)sulfur dioxide derivative mixture group,high-dose sulfur dioxide derivative mixture+N-acetylcysteine(NAC)group and normal saline control group(0.9%sodium chloride injection).Each group received intraperitone-al injection every day with a volume of 2 mL kg-1for four weeks.The high-dose+NAC group was given 200 mg kg-1NAC solution 30 min before each intraperitoneal injection.The activities of superoxide dismutase(SOD)andglutathione peroxidase(GSH-Px),contents of reduced glutathione(GSH)and malondialdehyde(MDA),and levelsof reactive oxygen species(ROS)of incisors tissue were determined by assay kit.The enamel organ was separatedfrom the root of the incisor,and the expression of key proteins in mitogen-activated protein kinase signal transduc-tion pathway and AE2 of enamel organ was determined by Western blot.The results showed that compared withthe control group,there was no statistical significance in the changes of antioxidant indexes and content of peroxideproducts in the incisor tissue of rats in the low-dose group,while SOD activity,GSH-Px activity and GSH contentin the incisor tissue of rats in the medium-dose group and the high-dose group were decreased and level of MDAand ROS were increased.The difference was statistically significant(P0.05).As for the protein expression,West-ern blot results showed that with the increase of sulfur dioxide derivative dose,the levels of p-p38 MAPK/p38MAPK and p-ERK/ERK did not change significantly in each group compared with the control group,while thelevels of p-JNK/JNK in medium and high-dose groups increased significantly and expression of AE2 protein in-creased with nuclear translocation distribution of AP-1 protein occurred(P0.05).NAC,a ROS inhibitor can im-prove the nuclear translocation of AP-1 protein and partly reverse the elevated p-JNK/JNK level and the up-regula-tion of AE2 protein expression induced by exposure to high dose of sulfur dioxide derivatives.These results sug-ge