6：2多氟烷基磷酸二酯通过Keap1/Nrf2/HO-1通路对大鼠和小鼠的甲状腺损伤效应比较研究

摘要/Abstract

摘要：

目的研究不同剂量6：2多氟烷基磷酸二酯（6：2 polyfluoroalkyl phosphate diester，6：2diPAP）暴露对大鼠和小鼠的甲状腺损伤，探究6：2diPAP对甲状腺功能障碍及氧化应激的影响及可能的机制。方法将雌性SD大鼠和雌性C57BL/6小鼠各15只，随机分为空白对照组、溶剂对照组，6：2diPAP低、中、高暴露组（0.5、5.0、50.0 μg/kg），每组3只，连续灌胃染毒21天。通过酶联免疫吸附试验法（enzyme-linked immunosorbnent assay，ELISA）检测血清中游离三碘甲腺原氨酸（free triiodothyronine，FT3）、游离甲状腺素（free thyroxine，FT4）、促甲状腺激素（thyrotropin，TSH）水平，化学比色法测定甲状腺组织中超氧化物歧化酶（superoxide dismutase，SOD）、过氧化氢酶（catalase，CAT）活性及丙二醛（malondialdehyde，MDA）含量，并利用western blot检测探讨6：2diPAP暴露致甲状腺损伤可能的作用机制。结果 6：2diPAP暴露可造成大鼠和小鼠甲状腺功能紊乱。ELISA检测结果显示，与空白对照组及溶剂对照组相比，6：2diPAP高暴露可显著降低大鼠血清中FT3[（5.49±0.70）pg/mL]和FT4[（8.66±0.43）pg/mL]水平，同时TSH[（9.13±1.33）ng/mL]含量上升（均P<0.05）。而小鼠上述激素指标未见统计学差异（P>0.05）。进一步分析甲状腺组织氧化应激指标发现，6：2diPAP高暴露可显著抑制大鼠甲状腺组织中SOD[（82.49±4.27）U/mgprot]和CAT[（1.26±0.19）U/mgprot]活性，并导致MDA[（2.34±0.24）μmol/gprot]浓度显著升高（均P<0.05）。小鼠SOD[（13.90±1.48）U/mgprot]、CAT[（9.72±1.14）U/mgprot]呈下降趋势且MDA[（2.15±0.10）μmol/gprot]呈上升趋势，但未见统计学差异（P>0.05）。Western blot检测结果表明，6：2diPAP高暴露可显著下调大鼠甲状腺组织中Kelch样ECH相关蛋白（kelch-like ECH-associated protein 1，Keap1）（0.73±0.12）、核因子E2-相关因子2（nuclear factor E2-related factor 2，Nrf2）（0.60±0.29）及血红素氧合酶-1（heme oxygenase-1，HO-1）（0.66±0.07）蛋白表达水平（P<0.05）。小鼠甲状腺组织Nrf2（0.55±0.27）蛋白表达亦显著降低（P<0.05）。结论 6：2diPAP暴露可能通过刺激氧化应激从而引发甲状腺功能紊乱，其中以大鼠表现更为敏感。其作用机制可能与Keap1/Nrf2/HO-1抗氧化防御通路受抑、抗氧化能力下降相关。

关键词: 多氟烷基磷酸二酯, 甲状腺损伤, 甲状腺激素, 氧化应激, Keap1/Nrf2/HO-1通路

Abstract:

Objective To investigate thyroid damage in rats and mice exposed to different doses of 6：2 polyfluoroalkyl phosphate diester（6：2diPAP），explore the effects of 6：2diPAP on thyroid dysfunction and oxidative stress and the possible mechanisms. Methods Fifteen female SD rats and 15 female C57BL/6 mice were randomly divided into a blank control group，a solvent control group，and a 6：2 diPAP low，medium，and high exposure group（0.5，5.0 and 50.0 μg/kg），with three mice in each group，and were dyed by gavage for 21 days. The levels of free triiodothyronine（FT3），free thyroxine（FT4），and thyrotropin（TSH） in serum were measured by enzyme-linked immunosorbnent assay（ELISA），and the activities of superoxide dismutase（SOD） and catalase（CAT） and the content of malondialdehyde（MDA） in thyroid tissues were determined by chemical assay kits，and the possible mechanism of action of thyroid damage caused by the exposure of 6：2diPAP was investigated using Western blot assay. Results Exposure to 6：2diPAP caused thyroid dysfunction in rats and mice. The ELISA results showed that，compared to the control and solvent control groups，high exposure to 6：2diPAP decreased serum levels of FT3[（5.49±0.70）pg/mL] and FT4[（8.66±0.43）pg/mL]，and increased TSH[（9.13±1.33）ng/mL] levels in rats（P<0.05）. In mice，no statistically significant differences were observed in these hormone indicators（P>0.05）.Further analysis showed that high exposure to 6∶2diPAP significantly inhibited SOD[（82.49±4.27）U/mgprot] and CAT[（1.26±0.19）U/mgprot] activities and led to increased MDA[（2.34±0.24）μmol/gprot] concentrations in rat thyroid tissue（P<0.05）. In mice，SOD[（13.90±1.48）U/mgprot] and CAT[（9.72±1.14）U/mgprot] showed a downward trend and MDA[（2.15±0.10）μmol/gprot] showed an upward trend，but the differences were not statistically significant（P>0.05）. Western blot results showed that 6∶2diPAP high exposure significantly down-regulated the expression levels of kelch-like ECH-associated protein 1（Keap1）（0.73±0.12），nuclear factor E2-related factor 2（Nrf2）（0.60±0.29），and heme oxygenase-1 （HO-1）（0.66±0.07） proteins in the thyroid tissues of rats（P<0.05）.Nrf2（0.55±0.27） protein expression in mouse thyroid tissue was also significantly decreased（P<0.05）. Conclusions 6∶2diPAP exposure may trigger thyroid dysfunction by stimulating oxidative stress，with rats showing greater sensitivity. The underlying mechanism of action may be related to the suppression of the Keap1/Nrf2/HO-1 antioxidant defense pathway and a consequent reduction in antioxidant capacity.

Key words: Polyfluoroalkyl phosphate diesters, Thyroid injury, Thyroid hormones, Oxidative stress, Keap1/Nrf2/HO-1 pathway

中图分类号:

R-332

张新轩, 齐鑫鑫, 从美丽, 刘金存, 孙红光, 刘涛. 6：2多氟烷基磷酸二酯通过Keap1/Nrf2/HO-1通路对大鼠和小鼠的甲状腺损伤效应比较研究. [J]职业与健康, 2026, 42(8): 1057-1064.

ZHANG Xinxuan, QI Xinxin, CONG Meili, LIU Jincun, SUN Hongguang, LIU Tao. Comparative study of thyroid damaging effects of 6：2 polyfluoroalkyl phosphate diester in rats and mice via the Keap1/Nrf2/HO-1 pathway. [J]OCCUPATION AND HEALTH, 2026, 42(8): 1057-1064.

图/表 5

图1 6：2diPAP暴露对大鼠及小鼠甲状腺组织病理学影响（HE，×400）注：图A（a~e）为大鼠甲状腺；图B（a~e）为小鼠甲状腺；a—高浓度组；b—中浓度组；c—低浓度组；d—溶剂对照组；e—空白对照组。

图2 6：2diPAP暴露对大鼠及小鼠脏器系数的影响（n=3）注：图A（a~b）大鼠脏器系数；图B（a~b）小鼠脏器系数。

图3 6：2diPAP 暴露对大鼠及小鼠甲状腺激素水平的影响（n=3）注：FT3—游离三碘甲状腺原氨酸；FT4—游离甲状腺素；TSH—促甲状腺素；图A（a~c）为大鼠甲状腺激素水平；图B（a~c）为小鼠甲状腺激素水平；a与空白对照组相比，P约0.05；b与溶剂对照组相比，P约0.05。

图4 6：2diPAP暴露对大鼠及小鼠甲状腺组织氧化损伤水平的影响（n=3）注：SOD—超氧化物歧化酶；CAT—过氧化氢酶；MDA—丙二醛；图A（a~c）为大鼠甲状腺组织氧化损伤水平；图B（a~c）为小鼠甲状腺组织氧化损伤水平；a与空白对照组相比，P约0.05；b与溶剂对照组相比，P约0.05。

图5 6：2diPAP暴露对大鼠及小鼠甲状腺组织相关蛋白表达水平的影响（n=3）注：Keap1—Kelch样ECH相关蛋白；Nrf2—核因子E2-相关因子2；HO-1—血红素氧合酶-1；β-actin—β-肌动蛋白；图A（a~d）为大鼠甲状腺组织蛋白表达水平；图B（a~d）为小鼠甲状腺组织蛋白表达水平；a与空白对照组相比，P<0.05；b与溶剂对照组相比，P<0.05。

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