磷酸三苯酯对小鼠胸腺组织免疫毒性作用与机制研究

Study on the immunotoxicity effect of triphenyl phosphate on thymus and its mechanism in mice

目的 观察磷酸三苯酯(TPHP)对小鼠胸腺组织的免疫毒性作用,并分析相关机制。方法 将无特定病原体级BALB/c小鼠随机分为对照组和低、中、高剂量组,每组12只,雌雄各半。4组小鼠分别予剂量为0、1、10和150 mg/kg体质量的TPHP灌胃,1次/d,连续60 d。染毒结束后,检测小鼠血常规,采集胸腺组织,计算胸腺脏器系数,观察胸腺组织病理学改变情况。采用实时定量聚合酶链式反应法检测胸腺组织中炎症、氧化应激、细胞自噬与凋亡相关基因的表达。结果 染毒期间,高剂量组雄鼠出现毛发状态较差、胡须脱落和易激惹;而雌鼠未见此现象。染毒结束时,各组小鼠体质量和胸腺脏器系数未见有统计学意义的改变;但高剂量组雄鼠胸腺组织出现细胞凋亡的改变。与对照组雄鼠比较,高剂量组雄鼠白细胞计数、淋巴细胞计数、中性粒细胞计数、红细胞分布宽度、血小板计数和血小板压积均下降(P值均<0.05)。低剂量组雄鼠胸腺组织中白细胞介素(Il)-1β、Il-6、过氧化氢酶(Cat)和P62的mRNA相对表达水平和B淋巴细胞瘤-2(Bcl-2)相关X蛋白(Bax)/Bcl-2比值均升高(P值均<0.05),中剂量组雄鼠胸腺组织中核因子E2相关因子2(Nrf2)、超氧化物歧化酶1(Sod1)、谷胱甘肽过氧化物酶亚基1(Gpx1)、P62的mRNA相对表达水平和Bax/Bcl-2比值均升高(P值均<0.05),高剂量组雄鼠胸腺组织中Nrf2、Cat、Sod1、Gpx1、P62、半胱氨酸天冬氨酸蛋白酶-3的mRNA相对表达水平和Bax/Bcl-2比值均升高(P值均<0.05)。与对照组雌鼠比较,低、中剂量组雌鼠胸腺组织中Il-1β mRNA相对表达水平和Bax/Bcl-2比值均升高(P值均<0.05),高剂量组雌鼠胸腺组织中干扰素-γ、Nrf2、Cat、P62、微管相关蛋白1轻链3的mRNA相对表达水平和Bax/Bcl-2比值均升高(P值均<0.05)。结论 TPHP暴露虽然对小鼠体质量、胸腺脏器系数和组织病理学尚未造成显著影响,但可引起胸腺组织氧化应激相关指标的变化,促进小鼠胸腺组织的细胞自噬、凋亡和炎症的发生;且存在性别差异。

Objective To investigate the immunotoxicity effect of triphenyl phosphate(TPHP) on thymus tissue of mice, and analyze the related mechanism. Methods Specific pathogen free BALB/c mice were randomly divided into control group, low-, medium-and high-dose groups, with 12 mice per group(equal gender distribution). Mice in these four groups were orally administered doses of 0, 1, 10, and 150 mg/kg body weight of TPHP daily for 60 days. After the exposure, the complete blood count of mice was detected, thymus tissue was collected, coefficient of thymus organs was calculated, and the histopathology changes of thymus were observed. Real-time quantitative polymerase chain reaction was used to assess the expression of genes related to inflammation, oxidative stress, cellular autophagy, and apoptosis in thymic tissues. Results During the exposure period, male mice in the high-dose group had poor fur condition, whisker loss, and increased irritability, while these phenomena were not observed in female mice. At the end of the exposure period, there were no significant changes in mice body weight or thymus organ coefficients among the groups. However, male mice in the high-dose group showed cellular apoptotic changes in the thymic tissue. The amount of white blood cell, lymphocyte, neutrophil granulocyte, red blood cell distribution width, platelet and the plateletcrit of male mice was lower in the high-dose group than that in the control group(all P<0.05).The relative mRNA expression of interleukin(Il)-1β, Il-6, catalase(Cat), P62, as well as the ratio of B-cell lymphoma 2(Bcl-2) associated X protein(Bax)/Bcl-2 in thymic tissue of male mice were higher in the low-dose group than that in the control group(all P<0.05). The relative mRNA expression of nuclear factor erythroid-2 related factor 2(Nrf2), superoxide dismutase 1(Sod1), glutathione peroxidase 1(Gpx1), P62, as well as the ratio of Bax/Bcl-2 in the thymic tissue of male mice were higher in the medium-dose group than that in the control group(all P<0.05). The relative mRNA expression of Nrf2, Cat, Sod1, Gpx1, P62, cysteinyl aspartate specific proteinase-3, as well as the ratio of Bax/Bcl-2 in the thymic tissue of male mice were higher in the high-dose group than that in the control group(all P<0.05). The relative mRNA expression of Il-1β and the ratio of Bax/Bcl-2 in thymic tissue of female mice were higher in the low-and medium-dose group(all P<0.05), while the relative mRNA expression of interferon-γ, Nrf2, Cat, P62, microtubule-associated protein light chain 3, as well as the ratio of Bax/Bcl-2 in thymic tissue of female mice were higher in the high-dose group than that in the control group(all P<0.05).Conclusion Although TPHP exposure had not significantly affected the body weight, thymus organ coefficient and histopathology of mice, it induced changes in oxidative stress-related indicators in thymic tissue, promoted cellular autophagy, apoptosis, and inflammation in the thymic tissue, with observed gender difference.