目的:观察低水平铅和1-硝基芘联合暴露对小鼠下丘脑维甲酸(RA)通路的表达及垂体损伤的影响。并探讨RA通路变化与下丘脑及垂体损害的关系。
方法:将84只4周龄ICR小鼠随机分为对照组,Pb~(2+)污染组(0.008mg/L),1-NP污染组(0.1mg/kg),低(0.008mg/LPb~(2+)+0.004mg/kg1-NP),培养基(0.008mg/LPb~(2+)+0.02mg/kg1-NP),高剂量共毒性组(0.008mg/LPb~(2+)+0.1mg/kg1-NP)按体重,每组14只小鼠。其中,Pb~(2+)由醋酸铅提供,添加到去离子水中,并由老鼠自由饮用,腹腔注射1-NP,通过腹膜内注射施用1-NP。记录每日水摄入量和食物摄入量。连续接触21天后,测量了体重,在光学显微镜下观察下丘脑和垂体的组织学变化,用原子吸收光谱法测定脑组织中的铅含量。实时荧光定量PCR检测维甲酸途径成员和c-JunN末端激酶基因(Jnks)的丰度,免疫印迹法检测乙醛脱氢酶2(ALDH2)的表达水平,细胞色素P450家族成员26A1(CYP26a1)蛋白。
结果:各组小鼠每周平均水摄入量和食物摄入量没有差异。高剂量共毒性组小鼠体重(27.4±1.9)g低于对照组(29.8±2.3)g(P<0.05)。中、高剂量共毒性组血清卵泡刺激素(FSH)水平(265.01±2.99),(260.42±3.61)pg/mL,分别)低于对照组((279.00±1.30)pg/mL,P<0.05)。各组含Pb~(2+)的脑组织中Pb~(2+)含量均高于对照组。在下丘脑和垂体组织中,Adh1,Adh2,Rar和Rxr的丰度,中、高剂量组的ALDH2水平高于对照组(P<0.05)。Cyp26a1基因丰度和蛋白水平在中、高剂量共毒组均低于对照组(P<0.05)。高剂量共毒性组的Jnks丰度高于对照组(P<0.05)。
结论:连续接触0.008mg/LPb~(2+)+0.1mg/kg1-NP21天可对小鼠下丘脑和垂体造成损伤,并激活RA信号通路。
OBJECTIVE: To observe the expression of the retinoic acid(RA) pathway in hypothalamus and pituitary damage induced by combined exposure of low-level lead and 1-nitropyrene in mice, and to explore the relationship between the changes of RA pathway and hypothalamus and pituitary damage.
METHODS: A total of 84 4-week-old ICR mice were randomly divided into the control group, Pb~(2+) tainted group(0.008 mg/L), 1-NP tainted group(0.1 mg/kg), low(0.008 mg/L Pb~(2+)+0.004 mg/kg 1-NP), medium(0.008 mg/L Pb~(2+)+0.02 mg/kg 1-NP), and high-dose co-toxicity group(0.008 mg/L Pb~(2+)+0.1 mg/kg 1-NP) according to body weight, with 14 mice in each group. Among them, Pb~(2+) was provided by lead acetate, added to deionized water and ingested by mice drinking freely, 1-NP was given by intraperitoneal injection, 1-NP was administered by intraperitoneal injection. Record daily water intake and food intake. After 21 consecutive days of exposure, body mass was measured, histological changes in the hypothalamus and pituitary were observed under an optical microscope, and lead content in brain tissue was measured by atomic absorption spectrometry. The real-time fluorescence quantitative PCR was used to detect the abundance of retinoic acid pathway members and c-Jun N-terminal kinases genes(Jnks), and the western blot method was used to detect expression levels of acetaldehyde dehydrogenase 2(ALDH2), cytochrome P450 family member 26A1(CYP26a1) proteins.
RESULTS: There was no difference in the mean weekly water intake and food intake of the mice in each group. The body weight of the high-dose co-toxicity group mice((27.4±1.9)g) was lower than that of the control group((29.8±2.3)g)(P<0.05). The level of serum follicle-stimulating hormone(FSH) in the middle and high dose co-toxicity groups((265.01±2.99), (260.42±3.61)pg/mL, respectively) was lower than that in the control group((279.00±1.30)pg/mL, P<0.05). The content of Pb~(2+) in the brain of each group containing Pb~(2+) was higher than that of the control group. In the hypothalamic and pituitary tissues, the abundance of Adh1, Adh2, Rar and Rxr, and ALDH2 levels in the medium and high dose co-toxicity groups were higher than those in the control group(P<0.05). Cyp26a1 gene abundance and protein levels were lower in the medium and high dose co-toxicity groups than in the control group(P<0.05). The abundance of Jnks in the high-dose co-toxicity group was higher than that in the control group(P<0.05).
CONCLUSIONS: Continuous exposure to 0.008 mg/L Pb~(2+)+0.1 mg/kg 1-NP for 21 days can cause damage to the hypothalamus and pituitary of mice, and activate the RA signaling pathway.