低水平铅和1-硝基芘联合暴露致小鼠下丘脑和垂体损伤中视黄酸通路的改变

Change of the retinoic acid pathway in hypothalamus and pituitary damage induced by combined exposure to low-level Pb2+and 1-nitropyrene in mice

目的观察低水平铅(Pb^(2+))和1-硝基芘(1-nitropyrene,1-NP)联合染毒致小鼠下丘脑和垂体损伤中视黄酸通路的表达,探讨该通路改变与下丘脑和垂体损伤的关系。方法将84只4周龄ICR小鼠按体重随机分为对照组、Pb^(2+)染毒组(0.008 mg/L)、1-NP染毒组(0.1 mg/kg)以及低(0.008 mg/L Pb^(2+)+0.004 mg/kg 1-NP)、中(0.008 mg/L Pb^(2+)+0.02 mg/kg 1-NP)、高剂量(0.008 mg/L Pb^(2+)+0.1 mg/kg 1-NP)联合染毒组,每组14只。其中Pb^(2+)由醋酸铅提供,添加于去离子水中,小鼠自由饮水;1-NP经腹腔注射。记录每日饮水量和摄食量;连续染毒21天后称体质量,光镜下观察下丘脑和垂体组织学改变、原子吸收法测定脑组织中铅含量;实时荧光定量PCR检测视黄酸通路成员及Jnks基因的丰度;Western Blot法检测乙醛脱氢酶2(acetaldehyde dehydrogenase 2,ALDH2)、细胞色素P450家族成员26A1(CYP26a1)蛋白的表达水平。结果各组小鼠每周平均饮水量和摄食量无差异。Pb^(2+)染毒各组小鼠脑组织Pb^(2+)含量高于对照组。高剂量联合染毒组小鼠体重[(27.4±1.9 g)]低于对照组[(29.8±2.3)g](P<0.05)。中、高剂量联合染毒组血清促卵泡激素水平[分别为(265.01±2.99)、(260.42±3.61)pg/mL]低于对照组[(279.00±1.30)pg/mL](P<0.05)。下丘脑和垂体组织中,中、高剂量联合染毒组Adh1、Adh2、Rar和Rxr基因丰度,ALDH2蛋白水平均高于对照组(P<0.05),低、中、高剂量联合染毒组CYP26a1基因丰度及蛋白水平均低于对照组(P<0.05),高剂量联合染毒组Jnks基因丰度均高于对照组(P<0.05)。结论0.008 mg/L Pb^(2+)+0.1 mg/kg 1-NP连续染毒21天可致小鼠下丘脑和垂体损伤,并激活视黄酸信号通路。

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 844-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).CONCLUSION 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.