Effect of maternal coal-burning fluorosis on neuronal nicotinic acetylcholine receptor protein and mRNA expression in brain tissue of offspring rats
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摘要:
目的 探讨母鼠燃煤氟中毒对仔鼠脑组织神经型尼古丁受体蛋白和mRNA表达的影响。 方法 将48只SD大鼠(雌鼠32只,雄鼠16只)按体重随机分为对照组、低、中、高剂量染氟组,每组8只,饲料含氟量分别为5.2、24.4、47.8、106.0 mg/kg。雄鼠不染氟,雌鼠从青春期开始染氟,3个月后雌雄大鼠按2 : 1合笼交配,仔鼠断乳后给予正常饲料。采用氟离子选择电极法检测仔鼠脑氟含量,以Morris水迷宫检测仔鼠学习记忆能力,蛋白印迹法测定仔鼠脑组织尼古丁受体的蛋白表达水平,实时荧光定量PCR检测仔鼠脑组织尼古丁受体的mRNA水平。 结果 与对照组比较,中、高剂量染氟组仔鼠脑氟含量增高(P < 0.05)。训练第3、4天,高剂量染氟组仔鼠的平均逃避潜伏期长于对照组(P < 0.05)。与对照组比较,中、高剂量染氟组仔鼠脑组织神经型尼古丁受体(nAChR)a7、a4、β2 mRNA水平降低(P < 0.05);各染氟组仔鼠脑组织nAChR a7、a4和β2蛋白表达均不同程度降低(P < 0.05)。 结论 母鼠燃煤氟中毒可引起仔鼠大脑神经型尼古丁受体蛋白和mRNA表达降低,可能是仔鼠学习记忆能力下降的机制之一。 -
关键词:
- 燃煤型氟中毒 /
- 仔鼠 /
- 神经型尼古丁受体(nAChR)
Abstract:Objective To study the effect of coal-burning fluorosis on protein and mRNA expression of neuronal nicotinic acetylcholine receptor (nAChR) in brain tissue of offspring rats. Methods Forty eight Sprague-Dawley (SD) rats (32 females and 16 males) were used in the study. The female rats were randomly divided into a control group and three (low, moderate, and high) fluoride treatment groups supplied with the feed containing fluoride at concentrations of 5.2, 24.4, 47.8, and 106.0 mg/kg continuously from puberty to lactation period. After 3-months′ fluoride treatment, the female rats of each groups were cohabited with 4 male rats. The females' offspring were fed with normal diet after weaning. Morris water maze test was performed for 30-day old offspring rats for evaluations of their learning and memory ability and the offspring rats′ brain tissues were collected for the determinations of nAChR protein with Western blot and nAChR mRNA with real-time fluorescent quantitative PCR. Results The brain tissue fluoride content of the offspring rats in moderate and high fluoride groups increased significantly compared to the offspring rats of the control group (both P < 0.05). On the 3rd and 4th day of Morris water maze test, the average escape latency of offspring rats of high fluoride group was significantly longer than that of the control offspring rats (P < 0.05). Significantly decreased expressions of nAChR a7, a4 and β2 mRNA in the offspring rats with moderate and high maternal fluoride exposure and nAChR a7, a4 and β2 protein in all the offspring rats with maternal fluoride exposure were detected in comparison with the offspring rats of control group (all P < 0.05). Conclusion Maternal fluoride exposure could reduce the expression of neural nicotine receptor protein and mRNA in brain tissue of offspring rats and the decreases may contribute to impacted learning and memory ability of the offspring rats. -
表 1 PCR引物
基因名称 引物序列 扩增片段
长度(bp)β-action 上游5′-CTGGAGAAACCTGCCAAGTATG-3′ 138 下游5′-GGTGGAAGAATGGGAGTTGCT-3′ a7 上游5′-GAAGTTTGGGTCCTGGTCCTATG-3′ 166 下游5′-GGTGACATCTGGGTATGGCTCT-3′ a4 上游5′-GACCCTGGTGACTACGAGAATGT-3′ 142 下游5′-TTCCGTCATAGAACAGGTGGG-3′ β2 上游5′-ATGCGAAGTGAGGATGATGACC-3′ 109 下游5′-TCCCAAAGACACAGACAAAGACA-3′ 表 2 母鼠染氟对仔鼠的平均逃避潜伏期影响(s,
$\bar x \pm s$ ,n = 10)组别(mg/kg) 第1天 第2天 第3天 第4天 对照组 56.13 ± 4.98 45.19 ± 5.20 30.26 ± 6.16 16.67 ± 4.43 染氟组 24.4 55.85 ± 5.41 46.91 ± 4.87 33.41 ± 5.49 19.09 ± 4.05 47.8 58.36 ± 6.47 48.39 ± 6.95 35.43 ± 5.63 23.32 ± 4.57 a 106.0 59.01 ± 6.60 50.06 ± 7.43 39.58 ± 6.99 ab 25.99 ± 5.88 ab 注:与对照组比较,a P < 0.05;与低剂量染氟组比较,b P < 0.05。 表 3 母鼠染氟对仔鼠脑组织nAChRS mRNA表达水平影响(
$\bar x \pm s$ ,n = 10)组别(mg/kg) a7 a4 β2 对照组 1.06 ± 0.10 1.05 ± 0.07 0.98 ± 0.07 染氟组 24.4 0.99 ± 0.05 0.98 ± 0.12 0.91 ± 0.13 47.8 0.88 ± 0.11 a 0.85 ± 0.20 a 0.85 ± 0.17 a 106.0 0.75 ± 0.18 abc 0.76 ± 0.17 ab 0.74 ± 0.08 ab 注:与对照组比较,a P < 0.05;与低剂量染氟组比较,b P < 0.05;与中剂量染氟组比较,c P < 0.05。 表 4 母鼠染氟对仔鼠脑组织nAChRS蛋白表达水平影响(
$\bar x \pm s$ ,n = 10)组别(mg/kg) a7 a4 β2 对照组 1.04 ± 0.14 0.77 ± 0.08 0.66 ± 0.08 染氟组 24.4 0.94 ± 0.06 a 0.65 ± 0.08 a 0.44 ± 0.09 a 47.8 0.72 ± 0.07 ab 0.37 ± 0.05 ab 0.40 ± 0.09 a 106.0 0.42 ± 0.08 abc 0.23 ± 0.05 abc 0.33 ± 0.06 ab 注:与对照组比较,a P < 0.05;与低剂量染氟组比较,b P < 0.05;与中剂量染氟组比较,c P < 0.05。 -
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