黄红茜, 李凤敏, 张爱华, 韩雪. 肠道菌群失调对甲基汞染毒小鼠组织汞及粪汞含量的影响[J]. 环境与职业医学, 2021, 38(5): 524-529. DOI: 10.13213/j.cnki.jeom.2021.20532
引用本文: 黄红茜, 李凤敏, 张爱华, 韩雪. 肠道菌群失调对甲基汞染毒小鼠组织汞及粪汞含量的影响[J]. 环境与职业医学, 2021, 38(5): 524-529. DOI: 10.13213/j.cnki.jeom.2021.20532
HUANG Hongqian, LI Fengmin, ZHANG Aihua, HAN Xue. Effects of intestinal flora dysregulation on mercury concentrations in tissues and feces in methylmercury treated mice[J]. Journal of Environmental and Occupational Medicine, 2021, 38(5): 524-529. DOI: 10.13213/j.cnki.jeom.2021.20532
Citation: HUANG Hongqian, LI Fengmin, ZHANG Aihua, HAN Xue. Effects of intestinal flora dysregulation on mercury concentrations in tissues and feces in methylmercury treated mice[J]. Journal of Environmental and Occupational Medicine, 2021, 38(5): 524-529. DOI: 10.13213/j.cnki.jeom.2021.20532

肠道菌群失调对甲基汞染毒小鼠组织汞及粪汞含量的影响

Effects of intestinal flora dysregulation on mercury concentrations in tissues and feces in methylmercury treated mice

  • 摘要: 背景

    肠道微生物可通过直接或间接作用影响重金属的代谢吸收,但其在甲基汞代谢吸收中的作用及机制尚不明确。

    目的

    探究肠道菌群对甲基汞吸收、分布和排泄的影响及可能的机制。

    方法

    20只雌性BALB/c小鼠随机分为空白组、菌群失调组、甲基汞组、菌群失调+甲基汞组(n=5)。对小鼠灌胃抗生素混合液(万古霉素100 mg·kg-1、硫酸新霉素200 mg·kg-1、甲硝唑200 mg·kg-1、氨苄青霉素200 mg·kg-1,以体重计,后同,1次·d-1,10 mL·kg-1)4 d构建肠道菌群紊乱模型。通过PCR验证细菌16S rRNA的V4区,判断菌群失调模型是否建立成功。对小鼠进行甲基汞染毒3 d(0.5 mg·kg-1,1次·d-1,灌胃量10 mL·kg-1),收集第一次甲基汞染毒后24、48、72h的小鼠粪便,实验结束处死小鼠,收集血、肝脏、肾脏、脑、结肠及盲肠内容物样本。原子荧光光谱分析法测定肝、肾、脑、血、粪便中的总汞含量;pH计测定结肠及盲肠内容物pH值;HE染色法观察肠道组织病理改变;反转录PCR法检测肠道紧密连接蛋白OccludinZo-1Claudin-1 mRNA表达水平。

    结果

    紫外光下细菌16S rRNA通用引物PCR扩增产物电泳结果显示,空白组小鼠具有良好的16S rRNA基因信号,抗生素处理的小鼠未检测到明显信号,表明抗生素处理清除了小鼠肠道内的细菌,即肠道菌群失调建模成功。第一次甲基汞暴露后24h、48h和72h,菌群失调+甲基汞组粪汞含量(90.39±27.56)、(366.75±81.82)、(641.33±354.24)ng·g-1低于甲基汞组(259.87±90.94)、(616.83±197.90)、(1322.50±377.77)ng·g-1P < 0.05)。菌群失调+甲基汞组肝、肾、脑、血总汞含量(4 090.38±929.44)、(8 539.97±1 242.64)、(1 348.24±336.69)、(40 290.00±5 515.11)ng·g-1高于甲基汞组(2 752.86±566.09)、(4 502.08±105.76)、(827.54±106.50)、(28 392.00±5 813.25)ng·g-1P < 0.05)。与未给抗生素小鼠比较,抗生素处理小鼠盲肠及结肠内容物pH值升高(P < 0.05)。HE染色显示菌群失调组具有肠绒毛坏死溶解,上皮细胞脱落,固有层轻微水肿等改变。与未给抗生素小鼠比较,抗生素处理小鼠肠道紧密连接蛋白OccludinZo-1Claudin-1 mRNA表达水平下降(P < 0.05)。

    结论

    肠道菌群可能是甲基汞代谢和汞循环的重要参与者。

     

    Abstract: Background

    Intestinal microorganisms can directly or indirectly affect the metabolism and absorption of heavy metals, but their roles and mechanisms in the metabolism and absorption of methylmercury are still unclear.

    Objective

    This experiment investigates the influences of intestinal flora on the absorption, distribution, and excretion of methylmercury and the possible mechanism.

    Methods

    Twenty female BALB/c mice were randomly divided into four groups (n=5) as follows: blank group, dysbacterial group, methylmercury group, and dysbacterial+methylmercury group. Mice were given antibiotic mixture for 4 d (vancomycin 100 mg·kg-1, neomycin sulfate 200 mg·kg-1, metronidazole 200 mg·kg-1, and ampicillin 200 mg·kg-1, in body weight, thereafter, once a day, 10 mL·kg-1) to establish an intestinal flora disorder model. The V4 region of 16S rRNA was detected by PCR to determine whether the bacterial flora disorder model was established successfully. The methylmercury group and the dysbacterial+methylmercury group were exposed to methylmercury for 3d (0.5mg·kg-1, once a day, 10mL·kg-1 by gavage). The feces of each group were collected 24, 48, and 72h after the first methylmercury exposure. At the end of the experiment, the mice were sacrificed, and samples of blood, liver, kidney, brain, colon, and cecum contents were collected. The total mercury levels in the liver, kidney, brain, blood, and feces were determined by atomic fluorescence spectrometry; the pH values of colon and cecum contents were tested with a pH meter; the intestinal pathological changes were observed after HE staining; the Occludin, Zo-1, and Claudin-1 (tight junction proteins) mRNA expression levels were measured by reverse transcription PCR.

    Results

    The results of electrophoresis of the PCR amplified products of bacterial 16S rRNA universal primers under ultraviolet light showed that the mice in the blank group had good 16S rRNA gene signals, while no obvious signal was detected in the antibiotic-treated mice, indicating that the antibiotic treatment eliminated the bacteria in the mouse intestine, and the model of intestinal flora disorder was successful. After 24, 48, and 72 h of the first methylmercury exposure, the fecal mercury levels of the dysbacterial+methylmercury group(90.39±27.56), (366.75±81.82), and (641.33±354.24) ng·g-1 were lower than the levels of the methylmercury group(259.87±90.94), (616.83±197.90), and (1322.50±377.77) ng·g-1 (P < 0.05). The total mercury levels in the liver, kidney, brain, and blood of the dysbacterial+ methylmercury group(4 090.38±929.44), (8 539.97±1 242.64), (1 348.24±336.69), and (40 290.00±5 515.11) ng·g-1 were higher than the levels of the methylmercury group(2 752.86±566.09), (4 502.08±105.76), (827.54±106.50), and (28 392.00±5 813.25) ng·g-1 (P < 0.05). Compared with the mice without antibiotics treatment, the pH values of the cecum and colon contents of the antibiotic-treated mice increased (P < 0.05). HE staining results showed that the dysbacterial mice had changes such as necrosis of intestinal villi, shedding of epithelial cells, and mild edema of the lamina propria. The Occludin, Zo-1, and Claudin-1 mRNA expression levels of the antibiotic-treated mice were decreased compared with the mice without antibiotics treatment (P < 0.05).

    Conclusion

    Intestinal flora may be an important participant in methylmercury metabolism and mercury cycle.

     

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