Effects of melatonin on learning and memory abilities of fluoride-exposed offspring rats
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目的 探讨褪黑素（melatonin，MEL）对氟暴露子代大鼠学习记忆能力的影响及肠道菌群在其中的作用。 方法 选取12只8周龄SD大鼠（雌鼠8只、雄鼠4只），体质量为180 ~ 220 g，采用随机数字表法分为对照组1和染氟组1，每组6只（雌∶雄= 2 ∶ 1），分别自由饮用纯净水和含100 mg/L氟化钠的纯净水。2个月后雌雄大鼠合笼饲养，将子代大鼠出生后（postnatal day，PND）第1天记为PND0。于PND21，采用成组设计将染氟组1子代大鼠分为染氟组（F组，n = 6）和染氟+ MEL组（FM组，n = 6），并继续饮水氟暴露；对照组1子代大鼠分为对照组（C组，n = 6）和MEL组（n = 6）。FM和MEL组给予20 mg/kg MEL灌胃，C和F组给予相同剂量生理盐水灌胃。于PND60，采用新事物识别实验和水迷宫实验观察子代大鼠学习记忆能力，蛋白质印迹法（Western blot，WB）检测子代大鼠海马组织脑源性神经营养因子（brain derived neurotrophic factor，BDNF）蛋白表达水平；并采用16S rDNA测序技术检测粪便样本中肠道菌群结构和组成变化情况。 结果 新事物识别实验结果显示，4组子代大鼠鉴别指数（discrimination index，DI）比较，差异有统计学意义（F = 3.95，P = 0.024），C、FM组DI均高于F组（均P < 0.05）。水迷宫实验结果显示，与C组比较，F组子代大鼠穿越平台次数较少，首次到达平台时间较长（均 P < 0.05）；与F组比较，FM组子代大鼠穿越平台次数较多，首次到达平台时间较短（均 P < 0.05）。WB结果显示，与C组（1.00 ± 0.07）比较，F组BDNF蛋白表达水平（0.68 ± 0.26）较低（ P < 0.05）；与F组比较，FM组BDNF蛋白表达水平（0.99 ± 0.14）较高（ P < 0.05）。Anosim相似性分析显示，4组子代大鼠肠道菌群结构和组成存在显著差异（ R = 0.395 062，P = 0.002）。肠道菌群物种分布特征结果显示，门水平上，与C组比较，F组拟杆菌门相对丰度从14.26%升至37.00%，厚壁菌门相对丰度从68.78%降至45.95%；与F组比较，FM组厚壁菌门相对丰度从45.95%升至65.26%，拟杆菌门相对丰度从37.00%降至23.00%。属水平上，与C组比较，F组乳杆菌属、杜氏菌属、HT002、UCG-005的相对丰度均较低，未分类的Muribaculaceae属相对丰度较高；与F组比较，FM组乳杆菌属、杜氏菌属、HT002、UCG-005的相对丰度均较高，未分类的Muribaculaceae属相对丰度较低。线性判别分析结果显示，候选单胞生糖菌属、毛螺菌属显著富集于C组，未分类的Muribaculaceae属、Muribaculum属显著富集于F组，异根瘤菌-新根瘤菌-副根瘤菌-根瘤菌属显著富集于FM组。 结论 MEL可以改善氟暴露所致的子代大鼠学习记忆能力损伤，可能与改变肠道菌群结构和组成有关。 Objective To investigate the effect of melatonin (MEL) on learning and memory abilities of fluoride-exposed offspring rats and the role of gut microbiota. Methods Twelve 8-week-old Sprague-Dawley (SD) rats (8 females and 4 males) with a body weight ranging from 180 to 220 g were selected and divided into control group 1 and fluoride-exposed group 1 using a random number table method, with 6 rats in each group (female ∶ male = 2 ∶ 1). They were free to drink purified water or purified water containing 100 mg/L sodium fluoride, respectively. After 2 months, male and female rats were raised together in cages, and the first postnatal day (PND) of the offspring rats was recorded as PND0. In PND21, the offspring rats of fluoride-exposed group 1 were divided into fluoride-exposed group (Group F, n = 6) and fluoride + MEL group (Group FM, n = 6) using a group design, and continued to be exposed to fluoride through drinking water. The offspring rats of control group 1 were divided into control group (Group C, n = 6) and MEL group (n = 6). The groups FM and MEL were given 20 mg/kg MEL by gavage, while the groups C and F were given the same dose of normal saline by gavage. In PND60, novel object recognition and Morris water maze tests were used to observe the learning and memory abilities of the offspring rats. Western blotting (WB) was used to detect the expression level of brain derived neurotrophic factor (BDNF) in the hippocampus of the offspring rats. And 16S rDNA sequencing technology was used to detect the changes in the structure and composition of gut microbiota in fecal samples. Results The results of novel object recognition test showed that there was a statistically significant difference in the discrimination index (DI) among the four groups of offspring rats (F = 3.95, P = 0.024). The DI in groups C and FM was higher than that of Group F (P < 0.05). The results of Morris water maze test showed that compared with Group C, the platform-crossing time of the offspring rats of Group F were less and they had a longer time to reach the platform for the first time ( P < 0.05). Compared with Group F, the platform-crossing time of the offspring rats of Group FM were increased and they had a shorter time to reach the platform for the first time ( P < 0.05). The WB results showed that compared with Group C (1.00 ± 0.07), the expression level of BDNF protein in Group F (0.68 ± 0.26) was lower ( P < 0.05). Compared with Group F, the expression level of BDNF protein in Group FM (0.99 ± 0.14) was higher ( P < 0.05). Anosim similarity analysis showed significant differences in the structure and composition of gut microbiota in the four groups of offspring rats ( R = 0.395 062, P = 0.002). The distribution characteristics of gut microbiota species showed that at the phylum level, compared with Group C, the relative abundance of Bacteroidetes in Group F increased from 14.26% to 37.00%, and the relative abundance of Firmicutes decreased from 68.78% to 45.95%. Compared with Group F, the relative abundance of Firmicutes in Group FM increased from 45.95% to 65.26%, and the relative abundance of Bacteroidetes decreased from 37.00% to 23.00%. At the genus level, compared with Group C, the relative abundance of Lactobacillus, Dubosiella, HT002 and UCG-005 in Group F was lower, while the relative abundance of unclassified Muribaculaceae was higher. Compared with Group F, the relative abundance of Lactobacillus, Dubosiella, HT002 and UCG-005 in Group FM was higher, while the relative abundance of unclassified Muribaculaceae was lower. The results of linear discriminant analysis revealed that the Candidatus-Saccharimonas and Incertae-Sedis were significantly enriched in Group C, unclassified Muribaculaceae and Muribaculum were significantly enriched in Group F, and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium were significantly enriched in Group FM. Conclusion MEL can improve the learning and memory impairment of offspring rats induced by fluoride exposure by changing the structure and composition of gut microbiota.
Objective To investigate the effect of melatonin (MEL) on learning and memory abilities of fluoride-exposed offspring rats and the role of gut microbiota. Methods Twelve 8-week-old Sprague-Dawley (SD) rats (8 females and 4 males) with a body weight ranging from 180 to 220 g were selected and divided into control group 1 and fluoride-exposed group 1 using a random number table method, with 6 rats in each group (female ∶ male = 2 ∶ 1). They were free to drink purified water or purified water containing 100 mg/L sodium fluoride, respectively. After 2 months, male and female rats were raised together in cages, and the first postnatal day (PND) of the offspring rats was recorded as PND0. In PND21, the offspring rats of fluoride-exposed group 1 were divided into fluoride-exposed group (Group F, n = 6) and fluoride + MEL group (Group FM, n = 6) using a group design, and continued to be exposed to fluoride through drinking water. The offspring rats of control group 1 were divided into control group (Group C, n = 6) and MEL group (n = 6). The groups FM and MEL were given 20 mg/kg MEL by gavage, while the groups C and F were given the same dose of normal saline by gavage. In PND60, novel object recognition and Morris water maze tests were used to observe the learning and memory abilities of the offspring rats. Western blotting (WB) was used to detect the expression level of brain derived neurotrophic factor (BDNF) in the hippocampus of the offspring rats. And 16S rDNA sequencing technology was used to detect the changes in the structure and composition of gut microbiota in fecal samples. Results The results of novel object recognition test showed that there was a statistically significant difference in the discrimination index (DI) among the four groups of offspring rats (F = 3.95, P = 0.024). The DI in groups C and FM was higher than that of Group F (P < 0.05). The results of Morris water maze test showed that compared with Group C, the platform-crossing time of the offspring rats of Group F were less and they had a longer time to reach the platform for the first time ( P < 0.05). Compared with Group F, the platform-crossing time of the offspring rats of Group FM were increased and they had a shorter time to reach the platform for the first time ( P < 0.05). The WB results showed that compared with Group C (1.00 ± 0.07), the expression level of BDNF protein in Group F (0.68 ± 0.26) was lower ( P < 0.05). Compared with Group F, the expression level of BDNF protein in Group FM (0.99 ± 0.14) was higher ( P < 0.05). Anosim similarity analysis showed significant differences in the structure and composition of gut microbiota in the four groups of offspring rats ( R = 0.395 062, P = 0.002). The distribution characteristics of gut microbiota species showed that at the phylum level, compared with Group C, the relative abundance of Bacteroidetes in Group F increased from 14.26% to 37.00%, and the relative abundance of Firmicutes decreased from 68.78% to 45.95%. Compared with Group F, the relative abundance of Firmicutes in Group FM increased from 45.95% to 65.26%, and the relative abundance of Bacteroidetes decreased from 37.00% to 23.00%. At the genus level, compared with Group C, the relative abundance of Lactobacillus, Dubosiella, HT002 and UCG-005 in Group F was lower, while the relative abundance of unclassified Muribaculaceae was higher. Compared with Group F, the relative abundance of Lactobacillus, Dubosiella, HT002 and UCG-005 in Group FM was higher, while the relative abundance of unclassified Muribaculaceae was lower. The results of linear discriminant analysis revealed that the Candidatus-Saccharimonas and Incertae-Sedis were significantly enriched in Group C, unclassified Muribaculaceae and Muribaculum were significantly enriched in Group F, and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium were significantly enriched in Group FM. Conclusion MEL can improve the learning and memory impairment of offspring rats induced by fluoride exposure by changing the structure and composition of gut microbiota.
FluorineMelatoninLearn and memory impairmentGut microbiota
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