维生素C对染Cr(VI)小鼠肠道菌群紊乱的影响
Effect of vitamin C on intestinal flora disorders in Cr(VI)-contaminated mice
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摘要:
背景 六价铬Cr(VI)暴露能引起肠道菌群结构紊乱,造成功能损伤。维生素C(VC)是人体必需的微量营养素之一,在促进肠道益生菌生长,改善肠道屏障,维持肠道菌群稳态中起重要作用。而VC对Cr(VI)暴露致肠道菌群紊乱的调节作用还有待研究。
目的 探讨VC对Cr(VI)暴露导致小鼠肠道菌群紊乱的影响。
方法 32只SPF级C57BL/6小鼠适应性喂养3 d后,随机分为正常对照组(Con组)、VC组、重铬酸钾(K2Cr2O7)组Cr(VI)组和VC+K2Cr2O7组VC+Cr(VI)组。于第4天早8:00,Con组小鼠灌胃给予双蒸水,腹腔注射双蒸水;VC组小鼠灌胃给予VC,腹腔注射双蒸水;Cr(VI)组小鼠灌胃给予双蒸水,腹腔注射K2Cr2O7溶液;VC+Cr(VI)组小鼠灌胃给予VC,腹腔注射K2Cr2O7溶液。VC给药量为200 mg·kg−1,K2Cr2O7给药量为1.25 mg·kg−1,连续处理45 d后处死小鼠,取结肠内容物于无菌冻存管中,每组收集3个重复样本。标记好后立即投入液氮中迅速冷冻。待所有样本收集完成后,转移至-80 ℃超低温冰箱中保存。结肠内容物样本通过高通量测序及生物信息学软件分析肠道菌群结构。
结果 与Con组相比,Cr(VI)暴露导致小鼠体重增加值减少。小鼠回肠组织发生病理改变,Cr(VI)组有明显炎症细胞浸润,VC+Cr(VI)组炎症细胞浸润减少。Cr(VI)组小鼠肠道菌群分类操作单元(OTU)数目改变。α多样性分析中,Cr(VI)暴露组Sobs指数平均为240.333±67.796,Chao指数为258.173±64.813,Ace指数为259.481±66.891,较Con组降低(
P <0.05),PD whole tree指数为27.863±2.399,较Con组升高(P <0.05),VC干预可改善Cr(VI)暴露导致上述指标的变化(P <0.05)。β多样性分析中,主坐标分析结果表明Cr(VI)组和Con组之间有明显分离,VC干预后,分离趋势有回调,差异减小。多样本相似性树状图结果显示正常对照组和VC组最先聚类在一起,然后与VC+Cr(VI)组聚成一类,最后再与Cr(VI)组聚成一类。Cr(VI)组小鼠肠道中的拟杆菌门、单糖菌门和软壁菌门丰度降低,厚壁菌门丰度升高;乳杆菌属、另枝菌属、拟杆菌属和瘤胃梭菌属丰度升高,其中拟杆菌属与Con组小鼠相比,丰度升高有统计学意义(P <0.05);而经VC干预后,上述肠道微生物中菌门及菌属的丰度变化均有所改善。结论 Cr(VI)暴露可导致小鼠肠道损伤及菌群结构紊乱,而VC干预可在一定程度上改善上述变化,使肠道菌群结构趋向正常。
Abstract:Background Hexavalent chromium Cr(VI) exposure can cause structural disruption of intestinal flora and functional impairment. Vitamin C (VC) is one of the essential micronutrients, which plays an important role in promoting the growth of intestinal probiotics, improving the intestinal barrier, and maintaining the homeostasis of intestinal flora. However, the regulatory effect of VC on the intestinal flora disorders caused by Cr(VI) exposure remains to be investigated.
Objective To investigate the effect of VC on intestinal flora disruption in mice due to Cr(VI) exposure.
Methods Thirty-two SPF-grade C57BL/6 mice were acclimatized and fed for 3 d and randomly divided into control (Con), VC, potassium dichromate K2Cr2O7, Cr(VI), and VC+K2Cr2O7 VC+Cr(VI) groups. At 8:00 a.m. on day 4, the Con group (double-distilled water given by gavage and injected intraperitoneally), the VC group (VC given by gavage and double-distilled water injected intraperitoneally), the Cr(VI) group (double-distilled water given by gavage and K2Cr2O7 solution injected intraperitoneally), and the VC+Cr(VI) group (VC given by gavage and K2Cr2O7 solution injected intraperitoneally) were treated. The dose of VC was 200 mg·kg−1, and the dose of K2Cr2O7 was 1.25 mg·kg−1. The mice were treated for 45 consecutive days and then executed, the contents of the colon were sampled in sterile freezing tubes, and three replicates were collected from each group. After labeling, the samples were immediately put into liquid nitrogen for rapid freezing. After all the samples were collected, they were transferred to a -80 ℃ ultra-low temperature refrigerator for storage. Samples of colon contents were analyzed for intestinal flora structure by high-throughput sequencing and bioinformatics software.
Results The Cr(VI) exposure resulted in reduced body weight gain values in mice compared to the Con group. Pathological changes occurred in the ileal tissue of mice, with significant inflammatory cell infiltration in the Cr(VI) group and reduced inflammatory cell infiltration in the VC+Cr(VI) group. The number of operational taxonomic units (OTUs) of intestinal flora was altered in the Cr(VI) group of mice. In the α diversity analysis, the mean Sobs index in the Cr(VI) group was 240.333±67.796, the Chao index was 258.173±64.813, and the Ace index was 259.481±66.891, which were significantly lower than those in the Con group (
P <0.05), the PD whole tree index in the Cr(VI) group was 27.863±2.399, which was significantly higher than that in the Con group (P <0.05), and the VC intervention significantly reversed the changes of the above indexes due to Cr(VI) exposure (P <0.05). In the β diversity analysis, the principal coordinates analysis (PCoA) results showed a significant separation between the Cr(VI) group and the Con group, and after the VC intervention, there was a retraction of the separation trend and the difference was reduced. The multi-sample similarity dendrogram results showed that the control and the VC groups clustered together first, then with the VC+Cr(VI) group, and finally with the Cr(VI) group. The abundances of Bacteroidetes, Saccharibacteria, and Tenericutes in the intestine of mice in the Cr(VI) group were decreased, and the abundance of Firmicutes was increased; the abundances ofLactobacillus ,Alistipes ,Bacteroides, andRuminiclostridium were also increased. Included among these,Bacteroides showed a significantly higher abundance compared to the control mice (P <0.05). Changes in the abundances of phyla and genera of the above mentioned gut microorganisms were reversed after the VC intervention.Conclusion Cr(VI) exposure can lead to intestinal damage and disorganization of the intestinal flora structure in mice, while VC intervention can ameliorate the above changes to a certain extent and normalize the intestinal flora structure.