富氢水对高氧环境小鼠肠道屏障和菌群的影响

Effect of hydrogen-rich water on intestinal barrier and gut microbiota in mice with hyperoxia

目的探讨富氢水(hydrogen-rich water)对高氧环境下小鼠肠道屏障和菌群的影响。方法选取SPF级C57BL/6雄性小鼠24只,随机分为4组(n=6):对照组(C组)、对照+富氢水组(CH组)、高氧组(H组)和高氧+富氢水组(HH组)。C组、CH组小鼠饲养于常氧环境,H组、HH组小鼠饲养于85%高氧环境。CH组和HH组小鼠每天给予富氢水0.1 mL/10 g灌胃2次,持续7 d;C组和H组小鼠给予等体积生理盐水。第7天采集小鼠粪便,心脏采血,处死动物并切取末端回肠组织5 cm。HE染色观察肠组织病理学变化,行肠黏膜损伤评分;透射电镜观察肠上皮细胞超微结构;测定肠组织丙二醛(MDA)水平,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活力,肠分泌型免疫球蛋白A(sIgA)水平;测定血清二胺氧化酶(DAO)水平;提取粪便DNA行肠道菌群检测和分类。结果与C组、CH组小鼠相比,H组肠黏膜损伤评分升高,肠道SOD、CAT活力下降,sIgA、MDA水平升高,血清DAO水平升高(均P<0.05);与H组小鼠相比,HH组肠黏膜损伤评分下降,MDA水平下降,肠道SOD、CAT活力升高,血清DAO水平下降(均P<0.05)。基因测序分析发现4组小鼠间肠道菌群多样性差异无统计学意义(P>0.05),但菌群的组成有明显差异:H组小鼠肠道菌群F16科、梭状芽胞杆菌属、葡萄球菌属和理研菌属富集;HH组小鼠肠道菌群蓝细菌门、双歧杆菌属和阿克曼菌属富集。结论高氧7 d后小鼠肠屏障功能受到氧化应激损伤,并影响了肠道菌群组成。富氢水可减轻高氧肠损伤,其机制与上调抗氧化酶SOD、CAT活力,降低氧化应激产物MDA水平,保持肠道机械屏障完整和肠道有益菌富集有关。

Objective To explore the effect of hydrogen-rich water on intestinal barrier and microbiota in mice with hyperoxia.Methods Twenty-four SPF C57 BL/6 mice were randomly divided into 4 groups(n=6),i.e.Control group(group C),Control+Hydrogen-rich water group(group CH),Hyperoxia group(group H),Hyperoxia+Hydrogen-rich water group(group HH).The mice in group C and CH were fed in normoxic environment,while those in group H and HH were kept in 85%hyperoxic environment.The mice in group CH and HH were given hydrogen-rich water(0.1 mL/10 g)by gavage twice a day for 7 days;Group C and group H were given the same volume of normal saline by gavage twice a day for 7 days.On the 7 th day,mice feces were collected,blood was collected from the heart,then the animals were sacrificed and 5 cm of terminal ileum tissue was excised.HE staining was used to observe the pathological changes of intestinal tissue,and intestinal mucosal injury was scored.The ultrastructure of intestinal epithelial cells was observed using transmission electron microscope.The activities of malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT)and the concentration of secretory immunoglobulin A(sIgA)in intestinal tissue were measured.The content of serum diamine oxidase(DAO)was determined;fecal DNA was extracted for intestinal microbiota detection.Results Compared with group C and CH,group H had higher intestinal mucosal injury scores,decreased intestinal SOD and CAT activities,increased sIgA and MDA levels,and increased serum DAO(all P<0.05).Compared with group H,the intestinal mucosal injury score,MDA content decreased,SOD and CAT activities increased and serum DAO decreased in group HH(all P<0.05).16 S RNA gene sequencing showed that there was no difference in the diversity of intestinal microflora among the four groups(P>0.05),but in the composition of intestinal microflora;in group H,F16,Clostridium,Staphylococcus and Rikenella were enriched,while in group HH,Cyanobacteria,Bifidobacterium and Akkermansia were enriched.Conclusion After 7 days of hyperoxia,the intestinal barrier function was damaged and the composition of intestinal microbiota was changed.The mechanism is related to the upregulation of the activities of antioxidant enzymes SOD and CAT,reduction of the level of MDA,maintenance of the integrity of intestinal mechanical barrier and enrichment of beneficial bacteria.