Study On The Mechanism Of Learning And Memory Impairment In Rat Exposed To Arsenic And Fluoride Based On Microbiome And Metabonomics

Objectives:In this study,the SD rats exposed to arsenic and/or fluoride from intrauterine to adult was used to explore the role of gut microbiota and metabolites in learning and memory impairment induced by arsenic and fluoride.Correlation analysis of differential gut microbiota and differential metabolites is aimed at exploring the mechanism of learning and memory impairment caused by arsenic and fluorine from a multi-omics level.Methods:According to body weight,the parent rats were randomly divided into control group(drinking water,con),low arsenic group(35mg/L,As＿(35)),high arsenic group(70mg/L,As＿(70)),low fluoride group(50mg/L,F＿(50)),high fluoride group(100 mg/L,F＿(100)),low arsenic and low fluoride group(35mg/L+50mg/L,As＿(35)F＿(50)),high arsenic and low fluoride group(70mg/L+50mg/L,As＿(70)F＿(50)),the low arsenic and high fluoride group(35mg/L+100 mg/L,As＿(35)F＿(100))and the high arsenic and high fluoride group(70mg/L+100 mg/L,As＿(70)F＿(100)).The exposure time of offspring was from gestation to postnatal day 90(PND 90).Morris water maze test was performed in PND 42 to evaluate the learning and memory ability of offspring(each group of eight).Urine,cerebellum and bone of PND 21、42、60 and 90offspring were collected to determine the content of arsenic and fluorine in urine and tissues to evaluate the excretion and accumulation of arsenic and fluorine.Hippocampus of PND 90offspring was stained with HE to observe the pathological changes of hippocampus.16Sr RNA and LC-MS were used to detect the changes of the gut microbiota characteristics and metabolic profile of the feces of PND90 day offspring rats.The data were analyzed using the SPSS 24.0.ANOVA with LSD(data conformed to the normal distribution and uniform variance)or Welch with Dunnett T3(data conformed to the normal distribution but uneven variance)was used to determine the difference among the groups,and the significance level was 0.05.Result:1.The results of Morris water maze showed that the average escape latency in exposed groups was higher than control group(p<0.05),the number of crossing platform and number of times into the target quadrant in exposed groups were lower than control group(p<0.05).Compared with the control group HE staining showed that,the number of neurons in hippocampal CA1 area in the exposed groups was reduced,accompanied by different degrees of nuclear lysis,nuclear destruction and vacuolar changes.The results showed that urinary arsenic concentration,urinary fluoride concentration,concentration of cerebellar arsenic and concentration of bone fluoride in exposed groups was significantly higher than unexposed groups(p<0.05).2.Arsenic and/or fluoride exposure can cause gut microbiota disorders of offspring rats.The dominant phyla and genera of the control group and the exposed groups were significantly different.The dominant phyla of the control group was Bacteroides(53.15%).The dominant phyla of the As＿(70),F＿(100),and As＿(70)F＿(100) was Firmicutes,and their proportions were 63.97%,61.51%and 65.13%respectively.In the control group,the dominant genus was Bacteroides(20.56%),in the As＿(70),the dominant genus was Lachnospiraceae＿NK4A136＿group(15.05%),and in the F＿(100),the dominant genus was Lactobacillus(15.10%),the dominant genus in the As＿(70)F＿(100) was Lactobacillus(18.26%).Theαdiversity andβdiversity are significantly different among the exposed groups and the control group.LEf Se analysis was used to screen bacteria such as Bacteroides,[Eubacterium]＿xylanophilum＿group,Lachnospiraceae＿NK4A136＿group,Roseburia,Prevotellaceae＿NK3B31＿group,Ruminococcus＿1 and Helicobacter.3.Exposure to arsenic and fluoride can cause changes in the metabolic profile of offspring,PCA and OPLS-DA showed that the metabolic profiles in the control group were significantly different from the exposed groups,KEGG functional enrichment of the differential metabolites revealed that the differential metabolites were mainly concentrated in the following metabolic pathways:Tryptophan metabolism,Lipoic acid metabolism,GABAergic synapse,Alanine,aspartate and glutamate metabolism,Arachidonic acid metabolism et al.4.Correlation analysis between gut microbiota and differential metabolites showed that significantly altered metabolites in the tryptophan metabolic pathway were correlated with differentialgutmicrobiota,[Eubacterium]＿xylanophilum＿group,Lachnospiraceae＿NK4A136＿group,Prevotellaceae＿NK3B31＿group was positively correlated with Xanthurenic acid,while 5-Hydroxyindoleacetic acid was negatively correlated with[Eubacterium]＿xylanophilum＿group,Lachnospiraceae＿NK4A136＿group and Prevotellaceae＿NK3B31＿group.The analysis of the relationship between the metabolite of the GABAergic synapse pathway and the different gut microbiota showed that there was positive correlation with the L-Glutamate,such as[Eubacterium]＿xylanophilum＿group,Lachnospiraceae＿NK4A136＿group and Prevotellaceae＿NK3B31＿group,succinic acid was negatively correlated with[Eubacterium]＿xylanophilum＿group.The correlation analysis between different metabolites and different gut microbiota in the pathway of lipoic acid metabolismshowedthat[Eubacterium]＿xylanophilum＿group,Lachnospiraceae＿NK4A136＿group and Prevotellaceae＿NK3B31＿group significant negative correlation with lipoic acid.Conclusions:1.Exposure to arsenic and/or fluorine can lead to learning and memory disorders accompanied by pathological changes in the hippocampus.2.The offspring rats exposured to arsenic and/or fluoride can lead to gut microbiota disorder,the relative abundance of Bacteroides,Ruminococcus＿1,[Eubacterium]＿xylanophilum＿group,Lachnospiraceae＿NK4A136＿group,Prevotellaceae＿NK3B31＿group and Helicobacter have changed significantly,the above gut microbiota can be used as the focus of follow-up research.3.Metabonomics analysis of faces from offspring rats suggests that the learning and memory impairment of arsenic and fluoride may be related to the Tryptophan metabolism,Lipoic acid metabolism,GABAergic synapse,Alanine,aspartate and glutamate metabolism,Arachidonic acid metabolism.4.The results of correlation analysis showed that there was a strong correlation between the selected bacteria and the different metabolites.