Effect Of Intestinal Flora-associated Tryptophan Metabolism On AD-like Changes Induced By Chronic Arsenic Exposure In Rats

Objective:To investigate the effect of arsenic on AD-like changes in offspring SD rats from intrauterine exposure to adulthood,and to further investigate the mechanism of AD-like changes induced by long-term arsenic exposure in rats through dual omics of intestinal flora and hippocampal tryptophan metabolism.Methods:The rats were exposed in utero until 185 days after birth and contaminated with free drinking water at doses of（0,30 mg/L,100 mg/L Na As O₂）.Offspring rats were divided into 12 each group（6 males and 6 females）.After completion of behavioral experiments and collection of feces,offspring were sacrificed and brain tissues were obtained.Two of each group were randomly selected for pathological examination;the remaining were used to detect the expression of TAU and p-TAU protein by Western blot,and the contents of Aβ_1-42 and quinolinic acid by ELISA.16S r DNA sequencing was used to detect the characteristics of intestinal flora in feces.LC-MS was used to detect targeted tryptophan metabolism in the hippocampus.Results:1.Effect of chronic arsenic exposure on AD-like changes in ratsDuring arsenic exposure,the body weight of offspring rats gradually increased from weaning to week 19,and tended to stabilize after week 19.At PND185,the brain coefficient of male rats in the high arsenic group was significantly lower than that of male rats in the control group（p<0.05）.In the Morris water maze,the escape latency was significantly higher in high arsenic group than in the control group（p<0.05）,and the number of platform crossings was significantly lower in both exposed groups than in the control group（p<0.05）.In the Y-maze,the correct rate of spontaneous alternation decreased in both exposure groups.HE staining and transmission electron microscopy found that the appearance of neuronal cell arrangement,deep staining and lipofuscin in the exposed groups,respectively,and the number of lipofuscin increased in the high arsenic group.In addition,p-TAU protein expression was significantly up-regulated in the exposed groups（p<0.05）.With the increase of arsenic dose,the phosphorylation rate of TAU protein and Aβ_1-42 content increased,and the high arsenic group was significantly higher than the control group（p<0.05）.2.The effect of chronic arsenic exposure on intestinal flora in rats by 16S amplicon sequencing analysisSequencing results revealed that chronic arsenic exposure could induce disturbances of the intestinal flora in rats.Further,LEf Se analysis was used to screen differential generas in the control and exposed groups,and a total of three differential generas（Eubacterium＿siraeum＿group,Roseburia,and Lachnospiraceae＿unclassified）were selected from the control and low arsenic group;a total of seven differential generas（Christensenellaceae＿R＿7＿group,Dubosiella,Tyzzerella,Eubacterium＿ruminantium＿group,Oscillibacter,Lachnospiraceae＿unclassified,and Ligilactobacillus）were selected from the control and high arsenic group.Using Spearman correlation analysis,it was found that there was a strong negative correlation between Firmicutes and Bacteroidota at the phylum level,and Ligilactobacillus,Clostridia＿UCG-014＿unclassified and Firmicutes＿unclassified,which were relatively abundant at the genus level,were more connected to other generas.3.The effect of chronic arsenic exposure on tryptophan metabolism in the hippocampus of rats by targeted metabolomics analysisArsenic exposure significantly decreased tryptophan content in hippocampus（p<0.05）.In the tryptophan-kynurenine metabolic pathway,the content of kynurenine in the high arsenic group was significantly higher than that in the control group（p<0.05）;the contents of its downstream metabolites picolinic acid and NAD⁺also showed different degrees of decrease in the exposed groups;further determination of quinolinic acid content by ELISA revealed that the content in the high arsenic group was significantly higher than that in the control and low arsenic group（p<0.01）.In the tryptophan-serotonin metabolic pathway,only 5-hydroxyindole-3-acetic acid content was significantly lower in the high arsenic group than in the control group（p<0.05）.Further calculation of the ratio between tryptophan,kynurenine,and serotonin revealed that kynurenine content/tryptophan content,serotonin content/tryptophan content,and kynurenine content/serotonin content were significantly lower in the control group than in the high arsenic group（p<0.05）.4.Combined analysis between different generas in intestinal flora and tryptophan metabolitesThe metabolites in kynurenine metabolism pathway were significantly correlated with most significantly different generas in the control and high arsenic group,of which quinolinic acid was positively correlated with Dubosiella and the correlation was the strongest,while tryptophan was negatively correlated with Dubosiella.Kynurenine was significantly associated with Acetivibrio,Lachnospiraceae＿unclassified and Eubacterium]＿siraeum＿group in the control and low arsenic group.Further,the generalized linear model was used to obtain that there there were two linear relationships between tryptophan and Ligilactobacillus,quinolinic acid and Dubosiella in the control group.Conclusions:1.Chronic arsenic exposure can damage neuronal cells,affect learning and memory ability and lead to AD-like changes in offspring rats.2.There was a significant correlation between tryptophan metabolites in hippocampus and intestinal flora,and arsenic-induced intestinal flora dysregulation and metabolite dysregulation of the tryptophan-kynurenine metabolic pathway may jointly lead to the appearance of AD-like changes in offspring rats.