Deciphering The Correlations Between Constipation And Aging By Metabolomics And Intestinal Microbiomics

Background:Constipation is a common gastrointestinal disease,with a long course of the disease and easy recrudescent.Worse,constipation will increase psychological distress,aggravate or induce other major diseases,such as gastrointestinal disorders,colon cancer,etc.Aging is a natural physiological process characterized by the decline of multiple organs and multiple levels of physiological dysfunctions.Aging can increase the susceptibility of bodies to chronic diseases,and even death.Epidemiological data show that the incidence of constipation in the elderly is more than 40%.Meanwhile,the occurrence of constipation is also significantly age-dependent.Therefore,based on individual experiences,clinical phenomena,and epidemiological data,aging and constipation tightly correlate.However,the scientific connotations of the correlation between constipation and aging are still unclear,that is,the correlations in phenotype,metabolites,mechanism of action need to be further studied.Both constipation and aging are the external embodiments of the internal microcosmic changes in the body’s physiological functions.Metabolomics reveals the final changes of small molecule metabolites and their biological systems in the body under physiological or pathological conditions.Gut microbiota,known as the“second brain”of the human body,plays an irreplaceable role in regulating physiological functions.Therefore,explaining the internal relationship between constipation and aging from the perspective of metabolites and intestinal microbiotas will provide technical supports for revealing the potential mechanism of both.This study aims to systematically explain the correlations between constipation and aging from the perspectives of phenotype,metabolomics,and microbiology.The results of this study will not only provide a new perspective for the understanding of constipation and aging in-depth,but also provide experimental supports for the research and development of drugs and foods to improve constipation in the elderly and clinical researches.Objective:1.To identify the phenotypes(i.e.behavioral and biochemical)correlations between constipation and aging;2.Based on ~1H NMR metabolomics,the correlations of metabolism levels between constipation and aging were determined;3.Based on 16S rRNA microbiome technology,the correlations of gut microbiota levels between constipation and aging were determined;4.Based on Spearman’s correlation network,the correlations between constipation and aging at phenotype level,metabolite level and gut microbiota level were further explained.Methods:1.Firstly,the Yang-deficiency constipation model induced by white vinegar and activated carbon-ice water and the aging model induced by D-galactose were replicated.2.The traditional behavioral indicators,including body-weight gains,food intakes,learning and memory abilities,gastrointestinal transport time,and small intestinal propulsion rates,were used to detect aging behavior and constipation behavior.Oxidative stress factors including superoxide dismutase,malondialdehyde,catalase,and glutathione reductase in the serum were used to study the degree of oxidative damage in aging rats and constipation rats.Changes in gastrointestinal functions in aging rats and constipation rats were detected by the contents of gastrointestinal hormones including somatostatin,motilin,and gastrin in serum.The caspase-3immunohistochemical assay was used to observe the degree of apoptosis in the hippocampus of aging rats and constipation rats.3.~1H NMR technique combined with multivariate statistical analysis was used to clarify the correlations among fecal metabolic profiles,differential metabolites and metabolic pathways of aging rats and constipation rats.4.16S rRNA sequencing technology was used to analyze the relationships among intestinal flora structure,differential microbiotas and functional pathways in cecal contents of aging rats and constipation rats.5.Based on the results of Spearman’s rank correlation,an integrated network for constipation and aging at 3 levels was constructed to visualize the associations of phenotype,metabolites and gut microbiota data.Results:1.After the administration of white vinegar and activated carbon-ice water,gastrointestinal transport time,small intestinal propulsion rates,and gastrointestinal hormones level of rats were significantly increased in the constipation group,suggesting that the constipation model was successfully replicated.After D-gal administration,the abilities of learning and memory and autonomous activities of aging rats decreased significantly while the apoptosis of CA3 region cells in the hippocampus and the oxidative damage of the body increased,indicating the success of aging model replication.2.Compared with the control rats,the learning and memory abilities of constipation rats decreased significantly while cell apoptosis and oxidative damage increased.The gastrointestinal motilities of aging rats decreased significantly and the gastrointestinal hormone secretion was disordered.These findings showed that the constipation rats also exhibited significant aging symptoms while the aging rats exhibited significant constipation symptoms.3.Compared with the control rats,the fecal metabolites of aging rats and constipation rats were significantly changed.Among them,22 and 23differential metabolites were significantly associated with constipation and aging,respectively.There are 16 differential metabolites and 8 metabolic pathways in common,which are mainly related to energy metabolism,amino acid metabolism,etc.4.Compared with the control rats,the cecal intestinal microbiotas of constipation rats and aging rats were significantly changed.The structures and functions of intestinal floras in aging rats were similar to those in constipation rats.And the abundances of Bacteroidetes and Firmicutes changed significantly.At the genus level,there were 6 intestinal bacteria both in the constipation and aging.And 5 intestinal bacteria genera were in common,including Lactobacillus,rc4-4,Adlercreutzia,Turicibacter,and Rothia.The the constipation-related and aging-related intestinal floras involved 16 and 12 functional pathways,respectively,with 8 in common.5.The“Behavior-biochemical indicator-metabolite-intestinal microbiota”network showed that the important parameters shared by both constipation and aging included CAT,MTL,glutamate,methionine,alanine,Rothia,and Turicibactar.Conclusion:The results preliminarily confirmed that the constipation rats induced by white vinegar and activated carbon-ice water showed significant aging symptoms,such as decreases in learning and memory abilities,an increase of cell apoptosis,aggravated oxidative damage in vivo,abnormal fecal metabolism,and disorder of intestinal floras.The aging rats induced by D-gal showed significant constipation symptoms,such as decreases in gastrointestinal motilities and gastrointestinal functions,abnormal fecal metabolism and disturbance of intestinal floras.The“Behavior-biochemical indicator-metabolite-intestinal microbiota”network showed correlations of constipation and aging at different levels.In this study,by combining macro and micro indexes,global and local indexes,traditional and modern indexes,various analytical methods were used to systematically expatiate the fact that constipation and aging are mutually causation and mutually influenced.The results of this study will deepen our understanding of constipation and aging,and also provide experimental supports and theoretical bases for further revealing their related mechanisms.