Investigation Of Intestinal Metabolite Alterations In Adenomyosis Mice And Evaluation Of Rhubarb-peach Kernel’s Regulatory Efficacy

Background Adenomyosis（AM）is a common medical condition characterized by the infiltration of endometrial glandular epithelial cells and mesenchymal cells into the myometrium.AM can cause a range of complications such as progressive dysmenorrhea,chronic pelvic pain,abnormal and excessive menstrual flow,as well as adverse effects on reproductive outcomes.Its onset is related to mechanisms such as hormonal disorders,chronic inflammation,and dysregulation of proliferation and apoptosis.Differential changes in intestinal metabolites have been shown to be involved in body hormone metabolism,immune response,and related to various inflammatory or invasive diseases.However,the pathogenesis of AM still lacks in-depth research.Currently,treating AM patients with fertility requirements is challenging.Blood-activating Traditional Chinese Medicines（TCMs）attract attention due to their ability to relieve clinical symptoms significantly without disturbing normal endocrine secretion.Rhubarb-Peach Kernel（RP）is a traditional herb-pair renowned for their ability to activate blood flow and dissolve blood stasis.RP is believed to enter the stomach and large intestine meridians when ingested.In addition to removing blood stasis,RP is also thought to have a cleaning effect on the intestines and regulate fluid metabolism.Despite its frequent clinical application in treating AM,there is still a need to confirm its regulatory effect on differential metabolic metabolites present in the intestines of individuals with AM.Objective The study utilized both liquid chromatography-mass spectrometry（LC-MS）and 16S r RNA sequencing techniques to investigate the differential metabolites present in the intestines of AM mice,as well as their potential interrelationship with intestinal microorganisms.Furthermore,LC-MS technology was employed to explore the regulatory effect of RP on these AM differential metabolites.Methods（1）Screening of differential metabolites in the gut of mice with adenomyosis:Sixteen female ICR mice,aged seven weeks,were randomly divided into the control group（n=8）and the model group（n=8）.The AM mouse model was established in the model group by using allogeneic pituitary transplantation,and pathological changes in the uterus of these mice were evaluated using hematoxylin-eosin（HE）staining.To identify fecal metabolites,LC-MS technology was employed,and metabolic pathway analysis was subsequently conducted.（2）Changes in gut microbiota of adenomyosis mice and their correlation with gut differential metabolites:16S r RNA sequencing technology was used to analyze the structural composition of intestinal microbiota in mice.Alpha and Beta diversity analyses were employed to evaluate the diversity of intestinal microbiota.The relative abundance of intestinal microbiota in mice was detected,and different bacteria at the phylum and genus levels between two groups of mice were screened.Spearman correlation analysis was also conducted to explore the correlation between differential metabolites and intestinal microbiota.（3）Exploring the interventional effect of RP on intestinal metabolism in adenomyosis:A total of 22 seven-week-old ICR female mice were randomly divided into the control group（n=9）,the model group（n=6）and the RP group（n=7）.The model group was orally administered with 0.3125 g·kg^-1 of RP granules,while the remaining two groups were administered with equal amounts of physiological saline.HE staining was used to observe the pathological changes in the uterus of the three groups of mice.LC-MS technology was employed to screen differential metabolites before and after treatment,and analysis of differential pathways was performed.Results（1）Metabolomics analysis showed that there were 60 differential metabolites in the intestine between the AM group and the control group.Multiple metabolic pathways,such as steroid hormone biosynthesis,cysteine and methionine metabolism,alanine,aspartate and glutamate metabolism,tryptophan metabolism,aminoacyl-t RNA biosynthesis,beta-Alanine metabolism,and primary bile acid biosynthesis,were found to be abnormal in AM mice.（2）Intestinal microbiota analysis showed that the relative abundance of Bacteroidetes and Bacteroides was decreased,while the relative abundance of Firmicutes,Firmicutes/Bacteroidetes,and Lactobacillus was increased in AM mice compared with normal mice.（3）Spearman correlation analysis showed that there was a certain correlation between the changes in the content of differential metabolites and the relative abundance of intestinal microbiota in AM mice.（4）RP can significantly improve the abnormality of 15 differential metabolites in AM mice,and its mechanism was related to the regulation of metabolic pathways such as tryptophan metabolism,beta-alanine metabolism,aminoacyl-t RNA biosynthesis,and primary bile acid biosynthesis.Conclusion（1）AM mice had unique gut microbes and metabolites,and there was a certain correlation between the change of metabolite content and the relative abundance of gut microorganisms.（2）RP may exert therapeutic effects on AM by regulating metabolic pathways such as tryptophan metabolism,beta-alanine metabolism,aminoacyl-t RNA biosynthesis,and primary bile acid biosynthesis.