The Role Of Homocysteine In Irritable Bowel Syndrome Induced By Early Stress And Its Methylation Mechanism

Irritable bowel syndrome(IBS)is a clinical syndrome of recurrent abdominal pain or discomfort associated with bowel habit or character changes as the main characteristics,which is a bowel disease of functional disorder,with high incidence,and brings bigger life burden for the characteristics of the recurrent population.However,the disease pathogenesis is unclear now.Epidemiological survey shows that stress can result in IBS symptoms attack or worse.Stress is a systemic non-specific adaptive response that occurs when the organism is stimulated by both internal and external environmental factors and social and psychological factors,and also called stress response.In recent years,it has been found that intestinal flora plays an increasingly important role in brain-gut axis.Dysbacteriosis may lead intestinal environment to the disorder of nutrients and amino acid metabolism,and abnormal metabolism of amino acids can lead to abnormal of active metabolites in the body to participate in the pathogenesis of IBS.Homocysteine(Hcy)as an active metabolite among methionine cycle,is a recognized risk factor for many diseases,it may play an important role in IBS injury,and the methyl group in the metabolic pathway may be involved in epigenetic mechanisms of IBS symptoms.Therefore,this study started from the new idea "from early stress damage,intestinal flora imbalance,amino acid metabolism changes to Hcy’s damage role",revealing the pathogenesis of IBS,and further explored the role of methylation mechanism mediated by Hcy in IBS epigenetics.This not only provide targets and scientific basis for studying the pathophysiology of IBS,and for clinical diagnosis and treatment,and also for the study of the pathogenesis of diseases induced by early damage stress and for certain enlightenment and scientific thought.Chapter One Changes of intestinal flora and amino acids metabolism in IBS rats induced by early stress.Objective: To detect the changes of intestinal flora and amino acids metabolism in IBS rats induced by early years stress,and to explore the role of intestinal flora imbalance and amino acids metabolism abnormality in IBS symptoms and their damage mechanism.Methods:(1)IBS animal model establishment and behavioral assessment: The IBS model of early stress was established by using maternal separation(MS)method.Using behavioral science experiments to evaluate and confirm the irritable bowel syndrome animal model,including colorectal expansion(CRD)-retracement reflection test for abdominal visceral sensitivity,defecate number in novelty environment for detecting function of intestinal transit,open field test and partial addicted to sugar water for mental behavior change,and colonic tissue HE staining.(2)Detection and analysis of intestinal flora.We use waste genomic DNA extraction kit for bacterial genome DNA of excrement,and synthesis,amplify and sequence primers with 16 S r DNA gene variable area,and use software to analyze the diversity of feces samples.(3)Amino acids detection: We use automatic amino acids analyzer to test the amino acid concentration of rat feces and serum samples.Results:(1)After maternal and infant separation intervention,the CRD capacity threshold of MS rats decreased(P<0.05)can indicate that the visceral sensitivity was enhanced.The increased number of defecation grains in MS rats(P<0.05)indicates that the intestinal transport function was enhanced.The duration of stay in the central region of MS rats was reduced(P<0.05),indicating that the anxiety behavior was more obvious.The decrease of the degree of biophilia in MS rats(P<0.05)indicated an increase in depressive behavior.No obvious morphological changes were observed in the colon HE staining of MS rats,which was consistent with the pathological features of IBS.The above shows that IBS rat model induced by the early stress was successfully established.(2)In the analysis of the Alpha diversity of intestinal flora,the Shannon index of IBS rats decreased compared with the normal control group,suggesting that the diversity of bacteria in IBS rats decreased.Lef Se analysis in Beta diversity index showed that the relative abundance of Lachnospiraceae,Bacteroides uniformis,Christensenellaceae,Deltaproteobacteria,Desulfovibrio and Desulfovibrionaceae in IBS rats was significantly higher than the normal control group.(3)The amino acid analysis showed that compared with the control group,the serine,methionine,threonine,proline and lysine were all significantly increased in IBS rats.Serum methionine,proline and phenylalanine increased significantly,while glycine and lysine decreased significantly.Conclusion:(1)Using maternal and infant separation method we established early stress induced IBS rat disease model.(2)The diversity of intestinal flora in IBS rats decreased,and some amino acid metabolism changes,and they may be involved in the pathogenesis of IBS.The significant changes of methionine indicated that the sulfur amino acids may play an important role in the pathogenesis of IBS.Chapter Two The role of Hcy in IBS induced by early stress and its effect on methylation levelObjective: To study the effect of Hcy on IBS induced by early stress,and then detect the effect of Hcy on methylation.Methods:(1)In order to study the damage of Hcy on IBS,we choosed early stress rat model to simulate IBS,the rats were divided into group C,group MS,group MS+Met and group MS+Vit.Plasma Hcy was detected by HPLC.(2)In order to study the damage of Hcy on IBS rats,we use ELISA to measure plasma SAM、SAH and the total level of DNA methylation in colon.The expression of DNMTs were tested by Westernblot,and the activity was measured by ELISA.However,the m RNA of DNMTs were measured by q PCR.Results:(1)Compared with group C,group MS shows a higher level in plasma Hcy.Based on the group MS,Met and vitamin may have a contrast function to regulate Hcy.Meanwhile,behavioral tests showed that the symptpm of intestine gets worsen in group MS+Met,however group MS+Vit relieved to group MS.The result above point out that Hcy may be the important mediating factor to IBS.(2)The methylation potential of group MS is much higher than group C,meanwhile,total DNA methylation level,the expression of DNMT3 b and the activity of DNMTs also showed a significant upper level.However,vitamin may overturn the outcome above.Conclusion: Hcy may be an important mediating factor to IBS,and the machenism may be closed to the methylation.Chapter Three: The molecular basis of IBS regulated by the methylation mechanism mediated by HcyObjective: To explore the molecular machenism of Hcy mediated methylation in IBS intestinal barrier dysfunction.Methods: In order to explore the damage effect of Hcy on Claudin-1 and Claudin-2 expression,we use q-PCR and Westernblot to measure both expression of the Clauding and m RNA level of Claudin-1/2 and Me CP2,then we use MSP to measure promotors methylation level of claudin-1 and claudin-2.Morever,we use Decitabine--the strong prohibitor of DNMTs,to down-regulate the activity of DNMTs in group MS,and measure the DNA methylation,both expression of the Claudin1/2 and m RNA level in the intestinal,as well as the expression of Me CP2.Results:Compared with group C,the level of Claudin-1 m RNA and protein expression in MS group decreased,while the gene promoter methylation level increased and the Claudin-2 m RNA level and protein expression level increased in MS Vit group.The decreased methylation level of gene promoter reversed the trend in MS Vit group.The higher the Hcy level,the higher the expression of Me CP2.Compared with MS group,the total DNA methylation level and the total activity of methyltransferase in MS+Dac group decreased significantly,and the methylation level of claudin-2 gene promoter decreased significantly.The higher the DNA methylation,the higher the expression of Me CP2.The corresponding m RNA level and protein expression level were significantly increased,indicating that methylation level plays a regulatory role in the methylation degree and expression level of claudin-1 and claudin-2 gene promoter.Conclusion:(1)Claudin-1 and Claudin-2 are the molecular basis of the Hcy damage effect,and the mechanism of Hcy mediated methylation may be involved in its regulation.(2)Hcy mediated methylation is involved in the regulation of Claudin-1 and Claudin-2 expression.