Study On The Therapeutic Effect And Potential Mechanism Of Probiotic Lactiplantibacillus Plantarum P9 On Patients With Chronic Constipation

Chronic constipation is a functional disease of the gastrointestinal tract caused by multiple factors,affecting about 15%of the world’s population,due to the change in living habits and increasing social pressure.Nearly half of the patients were dissatisfied with the treatment methods because of the differential efficacy and safety.Studies had shown that probiotics could improve defecation condition in constipated patients,while the current researches also have several problems such as inconsistent results due to the small scale,different intervention time,strain specificity,and dose differences.Therefore,large-scale and high-quality basic clinical research on specific strains is still worth ensuring the efficacy of probiotics.Lactiplantibacillus plantarum P9（P9）,isolated from the fermented sour porridge,could enter the human gastrointestinal tract in a living state and play the potential probiotic effects.Therefore,the study aims to explore the therapeutic effect of P9 in patients with chronic constipation and explore the underlying mechanism through a large-scale clinical trial.This is a single-center,randomized,double-blind,placebo-controlled study that included 163 patients with chronic constipation.After a 14-day screening period,patients were randomly divided into the P9 group（78 people）and the placebo group（85 people）.Patients in the P9 group were provided with P9(1×10¹¹ CFU/strip)after meal for 28consecutive days,and the follow-up period with no intervention was 14 days.The primary outcome measure during the trial was the mean weekly complete spontaneous bowel movement（CSBMs）,and the secondary outcome measures included the mean weekly spontaneous bowel movement（SBMs）,proportion of CSBMs≥3 per week,mean score of Bristol stool form,mean score of defecation difficulty and quality of life scores and emotional scores of patients with chronic constipation.Meanwhile,fecal metagenomics and untargeted metabolomics techniques were used to explore the mechanism of P9 in patients with chronic constipation.The main research results are as follows:（1）After continuous intake of P9 for 28 days in patients with chronic constipation,compared with the placebo group,the mean weekly CSBMs and SBMs were improved significantly（P<0.05）,And the mean weekly CSBMs still increased significantly on day 14after administration of P9 compared to the placebo group（P<0.05）.In addition,with continuous intake of P9 for 14 days,the level of worry and concerning events in patients was significantly down-regulated（P<0.05）,indicating that P9 intervention could significantly increase the frequency of defecation in patients with chronic constipation,and improve the quality of life score of patients with chronic constipation to a certain extent.（2）The results of fecal metagenomics showed that after 28 days of ingestion of P9,there was a certain classification trend in the patient’s gut microbiota structure（Stress=0.1933）,and the relative abundance of Prevotella,Faecalibacterium,Bacteroides,and Bifidobacterium were changed significantly in the gut microbiota of patients.Meanwhile,compared with the placebo group,the abundance of Lactiplantibacillus plantarum increased significantly（P<0.05）,the abundance of Alistipes Finegoldii,Bacteroides caccae,Oscillospiraceae sp900551995,Oscillospiraceae sp900763705 and several species belonging to Lachnospiraceae（Lachnospiraceae sp900066615,Lachnospiraceae sp900066375）decreased significantly（P<0.05）.Meanwhile,on day 14 after administration of P9,compared to the placebo group,the abundance of Enterocloster Aldenensis and Ruminococcus＿B gnavus in the P9 group increased significantly（P<0.05）,and the abundance of Lachnospiraceae sp003480725 and Lachnospiraceae sp900066615 decreased significantly（P<0.05）.In addition,Correlation analysis showed that the abundance of Alistipes Finegoldii was significantly negatively correlated to the mean weekly SBMs and the mean score of Bristol stool form（P<0.05）,Oscillospiraceae sp900551995 were significantly negatively correlated to the mean weekly CSBMs and SBMs（P<0.05）,and Ruminococcus＿B gnavus was significantly positively correlated to the mean weekly SBMs（P<0.05）,indicating that P9 could relieve constipation symptoms by regulating the gut microbiota in patients with chronic constipation.（3）The results of fecal untargeted metabolomics studies showed that after 28-day of P9intake,compared to the placebo group,the levels of p-Cresol,Succinic acid,Phosphorylcholine,and L-Palmitoylcarnitine decreased significantly（P<0.05）,the levels of O-Acetylcarnitine increased significantly（P<0.05）;on the day 14 after administration of P9,compared to the placebo group,the level of Trimethylamine decreased significantly and Homocitrulline increased significantly（P<0.05）.Correlation analysis showed that p-Cresol were significantly negatively correlated to the abundance of Lactiplantibacillus plantarum and significantly positively correlated to the abundance of Oscillospiraceae sp900763705,and Succinic acid was significantly positively correlated to the abundance of Oscillospiraceae sp900763705（P<0.05）,while Homocitrulline was significantly positively correlated to the abundance of Lachnospiraceae sp003480725（P<0.05）,indicating that P9play a crucial role in relieving constipation by regulating the gut microbiota,thereby regulating the levels of certain metabolites in the intestine.In addition,the main pathways for the enrichment of these differential metabolites include lysine degradation,phenylalanine metabolism,and various amino acid metabolisms（including the metabolism of alanine,tryptophan,glutamate,cysteine,and methionine,etc.）,suggesting that P9 may regulate the levels of metabolites such as p-Cresol and Succinic acid through these metabolic pathways to relieve chronic constipation.All in all,our study shows that the probiotic P9 regulates the gut microbiota of patients with chronic constipation by increasing beneficial bacteria,improving intestinal stability,and adjusting the overall balance of intestinal,which also provided energy to the intestinal peristalsis through lysine degradation,phenylalanine metabolism,and amino acid metabolism pathways,to relieve the symptoms of constipation in patients.The study laid a theoretical foundation for the mechanism research of P9 in alleviating chronic constipation and provide a new idea for the future development direction and research strategies.