The Effect Of Long-term Voyage Environment On The Intestinal Microecology Of Seafarers And Its Mechanism In The Intestine

Part 1 Effects of long-term voyages on the intestinal microbiota and metabolism of seafarers.Objective: To explore the effects of long-term voyages on the intestinal microbiota and metabolites of seafarers,and then to screen the key intestinal microbiota and metabolites related to the high incidence of digestive system diseases in long-term voyages.Methods: According to the inclusion and exclusion criteria,a total of 30 seafarers who participated in the six-month long-term voyage were included,and fecal samples were collected before and after the voyage,and the bacterial 16 S r RNA genome and LC-MS nontargeted metabolome sequencing were carried out to analyze the changes of intestinal microbiota and metabolites of the seafarers before and after the long-term voyage.Spearman correlation analysis was carried out on differential microbiota and differential metabolites,and Metabo Analyst and GMrepo databases were used to analyze the association among differential microbiota,differential metabolites,and digestive system diseases.The key intestinal microbiota and metabolites of the long-term voyage-related high-incidence digestive system diseases were screened.Results:(1)The effect of long-term voyage on the intestinal microbiota and microbiota function of seafarers.The composition of intestinal microbiota of seafarers before and after the long-term voyage changed significantly,especially change of Beta diversity.At the level of the genus,Holdemanella,Plesiomonas,Ruminococcaceae_UCG-008,etc.increased significantly,while Erysipelatoclostridium,Bilophila,Faecalitalea,etc.decreased significantly.The PICRUSt2 function prediction of intestinal microbiota showed that the functions of intestinal microbiota in folate biosynthesis,biotin metabolism,citrate cycle and glyoxylate and dicarboxylate cycle metabolism were significantly decreased after long-term voyage,while starch-sucrose metabolism,glycerolipid metabolism and pentose phosphate pathway were significantly increased.(2)Effect of long-term voyage on intestinal metabolites of seafarers.The intestinal metabolites of the seafarers changed significantly before and after the long-term voyage,especially the lysine,proline,citrulline and 2-hydroxycinnamic acid were significantly decreased after the long-term voyage.KEGG pathway analysis showed that the three major metabolic pathways(carbohydrate,amino acid,and lipid pathways)underwent significant changes in the long-term voyage,including arginine biosynthesis pathway,lysine degradation,phenylalanine metabolism,sphingolipid metabolism,pentose and glucuronate interconversions and glycine/serine/threonine metabolism pathway.(3)Association analysis of differential microbiota and differential metabolites associated with long-term voyages.Spearman correlation analysis of differential microbiota and differential metabolites showed that 6 differential metabolites(acetylcholine,D-glucuronic acid,citrulline,lysine,proline and 2-hydroxycinnamic acid)were significantly correlated with 14 differential microbiotas(e.g.,Bilophila,Plesiomonas,Holdemanella,etc.),which may affect human metabolic pathways(e.g.,protein digestion and absorption,arginine biosynthesis,etc.).(4)Further screening of the digestive system diseases,differential metabolites and intestinal microbiota related to long-term voyage based on public databases.The Metabo Analyst database was used to perform enrichment analysis on differential metabolites in the digestive system disease spectrum,and it was found that Irritable Bowel Syndrome(IBS)is the disease with the highest enrichment rate.Using the GMrepo database to analyze the correlation between differential microbiota and IBS,it was found that three bacterial genera(Holdemanella,Bilopila,and Bacteroides)were significantly correlated with IBS.Furthermore,according to the relative abundance and LDA value of the differential microbiota,Holdemanella biformis and four related differential metabolites(citrulline,lysine,proline and 2-hydroxycinnamic acid)were screened out,which were associated with IBS disease and used for follow-up research.Conclusion: The long-term voyage led to a decrease in the diversity of intestinal microbiota and changes in related metabolic pathways,among which four differential metabolites(citrulline,lysine,proline and 2-hydroxycinnamic acid)and one differential bacterium(H.Biformis)is highly correlated with long-term voyage-related IBS,which needs to be further studied.Part 2 The interaction study of four differential metabolites and the differential bacteria H.Biformis associated with long-term voyage-related IBS.Objective: To explore the interaction and regulatory mechanisms between the four differential metabolites(lysine,proline,citrulline or 2-hydroxycinnamic acid)and the differential bacteria H.Biformis,which were screened out in the Part 1 of the study.Methods: Spearman correlation analysis in the Part I of the study found that four differential metabolites(lysine,proline,citrulline,or 2-hydroxycinnamic acid)associated with long-term voyage-related IBS were negative correlated with H.Biformis in the abundance of expression.Further we co-cultured different concentrations of lysine,proline,citrulline and 2-hydroxycinnamic acid with H.Biformis.The effects of four differential metabolites on H.Biformis growth curves were observed.By LC-MS non-targeted metabolome sequencing of bacterial precipitation and bacterial supernatant after the intervention of lysine,proline,citrulline or 2-hydroxycinnamic acid,we analyzed the effects and possible regulatory mechanisms of four differential metabolites on H.Biformis.The bacterial supernatant of H.Biformis in the logarithmic phase of the growth culture and blank medium were sequenced by non-targeted metabolome,to observe the impact of H.Biformis on the four differential metabolites.Results: The growth curves of H.Biformis showed that four differential metabolites had an inhibitory effect on growth and reproduction of H.Biformis.The enrichment analysis of bacterial KEGG metabolic pathways showed that lysine,proline or citrulline down-regulated the energy metabolism pathways such as glycolysis pathway,pentose phosphate pathway and phosphotransferase system,while 2-hydroxycinnamic acid mainly down-regulated the energy metabolism pathways such as glycolysis pathway and pentose phosphate pathway,which inhibited the growth and reproduction of H.Biformis.H.Biformis has no effect on the production of four differential metabolites,and no activation of related metabolic pathways has been found in bacteria.Conclusion: Differential metabolites related to long-term voyage(lysine,proline,citrulline and 2-hydroxycinnamic acid)inhibit the growth and reproduction of H.Biformis by downregulating its energy metabolism pathways.H.Biformis has no effect on the production of four differential metabolites.Part 3 Effect of long-term voyage-related four differential metabolites and differential bacteria on human intestinal epithelial cells.Objective: To explore the effects of four differential metabolites(lysine,proline,citrulline,and 2-hydroxycinnamic acid)and H.Biformis in vitro screened out in Part 1 on human intestinal epithelial cells.Methods: Due to H.Biformis is an extremely anaerobic bacterium,and currently there are no conditions for co-culturing with cells.Therefore,in this study,cell intervention was performed with H.Biformis supernatant.Firstly,four differential metabolites(lysine,proline,citrulline and 2-hydroxycinnamic acid)and H.Biformis supernatant were added to human intestinal HIEC-6 cells and human colon NCM-460 cell culture medium,respectively,which were incubated for 24 h and then CCK-8 was used to observe their effects on cell viability and proliferation.IFN-γ pretreated human intestinal HIEC-6 cells and human colon NCM-460 cells were used to simulate the intestinal epithelial injury in IBS patients,and then intervened by four differential metabolites(lysine,proline,citrulline and 2-hydroxycinnamic acid)and H.Biformis supernatant.CCK-8 was used to detect the viability and proliferation status of cells.q PCR was used to detect the expression changes of proliferation-related genes.CalceinAM/PI staining method was used to detect cell viability and death.Flow cytometry(Annexin V-FITC/PI)was used to detect apoptosis.Flow cytometry(PI staining)was used to detect cell cycle changes.The scratch assay was used to detect the migration and healing rate of cells.q PCR,Western-blot,and immunofluorescence were used to detect TJP1 expression changes.Results: Lysine,proline,citrulline,2-hydroxycinnamic acid,and H.Biformis supernatant has no inhibitory effect on the viability of intestinal epithelial cells.Lysine,proline,citrulline,and H.Biformis supernatant significantly improved the growth inhibition of HIEC-6 and NCM-460 cells pretreated by IFN-γ,promoted the expression of proliferation-related genes such as PCNA,MKi67 or MCM6,improved cell migration and healing,and increased the expression level of TJP1,a junctional molecule between cells.2-Hydroxycinnamic acid had no significant effect on alleviating the migration and healing,and increasing the expression levels of TJP1 in intestinal epithelial cells pretreated by IFN-γ.Conclusion: Lysine,proline,citrulline,and H.Biformis supernatant can effectively ameliorate IBS-associated intestinal epithelial cell injury,including promoting cell proliferation,migration healing,and increasing intercellular TJP1 expression levels.Part 4 The effects of differential metabolites(lysine or proline)and differential bacteria H.Biformis on the long-term voyage-related IBS mouse models.Objective: We constructed long-term voyage-related IBS mouse models.Since citrulline-deficient feed is not yet available,this study mainly explored the effects of lysine,proline,and H.Biformis,which were effective in damaging intestinal epithelial cells,on body weight,fecal water content,intestinal permeability,intestinal barrier function and serum neurotransmitters in IBS mouse models.Methods: Low-concentration acetic acid enema combined with restraint stress was used to construct long-term voyage-related IBS mouse models in eight-week-old SPF grade C57BL/6J mice fed with lysine or proline deficient feed.The effects of amino acid deficiency on body weight,fecal moisture content,intestinal permeability,intestinal barrier function,and neurotransmitters in the IBS mouse models were observed.Furthermore,lysine(200 mg/kg/day)or proline(500 mg/kg/day)was given to mice by gavage to observe the effects of amino acid supplementation on body weight,fecal moisture content,intestinal permeability,intestinal barrier function and neurotransmitters in the IBS mouse models.Eight-week-old sterile C57BL/6J mice were subjected to low-concentration acetic acid enema combined with restraint stress to construct a long-term voyage-related IBS sterile mouse model,and H.Biformis bacterial solution gavage(108 CFU/ml/day×7 days)was performed to observe the changes of body weight,fecal water content,intestinal permeability,intestinal barrier function and neurotransmitters of the IBS mouse model compared with the control group(the same volume and frequency of bacterial liquid solvent).Results:(1)Construction of long-term voyage-related IBS mouse models.The fecal moisture content of mice was significantly increased by 2 % v/w and 3 % v/w acetic acid enema combined with restraint stress,but the mortality rate of mice under 3 % v/w acetic acid enema combined with restraint stress was significantly increased.Therefore,in this study,a mouse model of long-term voyage-related IBS was constructed by using 2 % v/w acetic acid enema combined with restraint stress for follow-up research.(2)Lysine or proline supplementation in the long-term voyage-related IBS mouse models.Lysine deficiency or proline deficiency can lead to further aggravation of IBS-related phenotypes in mice,including significant weight loss and significant increase in intestinal permeability.Supplementation of lysine or proline could significantly inhibit the weight loss and intestinal barrier disruption in IBS mice.Proline supplementation could significantly improve the wet stool of IBS mice,while lysine supplementation could not improve the wet stool of IBS mice.Replenishing lysine can significantly increase the levels of neurotransmitters such as N-acetylserotonin and L-tryptophan in the serum,while supplementing proline can significantly decrease the levels of abnormally elevated 3-methoxytyramine and 3-hydroxykynurenine in the serum.(3)Intervention of H.Biformis in sterile mouse model of long-term voyage-related IBS.Constructing a sterile mouse model of long-term voyage-related IBS,we found that oral administration of H.Biformis can significantly promote weight recovery in IBS sterile mice,significantly improve intestinal permeability in female IBS sterile mice,increase TJP1 expression levels in ileum tissue,and significantly increase neurotransmitters such as taurine,tryptophanol,and indoleacetic acid in serum.Conclusion: Lysine,proline,and H.Biformis have certain alleviating effects on the symptoms related to IBS mice,and have clinical application prospects for preventing or improving long-term voyage-related IBS.