Effectsof Heparin On Intestinal Bacteria And Intestinal Microbiota And The Action Mechanism

Heparin and heparan sulfate (HS) are linear polyanionicpolysaccharidesconsisting of uronic acid and glucosamine repeatingdisaccharide subunits. Both of them belong to the glycosaminoglycans (GAGs) family. In addition to heparin and HS, the GAGs family also includes hyaluronic acid, chondroitin sulfate, dermatan sulfate and keratin.Heparin and HS are the most complex members of the GAGs family. Heparinismainlysynthesized by and stored exclusively in mast cells, whereas HS is expressed, as a part of a proteoglycan, on various kinds of cell surfaces andthroughout tissue matrices. There are differences between the composition and length of the polysaccharide backbones of heparin and HS. Furthermore, variable patterns of substitution of thedisaccharide subunits with N-sulfate, O-sulfate and N-acetyl groups give rise to a large number of complex sequences to heparin or HS. Various structures of heparin and HS account for their abilities to bind many biological molecules, which confer many biological functions on them.Heparin and HS involve in many biological activities in vivo, such as anticoagulation, anti-infection, inflammation, metastasis of cancer, cell adhesion and differentiation, etc. There is increasing evidence that the adhesion, internalization, and transfer of pathogens could be inhibited by heparin or HS. It has been reported that the adhesion of entero-pathogenic bacteria, such as Staphylococcus aureus, Escherichia coli and Listeria monocylogenes, to enterocytes or mucus could be inhibited by exogenous heparin or HS. However, the effect of heparin on the adhesion of Salmonella spp. and Shigella spp. to enterocytes is still unknown. Here we have studied the effects of heparin on enterocyte and mucus binding of several selected entero-pathogenic bacteria, including entero-pathogenic E. coli(EPEC), S. aureus, Salmonella enterica, and Shigella flexneri, providing the basis for its further development as an anti-infection agent.Compared to the studies on the effect of heparin on pathogenic bacteria, investigations on the effect of heparin on probiotics are very limited. This paper was to study the effect of heparin on the adhesion of Lactobacillus rhamnosus GGto enterocytes and mucus and its subsequent colonizationin vitro.In previous studies on the anti-inflammation use of heparin, attentions were paid to the interactions between heparin and host cells, and the administration route was either subcutaneousinjection or intravenous injection. The effect of orally administered heparin on the intestinal microbiota is still unknown. Therefore, we further studied the effects of heparin on the intestinal microbiota in rats and proposed that heparin or its analogue could be used as an effective intestinal microbiota modulator based on the results.1The effects of heparin on the adhesion of several selected enterpathogenic bacteria and LGG to enterocytes or mucusin vitroObjective To studythe in vitro effects of heparin on the adhesion and biofilm formation of a Lactobacillus rhamnosus strain, and of several selected pathogens to enterocytes or mucus.Methods Intestinal enterocytes HT-29monolayer and immobilized mucus layer were adopted as models to evaluate the effects of heparin on the adhesion of tested enteropathogenicbacteria, including EPEC, S. aureus,S. enterica, and S. flexneri, as well as the common probiotics LGG to the enterocytes and mucus. The competition, displacement and resistance binding assays were also performed to explore if there was an additive effect between heparin and LGG on preventing the adhesion of EPEC.In addition, the effects of heparin on the colonization of LGG were evaluated throughits effects onLGG biofilm formation.Results Heparin blocked the adhesion of tested E. coli andS. aureus strains but did not block the adhesion of S. enterica, S. flexneri,and LGG to enterocytes and mucus. For EPEC binding, a dose dependent effect was observed. The heparin concentration of50μg/mLis efficient toinhibit the adhesion of selected pathogens. In the competitionand resistance binding assays, heparin contributed little to the adhesion inhibition effect on EPEC in the presence of high concentration of LGG while it did help the adhesion inhibition when the concentration of LGG was low. Heparin had no effect in displacement assays.Furthermore, heparin facilitated the colonization ofLGGby promoting the biofilm formationby30%.Conclusion Exogenous heparin mainly interfered with the binding process of EPEC to HT-29cells through competitive bingding to the bacteria ahesins. The high negative charge density of heparin is not necessary for the selective anti-adhesion but facilitate the biofilm formation. To our knowledge, this study is the first time to evaluate heparin as a supplement to probiotics with the most commonly used probiotic strain LGG as a model. Heparin has a potential as a supplementary treatment to probiotics for prophylaxis at the onset of infections.2The effects of oral administration of heparin or heparosan on the intestinal microbiota in ratsObjective To investigate the impact of exogenous heparin or heparosan on gut microbiota or its subpopulations.Methods Sprague-Dawley rats were randomly divided into five groups, i.e. the natural saline group, the high dose heparin group (10mg/kg), the low dose heparin group (5mg/kg), the high dose heparosan group (10mg/kg) and the low dose heparosan group (5mg/kg).All animals were orally administrated with the above mentioned various doses of heparin or heparosan on a daily basis for two weeks. Fresh fecal samples were collected before the treatment, and on the14th days of the treatment.16S rRNAs were amplified with PCR using universal or specificprimers. PCR amplicons were separated by denaturing gradient gel electrophoresis (DGGE). Principle component analysis was used to evaluate the different banding patterns between groups. Bands which showed different intensity between groups were excised from the gel and sequenced.Results The predominant bacterial communities in the feces of heparin-or heparosan-treated animals were different from those of the saline-treated animals, with increased Lactobacillus spp. and decreased Enterococcus sp.in heparin-or heparosan-treated animals Different DGGE banding patterns were also observed for the subpopulations of Lactobacillus and Bacteroides groups.Furthermore, no rectal bleeding was observed in any group.Conclusion Orally administered heparin or heparosan could act as an effective gut microbiota modulator by facilitating colonization of Lactobacillus and this modulation was helpful against infections and inflammatory bowel diseases. The results also showed that the modulation effect of heparosan on the gut microbiota was similar to that of heparin. Considering that heparosan has no risk of bleeding and can be obtained cost-effectively by bacterial fermentation, heparosan or heparosan-derived polysaccharide might show more advantage over heparin as novel drug to modulate the gut microbiota.