Analysis of the colonic mucosa associated microbiota (MAM) using brushing samples during colonic endoscopic procedures.

The mucosa-associated microbiota is an important component in human microbiota. The aim was to investigate mucosa-associated microbiota using brush samples during endoscopic procedures and compare with fecal microbiota. Seven patients who were planning to undergo a routine colonoscopy were recruited. Mucosal brushing samples were taken from 3 sites (terminal ileum, ascending and sigmoid colon), and a fecal sample was taken on the morning of colonoscopy. The samples were immediately placed in microcentrifuge tubes containing DNA stabilization reagent and analyzed using the next generation sequencer. The individual differences in microbiota were more evident than the differences of the sampling sites. Actinobacteria was more abundant and Bacteroidetes was less in the brush samples than those in the fecal samples. Taxonomic composition at the genus level and the proportion of genes responsible for some functions in the brushing samples tended to be different from those in the fecal samples. Bulleidia and Oribacteriumi were significantly more abundant and the proportions of genes responsible for transcription factors and phosphotransferase system were higher in ileal mucous than those in fecal samples. Brushing during colonoscopic procedure instead of using feces samples might be useful to analyze mucosa-associated microbiota.

Sishen Wan enhances intestinal barrier function via regulating endoplasmic reticulum stress to improve mice with diarrheal irritable bowel syndrome.

BACKGROUND: Diarrheal irritable bowel syndrome (IBS-D), characterized primarily by the presence of diarrhea and abdominal pain, is a clinical manifestation resulting from a multitude of causative factors. Furthermore, Sishen Wan (SSW) has demonstrated efficacy in treating IBS-D. Nevertheless, its mechanism of action remains unclear. METHODS: A model of IBS-D was induced by a diet containing 45 % lactose and chronic unpredictable mild stress. Additionally, the impact of SSW was assessed by measuring body weight, visceral sensitivity, defecation parameters, intestinal transport velocity, intestinal neurotransmitter levels, immunohistochemistry, and transmission electron microscopy analysis. Immunofluorescent staining was used to detect the expression of Mucin 2 (MUC2) and Occludin in the colon. Western blotting was used to detect changes in proteins related to tight junction (TJ), autophagy, and endoplasmic reticulum (ER) stress in the colon. Finally, 16S rRNA amplicon sequencing was used to monitor the alteration of gut microbiota after SSW treatment. RESULTS: Our study revealed that SSW administration resulted in reduced visceral sensitivity, improved defecation parameters, decreased intestinal transport velocity, and reduced intestinal permeability in IBS-D mice. Furthermore, SSW promotes the secretion of colonic mucus by enhancing autophagy and inhibiting ER stress. SSW treatment caused remodeling of the gut microbiome by increasing the abundance of Blautia, Muribaculum and Ruminococcus torques group. CONCLUSION: SSW can improve intestinal barrier function by promoting autophagy and inhibiting ER stress, thus exerting a therapeutic effect on IBS-D.

Could Microplastics Be a Driver for Early Onset Colorectal Cancer?

Introduction: The incidence of colorectal cancer in those under 50 years of age (early onset colorectal cancer (EOCRC)) is increasing throughout the world. This has predominantly been an increase in distal colonic and rectal cancers, which are biologically similar to late onset colorectal cancer (LOCRC) but with higher rates of mucinous or signet ring histology, or poorly differentiated cancers. The epidemiology of this change suggests that it is a cohort effect since 1960, and is most likely driven by an environmental cause. We explore the possible role of microplastics as a driver for this change. Review: The development of sporadic colorectal cancer is likely facilitated by the interaction of gut bacteria and the intestinal wall. Normally, a complex layer of luminal mucus provides colonocytes with a level of protection from the effects of these bacteria and their toxins. Plastics were first developed in the early 1900s. After 1945 they became more widely used, with a resultant dramatic increase in plastic pollution and their breakdown to microplastics. Microplastics (MPs) are consumed by humans from an early age and in increasingly large quantities. As MPs pass through the gastrointestinal tract they interact with the normal physiological mechanism of the body, particularly in the colon and rectum, where they may interact with the protective colonic mucus layer. We describe several possible mechanisms of how microplastics may disrupt this mucus layer, thus reducing its protective effect and increasing the likelihood of colorectal cancer. Conclusions: The epidemiology of increase in EOCRC suggests an environmental driver. This increase in EOCRC matches the time sequence in which we could expect to see an effect of rapid increase of MPs in the environment and, as such, we have explored possible mechanisms for this effect. We suggest that it is possible that the MPs damage the barrier integrity of the colonic mucus layer, thus reducing its protective effect. MPs in CRC pathogenesis warrants further investigation. Future directions: Further clarification needs to be sought regarding the interaction between MPs, gut microbiota and the mucus layer. This will need to be modelled in long-term animal studies to better understand how chronic consumption of environmentally-acquired MPs may contribute to an increased risk of colorectal carcinogenesis.

Systemic murine cathelicidin CRAMP safely attenuated colonic neutrophil infiltration in pigs.

Post-weaning diarrheic colitis, often caused by enteropathogens, are severe and potentially lethal diseases in young pigs. Conventional treatment with antibiotics is problematic due to increasing prevalence of multi-drug resistant bacteria. Few alternative treatments exist, so development of antibiotic-free therapies is urgently needed for livestock. Cathelicidin peptides, produced by epithelial cells and neutrophils, are microbicidal compounds capable of modulating innate immune and inflammatory responses. However, the effects of exogenous cathelicidin on gut homeostasis is poorly understood in pigs. We administered the murine cathelicidin CRAMP systemically to healthy pigs, to establish the peptide's safety and assess its ability to modulate colonic mucosal defenses. A single intraperitoneal injection of CRAMP was well tolerated up to two weeks and pigs remained clinically healthy. CRAMP caused some alteration of mucus glycosylation patterns in the colon by increasing sialylated mucins (P < 0.05) and decreased neutrophil influx close to the epithelium (P < 0.001). This study supports further investigation of CRAMP as an immunomodulatory treatment for infectious colitis in pigs.

Akkermansia muciniphila Protects Against Psychological Disorder-Induced Gut Microbiota-Mediated Colonic Mucosal Barrier Damage and Aggravation of Colitis.

Psychological disorders are associated with increased risk of severe inflammatory bowel disease (IBD) by causing gut microbiota dysbiosis and colonic mucosal barrier damage. However, the interaction between chronic restraint stress (CRS), gut microbiota composition, and colonic mucus remains unclear. We demonstrated that mice under CRS conditions exhibited alterations in microbiota composition, disruption of colonic mucus, and aggravation of colitis. In addition, the abundance of Akkermansia muciniphila was significantly decreased in mice under CRS and UC patients with depression, and positively associated with the expression of MUC2. After antibiotic treatment, the recipient mice colonized with CRS microbiota showed barrier defects and severe colitis. Administration of Akkermansia muciniphila was found to restore colonic mucus and modify the gut microbiota. We confirm that CRS-mediated gut microbiota dysbiosis results in colonic mucosal barrier damage and aggravation of colitis. Our results suggest that A. muciniphila is expected to be a potential probiotic to protect and treat colonic mucus that is involved in IBD with psychological disorders.

Rhubarb extract relieves constipation by stimulating mucus production in the colon and altering the intestinal flora.

BACKGROUND: Constipation, mainly characterized by the difficulty in defecation, is a clinical symptom caused by a variety of factors. It can be manifested as normal or slow colonic transport abnormalities, which can occur alone or concurrently with defecation disorders. As there is not uniform definition and assessment standard, no clear plan could be used for the treatment of constipation. Although rhubarb, a traditional Chinese medicine, plays a therapeutic role in diseases involving constipation symptoms, the detailed mechanism of it in treating constipation remains unclear. METHODS: A model of constipation-induced by diphenoxylate was prepared. Immunofluorescent staining was used to detect the expression of mucin 2 (MUC2), calnexin and chymase in colon. Western blotting was used to detect changes of tryptase and calnexin in the colon. And real-time polymerase chain reaction (PCR) was utilized to detect the changes of immunoglobulin-binding protein (Bip), X-box binding protein 1 (Xbp1) and C/EBP homologous protein (CHOP) of colonic goblet cells in mRNA levels. ELISA and biochemical kits were utilized to detect the changes of MUC2, Trefoil factor 3 (TFF3), acetylcholine, histamine and C-C motif chemokine ligand 5 (CCL5) in the colon. And the changes of colonic mucosa and intestinal flora of constipation model mice caused by rhubarb extract (RE) were analyzed to identify the mechanism of RE on the treatment of constipation. RESULTS: RE promotes the secretion of colonic mucus by recruiting mast cells and enhancing the content of histamine and Ach in the mice colon. In the process, RE causes up-regulation of Bip and CHOP mRNA expression and down-regulation of Xbp1 and Xbp1s mRNA expression that induces ER stress of colonic epithelium associated with changes in the intestinal flora diversity and short-chain fatty acids content. CONCLUSION: RE could relieve constipation by promoting the secretion of colonic mucus via mast cells activation and improving the intestinal microenvironment.

Dietary Modulation Alters Susceptibility to Listeria monocytogenes and Salmonella Typhimurium with or without a Gut Microbiota.

Food safety has considerably improved worldwide, yet infections with foodborne human enteric pathogens, such as Listeria spp. and Salmonella spp., still cause numerous hospitalizations and fatalities. Since dietary alterations, including fiber deficiency, might impact the colonization resistance mediated by the gut microbiome, studying the diet-microbiome-pathogen axis holds promise in further understanding the pathogenesis mechanisms. Using a gnotobiotic mouse model containing a 14-member synthetic human gut microbiota (14SM), we have previously shown that dietary fiber deprivation promotes proliferation of mucin-degrading bacteria, leading to a microbiome-mediated erosion of the colonic mucus barrier, which results in an increased susceptibility toward the rodent enteric pathogen Citrobacter rodentium. Here, we sought to understand how a low-fiber diet affects susceptibility to Listeria monocytogenes and Salmonella enterica serovar Typhimurium by using our 14SM gnotobiotic mouse model in BALB/c and C57BL/6 mouse backgrounds, respectively. Intriguingly, and in contrast to our results with C. rodentium, we observed that depriving mice of dietary fiber protected them from infections with both pathogens, compared to mice fed a standard chow. The microbiome delayed the overall pathogenicity compared to the onset of disease observed in germfree control mice. Nevertheless, we observed the same effect of diet on germfree mice, suggesting that the susceptibility is directly driven by the diet itself even in the absence of the gut microbiome. Our study points out an important observation, namely, that dietary fiber plays a crucial role in either the host's susceptibility, the virulence of these pathogens, or both. It would be judicious to design and interpret future studies on this basis. IMPORTANCE The human enteric pathogens Listeria monocytogenes and Salmonella Typhimurium are employed as classical models in rodent hosts to understand the pathogenesis mechanisms of foodborne pathogens. Research in the past decade has stressed the importance of the gut microbial composition in modulating susceptibility to these pathogens. The results of our study-using gnotobiotic mice and germfree control animals-additionally suggest that the dietary fiber components can dominate the impact of enteropathogenic virulence over the pathogenicity-modulating properties of the gut microbiome. The significance of our research is that there is a need to carefully choose a certain chow when performing the enteropathogen-associated mouse experiments and to cautiously match the rodent diets when trying to replicate experiments across different laboratories. Finally, our data underscore the importance of using germfree control animals to study these pathogens, as our findings would have been prone to misinterpretation in the absence of these controls.

1,25(OH)2D3 deficiency-induced gut microbial dysbiosis degrades the colonic mucus barrier in Cyp27b1 knockout mouse model.

BACKGROUND: The relationship between disturbances of the gut microbiota and 1,25(OH)2D3 deficiency has been established both in humans and animal models with a vitamin D poor diet or a lack of sun exposure. Our prior study has demonstrated that Cyp27b1 -/- (Cyp27b1 knockout) mice that could not produce 1,25(OH)2D3 had significant colon inflammation phenotypes. However, whether and how 1,25(OH)2D3 deficiency due to the genetic deletion controls the gut homeostasis and modulates the composition of the gut microbiota remains to be explored. RESULTS: 1,25(OH)2D3 deficiency impair the composition of the gut microbiota and metabolite in Cyp27b1 -/- mice, including Akkermansia muciniphila, Solitalea Canadensis, Bacteroides-acidifaciens, Bacteroides plebeius and SCFA production. 1,25(OH)2D3 deficiency cause the thinner colonic mucus layer and increase the translocation of the bacteria to the mesenteric lymph nodes. We also found 1,25(OH)2D3 supplement significantly decreased Akkermansia muciniphila abundance in fecal samples of Cyp27b1 -/- mice. CONCLUSION: Deficiency in 1,25(OH)2D3 impairs the composition of gut microbiota leading to disruption of intestinal epithelial barrier homeostasis and induction of colonic inflammation. This study highlights the association between 1,25(OH)2D3 status, the gut microbiota and the colonic mucus barrier that is regarded as a primary defense against enteric pathogens.

[Effect of Angelica sinensis radix on colonic morphology and mucus secretion of experimental XuexuBianmi model mice].

OBJECTIVE: To study the effects of Angelicaesinensis radix (Danggui) decoction on the therapeutic action and the colonic morphology and mucus secretion in XuexuBianmi model mice. METHODS: Sixty Kunming mice, maleandfemaleinhalf, were randomly divided into six groups according to gender and weight (n=10):normal control group, XuexuBianmi model group, positive control group, and high-dose, middle-dose and low-dose Danggui groups. Except the normal control group, the mice in the rest groups were orally administrated with diphenoxylate (DPN) and subcutaneously injected with acetylphenyhydrazine (APH) and intraperitoneally injected with cyclophosphamide (CPA) to copy XuexuBianmi model. Fromthe 14th day, the mice in Danggui groups were orally administered with different doses of Danggui decoction (16.67、8.33、4.17 g/kg), the mice in positive control group were orally administered with Changtongshu granule (5 g/kg), the mice in XuexuBianmi model group were administered with normal saline (NS) at the same volume (10 ml/kg), once a day for consecutive 28 days. The general status were observed, the first black defecation time (FBDT), the water content in stool and in colon in mice of all groups were tested. And the colon tissue was stained with hematoxylin-eosin (HE) and AB-PAS to observe the changes of colonic morphology and the mucus secretion. RESULTS: Compared with the normal control group, there appeared the XuexuBianmi syndromes in model group as follows, the defecation time (FBDT) was significantly prolonged, the water contentinstool and in colon were decreased (P<0.01), the colonic mucosa and gland were atrophied, mucous membrane layer was thinned (P<0.01), mucus secretion was decreased. Compared with the XuexuBianmimodel group, the Xuexu and Bianmi syndromes were improved, the FBDT was significantly shortened (P<0.05, P<0.01), the water content of colon was increased in the three groups of Danggui decoction (P<0.05, P<0.01). The water content of the stool was obviouslyincreased in groups of 16.67and 8.33kg.dDanggui decoction (P<0.05). The atrophy of colon mucosa and gland was improved, the mucus secretion was increased, and the colon lubrication function was improved in the three groups in different doses of Danggui decoction. CONCLUSIONS: Dangguican improve the mucosal atrophy, and increase the secretion of colonic mucus, then the stool is softened and promoted to discharge.

[Establishment and effect observation on three kinds of Ti-Xu with constipation rat models].

OBJECTIVE: To establish three types constipation models with the syndrome of Xue-Xu, Yin-Xu and Yang-Xu in rats, and to compare the difference of colonic motility, colonic water metabolism, colonic mucus secretion and the level of aquaporin-2 (AQP2). METHODS: Forty Sprague Dawley rats, male and female in half, were randomly divided into four groups:normal control group(N), Xue-Xu with constipation group(XC), Yin-Xu with constipation group(YC) and Yang-Xu with constipation group(YAC). Bloodletting and loperamide were used to induce the Xue-Xu with constipation in rats, thyroxin and loperamide were used to induce the Yin-Xu with constipation in rats, ice-water stimulation and loperamide were used to induce the Yang-Xu with constipation in rats. Bloodletting was coducted once a week,drug was ad-ministrated orally to rats once a day for continuous 42 days. Rats'state, body weights, stool character, transfer time between mouth and anal, small intestinal propulsion rate were measured. The colon tissues of rats were stained with alcian blue-periodic acid schiff (AB-PAS) to analyze the changes of mucus secretion. The aquaporin-2 expressions in proximal and distal colon of rats were measured by immunohistochemical method. RESULTS: Compared to normal control group, weight-increasing speed of the rats were reduced in three models. The order of indepen-dent movement change was YC, XC and YAC. Solid stool appeared at 30 d, fecal scores were increased. Transfer time between mouth and anal was significantly extended, small intestinal propulsion rate were significantly decreased(P < 0.05, P < 0.01). Water content in stool were significantly decreased in three type models(P < 0.05, P < 0.01), the content of water in colon were decreased in XC and YAC group (P < 0.05, P < 0.01). The colonic gland and goblet cells were narrowed in some extent, the mucus excretion were decreased. The levels of aquaporin-2 expressions in proximal and distal colon of rats were increased(P < 0.05, P < 0.01), increasing order of AQP2 expression in proximal colon was YAC, YC and XC, increasing order of AQP2 expression in distal colon was YAC, XC and YC. CONCLUSIONS: Long term stimulation of complex factors could induce Ti-Xu with constipation animal model, and there were some differences in colonic motility, water metabolism, colonic mucus secretion and AQP2 in rats.