Sucralose Promotes Colitis-Associated Colorectal Cancer Risk in a Murine Model Along With Changes in Microbiota.

Sucralose is a calorie-free high-intensity artificial sweetener that is widely used in thousands of foods and beverages all over the world. Although it was initially regarded as a safe, inert food additive, its adverse effect on gut microbiota and health has drawn more and more attention as evidence accumulates. Studies by us and others revealed that sucralose exacerbated gut damage and inflammation in animal models for inflammatory bowel disease (IBD), including those for both ulcerative colitis, and Crohn's disease. Our study demonstrated that sucralose greatly aggravated dextran sulfate sodium (DSS)-induced colitis along with causing changes in gut microbiota, the gut barrier and impaired inactivation of digestive proteases mediated by deconjugated bilirubin. It is well-documented that IBD greatly increases the risk of colorectal cancer (CRC), the globally third-most-common cancer, which, like IBD, has a high rate in the developed countries. Azoxymethane (AOM)/DSS has been the most commonly used animal model for CRC. In this study, we further explored the effect of sucralose on tumorigenesis and the possible mechanism involved using the AOM/DSS mouse model. First, 1.5 mg/ml sucralose was included in the drinking water for 6 weeks to reach a relatively stable phase of impact on gut microbiota. Then, 10 mg/kg AOM was administered through intraperitoneal injection. Seven days later, 2.5% DSS was put in the drinking water for 5 days, followed by 2 weeks without DSS. The 5 days of DSS was then repeated, and the mice were sacrificed 6 weeks after AOM injection. The results showed that sucralose caused significant increases in the number and size of AOM/DSS-induced colorectal tumors along with changes in other parameters such as body and spleen weight, pathological scores, mortality, fecal ß-glucuronidase and digestive proteases, gut barrier molecules, gut microbiota, inflammatory cytokines and pathways (TNFα, IL-1ß, IL-6, IL-10, and TLR4/Myd88/NF-κB signaling), and STAT3/VEGF tumor-associated signaling pathway molecules. These results suggest that sucralose may increase tumorigenesis along with dysbiosis of gut microbiota, impaired inactivation of digestive protease, damage to the gut barrier, and exacerbated inflammation.

The Functional Role of Fecal Microbiota Transplantation on Dextran Sulfate Sodium-Induced Colitis in Mice.

Increasingly studies revealed that dysbiosis of gut microbiota plays a pivotal role in the pathogenesis of ulcerative colitis (UC). Fecal microbiota transplantation (FMT) has drawn more and more attention and become an important therapeutic approach. This study aims to examine the facts about the effective components and look into potential mechanisms of FMT. Colitis was induced by 3% (w/v) dextran sulfate sodium (DSS) in drinking water for 7 days. Colitis mice were administered by oral gavage with fecal suspension, fecal supernatant, fecal bacteria, or boiling-killed fecal bacteria from healthy controls and the disease activity index was monitored daily. On the seventh day, mice were euthanized. The length, histological score, parameters related to inflammation, gut barrier functions of the colon, activities of digestive protease and ß-glucuronidase in feces were measured. All of the four fecal components showed certain degree of efficacy in DSS-induced colitis, while transplantation of fecal suspension showed the most potent effect as demonstrated by less body weight loss, lower disease activity scores, more expression of tight junction proteins and TRAF6 and IκBα, less expression of TNF-α, IL-1ß, IL-10, TLR-4, and MyD88 in gut tissue, as well as restoration of fecal ß-glucuronidase and decreases in fecal digestive proteases. These results provide a novel insight into the possible mechanism of FMT and may help to improve and optimize clinical use of FMT.

Unconjugated bilirubin alleviates experimental ulcerative colitis by regulating intestinal barrier function and immune inflammation.

BACKGROUND: Unconjugated bilirubin (UCB) is generally considered toxic but has gained recent prominence for its anti-inflammatory properties. However, the effects of it on the interaction between intestinal flora and organisms and how it influences immune responses remain unresolved. AIM: To investigate the role of UCB in intestinal barrier function and immune inflammation in mice with dextran-sulfate-sodium-induced colitis. METHODS: Acute colitis was induced by 3% (w/v) dextran sulfate sodium salt in drinking water for 6 d followed by untreated water for 2 d. Concurrently, mice with colitis were administered 0.2 mL UCB (400 µmol/L) by intra-gastric gavage for 7 d. Disease activity index (DAI) was monitored daily. Mice were sacrificed at the end of the experiment. The length of the colon and weight of the spleen were recorded. Serum level of D-lactate, intestinal digestive proteases activity, and changes to the gut flora were analyzed. In addition, colonic specimens were analyzed by histology and for expression of inflammatory markers and proteins. RESULTS: Mice treated with UCB had significantly relieved severity of colitis, including lower DAI, longer colon length, and lower spleen weight (colon length: 4.92 ± 0.09 cm vs 3.9 ± 0.15 cm; spleen weight: 0.33 ± 0.04 vs 0.74 ± 0.04, P < 0.001). UCB administration inactivated digestive proteases (chymotrypsin: 18.70 ± 0.69 U/g vs 44.81 ± 8.60 U/g; trypsin: 1.52 ± 0.23 U/g vs 9.05 ± 1.77 U/g, P < 0.01), increased expression of tight junction (0.99 ± 0.05 vs 0.57 ± 0.03, P < 0.001), decreased serum level of D-lactate (31.76 ± 3.37 µmol/L vs 54.25 ± 1.45 µmol/L, P < 0.001), and lowered histopathological score (4 ± 0.57 vs 7 ± 0.57, P < 0.001) and activity of myeloperoxidase (46.79 ± 2.57 U/g vs 110.32 ± 19.19 U/g, P < 0.001). UCB also regulated the intestinal microbiota, inhibited expression of tumor necrosis factor (TNF) α and interleukin 1ß (TNF-α: 52.61 ± 7.81 pg/mg vs 105.04 ± 11.92 pg/mg, interleukin 1ß: 13.43 ± 1.68 vs 32.41 ± 4.62 pg/mg, P < 0.001), decreased expression of Toll-like receptor 4 (0.61 ± 0.09 vs 1.07 ± 0.03, P < 0.001) and myeloid differentiation primary response gene 88 (0.73 ± 0.08 vs 1.01 ± 0.07, P < 0.05), and increased expression of TNF-receptor-associated factor 6 (0.79 ± 0.02 vs 0.43 ± 0.09 P < 0.05) and inhibitor of kappa B α (0.93 ± 0.07 vs 0.72 ± 0.07, P < 0.05) in the colon. CONCLUSION: UCB can protect intestinal barrier function, regulate normal intestinal homeostasis, and suppress inflammation via the Toll-like receptor 4/ nuclear factor-κB signaling pathway.

Unconjugated bilirubin ameliorates the inflammation and digestive protease increase in TNBS-induced colitis.

The authors previously demonstrated that unconjugated bilirubin (UCB) may inhibit the activities of various digestive proteases, including trypsin and chymotrypsin. The digestive proteases in the lower gut are important in the pathogenesis of inflammatory bowel diseases. The effects of UCB on the inflammation and levels of digestive proteases in feces of rats with colitis have not yet been revealed. The present study investigated the effect of UCB on the inflammatory status and levels of trypsin and chymotrypsin in the feces of rats with trinitrobenzenesulfonic acid (TNBS)­induced colitis. The data indicated that treatment with TNBS resulted in a marked reduction in weight gain, which was significantly alleviated in UCB­treated rats. Furthermore, UCB treatment alleviated the inflammation induced by TNBS, detected via macroscopic damage and microscopic inflammation scores, and pro­inflammatory markers including myeloperoxidase (MPO), tumor necrosis factor (TNF)­α and interleukin (IL)­1ß. Furthermore, rats with colitis demonstrated significant increases in fecal trypsin and chymotrypsin levels, whereas UCB treatment significantly alleviated these increases. A significant positive correlation was additionally revealed among the pro­inflammatory markers (MPO, TNF­α and IL­1ß) and fecal digestive proteases (trypsin and chymotrypsin) in colitis. The results of the present study demonstrated that UCB ameliorated the inflammation and digestive protease increase in TNBS-induced colitis.

Intraluminal nonbacterial intestinal components control gut and lung injury after trauma hemorrhagic shock.

OBJECTIVE: To test whether the mucus layer, luminal digestive enzymes, and intestinal mast cells are critical components in the pathogenesis of trauma shock-induced gut and lung injury. BACKGROUND: Gut origin sepsis studies have highlighted the importance of the systemic component (ischemia-reperfusion) of gut injury, whereas the intraluminal component is less well studied. METHODS: In rats subjected to trauma hemorrhagic shock (T/HS) or sham shock, the role of pancreatic enzymes in gut injury was tested by diversion of pancreatic enzymes via pancreatic duct exteriorization whereas the role of the mucus layer was tested via the enteral administration of a mucus surrogate. In addition, the role of mast cells was assessed by measuring mast cell activation and the ability of pharmacologic inhibition of mast cells to abrogate gut and lung injury. Gut and mucus injury was characterized functionally, morphologically, and chemically. RESULTS: Pancreatic duct exteriorization abrogated T/HS-induced gut barrier loss and limited chemical mucus changes. The mucus surrogate prevented T/HS-induced gut and lung injury. Finally, pancreatic enzyme-induced gut and lung injury seems to involve mast cell activation because T/HS activates mast cells and pharmacologic inhibition of intestinal mast cells prevented T/HS-induced gut and lung injury. CONCLUSIONS: These results indicate that gut and gut-induced lung injury after T/HS involves a complex process consisting of intraluminal digestive enzymes, the unstirred mucus layer, and a systemic ischemic-reperfusion injury. This suggests the possibility of intraluminal therapeutic strategies.

Why is damage limited to the mucosa in ulcerative colitis but transmural in Crohn's disease?

It has been a big puzzle as why the inflammation of ulcerative colitis (UC) is limited to the mucosa, while in Crohn's disease (CD) the inflammation is transmural and can be seen in all layers of the gut. Here, I give a tentative explanation extended from the unified hypothesis I proposed on the etiology of inflammatory bowel disease. This hypothesis suggested that both UC and CD are caused by weakening of the gut barrier due to damage of the protective mucus layer and the underlying tissue by the poorly inactivated digestive proteases resulting from a reduction of gut bacteria by dietary chemicals like saccharin and sucralose. However, the large amounts of bacteria in the colon make the recruitment of neutrophils and formation of crypt abscess the main manifestation of UC, while the infiltration of antigens and dietary particles in the small and large intestine mainly cause the recruitment of macrophages and formation of granulomas as the main manifestations in CD. The fast reacting and short life span of neutrophils make the fight and damage limited to the surface of the mucosa. In contrast, the long life span and constant movement of macrophages may bring the harmful agents deep into the tissue. Therefore, the pathogenesis of UC may be more like bacterial pneumonia, while CD may be more like pneumoconiosis or tuberculosis of the lung.

Etiology of inflammatory bowel disease: a unified hypothesis.

Inflammatory bowel disease (IBD), including both ulcerative colitis (UC) and Crohn's disease (CD), emerged and dramatically increased for about a century. Despite extensive research, its cause remains regarded as unknown. About a decade ago, a series of findings made me suspect that saccharin may be a key causative factor for IBD, through its inhibition on gut bacteria and the resultant impaired inactivation of digestive proteases and over digestion of the mucus layer and gut barrier (the Bacteria-Protease-Mucus-Barrier hypothesis). It explained many puzzles in IBD such as its emergence and temporal changes in last century. Recently I further found evidence suggesting sucralose may be also linked to IBD through a similar mechanism as saccharin and have contributed to the recent worldwide increase of IBD. This new hypothesis suggests that UC and CD are just two symptoms of the same morbidity, rather than two different diseases. They are both caused by a weakening in gut barrier and only differ in that UC is mainly due to increased infiltration of gut bacteria and the resultant recruitment of neutrophils and formation of crypt abscess, while CD is mainly due to increased infiltration of antigens and particles from gut lumen and the resultant recruitment of macrophages and formation of granulomas. It explained the delayed appearance but accelerated increase of CD over UC and many other phenomena. This paper aims to provide a detailed description of a unified hypothesis regarding the etiology of IBD, including the cause and mechanism of IBD, as well as the relationship between UC and CD.

Testosterone depletion or blockade in male rats protects against trauma hemorrhagic shock-induced distant organ injury by limiting gut injury and subsequent production of biologically active mesenteric lymph.

BACKGROUND: We tested the hypothesis that testosterone depletion or blockade in male rats protects against trauma hemorrhagic shock-induced distant organ injury by limiting gut injury and subsequent production of biologically active mesenteric lymph. METHODS: Male, castrated male, or flutamide-treated rats (25 mg/kg subcutaneously after resuscitation) were subjected to a laparotomy (trauma), mesenteric lymph duct cannulation, and 90 minutes of shock (35 mm Hg) or trauma sham-shock. Mesenteric lymph was collected preshock, during shock, and postshock. Gut injury was determined at 6 hours postshock using ex vivo ileal permeability with fluorescein dextran. Postshock mesenteric lymph was assayed for biological activity in vivo by injection into mice and measuring lung permeability, neutrophil activation, and red blood cell deformability. In vitro neutrophil priming capacity of the lymph was also tested. RESULTS: Castrated and flutamide-treated male rats were significantly protected against trauma hemorrhagic shock (T/HS)-induced gut injury when compared with hormonally intact males. Postshock mesenteric lymph from male rats had a higher capacity to induce lung injury, Neutrophil (PMN) activation, and loss of red blood cell deformability when injected into naïve mice when compared with castrated and flutamide-treated males. The increase in gut injury after T/HS in males directly correlated with the in vitro biological activity of mesenteric lymph to prime neutrophils for an increased respiratory burst. CONCLUSIONS: After T/HS, gut protective effects can be observed in males after testosterone blockade or depletion. This reduced gut injury contributes to decreased biological activity of mesenteric lymph leading to attenuated systemic inflammation and distant organ injury.