A Spore-Forming Probiotic Supplement Improves the Intestinal Immune Response and Protects the Intestinal Health During Recurrent Clostridioides difficile Colonization in Mice.

BACKGROUND: Around 15%-30% of patients develop recurrent Clostridioides difficile infection (CDI) as conventional therapies disrupt protective gut microbiota. We tested if supplementation with a spore-forming probiotic would protect intestinal health in a mouse model of recurrent CD colonization. METHODS: Methods: Female CF-1 mice were exposed to CD spores (4-log10 colony-forming units/10 µL) and then randomly assigned to receive either saline (CD-S) or probiotic (CD-PRO). Control mice received only saline (control). Following confirmation of initial CD colonization, mice were treated with vancomycin (10 days). After 5 days, mice recolonized with CD were treated again with vancomycin (10 days) and euthanized 5 days later. Fecal samples were collected at select time points for bacterial analysis. Following euthanasia, blood samples, cecum contents, and the intestine were collected for analysis. RESULTS: Probiotic supplementation mitigated the antibiotic-induced changes in cecum weight (P < .001). Probiotic-supplemented mice had increased messenger RNA expression of several immune parameters, accompanied by lower serum iron levels compared with CD-S mice (P < .05). Lower expressions of TNF α and calprotectin (P ≤ .05) were observed in CD-PRO mice compared with CD-S. The probiotics also supported the expression of intestinal tight junction proteins, which were diminished in the proximal colon of CD-S mice (P < .05). CONCLUSION: Mice supplemented with targeted spore-forming probiotics exhibited improved immune responses and nutrition immunity properties, which were linked with less inflammation and enhanced intestinal barrier proteins during recurrent CD colonization.

Dietary Synbiotic Supplementation Protects Barrier Integrity of Hepatocytes and Liver Sinusoidal Endothelium in a Mouse Model of Chronic-Binge Ethanol Exposure.

Alcohol overconsumption disrupts the gut microbiota and intestinal barrier, which decreases the production of beneficial microbial metabolic byproducts and allows for translocation of pathogenic bacterial-derived byproducts into the portal-hepatic circulation. As ethanol is known to damage liver sinusoidal endothelial cells (LSEC), here we evaluated dietary supplementation with a previously studied synbiotic on gut microbial composition, and hepatocyte and LSEC integrity in mice exposed to ethanol. We tested a chronic-binge ethanol feeding mouse model in which C57BL/6 female mice were fed ethanol (5% vol/vol) for 10 days and provided a single ethanol gavage (5 g/kg body weight) on day 11, 6 h before euthanasia. An ethanol-treatment group also received oral supplementation daily with a synbiotic; and an ethanol-control group received saline. Control mice were pair-fed and isocalorically substituted maltose dextran for ethanol over the entire exposure period; they received a saline gavage daily. Ethanol exposure decreased gut microbial abundance and diversity. This was linked with diminished expression of adherens junction proteins in hepatocytes and dysregulated expression of receptors for advanced glycation end-products; and this coincided with reduced expression of endothelial barrier proteins. Synbiotic supplementation mitigated these effects. These results demonstrate synbiotic supplementation, as a means to modulate ethanol-induced gut dysbiosis, is effective in attenuating injury to hepatocyte and liver endothelial barrier integrity, highlighting a link between the gut microbiome and early stages of acute liver injury in ethanol-exposed mice.

A Designer Synbiotic Attenuates Chronic-Binge Ethanol-Induced Gut-Liver Injury in Mice.

Gut dysbiosis and altered short-chain fatty acids are associated with ethanol-induced liver injury. SCFA are fermentation byproducts of the gut microbiota known to have many beneficial biological effects. We tested if a designer synbiotic could protect against ethanol-induced gut-liver injury. C57BL/6 female mice were exposed to chronic-binge ethanol feeding consisting of ethanol (5% vol/vol) for 10 days, followed by a single gavage (5 g/kg body weight) 6 h before euthanasia. A group of mice also received oral supplementation daily with a designer synbiotic, and another group received fecal slurry (FS); control animals received saline. Control mice were isocalorically substituted maltose dextran for ethanol over the entire exposure period. Ethanol exposure reduced expression of tight junction proteins in the proximal colon and induced hepatocyte injury and steatosis. Synbiotic supplementation not only mitigated losses in tight junction protein expression, but also prevented ethanol-induced steatosis and hepatocyte injury. Ethanol exposure also increased hepatic inflammation and oxidative stress, which was also attenuated by synbiotic supplementation. Mice receiving FS were not protected from ethanol-induced liver injury or steatosis. Results were associated with luminal SCFA levels and SCFA transporter expression in the proximal colon and liver. These results indicate supplementation with a designer synbiotic is effective in attenuating chronic-binge ethanol-induced gut-liver injury and steatosis in mice, and highlight the beneficial effects of the gut microbial fermentation byproducts.

Faecalibacterium prausnitzii and a Prebiotic Protect Intestinal Health in a Mouse Model of Antibiotic and Clostridium difficile Exposure.

BACKGROUND: Clostridium difficile (CD) infection (CDI) increases patient morbidity, mortality and healthcare costs. Antibiotic treatment induces gut dysbiosis and is both a major risk factor for CD colonization and treatment of CDI. Probiotics have been trialed to support commensal gut microbiota and reduce CDI. This study investigated commensal microbe Faecalibacterium prausnitzii (FP) and a prebiotic, both known to yield butyrate and be anti-inflammatory and immunomodulatory, on CD colonization and gut integrity in mice. METHODS: Mice were randomly grouped and supplemented daily with FP, prebiotic, FP + prebiotic, FP/prebiotic supernatant, or saline throughout the entire study. Following treatment with clindamycin for 3 days, mice were exposed to CD. Feces were collected at baseline, the day after antibiotic, and 1, 3, and 5 days after CD exposure and cultured for bacterial overgrowth and CD colonization. On days 1 and 5 after CD exposure, mice were randomly euthanized, and proximal colon was dissected for histological analysis and preparation of RNA for analysis of proinflammatory and anti-inflammatory cytokines. RESULTS: Although all mice exhibited bacterial overgrowth and CD colonization, bacterial burden resolved quicker in the FP + prebiotic group. This was associated with induction and resolution of innate immune responses, anion exchanger, and tight junction protein preservation in proximal colon. CD toxin virulence potential was questionable as expression of CD toxin B receptor was depleted in the FP + prebiotic group. CONCLUSION: Supplementation with anti-inflammatory butyrate-supporting commensal bacteria and prebiotic may support innate immune responses and minimize bacterial burden and negative effects during antibiotic and CD exposure.

Prophylactic tributyrin treatment mitigates chronic-binge ethanol-induced intestinal barrier and liver injury.

BACKGROUND AND AIM: Impaired gut-liver axis is a potential factor contributing to alcoholic liver disease. Ethanol depletes intestinal integrity and causes gut dysbiosis. Butyrate, a fermentation byproduct of gut microbiota, is altered negatively following chronic ethanol exposure. This study aimed to determine whether prophylactic tributyrin could protect the intestinal barrier and liver in mice during combined chronic-binge ethanol exposure. METHODS: C57BL/6J mice exposed to 5% v/v ethanol-containing diet for 10 days received a single ethanol gavage (5 g/kg) 9 h before euthanasia. Control mice were isocalorically pair-fed maltose dextrin for ethanol. Diets were supplemented (5 mM) with tributyrin or glycerol. Intestine and liver disease activity was assessed histologically. Protein and mRNA expression of tight junction (TJ) proteins, toll-like receptors, and tumor necrosis factor-alpha were assessed. Caco-2 monolayers with or without ethanol exposure and/or sodium butyrate were used to test butyrate's direct effects on intestinal integrity. RESULTS: Chronic-binge ethanol feeding impaired intestinal TJ protein co-localization staining; however, tributyrin co-treatment mitigated these effects. Ethanol depleted TJ and transepithelial electrical resistance in Caco-2 monolayers, but butyrate co-treatment reduced these effects. Hepatic toll-like receptor mRNA expression and tumor necrosis factor-alpha protein expression was induced by ethanol; however, the response was significantly dampened in mice co-treated with tributyrin. Tributyrin altered localization of both neutrophils and single hepatocyte death: Leukocytes and apoptotic hepatocytes localized predominantly around the portal tract in ethanol-only treated mice, whereas localization predominated around the central vein in ethanol-tributyrin mice. CONCLUSIONS: Prophylactic tributyrin supplementation mitigated effects of combined chronic-binge ethanol exposure on disruption of intestinal TJ localization and intestinal permeability and liver injury.

Ginkgo biloba Extract Prevents Female Mice from Ischemic Brain Damage and the Mechanism Is Independent of the HO1/Wnt Pathway.

It is well known that gender differences exist in experimental or clinical stroke with respect to brain damage and loss of functional outcome. We have previously reported neuroprotective properties of Ginkgo biloba/EGb 761® (EGb 761) in transient and permanent mouse models of brain ischemia using male mice, and the mechanism of action was attributed to the upregulation of the heme oxygenase 1 (HO1)/Wnt pathway. Here, we sought to investigate whether EGb 761's protective effect in ovariectomized female mice following stroke is also mediated by the HO1/Wnt pathway. Female mice were ovariectomized (OVX) to remove the protective effect of estrogen and were treated with EGb 761 for 7 days prior to inducing permanent middle cerebral artery occlusion (pMCAO) and allowed to survive for an additional 7 days. At day 8, animals were sacrificed, and the brains were harvested for infarct volume analysis, western blots, and immunohistochemistry. The OVX female mice treated with EGb 761 showed significantly lower infarct size as compared to Veh/OVX animals. EGb 761 treatment in female mice inhibited apoptosis by preventing caspase-3 cleavage and blocking the extrinsic apoptotic pathway. EGb 761 pretreatment significantly enhanced neurogenesis in OVX mice as compared to the Veh/OVX group and significantly upregulated androgen receptor expression with no changes in HO1/Wnt signaling. These results suggest that EGb 761 prevented brain damage in OVX female mice by improving grip strength and neurological deficits, and the mechanism of action is not through HO1/Wnt but via blocking the extrinsic apoptotic pathway.