Modulations of probiotics on gut microbiota in a 5-fluorouracil-induced mouse model of mucositis.

BACKGROUND AND AIM: Intestinal mucositis remained one of the most deleterious complications in cancer patients undergoing chemotherapy. 5-FU treatment was reported to affect the abundance of gut microbiota and cause mucositis, which might be ameliorated by probiotics. We investigate the potential changes of 5-FU treatment and the modulations of probiotics on gut microbiota in a mouse model. METHODS: Male BALB/c mice received either 5-FU or saline (S). They were separated and fed saline, Lactobacillus casei variety rhamnosus (Lcr) and Lactobacillus reuteri DSM 17938 (BG). Lcr and BG were simultaneously administered with 5-FU for 5 days. Stool specimens were collected for DNA extraction and pyrosequenced for bioinformatic analysis. RESULTS: Fecal microbial communities were obviously diverse. Bacteroides and Bacteroidaceae were the most abundant microbiota in FU.BG group while S24_7 was the most in S.S group. At phylum and class levels, abundances of Betaproteobacteria, Erysipelotrichi, Gammaproteobacteria, and Verrucomicrobia were significantly increased in the FU groups. Probiotics supplementation did increase the abundances of Enterobacteriales and Turicibacterales. We demonstrated that probiotics did modulate the abundance and diversity of gut microbiota. Bacterial motility proteins were found enriched and upregulated in the S.BG group. No mortality was noted. No bacterial translocation was found in spleen and blood among the six groups. CONCLUSION: Gut microbiota of mice undergoing chemotherapy exhibited a distinct disruption in bacterial composition. Probiotic did modulate the abundance and diversity of gut microbiota. This is the first study to analyze the effects and safety of Lactobacillus strains on 5-FU-induced mucositis systematically and assess changes in the intestinal microbiota after probiotic intervention.

Fecal Microbiota Transplantation Prevents Intestinal Injury, Upregulation of Toll-Like Receptors, and 5-Fluorouracil/Oxaliplatin-Induced Toxicity in Colorectal Cancer.

FOLFOX (5-fluorouracil, leucovorin, and oxaliplatin), a 5-fluorouracil (5-FU)-based chemotherapy regimen, is one of most common therapeutic regimens for colorectal cancer. However, intestinal mucositis is a common adverse effect for which no effective preventive strategies exist. Moreover, the efficacy and the safety of fecal microbiota transplants (FMT) in cancer patients treated with anti-neoplastic agents are still scant. We investigated the effect of FMT on FOLFOX-induced mucosal injury. BALB/c mice implanted with syngeneic CT26 colorectal adenocarcinoma cells were orally administered FMT daily during and two days after five-day injection of FOLFOX regimen for seven days. Administration of FOLFOX significantly induced marked levels of diarrhea and intestinal injury. FMT reduced the severity of diarrhea and intestinal mucositis. Additionally, the number of goblet cells and zonula occludens-1 decreased, while apoptotic and NF-κB-positive cells increased following FOLFOX treatment. The expression of toll-like receptors (TLRs), MyD88, and serum IL-6 were upregulated following FOLFOX treatment. These responses were attenuated following FMT. The disrupted fecal gut microbiota composition was also restored by FMT after FOLFOX treatment. Importantly, FMT did not cause bacteremia and safely alleviated FOLFOX-induced intestinal mucositis in colorectal cancer-bearing mice. The putative mechanism may involve the gut microbiota TLR-MyD88-NF-κB signaling pathway in mice with implanted colorectal carcinoma cells.

Membrane Proteomics of Impaired Energetics and Cytoskeletal Disorganization in Elderly Diet-Induced Diabetic Mice.

Diabetic cardiomyopathy is a well-recognized complication of diabetes, but its pathophysiology is unclear. We aimed to investigate the mechanisms underlying cardiac dysfunction in an elderly type 2 diabetic (T2DM) mouse model, using membrane proteomic analyses. Elderly mice were fed a high fat diet for 12 weeks to induce T2DM, and myocardial structure and function were assessed by echocardiography. Cardiomyocytes were isolated by Langendorff perfusion and subjected to iTRAQ-based quantitative membrane proteomic profiling, immunoblotting, and real-time quantitative reverse-transcriptase polymerase chain reaction. Compared to controls, elderly T2DM mice showed worse systolic function, more myocardial fibrosis and up-regulation of several heart failure markers (all p < 0.05). Cardiomyocyte membrane proteomic profiling revealed that 417 proteins had differential expressions related to perturbations in several biological processes in T2DM mice compared with the control. The most up-regulated proteins were involved in oxidative phosphorylation, whereas many down-regulated proteins were involved in cytoskeletal regulation. Differential protein expression correlated with myocardial systolic velocity by tissue Doppler. In addition, cardiomyocyte immunofluorescence staining showed greater disorganization of thick/parallel F-actin stress fibers and marked reduction in F-to-G-actin ratio in T2DM vs control (p < 0.05), which paralleled worsened myocardial systolic velocity. We concluded that cardiac contractile dysfunction in elderly T2DM mice was associated with impaired energetics and cytoskeletal disorganization.

Probiotic supplementation modifies the gut microbiota profile of very low birth weight preterm infants during hospitalization.

BACKGROUND: Probiotic supplementation is increasingly being given to very low birth weight (VLBW) preterm infants. This preliminary observational study aimed to investigate the effects of multiple-strain probiotics on the gut microbiota of VLBW preterm infants. METHODS: We collected meconium and stool samples on days 14, 30, and 60 after birth from 49 VLBW infants with a gestational age of <32 weeks. The infants were divided into the probiotics (n = 24) and control (n = 25) groups. The microbial composition and diversity in the gut of the two groups were analyzed using 16 S rRNA gene sequencing. RESULTS: The relative abundance of Bifidobacterium and Lactobacillus was significantly higher in the probiotics group than in the control group on days 14, 30, and 60 (Bifidobacterium: p = 0.002, p < 0.0001, and p < 0.0001, respectively; Lactobacillus: p = 0.012, p < 0.0001, and p < 0.0001, respectively). The control group exhibited a significantly higher proportion of participants with a low abundance (<1%) of Bifidobacterium or Lactobacillus on days 14, 30, and 60 than those in the probiotic group. Moreover, the probiotics group exhibited a significantly lower abundance of Klebsiella on days 14 and 30 (2.4% vs. 11.6%, p = 0.037; and 7.9% vs. 16.6%, p = 0.032, respectively) and of Escherichia-Shigella on day 60 than the control group (6.1% vs. 12.3%, p = 0.013). Beta diversity analysis revealed that the microbiota profile was clearly divided into two groups on days 30 and 60 (p = 0.001). CONCLUSION: Probiotic supplementation significantly increased the relative abundance of Bifidobacterium and Lactobacillus and inhibited the growth of potential pathogens. Furthermore, probiotic supplementation led to a distinct gut microbiota profile. Further research is needed to identify probiotic strains that exert significant influence on the gut microbiome and their long-term health implications in preterm infants.

Characteristics of Gut Microbiota in Small for Gestational Age Infants with Very Low Birth Weight.

Small for gestational age (SGA) birth is associated with high rates of mortality and morbidity in preterm infants. The aim of this preliminary observational study was to investigate the difference in gut microbiota between SGA and appropriate for gestational age (AGA) preterm infants with very low birth weight (VLBW). We included 20 VLBW preterm infants (SGA, n = 10; AGA, n = 10) in this study. Stool samples were collected on days 7, 14, and 30 after birth. We performed 16S ribosomal DNA sequencing to compare microbiota composition between both groups. The SGA group exhibited a lower abundance of Klebsiella on day 14 (SGA, 0.57%; AGA, 7.42%; p = 0.037). On day 30, the SGA group exhibited a lower abundance of Klebsiella (SGA 3.76% vs. AGA 16.05%; p = 0.07) and Enterobacter (SGA 5.09% vs. AGA 27.25%; p = 0.011) than the AGA group. Beta diversity demonstrated a separation of the bacterial community structure between both groups on day 30 (p = 0.019). The present study revealed that a distinct gut microbiota profile gradually develops in SGA preterm infants with VLBW during the early days of life. The role of changes in gut microbiota structure warrants further investigation.

Impact of Early Empiric Antibiotic Regimens on the Gut Microbiota in Very Low Birth Weight Preterm Infants: An Observational Study.

Frequent use of antibiotics in preterm infants disturbs their gut microbial balance. In this preliminary observational study, we investigated the effect of different antibiotic regimens, administered during the first week of life, on microbial composition and diversity in very low birth weight (VLBW) preterm infants. We performed fecal sampling of breastfed VLBW infants on days 7, 14, and 30. After excluding stool samples from infants who received probiotics or who were administered antibiotics beyond the age of 7 days, we compared gut microbiota profiles between infants receiving a combination of ampicillin and gentamicin for 3 days (AG group, n = 10) and those receiving a combination of ampicillin and cefotaxime for 7 days (AC group, n = 14) using 16S ribosomal DNA community profiling. We also assessed the changes over time in each group. Compared to the AG group, Enterococcus species were significantly more abundant in the AC group (P = 0.002), especially in 7-day samples (12.3 vs. 0.6%, respectively, P = 0.032). No difference was observed at phylum and genus level over time within each group. Species richness in the AC group decreased significantly in the 14-day (P = 0.038) and 30-day (P = 0.03) samples compared to that in the 7-day sample. The same was observed for microbial evenness; in contrast, no significant difference in Shannon index and beta-diversity was detected between the two groups. Controlling for relevant confounding variables did not change the results. In conclusion, different antibiotic regimens affect the early development of gut microbiota in VLBW preterm infants. Prolonged use of ampicillin and cefotaxime might result in overabundance of Enterococcus. However, given that no significant differences were observed in 1-month samples, bacterial genera appear to continue colonizing the gastrointestinal tract despite previous exposure to antibiotics. The clinical relevance of these findings should be elucidated by further studies.

Effects of Vitamin D-Deficient Diet on Intestinal Epithelial Integrity and Zonulin Expression in a C57BL/6 Mouse Model.

Background and Aims: Vitamin D (VD) plays an important role not only in mineral balance and skeletal maintenance but also in immune modulation. VD status was found correlated with the pathophysiology and severity of inflammatory bowel diseases and other autoimmune disorders. Epithelial barrier function is primarily regulated by the tight-junction (TJ) proteins. In this study, we try to establish an animal model by raising mice fed VD-deficient diet and to investigate the effects of VD-deficient diet on gut integrity and zonulin expression. Methods: Male C57BL/6 mice were administered either VD-deficient [VDD group, 25(OH)2D3 0 IU/per mouse] or VD-sufficient [VDS group, 25(OH)2D3 37.8 IU/per mouse] special diets for 7 weeks. Body weight and diet intake were recorded weekly. Serum VD levels were detected. After sacrifice, jejunum and colon specimens were collected. The villus length and crypt depth of the jejunum as well as mucosa thickness of the colon were measured. Various serum pro-inflammatory cytokines and intestinal TJ proteins were assessed. The serum level of zonulin and the mRNA expression of jejunum zonulin were also investigated. Results: We found that mice fed a VDD diet had a lower serum level of VD after 7 weeks (p < 0.001). VDD mice gained significant less weight (p = 0.022) and took a similar amount of diet (p = 0.398) when compared to mice raised on a VDS diet. Significantly decreased colon mucosa thickness was found in VDD mice compared with the VDS group (p = 0.022). A marked increase in serum pro-inflammatory cytokine levels was demonstrated in VDD mice. All relative levels of claudin (CLD)-1 (p = 0.007), CLD-3 (p < 0.001), CLD-7 (p < 0.001), and zonulin-1 (ZO-1, p = 0.038) protein expressions were significantly decreased in the VDD group when compared to the VDS group. A significant upregulation of mRNA expression of jejunum zonulin (p = 0.043) and elevated serum zonulin (p = 0.001) were found in the VDD group. Conclusions: We successfully demonstrated that VDD could lead to impaired barrier properties. We assume that sufficient VD could maintain intestinal epithelial integrity and prevent mucosal barrier dysfunction. VD supplementation may serve as part of a therapeutic strategy for human autoimmune and infectious diseases with intestinal barrier dysfunction (leaky gut) in the future. To our knowledge, this is the first study to demonstrate that VDD could lead to a significant upregulation in mRNA expression of the jejunum zonulin level and also a marked elevation of serum zonulin in a mouse model.

SCID/NOD mice model for 5-FU induced intestinal mucositis: Safety and effects of probiotics as therapy.

BACKGROUND: For chemotherapy patients, intestinal mucositis is a frequent complication. Previously, we evaluated the beneficial effect of oral probiotics in 5-Fluorouracil (5-FU) induced mucositis in BALB/c mice. Here, we used SCID/NOD mice instead to simulate the immunodeficiency of chemotherapy patients: first, to evaluate the safety of probiotic supplementation and second, to determine the probiotic effect in response to 5-FU intestinal mucositis. METHODS: Thirty-six SCID/NOD mice were injected with saline (three control groups) or 5-FU (three experimental groups) intraperitoneally daily for five days. Mice were given either oral saline daily, probiotic suspension of Lactobacillus casei variety rhamnosus (Lcr35, Antibiophilus™, France) or Lactobacillus acidophilus and Bifidobacterium bifidum (LaBi, Infloran™, Italy). Blood, liver, spleen, and lymph node tissue samples were evaluated for probiotic translocation via culture and Q-PCR. Weight change, diarrhea score, jejunal villus height (VH) and crypt depth (CD), and serum cytokine levels of TNF-α, IFNγ, IL-1ß, IL-6, IL-4, IL-10, IL-13, and IL-17 were also assessed. RESULTS: No weight loss was found in the SCID control group. Mean weight loss of 10.63 ± 0.87% was noted by day five in 5-FU group without probiotics but it was only 6.2 ± 0.43% if mice were given Lcr35 (p < 0.01) and 7.1 ± 1.80% (p < 0.01) if they were given LaBi. Diarrhea score of 5-FU group without probiotics was 2.0 ± 0.0 by day five, which dropped to 1.33 ± 0.17 (p < 0.05) and 1.42 ± 0.24 (p < 0.05) with Lcr35 and LaBi, respectively. Average VH significantly decreased and CD significantly increased in SCID mice given 5-FU. With probiotics, average CD improved (p < 0.05) while VH lengthened as well. Besides IL-13, all cytokine levels increased in 5-FU SCID mice. Both Lcr35 and LaBi significantly inhibited serum cytokines (p < 0.05). No probiotic strains were detected in blood cultures of any mice. CONCLUSION: Using SCID/NOD mice as a novel model for 5-FU induced intestinal mucositis, we find that probiotics Lcr35 and LaBi do not lead to bacteremia, can improve diarrhea and body weight, can restore jejunal crypt depth, and significantly inhibit cytokines TNF-α, IL-1ß, IFNγ, IL-6, IL-4, IL-10, and IL-17.

In vivo toxicologic study of larger silica nanoparticles in mice.

Silica nanoparticles (SiNPs) are being studied and used for medical purposes. As nanotechnology grows rapidly, its biosafety and toxicity have frequently raised concerns. However, diverse results have been reported about the safety of SiNPs; several studies reported that smaller particles might exhibit toxic effects to some cell lines, and larger particles of 100 nm were reported to be genotoxic to the cocultured cells. Here, we investigated the in vivo toxicity of SiNPs of 150 nm in various dosages via intravenous administration in mice. The mice were observed for 14 days before blood examination and histopathological assay. All the mice survived and behaved normally after the administration of nanoparticles. No significant weight change was noted. Blood examinations showed no definite systemic dysfunction of organ systems. Histopathological studies of vital organs confirmed no SiNP-related adverse effects. We concluded that 150 nm SiNPs were biocompatible and safe for in vivo use in mice.

Amelioration of Chemotherapy-Induced Intestinal Mucositis by Orally Administered Probiotics in a Mouse Model.

BACKGROUND AND AIMS: Intestinal mucositis is a frequently encountered side effect in oncology patients undergoing chemotherapy. No well-established or up to date therapeutic strategies are available. To study a novel way to alleviate mucositis, we investigate the effects and safety of probiotic supplementation in ameliorating 5-FU-induced intestinal mucositis in a mouse model. METHODS: Seventy-two mice were injected saline or 5-Fluorouracil (5-FU) intraperitoneally daily. Mice were either orally administrated daily saline, probiotic suspension of Lactobacillus casei variety rhamnosus (Lcr35) or Lactobacillus acidophilus and Bifidobacterium bifidum (LaBi). Diarrhea score, pro-inflammatory cytokines serum levels, intestinal villus height and crypt depth and total RNA from tissue were assessed. Samples of blood, liver and spleen tissues were assessed for translocation. RESULTS: Marked diarrhea developed in the 5-FU groups but was attenuated after oral Lcr35 and LaBi administrations. Diarrhea scores decreased significantly from 2.64 to 1.45 and 0.80, respectively (P<0.001). Those mice in 5-FU groups had significantly higher proinflammatory cytokine levels (TNF-α: 234.80 vs. 29.10, P<0.001, IL-6: 25.13 vs. 7.43, P<0.001, IFN-γ: 22.07 vs. 17.06, P = 0.137). A repairing of damage in jejunal villi was observed following probiotics administration. We also found TNF-α, IL-1ß and IL-6 mRNA expressions were up-regulated in intestinal mucositis tissues following 5-FU treatment (TNF-α: 4.35 vs. 1.18, IL-1ß: 2.29 vs. 1.07, IL-6: 1.49 vs. 1.02) and that probiotics treatment suppressed this up-regulation (P<0.05). No bacterial translocation was found in this study. CONCLUSIONS: In conclusion, our results show that oral administration of probiotics Lcr35 and LaBi can ameliorate chemotherapy-induced intestinal mucositis in a mouse model. This suggests probiotics may serve as an alternative therapeutic strategy for the prevention or management of chemotherapy-induced mucositis in the future.

In vitro prevention of salmonella lipopolysaccharide-induced damages in epithelial barrier function by various lactobacillus strains.

Background. Lactobacillus shows beneficial anti-inflammatory effects on Salmonella infection. The maintenance of the tight junction (TJ) integrity plays an importance role in avoiding bacterial invasion. Whether Lactobacillus could be used to regulate the TJ protein expression and distribution in inflamed intestinal epithelial cells was determined. Methods. Using the transwell coculture model, Salmonella lipopolysaccharide (LPS) was apically added to polarized Caco-2 cells cocultured with peripheral blood mononuclear cells in the basolateral compartment. LPS-stimulated Caco-2 cells were incubated with various Lactobacillus strains. TJ integrity was determined by measuring transepithelial electrical resistance across Caco-2 monolayer. Expression and localization of TJ proteins (zonula-occludens- (ZO-) 1) were determined by Western blot and immunofluorescence microscopy. Results. Various strains of Lactobacillus were responsible for the different modulations of cell layer integrity. LPS was specifically able to disrupt epithelial barrier and change the location of ZO-1. Our data demonstrate that Lactobacillus could attenuate the barrier disruption of intestinal epithelial cells caused by Salmonella LPS administration. We showed that Lactobacillus strains are associated with the maintenance of the tight junction integrity and appearance. Conclusion. In this study we provide insight that live probiotics could improve epithelial barrier properties and this may explain the potential mechanism behind their beneficial effect in vivo.

Inhibitory effects of Lactobacillus casei subsp. rhamnosus on Salmonella lipopolysaccharide-induced inflammation and epithelial barrier dysfunction in a co-culture model using Caco-2/peripheral blood mononuclear cells.

In this study, we investigated the anti-inflammatory and reinforcing barrier effects of Lactobacillus casei subsp. rhamnosus (Lcr35) on Caco-2 intestinal epithelial cells already exposed to Salmonella LPS. Using the Transwell co-culture model, Salmonella LPS was apically added to polarized Caco-2 cells co-cultured with peripheral blood mononuclear cells (PBMCs) in the basolateral compartment. LPS-stimulated Caco-2 cells were incubated with Lcr35 for 1, 6, 24 or 48 h. Apical inoculation of Lcr35 after 48 h significantly inhibited the basolateral secretion of interleukin-8 (IL-8) in the Caco-2/PBMC co-culture. The PCR analysis showed that Lcr35 significantly downregulated mRNA expression of monocyte chemoattractant protein 1 (MCP-1) (P<0.05) and had a trend of decreasing mRNA expression of IL-8 (P=0.05), but did not alter mRNA expression of transforming growth factor-beta1 in LPS-stimulated Caco-2 cells at 48 h after addition of Lcr35. Compared to non-LPS-pretreated controls, transepithelial electrical resistance (TEER) of the polarized Caco-2 cell monolayers pretreated with LPS for 48 h was decreased by 9.9 % (P<0.05). Additionally, compared to those cells only treated with LPS, apical co-incubation with Lcr35 showed biphasic TEER levels increased by 12.1 % (P<0.001), 5.7 % (P<0.05) and 86.8 % (P<0.001) in the Caco-2 cell monolayers compared to those without Lcr35 treatment after 1, 6 and 48 h, respectively. In conclusion, Lcr35 can exert anti-inflammatory effects and ameliorate barrier dysfunction in the Salmonella LPS-pretreated inflamed intestinal epithelium in vitro.