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The Role of Metal Oxide Nanoparticles, Escherichia coli, and Lactobacillus rhamnosus on Small Intestinal Enzyme Activity.

ABSTRACT: Engineered nanomaterials (ENMs) have become common in the food industry, which motivates the need to evaluate ENM effects on human health. Gastrointestinal (GI) in vitro models (e.g. Caco-2, Caco-2/HT29-MTX) have been used in nanotoxicology research. However, the human gut environment is composed of both human cells and the gut microbiota. The goal of this study is to increase the complexity of the Caco-2/HT29-MTX in vitro model by co-culturing human cells with the Gram-positive, commensal Lactobacillus rhamnosus or the Gram-negative, opportunistic Escherichia coli; with the hypothesis that the presence of bacteria would ameliorate the effects of exposure to metal oxide nanoparticles (NPs) such as iron oxide (Fe₂O₃), silicone dioxide (SiO₂), titanium dioxide (TiO₂), or zinc oxide (ZnO). To understand this relationship, Caco-2/HT29-MTX cell barriers were acutely co-exposed (4 hours) to bacteria and/or NPs (pristine or in vitro digested). The activity of the brush border membrane (BBM) enzymes intestinal alkaline phosphatase (IAP), aminopeptidase-N (APN), sucrase isomaltase (SI) and the basolateral membrane enzyme (BLM) Na⁺/K⁺ ATPase were assessed. Findings show that (i) the human digestion process alters the physicochemical properties of NPs, (ii) large agglomerates of NPs remain entrapped on the apical side of the intestinal barrier, which (iii) affects the activity of BBM enzymes. Interestingly, some NPs effects were attenuated in the presence of either bacterial strains. Confocal microscopy detected bacteria-NPs interactions, which may impede the NP-intestinal cell contact. These results highlight the importance of improving in vitro models to closely mimic the complexities of the human body.

SUBMITTER: Garcia-Rodriguez A

PROVIDER: S-EPMC8011031 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Probiotic could be a promising alternative to antibiotics for the prevention of enteric infections; however, further information on the dose effects is required. In this study, weanling piglets were orally administered low- or high-dose Lactobacillus rhamnosus ACTT 7469 (10(10) CFU/d or 10(12) CFU/d) for 1 week before F4 (K88)-positive Escherichia coli challenge. The compositions of faecal and gastrointestinal microbiota were recorded; gene expression in the intestines was assessed by real-time PCR; serum tumour necrosis factor-α (TNF-α) concentrations and intestinal Toll-like receptor 4 (TLR4) were detected by ELISA and immunohistochemistry, respectively. Unexpectedly, high-dose administration increased the incidence of diarrhoea before F4(+)ETEC challenge, despite the fact that both doses ameliorated F4(+)ETEC-induced diarrhoea with increased Lactobacillus and Bifidobacterium counts accompanied by reduced coliform shedding in faeces. Interestingly, L. rhamnosus administration reduced Lactobacillus and Bifidobacterium counts in the colonic contents, and the high-dose piglets also had lower Lactobacillius and Bacteroides counts in the ileal contents. An increase in the concentration of serum TNF-α induced by F4(+)ETEC was observed, but the increase was delayed by L. rhamnosus. In piglets exposed to F4(+)ETEC, jejunal TLR4 expression increased at the mRNA and protein levels, while jejunal interleukin (IL)-8 and ileal porcine β-defensins 2 (pBD2) mRNA expression increased; however, these increases were attenuated by administration of L. rhamnosus. Notably, expression of jejunal TLR2, ileal TLR9, Nod-like receptor NOD1 and TNF-α mRNA was upregulated in the low-dose piglets after F4(+)ETEC challenge, but not in the high-dose piglets. These findings indicate that pretreatment with a low dose of L. rhamnosus might be more effective than a high dose at ameliorating diarrhoea. There is a risk that high-dose L. rhamnosus pretreatment may negate the preventative effects, thus decreasing the prophylactic benefits against potential enteric pathogens. Our data suggest a safe threshold for preventative use of probiotics in clinical practice.

Project description:Enterotoxigenic Escherichia coli (ETEC) is a common enteric pathogen that causes diarrhoea in humans and animals. Lactobacillus rhamnosus LB1 (formerly named Lactobacillus zeae LB1) has been shown to reduce ETEC infection to Caenorhabditis elegans and Salmonella burden in pigs. This study was to evaluate the effect of L. rhamnosus LB1 on the gut health of lactating piglets that were challenged with ETEC. Six-four piglets at 7 days of age were equally assigned into 8 groups (8 piglets per group): 1) control group (basal diet, phosphate buffer saline); 2) CT group (basal diet + 40 mg/kg colistin); 3) LL group (basal diet + 1 × 107 CFU/pig/day LB1); 4) HL group (basal diet + 1 × 108 CFU/pig/day LB1); 5) ETEC group: (basal diet + ETEC challenged); 6) CT + ETEC group (basal diet + CT + ETEC); 7) LL + ETEC group (basal diet + 1 × 107 CFU/pig/day LB1 + ETEC); 8) HL + ETEC group (basal diet + 1 × 108 CFU/pig/day LB1 + ETEC). The trial lasted ten days including 3 days of adaptation. Several significant interactions were found on blood parameters, intestinal morphology, gene, and protein expression. ETEC infection disrupted the cell structure and biochemical indicators of blood, undermined the integrity of the intestinal tract, and induced oxidative stress, diarrhoea, intestinal damage, and death of piglets. The supplementation of L. rhamnosus LB1 alleviated ETEC's adverse effects by reducing pig diarrhoea, oxidative stress, and death, modulating cell structure and biochemical indicators of blood, improving the capacity of immunity and anti-oxidation stress of pigs, and restoring their intestinal integrity. At the molecular level, the beneficial effects of L. rhamnosus LB1 appeared to be mediated by regulating functional related proteins (including HSP70, Caspase-3, NLRP3, AQP3, and AQP4) and genes (including RPL4, IL-8, HP, HSP70, Mx1, Mx2, S100A12, Nrf2, GPX2 and ARG1). These results suggest that dietary supplementation of L. rhamnosus LB1 improved the intestinal functions and health of piglets.

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Project description:Escherichia coli is one of the most important pathogens that cause clinical mastitis in dairy cattle worldwide and lead to severe economic losses. Antibiotics are often used to treat this inflammatory disease; however, antimicrobial resistance and environmental pollution cannot be ignored. Probiotic is the best alternative; however, its mechanisms of action to prevent mastitis remain unclear. Moreover, the role of probiotics in regulating mitophagy, a selective autophagy that maintains mitochondrial quality, needs to be explored. E. coli infection induced NOD-like receptor family member pyrin domain-containing protein 3 (NLRP3) inflammasome assembly, Caspase-1 activation, and apoptosis in MAC-T cells. Infection also resulted in mitochondrial damage and subsequent increase in reactive oxygen species (ROS) production. Moreover, inhibition of ROS release by scavenger N-acetyl-L-cysteine (NAC) abrogated the importance of ROS in NLRP3 assembly and apoptosis in MAC-T cells. Pretreatment with Lactobacillus rhamnosus GR-1 (LGR-1), a probiotic, alleviated E. coli-induced NLRP3 inflammasome activation and apoptosis via ROS inhibition. Besides, E. coli infection inhibited mitophagy while LGR-1 pretreatment augmented PINK1/Parkin-mediated mitophagy activation, which further blocked ROS generation. To explore the effect of LGR-1 in vivo, a mouse mastitis model was established. The results showed that LGR-1 pretreatment had preventive and protective effects on E. coli induced mastitis, and could reduce cytokines levels such as IL-1β and TNF-α. In accordance with the results in vitro, E. coli can inhibit mitophagy and activate NLRP3 inflammasome and apoptosis, while LGR-1 can weaken the effect of E. coli. Taken together, our data indicated that LGR-1 pretreatment induced PINK1/Parkin-mediated mitophagy that eliminated damaged mitochondria and reduced ROS production and NLRP3 inflammasome activation, which subsequently decreased E. coli-induced apoptosis. To conclude, our study suggests that therapeutic strategies aiming at the upregulation of mitophagy under E. coli-induced mastitis may preserve mitochondrial function and provide theoretical support for the application of probiotics in bovine mastitis.

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The Role of Metal Oxide Nanoparticles, Escherichia coli, and Lactobacillus rhamnosus on Small Intestinal Enzyme Activity.

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