Safety assessment of the indigenous probiotic strain Lactiplantibacillus plantarum subsp. plantarum Kita-3 using Sprague-Dawley rats as a model.

Lactiplantibacillus plantarum subsp. plantarum Kita-3 is a candidate probiotic from Halloumi cheese produced by Mazaraat Artisan Cheese, Yogyakarta, Indonesia. This study evaluated the safety of consuming a high dose of L. plantarum subsp. plantarum Kita-3 in Sprague-Dawley rats for 28 days. Eighteen male rats were randomly divided into three groups, such as the control group, the skim milk group, and the probiotic group. Feed intake and body weight were monitored, and blood samples, organs (kidneys, spleen, and liver), and the colon were dissected. Organ weight, hematological parameters, serum glutamic oxaloacetic transaminase (SGOT), and serum glutamic pyruvic transaminase (SGPT) concentrations, as well as intestinal morphology of the rats, were measured. Microbial analyses were carried out on the digesta, feces, blood, organs, and colon. The results showed that consumption of L. plantarum did not negatively affect general health, organ weight, hematological parameters, SGOT and SGPT activities, or intestinal morphology. The number of L. plantarum in the feces of rats increased significantly, indicating survival of the bacterium in the gastrointestinal tract. The bacteria in the blood, organs, and colon of all groups were identified using repetitive-polymerase chain reaction with the BOXA1R primers and further by 16S rRNA gene sequencing analysis, which revealed that they were not identical to L. plantarum subsp. plantarum Kita-3. Thus, this strain did not translocate to the blood or organs of rats. Therefore, L. plantarum subsp. plantarum Kita-3 is likely to be safe for human consumption.

3,5,7,3',4'-Pentamethoxyflavone Enhances the Barrier Function through Transcriptional Regulation of the Tight Junction in Human Intestinal Caco-2 Cells.

The intestinal tight junction (TJ) barrier plays a pivotal role in the regulation of intestinal homeostasis. This study investigated the effects of 3,5,7,3',4'-pentamethoxyflavone (PMF), a major polymethoxyflavone found in black ginger, on TJ barrier regulation using intestinal Caco-2 cells. PMF treatment enhanced the TJ barrier integrity in Caco-2 cells, indicated by increased transepithelial electrical resistance (control, 1261 ± 36 Ω·cm2; 100 µM PMF, 1383 ± 55 Ω·cm2 at 48 h, p < 0.05) and decreased permeability to fluorescein-conjugated dextran (control, 24.2 ± 1.8 pmol/(cm2 × h); 100 µM PMF, 18.6 ± 1.0 pmol/(cm2 × h), p < 0.05). Immunoblot analysis revealed that PMF increased the cytoskeletal association and cellular expression of the TJ proteins, zonula occludens-1, claudin-3, and claudin-4 (e.g., occludin; control, 1.00 ± 0.2; 100 µM PMF, 3.69 ± 0.86 at 48 h, p < 0.05). Quantitative reverse transcriptase-polymerase chain reaction analysis and a luciferase promoter assay showed that PMF enhanced the transcription of occludin, claudin-3, and claudin-4. The promoter assay with site-directed mutagenesis indicated that PMF-induced occludin and claudin-3 transcription was mediated by transcription factors, KLF5 and EGR1, respectively, while PMF activated claudin-4 transcription through GATA1 and AP1. Taken together, the transcriptional regulation of TJ proteins is involved in PMF-mediated promotion of the intestinal barrier in vitro.

Resveratrol Ameliorates Intestinal Barrier Defects and Inflammation in Colitic Mice and Intestinal Cells.

This study is aimed to investigate the ameliorative effect of resveratrol in a dextran sodium sulfate (DSS)-induced colitis mouse model and intestinal Caco-2 cells, focusing on neutrophil infiltration and tight junction (TJ) barriers. DSS administration caused body weight loss (day8, control 104 ± 1, DSS 72 ± 2%, p < 0.05), shortening of colon length (control 5.1 ± 0.1, DSS 3.8 ± 0.1 cm, p < 0.05), pro-inflammatory cytokines increase-including interleukin (IL)-1ß (control 1.0 ± 0.2, DSS 58.5 ± 29.6 arbitrary unit (AU), p < 0.05), IL-6 (control 1.0 ± 0.3, DSS 312 ± 82 AU, p < 0.05), and chemokine motif ligand 2 (CXCL-2, a murine IL-8 homologue, control 1.0 ± 0.4, DSS 696 ± 262 AU, p < 0.05), decreased TJ proteins (e.g., occludin, control 1.0 ± 0.05, DSS 0.11 ± 0.03 AU, p < 0.05), and neutrophil infiltration (control 1.2 ± 0.2, DSS 25.9 ± 1.1 cells, p < 0.05). Supplemental resveratrol (0.1% (w/w) in the diet) partially or totally reversed these symptoms (body weight change 100 ± 1, colon length 4.6 ± 0.1; IL-1ß 5.9 ± 1.8, IL-6 10 ± 3, CXCL-2 14 ± 7, occludin 0.76 ± 0.06, neutrophil infiltration 9.3 ± 0.7, p < 0.05). Pretreatment of intestinal Caco-2 cells with resveratrol suppressed the TNF-α-induced production of IL-8 (control 1.00 ± 0.04, TNFα 3.40 ± 0.16, TNFα+Res 1.81 ± 0.28 AU, p < 0.05) and phosphorylation of the inflammatory signaling molecules including NF-κB, extracellular signal-regulated kinase and stress c-Jun N-terminal protein kinase. Collectively, the reduction of TJ barrier defect and IL-8 in intestinal cells, leading to reduced neutrophil infiltration into colonic tissues, appears to be one of the central mechanisms for the resveratrol-mediated effect.