Efeitos de probióticos e posbióticos na saúde óssea / Effects of probiotics and postbiotics in bone health

O envelhecimento da população tem sido acompanhado por um aumento significativo nas doenças crônicas, com destaque para a osteoporose. A busca por tratamentos alternativos tem levado ao estudo dos probióticos para prevenção e tratamento da perda óssea pós-menopáusica, com resultados encorajadores. Embora os benefícios dos probióticos sejam numerosos, eles são bactérias vivas e a administração de organismos vivos não é isenta de riscos. Os posbióticos são probióticos inativados ou seus produtos, e poucos são os estudos que avaliam seus efeitos ósseos. Nós revisamos a literatura sobre probióticos e posbióticos na saúde óssea e avaliamos os efeitos de Limosilactobacillus reuteri 6475 viável e inativado por calor em camundongos ovariectomizados. Mecanismos de ação semelhantes, como modulação da expressão de citocinas e ativação de sistemas de sinalização celular, são observados em probióticos e posbióticos, e os últimos têm vantagens como maior estabilidade e facilidade de incorporação em alimentos, embora seus efeitos a longo prazo, estabilidade e modo de ação ainda precisem ser estudados. Para que estas terapias sejam validadas clinicamente, é fundamental padronizar metodologias, aumentar o tamanho das amostras, realizar estudos randomizados e cegos, e definir detalhes como cepa, dose e duração do tratamento. Em um estudo in vivo, avaliamos os efeitos de L. reuteri (ATCC PTA 6475), e sua forma inativada (heat-killed) na perda óssea induzida por ovariectomia (ovx). Camundongos adultos, fêmeas, foram divididos aleatoriamente em quatro grupos: controle (sham); Ovx; Ovx + posbiótico; Ovx + probiótico. L. reuteri foi administrado aos grupos (109 UFC/dia) por gavagem. O tratamento se iniciou uma semana após a ovx e teve duração de 28 dias. Na análise por microscopia eletrônica de varredura, o probiótico manteve sua estrutura intacta, e no posbiótico foram observados alguns rompimentos na superfície celular. Foi avaliada a microarquitetura de fêmur, utilizando microtomografia computadorizada. Análise de expressão gênica em íleo foi feita para avaliar junções intercelulares intestinais e citocinas pró-inflamatórias, por meio dos marcadores Ocludina, Tnf-α e Il6. Os dados foram submetidos ao teste estatístico mais conveniente (α = 0,05). Os grupos Ovx apresentaram menor porcentagem de volume ósseo (BV/TV), menor número de trabéculas ósseas e maior porosidade total, em comparação ao grupo controle, porém os grupos que receberam L. reuteri apresentaram maior BV/TV quando comparados ao grupo Ovx. O tratamento com L. reuteri em suas duas formas levou à maior espessura trabecular, quando comparados ao controle e ao grupo Ovx. Todos apresentaram semelhança na expressão gênica da Ocludina, Tnf-α e Il-6 em intestino. Ambas as formas, viável e inativada por calor, preveniram a perda óssea induzida por depleção de estrógeno (AU)

The aging of the population has been accompanied by a significant increase in chronic diseases, with osteoporosis standing out. The search for alternative treatments has led to the study of probiotics for the prevention and treatment of postmenopausal bone loss, with encouraging results. Although the benefits of probiotics are numerous, they are live bacteria, and the administration of live organisms is not risk-free. Postbiotics are inactivated probiotics or their products, and few studies have evaluated their bone effects. We reviewed the literature on probiotics and postbiotics in bone health and assessed the effects of both viable and heat-killed Limosilactobacillus reuteri 6475 in ovariectomized mice. Similar mechanisms of action, such as the modulation of cytokine expression and activation of cellular signaling pathways, are observed in probiotics and postbiotics, and the latter have advantages such as greater stability and ease of incorporation into food, although their long-term effects, stability, and mode of action still need to be studied. To clinically validate these therapies, it is crucial to standardize methodologies, increase sample sizes, conduct randomized and blinded studies, and define details such as strain, dosage, and treatment duration. In an in vivo study, we evaluated the effects of L. reuteri (ATCC PTA 6475) and its heat-killed form on ovariectomy (ovx)-induced bone loss. Adult female mice were randomly divided into four groups: control (sham); OVX; OVX + postbiotic; OVX + probiotic. L. reuteri was administered to the groups (109 CFU/day) by gavage. Treatment began one week after ovx and lasted for 28 days. Scanning electron microscopy analysis showed that the probiotic maintained its intact structure, while some cell surface disruptions were observed in the postbiotic. Femur microarchitecture was evaluated using computerized microtomography. Gene expression analysis in the ileum was performed to assess intestinal intercellular junctions and pro-inflammatory cytokines, using markers Ocludin, Tnf-α, and Il-6. The data were subjected to the most appropriate statistical test (α = 0.05). The Ovx groups showed a lower percentage of bone volume (BV/TV), a lower number of trabecular bones, and greater total porosity compared to the control group. However, the groups that received L. reuteri showed higher BV/TV compared to the Ovx group. Treatment with both forms of L. reuteri led to greater trabecular thickness compared to the control and Ovx groups. All groups exhibited similarity in the gene expression of Ocludin, Tnf-α, and Il-6 in the intestine. Both viable and heatkilled forms prevented estrogen depletion-induced bone loss (AU)

Cytotoxic effect of proteinaceous postbiotic metabolites produced by Lactobacillus plantarum I-UL4 cultivated in different media composition on MCF-7 breast cancer cell

Aims: Lactobacillus sp. has capability of producing an array of bioactive metabolites that exhibit probiotic effects. Therefore, the objective of this study was to determine the cytotoxicity effect of proteinaceous postbiotic metabolites (PPM) produced by Lactobacillus plantarum I-UL4 cultivated in different media composition on MCF-7 breast cancer cell. Methodology and results: L. plantarum I-UL4 was cultivated in yeast extract and modified de Man, Rogosa and Sharpe broth containing Tween 80 (CRMRS+T80) or without Tween-80 (CRMRS-T80). Human breast adenocarcinoma cell (MCF-7) was employed as cancer cell in this study. Cytotoxicity and antiproliferative effects of PPM were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide assay and Trypan Blue Dye Exclusion assay, whereas Acridine Orange/Propidium Iodide staining was employed to determine the cytotoxicity mechanism. PPM produced in CRMRS+T80 exerted cytotoxicity in a time and dose dependent manner that was selective towards MCF-7 cancer cell. Profound cytotoxicity with the lowest IC50 concentration of 10.83 µg was detected at 72 h of incubation, whereas the most potent antiproliferative effect revealed by the lowest viable cell population was observed at 24 h of incubation. PPM cultivated in CRMRS+T80 induced 80.87% of apoptotic MCF-7 cells at 48 h of incubation. Conclusion, significance and impact of study: PPM of L. plantarum I-UL4 cultivated in different media composition induced different levels of MCF-7 cancer cell death. The percentage of apoptotic MCF-7 cells treated with PPM cultivated in CRMRS+T80 increased significantly (p < 0.05) from 24 to 48 h of incubation. The results obtained in this study have revealed the potential of PPM produced by L. plantarum I-UL4 as human health supplement and as anticancer preventive agent. Keywords: Lactobacillus plantarum I-UL4; cytotoxic effect; proteinaceous postbiotic metabolites; media composition; breast cancer

Effects of carbon and nitrogen sources on bacteriocin-inhibitory activity of postbiotic metabolites produced by Lactobacillus plantarum I-UL4

Aims: Postbiotic metabolites are metabolic compounds produced by probiotic lactic acid bacteria. These compounds produced by Lactobacillus sp. have been shown to be effective substitutes for in-feed antibiotic in livestock due to their broad inhibitory activity. Therefore, the aim of this study was to determine the effects of various carbon and nitrogen sources on the bacteriocin-inhibitory activity of postbiotic metabolites produced by Lactobacillus plantarum I-UL4. Methodology and results: The effects of various combinations of carbon and nitrogen sources on the bacteriocininhibitory activity (expressed as modified bacteriocin activity, MAU/mL) of postbiotic metabolites produced by L. plantarum I-UL4 were determined in basal media without micronutrients. The combination of glucose (20 g/L) and yeast extract (22 g/L) gave the best bacteriocin-inhibitory activity as compared to other combinations. Maximum bacteriocininhibitory activity of 1440 MAU/mL was achieved when 36.20 g/L of yeast extract was added as the sole nitrogen source in modified de Man, Rogosa and Sharpe (MRS) medium. The glucose concentration was further optimised to enhance the bacteriocin-inhibitory activity of the postbiotic metabolites. Lower bacteriocin-inhibitory activity was observed at 5, 10, 15 and 40 g/L in comparison to 20 g/L of glucose. Conclusion, significance and impact of study: Maximum bacteriocin-inhibitory activity of postbiotic metabolites was achieved at 1440 MAU/mL when 20 g/L of glucose and 36.20 g/L of yeast extract were added as the sole carbon and nitrogen sources respectively in the modified MRS medium. Optimisation of other micronutrients present in MRS media is necessary to further enhance the bacteriocin-inhibitory activity of postbiotic metabolites produced by L. plantarum IUL4.