Role of Probiotic Lactobacillus on the Physiology and Gastrointestinal Immunology in Response to Dietary status of Rat

Abstract

Background: The mammalian host colonizes by trillions of microbes, which inhabit the gastrointestinal tract in symbiotic relationship to their host. Different dietary habits strongly influence gut microbiota composition. The alterations in microbiota population, whether chronic or acute, are accompanied with alteration in the microbiome, their patterns and specific metabolic capabilities. Type 2 diabetes is characterized by peripheral insulin resistance is the most common form of diabetes. Besides immune and inflammatory mechanisms, other pathways may involve interaction between gut microbiota and metabolic syndrome. Bacterial secreted products reach the circulating blood, and the entire bacteria from intestinal microbiota can reach tissues where inflammation is stimulated. Animals models of diabetes connected with an altered microbiota composition in the development of obesity, insulin resistance, and diabetes in the host though increased energy harvest from the diet, altered fatty acid metabolism and composition in adipose tissue and liver, increased of lipopolysaccharide based Toll like receptor 4 and short chain fatty acid produced by gut microbiota. Probiotic Lactobacillus has previously been shown to exert beneficial metabolic effects. Probiotics are live micro-organisms that, when administered in adequate amounts, confer delaying effect on diabetes development.Aim: The present study was designed to understand the gut microbiota alteration following sugar rich diet and its affect on physiology and host gene expression and probiotic application for the restoration of altered physiology and gastrointestinal immunology. In this study, the effects Lactobacillus helveticus (InuJ), a new genetic engineered probiotic strain developed in the laboratory, was evaluated on insulin resistance and the immunomodulatory activity in high sucrose fed rats.Materials and Methods: The objective of the study was to screen the probiotic characterization lactobacilli isolates from the feces of male wistar rats. Various physiological features of the candidate probiotic isolates were preliminarily investigated, including tolerance to simulated gastric juice and bile salts, antimicrobial activity and mode of action, antibiotic susceptibility, presence of β-Galactosidase and in vitro aggregation. From selected probiotics which showed highest bacterial inhibition was used for genetic modification with incorporation of InuJ (Inulosucrase) gene and functional characterization of the recombinant product was monitored such as enzyme activity by SDS-PAGE, zymography and anion exchange chromatography. Gut Abstract microbiota, host gene expression (TLR2/TLR4) and inflammation (cytokines and NF-кB) associated with high sugar (fructose and sucrose) were investigated after 60 days of diet feeding.Fecal microflora enumeration and diversity by DGGE profiling were performed. The resultedrecombinant strain was investigated for antidiabetic potential determination in high sucrose fed rats following 60 days diet intake. The oral glucose tolerance, SCFA concentration in fecal sample was measured. The serum inflammatory response (IL-6, TNF-α, INF-γ, IL-10, MCP-1 and IL12P70) was measured. The TLR2, TLR4 and NF-кB mRNA expression in different tissue was also investigated. Results: Biochemical tests and 16S rRNA gene sequencing confirmed that isolated strains were Lactobacillus intestinalis PJ2, L. sakei PJ3, L. helveticus PJ4, and L. plantarum PJ7. Based on the results, L. helveticus PJ4 and L. plantarum PJ7 were ideal in vitro probiotic candidates. However L. helveticus showed higher inhibitory properties through bacteriocin like peptides was used for further experiments. The InuJ gene was incorporated in secretion vector pSLP111.3 (with xylA promoter, slpA signal sequence, and cell wall anchor protein) and electrotransformed in L. helveticus. Extracellular secretion of inulosucrase enzyme was confirmed by SDS-PAGE, Zymogram, DNSA method and HPAEC anion exchange chromatography. The enzyme activity was highest in 45-55 °C temperature and at PH 7.0. The bacteria coliforms and clostridium level was higher and Lactobacillus was lower in high fructose and high sucrose fed rats. Extensive diverse and densely populated bands were observed in sugar fed group. The band profiles of sugar fed group have clustered together. Elevated mRNA expression of TLR2, TLR4, NF-кB and GLUT5 were observed in sugar fed groups. Increased inflammation was confirmed by blood and tissue biochemical assay as well as in enhanced serum cytokines (IL-6, TNF-α, INF-γ and IL-12) in sugar fed diet groups.The levels of plasma glucose, liver injury markers, lipid profile in plasma and liver were significantly increased in high-sucrose-fed rats. However, after recombinant L. helveticus (InuJ) administration, the elevation of these parameters was significantly suppressed. Feeding of L.helveticus (InuJ) enhanced the butyrate production. The recombinant L. helveticus significantly reduced the adverse effect of main regulators of inflammatory cytokines like IL6 and TNF-α in high sugar fed type 2 diabetes. Besides that, the recombinant Lactobacillus was also reduces the other proinflammatory cytokines like MCP-1, INF-γ, and IL12p70 in sucrose fed rats. Gene expression analysis revealed that in the liver, distal ileum, proximal colon and adipose, the synbiotic Lactobacillus restored the HSD-dysregulated TLR2, TLR4 and NF-кB mRNAs toward a physiological level of expression. Recombinant L. helveticus is effective in reducing the severity of liver injury and insulin resistance, ameliorated hepatic steatosis associated with high sucrose intake, suggesting its possible therapeutic/preventive clinical utilization. Conclusion: Probiotics have wide therapeutic application. This study provided evidences clarifying the effectiveness of probiotic Lactobacillus with inulosucrase on reducing insulin resistance, hepatic steatosis, reducing the TLR2, TLR4 expression and also manages the suppression of inflammatory cytokines in high-sucrose-fed rats and suggested that Lactobacillus helveticus and inulosucrase (synbiotic) may be a promising therapeutic agent in treating type 2diabetes. Key words: Type 2 diabetes, Insulin Resistance, Gut microbiota, Probiotic, Lactobacillus, Inulosucrase, Prebiotics, Toll like receptors, NF-кB, cytokines, SCFA, Sugar rich diet.