Aplicação bioterapêutica de Lactobacillus delbrueckii CIDCA 133 selvagem e recombinante (pExu:hsp65) em modelo murino de mucosite intestinal

Abstract

Intestinal mucositis is an inflammation of the intestinal mucosa that occurs frequently in patients under treatment with anticancer drugs, such as 5-Fluorouracil (5-FU). Because it is a serious problem in clinical practice, there is a growing search for therapeutic strategies that aim to minimize this side effect. Probiotic microorganisms are considered a promising approach for this purpose, due to their anti-inflammatory capacity. As a way to potentiate the beneficial effects of these microorganisms, biotechnology and molecular biology tools have been developed and improved to build recombinant probiotic strains capable of producing and delivering therapeutic biomolecules to the host mucosal surfaces. In this context, the heat shock protein 65 Kda (Hsp65) of Mycobacterium leprae produced by probiotic bacteria has been showing good immunomodulatory characteristics. Thus, this work evaluated the molecular mechanisms involved with the enteroprotective effect of wild and recombinant Lactobacillus delbrueckii CIDCA 133 (pExu:hsp65) in a model of intestinal mucositis induced by the chemotherapy 5-FU (300mg/Kg). The main results obtained revealed that oral administration of wild-type CIDCA 133 attenuates 5-FU-induced epithelial and inflammatory damage through prevention of inflammatory cell infiltrate, reduced gene expression of markers involved in the activation of NF-κB signaling pathway (Tlr2, Tlr4, Nfkb1, Tnf, Il6, Il12 and Il1b) and positive regulation of the expression of immunoregulatory cytokine Il10. Furthermore, the beneficial effects of the strain can be attributed to its ability to increase gene expression of markers involved in the epithelial barrier, such as mucin 2 and firm junction proteins (occludin, claudin 2, zonulin, and JAM), collaborating to preserve intestinal permeability, and thus improving mucosal architecture and function. In parallel, using molecular biology and genetic improvement techniques, the eukaryotic expression vector pExu:hsp65 and the recombinant strain rCIDCA 133:HSP65 were constructed, which was also used in an in vivo assay using the same murine model of intestinal inflammation. Oral administration of the recombinant strain potentiated the effects of wild-type CIDCA 133, in which greater preservation of the intestinal epithelium destroyed by 5-FU was observed through reduced gene expression of Il1b and Il6, and increased gene expression of mucin 2, and also of the firm junction proteins claudin 1, claudin 2, and JAM. Delivery of the recombinant bacterium also potentiated the reduction of inflammatory infiltrate in the mucosa, intestinal permeability, and number of mucus-producing calliciform cells. Thus, in view of the promising results achieved, the present study paves the way for the consolidation of the Lactobacillus delbrueckii CIDCA 133 strain as a probiotic in addition to demonstrating the ability of this microorganism to efficiently deliver the eukaryotic expression vector pExu:hsp65 presenting itself as a promising therapeutic strategy for the prevention and treatment of intestinal mucositis, and other inflammatory diseases related to the gastrointestinal tract.