Irinotecano induz esteato-hepatite experimental pela translocação de bactérias intestinais: Efeito protetor de probióticos

Abstract

Non-alcoholic steatohepatitis (NASH) is a syndrome caused by the accumulation of fat and inflammatory cells in the liver. A significant portion of patients with metastatic colorectal cancer treated with adjuvant-irinotecan therapy develops NASH and this side effect reduces the effectiveness of surgical intervention. The general consensus is that the intestinal microbiota plays a key role in the pathogenesis of different NASH models. However, its involvement in irinotecan-induced NASH is still unknown. Thus, in the present study, we aimed to investigate the participation of the intestinal microbiota in the pathogenesis of NASH induced by irinotecan. Methods: Initially C57BL/6 mice were treated with an antibiotic combination (metronidazole, ciprofloxacin, vancomycin and imipenem) in drinking water (50mg/kg/day). On the 10th day after initiation of antibiotic therapy (ANTB) the animals were treated with irinotecan (50 mg/kg, 3x/week for 5 or 7 weeks, i.p.). In other protocol, the mice were treated with either saline or irinotecan (50 mg/kg, 3x/week for 5 weeks, i.p.) alone or in combination with daily administration of probiotic suspension (Simfort®, containing Lactobacillus acidophilus, Lactobacillus casei, Lactococcus lactis, Bifidobacterium bifidum and Bifidobacteririum lactis, 1x107 CFU/mL, v.o). After 5 or 7 weeks, peripheral blood samples were collected to assess bacteremia, to measure ALT (U/L) levels and to perform total leukocyte count. In the liver were analyzed the Kleiner scores (lobular inflammation, steatosis and balonization), inflammatory parameters (number of neutrophilic foci, IL-1β and TNF-α cytokines, and expression of Toll-like receptor 4 - TLR4 and nitric oxide synthase inducible - iNOS), lipid accumulation and bacterial translocation. Intestinal samples were collected for histopathological evaluation. Additionally, flow cytometry was performed to characterize the phenotype of splenic lymphocytes subpopulation. Results: Intestinal bacterial depletion abolished bacteremia and bacterial translocation to the liver related to irinotecan-induced NASH. In addition, a reduction in ALT serum levels (IRI: 25.5±3.2U/L, IRI+ANTB: 15.8±1.5U/L), number of inflammatory foci (IRI: 11.9±2,5; IRI+ANTB: 4.6±1.6), hepatic injury [IRI: 5 (3-7); IRI+PRO: 3(1-7)] accumulation of lipids in the liver (IRI: 49.9±2.5mg/g, IRI+ANTB: 37±3.7mg/g), and the alteration in the intestinal architecture [IRI: 9 (7-12); IRI+ANTB: 3 (2-6)] were also observed in the ANTB treated group vs. irinotecan group. In addition, local production of IL-1β (IRI: 978±55.6 pg/mL; IRI+ANTB: 748±29 pg/mL), and TNF-α (IRI: 1775±77 pg/mL; IRI+ANTB: 1565±28 pg/ml), iNOS (IRI: 2.6±0.2; IRI+ANTB: 1.6±0.2) and TLR4 expression (IRI: 1.11±0.1; IRI+ANTB: 0.33±0.2), and increased moist liver weight were also reduced in the ANTB group (IRI: 1962±165.3 mg/30g; IRI+ANTB: 1366±74.3 mg/30g). Furthermore, it was observed that the use of probiotics reduce diarrhea (IRI: 1.5±0.4; IRI+PRO: 0.2±0.1), hepatic [IRI: 4.5 (2-6); IRI+PRO: 3 (2-3)] and intestinal damage [IRI: 10 (7-12); IRI+PRO: 5 (3-8)] and reduce inflammatory foci number (IRI: 7.6±2; IRI+PRO: 2.7±0.6) induced by irinotecan. The hepatic lipids accumulation (IRI: 49.4±2.5 mg/g, IRI+PRO: 38±4.3mg/g), increased ALT activity (IRI: 63.2±7.6 U/L; IRI+PRO: 39.4±5.3 U/L) and the CD45+CD25+CCR6+ frequency spleens cells (%) (IRI: 39.7±2.4; IRI+PRO: 32.2±1.8) were also reduced by probiotics. Probiotics increase the % of splenic TCD4 lymphocytes when compared to the irinotecan group (IRI: 29±3.3; IRI+PRO: 40±1.3). Conclusion: Intestinal bacterial translocation to the liver is a key factor for the development of irinotecan-induced NASH. Additionally, the modulation of the microbiota through the use of probiotics might attenuate the NASH associated to irinotecan based chemotherapy. Financial support: CAPES, CNPq and FUNCAP.