Study On The Treatment Of Mice With Dextran Sulfate Sodium-induced Colitis With Human Interleukin 10 Secreted By Transformed Bifidobacterium

Objective:Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) which etiology has not yet been fully clarified. Cytokine interleukin-10 (IL-10) plays a central role in down-regulating inflammatory cascade in UC and is likely a candidate for therapeutic intervention. However, its intravenous administration is costly and inconvenient. Therefore, we established a novel IL-10 delivery system by transforming a hIL-10-containing plasmid into B. longum(BL-hIL-10) and investigated its effects on 5% dextran sulfate sodium (DSS)-induced ulcerative colitis in mice and the possible underlying mechanism.Methods:(1) hIL-10 was expressed and secreted into the culture supernatant of BL-hIL-10 after 0.2% L-arabinose induction in vitro as examined by Western blot, enzyme-linked immunosorbent assay (ELISA) and RT-PCR; Culture supernatants and bacterium pellets were collected after continuous culture for 12,24 and 36 h, respectively. (2) BL-hIL-10 culture supernatant were had the cytotoxic effect test examined with CCK cytotoxic kits. (3) The proinflammatory cytokines in BL-hIL-10 culture supernatant and lipopolysaccharide (LPS) induced THP-1 cells in vitro were examined by ELISA. (4) Oral administration of BL-hIL-10, BL and BLO to the model mice, evaluated colitis-activated NF-κB pathway measured by DNA-binding assay and colitis-elevated expression of pro-inflammatory cytokines by qRT-PCR and ELISA, and the expression of CD4⁺CD25⁺Foxp³⁺ Treg in blood and mesenteric lymph nodes measured by flow cytometry.Results:(1) The hIL-10 gene can increase the stability of plasmid vector in BL, hIL-10 was expressed and secreted into the culture supernatant of BL-hIL-10 after L-arabinose induction in vitro, and the levels of hIL-10 in both supernatant and cell pellet were similarly reached maximum at 24 h of culture. (2) addition of BL-hIL-10 culture supernatant had no cytotoxic effect and morphological alteration. (3) addition of BL-hIL-10 culture supernatant significantly inhibited the enhancement of proinflammatory cytokines by lipopolysaccharide (LPS) in THP-1 cells; (4) Oral administration of BL-hIL-10, BL and BLO alleviated colitis syndrome of the model mice, attenuated colitis-activated NF-κB pathway measured by DNA-binding assay and colitis-elevated expression of pro-inflammatory cytokines, and up-regulated CD4⁺CD25⁺Foxp³⁺Treg in blood and mesenteric lymph nodes measured by flow cytometry. (5) BL-hIL-10 also control inflammatory state and alleviate inflammatory response of the whole body were better than BL and BLO.Conclusion:In conclusion, BL-hlL-10 as a novel oral hIL-10 delivery system has been successfully established and oral administration of BL-hIL-10 alleviated inflammatory damage of colonic tissue in the model mice by blocking colitis-activated NF-κB pathway and up-regulating CD4⁺CD25⁺Foxp³⁺ Treg in blood and mesenteric lymph nodes in mice.