Fig. 2.
Prevention of DSS-colitis by administration of a RICK siRNA-expressing plasmid in NOD1 and NOD2-double deficient mice. NOD1 and NOD2-intact C57BL/6 mice (NOD1+/+NOD2+/+ mice) and NOD1 and NOD2-double deficient mice (NOD1−/−NOD2−/− mice) were treated with DSS (4%) in the drinking water from day 0 to day 6. Mice were administered HVJ-E-encapsulated RICK-siRNA expressing vector or control LUC-siRNA expressing vector via the intra-rectal route on days 0, 1 and 2. (A) Expression of RICK in colonic LPMCs (cLPMCs) in DSS-treated mice on day 8. Protein lysates were prepared from cLPMCs isolated from the colon of DSS-treated mice on day 8 and then subjected to immunoblotting. (B) Changes of body weight in mice. Results shown are values from pooled data derived from two independent experiments (NOD1+/+NOD2+/+ LUC-siRNA; n = 10, NOD1+/+NOD2+/+ RICK-siRNA; n = 10, NOD1−/−NOD2−/− LUC-siRNA; n = 10, NOD1−/−NOD2−/− RICK-siRNA; n = 8). Results are expressed as mean ± SEM. *P < 0.05, as compared with NOD1+/+NOD2+/+ HVJ-LUC siRNA group. ##P < 0.01, as compared with NOD1−/−NOD2−/− HVJ-LUC siRNA group. (C, D) H&E staining of colon tissue obtained from mice on day 8; pathology scores calculated from examination of tissues obtained from mice on day 8; results are expressed as mean ± SEM. Results shown were obtained from pooled tissues derived from two independent experiments. **P < 0.01, as compared with NOD1+/+NOD2+/+ HVJ-LUC siRNA group. ##P < 0.01, as compared with NOD1−/−NOD2−/− HVJ-LUC siRNA group. (E) Production of IL-12p40, TNF-α, IL-6 and IFN-γ by cLPMCs isolated from DSS-challenged mice on day 8; cLPMCs (1 × 106 per ml) were stimulated with FK565, MDP, PAM, LPS or anti-CD3 mAb for 48 h after which culture fluids were assayed for cytokine levels by ELISA, as indicated. Results are expressed as mean ± SEM. **P < 0.01, as compared with NOD1+/+NOD2+/+ HVJ-LUC siRNA group. #P < 0.05, ##P < 0.01, as compared with NOD1−/−NOD2−/− HVJ-LUC siRNA group.