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. 2011 Mar 24;2(3):e2. doi: 10.1038/ctg.2011.1

Figure 2.

Figure 2

mRNA and protein expression profiles in control and short interfering RNA (siRNA)-treated mice. Murine tumor necrosis factor-α (TNF-α) and murine cyclin D1 (CyD1) expression upon oral administration of three doses of nanoparticles-in-microsphere oral system (NiMOS; blank or encapsulating inactive (scrambled) siRNA, TNF-α siRNA, CyD1 siRNA, combined TNF-α/CyD1 siRNA) to animals under continuous dextran sulfate sodium (DSS) exposure. Each control or test group consisted of four to five animals. Gene mRNA levels were measured by real-time quantitative PCR analysis, normalized against L32 and depicted as relative gene expression of (a) murine TNF-α and (b) murine CyD1 mRNA performed on samples of the large intestines. Repeated oral administration of the respective siRNA and combination therapy loaded NiMOS led to a reduced mRNA expression of TNF-α and CyD1, respectively, compared with DSS control animals or animals treated with unloaded or scrambled siRNA loaded NiMOS study. (c) Quantitative determination of TNF-α in total cell lysates from colonic tissue by enzyme-linked immunosorbent assay. Levels of the protein were significantly reduced in CyD1 NiMOS groups on day 10 compared with all remaining treatment and control groups including the healthy control group. On day 12, TNF-α levels were reduced in all active siRNA groups compared with the inactive (scrambled) siRNA and blank NiMOS as well as untreated and control and DSS control groups. (d) Western blot of total cell lysates from intestinal tissue for determination of CyD1 with the loading control β-actin. Administration of CyD1 siRNA-loaded NiMOS led to reduction in CyD1 expression compared with the remaining control and test groups on days 10 and 12, whereas protein expression was reduced in TNF-α/CyD1 combined siRNA NiMOS only on day 10 and showed a slight increase on day 12. Values are represented as mean±s.d. (n=4–5). Control=naïve, no colitis; DSS control=colitis, no treatment; blank NiMOS=colitis, blank microspheres; scramble NiMOS=colitis, scrambled siRNA-containing microspheres; CyD1 NiMOS=colitis, CyD1 siRNA-containing microspheres; TNF-α NiMOS=colitis, TNF-α siRNA-containing microspheres; TNF/CyD1 NiMOS=colitis, microspheres containing and combination of both TNF-α and CyD1 siRNA. *P<0.05, vs. Scramble, Statistical comparison was performed on data sets of DSS control vs. TNF-α, Cyclin D1, and TNF-α/Cyclin D1 combination NiMOS, and between TNF-α, Cyclin D1, and TNF-α/Cyclin D1 combination vs. Scramble NiMOS group. Only significant differences are shown.