Effects of TGF-beta1 Ribbon Antisense on CCl(4)-induced Liver Fibrosis

Abstract

Ribbon-type antisense oligonucleotide to TGF-beta1 (TGF-beta1 RiAS) was designed and tested to prevent or resolve the fibrotic changes induced by CCl4 injection. When Hepa1c1c7 cells were transfected with TGF-beta1 RiAS, the level of TGF-beta1 mRNA was effectively reduced. TGF-beta1 RiAS, mismatched RiAS, and normal saline were each injected to mice via tail veins. When examined for the biochemical effects on the liver, TGF-beta1 mRNA levels were significantly reduced only in the TGF-beta1 RiAS-treated group. The results of immunohistochemical studies showed that TGF-beta1 RiAS prevented the accumulation of collagen and alpha-smooth muscle actin, but could not resolve established fibrosis. These results indicate that ribbon antisense to TGF-beta1 with efficient uptake can effectively prevent fibrosis of the liver.

Keywords: Cationic peptide; Liver cirrhosis; Ribbon antisense; Transforming growth factor-β1.

Fig. 1

Experimental schedule. For 8 weeks of experiment, 1 ml/kg (body weight) of CCl4 was intraperitoneally administered twice per week. RiAS to mouse TGF-β1 or mismatched RiAS (100 µg/30 g body weight) was also intravenously administered twice per week from the second week of experiment in the prevention group and mismatched group, and from the 5th week of experiment in the treatment group.

Fig. 2

(A) Schematic representation of ribbon-type antisense to TGF-β1 (TGF-β1 RiAS). The stem is formed by complementary sequences at both ends of each oligo. The 5' terminus of the stem has 4 bases of a single-stranded overhang of 5'-GATC-3'. Two TGF-β1 monomer molecules were ligated to generate a covalently closed molecule with diad symmetry. The RiAS oligos consist of two loops and an intervening stem. Each loop harbors an antisense sequence to TGF-β1. (B) Specific reduction of TGF-β1 mRNA by TGF-β1 RiAS. Hepa1c1c7 cells were transfected with DP complex and RT-PCR was conducted in order to determine the antisense activity of TGF-β1 RiAS. Transfection of TGF-β1 RiAS reduced TGF-β1 expression in Hepa1c1c7 cells. By way of contrast, however, when Hepa1c1c7 cells were treated with mismatched RiAS, TGF-β1 expression was not significantly affected. Mouse β-actin was as a control. Veh: vehicle, RiAS: TGF-β1 RiAS, MM: mismatched RiAS.

Fig. 3

(A) The DP complex mediated transfection in Hepa1c1c7 mouse hepatoma cells. Transfection of FITC-labeled TGF-β1 RiAS was conducted using cationic peptide. The DP complex was added to Hepa1c1c7 cells for 24 hours. Fluorescence signals are shown in the right panel. (B) Normal saline (sham) or 10 µg of FITC-labeled TGF-β1 RiAS as a form of naked DNA only and DP complex were injected through the tail veins of normal or cirrhotic mice. Tissue sections of mice liver were observed under a fluorescence microscope (×200).

Fig. 4

Histological observation of liver. Collagen deposition was detected as blue staining on Masson's trichrome staining (A). Immunohistochemistry for type I collagen (B) and α-smooth muscle actin (C). Staining was conducted with fixed and dehydrated tissues from mice treated with normal saline (CON), mismatched RiAS (MM), and TGF-β1 RiAS (Prevention and Treatment group). Stained tissues were mounted with a synthetic mounting solution for microscopic observation (×200).