Specific siRNA targeting the receptor for advanced glycation end products inhibits experimental hepatic fibrosis in rats

Abstract

Receptor for advanced glycation end products (RAGE) was studied in different stages of carbon tetrachloride induced hepatic fibrosis (HF), and effect of its gene silencing in the HF development was evaluated in rats. Silencing RAGE expression by specific siRNA effectively suppressed NF-kappaB activity, hepatic stellate cell activation, and accumulation of extracellular matrix proteins in the fibrotic liver, and also greatly improved the histopathology and the ultrastructure of liver cells. These effects may be partially mediated by the inhibition on IkappaBalpha degradation. RAGE gene silencing effectively prevented liver from fibrosis, therefore it offers a potential pharmacological tool for anti-HF gene therapy.

Keywords: Receptor for advanced glycation end products (RAGE); gene therapy; hepatic fibrosis (HF); hepatic stellate cells (HSCs); small interfering RNA (siRNA).

Figure 1.

Expression of GFP in pGCsi-R1 transfected HSC-T6 cells. GFP fluorescence in each group of the HSC-T6 cells was detected by fluorescence microscope and flow cytometry at 48 h after transfection. (A, B), bright light and fluorescence field views, respectively, of the same field in the untreated (blank) HSC-T6 cells; (C, D), bright light and fluorescence field views, respectively, of the same field in the pGCsi-R1 transfected HSC-T6 cells; (E), the percentage of the cells expressing GFP in the blank control group; (F), the percentage of the cells expressing GFP in the pGCsi-R1-transfected group. A-D, magnification of 100×.

Figure 2.

Effect of RAGE specific siRNA on the expression of RAGE in HSC-T6 cells under different concentrations and at various time points. (A, B), the expression of RAGE mRNA as the ratio to β-actin mRNA by real-time quantitative PCR; (C), the expression of RAGE proteins was determined by Western blot analysis; (D), the 46 KDa and 50 KDa RAGE proteins as the ratios to β-actin protein by densitometric scanning. The changes were expressed as percentages of the respective blank. *, † and ‡ indicate statistically significant differences. *P<0.05 vs. blank; †P<0.01 vs. 0.5 nM pGCsi-R2; ‡P<0.01 vs 1.0 nM pGCsi-R2

Figure 3.

Histological changes and Sirius red staining of rat liver fibrosis in different stages. (A, E), NC1; (B, F), FM1-2W; (C, G), FM1-4W; (D, H), FM1-6W; A-D:HE staining (200×); E-H:Sirius red staining (100×).

Figure 4.

Expression of RAGE mRNA and protein in rat liver tissues of CCl₄-induced fibrosis in different stages. (A), the expression of RAGE mRNA was determined by Northern blot analysis; (B), RAGE mRNA as the ratios to β-actin mRNA by densitometric scanning. (C), the expression of RAGE proteins was determined by Western blot analysis; (D), the 46 KDa and 50 KDa RAGE proteins as the ratios to β-actin protein by densitometric scanning. The changes were expressed as percentages of the respective NC1. *, † and ‡ indicate statistically significant differences. *P<0.01 vs. NC1; †P<0.01 vs. FM1-2W; ‡P<0.01 vs FM1-4W.

Figure 5.

Expression of RAGE siRNA in the rat liver. Representative Northern blot analysis of RAGE siRNA expression in each group of the rat liver.

Figure 6.

Effect of specific siRNA targeting RAGE on rat liver fibrosis by histological examination and Sirius red staining. (A, E), NC2; (B, F), FM2; (C, G), pGCsi-R1; (D, H), pGCsi-C. A-D: HE staining (200×); E-H: Sirius red staining (100×).

Figure 7.

Effect of specific siRNA targeting RAGE on the mRNA expression of RAGE, NF-κB, α-SMA and Collagen type I in rats. (A), the mRNA expression of RAGE, α-SMA, NF-κB and Collagen type I was determined by Northern blot analysis. (B), the mRNAs of RAGE, NF-κB, α-SMA and Collagen type I were expressed as the ratios to β-actin mRNA by densitometric scanning, respectively. The changes were expressed as percentages of the respective NC2. * and † indicate statistically significant differences. *P<0.01 vs. NC2; †P<0.01 vs. FM2.

Figure 8.

Effect of specific siRNA targeting RAGE on the expression of 46 kDa RAGE, 50 kDa RAGE, α-SMA, NF-κB, IκBα, and Collagen type I in rats. (A), the protein expression of 46 kDa RAGE, 50 kDa RAGE, α-SMA, NF-κB, IκBα and type I Collagen was determined by Western blot analysis. (B), the proteins of 46 KDa RAGE, 50 KDa RAGE, α-SMA, NF-κB, IκBα, and Collagen type I were expressed as the ratios to β-actin protein by densitometric scanning, respectively. The changes were expressed as percentages of the respective NC2. *, # and † indicate statistically significant differences. *P<0.01 vs NC2; ^#P <0.05, †P<0.01 vs. FM2.

Figure 9.

Effect of specific siRNA targeting RAGE on the expression of RAGE and α-SMA in fibrotic rat liver examined by immunohistological staining. Representative microphotographs of SP staining in livers were presented. (A, E), NC2; (B, F), FM2; (C, G), pGCsi-R1; (D, H), pGCsi-C. A-H: immunohistochemistey staining (200×). The increases of RAGE and α-SMA expression were observed in FM2 or pGCsi-C-treated livers. In contrast, RAGE and α-SMA expression were significantly decreased in pGCsi-R1-treated livers compared with FM2 or pGCsi-C-treated livers.

Figure 10.

EMSA for the NF-κB activity in the liver. Binding specificity was demonstrated by competition with excess unlabeled oligonucleotides containing the κB site and by the results of Supershift experiments. A: lane 1, 100 folded excess unlabeled NF-κB consensus sequence; lane 2, FM2; lane 3, pGCsi-C; lane 4, pGCsi-R1; lane 5, NC2; lane 6, normal saline treatment; B: Densitometric analysis of the results were expressed as percentages of the NC2. * and † indicate statistically significant differences. *P<0.01 vs NC2; †P<0.01 vs FM2.

Figure 11.

Effect of specific siRNA targeting RAGE on the changes in ultrastructure of rat liver. (A), HSCs in Disse's space and fenestrae formed in endothelial cells in the NC2 group; (B), HSCs, liver sinusoids and a few lipid droplets in HSCs in pGCsi-R1 group; (C), activated HSCs and a large amount of collagen fiber deposition in pGCsi-C group and the FM2 group. A: Magnification of 16000×; B: Magnification of 12000×; C: Magnification of 20000×; D: Magnification of 20000×; F:fat droplet; HSC: Hepatic stellate cell; H, hepatocyte; CF, collagen fiber; MFs: myofibroblast; E: endothelial cell.