Anti-fibrotic effects of specific-siRNA targeting of the receptor for advanced glycation end products in a rat model of experimental hepatic fibrosis

Abstract

Since the receptor for advanced glycation end products (RAGE)-ligand axis has been demonstrated to be important in fibrogenesis, rat models may be used to assess whether specific small interfering RNAs (siRNAs) that target RAGE are able to reduce the progression of hepatic fibrosis. However, the effect of RAGE-targeted siRNA on established hepatic fibrosis remains to be elucidated. In the present study, RAGE-specific siRNA expression vectors were constructed prior to the animal experiment. Sprague-Dawley rats were treated initially with olive oil (2 ml/kg) or 50% CCl4 (2 ml/kg; CCl4/olive oil=1:1) twice per week for six weeks to generate the fibrosis model. The rats were then treated with phosphate‑buffered saline, a RAGE-specific siRNA expression vector, at different doses or a non-specific siRNA expression vector twice weekly via tail vein injection for up to six weeks, and were sacrificed at week two, four or six. Compared with the control groups, RAGE‑specific siRNA therapy significantly decreased RAGE mRNA and protein expression in rat livers (P<0.01). Following six weeks of RAGE gene-silencing treatment, the liver function, which was assessed by analyzing serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin (TBIL), improved to varying degrees (P<0.01). The expression of nuclear factor-κB (NF-κB) significantly decreased following RAGE gene‑silencing therapy (P<0.01). In addition, the serum levels of inflammatory cytokines, including tumor necrosis factor‑α (TNF-α) and interleukin-6 (IL-6), and extracellular matrix (ECM) components, including hyaluronic acid (HA), laminin (LN) and procollagen type III (PCIII) also decreased (P<0.01). Furthermore, the expression of α-smooth muscle actin (α-SMA) and collagen I, which indicate the activation of hepatic stellate cells (HSCs), were downregulated following RAGE gene-silencing therapy (P<0.01). Furthermore, the inflammatory activity grade and fibrosis stage of rat livers also significantly improved compared with the control groups following RAGE gene-silencing therapy. Specific targeting of RAGE using siRNA may inhibit RAGE gene expression effectively in the rat hepatic fibrosis model and attenuate the progression of established hepatic fibrosis. This therapeutic effect may be mediated via inhibition of the expression of NF-κB. These findings suggest that RAGE may be a new target to prevent hepatic fibrosis.

Can Pharmacological Targeting of Advanced Glycation End Products Provide Protection Against Experimentally Induced Liver Fibrosis?

“…This is possibly achieved by acting upon many possible sites in the proposed pathway; first by preventing oxidative stress, second by inhibiting the synthesis of proinflammatory cytokines (TGF-b) and thirdly by combating extracellular matrix accumulation via upregulating TIMPs. Previous results have reported the ability of AGEs to induce oxidative stress as well as proinflammatory cytokines [34] and the ability of aminoguanidine to inhibit oxidative stress [35] and to decrease lung fibrosis [36].…”

“…The expression levels of RAGE together with IL-6, TNF-α, TGF-β1, connective tissue growth factor, laminin, hyaluronic acid and N-terminal procollagen III in the treated primary HSCs were significantly downregulated compared with those in the untreated. Thus, it can be deduced that RAGE-specific siRNAs inhibited the expression of RAGE in primary rat HSCs and inhibited the development of HF [36].…”

Cartilage Oligomeric Matrix Protein Participates in the Pathogenesis of Liver Fibrosis

“…7B). Finally, since several studies report that COMP is able to signal via Cd47 [46], Cd36 [47], Rage [48] and α v β 3 -Integrin [49] we analyzed their mRNA expression. Cd36 mRNA was up-regulated upon TAA-or CCl 4 -induced liver fibrosis in WT but not in Comp −/− mice (Fig.…”

Strategies to prevent and reverse liver fibrosis in humans and laboratory animals

“…(Zhang et al 2007a). Improved fibrosis and steatosis (Kitamura et al 2007) (d) Novel molecular target RAGE siRNA injected to CCl4-treated rats (Cai et al 2014) Inhibited RAGE expression, improved liver function, reduced inflammatory cytokines, and improved fibrotic stage Inhibited TG activation by propolis in TAA-treated rats (Chen et al 2008) Prevented the development of TAA-induced liver cirrhosis (e) Nanotechnology applications in liver fibrosis MSP-HSA coupled to liposomes and HVJ in CCl4-treated mice (Adrian et al 2007) M6P-HSA-HVJ liposomes accumulate in HSCs Delivered rosiglitazone by MSP-HAS liposomes in CCl4-treated rats (Patel et al 2012) Enhanced rosiglitazone liver uptake and disappeared from systemic circulation. Improved histopathological morphology, biochemical markers level, and decreased fibrosis grade RGD-labeled liposomes encapsulated IFNα-1b in BDL-treated rats (Du et al 2007) Accumulation of cRGD peptide-labeled liposomes in HSCs, reduced extent of liver fibrosis Vitamin A-coupled liposomes containing gp46 siRNA in DMN-treated rats (Sato et al 2008) Suppressed collagen secretion and fibrosis…”

“…Novel molecular targets: In vitro studies indicated that serum high mobility group box 1 (HMGB1) levels were positively correlated with TGF-β production and collagen deposition during fibrogenesis through receptor of advanced glycation end products (RAGE) pathway activation. CCl4-treated SD rats administered RAGE-specific siRNA twice weekly via tail vein injection for up to 6 weeks displayed significantly decreased levels of serum inflammatory cytokines, NFκB, procollagen III, and hepatic fibrosis, suggesting that HMGB1/RAGE may be a novel target to prevent liver fibrosis (Cai et al 2014). Tissue transglutaminase (tTG) was found to co-localize with collagen fibers to contribute to the development of liver fibrosis.…”