Inhibition of connective tissue growth factor by siRNA prevents liver fibrosis in rats

Abstract

Connective tissue growth factor (CTGF) is a highly profibrogenic molecule implicated in hepatic fibrogenesis. Small interfering RNA (siRNA) is an effective tool to silence gene expression post-transcriptionally. Therefore, we conducted an investigation to determine if intraportal vein siRNA injection targeting CTGF inhibits CTGF expression on rat liver in vivo and furthermore whether it protects the liver from liver fibrosis. Some rats received carbon tetrachloride (CCl4) by subcutaneous injections every three days for six consecutive weeks, and meantime they also obtained either siRNA (0.1 mg/kg) targeting CTGF, saline or a control siRNA by intraportal vein injection to rats' liver at the same pattern. Other rats received CCl4 by subcutaneous injection for 2 weeks, followed by CCl4 and CTGF siRNA intraportal vein injection for four more weeks. Intraportal vein injection of CTGF siRNA specifically reduced the expression of CTGF protein in rat liver, and these effects were maintained for 3 days. Six weeks after CCl4 injection, prominent upregulations were observed in the gene expressions of CTGF, type I, III collagen, laminin, tissue inhibitor metal proteinase-1 (TIMP-1) and transforming growth factor-beta1 (TGF-beta1) in saline or control siRNA-treated rats livers. Administrating CTGF siRNA for 4 or 6 weeks, by contrast, markedly attenuated the induction of CTGF, type I, III collagen, laminin, TIMP-1 and TGF-beta1 genes, whereas Smad2, 7 gene expression was not affected. The number of active hepatic stellate cells (HSCs) determined by the expression of alpha-smooth muscle actin was also significantly decreased. The CTGF siRNA treatment markedly reduced serum procollagen type III, hepatic hydroxyproline and liver fibrosis staging. Silencing CTGF expression with siRNA demonstrates therapeutic potential to prevent liver fibrosis by inhibiting HSC activation with consequent extracellular matrix accumulation and the upregulation of TGF-beta1 gene expression.