siRNA-mediated knockdown of connective tissue growth factor prevents N-nitrosodimethylamine-induced hepatic fibrosis in rats

Hepatic fibrosis is a dynamic process that involves the interplay of different cell types in the hepatic tissue. Connective tissue growth factor ( CTGF) is a highly profibrogenic molecule and plays a crucial role in the pathogenesis of hepatic fibrosis. The aim of the present investigation was three-fold. First, we studied the expression of CTGF in the cultured hepatic stellate cells using immunohistochemical technique. Second, we induced hepatic fibrosis in rats through serial intraperitoneal injections of N-nitrosodimethylamine ( NDMA; dimethylnitrosamine, DMN) and studied the upregulation of CTGF and TGF-beta 1 during hepatic fibrogenesis. Third, we downregulated CTGF expression using CTGF siRNA and examined the role of CTGF siRNA to prevent the progression of NDMA-induced hepatic fibrosis. The results depicted strong staining of CTGF in the transformed hepatic stellate cells in culture. Serial administrations of NDMA resulted in activation of hepatic stellate cells, upregulation of CTGF and TGF-beta 1 both at mRNA and protein levels and well-developed fibrosis in the liver. Immunostaining, Western blot analysis, semiquantitative and real-time RT-PCR studies showed downregulation of CTGF and TGF-beta 1 after treatment with CTGF siRNA. The results of the present study demonstrated that CTGF gene silencing through siRNA reduces activation of hepatic stellate cells, prevents the upregulation of CTGF and TGF-beta 1 gene expression and inhibits accumulation of connective tissue proteins in the liver. The data further suggest that knockdown of CTGF upregulation using siRNA has potential therapeutic application to prevent hepatic fibrogenesis.