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-beta1 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-beta1 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-beta1 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-beta1 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.
George
J
J
Department of Medicine, Division of Molecular Medicine, Columbia University, New York, NY, USA.
Tsutsumi
M
M
D000199:Actins / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor; D004128:Dimethylnitrosamine; D005355:Fibrosis; D020868:Gene Silencing; D015316:Genetic Therapy / Q000379:methods*; D017874:Immediate-Early Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D008099:Liver / Q000378:metabolism / Q000473:pathology*; D008106:Liver Cirrhosis, Experimental / Q000378:metabolism / Q000473:pathology*; D008297:Male; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods; D053773:Transforming Growth Factor beta1 / Q000032:analysis / Q000235:genetics / Q000378:metabolism
D000199:Actins; C526316:CCN2 protein, rat; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor; D004128:Dimethylnitrosamine