Modified synthetic siRNA targeting tissue inhibitor of metalloproteinase-2 inhibits hepatic fibrogenesis in rats.
OBJECTIVE
Fibrosis occurs in most chronic liver injuries and results from changes in the balance between synthesis and degradation of extracellular matrix (ECM) components. Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) are known to regulate the ECM turnover. We investigate the effect of modified synthetic small interfering RNA (siRNA) targeting TIMP-2 in rat model of liver fibrosis.


METHODS
Rat hepatic fibrosis was induced by CCl4 for 8 weeks. After the 2-week CCl4 injection period, rats in the three siRNA groups simultaneously received a different dosage (0.05, 0.1 and 0.2 mg.kg(-1), respectively) of modified synthetic siRNA targeting TIMP-2 via the tail vein every 3 days for 6 weeks. The pathological changes in liver tissues were observed by light microscopy and transmission electron microscopy. Portal vein pressure and proliferating cell nuclear antigen were measured. Expression of TIMP-2, MMP-2, MT1-MMP, MMP-13, hepatocyte growth factor, collagen type I, collagen type III and alpha-SMA were evaluated by quantitative real-time polymerase chain reaction or Western blotting or gelatin zymography.


RESULTS
Modified synthetic siRNA targeting TIMP-2 induced a dose-dependent inhibition of the TIMP-2 expression in the rat model of liver fibrosis with a similar trend in MMP-2 and MT1-MMP, but an increase in MMP-13. Rats administered siRNA targeting TIMP-2 showed promotion of ECM degradation, reduction in activated hepatic stellate cells and enhancement of hepatocyte regeneration. Furthermore, portal hypertension was also ameliorated after treatment with siRNA targeting TIMP-2.


CONCLUSIONS
Knock-down of TIMP-2 expression attenuates CCl4-induced liver fibrosis and is a potential pharmacological target for gene therapy in liver fibrosis.
Hu
Ying-Bin
YB
Department of Digestive Diseases, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kongjiang Road, Shanghai, 200092, P. R. China. huyingbin@gmail.com
Li
Ding-Guo
DG
Lu
Han-Ming
HM
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D001219:Aspartate Aminotransferases / Q000097:blood; D015415:Biomarkers / Q000097:blood; D049109:Cell Proliferation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D024061:Collagen Type III / Q000235:genetics / Q000378:metabolism; D005109:Extracellular Matrix / Q000648:ultrastructure; D015316:Genetic Therapy / Q000379:methods*; D006909:Hydroxyproline / Q000097:blood; D008099:Liver / Q000737:chemistry / Q000648:ultrastructure; D008106:Liver Cirrhosis, Experimental / Q000473:pathology / Q000628:therapy*; D008297:Male; D053509:Matrix Metalloproteinase 13 / Q000235:genetics / Q000378:metabolism; D053511:Matrix Metalloproteinase 14 / Q000235:genetics / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D034741:RNA, Small Interfering / Q000138:chemical synthesis / Q000737:chemistry / Q000627:therapeutic use*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000235:genetics
D000199:Actins; D000410:Alanine Transaminase; D001219:Aspartate Aminotransferases; D015415:Biomarkers; D024042:Collagen Type I; D024061:Collagen Type III; D006909:Hydroxyproline; D053509:Matrix Metalloproteinase 13; D053511:Matrix Metalloproteinase 14; D020778:Matrix Metalloproteinase 2; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D019716:Tissue Inhibitor of Metalloproteinase-2; C498862:Acta2 protein, rat