Ribozymes against TGFbeta1 reverse character of activated hepatic stellate cells in vitro and inhibit liver fibrosis in rats.
OBJECTIVE
Transforming growth factor beta (TGFbeta1) is considered the key mediator in the process of liver fibrosis. The purpose of this investigation was to evaluate the activity of ribozymes against TGFbeta1 in a cell-free system and activated hepatic stellate cells (HSCs), and antifibrotic effect in activated HSCs in vitro and in rats.


METHODS
Three ribozymes targeting against TGFbeta1 mRNA were designed, and then cloned into the U1 snRNA expression cassette. The chimeric ribozymes were selected for the analysis of their performances in activated HSCs through the detection of their cleavage activities in a cell-free system. After ribozyme-encoding plasmids had been transfected into HSC-T6 cells, the effects of ribozymes on activated HSCs were evaluated through the analysis of proliferation, activation and collagen deposition of HSC-T6. The adenoviral vector expressing the ribozymes was constructed, and then delivered into rat models of hepatic fibrosis induced by carbon tetrachloride.


RESULTS
TGFbeta1 expression was efficiently down-regulated in activated HSCs by U1 snRNA chimeric ribozymes which possessed perfect cleavage activity in a cell-free system. Further studies demonstrated that U1 snRNA chimeric ribozymes inhibited the synthesis of collagen I, reduced deposition of collagen I, suppressed BrdU incorporation, but had no effect on desmin and alpha-SMA expression in transfected HSC-T6 cells. Histological analysis demonstrated that the adenoviral vector expressing ribozyme (Rz803) could alleviate fibrotic pathology in rats treated with carbon tetrachloride.


CONCLUSIONS
The anti-TGFbeta1 ribozymes could reverse the character of activated HSCs in vitro and improve fibrotic pathology in vivo. It indicated that TGFbeta1 could be considered as a novel candidate for a therapeutic agent against hepatic fibrosis.
Song
Yu-Hu
YH
Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
Chen
Xiang-Long
XL
Kong
Xin-Juan
XJ
Liu
Nan-Zhi
NZ
Li
Wei
W
Wu
Xiao-Li
XL
Lin
Ju-Sheng
JS
Jin
You-Xin
YX
D000818:Animals; D001483:Base Sequence; D049109:Cell Proliferation / Q000187:drug effects; D002474:Cell-Free System; D002478:Cells, Cultured; D024042:Collagen Type I / Q000187:drug effects / Q000378:metabolism; D005822:Genetic Vectors; D007371:Interferon-gamma / Q000378:metabolism; D008099:Liver / Q000166:cytology*; D008103:Liver Cirrhosis / Q000139:chemically induced / Q000517:prevention & control*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016337:RNA, Catalytic / Q000494:pharmacology*; D012342:RNA, Small Nuclear / Q000235:genetics / Q000494:pharmacology; D051381:Rats; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D024042:Collagen Type I; D016337:RNA, Catalytic; D012342:RNA, Small Nuclear; D016212:Transforming Growth Factor beta; C405483:U1 small nuclear RNA; D007371:Interferon-gamma