Effects of salvianolic acid-B on TGF-beta 1 stimulated hepatic stellate cell activation and its intracellular signaling.
OBJECTIVE
To investigate hepatic stellate cells (HSC) responses at different differentiation stages on transforming growth factor-beta 1, and to elucidate the mechanisms of salvianolic acid - B (SA-B), a water soluble compound from Salvia miltiorrhiza, against hepatic fibrosis, relating to interference with TGF-beta 1 stimulated HSC activation and intracellular signal transduction via Smads.


METHODS
HSC was isolated from rat by in situ perfusion of liver and 8.2% nycondenz gradient centrifugation, and primarily cultured on uncoated plastic for 1 d, 4 d and 7 d respectively, which represented quiescent, intermediate and activated phenotypes. The cells were stimulated with 100 pmol/L TGF-beta 1, cell phenotypes were observed under inverted microscope, alpha-actin expression was checked by Western blot, and collagen secretion was measured with [(3)H] proline incorporation and collangenase digestion, then HSC at one definite differentiation stage that responded most sensitively to TGF-beta 1 was selected as the cell model for the following study. 0.1 micromol/L - 1 mmol/L SA-B was incubated with HSC and the cell proliferation was measured by intracellular [(3)H] thymidine pulse. SA-B was also incubated with TGF-beta 1 stimulated HSC, the collagen secretion was measured as above, alpha-actin and plasmin activator inhibitor-1 (PAI-1) were checked with Western blot, and alpha1 (I) procollagen mRNA levels were analyzed with Northern blot. The cytoplasmic and nuclear proteins were extracted, and cytoplasmic and nuclear Smad2, 3 expression and phosphorylation levels were measured with Western blot.


RESULTS
As culture duration prolonged, HSC phenotypes underwent activation gradually, accompanied by the increase of alpha-actin expression and collagen secretion. TGF-beta 1 increased the basal collagen levels at d1, d4 and d7 by 128.6%, 207.7% and 188.2% of the control respectively, while d4 HSC had the most sensitive response, and this intermediate HSC was used as cell model for the following study. Except 0.1 mmol/L-1 mmol/L SA-B caused parts of HSC death, 0.1 micromol/L-10 micromol/L SA-B had no influence on cell shape, but decreased HSC proliferation in a dose depend manner, by 76%, 60.1% and 47.8% of the control respectively. 1 micromol/L-10 micromol/L SA-B remarkably inhibited the collagen secretion of TGF-beta 1 stimulated HSC by 68.6% and 56.1% of the control, PAI-1 and alpha-actin expression, and down-regulated alpha 1 (I) pro-collagen gene expression. 0.1 micro mol/L approximately 10 micro mol/L SA-B decreased the cytoplasmic and nuclear Smad2, 3 protein expression, especially inhibited Smad2 phosphorylation and nuclear translocation.


CONCLUSIONS
SA-B obviously inhibits intermediate HSC proliferation, decreases TGF-beta 1 stimulated HSC activation and matrix protein and gene expression, and inhibited stimulated HSC Smad2, 3 protein expression, phosphorylation and nuclear translocation. The inhibition of TGF-beta 1 signaling in HSC and its biological responses is the important mechanism of SA-B against hepatic fibrosis.
Liu
Chenghai
C
Institute of Liver Diseases, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China.
Liu
Ping
P
Hu
Yiyang
Y
Zhu
Dayuan
D
D000199:Actins / Q000378:metabolism; D000818:Animals; D001572:Benzofurans / Q000494:pharmacology*; D015152:Blotting, Northern; D015153:Blotting, Western; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D024042:Collagen Type I / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D004365:Drugs, Chinese Herbal / Q000737:chemistry; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects*; D051899:Smad2 Protein; D051900:Smad3 Protein; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D053630:Depsides
D000199:Actins; D001572:Benzofurans; D003094:Collagen; D024042:Collagen Type I; D004268:DNA-Binding Proteins; D004365:Drugs, Chinese Herbal; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D051899:Smad2 Protein; D051900:Smad3 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C497784:Smad2 protein, rat; C497787:Smad3 protein, rat; C508962:Tgfb1 protein, rat; C076944:salvianolic acid B; D053630:Depsides