Effects of the tyrosine protein kinase inhibitor genistein on the proliferation, activation of cultured rat hepatic stellate cells.
OBJECTIVE
Hepatic stellate cell (HSC) plays a pivotal role in liver fibrosis and is considered as the therapeutic target for the treatment of hepatic fibrosis. Tyrosine protein kinase plays an important role in the proliferation, activation of HSC. The purpose of the study is to investigate the effects of the tyrosine protein kinase inhibitor genistein on the proliferation and activation of cultured rat HSC.


METHODS
Rat HSC were isolated from Wistar rats by in situ perfusion of collagenase and pronase and single-step density Nycodenz gradient. Culture-activated HSC were serum-starved and incubated with 10(-9) to 10(-5) mol/L concentration of genistein for 24, 48 or 72 h. In PDGF-induced HSC proliferation, HSC were stimulated with 10 microg.L(-1) PDGF-BB for 15 min, and then treated with genistein for the same time. Cell proliferation was measured by MTT assay and based on flow cytometric analysis of cell cycle. The a-smooth muscle actin (alpha-SMA) expression in HSC was studied with confocal laser microscopy and flow cytometry. c-fos, c-jun and cyclin D(1) expression in HSC was also detected by flow cytometry.


RESULTS
Genistein inhibited basal and PDGF-induced proliferation of HSC at the concentration of 10(-8) to 10(-5)mol/L, and treatment with 10(-7) mol/L concentration of genistein for 48 h inhibited the HSC proliferation significantly (the inhibition rate was 70.3 %, P<0.05). Immunofluorescence detected by confocal laser microscopy and flow cytometry showed that treatment with 10(-7) mol/L genistein for 48 h suppressed the expression of alpha-SMA significantly in HSC (the specific fluorescence intensity were 60.2+/-21.5 vs 35.3+/-11.6 and 12.8+/-10.4 vs 9.54+/-6.39, respectively, both P<0.05). The intensity of c-fos, c-jun and cyclin D(1) expression of HSCs treated with 10(-7)mol/L genistein for 48 h was also significantly decreased compared with the controls.


CONCLUSIONS
Genistein influences proliferation of HSC, suppresses the expression of alpha-SMA in HSC and t inhibits the intensity of c-fos, c-jun and cyclin D(1) expression of HSCs. Genistein has therapeutic potential against liver fibrosis.
Liu
Xiao-Jing
XJ
Laboratory of Department of Internal Medicine, West China Hospital, Sichuan University, 37 Wainan Guoxueshang, Chengdu 610041, Sichuan Province, China. xiaojingliu67@hotmail.com
Yang
Li
L
Mao
Yong-Qiu
YQ
Wang
Qiong
Q
Huang
Ming-Hui
MH
Wang
Yi-Ping
YP
Wu
Hong-Bin
HB
D000199:Actins / Q000378:metabolism; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology*; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D000199:Actins; D004791:Enzyme Inhibitors; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D019938:Cyclin D1; D019833:Genistein; D011505:Protein-Tyrosine Kinases