Effects of curcumin on peroxisome proliferator-activated receptor gamma expression and nuclear translocation/redistribution in culture-activated rat hepatic stellate cells.
BACKGROUND
The function of peroxisome proliferator-activated receptor gamma (PPARgamma) in hepatic fibrogenesis remains largely unknown. Curcumin is a natural substance extracted form Curcuma Longa Linn and has a variety of pharmacological effects. In this study, the effects of curcumin on the proliferation, activation and apoptosis of rat hepatic stellate cells (HSCs) through PPARgamma signaling were investigated.


METHODS
HSCs were isolated from the normal Sprague Dawley rats through in situ perfusion of the liver with Pronase E and density-gradient centrifugation with Nycodenz. Cells were treated with curcumin, troglitazone, salvianolic acid B or GW9662. The effect on HSCs proliferation was determined by MTT colorimetry. Total RNA was extracted by TRizol reagent and gene levels were determined by semi-quantitative RT-PCR. Total cellular and nuclear protein were isolated and separated by 10% sodium dodecy lsulfate polyacrylamide gel electrophoresis. Protein levels were determined by Western blot. Cell apoptosis was detected by Hoechst 33258 staining. PPARgamma subcellular distribution was detected by immunofluorescent staining. The activities of MMP-2 and 9 were measured by Gelatin zymograph assay.


RESULTS
Curcumin suppressed HSCs proliferation in a dose-dependent manner. As HSCs underwent gradual activation with culture prolongation the PPARgamma nuclear expression level decreased. Curcumin up-regulated PPARgamma expression and significantly inhibited the production of alpha-SMA and collagen I. PPARgamma is expressed in the cytoplasm and nucleus and is evenly distributed in HSCs, but accumulated in the nucleus of HSCs and disappeared from cytoplasm after curcumin treatment. Hoechst 33258 staining showed that curcumin induced the apoptosis of culture-activated HSCs and significantly increased pro-apoptotic Bax expression and reduced anti-apoptotic Bcl-2 expression. Cyclin D1 gene, activated NFkappaB p65 protein and TGFbetaR-I protein expression were down-regulated significantly by curcumin. The activities of MMP-2 and MMP-9 were enhanced significantly by curcumin.


CONCLUSIONS
Curcumin can inhibit the proliferation and activation of HSCs, induce the apoptosis of activated HSCs and enhance the activities of MMP-2 and MMP-9. The effects of curcumin are mediated through activating the PPARgamma signal transduction pathway and associated with PPARgamma nuclear translocation/redistribution.
Cheng
Yang
Y
Institute of Liver Diseases, Shuguang Hospital, Shanghai University of TCM, Shanghai 201203, China. chengyang@hotmail.com
Ping
Jian
J
Xu
Lie-Ming
LM
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D030201:Activin Receptors, Type I / Q000032:analysis; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003474:Curcumin / Q000494:pharmacology*; D019938:Cyclin D1 / Q000235:genetics; D008099:Liver / Q000166:cytology* / Q000378:metabolism*; D008103:Liver Cirrhosis / Q000188:drug therapy / Q000209:etiology*; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D047495:PPAR gamma / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000032:analysis; D015398:Signal Transduction; D051996:Transcription Factor RelA / Q000235:genetics; D051028:bcl-2-Associated X Protein / Q000032:analysis
D047495:PPAR gamma; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D051996:Transcription Factor RelA; D051028:bcl-2-Associated X Protein; D019938:Cyclin D1; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625843:Tgfbr1 protein, rat; D020780:Matrix Metalloproteinase 9; D003474:Curcumin