AIM: To study the molecular mechanisms of retinoic acid (RA) on prolix-feration and expression of cyclin-dependent kinase inhibitors (CKI), i.e.p16, p21 and p27 in cultured rat hepatic stellate cells (HSC) stimulated with transforming growth factor beta 1 (TGF-beta1). METHODS: HSC were isolated from healthy rat livers and cultured.After stimulated with 1mg/L TGF-beta1, subcultured HSC were treated with or without 1nmol/L RA. MTT assay, immunocytochemistry (ICC) for p16, p21, p27 and alpha-smooth muscle actin (alpha-SMA) protein, in situ hybridization (ISH) for retinoic acid receptor beta 2 (RAR-beta2) and p16, p21 and p27 mRNA and quantitative image analysis (partially) were performed. RESULTS: inhibited HSC proliferation (41.50%,P<0.05), decreased the protein level of alpha-SMA (55.09%, P<0.05), and induced HSC to express RAR-beta2 mRNA. In addition, RA increased the protein level of p16 (218.75%, P <0.05) and induced p21 protein expression; meanwhile, p27 was undetectable by ICC in both control and RA-treated HSC. However, RA had no influence on the mRNA levels of p16, p21 or p27 as determined by ISH. CONCLUSION: Up-regulation of p16 and p21 on post-transcriptional level may contribute, in part, to RA inhibition of TGF-beta 1-initiated rat HSC activation in vitro.

Hepatic stellate cells (HSCs) play key roles in hepatic fibrosis. One of the most striking alterations in activated HSCs is loss of cytoplasmic lipid droplets. However, the association of lipid storage with the activation of HSCs remains unclear. CCAAT/enhancer-binding proteins family (C/EBPs), especially C/EBP-alpha, controls differentiation of adipocytes. We suggested that C/EBP-alpha gene may be involved in HSCs activation. The present results showed that the expression levels of C/EBP-alpha and C/EBP-beta genes declined in activated HSCs. Over-expression of C/EBP-alpha gene in activated HSCs: (1) inhibited HSCs proliferation, extracellular matrix-producing, alpha-smooth muscle actin gene expression, and induced rebound of cytoplasmic lipid droplets; (2) reduced retinoic acid receptor-beta, C/EBP-delta and-beta gene expressions, but increased the active form C/EBP-beta PSer (105), and induced retinoid X receptor-alpha gene expression; and (3) did not affect the protein level of p16INK4a, p21Cip1/WAF1 or p27Kip1. In conclusions, C/EBP-alpha gene is involved in in vitro activation of rat HSCs.

BACKGROUND/AIM: In hepatic stellate cells isolated from rat fibrotic livers, the amount of retinoid X receptor-alpha (RXR-alpha) mRNA is greatly reduced. However, the effectiveness of retinoids in the treatment of liver fibrosis is controversial. We hypothesized that increasing the expression levels of RXR-alpha in livers will improve the response of liver fibrosis to retinoids treatment. METHODS: pTracer-CMV2 vector harboring both green fluorescent protein and RXR-alpha genes was given to mice with carbon tetrachloride (CCl(4))-induced liver fibrosis, by hydrodynamic-based in vivo transfection. Vitamin A was simultaneously administered to the mice. Sirius red staining and measurement of hydroxyproline content were performed to evaluate liver fibrosis. The incorporation of 5-bromo-2-deoxyribouridine (BrdU) was carried out to determine liver cell proliferation. RESULTS: Successful transfection and expression of exogenous RXR-alpha gene in the liver was determined by observance of green fluorescence under a confocal microscope, and detection of RXR-alpha protein by immunohistochemistry. Hepatic fibrosis, evaluated by both Sirius red staining with image analysis and quantity of hydroxyproline in livers of RXR-alpha-transfected group, tapered off remarkably. The hydroxyproline content and Sirius red-positive staining area on liver sections from RXR-alpha-transfected mice decreased by 34.3% and 54.63%, respectively, compared with the control group receiving empty vector. The labeling index of BrdU in non-parenchymal cells was much lower in livers from the RXR-alpha-transfected group than that of empty vector-transfected group. CONCLUSIONS: Hydrodynamic-based in vivo transfection of the RXR-alpha gene can enhance the vitamin A-induced attenuation of liver fibrosis in mice. One of the possible mechanisms of action for this gene treatment is inhibition of non-parenchymal cell proliferation mainly composed of hepatic stellate cells in fibrotic livers.

OBJECTIVE: To study the effect of retinoid receptor alpha (RXRalpha) transfection plus treatment with the RXRalpha ligand, 9-cis-RA, on the proliferation and phenotype of platelet-derived growth factor (PDGF) activated hepatic stellate cells (HSCs). METHODS: PDGF activated rat hepatic stellate cells were transfected with eukaryotic expression vector pcDNA3.1-human RXRalpha, and confirmed by Western blot. Proliferation of transfected HSC was assayed by bromodeoxyuridine (BrdU) incorporation as well as MTT, and the phenotype (alpha-smooth muscle actin, desmin) was observed by immunocytochemistry with image analysis. RESULTS: Transfection of the RXRalpha gene and treatment with ligand 9-cis-RA of PDGF-activated HSCs extended the increased expression of RXRalpha protein for at least 168 hours. Cell proliferation and expressions of alpha-smooth muscle actin (alpha-SMA) and desmin were blocked, compared with groups of sham-transfected, PDGF-activated, no transfection, no ligand treatment, and irrelevant ligand treated HSCs. CONCLUSION: Transfection with the RXRalpha gene followed by 9-cis-RA ligand treatment will inhibit the proliferation and reverse the phenotype of activated HSC.

OBJECTIVE: To study the effect of RAR-beta transfection plus treatment with the corresponding ligand ATRA on the proliferation and phenotype of platelet-derived growth factor (PDGF)-activated hepatic stellate cells (HSC). METHODS: PDGF-activated hepatic stellate cells of rats were transfected with eukaryotic expression vector pCMV-script-RAR-beta, which was verified by western blot. The proliferation of transfected HSC was assayed by western blot incorporation as well as MTT methods. Their phenotype (alpha-SMA and desmin) was observed by immunocytochemistry assay with image analysis and RAR-beta protein expression was detected by western blot. RESULTS: Transfection of RAR-beta gene and treatment with ligand ATRA could increase the expression of RAR-beta protein for at least 144h and inhibit the proliferation and the expression of alpha-SMA and desmin in PDGF-activated HSC. Significant statistical differences were perceived comparing with sham-transfected, only-PDGF treated, non-ligand treated and irrelevant ligand-treated HSC. CONCLUSIONS: Transfected with RAR-beta gene as well as using related ligand ATRA could suppress the proliferation and reverse the activation phenotype of activated HSC.