OBJECTIVE: To study the effects of retinoic acid on regulation of expressions of cyclin-dependent kinase inhibitors, i.e. p16, p21 and p27 in cultured rat hepatic stellate cells. METHODS: Stellate cells were isolated from healthy rat livers and cultured. After stimulated with 1 ng/ml transforming growth factor beta 1, subcultured hepatic stellate cells were treated with or without 1 nmol/ml retinoic acid. MTT assay, immunocytochemistry for p16, p21, p27 and alpha-smooth muscle actin protein, in situ hybridization for retinoic acid receptor beta 2 and p16, p21 and p27 mRNA and quantitative image analysis (partially) were performed. RESULTS: Retinoic acid markedly inhibited hepatic stellate cells proliferation (41.50%, P<0.05), decreased the protein level of alpha-smooth muscle actin (55.09%, P<0.05), and induced hepatic stellate cells to express retinoic acid receptor beta 2 mRNA. In addition, retinoic acid increased the protein level of p16 (218.75%, P <0.05) and induced p21 protein expression; meanwhile, p27 was undetectable by immunocytochemistry in both control and retinoic acid-treated hepatic stellate cells. However, retinoic acid had no influence on the mRNA level of p16, p21 or p27 as determined by in situ hybridization. CONCLUSIONS: Up-regulation of p16 and p21 on post-transcriptional level may contribute, in part, to retinoic acid inhibition of transforming growth factor beta 1-initiated rat hepatic stellate cells activation in vitro.

OBJECTIVE: To investigate the effects of PDGF on the proliferation of rat HSC, its gene expression of collagens and the autocrine excretion of PDGF in vitro. METHODS: (3)H-TdR incorporation was used to estimate the DNA synthesis elicited by PDGF, TGF-beta1 and EGF in HSC cultured in an absolutely serum-free medium. Northern blot was employed to detect the levels of mRNA expressions of type I and III procollagens and PDGF induced by PDGF added in the same medium for HSC. RESULTS: Both PDGF and EGF enabled to stimulate the proliferation of HSC in a dose-dependent manner, and the effect of the former was stronger. The proliferation was dose-dependently increased in PDGF and EGF with a dosage between 0.5 and 10 ng/ml, and the synthesis of DNA became saturated when the dosage was over 10 ng/ml. TGF-beta1 gave no effects on the proliferation of HSC under current experimental condition. Furthermore, PDGF was able to enhance the mRNA expression for type I and III procollagens and PDGF itself. The promoting effect for the expressions of type I and III procollagen mRNAs by PDGF was bi-phasic between 6 to 48 hours of the cultivation. For type I procollagen, mRNA expression began to increase 12 hours after PDGF stimulation, and the increase became prominent at 48 hours. For type III procollagen, an increase of mRNA expression was seen before the stimulation of PDGF, and the expression level was comparatively increased 48 hours later. The mRNA expression of PDGF was increased by 2-3 times 6 hours after PDGF stimulation, and back to the original level 24 hours later. CONCLUSIONS: PDGF enabled to promote the proliferation of HSC and expression of collagens in HSC. The bi-phasic effect on the expression of collagens may be interrelated with the autocrine excretion of PDGF.

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 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.