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.

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.

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.

OBJECTIVE: To study the effects of heparin on the growth, extracellular matrix and matrix metalloproteinase (MMP) gene expression in rat hepatic stellate cells (HSC). METHODS: Activated HSC was treated by heparin or fetal calf serum without heparin. The cell growth was evaluated by actual cell count and BrdU-labelled immunocytochemical stain. The gene expressions of type I and IV procollagen, fibronectin, MMP-2 and membrane type matrix metalloproteinase (MT-MMP) were investigated by immunocytochemical stain and digoxigenin-labeled in situ hybridization technique, respectively. In addition, the gelatinase activity of MMP-2 was examined by zymography. RESULTS: Heparin could obviously reduce HSC growth, inhibit the synthesis of type I procollagen and fibronectin protein, and the gene expressions of type I procollagen, fibronectin and MT-MMP. The expressions of type IV procollagen, MMP-2 and MMP-2 activity were not affected by heparin. CONCLUSION: The results demonstrate that heparin can inhibit HSC proliferation, down-regulate interstitial collagen synthesis and inhibit MT-MMP gene expression.

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.