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Hepatic fibrosis is a significant health problem worldwide for which no effective therapy exists. Hepatic stellate cell (HSC) plays a critical role in the live fibrosis. HSC has dominated studies which are about mechanisms of liver fibrosis over the last two decades. Following hepatic injury, HSC becomes activated, proliferate and produce extracellular matrix (ECM), of which type I collagen predominates. Initiating events in stellate cell activation occurs on a background of progressive changes which is in the surrounding ECM and the ECM is within the subendothelial space of Disse. Over time, the subendothelial matrix compositions change. At first, the ECM is one comprised of type IV collagen, heparan sulfate proteoglycan, and laminin (the classic constituents of a basal lamina). Following it becomes one rich in fibril-forming collagens, particularly types I and III.Raf/extracellular-signal-regulated kinase (ERK) and focal adhesion kinase (FAK)- phosphatidylinositol 3-linase (PI3K)/Akt/70-kDa ribosomal S6 kinase (p70S6K) related signaling pathways, which regulate the transcription of profibrogenic genes, protein synthesis, cell proliferation, cell cycle control and apoptosis. The phosphatidylinositol 3-kinase (PI3K) is activated by FAK after platelet-derived growth factor (PDGF) treatment of HSC.The endocannabinoid system is made up of the cannabinoid receptors, their endogenous ligands (or endocannabinoids), and the proteins regulating their synthesis and inactivation. Two cannabinoid receptors, cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2) have been cloned and characterized. Rimonabant (SR141716) is the first selective and orally active antagonist of the brain cannabinoid receptor. The endocannabinoid has been shown to inhibit the growth of tumor cells in culture and animal models by modulating key cell signaling pathway. Recently, it has been demonstrated that endocannabinoids are involved in the regulation of fibrogenesis in the liver. CB2-/- mice show increased fibrogenesis in response to CCl4 injection, whereas CB1-/- mice have decreased hepatic fibrogenesis. There are many studies about CB1 and liver fibrosis. However, the pharmacological effect of rimonabant on HSC proliferation, apoptosis, collagen secretion and the mechanisms by which rimonabant regulates fibrogenesis are not well characterized in HSC in vitro.Objective: We investigated the HSC-T6 cell and primary HSC proliferation,HSC-T6 cell apoptosis and collagen secretion, even though the cells were stimulated by 10% fetal bovine serum (FBS). We also investigated the relevant mechanism contained inhibiting the phosphorylations of FAK and ERK.Methods: proliferation was evaluated by 3-(4,5-dimethyl)2,5- diphenyl- 2H- tetrazolium bromide (MTT) essay. CB1, FAK and ERK mRNA transcriptions were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay and the FAK, p-FAK, ERK, p-ERK protein expressions were investigated by Western blot. Cell apoptosis, caspase-3 protein expression and cell cycle were detected by flow cytometry or transmission electron microscopy (TEM). Caspase-3 activity was measured using Caspase-3 Activity Assay Kit. Cell viability was detected by 3-(4, 5-dimethyl)2,5- diphenyl-2H-tetrazolium bromide (MTT) assay. Hyaluronic acid (HA), procollagen type III (PCIII),type IV collagen (collagen IV) and laminim (LN) were evaluated by radioimmunoassay.Results: 1 Rimonabant inhibited the proliferation of primary HSC and HSC-T6 cells in a concentration dependent manner. Cell viability was significantly decreased in both HSC-T6 cells and primary HSC treated with rimonabant (5, 10, 20μM) compared with the vehicle (HSC-T6 cells, 0.86±0.06, 0.79±0.13, 0.20±0.04 vs 1.00±0.00, primary HSC, 0.83±0.13, 0.67±0.09, 0.62±0.10 vs 1.00±0.00, P < 0.05, n=3). The similar decrease was found in 10% FBS stimulated HSC treated with rimonabant (5, 10 and 20μM). G2/M phase cells decreased and G0/G1 phase cells increased in HSC-T6 cells treated with rimonabant (5, 10 and 20μM) for 12 h compared with the vehicle (G2/M phase cells, 34.33%±7.82%, 21.33%±7.07%, 20.13%±0.99% vs 40.57%±5.48%, G0/G1 phase cells, 50.80%±3.13%, 62.70%±0.95%, 67.60%±0.70% vs 45.33%±2.51%, P < 0.05, n=3). G2/M phase cells decreased and G0/G1 phase cells increased in HSC-T6 cells treated with 10μM rimonabant for 6, 24 and 48 h compared with the vehicle (G2/M phase cells, 22.57%±0.86%, 9.40%±1.77, 6.70%±5.03% vs 29.03%±5.87%, G0/G1 phase cells, 51.97%±1.53%, 66.93%±2.59%, 70.67%±1.60% vs 49.33%±2.36%, P < 0.05, n=3). We found a decrease in G2/M phase cells in the 10% FBS + rimonabant (20μM) group compared with the 10% FBS group (23.47%±6.06% vs 32.60%±5.60%, P < 0.05, n=3).2 TEM revealed that HSC-T6 cells in the 20μM rimonabant group and 10% FBS +20μM rimonabant group exhibited the shrunken chromatin which is also aggregated and condensed along the inside nuclear membrane. The morphologies of the cell nuclei were crescent-shaped or spherical. Dilated endoplasmic reticulums, vanished cell surface microvilli, folded nuclear membranes and osteoporosis, swollen mitochondria were observed in apoptotic cells. The apoptosis rate of HSC-T6 cells treated by rimonabant (5, 10 and 20μM) increased to 24.33%±1.21%, 27.80%±0.90%, 35.70%±0.90% compared with 22.67%±0.47% in the vehicle group. The apoptosis rate was increased in HSC-T6 cells treated with 10μM rimonabant for 6, 24 and 48 h compared with the vehicle. The apoptosis rates in 10% FBS +20μM rimonabant group are significantly increased compared with 10% FBS group. The caspase-3 in the groups treated with rimonabant (5, 10 and 20μM) for 12 h increased by 34.78%, 88.83%, 143.45% compared with the vehicle group. Similarly, the caspase-3 protein expression in the groups treated with 10μM rimonabant for 6, 24 and 48 h increased by 33.33%, 112.50%, 220.83% compared with the vehicle group. The increase of caspase-3 protein expression remained significant in the 10% FBS + 20μM rimonabant group compared with the 10% FBS group. The enzymatic activity of caspase-3 was increased by rimonabant in a concentration dependent manner in the groups treated with rimonabant (5, 10 and 20μM) compared with the vehicle group.3 The secretions of HA, PC III, collagen IV and LN were significantly inhibited in the groups treated with rimonabant (5, 10 and 20μM) for 12 h compared with the vehicle group. The inhibition of HA, PC III, collagen IV and LN secretions were evident in the groups treated by 10μM rimonabant for 24 and 48 h compared with the vehicle group. HA, PC III, collagen IV and LN secretions increased significantly in the 10% FBS + 20μM rimonabant group compared with the 10% FBS group.4 The CB1 mRNA expression was decreased significantly in the groups treated with rimonabant (5, 10 and 20μM) for 12 h compared with the vehicle group (0.70±0.60, 0.683±0.07, 0.19±0.03 vs 1.00±0.00, P < 0.05, n=3). There was a most significant reduction noted at the 20μM dose level. Compared with the vehicle group, rimonabant inhibited the CB1 mRNA expression in the groups treated by 10μM rimonabant for 24 and 48 h (0.65±0.04, 0.58±0.22 vs 1.00±0.00, P < 0.05, n=3). Interestingly, the treatments with rimonabant (20μM) inhibited the CB1 mRNA expression in HSC-T6 cells stimulated with 10% FBS, compared with the 10% FBS group (0.35±0.05 vs 1.20±0.14, P < 0.05, n=3). The FAK and ERK mRNA levels were unchanged in HSC-T6 cells treated by rimonabant. Rimonabant inhibited FAK and ERK phosphorylation in the groups treated with rimonabant (10 and 20μM) for 12 h compared with the vehicle group. The inhibitory effect was the most significant in the 20μM rimonabant group. The level of FAK and ERK phosphorylation decreased in the groups treated by 10μM rimonabant for 24 and 48 h compared with the vehicle group. Total FAK and ERK levels were unchanged in HSC-T6 cells treated by rimonabant. The inhibition of ERK phosphorylation and FAK phosphorylation remained significant in the 10% FBS + 20μM rimonabant group compared with the 10% FBS group.Conclusion: Rimonabant reduced HSC proliferation, induced HSC apoptosis and inhibited its collagen secretion through inhibiting the phosphorylation of FAK and ERK and down-regulating the CB1 mRNA expression, even though the cells were stimulation by 10% FBS.
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