Effects of Ginkgo biloba extract on cell proliferation, cytokines and extracellular matrix of hepatic stellate cells.

BACKGROUND/AIMS: Hepatic fibrosis is the common wound-healing response to chronic liver injury. Ginkgo biloba extract (GbE) has been indicated to reverse hepatic fibrosis and exhibit therapeutic effects both in vitro and in vivo. This study aimed to investigate the underlying mechanism of GbE using HSC-T6 cells, a subline of hepatic stellate cells (HSC) as a model. METHODS: HSC-T6 cells were seeded into six-well plates and allowed to attach overnight. After exposure to different concentrations of GbE761 for 24 or 48 h, cell cycle analysis, semiquantitative RT-PCR, Western blotting analysis and analysis of ECM secretion were performed. RESULTS: It was revealed that GbE (1, 10, 100, 500 mg/l) suppressed HSC proliferation and caused G0/G1 phase arrest in a concentration-dependent manner. RT-PCR and Western blot assays were applied to detect the decline of transforming growth factor beta1(TGF-beta1) and connective tissue growth factor (CTGF) in both mRNA and protein levels after GbE treatment in HSC-T6 cells for 24 or 48 h. Meanwhile, GbE inhibited the synthesis of type I and type III collagens. Secretion of some extracellular matrix (ECM) proteins, such as type III procollagen (PC III), type IV collagen (collagen IV), laminin (LN), hyaluronic acid (HA), were all decreased in supernatant of GbE treated HSC cells. CONCLUSIONS: Our results suggest that GbE confers its anti-fibrosis effects through inhibiting HSC proliferation, reducing TGF-beta1 and CTGF expression and consequently suppressing the collagen production and ECM secretion.     

Inhibitory effect of human interferon-beta-1a on activated rat and human hepatic stellate cells

Background and Aims: Hepatic stellate cells (HSC) are the primary cell type mediating hepatic fibrosis. Although known for its antiviral effects, the inhibitory effects of interferon‐beta (IFN‐β) on HSC treatment have not yet been established. Methods: Both human and rat activated HSC cell lines were incubated with increasing concentrations of recombinant human IFN‐β1a (rhIFN‐β1a) for 24, 48 or 72 h. The effects of rhIFN‐β1a on α‐smooth muscle actin (α‐SMA), collagen types I and III, transforming growth factor‐β1 (TGF‐β1), platelet‐derived growth factor‐BB (PDGF‐BB), and mothers against decapentaplegic homolog (Smad4, Smad7) expression in HSC were examined using Western blotting and immunocytochemistry. Proliferation of HSC was evaluated via bromodeoxyuridine assay. Results: rhIFN‐β1a treatment had a dose‐dependent, inhibitory effect on α‐SMA and collagen type I protein expression. In addition, rhIFN‐β1a decreased the expression of collagen type III, TGF‐β1, PDGF‐BB and Smad4 protein expression in HSC compared with untreated cells. We also observed increased Smad7 protein expression and decreased proliferation in rhIFN‐β1a‐treated HSC. Conclusions: Our data suggest that rhIFN‐β1a treatment decreased α‐SMA and collagen expression and inhibited the activation of HSC through the inhibition of the TGF‐β and PDGF pathways.     

软肝化坚颗粒调节肝星状细胞分泌胶原及相关细胞因子的研究

目的:观察软肝化坚颗粒药物血清对肝星状细胞(HSC)分泌Ⅰ、Ⅲ型胶原及肝纤维化相关细胞因子的影响.方法:通过灌胃制备小鼠软肝化坚颗粒及秋水仙碱药物血清.将HSC-T6细胞分为3组,分别加入含10％软肝化坚颗粒血清、10％秋水仙碱血清和10％正常小鼠血清的培养基,培养24小时后,收集培养上清.采用ELISA法检测Ⅰ型胶原、转化生长因子β1 (TGF-β1)、瘦素(LP)及血小板衍生生长因子(PDGF)的含量;采用放射免疫法检测Ⅲ型胶原含量.结果:与正常血清对照组相比,秋水仙碱血清组和软肝化坚颗粒血清组培养上清中Ⅰ型胶原含量均显著降低,P值均为0.000;Ⅲ型胶原的含量也均显著降低,P值分别为0.005和0.001.与正常血清对照组相比,秋水仙碱血清组培养上清中TGF-β1、LP和PDGF的含量均明显降低,P值均为0.000;软肝化坚血清组也均显著降低,P值均为0.000;软肝化坚颗粒血清组与秋水仙碱血清组相比,培养上清中TGF-β1、LP的含量均明显降低,P值分别为0.006和0.043.结论:软肝化坚颗粒可抑制活化的HSC产生TGF-β1、PDGF及LP等细胞因子,从而对HSC分泌胶原产生抑制作用.     

Melatonin prevents H2O2-induced activation of rat hepatic stellate cells

Considerable evidence shows therapeutic effects of melatonin on liver injury and the involvement of hepatic stellate cells (HSCs) in vivo. In the present studies, we investigate the protective effect of melatonin on H2O2-induced activation of HSCs in vitro. Compared with that in control HSCs, synthesis of collagen type I was increased in H2O2-treated cells. Melatonin pretreatment significantly inhibited the above effects of H2O2 in HSCs. CCAAT/enhancer-binding protein alpha (C/EBP-alpha), which could partially reverse the phenotype of activated HSCs, augmented in HSCs pretreated with melatonin. Moreover, secretion of the most important fibrotic cytokine transforming growth factor beta 1 (TGF-beta1) diminished in melatonin-pretreated HSCs. These results suggest that melatonin prevents H2O2-induced activation of HSCs and that the mechanism involves, at least in part, differential regulation of TGF-beta1 and C/EBP-alpha gene expression.     

Effects of rhubarb on migration of rat hepatic stellate cells

Background and Aims:  In chronic liver injury, hepatic stellate cells (HSCs) acquire an activated phenotype, migrate to the injured region in response to chemotactic factors, and produce extracellular matrix proteins including collagen. In this study, we investigated the effects of rhubarb ( Rheum palmatum L.) on transforming growth factor (TGF)-β1-induced expressions of α-smooth-muscle actin (SMA) and collagen, and the migration of HSCs.
Methods:  HSC-T6, a cell line of rat HSCs, was used in the in vitro experiments. An enzyme-linked immunosorbent assay and Sircol red assay were used to detect the expressions of α-SMA and collagen, respectively. HSC-T6 migration was assayed with a transwell apparatus. Phosphorylations of Smad2/3 and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK) 1/2, p38, and c-jun N-terminal kinase (JNK), were analyzed with Western blotting. Matrix metalloproteinase (MMP)-2 activity was examined by gelatin zymography.
Results:  The results revealed that a rhubarb extract concentration-dependently attenuated TGF-β1-induced α-SMA and collagen expressions and migration of HSCs. The inhibitory effect of rhubarb was associated with (i) down-regulation of the phosphorylation of Smad2/3 and JNK, and (ii) attenuation of MMP-2 activity. Within the working concentrations used, the rhubarb extract did not affect cell viability of HSCs.
Conclusion:  The results suggest that rhubarb attenuated TGF-β1-mediated migration of HSCs possibly by interfering with Smad2/3 phosphorylation, the MAPK pathway, and MMP-2 activity.     

雷帕霉素诱导大鼠肝星状细胞凋亡的研究

目的观察雷帕霉素(RAPA)对大鼠肝星状细胞系rHSCs-99增殖、凋亡的影响。方法将rHSCs-99分成4组:对照组(A组),RAPA 50 nmol/L组(B组),RAPA 100 nmol/L组(C组),RAPA 150 nmol/L组(D组)。RAPA作用72 h后,分别用MTT法检测rHSCs-99增殖,ELISA法检测I型胶原表达,流式细胞仪Annexin-V FICT/PI法检测细胞凋亡情况,Wright-Giemsa染色法观察细胞形态学改变,细胞免疫组化法检测Fas、P53与Bcl-2的表达变化。结果 RAPA作用后,rHSCs-99增殖受到抑制,I型胶原含量降低,凋亡率增高,Fas、P53表达增多,Bcl-2表达减少。结论 RAPA能抑制rHSCs-99的增殖及I型胶原合成。促进rHSCs-99凋亡,其机制可能是通过开启Fas/FasL凋亡途径及上调凋亡相关基因P53、下调Bcl-2的表达来实现。     

粉防己碱对大鼠肝星状细胞跨膜信号转导的影响

粉防己碱（Tet）是抗肝纤维化的有效药物，这一作用可能与Tet对肝星状细胞（HSC）活化的直接抑制作用有关。最近在动物实验发现较低浓度Tet（2.5-5mg/kg）亦有较强的抗肝纤维化作用，为进一步了解低浓度Tet抗肝纤维化的作用机制，本研究观察低剂量Tet对静止期HSC培养活化和转化生长因子β1（TGFβ1）促其活化作用影响，以及此过程中TGFβ和其下游Smads信号蛋白的表达变化。     

β-榄香烯对肝星状细胞凋亡相关蛋白Bcl-2家族的影响

[目的]研究β-榄香烯对肝星状细胞(HSC)凋亡及凋亡相关蛋白的影响。[方法]传代培养的大鼠HSC系HSC-T6与不同剂量β-榄香烯(终浓度为10、5、2.5 mg/L)共同培养48 h后,应用Annexin-V-碘化丙锭染色检测β-榄香烯对HSC凋亡的影响,免疫组化检测凋亡相关蛋白BcL-2/Bax。[结果]β-榄香烯可使HSC凋亡率明显增多,同时可使凋亡抑制分子Bcl-2表达下调,促凋亡分子Bax表达增强(P<0.01)。[结论]β-榄香烯可通过下调Bcl-2/Bax比率,促进HSC凋亡。     

Apoptosis of hepatic stellate cells: involvement in resolution of biliary fibrosis and regulation by soluble growth factors

BACKGROUND: Activated hepatic stellate cells (HSC) are central to the pathogenesis of liver fibrosis, both as a source of fibrillar collagens that characterise fibrosis and matrix degrading metalloproteinases and their tissue inhibitors, the TIMPs. AIMS: To test the hypothesis that HSC apoptosis is critical to recovery from biliary fibrosis and that soluble growth factors may regulate HSC survival and apoptosis. METHODS: Rats (n=15) were subjected to bile duct ligation for 21 days, after which biliodigestive anastomosis was undertaken (n=13). Livers were harvested at fixed time points of recovery for periods of up to 42 days. Numbers of activated HSCs were quantified after alpha smooth muscle actin staining and HSC apoptosis was detected by terminal UDP-nick end labelling (TUNEL) staining and quantified at each time point. HSC apoptosis was quantified in vitro in the presence or absence of insulin-like growth factor (IGF)-1, IGF-2, platelet derived growth factor (PDGF), and transforming growth factor beta1 (TGF-beta1). RESULTS: Following biliodigestive anastomosis after 21 days of bile duct ligation, rat liver demonstrated a progressive resolution of biliary fibrosis over 42 days, associated with a fivefold decrease in activated HSC determined by alpha smooth muscle actin staining. TUNEL staining indicated that loss of activated HSC resulted from an increase in the rate of apoptosis during the first two days post biliodigestive anastomosis. Serum deprivation and culture in the presence of 50 microM cycloheximide was associated with an increase in HSC apoptosis which was significantly inhibited by addition of 10 ng/ml and 100 ng/ml IGF-1, respectively (0.05>p, n=5). In contrast, 1 and 10 ng/ml of TGF-beta1 caused a significant increase in HSC apoptosis compared with serum free controls (p<0.05, n=4). PDGF and IGF-2 were neutral with respect to their effect on HSC apoptosis. CONCLUSION: HSC apoptosis plays a critical role in the spontaneous recovery from biliary fibrosis. Both survival and apoptosis of HSC are regulated by growth factors expressed during fibrotic liver injury.     

Melatonin suppresses activation of hepatic stellate cells through RORα‐mediated inhibition of 5‐lipoxygenase

Liver fibrosis is scar tissue resulting from an uncontrolled wound‐healing process in response to chronic liver injury. Liver damage generates an inflammatory reaction that activates hepatic stellate cells (HSC) that transdifferentiate from quiescent cells that control retinol metabolism to proliferative and migratory myofibroblasts that produce excessive amounts of extracellular matrix proteins, in particular collagen 1a1 (COL1A1). Although liver fibrosis is reversible, no effective drug therapy is available to prevent or reverse HSC activation. Melatonin has potent hepatoprotective properties in a variety of acute and chronic liver injury models and suppresses liver fibrosis. However, it remains unclear whether melatonin acts indirectly or directly on HSC to prevent liver fibrosis. Here, we studied the effect of melatonin on culture‐activated rat HSC. Melatonin dose‐dependently suppressed the expression of HSC activation markers Col1a1 and alpha‐smooth muscle actin (αSMA, Acta2), as well as HSC proliferation and loss of lipid droplets. The nuclear melatonin sensor retinoic acid receptor‐related orphan receptor‐alpha (RORα/Nr1f1) was expressed in quiescent and activated HSC, while the membranous melatonin receptors (Mtrn1a and Mtrn1b) were not. The synthetic RORα agonist SR1078 more potently suppressed Col1a1 and αSma expression, HSC proliferation, and lipid droplet loss, while the RORα antagonist SR1001 blocked the antifibrotic features of melatonin. Melatonin and SR1078 inhibited the expression of Alox5, encoding 5‐lipoxygenase (5‐LO). The pharmacological 5‐LO inhibitor AA861 reduced Acta2 and Col1a1 expression in activated HSC. We conclude that melatonin directly suppresses HSC activation via RORα‐mediated inhibition of Alox5 expression, which provides novel drug targets to treat liver fibrosis.     

肝素对肝纤维化大鼠肝星状细胞增殖及分泌的影响及其作用机制

目的观察肝素在体外对肝纤维化大鼠肝星状细胞增殖及分泌的影响,并探讨其作用机制。方法应用Nycodenz分离肝纤维化大鼠的肝星状细胞置培养板中,并分为三组。A、B组分别加入1000μg/ml及2000μg/ml肝素,C组不用药。应用MTT比色法检测各组肝星状细胞的增殖状况,免疫细胞化学检测各组肝星状细胞中α-平滑肌肌动蛋白(α-SMA)、转化生长因子-β1(TGF-β1)、层粘连蛋白(LN)的表达。结果OD值A组为0.0626±0.0137,B组为0.0746±0.0131,C组为0.1106±0.0198,A、B组均显著低于C组(P均<0.05),B组低于A组(P>0.05);C组肝星状细胞胞质中可见α-SMA、TGF-β1和LN明显表达,A、B组各指标表达均低于C组(P分别<0.01和<0.05),A组表达低于B组(P<0.05)。结论肝素可抑制肝纤维化大鼠肝星状细胞的增殖及胶原分泌,作用机制可能是抑制肝星状细胞表达TGF-β1。     

神经生长因子抑制肝星状细胞增殖的观察

目的 观察神经生长因子(NGF)对大鼠肝星状细胞(HSC)增殖的抑制作用,并探讨其作用机制. 方法 体外培养大鼠肝星状细胞株HSC-T6,将HSC-T6与不同浓度NGF孵育后,用XTT比色法检测NGF对HSC增殖的影响,流式细胞术分析NGF对HSC细胞周期的影响,透射电镜观察经100 ng/ml NGF作用24h后HSC形态学变化.结果 (100、200、400) ng/ml浓度时NGF对HSC的抑制作用经XTT法测得A值分别为0.66±0.03、0.69±0.03和0.66±0.03,与对照组(0.73±0.01)比较,差异具有统计学意义(P＜0.05),但无浓度依赖性(P＞0.05).100、200、400ng/ml NGF作用于HSC 24h后,G2期比例分别为14.83％±5.41％、14.73％±2.50％和14.87％±2.06％,与对照组(7.47％±4.39％)比较,明显增加(P＜0.05),透射电镜可以见到细胞凋亡的形态学变化. 结论 NGF可抑制HSC增殖,通过使HSC细胞周期停滞于G2期而抑制HSC增殖可能为其作用机制之一;经NGF作用的大鼠肝星状细胞可出现明显的增殖受抑、细胞凋亡的形态学改变.