Cell survival,activation and apoptosis of hepatic stellate cells: modulation by extracellular matrix proteins

Aim: Cytokines and growth factors released by various hepatic cells exert both paracrine and autocrine effects on hepatic stellate cell (HSC) activation during liver injury. The aim of the present study was to examine whether the surrounding extracellular matrix (ECM) influences the activation, transdifferentiation and survival of HSCs. Methods: An in vitro model system of isolated HSCs maintained in culture on different matrix protein substrata was employed. Results: The rate of loss of HSC‐specific retinol uptake activity and gain of myofibroblast‐like activity such as ³⁵[S] proteoglycan synthesis varied in cells maintained on different matrix proteins and was in the order collagen I > collagen IV ≥ laminin. ³[H]‐thymidine incorporation by HSCs maintained on different matrix proteins varied and was in the order collagen I > collagen IV > laminin. MTT assay revealed that the growth inhibition in response to curcumin was significantly low in cells maintained on collagen I. Apoptotic marker activities such as DNA fragmentation, 4′,6′‐diamidino‐2‐phenylindole dihydrochloride (DAPI) staining, annexin staining and caspase‐3 activities showed that cells maintained on collagen I showed minimal apoptosis than those maintained on collagen IV, laminin and polylysine, showing the influence of ECM on HSC apoptosis. Experiments using blocking antibodies showed that the collagen I effect was mediated through α₂β₁ integrin. Conclusions: These results indicate that ECM influences activation, transdifferentiation and survival of HSCs, and suggest that apart from diffusible factors, the surrounding ECM also influences HSC behavior critical in both the progression of the fibrosis and the restitution of the liver during recovery after hepatic injury.     

姜黄素对肝星状细胞增殖及分泌细胞外基质的影响

观察姜黄素对大鼠肝星状细胞 (HSC)增殖及分泌细胞外基质的影响。用不同浓度的姜黄素处理大鼠肝星状细胞 ;以MTT比色法测定细胞的增殖 ;放射免疫法测定透明质酸和层粘连蛋白 ;酶联免疫吸附法 (ELISA)测定Ⅰ型胶原的水平。姜黄素可剂量依赖性地抑制HSC增殖 ,不同程度抑制Ⅰ型胶原、透明质酸和层粘连蛋白分泌。姜黄素可显著抑制HSC的增殖及分泌细胞外基质。     

龙血素B对肝星状细胞增殖及细胞外基质分泌的影响

目的观察龙血素B对大鼠肝星状细胞(HSC)增殖及细胞外基质分泌的影响。方法用不同浓度的龙血素B处理大鼠HSC;MTT法计算药物的抑制率;放射免疫法测定细胞上清中透明质酸(HA)、层粘连蛋白(LN)和Ⅳ型胶原(Ⅳ-C)。结果随药物浓度的升高,龙血素B对HSC-T6增殖的抑制作用逐渐增强,有明显的量效关系。细胞上清中HA、LN和Ⅳ-C的含量与对照组相比降低,其中龙血素B浓度为0.300μg/μL时降低最明显。结论龙血素B可抑制HSC-T6增殖,并不同程度抑制HA、LN和Ⅳ-C的分泌。     

Inhibition of hepatic stellate cells proliferation by mesenchymal stem cells and the possible mechanisms

Aim: During fibrosis, hepatic stellate cells (HSCs) undergo a complex activation process characterized by increased proliferation and extracellular matrix deposition. Previous studies have suggested that mesenchymal stem cells (MSCs) may ameliorate fibrogenesis and represent a promising strategy for cell therapy. However, the underlying mechanisms are not fully understood. Methods: Hepatic stellate cells were treated with or without MSCs. Then cell proliferation and cell cycle were analyzed. Production of soluble factors by MSCs and its relation with cell proliferation suppression was evaluated by transwell co‐culture and RNA interference. Effects of MSCs on the gene expression of collagen were also evaluated. Results: MSCs induced G₀/G₁ arrest of HSCs growth partly through secreting soluble factors TGF‐β3 and HGF, which resulted in up‐regulation of p21^Cip1 and p27^Kip1 expression and down‐regulation of cyclinD1. MSCs inhibited the phosphorylation of extracellular signal‐regulated kinase (ERK) 1/2 and reduced gene expression of collagen type I and III. MSCs did not reverse the proliferation and collagen type I gene expression of HSCs provoked by PDGF. Conclusions: The growth inhibition of HSCs induced by MSCs through an arrest in the G₀/G₁ phase of the cell cycle is partially mediated by secretion of TGF‐β3 and HGF. MSCs inhibit HSCs activation through decreasing phosphorylation of extracellular signal‐regulated kinase (ERK) 1/2. These results further support MSCs may be used as a novel therapy for treating fibrotic diseases in human.     

Contribution of hepatic stellate cells and matrix metalloproteinase 9 in acute liver failure

Background/Aims: Fulminant hepatitis or acute liver failure (ALF), initiated by viral infection or hepatic toxin, is a devastating medical complication without effective therapeutic treatment. In this study, we addressed the potential roles of hepatic stellate cells (HSCs) and their produced matrix metalloproteinases (MMPs) in development of ALF. Methods: Mice were given lipopolysaccharide (LPS) and beta‐galactosamine (GA) or carbon tetrachloride to create ALF and establish the association of IL‐1, MMP‐9, and caspase‐3 in acute liver failure. Results: In response to the hepatic toxin, IL‐1 and MMP‐9 were promptly induced within 1 hour, followed by caspase‐3 activation at 2 hours, and dehiscence of sinusoids at 4 hours, and consequent lethality. In contrast, MMP‐9 knockout mice were resistant to lethality and absent of caspase‐3 activation, demonstrating an MMP‐9‐dependent activation of caspase in vivo. Further, IL‐1‐receptor knockout mice were resistant to lethality in MMP‐9 dependent manner, indicating a causative relationship. Although many hepatic cells are capable to produce MMP‐9 in vitro, HSCs were demonstrated here as the major hepatic cells to express MMP‐9 in liver injury. To recapitulate the sinusoidal microenvironment we cultured primary HSCs in 3‐dimensional ECM. In response to IL‐1, massive MMP‐9 was produced by the 3D culture concomitantly with degradation of type‐IV collagen. Conclusions: Based on these evidences, we propose a novel model to highlight the initiation of acute liver failure: IL‐1‐induced MMPs by HSCs within the space of Disse and thereafter ECM degradation may provoke the collapse of sinusoids, leading parenchymal cell death and loss of liver functions.     

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.     

Effects of Fuzhenghuayu decoction on collagen synthesis of cultured hepatic stellate cells, hepatocytes and fibroblasts in rats

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Liver fibrogenesis and the role of hepatic stellate cells: New insights and prospects for therapy

Hepatic fibrosis is a wound-healing response to chronic liver injury, which if persistent leads to cirrhosis and liver failure. Exciting progress has been made in understanding the mechanisms of hepatic fibrosis. Major advances include: (i) characterization of the components of extracellular matrix (ECM) in normal and fibrotic liver; (ii) identification of hepatic stellate cells as the primary source of ECM in liver fibrosis; (iii) elucidation of key cytokines, their cellular sources, modes of regulation, and signalling pathways involved in liver fibrogenesis; (iv) characterization of key matrix proteases and their inhibitors; (v) identification of apoptotic mediators in stellate cells and exploration of their roles during the resolution of liver injury. These advances have helped delineate a more comprehensive picture of liver fibrosis in which the central event is the activation of stellate cells, a transformation from quiescent vitamin A-rich cells to proliferative, fibrogenic and contractile myofibroblasts. The progress in understanding fibrogenic mechanisms brings the development of effective therapies closer to reality. In the future, targeting of stellate cells and fibrogenic mediators will be a mainstay of antifibrotic therapy. Points of therapeutic intervention may include: (i) removing the injurious stimuli; (ii) suppressing hepatic inflammation; (iii) down-regulating stellate cell activation; and (iv) promoting matrix degradation. The future prospects for effective antifibrotic treatment are more promising than ever for the millions of patients with chronic liver disease worldwide.     

Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo

BACKGROUND: Following liver injury, hepatic stellate cells (HSC) transform into myofibroblast-like cells (activation) and are the major source of type I collagen and the potent collagenase inhibitors tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) in the fibrotic liver. The reproductive hormone relaxin has been reported to reduce collagen and TIMP-1 expression by dermal and lung fibroblasts and thus has potential antifibrotic activity in liver fibrosis. AIMS: To determine the effects of relaxin on activated HSC. METHODS: Following isolation, HSC were activated by culture on plastic and exposed to relaxin (1-100 ng/ml). Collagen deposition was determined by Sirius red dye binding and radiolabelled proline incorporation. Matrix metalloproteinase (MMP) and TIMP expression were assessed by zymography and northern analysis. Transforming growth factor beta1 (TGF-beta1) mRNA and protein levels were quantified by northern analysis and ELISA, respectively. RESULTS: Exposure of activated HSC to relaxin resulted in a concentration dependent decrease in both collagen synthesis and deposition. There was a parallel decrease in TIMP-1 and TIMP-2 secretion into the HSC conditioned media but no change in gelatinase expression was observed. Northern analysis demonstrated that primary HSC, continuously exposed to relaxin, had decreased TIMP-1 mRNA expression but unaltered type I collagen, collagenase (MMP-13), alpha smooth muscle actin, and TGF-beta1 mRNA expression. CONCLUSION: These data demonstrate that relaxin modulates effective collagen deposition by HSC, at least in part, due to changes in the pattern of matrix degradation.     

肝素对大鼠肝星状细胞生长、细胞外基质和基质金属蛋白酶基因表达的影响

目的 探讨肝素对ＨＳＣ生长、细胞外基质和基质金属蛋白酶基因表达的影响。方法 用肝素和不用肝素对活化的ＨＳＣ进行处理,以直接细胞计数和ＢｒｄＵ标记免疫细胞化学染色检测细胞的生长情况。用免疫细胞化学染色和细胞闰核酸分子杂交分别检测Ｉ、Ⅳ型前胶原蛋白、纤连蛋白和Ⅰ、Ⅳ型胶原、纤连蛋白、基质金属蛋白酶－２及膜型基质金属蛋白酶基因的表达,并用酶图法检测基质金属蛋白酶－２的活性。结果 肝素使血清所致的ＨＳＣ生     

Hepatic stellate cells and extracellular matrix in hepatocellular carcinoma: more complicated than ever

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third leading cause of cancer death. Recent epidemiological data indicate that the mortality rate of HCC will double over the next decades in the USA and Europe. Liver cancer progresses in a large percentage of cases during the clinical course of chronic fibro‐inflammatory liver diseases leading to cirrhosis. Therefore, HCC development is regarded as the result of different environmental risk factors each involving different genetic, epigenetic‐ and chromosomal alterations and gene mutations. During tumour progression, the malignant hepatocytes and the activated hepatic stellate cells are accompanied by cancer‐associated fibroblasts, myofibroblasts and immune cells generally called tumour stromal cells. This new and dynamic milieu further enhances the responsiveness of tumour cells towards soluble mediators secreted by tumour stromal cells, thus directly affecting the malignant hepatocytes. This results in altered molecular pathways with cell proliferation as the most important mechanism of liver cancer progression. Given this contextual complexity, it is of utmost importance to characterize the molecular pathogenesis of HCC, and to identify the dominant pathways/drivers and aberrant signalling pathways. This will allow an effective therapy for HCC that should combine strategies affecting both cancer and the tumour stromal cells. This review provides an overview of the recent challenges and issues regarding hepatic stellate cells, extracellular matrix dynamics, liver fibrosis/cirrhosis and therapy, tumour microenvironment and HCC.     

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。     

木犀草素抑制肝星状细胞增殖及其胶原合成

目的 研究木犀草素对体外培养的肝星状细胞(hepatic stellate cells,HSC)增殖及其胶原表达、合成的影响。方法 从Wistar大鼠肝脏分离培养HSC,并用~3H-TdR和~3H-pro同位素掺入实验,基因探针原位杂交等技术研究了木犀草素对HSC增殖、胶原基因表达合成的影响。结果 当木犀草素的浓度分别达到10 μmol/L和20 μmol/L 时抑制HSC增殖(t=2.542,P＜0.05)和胶原合成(t=3.650,P＜0.01),其作用具有剂量依赖关系;25 μmol/L木犀草素使Ⅰ、Ⅲ型前胶原mRNA的表达降低,其中Ⅰ型前胶原基因表达降低具有统计学差异(x~2=6.850,P＜0.01)。结论 木犀草素在体外抑制HSC增殖和胶原表达合成,在体内可能会具有预防或冶疗肝纤维化的作用。     

洛伐他丁对肝星状细胞增殖及细胞外基质分泌的影响

目的 观察洛伐他丁对肝星状细胞增殖及细胞外基质分泌的影响,并探讨其作用机制。方法 用不同浓度的洛伐他丁和胆固醇合成过程中产生的非脂性中间产物香叶基香叶基焦磷酸处理大鼠肝星状细胞株;用MTT法检测细胞增殖,流式细胞仪检测细胞周期,ELISA检测细胞外基质IV型胶原和层粘连蛋白,免疫细胞化学结合计算机图文分析系统检测c—jun、c-fos基因表达。结果 洛伐他丁可剂量依赖性地抑制肝星状细胞增殖（对照组A值24 h和48 h分别为0.736±0.090和0.972±0.097,洛伐他丁浓度在10μmol/L时24h和48h分别为0.602±0.049和0.785±0.028,两组比较差异有显著性）,影响其细胞周期,使G0/G1期细胞增多,S期细胞减少,并明显抑制c-jun、c—fos表达（洛伐他丁浓度在 50μmol/L时分别较对照组降低51.5％和 54.5％）,抑制IV型胶原和层粘连蛋白分泌（P<0.01）。而香叶基香叶基焦磷酸可部分拮抗洛伐他丁的上述抑制作用。结论 洛伐他丁可显著抑制肝星状细胞增殖及细胞外基质分泌,其机制可能与抑制香叶基香叶基焦磷酸产生而阻止信号转导有关。     

High glucose stimulates hepatic stellate cells to proliferate and to produce collagen through free radical production and activation of mitogen-activated protein kinase.

BACKGROUND: Nonalcoholic steatohepatitis is a clinicopathologic condition that may progress to liver fibrosis. Hyperglycemia is supposed to be one of the factors inducing hepatic fibrogenesis, but the mechanism has not been fully clarified. Oxidative stress is increasingly found in patients with diabetes/hyperglycemia in which conditions reactive oxygen species (ROS) are produced. METHODS: We performed experiments using hepatic stellate cells (HSCs) in culture in order to confirm the effect of high glucose concentrations on cell proliferation, type I collagen production, ROS production and activation of mitogen-activated protein (MAP) kinase pathway. RESULTS: High glucose stimulated cell growth of HSCs and up-regulated the levels of activated/phosphorylated extracellular signal-regulated kinase 1/2 and free radical production in HSCs. The MAP kinase phosphorylation and cell proliferation were suppressed by diphenylene iodonium chloride, an NADPH oxidase inhibitor, and by calphostin C, a protein kinase C (PKC)-specific inhibitor. Increased type I collagen mRNA and protein levels were also observed in HSCs at high glucose concentrations. CONCLUSIONS: Our findings indicate that high glucose concentrations may stimulate ROS production through PKC-dependent activation of NADPH oxidase, and induce MAP kinase phosphorylation subsequent to proliferation and type I collagen production by HSCs.     

Specific shRNA targeting of FAK influenced collagen metabolism in rat hepatic stellate cells

AIM:To investigate the effects and mechanism of disruption of focal adhesion kinase(FAK) expression on collagen metabolism in rat hepatic stellate cells(HSC).METHODS:The plasmids expressing FAK short hairpin RNA(shRNA) were transfected into HSC-T6 cells,and the level of FAK expression was determined by both real-time quantitative polymerase chain reaction(QPCR) and Western blotting analysis.The production of type collagen and type collagen in FAK-disrupted cells was analyzed by real-time Q-PCR.The level of ...