Inhibitory effects of prostaglandin E_1 on activation of hepatic stellate cells in rabbits with schistosomiasis

BACKGROUND: Liver fibrosis is the result of an imbalance between synthesis and degradation of extracellular matrix proteins of the liver. At the cellular and molecular levels, this progressive process is mainly characterized by activation of hepatic stellate cells (HSCs). Schistosoma japonica is one of the most prevalent causes of liver fibrosis in China. It is characterized by hepatocyte damage, inflammation, and chronic parasite egg-induced granuloma formation leading to fibrosis. This study aimed to investigate the inhibitory effects of prostaglandin E1 (PGE1) on activation of HSCs and the alteration of type Ⅰ and Ⅲ collagen in rabbits with schistosomiasis. The study may promote the clinical application of praziquantel and PGE1 as a combined therapy to reverse hepatic fibrosis caused by schistosomiasis. METHODS: Rabbits were percutaneously infected with cercaria of S. japonicum. Seven rabbits were subjected to intravenous injections of PGE1 (2.5 μg/kg daily) from days 60 to 120 after infection. The ultrastructural changes in activated HSCs were observed under transmission electron microscopy. The expression of α-smooth muscle actin (α-SMA) was detected by immunohistochemistry. Fibril-forming collagens were detected by picrosirius staining. RESULTS: Activation of HSCs was a characteristic alteration in schistosome-induced hepatic fibrosis. The expression of contraction-related α-SMA and thecontent of collagens were increased. Exogenous PGE1 markedly inhibited the activation of HSCs and reduced the expression of α-SMA around the hepatic sinusoids (P<0.01). The contents of type Ⅰ and Ⅲ collagens were significantly attenuated. The ratio of staining area to the whole field (10×3.3) under a polarized light microscope in the untreated and treated groups was 37.25±9.71 vs. 13.38±4.24 (P<0.01) and 9.66±3.52 vs. 6.23±1.81 (P<0.05), respectively. CONCLUSIONS: Activation of HSCs may play a key role in the progress of schistosome-induced hepatic fibrosis. PGE1 effectively protects rabbit liver from fibrosis, at least in part by inhibiting the activation of HSCs.     

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.     

Effect of IL-4 and IL-13 on collagen production in cultured LI90 human hepatic stellate cells.

BACKGROUND: Recently, it has been reported that interleukin 4 (IL-4) and 13 (IL-13) directly activate fibroblasts and promote fibrosis. In the process of hepatic fibrosis, the effects of these cytokines on hepatic stellate cells (HSCs) are not well known. METHODS: We evaluated the effects of IL-4 and IL-13 on the collagen production and the proliferation of LI90, a hepatic stellate cell line. We also examined whether interferon (IFN) interferes with the expression of collagen, since IFN has been reported to clinically suppress hepatic fibrosis. RESULTS: The receptor complex for IL-4 and IL-13 was IL-4Ralpha/IL-13Ralpha1 on LI90 cells, and the phosphorylation of Stat6 was induced by IL-4 and IL-13. The treatment of LI90 cells with IL-4 or IL-13 increased the production of collagen I protein levels by nearly three times in comparison with untreated cells. Collagen mRNA levels were increased roughly 10-fold by IL-4 and 100-fold by IL-13. Interestingly, BrdU incorporation in LI90 cells was decreased by IL-4 or IL-13 treatment. Furthermore, induction of collagen I production by these cytokines was blocked by IFNalpha or IFNbeta treatment, although neither treatment alone suppressed collagen production. CONCLUSIONS: Our data suggested that IL-4 and IL-13 directly affected HSCs by increasing collagen production and suppressing cell proliferation. The anti-fibrogenetic effect of IFN may be due in part to the blockade of IL-4 and IL-13 stimulation of HSCs.     

Angiotensin II stimulates the synthesis of nucleic acid,protein and collagen of cultured hepatic stellate cells in rats

OBJECTIVE: To investigate the effects of different doses of angiotensin II (AngII) on the proliferation of hepatic stellate cells (HSC) and collagen synthesis in rats. METHODS: Pronase E and collagen I were used to isolate the HSC, and ³H‐TdR,³H‐Leu and ³H‐pro incorporation methods were used to evaluate the effects of the different doses of AngII on HSC DNA, protein and proline synthesis. RESULTS: It was found that 10^‐9?10^?6 mol/L AngII could increase the ³H‐TdR incorporation rate of the HSC (P < 0.05), but only 10^?6 mol/L and 10^?7 mol/L AngII could significantly increase the ³H‐Leu incorporation rate of the HSC (P < 0.01); 10^?9?10^?6 mol/L AngII caused a significant increment of the ³H‐pro incorporation rate of the HSC in a dose‐dependent manner. CONCLUSIONS: The appropriate dose of AngII may promote HSC proliferation and collagen synthesis.     

Inhibition of hepatic stellate cell activation following Yinchenhao decoction administration to dimethylnitrosamine-treated rats

Aim: In an effort to investigate the mechanism by which Yinchenhao decoction (YCHD) acts on liver injury, we investigated the potential antifibrogenic effects of YCHD in an experimental liver fibrosis rat model, with special focus on the mechanisms inhibiting the activation and promoting apoptosis of hepatic stellate cells (HSC). Methods: The rats were initially randomized into two groups: the control (n = 10) and dimethylnitrosamine‐treated (DMN; n = 30) groups. DMN (10 mg/kg body weight) was administered intraperitoneally to the DMN‐treated rats for three consecutive days each week. At the end of the second week, three rats from the control and six rats from the DMN‐treated groups were killed for the fibrosis development assessment. The remaining DMN rats were further randomized into two groups: the DMN–water group (n = 12) and the DMN–YCHD group (n = 12). Both groups continued to receive weekly DMN treatment for another 2 weeks in addition to daily administration of either water or YCHD, which were given intragastrically at a dose of 0.418 g/100 g body weight. Results: Hepatic hydroxyproline content decreased and had improved histopathology in the DMN–YCHD rats. Compared to the DMN group, α‐smooth muscle actin (SMA) and CD68 expression in the DMN–YCHD group was reduced significantly; however, α‐SMA‐positive HSC apoptosis was not observed by confocal microscopy; Fibrogenic proteins (tissue inhibitor matrix proteinases‐1 and 2 and matrix metalloproteinase [MMP]‐2/14) and cytokines (tumor necrosis factor‐α and transforming growth factor‐β₁) were decreased; MMP‐9 was significantly upregulated. Conclusion: Yinchenhao administration attenuates liver fibrosis at least in part by inhibiting HSC activation directly, rather than promoting cell apoptosis of activated HSC, and the suppressive activation of Kupffer cells.     

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.     

Molecular mechanism of hepatic stellate cell activation and antifibrotic therapeutic strategies

Activation of hepatic stellate cells (HSCs) is the dominant event in liver fibrosis. The early events in the organization of HSC activation have been termed initiation. Initiation encompasses rapid changes in gene expression and phenotype that render the cells responsive to cytokines and other local stimuli. Cellular responses following initiation are termed perpetuation, which encompasses those cellular events that amplify the activated phenotype through enhanced growth factor expression and responsiveness. Multiple cells and cytokines play a part in the regulation of HSC activation. HSC activation consists of discrete phenotype responses, mainly proliferation, contractility, fibrogenesis, matrix degradation, chemotaxis and retinoid loss. Currently, antifibrotic therapeutic strategies include inhibition of HSC proliferation or stimulation of HSC apoptosis, downregulation of collagen production or promotion of its degradation, administration of cytokines, and infusion of mesenchymal stem cells. In this review, we summarize the latest advances in our understanding of the mechanisms of HSC activation and possible antifibrotic therapeutic strategies.     

大麻素受体2介导的N-花生四烯酸氨基乙醇对肝星状细胞增殖和活化的影响

目的 观察内源性大麻素N-花生四烯酸氨基乙醇(AEA)及大麻素受体(CBR)2对肝星状细胞(HSC)增殖活化的影响,以探讨内源性大麻素及其受体系统在肝纤维化发展中的作用.方法 采用免疫荧光观察血小板衍生生长因子(PDGF)刺激前后HSC中CBR1和CBR2的表达.Western blot、PCR法观察不同浓度AEA及CBR2拮抗剂AM630对PDGF刺激下HSC增殖及活化的影响,同时用四甲基偶氮唑盐、流式细胞仪分析AEA对HSC活力及凋亡的影响.结果 HSC中CBR2的表达较CBR1高(F=116.797,P<0.01),且PDGF刺激后CBR2的表达明显增强(F=7.878,P<0.05).AEA可剂量依赖地抑制HSC的增殖,在浓度为10,20、50μmol/L时抑制率分别为7.12%±0.34%、12.52%±0.78%、80.13%±1.57%,差异有统计学意义(F=533.41,P<0.01);但对HSC凋亡的影响不明显.同时AEA可抑制HSC的活化指标α-平滑肌肌动蛋白、转化生长因子β1、Ⅰ型胶原、Ⅲ型胶原及基质金属蛋白酶抑制因子等的表达,但这种抑制作用在给予CBR2拮抗剂AM630后明显减弱,差异有统计学意义(P<0.05).结论 CBR2在AEA引起的HSC增殖及活化抑制中起关键作用,AEA和CBR2可望成为肝纤维治疗的新靶点.     

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

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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。     

肝星状细胞在逆转肝纤维化中的作用

肝纤维化是一种能够导致门静脉高压、肝硬化、肝衰竭的严重疾病。已经发现肝星状细胞的活化是引起肝纤维化的中心环节,因此抑制肝星状细胞的活化、加速肝星状细胞的清除有望逆转肝纤维化。本文将对活化的肝星状细胞的凋亡、衰老以及清除的研究进展作综述,阐明肝星状细胞在肝纤维化过程中所起的作用及相关机制。     

MicroRNAs在肝星状细胞活化过程中表达差异谱的研究

目的 应用MicroRNAs基因芯片技术筛选静止和活化肝星状细胞差异表达的MicroRNAs.方法 用链酶蛋白酶和胶原酶原位灌流,Nycodenz密度梯度离心分离大鼠HSCs,并进行体外培养,借助荧光倒置显微镜观察细胞形态,MicroRNAs基因芯片技术和荧光定量PCR检验MicroRNAs在HSCs静止期(2天)和活化期(14天)的表达.结果 HSCs的miRNAs表达谱中差异表达miRNAs共21个,其中上调12个,下调9个(P＜0.01);荧光定量RT-PCR证实其表达水平在静止和活化期肝星状细胞中均有差异(P＜0.01).结论 MicroRNAs基因芯片筛选差异表达miRNAs可望为肝纤维化的基因治疗提供全新的靶标和策略,为肝纤维化的发病机制和诊断治疗研究提供了新的思路.     

Advanced glycation end products enhance the proliferation and activation of hepatic stellate cells

Background Advanced glycation end products (AGEs), final reaction products of protein with sugars, are known to contribute to diabetes-related complications. We have recently demonstrated high levels of serum AGEs in patients with nonalcoholic steatohepatitis (NASH). However, direct evidence for the participation of AGEs in hepatic infl ammation and fibrosis has not been shown. To explore the pathogenesis of NASH, we examined the biological infl uence of AGEs on hepatic stellate cells (HSCs) in vitro. Methods An established human HSC line, LI90, was exposed to a glyceraldehyde-derived-AGE (glycer-AGE), and the phenotypical changes of the LI90 cells were investigated. Intracellular formation of reactive oxygen species (ROS) was measured using a fl uorescent probe. Cell proliferation was examined by MTS assay. Fibrogenic marker gene expression was analyzed by quantitative real-time polymerase chain reaction. The production of monocyte chemoattractant protein 1 (MCP-1) was assessed by enzyme-linked immunosorbent assay. Results The expression of AGE receptor was confirmed in LI90 cells at the mRNA and protein levels. In addition to increasing intracellular ROS generation, glycer-AGE upregulated fibrogenic genes such as those encoding for α-smooth muscle actin, transforming growth factor-β1, and collagen type Iα2. The expression of MCP-1 mRNA in LI90 cells as well as its secretion into the culture medium was significantly increased in response to AGEs. These changes were attenuated by treatment with the antioxidant N-acetylcysteine. Conclusions These data indicate that AGEs induce ROS generation and intensify the proliferation and activation of HSCs, supporting the possibility that antioxidants may represent a promising treatment for prevention of the development of hepatic fibrosis in NASH.     

Fenretinide stimulates the apoptosis of hepatic stellate cells and ameliorates hepatic fibrosis in mice

Aim: To investigate whether fenretinide, a clinically proved apoptosis‐inducing chemopreventive agent in tumor cells, can induce apoptosis in hepatic stellate cells (HSCs) and resolve hepatic fibrosis. Methods: CCl₄‐induced liver fibrosis in mice and rat activated hepatic stellate cells (HSC‐T6) as well as hepatocytes (BRL‐3A) were studied. Results: The duplex staining of proliferating cell nuclear antigen and α‐ smooth muscle actin or terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labeling and α‐ smooth muscle actin demonstrated that fenretinide executed its anti‐fibrosis effect in liver by inducing apoptosis rather than inhibiting proliferation of HSCs, while it had no apparently apoptotic effect on hepatocytes. Fenretinide could elicit apoptosis of HSC‐T6 in vitro at the concentration range from 0.5 to 5 µM, but at higher concentrations ≥5 µM was required to induce apoptosis in hepatocytes (BRL‐3A). Conclusion: Further studies using malondialdehyde measurement, Western blot, antioxidant, inhibitors for p53, caspase 8 and 9 – as well as anti‐Fas neutralizing antibody – have shown that in HSC‐T6, fenretinide‐induced apoptosis involves a reactive oxygen species (ROS)‐generated, P53‐independent, mitochondria‐associated intrinsic pathway, whereas in hepatocytes (BRL‐3A), a ROS‐generated, P53‐dependent, Fas‐related extrinsic pathway is triggered only at high concentration.     

Somatostatin inhibits pro-inflammatory cytokine secretion from rat hepatic stellate cells.

BACKGROUND/AIMS: Activated hepatic stellate cells (HSCs) have been implicated in hepatic fibrosis. Somatostatin (SOM) has an immunomodulatory role. The aim of this study was to assess the secretion of pro-inflammatory cytokines by HSCs and to determine the effect of SOM on the secretion of these mediators. METHODS: Activated rat HSCs were evaluated for their secretion of IL-1beta, IL-8, and TNF-alpha using ELISA. RNA protection assay was used to determine cytokine mRNA levels. The expression of chemokine and cytokine mRNA and the secretion of these mediators were assessed following incubation with SOM or octreotide. RESULTS: HSCs spontaneously secreted IL-1beta, IL-8, and TNF-alpha. This secretion was augmented following stimulation by IL-1beta and TNF-alpha. SOM inhibited the spontaneous and TNF-alpha-induced secretion of IL-1beta, IL-8, and TNF-alpha and suppressed the expression of IL-1beta and TNF-alpha mRNA. Octreotide suppressed the secretion of IL-1beta and IL-8. CONCLUSIONS: These observations indicate that SOM exerts an inhibitory immunomodulatory effect on HSCs.