The tumor suppressor protein PTEN inhibits rat hepatic stellate cell activation

Background  Following a fibrogenic stimulus, the hepatic stellate cell (HSC) transforms from a quiescent to an activated cell type associated with increased proliferation, collagen and smooth muscle α-actin (αSMA) expression. Phosphatase and Tensin Homolog Deleted on Chromosome Ten (PTEN), a tumor suppressor phosphatase, has been shown to play a role in several nonmalignant diseases. Here, we investigated the role of PTEN during HSC activation. Methods  Rat HSCs 2 days after isolation were transduced with adenoviruses expressing either the wild-type (WT) or a dominant negative form of PTEN, and culture-associated activation of HSCs, including morphological changes, expression of αSMA and α1(I) collagen, and cell proliferation, were evaluated. Apoptosis of HSCs was detected by measuring activity of caspase 3/7. Phosphorylation status of Akt, p70^S6K, and Erk was detected by Western blotting. Results  Overexpression of WT-PTEN inhibited phenotypic changes were associated with HSC activation, including morphological changes, expression of αSMA and α1(I) collagen, and HSC proliferation, including cyclin D1 expression. WT-PTEN expression also induced apoptosis in HSCs with increased caspase 3/7 activity. Expression of WT-PTEN also caused decreased activation of Akt, p70^S6K, and Erk signaling pathways. Conclusions  Taken together, these findings show that PTEN represents an important negative regulator for transactivation of HSCs. This may have important implications for the design of therapeutic strategies to prevent the progression of liver fibrosis.     

Effect of protein kinase C activation and inhibition on rat hepatic stellate cell activation

Protein kinase C (PKC) may play a role in the intracellular signaling pathways responsible for transforming hepatic stellate cells into myofibroblasts. This study examined the effects of inhibitors and activators of PKC on hepatic stellate cell activation. Stellate cells isolated from normal rats were incubated with either 10^–5 M chelerythrine, 10^–7 M bisindolylmaleimide I hydrochloride (BIM), or 10^–6 M staurosporine (PKC inhibitors), or 10^–7 M phorbol myristate acetate (PMA) or 10^–6 M thymeleatoxin (PKC activators). Chelerythrine suppressed -smooth muscle actin expression and proliferation by 49% and 33%, respectively. BIM inhibited -smooth muscle actin expression by 60%, but had no significant effect on proliferation. Staurosporine decreased proliferation by 86% and completely prevented -smooth muscle actin expression. PKC activators had divergent effects on proliferation and -smooth muscle actin expression. PMA and thymeleatoxin caused a 2.8- to 3.2-fold increase in proliferation, while suppressing -smooth muscle actin expression by 50–70%. The demonstration that hepatic stellate cell activation can be suppressed by PKC inhibitors suggests a role for PKC in the regulation of hepatic stellate cell activation.     

Effects of lipopolysaccharides stimulated Kupffer cells on activation of rat hepatic stellate cells

AIM:To study the effects of Kupffer cell-conditioned medium (KCCM) derived from lipopolysaccharide (LPS) treatment on proliferation of rat hepatic stellate cells (HSC).METHODS:HSC and Kupffer cells were isolated from the liver of Wistar rats by in situ perfusion with pronase and collagenase and density gradient centrifugation with Nycodenz and cultured. KCCM was prepared and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to detect HSC proliferation. The content of type Ⅳ collagen and laminin secreted by HSC in the HSC-conditioned medium was determined by radioimmunoassay.TGF-β1 production in the KCCM was detected by enzymelinked immunosorbent assay (ELISA).RESULTS:HSC and Kupffer cells isolated had high purity.One microgram per mililiter LPS-activated KCCM and unstimulated KCCM could significantly promote HSC proliferation [0.132±0.005 and 0.123±0.008 vscontrol group (0.100±0.003), P<0.01], and there was a difference between them (P<0.05). Ten microgram per mililiter LPS-activated KCCM (0.106±0.010) was unable to promote HSC proliferation (P>0.05). Adding anti-TGF-β1 antibodies could suppress the proliferation promoted by unstimulated KCCM and LPS(1μg/ml)-activated KCCM (0.109±0.009 vs 0.123±0.008,0.115±0.008 vs 0.132±0.005, P<0.01). LPS (1μg/ml or 10μg/ml) could not promote HSC proliferation immediately (0.096±0.003 and 0.101±0.004 vs 0.100±0.003, P>0.05). There was a parallel behavior between HSC proliferation and increased ECM level. One microgram per mililiter LPS-activated KCCM contained a larger amount of TGF-β1 than unstimulated KCCM.CONCLUSION:The technique for isolation of HSC and Kupffer cells described here is simple and reliable. KCCM stimulated by LPS may promote HSC proliferation and collagen accumulation, which are associated with hepatic fibrogenesis.     

去甲肾上腺素对大鼠肝星状细胞作用的实验研究

目的 探讨交感神经系统在肝纤维化发生和发展中的作用. 方法采用免疫荧光和RT-PCR检测体外培养的肝星状细胞(HSC)中α1、β2-肾上腺素能受体的表达;用四甲基偶氮唑盐法检测不同浓度的去甲肾上腺素(NE)对HSC增殖活性的影响.同时用RT-PCR检测受NE作用后HSC的活化指标胶原蛋白-1、转化生长因子β(TGF β)及α-平滑肌动蛋白(α-SMA)的表达.用高效液相色谱-电化学法测定活化的HSC中交感神经递质NE的水平. 结果α1和β2-肾上腺素能受体表达于HSC的胞膜和胞质内;NE可呈剂量依赖性地促进HSC增殖,在浓度为100μmol/L时达到最大效应,F=140.464,P<0.05,差异有统计学意义.以NE 100 μmol/L作用细胞24h后,可显著促进反应HSC活化的指标上升,胶原蛋白-1表达为0.3022±0.0610,TGF β表达为2.2080±0.2151,α-SMA mRNA表达为0.5469±0.0108,与对照组胶原蛋白-1(0.1040±0.0556)、TGF β(1.1190±0.0070)、α-SMA mRNA表达(0.0759±0.0449)比较,t值分别为-4.160、-8.763和-17.651,P值均<0.05,差异均有统计学意义.HSC可以合成并释放NE,且受血小板衍生生长因子(10ng/ml)刺激后HSC中NE含量为(14.24±0.21)ng/ml,对照组为(11.34±0.15)ng/ml,两组比较,t=-32.907,P<0.05,差异有统计意义.结论 抑制交感神经系统使HSC活性降低对临床上治疗肝纤维化有一定的指导意义.     

一种可靠的大鼠肝星状细胞分离及培养方法

目的探讨一种高效、经济、稳定的肝星状细胞的分离方法,为肝纤维化研究提供细胞模型。方法应用链霉蛋白酶、胶原酶灌流,以Nycodenz为分离介质,采用密度梯度离心法分离大鼠的肝星状细胞。结果肝星状细胞的获得率为(3.2±0.67)×10~7/只、存活率为90%以上。结论该法是一种较理想的分离肝星状细胞的方法。     

整合素与肝星状细胞活化

整合素(integrin)参与细胞和细胞外基质(ECM)、细胞和细胞间的黏附,介导与细胞活化、增殖、运动和凋亡等有关的信号,在免疫调节、肿瘤浸润、损伤修复中起重要作用。慢性肝损伤中静止的肝星状细胞(HSC)活化、增殖、表型转变,ECM异常沉积,星状细胞整合素表达水平和受体亲和力改变,并与生长因子协同作用参与肝纤维化的分子机制。一、整合素家族(一)整合素分子结构整合素(或整合素受体)是由α、β两个亚基非共价结合成的异源二聚体细胞表面糖蛋白。目前已发现8种类型的β亚基和18种α亚基,装配成至少24种不同的亚型受体,分布有选择性和时序…     

Altered activation of rat hepatic cyclic AMP-dependent protein kinases with increasing age

The activity ratio of hepatic cyclic AMP-dependent protein kinase has been found to change with increasing age in male, Long-Evans rats. In rats 35 days old (135 g), the activity ratio (cyclic AMP-dependent protein kinase activity measured in the absence and presence of exogenous cyclic AMP) was 0.76, it increased to 0.92 in rats 75 days old (315 g) and to 0.98 in rats 305 days old (646 g). This change in the activity ratio seems to correlate with the ability of aging animals to respond to a stimulus, and may be a primary mechanism in the biochemistry of aging.     

Effects of the tyrosine protein kinase inhibitor genistein on the proliferation,activation of cultured rat hepatic stellate cells

AIM：Hepatic stellate cell(HSC)plays a pivotal role in liver fibrosis and is considered as the therapeutic target for the treatment of hepatic fibrosis,Tyrosine protein kinase plays an important role in the proliferation,activation of HSC.The purpose of the study is to investigate the effects of the tyosine protein kinase inhibitor genistein on the proliferation and activation of cultured rat HSC.METHODS:Rat HSC were isolated from Wistar rats by in situ perfusion of collagenase and pronase and single-step density Nycodenz gradient,Culture-activated HSC were serum-starved and incubated with10^-9to10^-5mol/L concentration of genistein for 24,48or 72h,In PDGF-induced HSC proliferation,HSC were stimulated with10μg&#183;L^-1PDGF-BBfo15min,and thentreated with genistein for the same time.Cell proliferation was measured by MПassay and based on flow cytometric analysis of cell cycle.The a-smooth muscle actin(α-SMA）expression in HSC was studied with confocallaser microscopy and flow cytometry.c-fos,c-jun and cyclinD1expression in HSCwas also detected by flow cytometry.RESULTS:Genistein inhibited basal and PDGF-induced proliferation of HSCat the concentration of 10^-8to10^-5mol/L,and treatment with10^-7mol/L concentration of genistein for 48h inhibited the HSCproliferation significantly(the inhibition rate was 70.3%,P&lt;0.05).Immunofluorescence detected by confocal laser microscopy and flow cytometry showed that treatment with10^-7mol/L genistein for48h suppressed the expression of α-SMA significantly in HSC(the specific fluorescence intensity were60.2&#177;21.5vs35.3&#177;11.6and12.8&#177;10.4vs9.54&#177;6.39,respectively,bothP&lt;0.05).The intensity of c-fos,c-jun and cyclinD1 expression of HSCs treated with 10^-7mol/L genistein for 48h was also significantly decreased compared with the controls.CONCLUSION;Genistein influences proliferation of HSC,suppresses the expression of α-SMA in HSC and tinhibits the intensity of c-fos,c-jun and cyclinD1 expression of HCSs,Genistein has therapeutic potential against liver fibrosis.     

肝星状细胞激活的研究进展

肝纤维化(HF)是各种慢性肝病共有的病理改变,其特点是以胶原为主的细胞外基质(ECM)在肝内过度沉积。肝星状细胞(HSC)激活、增殖、迁移、合成和分泌大量ECM是HF形成和发展的中心环节与细胞学基础。在此过程中,许多细胞因子(CK)、氧化应激产物、ECM组构的广泛改变、化学分子、细胞周期调控因子以及核转录因子(NF)参与HSC的激活。     

Notch3 regulates the activation of hepatic stellate cells

AIM: To investigate whether Notch signaling is involved in liver fibrosis by regulating the activation of hepatic stellate cells (HSCs).METHODS: Immunohistochemistry was used to detect the expression of Notch3 in fibrotic liver tissues of patients with chronic active hepatitis. The expression of Notch3 in HSC-T6 cells treated or not with transforming growth factor (TGF)-β1 was analyzed by immunofluorescence staining. The expression of Notch3 and myofibroblastic marker α-smooth muscle actin (α-SMA) and collagen I in HSC-T6 cells transfected with pcDNA3.1-N3ICD or control vector were detected by Western blotting and immunofluorescence staining. Moreover, effects of Notch3 knockdown in HSC-T6 by Notch3 siRNA were investigated by Western blotting and immunofluorescence staining.RESULTS: The expression of Notch3 was significantly up-regulated in fibrotic liver tissues of patients with chronic active hepatitis, but not detected in normal liver tissues. Active Notch signaling was found in HSC-T6 cells. TGF-β1 treatment led to up-regulation of Notch3 expression in HSC-T6 cells, and over-expression of Notch3 increased the expression of α-SMA and collagen I in HSC-T6 without TGF-β1 treatment. Interestingly, transient knockdown of Notch3 decreased the expression of myofibroblastic marker and antagonized TGF-β1-induced expression of α-SMA and collagen I in HSC-T6.CONCLUSION: Notch3 may regulate the activation of HSCs, and the selective interruption of Notch3 may provide an anti-fibrotic strategy in hepatic fibrosis.     

辛伐他汀抑制肝星状细胞活化及其对腺苷酸活化蛋白激酶活性的影响

目的 研究辛伐他汀治疗非酒精性脂肪性肝纤维化的作用机制.方法 高脂饲料喂养大鼠复制非酒精性脂肪性肝纤维化模型,辛伐他汀灌胃治疗后,病理学染色观察肝组织病理改变,RT-PCR和Western blot法测定肝组织腺苷酸活化蛋白激酶(AMPK)α的mRNA和蛋白质表达水平,同时测定血清胆固醇(TC)、甘油三酯(TG)、ALT、AST和血清肿瘤坏死因子(TNF)α水平.体外用促进脂肪细胞分化的培养液诱导人肝星状细胞株LX-2获得静止表型,分别用转化生长因子β1(TGFβ1)、辛伐他汀、TGFβ1+辛伐他汀处理静止型LX-2细胞,RT-PCR和Western blot法检测AMPKα的mRNA及蛋白质表达变化.多组间数据比较用单因素方差分析或析因设计方差分析,两组间数据比较用q检验. 结果 高脂饮食24周成功复制大鼠非酒精性脂肪性肝纤维化模型.随造模时间的延长,肝组织炎症、脂肪变和纤维化程度加重,血清TC、TG、ALT、AST和TNFα水平逐渐升高(P值均＜0.01),肝组织AMPKα的mRNA相对表达量及活性逐渐降低(P＜0.01或P＜0.05).与模型对照组相比,辛伐他汀治疗组TC、TG、ALT、AST和TNF α[水平明显下降(P值均＜0.05),AMPKα mRNA(0.31±0.05比0.24±0.07)和AMPK α[活性(0.45±0.07比0.27±0.07)明显升高(q值分别为3.22和6.44,P＜0.01或P＜0.05);肝星状细胞的AMPKα活性明显增强(静止型为0.93±0.10比0.72±0.09,活化型为0.72±0.10比0.54±0.10,q值分别为7.00和6.00,P值均＜0.01),α-平滑肌动蛋白的mRNA和蛋白质表达明显减少(分别为0.30±0.02比0.36±0.02和0.30±0.03比0.38±0.02,q值分别为11.245和11.216,P值均＜0.01),I型胶原的mRNA和和蛋白质表达也明显减少(分别为0.30±0.03比0.37±0.03和0.25±0.03比0.33±0.03,q值分别为8.791和11.163,P值均＜0.01).结论 辛伐他汀可以通过提高AMPK活性,抑制肝星状细胞活化,延缓非酒精性脂肪性肝纤维化的发生和发展.     

大黄蔗虫丸对大鼠肝星状细胞活化及增殖的影响

目的观察大黄蔗虫丸对大鼠肝星状细胞活化及增殖的影响。方法用链酶蛋白酶和胶原酶原位灌注消化正常大鼠肝脏,Nycodenz密度梯度离心分离肝星状细胞(HSC),在制备大黄蔗虫丸大鼠药物血清的基础上,温育HSC。MTT比色法测定细胞增殖,流式细胞仪检测细胞凋亡及增殖周期情况。结果药物血清对体外培养的HSC有抑制作用,且存在浓度剂量依赖关系,但作用较弱,需在20%以上方见到显著性差异(P<0.05),对HSC凋亡及细胞增殖周期则无显著影响(P>0.05)。结论大黄蔗虫丸对HSC增殖有一定抑制作用,但可能不是其抗肝纤维化作用的主要途径,其抗肝纤维化作用与诱导细胞凋亡无关。     

雌二醇对大鼠体外培养肝星状细胞影响作用的实验研究

17β—雌二醇(E2)可能在肝纤维化、肝硬化发展过程中发挥作用。现探讨E2在肝纤维化形成中的作用及其机制。     

Role of hepatic stellate cell/hepatocyte interaction and activation of hepatic stellate cells in the early phase of liver regeneration in the rat

BACKGROUND/AIMS: Hepatic stellate cells (HSCs) are known to play a role in hepatic regeneration. We investigated hepatocyte/HSC interaction and HSC activation at various times after 70% partial hepatectomy (PHx) in the rat. METHODS: The hepatic microcirculation was studied using intravital fluorescence microscopy (IVFM). Desmin and alpha-SMA within liver tissue were detected by immunohistochemistry. In isolated parenchymal liver cells (PLCs) and HSCs, double immunostaining was used to identify activated HSC. RESULTS: Using IVFM, hepatocyte-clusters were often seen in vivo at 3 days after PHx (PHx3). Distance between HSC fell from 61.7+/-2.1 microm in controls to 36.1+/-1.4 microm (P<0.001) while the HSC/hepatocyte ratio rose (0.71+/-0.01 to 1.08+/-0.03; P<0.001). In >80% of in vivo microscopic fields in the PHx3 group, clusters of HSCs were observed especially near hepatocyte-clusters. At PHx1 and PHx3, >20% of cells in the PLC-fraction were HSCs which adhered to hepatocytes. At PHx3, in addition to desmin staining, isolated HSCs were also positive for BrdU and alpha-SMA, and formed clusters. HSCs in the HSC-fraction were only positive for desmin which indicated that adherence to hepatocytes is required for HSC activation. CONCLUSIONS: Our data suggest that HSCs are activated by adhering to hepatocytes in the early phase of liver regeneration.     

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.     

肝星状细胞激活与信号转导

肝纤维化是多种慢性肝病向肝硬化发展的必经阶段,是所有慢性肝病的共同病理基础.目前认为肝星状细胞的激活是肝纤维化形成的关键,各种致病因素作用下引起肝损伤,释放多种细胞因子,如转化生长因子β、血管紧张素、瘦素,通过各种信号转导使肝星状细胞激活.本文就肝星状细胞激活过程中一些重要的膜受体、核受体信号转导途径及其研究进展作一综述.     

肝星状细胞活化过程中的信号转导

肝星状细胞(HSC)是肝脏的主要纤维生成细胞,其活化增殖,进而产生大量细胞外基质,是肝纤维化形成的中心环节。随着对肝纤维化的深入研究,HSC活化过程中所涉及的信号转导已成为研究热点。归纳了HSC活化过程中所涉及的主要信号转导,并介绍了近年来的相关研究进展。相信随着对这些信号转导的深入研究,肝纤维化的防治必将取得新突破。