罗格列酮抑制体外大鼠肝星状细胞活化

目的探讨罗格列酮（PPARγ配体）对肝星状细胞（HSCs）作用的机制。方法将原代HSCs随机分为3组：对照组;TGF-β1（5μg/L）组;TGF-β1加10μmol/L罗格列酮组。加药后48h用RT-PCR法检测细胞Ⅰ型前胶原的表达。用Western blot方法检测细胞SMAD3、SMAD4、SMAD7、α-平滑肌肌动蛋白（α-SMA）和Ⅰ型胶原的表达。免疫荧光化学标记,共聚焦显微镜下观察α-SMA的表达。用四甲基偶氮唑盐（MTT）法检测细胞的增殖。结果TGF-β1明显促进HSCs的增殖,促进HSCs表达Ⅰ型胶原和α-SMA（P〈0.01）;罗格列酮显著降低TGF-β1的作用（P〈0.01）。各组细胞SMAD3、SMAD4、SMAD7的表达无明显差异。结论PPARγ配体可以抑制TGF-β1对HSCs的活化作用。     

Linagliptin alleviates hepatic steatosis and inflammation in a mouse model of non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is a primary cause of cirrhosis and hepatocellular carcinoma. Dipeptidyl peptidase (DPP)-4 inhibitors are established therapies for type 2 diabetes and although DPP-4 inhibitors can reduce hepatic steatosis, their impact on local inflammation and fibrosis in NASH remains unknown. Using two different experimental treatment regimens (4- and 2-week treatments) in streptozotocin-treated neonatal mice on a high-fat diet, we show that the DPP-4 inhibitor linagliptin (10 and 30 mg/kg) significantly attenuated the NAS score from 4.9 ± 0.6 to 3.7 ± 0.4 and 3.6 ± 0.3, respectively, in the 4-week study. In the 2-week study, linagliptin 10 mg/kg significantly reduced NAS score from 4.1 ± 0.4 to 2.4 ± 0.4. Telmisartan was used as a positive control in both studies and lowered NAS score to 1.9 ± 0.7 and 1.4 ± 0.3, respectively. Due to streptozotocin treatment, elevated glucose levels were unchanged by either drug treatment. Further, linagliptin 10 mg/kg significantly reduced mRNA levels of SOCS-3 (from 1.68 ± 0.2 to 0.83 ± 0.08), IFN-γ (from 4.0 ± 0.5 to 2.3 ± 0.3), and TNF-α (from 5.7 ± 0.5 to 2.13 ± 0.3). The latter observation was confirmed by immunohistochemistry of TNF-α in liver specimens. In addition, using microautoradiography, we showed that the distribution of radiolabeled linagliptin was heterogeneous with the highest density associated with interlobular bile ducts and portal tracts (acini). In conclusion, these studies confirm that linagliptin has high exposure in hepatic tissue and has both anti-inflammatory and anti-steatotic activity in NASH.     

Hepatic stellate cell activation in nonalcoholic steatohepatitis and fatty liver

Factors associated with the development of fibrosis in nonalcoholic steatohepatitis (NASH) are largely unknown, although an association with increased hepatic iron has been suggested. Hepatic stellate cells are the principal collagen-producing cells in many liver diseases and when activated express alpha-smooth muscle actin (alpha-SMA). Hepatic stellate cell activation and association with fibrosis, necroinflammatory activity, steatosis, and stainable iron in 60 cases of NASH and 16 cases of steatosis were evaluated. All 76 patients were obese or had other risk factors for NASH. All biopsy specimens were stained for alpha-smooth muscle actin to evaluate the pattern of hepatic stellate cell activation and were evaluated for inflammatory activity (0 to 3), fibrosis (0 to 4), and stainable iron stores (0 to 4). The zonal location of activated stellate cells was recorded, and the degree of activation was graded as high-grade or low-grade based on the percentage of lobular alpha-SMA+ cells. Activated stellate cells were identified in the hepatic lobule in 74 of 76 biopsy specimens and graded as low-grade in 26 and high-grade in 48. Zone 3 was involved in 72 of 74 positive cases, and in 33 cases, the activated stellate cells were preferentially located in zone 3. The degree of stellate cell activation correlated with fibrosis but not with inflammatory activity, severity of steatosis, or stainable iron. In most cases, the degree of stellate cell activation paralleled the degree of hepatic fibrosis, but in 25 cases, the degree of hepatic stellate cell activation was greater than expected, raising the question of whether such patients are at risk for disease progression.     

碧萝芷通过下调ERK磷酸化及自噬水平抑制TGF-β1诱导的肝星状细胞活化

目的:探究碧萝芷对转化生长因子β1(TGF-β1)诱导的肝星状细胞活化的影响。方法:5μg/L TGF-β1和不同浓度碧萝芷(0、10、25、50 mg/L)分别作用于LX-2细胞,在有或无自噬抑制剂3-MA和ERK抑制剂PD98059的情况下,用MTT法检测细胞活力的变化,Western blot实验检测α-SMA、ColⅠ、TIMP-1、LC3-Ⅱ/Ⅰ、beclin1、p-ERK1/2和ERK1/2蛋白水平的变化。结果:与对照组相比,5μg/L TGF-β1组的LX-2细胞活力以及α-SMA、ColⅠ、TIMP-1、LC3-Ⅱ/Ⅰ、beclin 1、p-ERK1/2和ERK1/2蛋白水平明显增加(P0.05)。而碧萝芷预处理能逆转上述效应,并呈现一定的剂量依赖性,50 mg/L碧萝芷的抑制效果最为显著(P0.05)。而且,与TGF-β1组相比较,50 mg/L碧萝芷、5 mmol/L 3-MA或者20μmol/L PD98059预处理下,TGF-β1诱导的LX-2细胞活力以及α-SMA和LC3-Ⅱ/Ⅰ蛋白表达均明显下调(P0.05)。结论:碧萝芷通过下调ERK磷酸化及自噬水平抑制TGF-β1诱导的肝星状细胞活化。     

脂肪特异性蛋白27对大鼠肝星状细胞活化的影响

 目的：探讨脂肪特异性蛋白27（Fsp27）对肝星状细胞（HSCs）增殖和活化的影响及其对纤维化相关蛋白的调节作用。方法：从SD大鼠肝脏提取HSCs并培养,采用实时荧光定量PCR、免疫荧光染色和Western blotting检测原代HSCs和活化HSCs中Fsp27 mRNA和蛋白的表达。构建携带Fsp27基因的慢病毒,转染活化的HSCs并继续培养72 h,通过CCK-8比色法检测Fsp27对HSCs增殖的影响;Western blotting检测HSCs中α-平滑肌肌动蛋白（α-SMA）的表达,了解HSCs的活化状态;实时荧光定量PCR检测Fsp27对HSCs中纤维化相关蛋白［包括基质金属蛋白酶2（MMP-2）、金属蛋白酶组织抑制物1（TIMP-1）和转化生长因子β1（TGF-β1）］ mRNA表达的影响。结果：成功分离大鼠原代HSCs。Fsp27在原代HSCs和活化HSCs中的表达差异显著（P<0.01）;活化HSCs成功转染携带Fsp27基因的慢病毒后继续培养72 h,与对照组比较,HSCs的活化与增殖被明显抑制（P<0.05）;Fsp27促进MMP-2 mRNA的表达（P<0.05）,降低TIMP-1和TGF-β1 mRNA的表达（P<0.05）。结论：Fsp27可抑制HSCs的增殖和活化,并调节纤维化相关蛋白的表达。Fsp27的作用可能与其维持HSCs静息状态细胞表型有关。     

Expression of cytoskeletal proteins in hepatic stellate cells isolated from normal and cirrhotic rat liver

Hepatic stellate cells (HSC) are located in Disse spaces of normal rat liver. In their quiescent state they serve as a storage site for vitamin A. In fibrotic liver they become activated, proliferate and they undergo transdifferentiation into myofibroblast-like cells. Changes in the cell phenotype are accompanied by changes in the cellular cytoskeleton. We have studied the expression of alpha-smooth muscle actin and intermediate filament proteins vimentin, desmin and glial fibrillary acidic protein (GFAP) by immunocytochemistry in HSC cultured for 2 or 7 days after isolation. Normal or cirrhotic rat liver was perfused with solutions of pronase and collagenase and HSC were isolated by density gradient centrifugation of the resulting cell suspension. Liver cirrhosis was produced in rats by repeated carbon tetrachloride administration. Vimentin was detected in all cells from normal and cirrhotic liver. The concentration of desmin in the cells from cirrhotic liver was slightly higher than that in normal cells and it increased with time in culture. GFAP could be detected only in normal cells 2 days after their isolation. In contrast, alpha smooth muscle actin (alpha-SMA) was absent from normal cells at this time but its expression was pronouced later. In most cells from cirrhotic liver this antigen was already present on the second day of culture and its expression further increased.     

Inhibition of connective tissue growth factor suppresses hepatic stellate cell activation in vitro and prevents liver fibrosis in vivo

Activation of hepatic stellate cells (HSC) represents a critical event in fibrosis, and connective tissue growth factor (CTGF) plays a profibrotic activity and a key factor in the pathogenesis of tissue fibrosis. The current study aimed to determine whether lentivirus-mediated short hairpin RNA (shRNA)–targeted CTGF downregulates the CTGF expression and furthermore whether it suppresses the activation and proliferation of HSC in vitro and prevents liver fibrosis in vivo. HSC-T6 cells were treated with recombinant lentivirus carrying CTGF siRNA. Real-time PCR, Western blotting, MTT, and flow cytometry were performed to investigate the activation and proliferation of HSC-T6 cells in response to CTGF silence. CCl₄-induced rats were received lentivirus containing CTGF siRNA by intraportal vein injection. Levels of liver fibrosis were assessed by biochemical and histopathologic examinations. Recombinant lentivirus containing CTGF siRNA could effectively and specifically downregulate the expression of CTGF in both HSC-T6 cells and CCl₄-induced rats with liver fibrosis. Blockade of CTGF resulted in significant inhibition of HSC activation and proliferation with decrease in TIMPs, MMP2, MMP9, and collagen I, as well as increase in cells in S phase. Silencing CTGF expression with siRNA prevented liver fibrosis in CCl₄-induced rat model. These findings indicated that CTGF plays a key role in the pathogenesis of liver fibrosis and lentiviral-mediated CTGF siRNA has the potential to be an effective treatment for liver fibrosis.     

Copper-induced hepatotoxicosis with hepatic stellate cell activation and severe fibrosis in North Ronaldsay lambs: a model for non-Wilsonian hepatic copper toxicosis of infants

Copper-sensitive North Ronaldsay sheep represent a possible model for certain hepatic-overload syndromes of infancy and childhood that are clinically, pathologically and genetically distinct from Wilson's disease. The purpose of this study was to simulate in artificially reared lambs the syndrome produced by copper exposure in susceptible human infants. Twenty four North Ronaldsay lambs were assigned to three groups of eight animals, namely, an unsupplemented control group and two trial groups given milk replacer to which copper (CuSO4) had been added at the rate of 5 mg/litre and 10 mg/litre. Four lambs from each group were killed at 40 or 69 days. Livers were fixed in 10% formalin and analysed for copper by mass spectrometry. Paraffin wax-embedded sections were stained with rhodanine for copper and labelled immunohistochemically for alpha smooth muscle actin (ASMA). At 40 days the maximum amounts of copper in the livers of both copper-supplemented groups was 1466-1605 microg/g dry weight (control group 172-201 microg/g Cu dry weight). Histochemically, copper was demonstrated within hepatocytes, together with marked apoptosis. At 69 days there was a florid pericellular fibrosis complemented by strong ASMA immunolabelling, confirming phenotypic modulation of hepatic stellate cells. Such primary copper-induced fibrogenesis confirms the unique status of this animal model in respect of childhood copper toxicosis.     

Homocysteine enhances cell proliferation in hepatic myofibroblastic stellate cells

Homocysteine is an intermediate in sulfur amino acid metabolism, which takes place mainly in the liver. Recent studies have shown that hyperhomocysteinemia in patients and murine models develop hepatic fibrosis. To define mechanisms underlying homocysteine-induced hepatic fibrosis, the effect of homocysteine on hepatic stellate cell (HSC) proliferation was examined. In the present study, homocysteine promoted proliferation in myofibroblastic HSCs. Homocysteine elicited a transient formation of reactive oxygen species (ROS). The initial ROS activated extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, which were involved in the activation of NAD(P)H oxidases and the generation of more ROS. The activation of NAD(P)H oxidases resulted from upregulation of the expression of p22(phox) and the phosphorylation of p47(phox). The ROS derived from NAD(P)H oxidases activated the PI3K/Akt pathway, thus promoting cellular proliferation in HSCs. These findings provide a mechanistic explanation for the development and progression of hepatic fibrosis in hyperhomocysteinemia.     

Oxidative stress effect on the activation of hepatic stellate cells

Collagen is the most excessive extracellular matrix protein in hepatic fibrosis. Activated, but not quiescent, hepatic stellate cells (HSCs) have a high level of collagen and a smooth muscle actin (alpha SMA) expression. HSCs play a key role in the pathogenesis of hepatic fibrosis. We analyzed a mechanism leading to HSC activation by evaluating the role of oxidative stress and the expression of NFkB. In vitro study HSCs were proliferated (PCNA:2% vs 68%) and activated (alpha SMA: 5% vs 78%) by ascorbate/FeSO4, and HSCs activated by type I collagen were blocked (PCNA: 97% vs 4%, a SMA: 86% vs 9%) by a-tocopherol. In vivo study means of a SMA positive cells in liver at 400 x HPF were 48.3+/-5.2 and 15.2+/-1.8 and [3H]thymidine uptake of HSC was 529.2+/-284.8 cpm and 223.0+/-86.3 cpm in control and a-tocopherol treated group respectively at 32 hours after CCl4 injection. Nuclear extracts from activated, but not from quiescent, HSCs formed a complex with the NFkB cognate oligonucleotidesand alpha-tocopherol inhibited this bindings. This study indicates that oxidative stress plays an essential role through the induction of NFkB on HSC activation.     

奥曲肽抑制骨桥蛋白刺激的肝星状细胞增殖

目的:探讨奥曲肽对骨桥蛋白(OPN)刺激的肝星状细胞(HSCs)增殖的影响。方法:体外培养HSCs,MTT法测定HSCs增殖,Westernblotting测定细胞外信号调节激酶1(ERK1)蛋白表达。结果:①干预24h,奥曲肽2.5、5.0和10.0mg/L组吸光度(A)值均显著低于OPN组,抑制率分别是18.75%、37.50%和40.63%,P<0.05;10.0mg/L组作用12、24和48h的抑制率分别是34.38%、40.63%和53.13%,P<0.05。②对照组有ERK1蛋白表达,OPN刺激24h后,ERK1蛋白含量较对照组高95.62%;3个剂量奥曲肽组ERK1蛋白含量则分别降低16.74%、34.30%和65.72%。结论:奥曲肽对OPN刺激的HSCs增殖有明显的抑制作用,在一定范围内,呈剂量依赖性和时间依赖性;该作用与其抑制ERK1蛋白表达有关。     

Study on the in vitro and in vivo activation of rat hepatic stellate cells by Raman spectroscopy

The feasibility of a novel and efficient diagnostic method for liver fibrosis using Raman spectroscopy is studied. Confocal Raman spectroscopy (CRS) is utilized to monitor the molecular changes of hepatic stellate cells (HSCs) in vitro as well as in vivo activation. In vitro activation was induced by growth in uncoated plastic plates, while the in vivo activation is induced by a single intraperitoneal injection of carbon tetrachloride (CCl(4)). The biochemical changes of HSCs during activation such as the loss of retinoid, the increase of alpha-helical protein, and the increased production of extracellular matrix proteins are observed by CRS. A user-friendly autoclassifying system is also developed to classify Raman spectra of liver injury tissues with a 90% accuracy rate. Raman spectroscopy combined with a fiber optical probe could be potentially accomplished for in vivo detection, which can lead to a novel and efficient diagnosis for liver fibrosis.     

骨髓间质干细胞抑制肝星状细胞增殖与活化的体外研究

目的：探讨骨髓间质干细胞（MSCs）对活化态肝星状细胞（HSCs）增殖的影响。 方法： 分别从骨髓和肝脏分离纯化培养大鼠MSCs及HSCs，塑料板传代培养激活HSCs；在半透膜（transwell insert）上接种MSCs，在6孔塑料培养板上接种HSCs，建立上下双层细胞共培养体系；大鼠正常肝细胞系（BRLs）及HSCs培养分别作为对照。免疫细胞化学检测平滑肌激动蛋白（α-SMA）与结蛋白的表达，IBAS 2.5软件分析阳性染色表达量。 结果： HSCs与MSCs共培养24 h，HSCs表现轻度增殖抑制，随着培养时间延长，HSCs增殖活性受抑制更明显，在48 h和72 h的抑制率分别达15.7%与30.3%，与BRLs共培养体系比较有显著差异；与MSCs共培养72 h, HSCs表达α-SMA量明显低于两个对照组BRLs及HSCs培养体系（50.2% vs 90.2%、95.6%, P＜0.01）；而结蛋白表达的量在3组共培养体系中均无显著差异。 结论： MSCs具有分泌细胞因子抑制HSCs增殖活性的潜能,在治疗肝纤维化中可能发挥作用。     

槲皮素抑制离体大鼠肝星状细胞增殖

目的：研究酪氨酸蛋白激酶抑制剂槲皮素对肝星状细胞增殖的影响。方法：用链酶蛋白酶和胶原酶原位灌流消化正常大鼠肝脏，Nycodenz密度梯度离心分离肝星状细胞，采用MTT比色法，流式细胞术检测细胞增殖水平。结果：槲皮素处理组可显著地抑制肝星状细胞的增殖并呈剂量依赖性关系；可使G0／G1期细胞增多，S期细胞减少。结论：槲皮素可明显抑制肝星状细胞增殖。     

Smad expression of hepatic stellate cells in liver cirrhosis in vivo and hepatic stellate cell line in vitro

Smad expressions, signaling mediators of transforming growth factor-beta (TGF-beta) superfamily of cytokines, were investigated in paraffin-embedded tissue sections of liver cirrhosis due to the hepatitis C virus infection and in the hepatic stellate cell (HSC) line in vitro. Smad 2/3, 4 and 7 was expressed in the nucleus of the HSC in the cirrhotic liver, while the expression was weak in the non-cirrhotic liver. TGF-beta1 expression in the HSC of the cirrhotic liver was strong, while the expression was weak in the non-cirrhotic liver. In situ hybridization also demonstrated the Smad signalings in the HSC of the cirrhotic liver, which confirmed the results of the Smad expressions by immunohistochemistry. The HSC line showed a cytoplasmic and a weak nuclear expression of Smads without TGF-beta1 stimulation, while these cells showed a strong Smad expression in the nucleus by TGF-beta1 stimulation. Immunocytochemical assay demonstrated that the TGF-beta1 stimulation induced the increase of the Smad expressions and the decrease of the autocrine TGF-beta1 in the HSC line. In situ hybridization assay also demonstrated an increase of the Smad mRNA signalings by TGF-beta1 stimulation in vitro. These observations suggest that the Smad expressions increase in the nucleus of the HSC in the cirrhotic liver and that the TGF-beta1 stimulation induces the Smad expression.     

大鼠骨髓间充质干细胞体外诱导肝星状细胞系凋亡

目的 研究大鼠骨髓间充质干细胞（MSCs）体外诱导大鼠肝星状细胞（HSCs）凋亡及其机制。方法 分离培养MSCs, HSC-T6系及纤维原细胞系冻融后传代使用。用6孔塑料培养板,建立上下双层细胞共培养体系。分3组：⑴空白对照组⑵阴性对照组⑶实验组。以上体系培养观察24、48和72h,于倒置相差显微镜下动态观察HSCs细胞形态;免疫组化法检测HSCs a-SMA表达;WST-8法检测HSCs增殖抑制率;流式细胞仪Annexin-V-FITC/PI双染法和DNA凝胶电泳（DNA Ladder ）检测HSCs细胞凋亡;RT-PCR 、Western blot检测HSCs Caspase-3、Bax基因mRNA和蛋白表达。结果 实验组出现明显的DNA Ladder,24h后HSCs 增殖抑制率、凋亡率和Caspase-3, Bax mRNA和蛋白表达呈时间依赖性,显著高于对照组。（P<0.01）结论 MSCs可在体外抑制HSCs增殖,可能通过旁分泌途径诱导HSCs凋亡,其凋亡发生是通过上调Caspase-3、Bax表达发挥作用,本研究支持MSCs通过抑制HSCs产生抗肝纤维化机制。     

Activation of cultured rat hepatic stellate cells by tumoral hepatocytes

Hepatocellular carcinoma (HCC) is the main type of primary liver cancer, and it develops from hepatocytes. The stroma of HCC is infiltrated by myofibroblasts. In other settings, such as liver fibrosis, myofibroblasts are derived mainly from the activation of hepatic stellate cells (HSC). In this study, we investigated whether tumoral hepatocytes were able to activate HSC. HSC were isolated from normal rats and were plated in dishes coated with Matrigel, to prevent their spontaneous activation. HSC were exposed to conditioned medium (CM) from the rat HCC lines Fao and H5. Tumor cell CM elicited major morphologic changes, such as spreading and generation of cytoplasmic processes. Fao and H5 CM increased HSC proliferation to 1.60 and 1.76 times control values, respectively. The expression of alpha-smooth muscle actin was low or undetectable in control cells and was markedly increased by both tumor cell CM but not by normal rat hepatocyte CM. Desmin expression was also enhanced. Gelatinase A secretion was significantly increased 1.20-fold by Fao CM and 1.55-fold by H5 CM. Expression of beta-type platelet-derived growth factor receptor mRNA was increased 5.8-fold by H5 CM but was decreased to 13% of control levels by Fao CM. HSC activation by tumor cell CM was not prevented by urokinase or matrix metalloproteinase inhibitors, suggesting that Matrigel degradation was not central to the activation process. Finally, a blocking antibody to transforming growth factor-beta1 did not impede Fao CM-induced activation but significantly blocked the increase in matrix metalloproteinase-2 expression induced by H5 CM. Our results show that tumoral rat hepatocyte CM is able to induce the activation of rat HSC in culture. The lack of induction of beta-type platelet-derived growth factor receptor mRNA by Fao CM indicates that, in some cases, tumor-induced activation differs from classic fibrosis-type activation. Our data thus suggest that HSC recruitment and activation in HCC could be under the control of tumor cells.