RNAi抑制肝星状细胞TIMP-1基因表达及对细胞生物学行为的影响

研究了siRNA对大鼠肝星状细胞（CFsC）TIMP-1基因表达及对细胞生物学行为的影响．用RT—PCR方法获得带有T7启动子的TIMP-1全基因序列,体外转录法获得TIMP-1dsRNA,Dicer酶切后得到siRNA,用质脂体将siRNA转染至CFSC．RT—PCR检测TIMP-1mRNA的表达,MTT方法检测细胞生长,流式细胞仪检测细胞凋亡．结果成功获得TIMP-IsiRNA,将TIMP-1siRNA转染至CFSC12、24、48h后,与对照相比,TIMP-1mRNA的表达逐渐下降．经OuanityOne（BIO—RAD,USA）分析后,其抑制率分别为49.18％、58．72％和64．73％;siRNA转染CFSC细胞后,CFSC生长受到抑制,细胞凋亡不明显．实验结果表明体外转录法得到的siRNA能有效地降低大鼠CFSC中TIMP-1的表达．明显抑制CFSC的增殖,但不直接引起CFSC的凋亡。     

丹参单体IH764-3通过下调H2O2刺激的肝星状细胞FAK水平影响MMP-13和TIMP-1的表达

目的：研究丹参单体IH764—3对H2O2刺激的肝星状细胞（HSC）基质金属蛋白酶-13（MMP-13）、基质金属蛋白酶组织抑制因子-1（TIMP-1）表达的影响以及此过程中粘着斑激酶（FAK）的变化。方法：应用RT-PCR方法检测MMP-13及FAKmRNA表达,原位杂交方法检测TIMP-1mRNA水平,Western blotting技术检测FAK及TIMP-1蛋白表达。结果：IH764—3干预组的MMP-13mRNA在2h的表达强度明显上调,而TIMP-1mRNA表达明显受抑,FAKmRNA表达强度明显下调;IH764—3干预24h组FAK及TIMP-1蛋白表达受抑制。结论：丹参单体IH764—3可以诱导MMP-13表达,抑制TIMP-1表达,下调FAK表达是其中的机制之一。     

酶谱法测定肝星状细胞合成的基质金属蛋白酶活性

目的：建立检测肝星状细胞（HSC）合成的基质金属蛋白酶（MMPs）活性的方法。方法：用含SDS－明胶的聚丙烯酰胺凝胶电泳为基础的酶谱法,检测体外培养的HSC所合成的MMPs活性。结果：酶谱法能灵敏地检测到HSC细胞内和分泌到培养基中的MMP－2和MMP－9,培养基中MMP－2活性高子MMP－9,并观察到药物作用以后酶活性的改变。结论：酶谱法是研究HSC调节肝纤维化过程中细胞外基质（ECM）代谢的有效手段。     

中药乙肝散逆转肝纤维化过程中肝组织TIMP-1及MMP-2表达变化的实验研究

目的探讨中药乙肝散逆转免疫性肝纤维化过程中肝组织TIMP-1(金属蛋白酶组织抑制因子-1) 及MMP-2(间质金属蛋白酶-2)表达的变化.方法雄性Wistar大鼠尾静脉注射白蛋白建立免疫损伤性肝纤维化模型后,分组加用不同浓度的乙肝散颗粒饲料喂养12周,流式细胞仪定量分析大鼠肝组织TIMP-1和MMP-2基因表达量,观察大鼠肝组织病理、苦味酸-天狼红染色、Ⅳ胶原免疫组化、α-SMA(α平滑肌肌动蛋白)免疫组化、肝匀浆Hyp(羟脯氨酸).结果乙肝散应用组肝组织TIMP-1表达量较模型对照组明显降低(P<0.01),MMP-2变化无统计学差异;同时肝组织Ⅰ胶原、Ⅲ胶原、Ⅳ胶原水平和Hyp含量明显降低(P<0.01或P<0.05).结论中药乙肝散在逆转肝纤维化过程中对抑制肝组织TIMP-1基因表达具有一定作用,TIMP-1在肝纤维化的逆转中起重要作用.     

肺结核并发纤维化患者BALF和血清MMP及TIMP-1检测的临床意义

目的:探讨肺结核并发纤维化患者支气管肺泡灌洗液(BALF)及血清中基质金属蛋白9(MMP-9)及基质金属蛋白酶组织抑制剂l(TIMP-1)含量与肺纤维化及肺功能的关系.方法:酶联免疫(ELISA)法检测我院2009年3月至2009年9月68例肺结核并发纤维化患者(纤维化组)及30例无肺部疾病患者(对照组)的支气管肺泡灌洗液及血清中MMP-9及TIMP-1的含量,同时进行肺功能检查.结果:纤维化组患者的BALF及血清MMP-9的含量与对照组怠者相比无统计学意义,但纤维化组患者的BALF及血清TIMP-1的含量显著高于对照组(P＜0.05),且纤雏化组患者的BALF及血清MMP-9/TIMP-1比值低于对照组(P＜0.05).患者的动脉血氧分压、肺活量等肺功能指标与TIMP-1呈正相关,而与MMP-9/TIMP-1比值呈负相关(P＜0.05).结论:肺结核患者肺纤维化的发生可能与TIMP-1水平升高及MMP-9/TIMP-1比值降低相关,降低TIMP-9的水平可能是防止肺结核惠者并发肺纤维化的一种新途径.     

基质金属蛋白酶及其抑制剂在肝纤维化中的表达变化

目的 观察肝纤维化形成过程中基质金属蛋白酶MMP-1及其抑制剂TIMP-1的表达变化,从细胞外基质降解代谢的角度研究四氯化碳(CCl4)中毒性肝纤维化发生的机制.方法 雄性Wistar大鼠20只,分为正常组和肝纤维化模型组.肝纤维化组采用CCl4、饮酒、高脂低蛋白饮食等复合病因刺激制备肝纤维化动物模型,造模时间为8周.实验结束后测定肝脏指数、血清透明质酸(HA)、谷丙转氨酶(ALT)及尿羟脯氨酸(HYP)排出量,光镜下观察肝组织纤维化程度,并用免疫组化SABC法检测肝组织中Ⅰ、Ⅲ型胶原蛋白及MMP-1、TIMP-1的表达,同时用荧光实时定量PCR(RT-PCR)的方法检测肝组织中MMP-1、TIMP-1 mRNA的表达.结果 与正常对照组比较,肝纤维化模型组大鼠肝脏指数、血清HA及ALT显著增高,尿羟脯氨酸的排出量明显增加,病理组织学检查发现肝组织内纤维结缔组织增生明显,有假小叶形成;免疫组化的结果显示肝组织内Ⅰ、Ⅲ型胶原蛋白、MMP-1及TIMP-1的表达较正常组显著增加.结论 肝组织中MMP-1及TIMP-1的表达变化可能是导致肝纤维化的重要机制之一.     

胃癌组织中MMP-7和TIMP-1的表达及意义

目的:探讨基质金属蛋白酶-7(MMP-7)及其组织抑制剂-1(TIMP-1)与胃癌发生发展的关系.方法:采用免疫组化技术检测46例胃癌组织和相应癌旁组织中MMP-7和TIMP-1的表达,结合病人临床病理资料进行综合分析.结果:胃癌组织中MMP-7阳性表达率(60.87%)显著高于相应癌旁组织,差异有统计学意义(P<0.05);其表达与淋巴结转移(P<0.05)相关.胃癌组织中TIMP-1的阳性表达率(93.48%)明显高于相应癌旁组织(63.04%),差异有统计学意义(P<0.05);其表达与淋巴结转移相关(P<0.05).结论:MMP-7与胃癌的侵袭转移有关,TIMP-1有可能成为评价胃癌恶性生物学行为的指标.     

肝组织TGF-β1、TIMP-1及MMP-2表达与纤维化关系的实验研究

目的探讨免疫性肝纤维化肝组织TGF-β1、TIMP-1及MMP-2表达与肝纤维化间的关系.方法 24只雄性Wistar大鼠尾静脉注射白蛋白建立实验性免疫性肝纤维化模型,检测血清HA、ALT、AST、ALB,流式细胞仪定量分析肝组织TGF-β1、TIMP-1、MMP-2和α-SMA基因表达量,观察大鼠肝组织病理、肝组织Pollak三重染色和Gomori银染色、苦味酸-天狼红染色、Ⅳ型胶原免疫组化、α-SMA免疫组化、肝匀浆Hyp测定.结果随着肝纤维化程度的加重血清HA、ALT、AST、肝组织匀浆Hyp的浓度和TGF-β1、TIMP-1及α-SMA基因表达量均增加, MMP-2于肝纤维化早期明显升高,晚期明显降低.结论实验性肝纤维化过程中TGF-β1、TIMP-1促进肝纤维化的进展,MMP-2抑制其进展.     

通心络胶囊对心肌梗死模型大鼠MMP-2和TIMP-1表达的影响

目的:探讨通心络胶囊对心肌梗死大鼠基质金属蛋白酶-2(MMP-2)及基质金属蛋白酶抑制剂1(TIMP-1)的表达、心脏结构和功能改变的影响.方法:取SD大鼠24只,随机分成假手术组(SH group,n=8),心肌梗死模型组(MI group,n=8),用药组(Treated group,n=8).术后4w,测量左室收缩压(LVSP)、左室舒张末压(LVEDP)、左室上升最大速率(+dP/dtmax)、左室下降最大速率(-dP/dtmax);测定全心重(THW)及左心室称重(LVW),计算THW/体重(BW)、LVW/BW值和心肌梗死面积;用酶联免疫吸附法(ELISA)检测血清MMP-2及TIMP-1水平.结果:与SH组比较,MI组心室重量增加,心室功能显著降低,MMP-2升高,TIMP-1降低;与MI组比较,Treated组心室重量降低,心室功能显著增高,MMP-2减少,TIMP-1增加.结论:大鼠心肌梗死后,通心络胶囊能降低血清MMP-2水平,升高TIMP-1水平,抑制左室重构、改善心功能.     

栀子苷通过TGF-β1/Smad信号通路抑制肝纤维化和肝星状细胞活化

本研究旨在观察栀子苷对肝纤维化和肝星状细胞活化的影响,并探讨其可能机制。人肝星状细胞LX-2用5 ng/mL转化生长因子-β1 (transforming growth factor-β1, TGF-β1)处理,并用不同浓度(0, 1, 2.5, 5, 10, 20, 40, 60, 80, 100μmol/L)的栀子苷联合培养,MTT法进行细胞活力测定,LX-2分别经5 ng/mL TGF-β1、TGF-β1+栀子苷(20μmol/L)处理后,用qPCR和Western blot分别检测I型胶原蛋白(collagen I)、纤连蛋白(fibronectin)、α平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)、p-Smad2和p-Smad3基因和蛋白的表达。BALB/c小鼠用CCl₄ (25%, 1 mL/kg)诱导肝纤维化,设立对照组、CCl₄组和CCl₄+栀子苷组(40 mg/kg),4周后检测肝功能及血清肝纤维化指标,用HE和Masson染色进行组织学观察,免疫组织化学分析组织α-SMA...     

Induction of TIMP-1 expression in rat hepatic stellate cells and hepatocytes: a new role for homocysteine in liver fibrosis.

Elevated plasma levels of homocysteine have been shown to interfere with normal cell function in a variety of tissues and organs, such as the vascular wall and the liver. However, the molecular mechanisms behind homocysteine effects are not completely understood. In order to better characterize the cellular effects of homocysteine, we have searched for changes in gene expression induced by this amino acid. Our results show that homocysteine is able to induce the expression and synthesis of the tissue inhibitor of metalloproteinases-1 (TIMP-1) in a variety of cell types ranging from vascular smooth muscle cells to hepatocytes, HepG2 cells and hepatic stellate cells. In this latter cell type, homocysteine also stimulated alpha 1(I) procollagen mRNA expression. TIMP-1 induction by homocysteine appears to be mediated by its thiol group. Additionally, we demonstrate that homocysteine is able to promote activating protein-1 (AP-1) binding activity, which has been shown to be critical for TIMP-1 induction. Our findings suggest that homocysteine may alter extracellular matrix homeostasis on diverse tissular backgrounds besides the vascular wall. The liver could be considered as another target for such action of homocysteine. Consequently, the elevated plasma levels of this amino acid found in different pathological or nutritional circumstances may cooperate with other agents, such as ethanol, in the onset of liver fibrosis.     

Mechanistic insights into the effects of SREBP1c on hepatic stellate cell and liver fibrosis

Sterol regulatory element‐binding protein 1c (SREBP1c) plays key roles in maintenance of hepatic stellate cell (HSC) quiescence. The present researches investigated the mechanisms underlying the effects of SREBP1c on HSCs and liver fibrogenesis by HSC‐targeted overexpression of the active SREBP1c using adenovirus in vitro and in vivo. Results demonstrated that SREBP1c exerted inhibitory effects on TAA‐induced liver fibrosis. SREBP1c down‐regulated TGFβ1 level in liver, reduced the receptors for TGFβ1 and PDGFβ, and interrupted the signalling pathways of Smad3 and Akt1/2/3 but not ERK1/2 in HSCs. SREBP1c also led to the decreases in the protein levels of the bromodomain‐containing chromatin‐modifying factor bromodomain protein 4, methionine adenosyltransferase 2B (MAT2B) and TIMP1 in HSCs. In vivo activated HSCs did not express cyclin D1 and cyclin E1 but SREBP1c down‐regulated both cyclins in vitro. SREBP1c elevated PPARγ and MMP1 protein levels in the model of liver fibrosis. The effect of SREBP1c on MAT2B expression was associated with its binding to MAT2B1 promoter. Taken together, the mechanisms underlying the effects of SREBP1c on HSC activation and liver fibrosis were involved in its influences on TGFβ1 level, the receptors for TGFβ1 and PDGFβ and their downstream signalling, and the molecules for epigenetic regulation of genes.     

Addressing liver fibrosis with liposomes targeted to hepatic stellate cells

Liver fibrosis is a chronic disease that results from hepatitis B and C infections, alcohol abuse or metabolic and genetic disorders. Ultimately, progression of fibrosis leads to cirrhosis, a stage of the disease characterized by failure of the normal liver functions. Currently, the treatment of liver fibrosis is mainly based on the removal of the underlying cause of the disease and liver transplantation, which is the only treatment for patients with advanced fibrosis. Hepatic stellate cells (HSC) are considered to be key players in the development of liver fibrosis. Chronically activated HSC produces large amounts of extracellular matrix and enhance fibrosis by secreting a broad spectrum of cytokines that exert pro-fibrotic actions in other cells, and in an autocrine manner perpetuate their own activation. Therefore, therapeutic interventions that inhibit activation of HSC and its pro-fibrotic activities are currently under investigation worldwide. In the present study we applied targeted liposomes as drug carriers to HSC in the fibrotic liver and explored the potential of these liposomes in antifibrotic therapies. Moreover, we investigated effects of bioactive compounds delivered by these liposomes on the progression of liver fibrosis. To our knowledge, this is the first study demonstrating that lipid-based drug carriers can be selectively delivered to HSC in the fibrotic liver. By incorporating the bioactive lipid DLPC, these liposomes can modulate different processes such as inflammation and fibrogenesis in the fibrotic liver. This dual functionality of liposomes as a drug carrier system with intrinsic biological effects may be exploited in new approaches to treat liver fibrosis.     

CDH11 promotes liver fibrosis via activation of hepatic stellate cells

Liver fibrosis, an important health condition associated with chronic liver injury that provides a permissive environment for cancer development, is characterized by the persistent deposition of extracellular matrix components that are mainly derived from activated hepatic stellate cells (HSCs). CDH11 belongs to a group of transmembrane proteins that are principally located in adherens junctions. CDH11 mediates homophilic cell-to-cell adhesion, which may promote the development of cirrhosis. The goal of this study was to determine whether CDH11 regulates liver fibrosis and to examine its mechanism by focusing on HSC activation. Here we demonstrate that CDH11 expression is elevated in human and mouse fibrotic liver tissues and that CDH11 mediates the profibrotic response in activated HSCs. Our data indicate that CDH11 regulates the TGFβ-induced activation of HSCs. Moreover, cells from CDH11 deficient mice displayed decreased HSC activation in vitro, and CDH11 deficient mice developed liver fibrogenesis in response to chronic damage induced by CCl₄ administration. In addition, CDH11 expression was positively correlated with liver fibrosis in patients with cirrhosis, and could therefore be a prognostic factor in patients with liver fibrosis. Collectively, our findings demonstrate that CDH11 promotes liver fibrosis by activating HSCs and may represent a potential target for anti-fibrotic therapies.     

MyD88 in hepatic stellate cells enhances liver fibrosis via promoting macrophage M1 polarization

During liver fibrosis, quiescent HSCs (qHSCs) are activated to become activated HSCs (aHSCs)/myofibroblasts. The signal adapter MyD88, an essential component of TLR signaling, plays an important role in liver fibrosis. However, far less is known about the specific effects of MyD88 signaling in both qHSCs and aHSCs in the progress of liver fibrosis. Here, we used a CCl₄-induced mouse fibrosis model in which MyD88 was selectively depleted in qHSCs (GFAP^MyD88−/− mice) or aHSCs (α-SMA^MyD88−/− mice). MyD88 deficiency in qHSCs or aHSCs attenuated liver fibrosis in mice and inhibited α-SMA-positive cell activation. Inhibition of MyD88 in HSCs decreased α-SMA and collagen I levels, inflammatory cell infiltration, and pro-inflammatory gene expression. Furthermore, MyD88 signaling in HSCs increased the secretion of CXCL10, which promoted macrophage M1 polarization through CXCR3, leading to activation of the JAK/STAT1 pathway. Inhibition of CXCL10 attenuated macrophage M1 polarization and reduced liver fibrosis. Thus, MyD88 signaling in HSCs crucially contributes to liver fibrosis and provides a promising therapeutic target for the prevention and treatment of liver fibrosis.Subject terms: Mechanisms of disease, Kupffer cells     

Follistatin attenuates early liver fibrosis: effects on hepatic stellate cell activation and hepatocyte apoptosis

Activin A, a member of the transforming growth factor-beta superfamily, is constitutively expressed in hepatocytes and regulates liver mass through tonic inhibition of hepatocyte DNA synthesis. Follistatin is the main biological inhibitor of activin bioactivity. These molecules may be involved in hepatic fibrogenesis, although defined roles remain unclear. We studied activin and follistatin gene and protein expression in cultured rat hepatic stellate cells (HSCs) and in rats given CCl4 for 8 wk and examined the effect of follistatin administration on the development of hepatic fibrosis. In activated HSCs, activin mRNA was upregulated with high expression levels, whereas follistatin mRNA expression was unchanged from baseline. Activin A expression in normal lobular hepatocytes redistributed to periseptal hepatocytes and smooth muscle actin-positive HSCs in the fibrotic liver. A 32% reduction in fibrosis, maximal at week 4, occurred in CCl4-exposed rats treated with follistatin. Hepatocyte apoptosis decreased by 87% and was maximal at week 4 during follistatin treatment. In conclusion, activin is produced by activated HSCs in vitro and in vivo. Absence of simultaneous upregulation of follistatin gene expression in HSCs suggests that HSC-derived activin is biologically active and unopposed by follistatin. Our in vivo and in vitro results demonstrate that activin-mediated events contribute to hepatic fibrogenesis and that follistatin attenuates early events in fibrogenesis by constraining HSC proliferation and inhibiting hepatocyte apoptosis.     

Curcumin attenuates angiogenesis in liver fibrosis and inhibits angiogenic properties of hepatic stellate cells

Hepatic fibrosis is concomitant with sinusoidal pathological angiogenesis, which has been highlighted as novel therapeutic targets for the treatment of chronic liver disease. Our prior studies have demonstrated that curcumin has potent antifibrotic activity, but the mechanisms remain to be elucidated. The current work demonstrated that curcumin ameliorated fibrotic injury and sinusoidal angiogenesis in rat liver with fibrosis caused by carbon tetrachloride. Curcumin reduced the expression of a number of angiogenic markers in fibrotic liver. Experiments in vitro showed that the viability and vascularization of rat liver sinusoidal endothelial cells and rat aortic ring angiogenesis were not impaired by curcumin. These results indicated that hepatic stellate cells (HSCs) that are characterized as liver‐specific pericytes could be potential target cells for curcumin. Further investigations showed that curcumin inhibited VEGF expression in HSCs associated with disrupting platelet‐derived growth factor‐β receptor (PDGF‐βR)/ERK and mTOR pathways. HSC motility and vascularization were also suppressed by curcumin associated with blocking PDGF‐βR/focal adhesion kinase/RhoA cascade. Gain‐ or loss‐of‐function analyses revealed that activation of peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) was required for curcumin to inhibit angiogenic properties of HSCs. We concluded that curcumin attenuated sinusoidal angiogenesis in liver fibrosis possibly by targeting HSCs via a PPAR‐γ activation‐dependent mechanism. PPAR‐γ could be a target molecule for reducing pathological angiogenesis during liver fibrosis.