选择性COX-2抑制剂、PGE1对肝硬化大鼠VEGF和CTGF表达的影响

目的探讨环氧合酶-2（COX-2）抑制剂、PGE1对肝硬化大鼠肝脏血管内皮生长因子（VEGF）和结缔组织生长因子（CT-GF）表达的影响。方法用50％CCl4（CCl4／橄榄油：体积比1／1）腹腔注射诱导SD大鼠肝硬化模型,每周2次,共8周。52只SD大鼠随机分为4组：正常对照组（n=10）腹腔注射橄榄油;模型对照组（n=14）,在诱导模型的同时给予等体积生理盐水灌胃,每日1次;选择性COX-2抑制剂罗非昔布组（n=14）,按10mg·kg^-1·d^-1灌胃;米索前列醇即PGE,组（n=14）,按每只大鼠10μg·d^-1灌胃。8周末处死大鼠留取肝组织,通过Westernblot和免疫组织化学方法检测肝组织VEGF和CTGF表达和定位。结果随着CCl4诱导大鼠肝硬化的形成,肝组织VEGF和CTGF的表达增加,与肝硬化模型对照组（安慰剂）比较,选择性COX-2抑制剂罗非昔布能显著降低肝脏VEGF和CTGF蛋白表达水平,而PGE1对VEGF无影响,CTGF显著降低。结论肝纤维化形成过程中,肝组织VEGF和CTGF的表达增加,选择性COX-2抑制罗非昔布能在体内抑制肝组织CTGF和VEGF的表达,这可能是罗非昔布抗肝纤维化的分子机制。     

选择性环氧合酶-2抑制剂塞莱希布对大鼠肝纤维化的作用

目的:探讨选择性COX-2抑制剂塞莱昔布对CCl4诱导的大鼠肝纤维化的作用.方法:5-6周龄的♂SD大鼠50只,随机分为6组.A组(肝纤维化模型组):10只,50%CCl4橄榄油溶液1mL/kg,每周2次皮下注射,同时给予生理盐水灌胃;B组(早期治疗组):10只,造模同时给予塞莱昔布15mg/kg溶于生理盐水中灌胃,每天1次;C组(中期治疗组):10只,造模同时给予生理盐水灌胃,第3周起改为塞莱昔布灌胃;D组(晚期治疗组):10只,造模同时给予生理盐水灌胃,第5周起改为塞莱昔布灌胃;E组:5只,给予相同体积的橄榄油皮下注射,同时给予相同剂量的塞莱昔布灌胃;F组:5只,给予相同体积的橄榄油皮下注射和生理盐水中灌胃.上述处置共8wk.实验结束后腹主动脉取血,取肝组织,检测血清谷丙转氨酶(ALT)、透明质酸(HA)、层粘连蛋白(LN)水平;病理组织学HE染色及Masson染色观察肝纤维化严重程度并评分,免疫组织化学方法检测肝组织Ⅰ胶原、α-SMA、COX-1、COX-2的表达.结果:A组与F组相比,可见显著的肝纤维化改变(P<0.01);与A组相比,B组病理染色可见肝纤维化程度明显减轻(P<0.01),血清ALT、...     

沉默结缔组织生长因子对大鼠肝纤维化的影响

目的 研究结缔组织生长因子(CTGF)小干扰RNA(siRNA)在CCl4诱导的大鼠肝纤维化模型中的抗肝纤维化作用及其对肝星状细胞(HSC)激活的影响.方法 雄性SD大鼠24只,随机分成4组.模型组:皮下注射CCl4及经门静脉注射等渗盐水,每3 d 1次,连续6周(给药频率与时间,下同);CTGF siRNA干预组:皮下注射CCl4及经门静脉注射CTGF siRNA ;对照siRNA干预组:皮下注射CCl4及经门静脉注射对照siRNA;正常对照组:经门静脉注射等渗盐水.用HE染色和Sirius red染色评估大鼠肝组织学变化,Western blot检测肝组织CTGF及α平滑肌肌动蛋白(α-SMA)的表达,免疫组织化学染色评估肝组织α-SMA阳性细胞数量.对实验数据用单因素方差分析或Kruskal-Wallis检验分析.结果 模型组及对照siRNA组大鼠肝组织CTGF及α-SMA蛋白表达显著上调,α-SMA染色阳性细胞数量明显增加.与模型组相比,CTGF siRNA组CTGF及α-SMA蛋白表达分别下调95%±2%和86%±11%(F值分别为21.234和12.473,P值均＜0.01);肝组织α-SMA染色阳性细胞数减少76%±9%(F=9.179,P＜0.01);组织学检查显示肝纤维化程度明显减轻.结论 siRNA沉默CTGF基因能显著减轻大鼠实验性肝纤维化,减少活化HSC数量可能是其改善肝纤维化的主要机制之一.     

The anti‐inflammatory effect of celecoxib does not prevent liver fibrosis in bile duct‐ligated rats

Background/Aims: Celecoxib was used in the treatment of inflammation in patients with cirrhosis. However, data on the progression of liver fibrosis after treatment by celecoxib are not available. This study aims to elucidate the effects of celecoxib on cholestatic liver fibrosis in rats. Methods: Rats underwent bile duct ligation (BDL) for 1 or 2 weeks to induce hepatic fibrosis. Celecoxib was introduced on day 1 after operation. The effects of celecoxib were assessed by comparison of the severity of hepatic fibrosis. Results: Infiltration of inflammatory cells and proliferation of bile ducts was seen after 1 week of BDL and fibrosis was induced after 2 weeks. Reduced alanine aminotransferase (ALT) levels and blunted expression of inflammatory factors [tumour necrosis factor‐α, interleukin (IL)‐1β and IL‐6] were seen in the liver of BDL‐treated rats that received celecoxib at week 1. Although celecoxib was sufficient in suppressing the cyclo‐oxygenase (COX)‐2 expression in the control organ (kidney), it failed to suppress the enhanced hepatic COX‐2 expression. At week 2, celecoxib did not alter the ALT level, the severity of fibrosis and hepatic collagen contents. This was associated with unchanged α‐smooth muscle actin protein expression and tissue inhibitor of metalloproteinase‐2 (TIMP‐2), matrix metalloproteinase (MMP)‐2 and MMP‐9 mRNA expressions in the liver. Celecoxib had no effect on the BDL‐dependent increase in bilirubin levels at any time point. Conclusions: The present study provides morphological and molecular biological evidences for the role of celecoxib in cholestatic liver fibrosis. Celecoxib protects against hepatic inflammation in the early stage of BDL rats, but does not have an effect on liver fibrosis.     

Antifibrotic activity of rofecoxib in vivo is associated with reduced portal hypertension in rats with carbon tetrachloride-induced liver injury

BACKGROUND AND AIM: Upregulation of cyclooxygenase-2 (COX-2), an inducible enzyme that is actively involved in inflammation and wound healing, has been found in cirrhotic livers. The aim of this study was to investigate the effects of selective inhibition of COX-2 on the development of liver cirrhosis and portal hypertension in rats. METHODS: Liver cirrhosis was induced by carbon tetrachloride (CCl(4)) in Sprague-Dawley rats. Rofecoxib, a highly selective COX-2 inhibitor, was orally administered to rats at a dose of 10 mg/kg/day. Portal pressure was measured at 8 weeks post CCl(4) administration with the catheterization method followed by the harvesting of liver samples. Liver histopathology was analyzed with hematoxylin and eosin and Masson's trichrome staining. The activated, alpha smooth muscle actin (alpha-SMA) positive hepatic stellate cells (HSCs) and the protein levels of collagen types I, III, IV, as well as laminin and two fibrogenic mediators, vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) in the livers, were detected with immunohistochemical staining and western blot methods, respectively. The level of hepatic thromboxane B(2) (TXB(2)), a potent vasoconstrictive substance derived from COX, was measured with enzyme immunoassay. RESULTS: Oral administration of rofecoxib decreased portal pressure in rats that were treated with CCl(4) for 8 weeks. This was associated with a marked reduction in collagen accumulation and TXB(2) level in the rat livers. In addition, rofecoxib administration was found to reduce the number of activated HSCs and to downregulate hepatic protein levels of three detected types of collagen, laminin, VEGF and CTGF in CCl(4)-treated rats. CONCLUSIONS: COX-2 is involved in the fibrogenesis of livers and the formation of portal hypertension in CCl(4)-treated rats. Selective inhibition of COX-2 by rofecoxib reduces portal hypertension and this is associated with antifibrotic activity as well as a reduction of COX-2-derived vasoactive substance.     

Inhibition of rat liver fibrogenesis through noradrenergic antagonism

The effect of adrenergic innervation and/or circulating catecholamines on the function of liver fibrogenic cells is poorly understood. Our aim was to investigate the effects of noradrenergic antagonism on carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Two weeks of CCl4 induced an approximately 5-fold increase in the area of fibrosis as compared with controls. The addition of 6-hydroxydopamine (OHDA), a toxin that destroys noradrenergic fibers, decreased fibrosis by 60%. After 6 weeks of CCl4, the area of fibrosis increased about 30-fold in CCl4-treated animals and was decreased by 36% with OHDA. At 2 weeks, OHDA abrogated the CCl4-induced increase in mRNA level of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), an inhibitor of extracellular matrix degradation, and it greatly reduced it at 6 weeks. Finally, when rats treated with CCl4 for 2 weeks also received prazosin, an antagonist of alpha1-adrenergic receptors, fibrosis was decreased by 83%. In conclusion, destruction of noradrenergic fibers or antagonism of noradrenergic signaling through alpha1 receptors inhibited the development of liver fibrosis. Because adrenoreceptor antagonists have a very sound safety profile, they appear as attractive drugs to reduce liver fibrogenesis.     

沉默结缔组织生长因子对大鼠肝纤维化及细胞外基质积聚的影响

目的 研究结缔组织生长因子(CTGF)小干扰RNA(siRNA)在CCl4诱导的大鼠肝纤维化模型中的抗肝纤维化作用及其对细胞外基质积聚的影响.方法 雄性SD大鼠30只,随机分成5组.模型组:皮下注射CCl4及经门静脉注射等渗盐水,3 d 1次,连续6周;预防组:皮下注射CCl4及经门静脉注射CTGF siRNA,连续6周;治疗组:皮下注射CCl4 2周,随后给予CTGF siRNA及CCl4 4周;对照siRNA干预组:皮下注射CCl4及经门静脉注射对照siRNA,连续6周;空白对照组:经门静脉注射等渗盐水6周.用HE和Sirius red染色评估大鼠肝组织学变化,RTPCR或(和)Western blot检测肝组织CTGF、Ⅰ与Ⅲ型胶原及层黏连蛋白的表达,放射免疫法检测外周血Ⅲ型前胶原及透明质酸含量.结果 模型组及对照siRNA组大鼠肝组织CTGF、Ⅰ与Ⅲ型胶原及层黏连蛋白基因表达显著上调;与模型组相比,预防组及治疗组由CCl4诱导的CTGF mRNA和蛋白表达及Ⅰ与Ⅲ型胶原、层黏连蛋白mRNA表达分别下调76%±8%、95%±2%、74%±8%、78%±8%、31%±7%和80%±3%、93%±3%、57%±6%、59%±10%、43%±9%(F值分别为68.630,21.234,24.219,16.315和9.716,P值均＜0.01),肝纤维化程度明显减轻,大鼠外周血Ⅲ型前胶原和透明质酸含量也显著降低.结论 经门静脉注射CTGF siRNA能显著抑制实验大鼠肝CTGF基因表达,由此通过阻止细胞外基质积聚而缓解肝纤维化.     

Prostaglandin E2 inhibits transforming growth factor beta 1-mediated induction of collagen alpha 1(I) in hepatic stellate cells

BACKGROUND/AIMS: Cyclooxygenase-2 (COX-2) has been implicated in a number of hepatic stellate cell (HSC) functions but its relationship to transforming growth factor-beta 1 (TGF-beta 1)-mediated fibrogenesis is unknown. We assessed the impact of COX-2 inhibition and PGE(2) on the regulation of TGF-beta 1-stimulated matrix synthesis in an immortalized human HSC line, LX-1 and corroborated these findings in primary stellate cells. METHODS: Expression of COX-2 was assessed by Western blotting and real time quantitative PCR. The effect of NS398, a selective COX-2 inhibitor, and PGE(2) on TGF-beta 1-mediated fibrogenesis was examined by measuring mRNA levels of collagen alpha1(I). PGE(2) receptor expression was analyzed by RT-PCR. RESULTS: Under basal conditions, NS398 suppressed PGE(2) synthesis and induced collagen alpha 1(I) whereas exogenous PGE(2) suppressed expression of collagen alpha1(I). TGF-beta 1 induced COX-2 mRNA, COX-2 protein and PGE(2) biosynthesis. Importantly, TGF-beta 1-mediated induction of collagen alpha 1(I) was markedly suppressed by the addition of exogenous PGE(2). All four major PGE(2) receptors were expressed in LX-1 cells. CONCLUSIONS: These results suggest that COX-2-derived PGE(2) inhibits both basal and TGF-beta 1-mediated induction of collagen synthesis by HSC. Based on these findings, it will be important to determine whether inhibiting COX-derived PGE(2) synthesis alters the progression of liver fibrosis in vivo.     

Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis

BACKGROUND: It has been shown that expression of the potent angiogenic factor, vascular endothelial growth factor (VEGF), and its receptors, flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2), increased during the development of liver fibrosis. AIMS: To elucidate the in vivo role of interaction between VEGF and its receptors in liver fibrogenesis. METHODS: A model of CCl(4) induced hepatic fibrosis was used to assess the role of VEGFR-1 and VEGFR-2 by means of specific neutralising monoclonal antibodies (R-1mAb and R-2mAb, respectively). R-1mAb and R-2mAb were administered after two weeks of treatment with CCl(4), and indices of fibrosis were assessed at eight weeks. RESULTS: Hepatic VEGF mRNA expression significantly increased during the development of liver fibrosis. Both R-1mAb and R-2mAb treatments significantly attenuated the development of fibrosis associated with suppression of neovascularisation in the liver. Hepatic hydroxyproline and serum fibrosis markers were also suppressed. Furthermore, the number of alpha-smooth muscle actin positive cells and alpha1(I)-procollagen mRNA expression were significantly suppressed by R-1mAb and R-2mAb treatment. The inhibitory effect of R-2mAb was more potent than that of R-1mAb, and combination treatment with both mAbs almost completely attenuated fibrosis development. Our in vitro study showed that VEGF treatment significantly stimulated proliferation of both activated hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC). VEGF also significantly increased alpha1(I)-procollagen mRNA expression in activated HSC. CONCLUSIONS: These results suggest that the interaction of VEGF and its receptor, which reflected the combined effects of both on HSC and SEC, was a prerequisite for liver fibrosis development.     

Tissue inhibitor of metalloproteinases-1 promotes liver fibrosis development in a transgenic mouse model

Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been shown to be increased in liver fibrosis development both in murine experimental models and human samples. However, the direct role of TIMP-1 during liver fibrosis development has not been defined. To address this issue, we developed transgenic mice overexpressing human TIMP-1 (hTIMP-1) in the liver under control of the albumin promoter/ enhancer. A model of CCl(4)-induced hepatic fibrosis was used to assess the extent of fibrosis development in TIMP-1 transgenic (TIMP-Tg) mice and control hybrid (Cont) mice. Without any treatment, overexpression of TIMP-1 itself did not induce liver fibrosis. There were no significant differences of pro-(alpha1)-collagen-I, (alpha2)-collagen-IV, and alpha-smooth muscle actin (alpha-SMA) mRNA expression in the liver between TIMP-Tg and Cont-mice, suggesting that overexpression of TIMP-1 itself did not cause hepatic stellate cell (HSC) activation. After 4-week treatment with CCl(4), however, densitometric analysis revealed that TIMP-Tg-mice had a seven-fold increase in liver fibrosis compared with the Cont-mice. The hepatic hydroxyproline content and serum hyaluronic acid were also significantly increased in TIMP-Tg-mice, whereas CCl(4)-induced liver dysfunction was not altered. An active form of matrix metalloproteinases-2 (MMP-2) level in the liver of TIMP-Tg-mice was decreased relative to that in Cont-mice because of the transgenic TIMP-1. Immunohistochemical analysis revealed that collagen-I and collagen-IV accumulation was markedly increased in the liver of CCl(4)-treated TIMP-Tg-mice with a pattern similar to that of alpha-SMA positive cells. These results suggest that TIMP-1 does not by itself result in liver fibrosis, but strongly promotes liver fibrosis development.     

抗结缔组织生长因子小分子干扰RNA防治大鼠肝纤维化的研究

目的 探讨经腹腔注射抗结缔组织生长因子(CTGF)小分子干扰RNA(siRNA)是否能抑制大鼠体内肝脏CTGF基因表达及防治大鼠肝纤维化.方法 雄性SD大鼠30只,随机分成5组,分别为模型组腹腔注射40%CCl4 (3 mL/kg)及尾静脉注射生理盐水,1次/3 d,连续8周;预防组腹腔注射40%CCl4 及尾静脉注射...     

Antifibrotic effects of a tissue inhibitor of metalloproteinase-1 antibody on established liver fibrosis in rats

Liver fibrosis is characterized by increased synthesis, and decreased degradation, of extracellular matrix (ECM) within the injured tissue. Decreased ECM degradation results, in part, from increased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), which blocks matrix metalloproteinase (MMP) activity. TIMP-1 is also involved in promoting survival of activated hepatic stellate cells (HSCs), a major source of ECM. This study examined the effects of blocking TIMP-1 activity in a clinically relevant model of established liver fibrosis. Rats were treated with carbon tetrachloride (CCl(4)), or olive oil control, for 6 weeks; 24 days into the treatment, the rats were administered a neutralizing anti-TIMP-1 antibody derived from a fully human combinatorial antibody library (HuCAL), PBS, or an isotype control antibody. Livers from CCl(4)-treated rats exhibited substantial damage, including bridging fibrosis, inflammation, and extensive expression of smooth muscle alpha-actin (alpha-SMA). Compared to controls, rats administered anti-TIMP-1 showed a reduction in collagen accumulation by histological examination and hydroxyproline content. Administration of anti-TIMP-1 resulted in a marked decrease in alpha-SMA staining. Zymography analysis showed antibody treatment decreased the activity of MMP-2. In conclusion, administration of a TIMP-1 antibody attenuated CCl(4)-induced liver fibrosis and decreased HSC activation and MMP-2 activity.     

Halofuginone to prevent and treat thioacetamide-induced liver fibrosis in rats

Hepatic fibrosis is associated with activation of hepatic stellate cells (HSC), the major source of the extracellular matrix (ECM) proteins. The predominant ECM protein synthesized by the HSC is collagen type I. We evaluated the effect of halofuginone-an inhibitor of collagen synthesis-on thioacetamide (TAA)-induced liver fibrosis in rats. In the control rats the HSC did not express smooth muscle actin, collagen type I gene, or tissue inhibitor of metalloproteinases-2 (TIMP-2), suggesting that they were in their quiescent state. When treated with TAA, the livers displayed large fibrous septa, which were populated by smooth muscle actin-positive cells expressing high levels of the collagen alpha1(I) gene and containing high levels of TIMP-2, all of which are characteristic of advanced fibrosis. Halofuginone given orally before fibrosis induction prevented the activation of most of the stellate cells and the remaining cells expressed low levels of collagen alpha1(I) gene, resulting in low levels of collagen. The level of TIMP-2 was almost the same as in the control livers. When given to rats with established fibrosis, halofuginone caused almost complete resolution of the fibrotic condition. The levels of collagen, collagen alpha1(I) gene expression, TIMP-2 content, and smooth muscle actin-positive cells were as in the control rats. Halofuginone inhibited the proliferation of other cell types of the fibrotic liver in vivo and inhibited collagen production and collagen alpha1(I) gene expression in the SV40-immortalized rat HSC-T6 cells in vitro. These results suggest that halofuginone may become an effective and novel mode of therapy in the treatment of liver fibrosis.     

沉默结缔组织生长因子对鼠转化生长因子β/Smads信号的影响

目的 研究小干扰RNA(siRNA)沉默结缔组织生长因子(CTGF)对大鼠转化生长因子(TGF)β/Smads信号通路的影响.方法 将化学合成CTGF siRNA转染肝星状细胞(HSC)T6和经门静脉注入CCl4诱导6周的肝纤维化大鼠,设空白及随机siRNA对照,抽提HSC T6及大鼠肝组织总RNA和蛋白质,应用Western blot和RT-PCR检测HSC T6及肝组织CTGF及TGF β1,Smad2、3、7蛋白质和基因表达. 结果与空白对照组相比,siRNA能显著下调HSC T6 CTGF蛋白表达,以48 h最明显,CTGF蛋白表达下调94%±4%(t=46.196,P<0.01),而TGF β1、Smad2,3,7 mRNA表达差异无统计学意义;模型组及对照siRNA组,CCl4诱导的大鼠肝组织CTGF和TGF β1蛋白表达明显上调,与模型组相比,CTGF siRNA组大鼠肝组织CTGF及TGF β1蛋白表达分别下调95%±2%(F=21.234,P<0.01)和74%±8%(F=13.464,P<0.05),但Smad2和Smad7蛋白表达无明显改变. 结论沉默CTGF基因表达对大鼠肝TGF β/Smads信号具有阻抑作用.     

Expression of matrix metalloproteinase-2 and -14 persists during early resolution of experimental liver fibrosis and might contribute to fibrolysis.

BACKGROUND/AIMS: Resolution of liver fibrosis is possible but the identity of the matrix metalloproteinases (MMPs) which degrade the accumulated collagens is uncertain. We examined MMP-2 and MMP-14 expression in established and resolving fibrosis to assess their role in resolution of liver fibrosis. METHODS: MMP and tissue inhibitor of metalloproteinase (TIMP)-2 expression in liver extracts was examined by ribonuclease protection assay, Western blotting and gelatin zymography. MMP activity was examined by (14)C gelatin degradation. RESULTS: In human cirrhotic liver, MMP-14 mRNA was increased to 230-330% of normal liver expression. Both 63 kDa proenzyme and 60 kDa activated form were present. Cirrhotic livers had 270-320% of normal liver expression of MMP-2 protein with 20-25% being the 62 Da activated form. Protein and mRNA for MMP-2 and MMP-14 progressively increased during 8 weeks of CCl(4) treatment in rats. Between 3 and 7 days of resolution from CCl(4) liver fibrosis, MMP-2 and MMP-14 persisted at elevated levels. Gelatinolytic activity in liver homogenates peaked at 7 days of recovery, being 140% above that in livers at peak fibrosis. CONCLUSIONS: Increased expression and activation of MMP-2 and -14 occurs even under conditions of elevated TIMPs during liver fibrogenesis. During liver fibrosis resolution, as TIMP expression decays, the persistence of MMP-2 and MMP-14 may permit collagen degradation.     

Expression and location of Smad2, 4 mRNAs during and after liver fibrogenesis of rats

AIM: To investigate the location alteration of Smad2 and Smad4 mRNAs in the liver during and after fibrogenesis in rats. METHODS: Eighty male Wistar rats weighing approximately 200 g each were used. The rat models of experimental hepatic fibrosis were established by injection with carbon tetrachloride (CCl4), normal rats and rats were injected with olive oil and served as control groups. In situ hybridization(ISH) was used to detect the Smad2 and Smad4 mRNA in liver. RESULTS: In situ hybridization showed Smad2 and Smad4 mRNA expressions in the cytoplasm of hepatic stellate cells (HSC), fibroblasts and myofibroblasts around the central vein and hepatic sinus during and after fibrogenesis. Expression of Smad2, 4 mRNA was higher than that in normal and control rats. CONCLUSION: In the process of and after hepatic fibrosis formation, HSC, fibroblasts and myofibroblasts are the major cells that express Smad2 and Smad4. The more serious the hepatic fibrosis is in the injured liver, the higher the level of Smad2 and Smad4 gene expression is during and after fibrogenesis respectively.