Effects of estradiol on liver estrogen receptor-α and its mRNA expression in hepatic fibrosis in rats

AIM: Estradiol treatment regulates estrogen receptor (ER) level in normal rat liver. However, little information is available concerning the role of estrogen in regulating liver ER in hepatic fibrosis in rats. The present study was conducted to determine whether estradiol treatment in CCl4-induced liver fibrosis of female and ovariectomized rats altered liver Erα and its mRNA expression, and to investigate the possible mechanisms.METHODS: Seventy female rats were divided into seven groups with ten rats in each. The ovariectomy groups were initiated with ovariectomies and the sham operation groups were initiated with just sham operations. The CCl4 toxic fibrosis groups Received 400 mL/L CCl4 subcutaneously at a dose of 2 mL/kg twice weekly. Estrogen groups were treated subcutaneously with estradiol 1 mg/kg, the normal control group and an ovariectomy group Received injection of peanut oil vehicle twice weekly. At the end of 8 weeks, all the rats were killed to detect their serum and hepatic indicators,their hepatic collagen content, and liver ER and ER mRNA expression.RESULTS: Estradiol treatment in both ovariectomy and sham ovariectomy groups reduced liver levels of ALT (from 658±220 nkat/L to 311±146 nkat/L and 540±252 nkat/L to 314±163 nkat/L, P＜0.05) and AST (from 697±240 nkat/L to 321±121 nkat/L and 631±268 nkat/L to 302±153 nkat/L,P＜0.05), increased serum nitric oxide (NO) level (from 53.7±17.1 μmol/L to 93.3±24.2 μmol/L and 55.3±23.1 μmol/L to 87.5±23.6 μmol/L, P＜0.05) and hepatic nitric oxide synthase (NOS) activity (from 1.73±0.71 KU/g to 2.49±1.20 KU/g and1.65±0.46 KU/g to 2.68±1.17 KU/g, P＜0.05), diminished the accumulation of hepatic collagen, decreased centrolobular necrotic areas as well as the inflammatory reaction in rats subjected to CCl4. The positive signal of ER and ER mRNA distributed in parenchymal and non-parenchymal hepatic cells, especially near the hepatic centrolobular and periportal areas. Ovariectomy decreased ER level (from 10.2±3.2 to4.3±1.3) and ER mRNA expression (from 12.8±2.1 to 10.9±1.3)significantly (P＜0.05). Hepatic ER and ER mRNA concentrations were elevated after treatment with estradiol in both ovariectomy (15.8±2.4, 20.8±3.1) and sham ovariectomy(18.7±3.8, 23.1±3.7) fibrotic groups (P＜ 0.05).CONCLUSION: The increase in hepatic ER and mRNA expression may be part of the molecular mechanisms underlying the suppressive effect of estradiol on liver fibrosis induced by CCl4 administration.     

补肾柔肝方对二甲基亚硝胺诱导大鼠肝纤维化后CTGF mRNA表达的影响

目的:探讨中药复方补肾柔肝方对二甲基亚硝胺(dimethylnitrosamine,DMN)诱导大鼠肝纤维化后肝脏组织结缔组织生长因子(connective tissue growth factor,CTGF) mRNA的表达影响,以了解其对肝纤维化的治疗作用和分子机制.方法:♂ Wjstar大鼠40只,随机分为正常对照组(n=10)、模型组(n=15)及治疗组(n=15).模型组和治疗组以10 mg/kg的剂量腹腔注射DMN,每天1次,每周连续3 d,共4 wk_模型组在造模结束后给予生理盐水ig,而治疗组在造模结束后给补肾柔肝方ig进行治疗干预,用药4 wk第8周末处死全部大鼠,用放射免疫试剂盒方法检测血清胶原成分HA、LN及Ⅳ-C的含量;用HE和天狼猩红染色法观察肝组织的炎症及纤维增生情况.并采用RT-PCR半定量方法,探讨大鼠肝组织CTGFmRNA的表达水平.结果:治疗组大鼠一般状态明显好于模型组:正常组大鼠无死亡,模型组死亡率为40%,治疗组死亡率20%.模型组血清胶原成分HA、LN及Ⅳ-C的含量较正常组显著增高,治疗组较模型组显著下降(HA:319.75±63.23 pg/L vs 434.44±98.81 pg/L;LN:44.83±4.09 pg/L vs 70.67±6.32 pg/L:Ⅳ-C:52.79±5.71 pg/L vs 79.39±10.52 pg/L,均P<0.01).DMN可以成功诱导大鼠肝纤维化,模型组肝组织CTGF mRNA表达明显增强,中药复方治疗组与模型组相比明显减弱(CTGF/β-actin:0.76±0.10 vs1.08±0.17,P<0.01),而正常对照组表达较少.结论:CTGF mRNA的表达可能与肝纤维化的发生密切相关,中药复方补肾柔肝方具有较好的抗肝纤维化作用,可以显著抑制大鼠肝组织CTGF mRNA的表达.     

血吸虫病肝纤维化过程中肝脏前胶原mRNA的表达变化研究

目的阐明血吸虫病肝纤维化过程中,肝脏前胶原基因表达的动态变化规律。方法应用Ｎｏｒｔｈｅｒｎ核酸分子杂交技术,检测血吸虫感染小鼠在肝纤维化过程中肝脏前胶原ｍＲＮＡ表达量的变化。结果小鼠感染血吸虫后６周时,肝内α１（Ⅰ）和α１（Ⅲ）前胶原ｍＲＮＡ的表达量即有显著增加,８周时,α１（Ⅰ）前胶原ｍＲＮＡ的表达量已达高峰,α１（Ⅲ）和α１（Ⅵ）前胶原ｍＲＮＡ的表达高峰在１０周时出现。１２～１６周时,三者的表达量略有下降,２０周时均又回升。在整个肝纤维化过程中,α１（Ⅰ）前胶原ｍＲＮＡ呈优势表达,α１（Ⅲ）前胶原ｍＲＮＡ次之,α１（Ⅵ）前胶原ｍＲＮＡ的表达量较前二者稍低,但其表达水平的增幅最大。结论肝脏前胶原ｍＲＮＡ表达水平的检测能敏感地反映肝纤维化的趋向,α１（Ⅵ）前胶原ｍＲＮＡ的表达在肝纤维化形成中可能起重要作用。     

Effects of tobacco smoke condensate on estrogen receptor-alpha gene expression and activity

Metallo-estrogens are a new class of potent environmental estrogens. This study investigates whether tobacco smoke condensate (TSC), which contains metals and metalloids, elicits estrogen-like effects at environmentally relevant doses. Treatment of human breast cancer cells, MCF-7, with 40 microg/ml TSC resulted in a 2.5-fold stimulation of cell growth. TSC decreased the concentration of estrogen receptor (ER)-alpha protein and mRNA (63 and 62%, respectively), and increased the expression of the estrogen-regulated genes, progesterone receptor and pS2 (5- and 2-fold, respectively). In addition, TSC activated ER-alpha in COS-1 or CHO cells transiently transfected with wild-type ER-alpha and an ERE-CAT or an ERE-luciferase reporter gene (11- and 6-fold, respectively). TSC also activated a chimeric receptor (GAL-ER) containing the hormone binding domain of ER-alpha (3.5-fold). It blocked the binding of estradiol to the receptor without altering the affinity of estradiol (K(d) = 2.2-6.8 x 10(-10) m). Transfection assays with ER-alpha mutants identified C381, C447, H524, N532, E523, and D538 in the hormone binding domain as important for activation by TSC. In ovariectomized rats, low doses of TSC [10 or 20 mg/kg body weight (bw)] increased uterine wet weight (1.7- and 2.1-fold), and induced the expression of progesterone receptor and complement C3 in the uterus (2- and 26-fold) and mammary gland (4.4- and 15-fold). Both the in vitro and in vivo TSC effects were blocked by the antiestrogen ICI 182,780, suggesting the involvement of ER. Collectively, these results provide strong evidence that low doses of TSC, acting through the hormone binding domain, exert estrogen-like effects in cell culture and animals.     

雌二醇对肝纤维化大鼠Ⅰ,Ⅲ型胶原及TGFβ1表达的影响

目的:观察雌二醇对肝纤维化大鼠肝脏胶原沉积和转化生长因子β_1(TGF β_1)表达的影响,研究雌激素对肝纤维化形成的抑制作用,并探讨其可能的机制。方法:设立模型组、治疗对照组、雌二醇组和正常对照组,以四氯化碳复合因素诱导大鼠肝纤维化动物模型,雌二醇组在四氯化碳应用的同时皮下注射苯甲酸雌二醇1mg/kg,2次/wk,共8wk。大鼠肝脏HE染色与Masson染色,分级观察肝组织的炎性坏死与胶原纤维沉积变化,并观察对大鼠肝纤维化形成过程中肝脏表达Ⅰ,Ⅲ型胶原蛋白及对TGF β_1的影响。结果:与正常对照组比较,四氯化碳模型大鼠出现典型的肝纤维化表现,肝脏胶原纤维间隔广泛形成,肝小叶与肝窦内胶原增生沉积明显,Ⅰ,Ⅲ型胶原(0.58±0.26vs 6.34±2.24,1.07±0.49 vs 5.28±1.28,P值均<0.001)及TGF β_1基因表达明显增多;雌二醇应用可以明显减轻肝脏内胶原纤维增生沉积P<0.05),抑制肝脏Ⅰ、Ⅲ型胶原蛋白(2.47±0.76 vs 6.34±2.24,3.02±1.20 vs5.28±1.28,P值均<0.05)及TGF β_1的合成表达。结论:雌二醇可抑制肝纤维化大鼠肝脏Ⅰ,Ⅲ型胶原蛋白及TGF β_1的合成表达,发挥对肝纤维化的抑制作用。     

大黄素抗肝纤维化作用的实验研究

目的 研究大黄素抗肝纤维化作用。方法 采用40％四氯化碳给大鼠皮下注射制备肝纤维化模型并以小、中和大剂量大黄素（20mg/kg、40mg/kg和80mg/kg体重）干预,测定肝功能、血清透明质酸、层粘连蛋白及肝组织胶原蛋白,并通过光镜观察肝组织病理变化,免疫组织化学法检测肝组织α－肌蛋白表达。结果 大黄素组较模型组：（1）肝功能明显改善：谷氨酸转氨酶及碱性磷酸酶显著降低,总蛋白及白蛋白显著升高;（2）血清透明质酸及层粘连蛋白显著降低;（3）肝组织胶原蛋白含量明显减少;（4）肝组织纤维化程度明显改善;（5）肝组织α－肌动蛋白表达减少。结论 大黄素具有抗肝纤维化作用。     

纤维化大鼠肝星状细胞IL-10/IL-10R的表达及IL-10干预的影响

目的研究实验性肝纤维化大鼠肝星状细胞白介素10(IL-10)/白介素10受体(IL-10R)表达及IL-10干预对其表达的影响。方法60只清洁级SD大鼠,随机分为正常对照(A组)、肝纤维化(B组)和IL-10干预组(C组)。B组和C组以CCl4腹腔内注射构建肝纤维化模型,C组并予IL-10腹腔内注射干预造模。于第7周和第11周分别随机选取一批大鼠,分离肝星状细胞,抽提总RNA,以半定量RT-PCR法检测各组HSC中IL-10和IL-10R mRNA表达情况。结果病理组织学证实肝纤维化模型构建成功,原代HSC分离培养成功,IL-10干预可以减轻其肝纤维化程度。肝纤维化时IL-10和IL-10R的mRNA表达均较正常组有所增强,但随着肝纤维化进展,IL-10的表达逐渐减弱。IL-10干预可以使二者的表达上调,以第11周时变化最为显著。结论大鼠肝HSC可表达IL-10及IL-10R,IL-10可作用于HSC而发挥抗肝纤维化作用。     

螺内酯防治实验性大鼠肝纤维化作用研究

探讨螺内酯对肝纤维化的防治作用。雄性SD大鼠46只,随机分成对照组(6只);模型组(30只),复合因素制成肝纤维化模型;螺内酯预防组(10只),造模方法同模型组,螺内酯每天100mg·11ml灌胃。于第8W末,将模型组(16只)再随机分为A组(肝硬化组)8只,用等量自来水灌胃;B组(螺内酯治疗组)8只,螺内酯灌胃8w。分别观察肝内纤维组织变化。显示螺内酯预防组肝脏I、Ⅲ型胶原、纤维连接蛋白、层粘蛋白增生明显减低(PO.05)。     

自拟中药对CCl4诱导的肝纤维化大鼠肝组织Smad3表达的影响

目的研究自拟中药对CCl4诱导的大鼠肝纤维化肝脏Smad3表达的影响，探讨其抗纤维化的作用机制。方法Wistar大鼠40只，随机分成正常对照组、模型组、自拟中药组、鳖甲软肝片组，采用CCl4背部皮下注射构建肝纤维化模型，行HE和VG染色，光镜下观察肝组织肝纤维化程度。同时免疫组化SABC方法检测各组Smad3的表达。结果与正常对照组比，模型组Smad3表达明显增强，自拟中药组大鼠肝脏Smad3的表达（0．279±0．085对0．885±0．904，P〈0．05）显著下调。另外，自拟中药组大鼠肝脏肝纤维化病理变化显著改善。结论自拟中药对CCl4诱导的大鼠肝纤维化模型肝脏Smad3表达有明显的下调作用，可能是其抗肝纤维化的主要作用机制之一。     

红景天甙对肝纤维化大鼠肝组织ROCK表达的影响

目的:研究红景天甙对肝纤维化大鼠肝组织ROCK基因表达的影响,了解其干预肝纤维化的机制.方法:健康雄性SD大鼠,随机分为3组:对照组( n = 10),红景天甙干预组( n = 40)和肝纤维化模型组( n = 40).大鼠肝纤维化采用CCl4皮下注射法(300 mL/L,3 mL/kg,每周2次,共8 wk)诱导.红景天甙干预采用腹腔注射法,剂量5 mg/kg,每周2次.肝脏胶原沉积状况采用Masson胶原染色以及HE染色观察,ROCKⅠ、ROCKⅡ表达水平分别采用原位杂交(in situ hybridization,ISH) 和免疫组化(immunohisto chemistry,IH)方法检测.实验图像经电子计算机扫描,数据采用专业图像分析软件统计分析.结果:肝纤维化大鼠肝脏肝细胞坏死、再生明显,假小叶形成,胶原纤维沉积明显增加,肝实质结构紊乱.红景天甙干预组大鼠肝组织胶原沉积明显减少,组织学积分平均胶原面积降低(2.1±0.3 vs 3.6±0.8,74.82±21.51μm2 vs 290.86±89.37 μm2,均P<0.05).与模型组比较,红景天甙干预性治疗组ROCKⅠ,Ⅱ蛋白表达水平均明显下降(0.203±0.068 vs0.357±0.182,0.237±0.056 vs 0.394±0.238,均P<0.05),ROCKⅠ,Ⅱ mRNA表达水平明显下降(0.197±0.019 vs 0.394±0.238,0.185±0.031 vs 0.279±0.112,均P<0.01).结论:红景天甙可能通过调节Rho-ROCK信号传导通路,减少肝脏胶原纤维的合成与沉积,有效干预CCl4诱导的大鼠肝纤维化.     

反义寡核苷酸对大鼠纤维化肝组织c-myb表达的影响

转录因子c-myb是一种与转录活性有关的核因子,在多种组织纤维化包括肝纤维化的形成过程中起重要作用。本研究中探讨了反义c-myb寡核苷酸对在体肝纤维化肝组织c-myb表达及肝纤维化的影响。     

Suppressive effects of estradiol on dimethylnitrosamine-induced fibrosis of the liver in rats

As a model for the analysis of the fibrosuppressive role of estradiol, hepatic fibrosis was induced in male and female rats by the administration of a single dose of dimethylnitrosamine (DMN). The fibrotic response of the male liver after DMN treatment was significantly stronger than that of the female liver. In the male DMN model, estradiol reduced hepatic mRNA for type I and III procollagens and the tissue inhibitor of metalloproteinase-1 (TIMP-1), as well as deposition of type I and III collagen protein total hepatic collagen and malondialdehyde (MDA), a product of lipid peroxidation. Concomitant administration of a neutralizing antibody against rat estradiol enhanced fibrogenesis, as judged by the same parameters. Ovariectomy in the female model had a fibrogenic effect, inducing the hepatic expression of both types of procollagen and TIMP-1; in addition, the number of alpha-smooth muscle actin (alpha-SMA)-positive cells in the liver increased; estradiol replacement was fibrosuppressive in the castrated-female model. In rat hepatic stellate cells incubated in primary culture with estradiol, cell number, type I collagen production, and alpha-SMA expression were all reduced. These findings suggest that estradiol suppressed the induction of hepatic fibrosis, and may in part underlie the more rapid progression in males of hepatic fibrosis and its complications.     

肝纤维化发生发展过程中纤溶酶原激活物抑制剂-1 mRNA及其蛋白表达

[目的]探讨纤溶酶原激活物抑制剂-1（PAI-1）mRNA及其蛋白表达在大鼠肝纤维化发生发展中的作用。[方法]采用复合致病因子四氯化碳、高胆固醇、乙醇喂养大鼠6周、8周，复制肝纤维化、肝硬化动物模型。动物随机分为正常组、肝纤维化组、肝硬化组。测定血浆转氨酶、总胆红素（TB）、透明质酸（HA）以及肝组织内羟脯氨酸（Hyp）水平；免疫组化测定PAI-1、廿平滑肌肌动蛋白（α-SMA）、基质金属蛋白酶抑制剂-1（TIMP-1）、转化生长因子β1（TGF-β1）蛋白表达；RT-PCR测定PAI-1、TGF-β1 mRNA表达。[结果]与正常组相比，肝纤维化、肝硬化组血浆转氨酶、TB、HA、肝组织内Hyp均明显增加。随着肝纤维化的发展，肝组织静SMA、TIMP-1、TGF-β1蛋白表达明显增加，PAI-1、TGF-β1 mRNA表达增加。[结论]肝组织PAI-1 mRNA及其蛋白表达在肝纤维化发生发展过程中发挥着重要作用。减少PAI-1 mRNA及其蛋白表达，可能会减缓肝纤维化的发生发展。     

动态观察CD44及其配体骨调素在大鼠酒精性肝纤维化中的表达

目的动态观察 CD44及其配体骨调素(OPN)在大鼠酒精性肝纤维化形成过程巾的表达并初步探讨其作用。方法用白酒-玉米油-吡唑混合液灌胃建立酒精性肝纤维化大鼠模型,分别于第2、4、8、12、16周处死大鼠,取肝组织作常规 H-E、Masson 胶原染色测定胶原纤维面积百分比,通过免疫组化染色、原位杂交技术动态观察和定量分析肝组织中 CD44、OPN 分布状况。结果灌胃4周时,大鼠肝组织开始出现 CD44和 OPN 表达,表达量分别为7.81±1.20和2.83±0.60,并随时间延长逐渐增多,各时间点差异有统汁学意义(P<0.05),16周时 CD44、OPN 表达量分别为18.21±1.13和21.69±3.47。大鼠肝组织中胶原面积与 CD44和 OPN 强度呈正相关,相关系数分别为0.91和0.88。结论乙醇可使肝组织表达 CD44和 OPN,并且 CD44、OPN 的表达可能与肝纤维化有关。     

壮肝逐瘀煎对肝纤维化大鼠肝转化生长因子β1及其βⅠ型和Ⅱ型受体mRNA表达的影响

[目的]研究中药壮肝逐瘀煎对肝纤维化(hepatic fibrosis,HF)大鼠转化生长因子β1(TGF-β1)及转化生长因子βⅠ、Ⅱ型受体(TβRⅠ/Ⅱ)mRNA表达的影响,探讨其抗HF的作用机制。[方法]采用四氯化碳(CCl4)复合因素HF大鼠模型,予壮肝逐瘀煎灌胃,取血清及肝组织,双抗体夹心ABC-ELISA法测定血清TGF-β1水平,实时荧光定量PCR技术对肝组织TβRⅠ/ⅡmRNA进行定量分析。[结果]观察12周后,与病理模型组大鼠比较,壮肝逐瘀煎治疗组肝小叶结构趋于正常,纤维间隔明显变薄,TGF-β1及TβRⅠ/ⅡmRNA明显降低(P<0.01)。[结论]壮肝逐瘀煎能够显著改善HF大鼠肝组织的病理变化,具有明显的抗HF作用,其作用机制可能与壮肝逐瘀煎抑制TGF-β1及TβRⅠ/ⅡmRNA表达有关。     

大麻素受体1和细胞外信号调节激酶在肝纤维化小鼠肝组织中的表达及意义

目的研究大麻素受体1(CB1)mRNA在肝纤维化形成过程中表达的变化,从基因转录水平探讨其与细胞外信号调节激酶(ERK)的关系。方法采用10%四氯化碳腹腔注射制备肝纤维化模型,分别于造模第2、4、6、8周留取小鼠的肝组织及血清。通过病理对肝组织进行形态学观察,荧光定量RT-PCR检测CB1 mRNA和ERK mRNA水平,生化法检测血清中ALT和AST的含量,放射免疫法检测血清中透明质酸(HA)的含量。结果与正常对照组相比,各模型组小鼠肝组织中CB1 mRNA和ERK mRNA含量显著升高(P<0.05),而且随造模时间的延长,CB1 mRNA和ERK mRNA含量亦逐渐增高,各组之间差异有统计学意义(P<0.05)。各模型组小鼠血清中ALT、AST及HA的水平随造模时间延长而不断升高,各组间差异具有统计学意义(P<0.05)。CB1 mRNA的含量不但与血清中ALT、AST、HA的含量呈显著正相关(r=0.741、0.763、0.769,P均<0.01),而且与肝组织中ERK mRNA含量也呈显著正相关(r=0.789,P均<0.01)。结论CB1可能通过激活ERK,以ERK通路诱导肝星状细胞(HSC)增殖,促进肝纤维化的形成。