奥曲肽抗肝纤维化的实验研究

目的 观察奥曲肽(Oct)对大鼠实验性肝纤维化的治疗效果,并探讨其作用机制。方法 用四氯化碳诱导大鼠肝纤维化模型,将实验动物随机分为正常对照组、治疗前模型组、治疗后模型组和Oct治疗组。Oct治疗组给予Oct(50ng/100g)皮下注射,每日2次,连续用药30d,分别用放射免疫法检测血清层黏连蛋白(LN)、Ⅲ型前胶原(PC Ⅲ)及透明质酸(HA)。VG染色法组织切片观察组织病理变化,免疫组织化学法检测肝组织平滑肌肌动蛋白(α-SMA)和转化生长因子β_1(TGFβ_1)表达及逆转录聚合酶链反应法检测Ⅰ型和Ⅲ型前胶原mRNA表达。结果 治疗前和治疗后模型组大鼠血清HA(ng/L)为121.8±9.5和110.3±13.4,正常对照组为33.1±3.7、LN(μg/L)为85.7±12.1和78.2±7.9,正常对照组为37.1±6.3、PC Ⅲ(ng/L)为35.9±3.5和33.7±2.6,正常对照组为15.6±2.8。Oct组大鼠血清HA为55.8±7.2、LN为43.1±3.4、PC Ⅲ为27.8±3.4,与模型组大鼠比较,差异有显著性,t=2.76～11.07,P<0.05。Oct能显著降低纤维化大鼠肝组织纤维化积分,下调α—SMA和TGFβ_1蛋白质及I型和III前胶原mRNA表达水平。结论 Oct抑制肝星状细胞激活和转化、下调TGFβ_1蛋白质及I型和III型前胶mRNA表达而发挥抗肝纤维化作用。     

丹酚酸B对肝纤维化大鼠TGF-β1、MMP-2和TIMP-2表达的影响

目的:观察丹酚酸B盐(salvianolic acid B,SA-B)对肝纤维化大鼠肝组织转化生长因子-β1 (TGF-β1)、基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶组织抑制因子-2(TIMP-2)表达的影响,并探讨SA-B抗肝纤维化的可能机制.方法:♂SD大鼠30只随机分为正常对照组、模型组和丹酚酸B治疗组,以5 mL/L的二甲基亚硝胺(DMN)建立肝纤维化模型,治疗组在造模4 wk后给予SA-B治疗4 wk.应用HE,Masson染色观察肝组织病理纤维化分级,全自动生化分析仪检测ALT、AST和Alb,放免法检测HA和LN,S-P免疫组织化学方法检测TGF-β1、MMP-2和TIMP-2蛋白质的表达.结果:与模型组相比,SA-B能改善肝纤维化大鼠肝脏病理组织学结构,治疗组血清ALT、AST、HA和LN水平明显减低(87.0±28.7 U/L vs 190.4±27.4 U/L.85.6±25.3 U/L vs 178.2±15.9 U/L,179.7±32.8 mg/L vs 433.3±86.1 mg/L,135.6±21.1 mg/L vs 224.7±29.2 mg/L,均P<0.01),经SA-B干预后TGF-β1和TIMP-2表达明显下降,与模型组相比有显著性差异(18.53±2.54 vs 12.78±2.65,21.88±3.83 vs 14.69±4.51,均P<0.01),而MMP-2表达水平无明显变化.结论:SA-B能改善肝纤维化大鼠肝脏病理组织学结构,可能通过抑制TGF-β1和TIMP-2表达而促进肝纤维化的逆转.     

β胡萝卜素对酒精性肝纤维化的防治效果

目的:研究β胡萝卜素防治酒精性肝纤维化患者的效果.方法:203例受试者,其中酒精性肝纤维化67例,非酒精性肝纤维化74例,慢性肝炎肝纤维化患者62例.另选60例健康体检者.采用肝纤维化血清透明质酸(HA)、层黏蛋白(LN)、Ⅳ型前胶原(Ⅳ-C)、Ⅲ型前胶原肽(PCⅢ)、血清结缔组织生长因子(CTGF)、血小板衍生生长因子-BB(PDGF-BB)、金属蛋白酶抑制剂-1(TIMP-1)、转化生长因子-β1(TGF-β1)、彩超进行组内组间比较,分析β胡萝卜素防治酒精性肝纤维化患者的效果.结果:HA、LN、Ⅳ-C、PCIII在肝纤维化S0-S1、S2-S4期患者治疗前较健康体检者均有显著性差异(均P<0.01).治疗后,除Ⅳ-C(μg/L)S2-S4期患者较健康体检者有统计学差异外(95.57±15.47,100.16±13.70,96.89±16.41vs84.05±24.16,均P<0.05),其他均无差异.CTGF、PDGF-BB、TIMP-1、TGF-β1在肝纤维化S0-S1、S2-S4期患者治疗较健康体检者均有统计学差异(均P<0.01).治疗后,除CTGF(μg/L)、PDGF-BB(μg/L)S2-S...     

丹芍化纤胶囊对肝纤维化大鼠PDGFR-β和p-ERK1/2的影响

目的:通过观察丹芍化纤胶囊对肝纤维化大鼠PDGFR-β和p-ERK1/2的影响,探索其抗肝纤维化的可能机制.方法:♂SD大鼠55只,体质量180-220g,随机分为正常组、模型组、预防组.除正常组外,其余均采用四氯化碳、高脂饮食及乙醇复合因素复制肝纤维化模型,造模同时预防组每日一次予以0.8 g/kg丹芍化纤自来水悬液灌胃,模型组、正常组予以等体积自来水灌胃,持续8 wk.造模结束后肝组织HE染色病理检查,免疫组化检测肝组织PDGFR-β、蛋白印迹检测肝组织p-ERK1/2在各组表达,生化法检测各组血清透明质酸(HA)、层黏蛋白(LN)、Ⅲ型前胶原(PⅢP)、白蛋白(ALB)、总蛋白(TP),计算白球比(A/G).结果:造模8 wk后经病理学证实模型大鼠形成典型的肝纤维化,模型成功.与正常组相比,模型组肝组织PDGFR-β、p-ERK1/2及血清HA、LN、PⅢP均有显著升高,ALB、A/G显著降低(PDGFR-β:184.6±8.5 vs 89.6±5.8,P<0.05;p-ERK1/2:360.0±14.5 vx 15.4±2.1,P<0.05;HA:517.5±91.5μg/L vs 254.4±33.1μg/L,P<0.05;LN:58.4±11.3μg/L vs 37.3±9.8μg/L,P<0.05:PⅢP:36.9±5.6μg/L vs 4.7±1.5μg/L,P<0.05;ALB:27.4±4.9 g/L vs 42.1±1.6 g/L,P<0.05;A/G:0.89±0.08 vs 1.38±0.09,P<0.05);与模型组相比,预防组肝组织PDGFR-β、p-ERK1/2及血清HA、LN、PⅢP均有显著降低,ALB、A/G显著升高(PDGFR-β:91.1±6.3 vs 184.6±8.5,P<0.05;p-ERK1/2:253.8±18.2 vs 360.0±14.5,P<0.05;HA:322.9±41.4μg/L vs 517.5±91.5μg/L,P<0.05;LN:46.0±9.4μg/L vs 58.4±11.3μg/L,P<0.05;PⅢP:14.5±2.4μg/L vs 36.9±5.6μg/L,P<0.05;ALB:37.2±2.8g/L vs 27.4±4.9g/L,P<0.05;A/G:1.18±0.13 vs 0.89±0.08,P<0.05).结论:PDGFR-β及p-ERK1/2在肝纤维化形成中起重要作用,丹芍化纤胶囊具有良好的预防肝纤维化形成的作用,其降低PDGFR-β及p-ERK1/2在肝组织的表达可能是其作用机制之一.     

丹芍化纤胶囊对肝纤维化大鼠PDGFR-β和p-ERK1/2的影响

目的通过观察丹芍化纤胶囊对肝纤维化大鼠PDGFR-β和p-ERK1/2的影响,探索其抗肝纤维化的可能机制.方法♂SD大鼠55只,体质量180-220 g,随机分为正常组、模型组、预防组.除正常组外,其余均采用四氯化碳、高脂饮食及乙醇复合因素复制肝纤维化模型,造模同时预防组每日一次予以0.8 g/kg丹芍化纤自来水悬液灌胃,模型组、正常组予以等体积自来水灌胃,持续8 wk.造模结束后肝组织HE染色病理检查,免疫组化检测肝组织PDGFR-β、蛋白印迹检测肝组织p-ERK1/2在各组表达,生化法检测各组血清透明质酸(HA)、层黏蛋白(LN)、Ⅲ型前胶原(PⅢP)、白蛋白(ALB)、总蛋白(TP),计算白球比(A/G).结果造模8 wk后经病理学证实模型大鼠形成典型的肝纤维化,模型成功.与正常组相比,模型组肝组织PDGFR-β、p-ERK1/2及血清HA、LN、PⅢP均有显著升高,ALB、A/G显著降低(PDGFR-β1 84.6±8.5 vs 89.6±5.8,P＜0.05;p-ERK1/2360.0±14.5 vs 15.4±2.1,P＜0.05;HA517.5±91.5 μg/L vs 254.4±33.1 μg/L,P＜0.05;LN58.4±11.3 μg/L vs 37.3±9.8 μg/L,P＜0.05;PⅢP36.9±5.6 μg/L vs 4.7±1.5 μg/L,P＜0.05;ALB27.4±4.9 g/L vs 42.1±1.6 g/L,P＜0.05;A/G0.89±0.08 vs 1.38±0.09,P＜0.05);与模型组相比,预防组肝组织PDGFR-β、p-ERK1/2及血清HA、LN、PⅢP均有显著降低,ALB、A/G显著升高(PDGFR-β91.1±6.3vs 184.6±8.5,P＜0.05;p-ERK1/2253.8±18.2vs 360.0±14.5,P＜0.05;HA322.9±41.4 μg/Lvs 517.5±91.5 μg/L,P＜0.05;LN46.0±9.4μg/L vs 58.4±11.3 μg/L,P＜0.05;PⅢP14.5±2.4 μg/L vs 36.9±5.6 μg/L,P＜0.05;ALB37.2±2.8 g/L vs 27.4±4.9 g/L,P＜0.05;A/G1.18±0.13 vs 0.89±0.08,P＜0.05).结论PDGFR-β及p-ERK1/2在肝纤维化形成中起重要作用,丹芍化纤胶囊具有良好的预防肝纤维化形成的作用,其降低PDGFR-β及p-ERK1/2在肝组织的表达可能是其作用机制之一.     

培哚普利和缬沙坦对肝纤维化大鼠TGF β1及其Ⅱ型受体mRNA与Smad3、7表达的影响

目的观察培哚普利和缬沙坦抗大鼠肝纤维化的疗效和对转化生长因子β1(TGFβ1)及其Ⅱ型受体(TGFβ1RⅡ)mRNA与Smad3、smad17的影响。方法大鼠随机分为止常对照组，模型组、培哚普利治疗组和缬沙坦治疗组。四氯化碳皮下注射诱导大鼠肝纤维化模型，治疗组于造模第4周开始分别予以培哚普利和缬沙坦灌胃。采用RT-PCR检测肝组织TGFβ1与TGFβ1RⅡ mRNA；免疫组织化学技术检测TGFβ1、Smad3及smad7在肝内的表达及定位，检测肝组织病理改变，检测肝组织羟脯氢酸和血清透明质酸。结果与模型组大鼠比较，经培哚普利或缬沙坦治疗大鼠肝内TGFβ1与TGFβ1RⅡ mRNA表达明显下降、以及smad3蛋白表达明显降低，而Smad7的表达增加。TGFβ1与Smad3的免疫阳性反应信号主要位于纤维间隔中的细胞浆，Smad7主要在肝细胞浆表达，上述物质在两种药物组之间表达差异无显著性。培哚普利或缬沙坦治疗后肝组织羟脯氨酸及血清透明质酸含量较模型组显著降低；肝小叶结构均趋于正常，纤维间隔明显变溥。结论培哚普利或缬沙坦均能有效地减轻肝纤维化大鼠的肝脏损伤及纤维化程度，其机制可能与直接或间接抑制肝内TGFβ1与TGFβ1RⅡ mRNA及Smad3表达，并促进Smad7表达有关。     

柴胡疏肝散抗肝纤维化的实验研究

目的探讨柴胡疏肝散对肝纤维化大鼠模型α-SMA、TGF-β1的影响及其抗纤维化的机制。方法采用40?l4皮下注射，制备肝纤维化模型并以柴胡疏肝散干预，测定肝功能、血清透明质酸（HA）、层黏连蛋白（LN）及肝组织羟脯氨酸（HYP），光镜观察肝组织病理变化，免疫组织化学法检测肝组织α-SMA、TGF-β1表达，RT-PCR检测TGF-β1 mRNA的表达。结果柴胡疏肝散组较模型组：肝功能明显改善，血清HA及LN显著降低，肝组织HYP含量明显少，肝组织纤维化程度明显改善，肝组织α-SMA及TGF-β1表达减少。结论柴胡疏肝散对CCl4诱导的大鼠肝纤维化有良好的防治作用。     

银杏叶提取物与秋水仙碱对大鼠肝纤维化预防作用的比较

目的比较银杏叶提取物(GBE)和秋水仙碱对实验性大鼠肝纤维化的预防作用。方法SD雄性大鼠40只随机分为4组:正常组(n=10)、模型组(n=10)、秋水仙碱预防组(n=10)及GBE预防组(n=10)。模型组、秋水仙碱预防组及GBE预防组给予500 mL/L CCl4腹腔注射,1 mL/kg,每周2次,共8周,秋水仙碱预防组每天同时给予秋水仙碱灌胃,0.2 mg/kg,GBE预防组每天同时给予GBE灌胃,0.3 g/kg。实验结束后,心脏取血分离血清行肝功能生化指标检测,处死动物取肝脏甲醛固定,常规行HE染色,免疫组化检测α-SMA和TGF-β1。结果光镜下组织学检查纤维化分级GBE预防组低于秋水仙碱预防组(P0.05),肝功能生化指标检测GBE预防组优于秋水仙碱预防组[ALT:(168.4±34.6)U/Lvs(210.6±40.8)U/L;AST:(318.8±62.5)U/Lvs(511.2±53.2)U/L;ALB:(31.0±2.1)g/Lvs(28.1±2.0)g/L;P均0.05],免疫组化检测α-SMA及TGF-β1蛋白表达GBE预防组低于秋水仙碱预防组(α-SMA:29.3±1.5vs5.1±2.2;TGF-β1:14.5±0.9vs28.6±0.9)。结论GBE预防大鼠肝纤维化的作用优于秋水仙碱,GBE作为一种新的抗肝纤维化药物有很好的研究和应用前景。     

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.     

地龙2号抑制大鼠肝纤维化的研究

目的 研究地龙2号抑制大鼠肝内纤维组织形成的作用。方法 雄性Wistar大鼠,随机分成6组:正常组:不做药物处理;阴性对照组:皮下注射花生油,单灌蒸水;模型组:皮下注射CCl4,每日单蒸水灌胃;阳性对照组:造模方法同模型组,用秋水仙碱每日0.1 mg/kg灌胃;地龙大剂量组:造模同时用地龙2号每日50mg/kg灌胃;地龙小剂量组:造模同时用地龙2号每日25mg/kg灌胃。于8周末处死大鼠,取血测血清ALT、AST、AST/ALT,放射免疫法检测透明质酸(HA)和层黏连蛋白(LN),取肝组织作HE染色,病理分级。结果 地龙2号组与模型组相比,病理示肝纤维化程度明显减轻(P<0.05),肝细胞损害亦轻(AST/ALT大剂量组P<0.05,小剂量组P<0.01),且反应肝纤维化的指标HA及LN均显著降低(P<0.01)。结论 地龙2号有明显抑制大鼠肝纤维化形成的作用。     

ACEI attenuates the progression of CCl4-induced rat hepatic fibrogenesis by inhibiting TGF-β1, PDGF-BB, NF-κB and MMP-2,9

AIM: Angiotensin II has pro-fibrotic function in the liver. Blockade of the renin-angiotensin-aldosterone-system (RAAS) attenuates hepatic fibrosis. The aim of the present study was to determine the mechanism of angiotensin-converting enzyme inhibitor (ACEI) on the progression of rat hepatic fibrosis. METHODS: Forty male Wistar rats were divided into three groups. Model group (Mo): The rats were injected subcutaneously with 40% of CCl(4) 0.25 mL/100 g. Perindopril group (Pe): The rats were injected subcutaneously with 40% of CCl(4). Perindopril, equivalent to 2 mg/(kg.d), was administrated. Control group (Nc): the rats were treated with olive oil only. After 4 and 6 wk, the rats were killed. The liver sections were stained with Masson. The protein expressions of AT1R, TGF-beta1 and PDGF-BB were examined by Western blot. Nuclear factor kappaB (NF-kappaB) DNA binding activity was examined by EMSA (Electrophoretic gel mobility shift assay). Matrix metalloproteinase-2,9 (MMP-2,9) activity was assessed by zymography. Serum laminin (LN) and hyaluronic acid (HA) were measured using radioimmunoassays. RESULTS: Using Western blot, we clearly provided direct evidence for the expression of AT1R in liver. The expression was up-regulated when fibrogenesis occurred. Perindopril treatment significantly reduced mean fibrosis score, protein levels of AT1R, TGF-beta1 and PDGF-BB, serum levels of HA and LN, and the activity of MMP-2,9. NF-kappaB DNA binding activity markedly increased in model group, perindopril treatment considerably reduced NF-kappaB DNA binding activity. CONCLUSION: Perindopril attenuates CCl(4)-induced hepatic fibrogenesis of rat by inhibiting TGF-beta1, PDGF-BB, NF-kappaB and MMP-2,9.     

ACEI attenuates the progression of CCl4-induced rat hepatic fibrogenesis by inhibiting TGF-β1, PDGF-BB, NF-κB and MMP-2,9

AIM: Angiotensin Ⅱ has pro-fibrotic function in the liver.Blockade of the renin-angiotensin-aldosterone-system (RAAS) attenuates hepatic fibrosis. The aim of the present study was to determine the mechanism of angiotensinconverting enzyme inhibitor (ACEI) on the progression of rat hepatic fibrosis.METHODS: Forty male Wistar rats were divided into three groups. Model group (Mo): The rats were injected subcutaneously with 40% of CCl4 0.25 mL/100 g. Perindopril group (Pe): The rats were injected subcutaneously with administrated. Control group (Nc): the rats were treated with olive oil only. After 4 and 6 wk, the rats were killed.The liver sections were stained with Masson. The protein expressions of AT1R, TGF-β1 and PDGF-BB were examined by Western blot. Nuclear factor κB (NF-κB) DNA binding activity was examined by EMSA (Electrophoretic gel mobility shift assay). Matrix metalloproteinase-2,9(MMP-2,9) activity was assessed by zymography. Serum laminin (LN) and hyaluronic acid (HA) were measured using radioimmunoassays.RESULTS: Using Western blot, we clearly provided direct evidence for the expression of AT1R in liver. The expression was up-regulated when fibrogenesis occurred. Perindopril treatment significantly reduced mean fibrosis score,protein levels of AT1R, TGF-β1 and PDGF-BB, serum levels of HA and LN, and the activity of MMP-2,9. NF-κB DNA binding activity markedly increased in model group, perindopril treatment considerably reduced NF-κB DNA binding activity.CONCLUSION: Perindopril attenuates CCl4-induced hepatic fibrogenesis of rat by inhibiting TGF-β1, PDGF-BB,NF-κB and MMP-2,9.     

八项肝纤维化血清标志物比较研究

目的比较血清血小板衍生生长因子-BB(PDGF-BB)、转化生长因子-β1(TGF-B1)、基质金属蛋白酶抑制剂-1(TIMP-1)、基质金属蛋白酶-1(MMP-1)、透明质酸(HA)、Ⅲ型前胶原(PC Ⅲ)、Ⅳ型胶原(C Ⅳ)和层黏连蛋白(LN)及外周血单个核细胞(PBMC)内TIMP-1 mRNA、MMP-1 mRNA在肝纤维化中的诊断价值。方法常规肝穿活检、组织病理学诊断；RT-PCR检测PBMCs中MMP-1 mRNA、TIMP-1 mRNA水平；酶标法检测血清PDGF-BB、TGF-β1、TIMP-1和MMP-1含量；放射免疫法检测血清HA、PC Ⅲ、C-Ⅳ和LN含量。结果经ROC曲线分析，血清PDGF-BB、TIMP-1、HA、PC Ⅲ、C-Ⅳ、LN和TIMP-1 mRNA的AUC分别为0．985、0．726、0．318、0．728、0．727、0．583、0．463、0．876；血清PDGF-BB和PBMCs中TIMP-1 mRNA的灵敏度和特异度分别为90％、95％，73．7％、100％；两者联合检测的灵敏度为97．4％，特异度为95．0％。结论八项指标中，血清PDGF-BB的诊断价值最大。在筛选肝纤维化患者时，以血清PDGF-BB、PBMC中TIMP-1 mRNA联合检测最佳。     

Diagnostic value of platelet derived growth factor-BB, transforming growth factor-β1,matrix metalloproteinase-1, and tissue inhibitor of matrix metaUoproteinase-1 in serum and peripheral blood mononuclear cells for hepatic fibrosis

AIM: Noninvasive diagnosis of hepatic fibrosis has become the focus because of the limited biopsy, especially in the surveillance of treatment and in screening hepatic fibrosis. Recently, regulatory elements involved in liver fibrosis, such as platelet derived growth factor-BB (PDGF-BB), transforming growth factor-β1(TGF-β1), matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), have been studied extensively. To determine whether these factors or enzymes could be used as the indices for the diagnosis of hepatic fibrosis, we investigated them by means of receiver operating characteristic (ROC) curve. METHODS: Serum samples from sixty patients with chronic viral hepatitis B and twenty healthy blood donors were assayed to determine the level of PDGF-BB, TGF-β1, MMP-1, and TIMP-1 with ELISA, and HA, PCIII, C-IV, and LNlevel with RIA. The message RNA (mRNA) expression of TIMP-1 and MMP-1 in peripheral blood mononuclear cells (PBMCs) was detected by RT-PCR and Northern blot hybridization. Liver biopsy was performed in all patients. The biopsy samples were histopatholocjically examined. The trial was double-blind controlled. RESULTS: The serum level of PDGF-BB, TIMP-1, the ratio of TIMP-1 and MMP-1 (TIMP-1/MMP-1), mRNA expression of TIMP-1 (TIMP-lmRNA), and the ratio of TIMP-lmRNA and MMP-lmRNA (TIMP-lmRNA/MMP-lmRNA) in patients was significantly higher than those in the healthy blood donors (t=2.514-11.435, P=0.000-0.016). The serum level of PDGF-BB, TIMP-1, TIMP-1/MMP-1, and TIMP-lmRNA was positively correlated with fibrosis stage and inflammation grade (r=0.239-0.565, P=0.000-0.033), while the serum level of MMP-1 was negatively correlated with fibrosis stage and inflammation grade, and TIMP-lmRNA/MMP-lmRNA was positively correlated with inflammation grade. Through the analysis by ROC curve, serum PDGF-BB was the most valuable marker, and its sensitivity was the highest among the nine indices. The markers with the highest specificity were TIMP-lmRNA and TIMP-lmRNA/MMP-lmRNA in PBMCs. The area under the curve (AUC) of PDGF-BB, TIMP-lmRNA, TIMP-lmRNA/MMP-lmRNA, TIMP-1/MMP-1, HA, PCIII, TIMP-1, C-IV, and LN was 0.985, 0.876, 0.792, 0.748, 0.728, 0.727, 0.726, 0.583, and 0.463, respectively. The sensitivity and the specificity in the parallel test was 99.0% and 95.0 % when serum PDGF-BB, TIMP-lmRNA and TIMP-lmRNA/MMP-lmRNA was detected simultaneously. CONCLUSION: Serum level of PDGF-BB, TIIVIP-lmRNA, TIMP-lmRNA/MMP-lmRNA in PBMCs, and serum level of TIMP-1 and TIMP-1/MMP-1 can be used as the indices for the diagnosis of hepatic fibrosis, but the former three are more useful. The combination of serum PDGF-BB, TIMP-lmRNA and TIMP-lmRNA/MMP-lmRNA in PBMCs is even more efficient in screening liver fibrosis.     

Effects of AT1 receptor antagonist, losartan, on rat hepatic fibrosis induced by CCl(4)

AIM:To investigate effect of losartan, an AT1 receptor antagonist, on hepatic fibrosis induced by CCl(4); and to determine whether or not AT1 receptors are expressed on hepatic stellate cells. METHODS AND RESULTS:Fifty male Sprague-Dawley rats, weighing (180 plus minus20)g, were randomized into five groups (control group, model group, and three losartan treated groups), in which all rats were given the subcutaneous injection of 40% CCl(4)(every 3 days for 6 weeks) except for rats of control group. Rats of losartan-treated groups were treated with losartan (20 mg/kg, 10 mg/kg, 5 mg/kg, daily gavage). After 6 weeks liver tissue and serum samples of all rats were examined. Serum hyaluronic acid (HA), procollagen type III (PC III) were detected by radioimmunoassays. van Giesion collagen staining was used to evaluate the extracellular matrix of rats with liver fibrosis. The expression of AT1 receptors, transforming growth factor-beta (TGF-beta), and alpha-smooth muscle actinalpha-SMA) in liver tissue were determined by immunohistochemical techniques. Compared with model group, serum ALT and AST of losartan-treated groups were significantly reduced (italic>t = 4.20,P < 0.01 and italic>t = 4.57,P < 0.01). Serum HA and PC III also had significant differences (italic>t = 3.53,P<0.01 and t=2.20, P<0.05). The degree of fibrosis was improved by losartan and correlated with the expressions of AT1 receptors, TGF-beta, and alpha-SMA in liver tissue.CONCLUSION:AT1 receptor antagonist, losartan, could limit the progression of the hepatic fibrosis induced by CCl(4). The mechanism may be related to the decrease in the expression of AT1 receptors and TGF-beta, ameliorating the injury of hepatocytes; activation of local renin-angiotensin system might relate to hepatic fibrosis; and during progression of fibrosis, activated hepatic stellate cells might express AT1 receptors.     

Effects of perindopril and valsartan on expression of transforming growth factor-beta-Smads in experimental hepatic fibrosis in rats

BACKGROUND: Previous studies have shown that the renin-angiotensin system (RAS) plays an important role in the pathogenesis of hepatic fibrosis, and blockers of the RAS may be active as an antifibrogenic goal. However, the potential role of RAS inhibition on expression transforming growth factor (TGF)-beta-Smads in hepatic fibrosis remains unknown. The aim of this study was to investigate the effect and mechanism of an angiotensin-converting enzyme inhibitor (perindopril) and an angiotensin II receptor blocker (valsartan) on TGF-beta1 and TGF receptor II (TRII) mRNA, Smad3 and Smad7 in fibrotic hepatic livers in rats. METHODS: Sixty Wistar rats were randomly divided into four study groups (n = 15 for each group), including normal controls, hepatic fibrosis models, and two treated groups with either perindopril or valsartan, starting from the fourth week after being exposed to carbon tetrachloride (CCl(4)) for 4 weeks. The levels of TGF-beta and TRII mRNA in liver tissue were analyzed by RT-PCR. The expressions of TGF-beta1, Smad3 and Smad7 in liver tissues were evaluated by immunohistochemistry. The liver histopathology was examined by hematoxylin and eosin (HE) staining and by electron microscopy, respectively. The liver function and serum hyaluronic acid were also assayed by biochemistry and radioimmunoassay. RESULTS: Compared with the hepatic fibrosis models, the levels of TGF-beta1, TRII mRNA and the expression Smad3 significantly decreased in the two treated groups, and the expression of Smad7 was significantly increased in the liver of rats treated with perindopril or valsartan (P < 0.05 or P < 0.01). The histological changes and ultrastructure of fibrotic liver, liver function and hyaluronic acid also remarkably improved in the treated rats. CONCLUSIONS: The angiotensin-converting enzyme inhibitors perindopril and valsartan have a protective effect on liver injury and can ameliorate hepatic fibrosis in rats induced by CCl(4). The mechanisms may be associated with their effects of down-regulating TGF-beta1, TRII mRNA and smad3, and up-regulating Smad7.     

Diagnostic value of platelet derived growth factor-BB, transforming growth factor-β1,matrix metalloproteinase-1, and tissue inhibitor of matrix metalloproteinase-1 in serum and peripheral blood mononuclear cells for hepatic fibrosis

AIM: Noninvasive diagnosis of hepatic fibrosis has become the focus because of the limited biopsy, especially in the surveillance of treatment and in screening hepatic fibrosis.Recently, regulatory elements involved in liver fibrosis, such as platelet derived growth factor-BB (PDGF-BB), transforming growth factor-β1 (TGF-β1), matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), have been studied extensively. To determine whether these factors or enzymes could be used as the indices for the diagnosis of hepatic fibrosis, we investigated them by means of receiver operating characteristic (ROC) curve.METHODS: Serum samples from sixty patients with chronic viral hepatitis B and twenty healthy blood donors were assayed to determine the level of PDGF-BB, TGF-β1, MMP-1, and TIMP-1 with ELISA, and HA, PCIII, C-IV, and LN level with RIA. The message RNA (mRNA) expression of TIMP-1 and MMP-1 in peripheral blood mononuclear cells (PBMCs) was detected by RT-PCR and Northern blot hybridization. Liver biopsy was performed in all patients.The biopsy samples were histopathologically examined. The trial was double-blind controlled.RESULTS: The serum level of PDGF-BB, TIMP-1, the ratio of TIMP-1 and MMP-1 (TIMP-1/MMP-1), mRNA expression of TIMP-1 (TIMP-1mRNA), and the ratio of TIMP-1mRNA and MMP-1mRNA (TIMP-1mRNA/MMP-1mRNA) in patients was significantly higher than those in the healthy blood donors (t=2.514-11.435, P=0.000-0.016). The serum level of PDGF-BB, TIMP-1, TIMP-1/MMP-1, and TIMP-1mRNA was positively correlated with fibrosis stage and inflammation grade (r=0.239-0.565, P=0.000-0.033), while the serum level of MMP-1 was negatively correlated with fibrosis stage and inflammation grade, and TIMP-1mRNA/MMP-1mRNA was positively correlated with inflammation grade. Through the analysis by ROC curve, serum PDGF-BB was the most valuable marker, and its sensitivity was the highest among the nine indices. The markers with the highest specificity were TIMP-1mRNA and TIMP-1mRNA/MMP-1mRNA in PBMCs. The area under the curve (AUC) of PDGF-BB, TIMP-1mRNA, TIMP-1mRNA/MMP-1mRNA, TIMP-1/MMP-1, HA,PCIII, TIMP-1, C-IV, and LN was 0.985, 0.876, 0.792, 0.748,0.728, 0.727, 0.726, 0.583, and 0.463, respectively. The sensitivity and the specificity in the parallel test was 99.0 %and 95.0 % when serum PDGF-BB, TIMP-1mRNA and TIMP1mRNA/MMP-1mRNA was detected simultaneously.CONCLUSION: Serum level of PDGF-BB, TIMP-1mRNA,TIMP-1mRNA/MMP-1mRNA in PBMCs, and serum level of TIMP-1 and TIMP-1/MMP-1 can be used as the indices for the diagnosis of hepatic fibrosis, but the former three are more useful. The combination of serum PDGF-BB, TIMP-1mRNA and TIMP-1mRNA/MMP-1mRNA in PBMCs is even more efficient in screening liver fibrosis.     

Antifibrotic effects of interleukin-10 on experimental hepatic fibrosis

BACKGROUND/AIMS: To study the effects of interleukin-10 on hepatic stellate cells and liver tissue in experimental rats hepatic fibrosis. METHODOLOGY: Rat hepatic fibrosis model induced by carbon tetrachloride was established. Liver tissues were harvested from the rats administered CCl4 with or without IL-10 treatment and the animals of the control group. The expression of TGF-beta1, MMP-2 and TIMP-1 in the liver tissues was measured by S-P immunohistochemistry. In addition, another model was established; HSCs in rats in each group were isolated. RT-PCR was employed to analyze TGF-beta1, MMP-2 and TIMP-1 mRNA expression in cells and immunocytochemistry was performed to detect protein expression of alpha-SMA, NF-kappaB, TGF-beta1, MMP-2 and TIMP-1 in HSCs. RESULTS: Rat hepatic fibrosis was developed successfully. The fibrosis changes were partially reversed by simultaneous administration of IL-10. The positive signals of TGF-beta1, MMP-2 and TIMP-1 were observed more frequently (P<0.05) in the CCl4-treated group compared to those in the IL-10-treated group and the control group. HSCs were successfully isolated. TGF-beta1, MMP-2 and TIMP-1 mRNA in HSCs increased obviously during the course of hepatic fibrosis, and their levels were decreased after the treatment with IL-10 (P<0.05). The immunocytochemistry positive levels for TGF-beta1, MMP-2, TIMP-1, alpha-SMA and NF-kappaB in the fibrogenesis group were increased significantly compared to the normal group (P<0.01). The positive signals decreased significantly (P<0.05) after the treatment with IL-10. CONCLUSIONS: The expression of TGF-beta1, MMP-2 and TIMP-1 increased in liver or in HSC of hepatic fibrosis rats and decreased after treatment with IL-10. The IL-10 could inhibit the activation of HSCs and make an antifibrogenic process come into effect in this way.     

肝纤维化血清五项标志物的诊断意义

目的探讨慢性肝炎患者血清透明质酸(HA)、Ⅲ型前胶原(PCⅢ)、Ⅳ型胶原(CⅣ)、层粘蛋白(LN)和转化生长因子β1(TGFβ1)对肝纤维化的诊断意义.方法检测116例病毒性肝炎患者血清HA、PCⅢ、CⅣ、LN、TGFβ1水平、并与其中87例慢性肝炎患者的肝组织病理作对比.结果血清HA与肝组织炎症活动度呈较弱的正相关(r＝0393,P<0.05),血清HA、PCⅢ、LN、TGFβ1与肝纤维化程度呈中等程度的正相关(r分别为0584、0454、0441和0612,P<005),血清CⅣ与之则呈较弱的正相关(r=0.319,P<0.05).血清HA诊断肝硬化的AUC明显大于血清PCⅢ、CⅣ、LN、TGFβ1者(AUC=0.904vs0.784、0.815、0.805、0828.P<0.05)血清HA、LN、TGFβ1判断S2期以上肝纤维化的ROC曲线下面积(AUC)明显大于血清PCⅢ、CⅣ者(AUC=0849、0.819、0836vs0702、0721,P<0.05).联合五项指标估计肝纤维化程度,判别分析只选人血清HA和TGFβ1.若将肝纤维化程度S1、S2、S3不作区分,判别效果中各期的差异有显著性(P<005).正确预测率为72.90%.结论五项指标均有助于诊断肝硬化和判断S2期以上肝纤维化,前者应选择血清HA.后者则可选择血清HA、LN或TGFβ1;估计肝纤维化程度以血清HA和TGFβ1同时检测为佳,但仅有助于估计慢性肝炎患者是"无肝纤维化”、"处于肝纤维化阶段”或"肝硬化”,而不能对肝纤维化程度进行精确估计.因而不能取代肝组织病理活检.