Peroxisome proliferator activated receptor-γ in pathogenesis of experimental fatty liver disease

AIM: To study the expression of peroxisome proliferator activated receptor-γ (PPARγ) in the liver of rats with fatty liver disease (FLD) and to explore the role of PPARγ in the pathogenesis of FLD to provide the basis for using PPARγligand to treat patients with FLD.METHODS: Forty Wistar rats were divided into 4 groups of ten rats each randomly: normal group (group A), alcohol group (group B), fat-rich diet group (group C), alcohol and fat-rich diet group (group D). The rats were sacrificed at the end of the 16th week from the feeding day. Alanine aminotransferase (ALT), tumor necrosis factor-alfa (TNFα)in serum and malondialdehyde (MDA) in liver homogenate were determined; livers were collected for observing pathologic changes by HE, Sudan Ⅳ, Masson stain under microscope. The morphologic results were analyzed by picture quantitative analysis technique. The changes of ultrastructure were also examined under electron microscope.The expression of PPARγ in liver was detected by immunohistochemistry and RT-PCR. The correlations between the expression of PPARγ and biochemical indexes, and liver histology were analyzed.RESULTS: The steatosis, inflammation, necrosis and fibrosis were present in livers of different experimental groups,especially in livers of alcohol and fat-rich diet group. The content of immunodetectable PPARγ was decreasedremarkably in the livers of model rats (group B-D); the level in alcohol and fat-rich diet group (3.43±1.48) was significantly lower than that in normal group (18.34±3.73), alcohol group (8.82±2.52) and fat-rich diet group (11.73±2.51) (all P＜0.01).The level of PPARγ mRNA was also lower in the livers of model rats (group B-D) than in'livers of controls. The expression of PPARγ in rat liver correlated negatively with the degree of its inflammation, necrosis and fibrosis, as well as the level of serum TNFα and the content of MDA in liver homogenates, but not with steatosis or serum ALT.CONCLUSION: Decreased expression of PPARγ may play an important role in the development of hepatocellular inflammation, necrosis and fibrosis of rats with FLD. Thus,activating PPARγ by its ligand can be anticipated to provide a therapy target for FLD.     

大鼠肝组织核因子-κB活性变化与过氧化物酶体增殖物激活受体γ表达的关系

目的 观察不同病因所致的脂肪性肝病大鼠肝组织核因子-κB(NF-κB)的活性变化,过氧化物酶体增殖物激活受体γ(PPAR γ)的表达,及其相互关系在脂肪性肝病炎症反应中的作用。方法40只Wistar 大鼠随机分为正常对照组、酒精组、高脂组和酒精加高脂4组,每组10只大鼠,16周断头处死,用HE、苏丹Ⅳ、Masson三色染色观察肝组织光镜下的病理改变和超微结构的变化。用电泳迁移率分析、逆转录聚合酶链反应观察各组大鼠肝组织NF-κB的活性变化与PPAR γ mRNA的表达、各生物化学指标间的相互关系。 结果 模型组大鼠肝组织均表现有不同程度的脂肪变性、炎症、坏死和纤维化,以酒精加高脂组病理损害最重。酒精组和酒精加高脂组的NF-κB活性(142±16.32,238±19.14)明显高于正常对照组(73±9.24,F值分别为6.36、17.93,P值均<0.01)和单纯高脂组(84±10.38,F值分别为5.96、16.20,P值均<0.01),酒精加高脂组的NF-κB活性显著高于酒精组(F=6.23,P<0.01),而高脂组和正常对照组比较差异无统计学意义。酒精组、高脂组和酒精加高脂组大鼠肝组织PPAR γmRNA的表达(0.2530±0.069,0.3647±0.082,0.1226±0.054)均较正常对照组(0.8097±0.094)有不同程度的减弱(F值分别为15.43、7.24、21.45,P值均<0.01)。相关分析显示:酒精组和酒精加高     

非酒精性脂肪性肝病大鼠肝脏解偶联蛋白2表达及其与能量贮备的关系

目的探讨非酒精性脂肪性肝病(NAFLD)大鼠肝脏线粒体解偶联蛋白2(UCP2)表达及其与能量贮备的关系。方法模型组SD大鼠给予高脂肪高胆固醇饮食饲养,分批于实验第8、12、16、24 周处死,同期设普通饮食饲养大鼠作对照。免疫组织化学和逆转录聚合酶链反应(RT-PCR)检测肝脏UCP2 mRNA转录及其蛋白表达。荧光测定法检测肝脏三磷酸腺苷(ATP)含量。结果模型组大鼠8周呈现单纯性脂肪肝,12-24周从脂肪性肝炎进展为脂肪性肝炎伴肝纤维化。免疫组织化学和RT-PCR显示,随着造模时间延长,模型组肝脏UCP2表达逐渐增强,UCP mRNA转录于24周达高峰,较对照组升高4.2倍, t=16.474,P<0.01;模型组肝脏ATP含量则随造模时间延长而逐渐减低,24周为(1.99±0.66) ×108μmol/g,对照组为(2.97±0.48)×108μmol/g,t=3.248,P<0.01。模型组肝脏UCP2 mRNA 转录的相对数值与其ATP含量呈密切负相关,r=-0.93,P<0.01。结论持续24周高脂饮食成功复制大鼠NAFLD模型,模型大鼠肝脏UCP2表达增强而ATP含量减少,两者之间关系密切。     

细胞凋亡在大鼠酒精和高脂饮食诱导的脂肪性肝病中的作用

目的探讨在实验性脂肪性肝病动物模型中肝细胞凋亡的作用。方法雄性Wistar大鼠46只，分为对照组、酒精组、高脂组和高脂加酒精组。制模成功后取血测血清ALT、AST、CHO、TG、FFA；PCR法和免疫组化法测定肝组织caspase-3的基因与蛋白表达；TUNEL法测定肝细胞凋亡指数；电镜下观察肝细胞凋亡情况。结果酒精组、高脂组、酒精加高脂组细胞凋亡指数分别为2．16±0．43、2．33±0．60和3．93±0．80，显著高于正常对照组（1．070．27，P〈0．05）；实验组caspase-3无论是基因还是蛋白表达均较对照组升高（P〈0．05），其中以酒精加高脂组增加最明显。析因分析显示，加用与不加用酒精或高脂饮食对caspase-3及其基因表达有显著性影响（P=0．00）；高脂饮食与酒精交互效应显著（P〈0．05）。结论酒精和高脂均能诱导肝细胞发生凋亡，同时酒精和高脂同时作用于肝脏加重肝细胞caspase-3的表达，两者具有协同损伤肝脏的作用。     

己酮可可碱对非酒精性脂肪性肝炎大鼠肝脏核因子-κB信号通路及胰岛素抵抗的干预作用

目的观察己酮可可碱对非酒精性脂肪性肝炎大鼠肝脏核因子-kB（NF—kB）信号通路及胰岛素受体底物（IRS）-1、IRS-2和葡萄糖转运子2（GLUT2）表达的影响。方法24只SD大鼠高脂饮食饲养4周后，随机分为模型组和干预组，于实验第24周处死，并设普通饮食饲养大鼠6只作对照组。电泳迁移率分析检测肝脏NF—kB活性，Westernblot检测肿瘤坏死因子（TNF）α和KB抑制蛋白α（IkBα）表达，逆转录聚合酶链反应检测肝脏IRS—1、IRS12和GLUT2mRNA表达。结果与对照组相比，模型组肝脏NF-kB和TNFα显著增加，IkBα显著降低，伴IRS-2表达显著增加；与模型组相比，干预组NF—kB和TNFα显著降低，IkBα有所增高，而IRS-2表达显著减少；肝脏IRS—1和GLUT2表达在3组之间差异均无统计学意义。结论己酮可可碱可能通过影响肝脏NF—kB信号通路和增加肝细胞IRS-2表达，从而有助于改善非酒精性脂肪性肝炎大鼠肝脏胰岛素抵抗。     

非酒精性脂肪性肝病大鼠血浆前列环素与血栓素的变化及其与肝脏损伤的关系

目的 探讨非酒精性脂肪性肝病(NAFLD)大鼠血浆前列环素(PG12)和血栓索(TX)A2的动念变化及其与肝组织学改变之间的关系。 方法 48只模型组SD大鼠给予高脂肪高胆固醇饮食饲养,分批于第8、12、16、24周处死,24只正常饮食大鼠作对照。放射免疫法检测血浆PGI 2和TXA 2的稳定代谢产物6酮-前列环素1α(PGF1 α)和TXB2含量,光镜观察肝组织切片病理学改变。 结果 模型组大鼠8周呈现单纯性脂肪肝,12～24周从脂肪性肝炎进展至脂肪性肝纤维化。模型组大鼠血浆TXB 2在造模第8、24周分别为(52.4±3.15)ng/L和(117.7±7.47)ng/L,对照组为(41.1±1.45)ng/L,t值为9.12和31.34,P<0.01和P<0.001。 血浆PGF1 α水平在造模8、24周分别为(31.1±1.6)ng/L和(3.4±2.4)ng/L,对照组为(36.5±1.7)ng/L,t值为6.27和34.62,P<0.01和,P<0.001。模型组大鼠血浆TXB2和PGF1 α水平分别与其肝组织损伤程度呈显著正相关(r=0.537,P<0.001)及负相关(r=-0.452,P<0.01)。 结论 持续24周的高脂饮食可以成功复制大鼠NAFLD模型,模型大鼠血浆TXA 2与PGI 2平衡失调,可能参与NAFLD的发病。     

Changes in the markers related to collagen synthesis in the liver of chronically alcohol treated rats

In order to clarify the roles of Ito cells in the development of alcoholic fibrosis, markers related to collagen synthesis in the liver were analyzed in chronically alcohol treated rats. The livers were obtained from rats fed a diet containing alcohol (alcohol group) and those fed a control diet (control group) for 4 weeks. Prolyl hydroxylase (PH) activity in the whole liver tissue did not differ in the alcohol and control groups. However, the activity in the isolated Ito cells was significantly higher in the alcohol group than in the control group. Immunoreactive PH beta-subunit contents in the liver and serum were significantly higher in the alcohol group than in the control group. Hydroxyproline contents in the livers did not differ in either groups. Immunohistochemically, type IV collagen and laminin were clearly stained along with the sinusoid in the livers of the alcohol group. However, the staining reactions were very weak in the control group. Staining reactions to types I and III collagen were very weak or almost absent in the livers of both groups. Desmin-positive cells, along with the sinusoid, increased significantly in the alchol group, especially at the centrilobular area, suggesting that the number of Ito cell increase in the centrilobular areas of the alcohol treated rats. These results suggest that type IV collagen and laminin synthesis increase in the Ito cells of chronically alcohol treated rats, although clear evidence of hepatic fibrosis was not obtained. This increase may be related to capillarization of the sinusoids and finally to the development of perisinusoidal fibrosis in alcoholics.     

Changes in the markers related to collagen synthesis in the liver of chronically alcohol treated rats.

In order to clarify the roles of Ito cells in the development of alcoholic fibrosis, markers related to collagen synthesis in the liver were analyzed in chronically alcohol treated rats. The livers were obtained from rats fed a diet containing alcohol (alcohol group) and those fed a control diet (control group) for 4 weeks. Prolyl hydroxylase (PH) activity in the whole liver tissue did not differ in the alcohol and control groups. However, the activity in the isolated Ito cells was significantly higher in the alcohol group than in the control group. Immunoreactive PH beta-subunit contents in the liver and serum were significantly higher in the alcohol group than in the control group. Hydroxyproline contents in the liver did not differ in either groups. Immunohistochemically, type IV collagen and laminin were clearly stained along with the sinusoid in the livers of the alcohol group. However, the staining reactions were very weak in the control group. Staining reactions to types I and III collagen were very weak or almost absent in the livers of both groups. Desmin-positive cells, along with the sinusoid, increased significantly in the alcohol group, especially at the centrilobular area, suggesting that the number of Ito cell increase in the centrilobular areas of the alcohol treated rats. These results suggest that type IV collagen and laminin synthesis increase in the Ito cells of chronically alcohol treated rats, although clear evidence of hepatic fibrosis was not obtained. This increase may be related to capillarization of the sinusoids and finally to the development of perisinusoidal fibrosis in alcoholics.     

大鼠非酒精性脂肪性肝病转化生长因子β1/Smad4信号通路表达的研究

目的 观察转化生长因子(TGF)β1、转化生长因子β受体(TβR)-Ⅰ、TβR-Ⅲ和胞内Smad4信号通道蛋白以及转录因子激活剂蛋白-1(AP-1)家族中Jun蛋白质因子在大鼠非酒精性脂肪性肝病(NAFLD)中的表达.探讨NAFLD发生肝纤维化的可能机制.方法 将18只大鼠分为对照组与模型组,每组9只.对照组喂饲普通饲料,模型组喂饲高脂饲料(88%标准普通饲料+10%猪油+2%胆固醇)造模.在20周处死所有大鼠,免疫组化法检测TGFβ1、Smad4的表达;RT-PCR检测肝组织TGFβ1、TβR-Ⅰ、TβR-Ⅱ的表达;Western印迹法测定磷酸化C-Jun蛋白的表达.结果 成功建立NAFLD动物模型,免疫组化法检测结果显示,TGFβ1和Smad4在对照组大鼠肝实质细胞的胞质内均有少量表达.TGFβ1在模型组大鼠肝实质细胞胞质、肝血窦周围的表达明显增强,尤其在汇管区更为明显.Smad4在模型组大鼠肝实质细胞胞质表达亦明显增强,而在汇管区的表达更为明显.RT-PCR检测结果显示,模型组大鼠肝组织TGFβ1、TβR-Ⅰ、TβR-ⅡmRNA的A值分别为0.46±0.12、5.24±2.70和3.35±1.95,明显高于对照组(0.21±0.09、1.36±0.77和0.52±0.19,P值均＜0.01).Western印迹检测显示,模型组磷酸化C-Jun蛋白表达同内参灰度值比为0.93±0.41,较对照组显著增高(0.32±0.25,P=0.001).结论 TGFβ1/Smad4信号通路可能参与了NAFLD进展为肝纤维化的过程.阻断TGFβ/Smad4信号传导通路,可开辟治疗NAFLD新的思路.     

己酮可可碱对非酒精性脂肪肝大鼠肝组织线粒体解耦联蛋白表达的影响

目的 探讨己酮可可碱(PTX)对非酒精性脂肪肝性肝病(NAFLD)大鼠线粒体解耦联蛋白(UCP)-2 mRNA表达的影响. 方法 SD大鼠60只,正常喂养1周后随机分为3组,每组20只.其中对照组20只,以普通饲料喂养,实验组和干预组各20只,以高脂饲料喂养,干预组高脂饮食4周后,在饮水中加用已酮可可碱(PTX)(100 mg·Kg-1·d-1),于第24周处死,采用反转录聚合酶链反应(RT-PCR)技术检测肝脏UCP-2mRNA的表达. 结果 对照组大鼠肝脏UCP2mRNA呈微量表达(1.2±0.1),实验组大鼠肝脏UCP2mRNA呈强表达(4.0±0.3),干预组大鼠肝脏UCP2mRNA呈弱表达(3.0±0.2),3组间UCP-2mRNA表达差异有统计学意义(F=160.67,P＜0.01). 结论 己酮可可碱可下调NAFLD大鼠肝细胞UCP-2mRNA的表达.     

基质金属蛋白酶-13在大鼠酒精性肝纤维化表达的研究

目的 观察大鼠酒精性肝纤维化形成过程中MMP—13的表达,探讨MMP—13在酒精性肝纤维化发生、发展中的作用。 方法 56％(v/v)的白酒平均以7 g/kg的剂量每日早晨灌胃1次制备肝纤维化模型,灌胃4周、12周及24周采用股静脉放血法分别处死大鼠,采用半定量逆转录聚合酶链反应(RT—PCR)检测MMP—13 mRNA的表达。 结果 研究表明,正常肝脏表达MMP—13 mRNA(0.24±0.41),白酒灌胃4周后其mRNA表达逐渐上升(0.62±0.54),但与对照组相比差异无显著性,至12周肝组织中MMP—13 mRNA表达较正常组显著增加(1.6 5±0.47),两组比较差异有显著性(t=-4.36 3,P<0.01)。而至24周,大鼠肝组织中MMP—13 mRNA表达(0.39±0.25)又下降至与正常组相似,两组比较差异无显著性。 结论 MMP—13可能参与酒精性肝纤维化早期基质的降解,且其表达存在明显的窗口期。     

In vivo effects of Chinese herbal recipe, Danshaohuaxian, on apoptosis and proliferation of hepatic stellate cells in hepatic fibrotic rats

AIM: To investigate the effects of Danshaohuaxian (DSHX), a Chinese herbal recipe, on the apoptosis and cell cycles of hepatic stellate cells (HSCs) in rat hepatic fibrosis and its possible mechanisms. METHODS: Seventy-six male Wistar rats were randomly divided into normal control group, hepatic fibrosis group, non-DSHX-treated group and DSHX-treated group. Except for the normal control group, rat hepatic fibrotic models were induced by subcutaneous injection of carbon tetrachloride (CCl4), drinking alcohol, giving diet of hyperlipid and hypoprotein for 8 wk. When the hepatic fibrotic models were produced, 12 rats of hepatic fibrosis group (15 rats survived, others died during the 8 wk) were sacrificed to collect blood and livers. HSCs were isolated from the other 3 rats to detect the apoptotic index (AI) and cell cycles by flow cytometry. DSHX was then given to the DSHX-treated group (1.0 g/kg, PO, daily) for 8 wk. At the same time, normal control group and non-DSHX-treated group were given normal saline for 8 wk. At end of the experiment, some rats in these three groups were sacrificed to collect blood and livers, the other rats were used for HSC isolation to detect the apoptotic index (AI) and cell cycles. Then the liver index, serum hyaluronic acid (HA) and alanine aminotransferase (ALT), degree of hepatic fibrosis, urinary excretion of hydroxyproline (Hyp) and expression of collagen types Ⅰ and Ⅲ (COL Ⅰ and Ⅲ) in these four groups were detected respectively. RESULTS: Compared with the indexes of the hepatic fibrosis group and non-DSHX-treated group, the DSHX-treated group revealed a liver index of (0.0267±0.0017 vs 0.0423±0.0044, 0.0295±0.0019, P<0.05), levels of serum HA (200.78±31.71 vs 316.17±78.48, 300.86±72.73, P<0.05) and ALT(93.13±5.79 vs 174.5±6.02, 104.75±6.54, P<0.01), and stage of hepatic fibrosis (1.30 vs 4.25, 2.60, P<0.01) all reduced. The urinary excretion of Hyp increased (541.09±73.39 vs 62.00±6.40, 182.44±30.83, P<0.01), the COL Ⅰ and Ⅲ expression decreased (COL I: 1.07±0.96 vs 4.18±2.26, 3.22±1.44, P<0.01; COL Ⅲ: 1.09±0.58 vs 3.04±0.62, 2.23±0.58, P<0.01), the HSCs apoptotic index of HSCs (7.81±0.47 vs 1.63±0.25, 1.78±0.4, P<0.05) and the ratio of G0-G1 phase cells increased (94.30±1.33 vs 62.27±17.96, 50.53±2.25, P<0.05). The ratios of S-phase cells (3.11±1.27 vs 9.83±1.81, 11.87±1.9, P<0.05) and G2-M phase cells (2.58±0.73 vs 23.26±10.95, 13.60±1.15, P<0.01) declined. CONCLUSION: DSHX capsule shows certain therapeutic effects on hepatic fibrosis in rats and inhibits abnormal deposition of COL I and III in rat livers by promoting the apoptosis of HSCs and preventing their proliferation.     

Effect of Sinai san decoction on the development of non-alcoholic steatohepatitis in rats

AIM: To explore the effect of Sinai san decoction on the development of non-alcoholic steatohepatitis induced by CCL4 combined with a fat-rich diet in rats. METHODS: Twenty-seven Sprague-Dawley rats were divided into three groups randomly: control group (n=9), model group (n = 9) and treatment group (n = 9). The rats of model group and treatment group were given small dosage of CCL4 combined with a fat-rich diet, and those of control group were given normal diet. After four weeks of fat-rich diet feeding, the rats of treatment group were given Sinai san decoction. The serum levels of aminotransferase and lipid were measured, and the pathology of livers was observed by HE staining after the rats were sacrificed at eight weeks. RESULTS: The rats' livers presented the pathology of steatosis and inflammation with higher serum levels of ALT and AST in the model group. In the treatment group the serum ALT and AST levels decreased significantly and were close to the control group. The hepatic inflammation scores also decreased markedly, but were still higher than those of control group. And the degree of hepatocyte steatosis was similar to that of model group. CONCLUSION: Sinai san decoction may ameliorate the hepatic inflammation of rats with steatohepatitis induced by small dosage of CCL4 combined with a fat-rich diet, but does not prevent the development of hepatocyte steatosis.     

Effect of Danshao Huaxian capsule on expression of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in fibrotic liver of rats

AIM: To investigate the effects of Danshao Huaxian (DSHX) capsules, a preparation of traditional Chinese medicine, on the expression of matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the fibrous livers of rats. METHODS: Eighty male Wistar rats were randomly divided into normal control group (group A), CCl4-induced hepatic fibrosis group (group B), non-DSHX-treated group (group C), low dose-treated group (group D), and high dose-treated group (group E). Fibrous liver models in rats were induced by subcutaneous injection of CCl4, oral administration of alcohol and high-lipid/low-protein diet for 8 wk. After the models were established, the rats in groups D and E were orally given a low dose (0.5 g/kg) and a high dose (1.0 g/kg) of DSHX daily for 8 wk, respectively. Then, the liver indexes, serum hyaluronic acid (HA) and alanine aminotransferase (ALT) were examined. The degree of hepatic fibrosis was evaluated by optical microscopy. Hydroxyproline (Hyp) in the urine was determined, and the expression of MMP-1 and TIMP-1 was detected by immunohistochemical techniques. RESULTS: In groups D and E, the liver indexes, levels of serum HA and ALT reduced and development of hepatic fibrosis weakened significantly. The urinary Hyp and expression of MMP-1 in the liver tissues elevated, but the expression of TIMP-1 decreased obviously, as compared to groups B and C. CONCLUSION: DSHX enhances the expression of MMP-1 but decreases that of TIMP-1 in liver tissues of CCl4-induced hepatic fibrotic rats, which may result in its elevated activity that contributes to fighting against hepatic fibrosis.     

罗格列酮对大鼠非酒精性脂肪性肝炎逆转机制的研究

目的 探讨过氧化物酶体增殖物激活受体γ（PPAR-γ）激动剂罗格列酮对库普弗细胞中LPS诱导的IκB激酶-β（IKK-β）表达以及对NASH大鼠肝组织核因子-κB（NF-κB）活性和环氧合酶-2（COX-2）表达水平的影响及意义。方法 采用Ⅳ型胶原酶消化和密度梯度离心的方法分离纯化正常Wistar大鼠库普弗细胞,并应用LPS（1μg／ml）及不同浓度的罗格列酮（10 nmol／L和50 nmol／L）干预培养,收集细胞及培养上清液。体内实验：健康雄性Wistar大鼠随机分为正常组和模型组,后者以高脂饲料（2％胆固醇＋10%猪油＋5％玉米油）喂养复制NASH大鼠动物模型,造模成功后,分别以1 mg·kg^-1·d^-1、4mg·kg^-1·d^-1剂量的罗格列酮和等容积等渗盐水灌胃,实验结束后采集大鼠血清和肝组织标本。RT-PCR检测库普弗中细胞IKK-β、肝组织中COX-2 mRNA的表达,Western blot和电泳迁移率改变分析（EMSA）分别检测库普弗细胞中IKK-β蛋白表达和肝组织NF-κB活性变化,ELISA检测细胞培养上清液及血清中TNFα水平。结果 LPS可显著增高体外培养库普弗细胞中IKK-βmRNA、蛋白的表达及上清液中TNFα水平。模型组大鼠肝组织COX-2 mRNA和蛋白的表达均较正常对照组增强,NF-κB活性与COX-2的表达和血清TNFα水平呈正相关。罗格列酮体外可抑制LPS诱导库普弗细胞中IKK-βmRNA和蛋白的表达,体内通过抑制肝组织NF-κB活化,减少COX-2表达,均表明罗格列酮可通过抑制炎症细胞活化,下调炎症介质基因表达,从而减少了NFα等炎症介质合成、释放,抑制肝脏炎症反应。结论 PPAR-γ特异性激动剂罗格列酮通过干预IKK-β／IκB／NF-κβB／TNFα信号通路而发挥抗炎作用,从细胞分子水平逆转NASH大鼠肝组织的炎症反应,这为临床上有效治疗NASH提供了新思路。     

大鼠非酒精性脂肪性肝纤维化形成过程中骨桥蛋白的表达及其意义

目的 通过观察非酒精性脂肪性肝纤维化形成过程中肝组织内骨桥蛋白(OPN)的变化规律,探讨OPN在非酒精性脂肪性肝纤维化形成过程中的作用. 方法选用纯系Wistar雄性大鼠56只,体质量180～200g,随机分为正常对照组和高脂饮食4、8、12,16、20、24周组(其中每组各8只).肝组织常规进行HE及Masson三色胶原染色,免疫组织化学染色检测肝组织α-平滑肌肌动蛋白的表达,RT-PeR、Western blot观察肝组织中OPN动态变化.结果 与正常对照组相比,高脂饲料组大鼠肝脏内OPN含量明显升高,随着脂肪肝程度的加重,OPN表达逐渐增强,mRNA及蛋白质表达比较,F值分别为7.30和7.15,尸值均<0.01;与α-平滑肌肌动蛋白表达及肝组织胶原纤维面积百分比呈显著正相关(r值分别为0.94和0.82,P值均<0.01).结论 在非酒精性脂肪性肝纤维化形成过程中,OPN在肝组织中表达显著上调,可能在非酒精性脂肪性肝纤维化的形成过程中发挥重要作用.     

Effects of Guiyuanfang and autologous transplantation of bone marrow stem cells on rats with liver fibrosis

AIM: To investigate the therapeutic effects of Guiyuanfang and bone marrow stem cells (BMSCs) on rats with liver fibrosis. METHODS: Liver fibrosis model was induced by carbon tetrachloride, ethanol, high lipid and assessed biochemically and histologically. Liver function and hydroxyproline contents of liver tissue were determined. Serum hyaluronic acid (HA) level and procollagen Ⅲ level were performed by radioimmunoassay. The VG staining was used to evaluate the collagen deposit in the liver. Immunohistochemical SABC methods were used to detect transplanted BMSCs and expression of urokinase plasminogen activator (uPA). RESULTS: Serum transaminase level and liver fibrosis in rats were markedly reduced by Guiyuanfang and BMSCs. HA level and procollagen Ⅲlevel were also reduced obviously, compared to model rats (HA: 47.18±10.97 ng/mL, 48.96±14.79 ng/mL; PCⅢ: 22.48±5.46 ng/mL, 26.90±3.35 ng/mL; P<0.05). Hydroxyproline contents of liver tissue in both BMSCs group and Guiyuanfang group were far lower than that of model group (1 227.2±43.1 μg/g liver tissue, 1390.8±156.3 μg/g liver tissue; P<0.01). After treatment fibrosis scores were also reduced. Both Guiyuanfang and BMSCs could increase the expression of uPA. The transplanted BMSCs could engraft, survive, and proliferate in the liver. CONCLUSION: Guiyuanfang protects against liver fibrosis. Transplanted BMSCs may engraft, survive, and proliferate in the fibrosis livers indefinitely. Guiyuanfang may synergize with BMSCs to improve recovery from liver fibrosis.     

Effect of ginkgo biloba extract on livers in aged rats

AIM: To investigate the protective effect of ginkgo biloba extract (GBE) on livers of aged rats and the associated mechanisms. METHODS: Two-mo- and 20-mo-old rats were treated with GBE/saline for 3 mo. Liver tissue samples from 5-mo-old rats treated with saline (group Y) and 23-mo-old rats treated with GBE (group E) or saline (group N) were used for histopathological examinations (hematoxylin-eosin and Masson staining, Lipofuscin staining-Schmorl staining) and determination of expression of tissue inhibitor-1 of metalloproteinase (TIMP-1) and the level of malondialdehyde (MDA), glutathione peroxidase (GPx) and superoxide dismutase (SOD). Blood samples were collected for determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) and albumin. RESULTS: Microscopic studies with Masson staining revealed mild liver fibrosis in aged rats (group N), while the livers of aged rats receiving GBE (group E) showed amelioration in fibrosis (2.2±0.1 vs2.8±0.1, P＜0.01) and deposition of lipofuscin (33.7±5.3 vs62.8±5.7, P＜0.01). The expression of TIMP-1 and the level of liver MDA (1.0±0.1 vs1.2±0.2,P＜0.05) also decreased but the activity of GPx (97.1±15.3 vs61.8±14.5, P＜0.01) increased in group E. Compared with group Y, the level of liver MDA (0.8±0.1 vs1.2±0.2, P＜0.01), lipofuscin (32.4±6.0 vs 62.8±5.7, P＜0.01) and TIMP-1 expression were increased, while the activity of GPx (103.2±17.6 vs61.8±14.5, P＜0.01) and SOD (16.7±4.4 vs11.8±3.9, P＜0.05) was decreased in group N. There was no difference in liver function among these three groups. CONCLUSION: GBE has protective effects on aging liver. The possible mechanisms might be its antioxidant activity and inhibition of TIMP-1 expression.     

Expression of TIMP-1 and TIMP-2 in rats with hepatic fibrosis

AIM: To investigate the location and expression of TIMP-1 and TIMP-2 in the liver of normal and experimental hepatic fibrosis in rats. METHODS: The rat models of experimental immunity hepatic fibrosis (n=20) were prepared by the means of immunologic attacking with human serum albumin (HSA),and normal rats (n=10) served as control group. Both immunohistochemistry and in situ hybridization methods were respectively used to detect the TIMP-1 and TIMP-2 mRNA and related antigens in liver. The liver tissue was detected to find out the gene expression of TIMP-1 and TIMP-2 with RT-PCR. RESULTS: The TIMP-1 and TIMP-2 related antigens in livers of experimental group were expressed in myofibroblasts and fibroblasts (TIMP-1: 482&#177;65 vs 60&#177;20; TIMP-2:336&#177;48 vs 50&#177;19, P&lt;0.001). This was the most obvious in portal area and fibrous septum. The positive signals were located in cytoplasm, not in nucleus. Such distribution and location were confirmed bysitu hybridization (TIMP-1/β-actin: 1.86&#177;0.47 vs 0.36&#177;0.08; TIMP-2/β-actin: 1.06&#177;0.22 vs 0.36&#177;0.08,P&lt;0.001). The expression of TIMP-1 and TIMP-2 was seen in the liver of normal rats, but the expression level was very low. However, the expression of TIMP-1 and TIMP-2 in the liver of experimental group was obviously high. CONCLUSION: In the process of hepatic fibrosis, fibroblasts and myofibroblasts are the major cells that express TIMPs.The more serious the hepatic fibrosis is in the injured liver,the higher the level of TIMP-1 and TIMP-2 gene expression.