软肝冲剂对肝纤维化大鼠组织金属蛋白酶抑制剂的影响

目的：探讨软肝冲剂治疗肝纤维化和肝硬化的机制是否与抑制TIMP-1的表达有关。方法：用CCl4诱导大鼠的肝纤维化模型（病理分期为0-Ⅴ期），将大鼠随机分成两组，分别用软肝冲剂和生理盐水治疗2个月后。对肝组织TIMP-1mRNA的表达进行分析，并检测羟脯氨酸（Hyp）的量，同时检测血清中TIMP-1和TGF-β1的浓度。结果：软肝冲剂能够降低大鼠肝组织中TIMP-1mRNA的表达和胶原蛋白的含量，同时血清中TIMP-1和TGF-β1较CCl4诱导的模型组和生理盐水治疗组显著降低（P〈0．05）。结论：软肝冲剂治疗肝纤维化可能通过阻止TIMP-1的表达起作用。     

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.     

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.     

牛磺酸对四氯化碳诱导大鼠肝纤维化的抑制作用

目的研究牛磺酸的体内抗肝纤维化作用。方法用四氯化碳（ＣＣｌ４）复制大鼠肝纤维化模型;造模开始或造模６周后给予牛磺酸;实验结束后分别测定肝功能、纤维化指标（透明质酸和层粘连素）、肝羟脯氨酸及Ⅰ、Ⅲ型前胶原ｍＲＮＡ含量,并作肝病理检查。结果牛磺酸可显著减轻ＣＣｌ４肝纤维化程度,能明显降低肝羟脯氨酸和Ⅰ、Ⅲ型前胶原ｍＲＮＡ含量,降低血清透明质酸和层粘连素水平,改善肝功能,组织学检查亦显示具有抗肝纤维化作用。结论牛磺酸在体内具有抗肝纤维化的作用,可望用于肝纤维化的防治。     

Ginkgo biloba extract reverses CCl4-induced liver fibrosis in rats

AIM: To study the reversing effect of Ginkgo biloba extract (GbE) on established liver fibrosis in rats. METHODS: Following confirmation of CCI4-induced liver fibrosis, GbE or saline was administrated to the rats for 4 weeks. The remaining rats received neither CCI 4 norGbE as normal control. The four groups were compared in terms of serum enzymes, tissue damage, expression of αSMA and tissue inhibitor-1 of metalloproteinase (TIMP-1) and metalloproteinase-1 (MMP-1). RESULTS: Compared with saline-treated group, liver fibrosis rats treated with GbE had decreased serum total bilirubin (P&lt;0.01) and aminotransferase levels (P&lt;0.01) and increased levels of serum albumin (P&lt;0.01). Microscopic studies revealed that the livers of rats receiving GbE showed allieviation in fibrosis (P&lt;0.05) as well as expression of αSMA (P&lt;0.01). The liver collagen and reticulum contents were lower in rats treated with GbE than saline-treated group (P&lt;0.01). RT-PCR revealed that the level of TIMP-1 decreased while the level of MMP-1 increased in GbE group. CONCLUSION: Administration of GbE improved CCI4-induced liver fibrosis. It is possibly attributed to its effect of inhibiting the expression of TIMP-1 and promoting the apoptosis of hepatic stellate cells.     

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.     

Therapeutic effect of interleukin-10 on CCl4-induced hepatic fibrosis in rats

AIM: To study the therapeutic effect of exogenous interleukin-10 on CCl4-induced hepatic fibrosis in rats and its possible mechanisms. METHODS: Fourty-seven SD rats were randomly divided into control group (group N) and CCl4-induced hepatic fibrosis model group (group C). After CCl4 was given for 9 wk, the model group was divided into three groups. Rats in group M were put to death immediately,rats in group T were treated with IL-10 for another three wk and then put to death, rats in group R recovered after three weeks and were then killed. The degree of hepatic fibrosis was measured by HE staining and histological activity index (HAI). Histological activity index (HAI), change of collagen types I and III were measured by Picrosirius staining. The expression of TNF-alpha, MMP-2 and TIMP-1 in liver tissue was measured by S-P immunohistochemistry. RESULTS: CCl4- induced experimental rat hepatic fibrosis model was established successfully. The degree of hepatic fibrosis was markedly lower in group T than in groups M and R, and there was no difference between the two groups.The expression of collagen types I and III was significantly suppressed in group T and was slightly suppressed in groups M and R. The positive levels of TNF-alpha, MMP-2 and TIMP-1 in group M increased significantly compared to those in group N (P<0.01). The positive signals decreased significantly in groups T and R (P<0.01),but positive score was significantly lower in group T than in group R (P<0.01). CONCLUSION: Exogenous IL-10 can reverse CCl4-induced hepatic fibrosis in rats. IL-10 may exert its reversible effects on hepatic fibrosis by blocking CCl4-induced inflammation,inhibiting expression of MMP-2 and TIMP-1 and promoting resolution of collagen types I and III.     

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.     

Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo

BACKGROUND: Following liver injury, hepatic stellate cells (HSC) transform into myofibroblast-like cells (activation) and are the major source of type I collagen and the potent collagenase inhibitors tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) in the fibrotic liver. The reproductive hormone relaxin has been reported to reduce collagen and TIMP-1 expression by dermal and lung fibroblasts and thus has potential antifibrotic activity in liver fibrosis. AIMS: To determine the effects of relaxin on activated HSC. METHODS: Following isolation, HSC were activated by culture on plastic and exposed to relaxin (1-100 ng/ml). Collagen deposition was determined by Sirius red dye binding and radiolabelled proline incorporation. Matrix metalloproteinase (MMP) and TIMP expression were assessed by zymography and northern analysis. Transforming growth factor beta1 (TGF-beta1) mRNA and protein levels were quantified by northern analysis and ELISA, respectively. RESULTS: Exposure of activated HSC to relaxin resulted in a concentration dependent decrease in both collagen synthesis and deposition. There was a parallel decrease in TIMP-1 and TIMP-2 secretion into the HSC conditioned media but no change in gelatinase expression was observed. Northern analysis demonstrated that primary HSC, continuously exposed to relaxin, had decreased TIMP-1 mRNA expression but unaltered type I collagen, collagenase (MMP-13), alpha smooth muscle actin, and TGF-beta1 mRNA expression. CONCLUSION: These data demonstrate that relaxin modulates effective collagen deposition by HSC, at least in part, due to changes in the pattern of matrix degradation.     

肝复康对肝纤维化大鼠肝组织NF-κB、MMP-2和TIMP-2表达的影响

目的研究中药肝复康对肝纤维化大鼠肝脏NF-κB、基质金属蛋白酶-2（MMP-2）和金属蛋白酶组织抑制因子2（TIMP-2）基因表达的影响。方法随机将SD大鼠分为正常对照组、模型组、小剂量、中剂量和大剂量治疗组,制备CCl4肝纤维化模型,自第9周起,给予中药肝复康治疗至第20周。采用Western-Blot法检测NF-κB表达;采用RT-PCR法观察NF-κB、MMP-2和TIMP-2 mRNA水平。结果模型组大鼠肝组织NF-κB、MMP-2和TIMP-2表达增加,肝复康可明显抑制上述因子的表达（P〈0.01）,且三者mRNA水平在各组间呈显著正相关。结论肝复康治疗肝纤维化可能与其抑制NF-κB、MMP-2和TIMP-2的表达有关。     

An herbal formula, CGX, exerts hepatotherapeutic effects on dimethylnitrosamine-induced chronic liver injury model in rats

INTRODUCTION Several pathologic conditions, including chronic alcohol consumption, viral B or C hepatitis, and constant cholestasis, induce chronic injury to liver tissue. Moreover, these chronic liver disorders lead progressively to fibrosis or liver cir…     

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.     

Taurine inhibits deposition of extracellular matrix in experimental liver fibrosis in rats]

OBJECTIVE: To study the effect of taurine on liver fibrosis and its mechanism. METHODS: Fibrosis was induced by the administration of carbon tertrachloride(CCl4) in rats. Some of the animals were treated with taurine. The rats were killed after 12 weeks of CCl4 treatment. Depositions of type I, III and IV collages, laminin and hyaluronic acid were studied in liver sections by immunohistochemical technique using specific antibody. The hepatic contents of type I, III procollage and tissue inhibitor of metalloproteinase-1(TIMP-1) mRNA were determined by Northern blot hybridization. RESULTS: A significant elevations of hepatic collagen I, III, IV, laminin and hyaluronic acid were observed after 12 weeks of liver injury in animals without taurine treatment, and a definite increase in the amounts of hepatic type I, III procollagen and TIMP-1 mRNA was noted. Taurine prevented increases in type I, III procollagen mRNA expression as well as the accumulation of the collagens, laminin and hyaluronic acid in the liver. CONCLUSION: The data indicate that taurine has a protective effect in CCl4-induced hepatic fibrosis. The results suggest taurine might be of potential value in clinical practice.     

Rat liver fibrosis regresses better with pegylated interferon alpha2b and ursodeoxycholic acid treatments than spontaneous recovery.

BACKGROUND/AIMS: Fibrosis and cirrhosis are common complications of chronic liver diseases. An imbalance between fibrogenesis and fibrolysis results in scarring of the liver parenchyma. We aimed to investigate the possible antifibrotic effectiveness of a newly modified interferon molecule peginterferon alpha2b (PEG-IFNalpha2b) which has better antiviral activity, and ursodeoxycholic acid (UDCA). METHODOLOGY: Liver fibrosis was established on 60 male Sprague Dawley rats with CCl4 in 12 weeks. After cessation of CCl4 Group I was left for spontaneous recovery. Group II was treated with PEG-IFN 1.5 microg/kg/week, Group III with UDCA 25 mg/kg/day and Group IV with combination of both drugs. All rats were killed at week 16. Histopathologic fibrosis scores, tissue hydroxyproline, TIMP-1 and MMP-13 levels were determined. Hepatic stellate cell apoptosis was detected by dual staining with TUNEL technique and anti-alpha smooth muscle actin. RESULTS: Fibrosis scores were lower in Group II, III and IV than Group I (p<0.05 for group I vs. II and III; p<0.01 for group I vs. IV). Tissue hydroxyproline levels were significantly decreased in Group II, III and IV when compared to Group I (p<0.05 for group I vs. II, p<0.01 for group I vs. III and IV). Lower liver TIMP-1 and higher MMP-13 levels were measured in Group II, III, and Group IV than Group I (p<0.01 for TIMP-1 and p<0.01, for MMP). Activated HSC apoptosis was significantly increased in Group II, III and IV when compared to Group I (p<0.01, for all). There was significantly higher apoptosis in Group II than Group III and IV (p<0.01). CONCLUSION: Treatment with both PEG-IFNalpha2b and UDCA improved CCl4 induced rat liver fibrosis. Significantly higher effects were obtained using these agents in combination.     

Adeno-associated virus mediated interferon-gamma inhibits the progression of hepatic fibrosis in vitro and in vivo

AIM: To investigate the effects of adeno-associated virus (AAV) mediated expression of human interferon-γ for gene therapy in experimental hepatic fibrosis in vitro and in vivo. METHODS: We constructed the recombinant AAV encoding human INF-γ (rAAV- INF-γ) and took the primary rat hepatic stellate cells and carbon tetrachloride induced rats as the experimental hepatic fibrosis model in vitro and in vivo. Immunocytochemistry analysis was used to reveal the expression of α-SMA, the marker protein expressed in hepatic stellate cells. The mRNA expression of TGF-β, TIMP-1, and MMP-13 were analyzed by RT-PCR method. In vivo study, the hydroxyproline content in liver and serum AST, ALT were also detected. RESULTS: In vitro study, AAV vector could mediated efficient expression of human INF-γ, which inhibit the activation of hepatic stellate cells, decrease the expression of α-SMA and mRNA of TIMP-1, TGF-β, with the MMP-13 unchanged. In vivo study, the histological examination revealed that rAAV- INF-γ could inhibit the progression of the hepatic fibrosis. In the rAAV-INF-γ induced group, the hydroxyproline content and serum AST, ALT level were decreased to 177±28 μg/g wet liver, 668.5±140.0, 458.4±123.5 U/L, compare with the fibrosis control group 236±31 μg/g wet liver, 1 019.1±276.3, 770.5±154.3 U/L, respectively (P<0.01). mRNA expression of TIMP-1 in the rAAV-INF-γ induced rat liver was decreased while no significant change was observed in TGF-β and MMP-13. CONCLUSION: All these results indicated that rAAV-INF-γ has potential effects for gene therapy of hepatic fibrosis, which could inhibit the progression of hepatic fibrosis.     

培哚普利抗实验性大鼠肝纤维化的实验研究

目的 研究血管紧张素转化酶抑制剂-培哚普利对实验性肝纤维化的影响,并探讨其作用机制。 方法 取雄性大鼠40只,随机分为3组。模型组:40％ CCl4油0.25 g/L皮下注射,每周2次;培哚普利治疗组:40％CCl4油0.25 g/L皮下注射,2次/周,同时予以培哚普利2 mg/kg灌胃,1次/d。对照组:橄榄油皮下注射。于第4、6周取材。光镜下动态观察组织学改变,检测血管紧张素Ⅱ1型受体(A T 1 R)、转化生长因子β1(TGF—β1)、血小板衍生性生长因子-BB(PDGF—BB)蛋白的表达、金属蛋白酶-2(MMP-2)的活性和血清透明质酸(HA)、层黏连蛋白(LN)的含量。 结果 培哚普利治疗组肝组织炎症程度明显减轻,纤维间隔较为细小,血清HA、LN(μg/L)含量分别为82.07±13.66和94.84±3.54,模型组分别为147.86±18.92和113.72±2.14,t=2.27～2.87,P<0.05;培哚普利治疗组AT1R mRNA和蛋白质表达水平、TGF-β1和PDGF—BB蛋白质的表达低于模型组;模型组MMP-2活性高于培哚普利组。 结论 培哚普利对实验性大鼠肝纤维化具有抑制作用。     

秋水仙碱对肝纤维化大鼠肝脏基质金属蛋白酶-1及其抑制因子-1表达的影响

目的 观察秋水仙碱对纤维化肝脏基质金属蛋白酶-1(MMP-1)、基质金属蛋白酶组织抑制因子-1(TIMP-1)表达的影响,从胶原降解的角度探讨秋水仙碱对肝纤维化有无逆转作用及可能存在的机制.方法 制备免疫性大鼠肝纤维化模型,并给予秋水仙碱治疗;通过RT-PCR检测MMP-1、TIMP-1的表达,并作Ⅰ、Ⅲ型胶原的免疫组化以及Masson胶原染色.结果 发现秋水仙碱对肝纤维化大鼠MMP-1的表达无明显影响(P>0.05),但可以抑制TIMP-1的表达(P<0.05),促进Ⅰ、Ⅲ型胶原的降解(P<0.05);然而在病理形态学的观察中,未发现秋水仙碱治疗组与肝纤维化模型组之间存在的显著性差异(P>0.05).结论 秋水仙碱可以抑制纤维化肝脏TIMP-1的表达,从而增强间质胶原酶的活性,促进Ⅰ、Ⅲ型胶原的降解,产生抗肝纤维化的作用,但其作用有限.