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.     

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。     

Efficacy of Chinese medicine Yi-gan-kang granule in prophylaxis and treatment of liver fibrosis in rats

AIM: To investigate the efficacy of a Chinese medicine, Yi-gan-kang granule (granules for benefiting the liver), in prophylaxis and treatment of liver fibrosis in rats and its possible mechanism. METHODS: One hundred and forty Sprague-Dawley rats were randomly divided into seven groups (20 each): group 1, blank control group without any interference during the study; group 2, CCI4-induced liver fibrosis group; group 3, pig serum-induced liver fibrosis group; group 4, prophylaxis group of CCl4-induced liver fibrosis by Yi-gan-kang; group 5, prophylaxis group of pig serum-induced liver fibrosis by Yi-gan-kang; group 6, treatment group of CCI4-induced liver fibrosis by Yi-gan-kang; group 7, treatment group of CCI4-induced liver fibrosis by Yi-gan-kang. At wk 6,10,14 and 20 (baseline for CCl4., or big serum induction), five rats in each group were anesthetized and their livers were removed for pathological studies including immunohistochemical studies for α-SMA, type I collagen and In situ hybridization of tissue inhibitor of metalloproteinase-1 (TTMP-1) mRNA of hepatic stellate cells (HSCs). Anti-lipid peroxidation in isolated mitochondria and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) colorimetric assay for proliferation and terminal deoxynucleotidyl transferase-medicated dUTP-biotin nick end-labeling (TUNEL), flow cytometry and electron microscopy for apoptosis in isolated HSCs were also studied. RESULTS: The mean number of pseudolobuli at wk 10, 14 and 20 in the prophylaxis group was significantly less than that in the control group (P<0.05 or 0.01). The effect of prophylaxis at wk 14 in CCI4 rats and at wk 10 in pig serum-induced rats was much better than that of treatment group (P<0.01). The thickness (in μm) of fibers both in pig serum-induced prophylaxis and in treatment groups at wk 14 and. 20 was significantly less than that in control group (P<0.05). The number of fibers both in prophylaxis and in treatment groups from wk 10 or 14 to 20 was significantly less than that in control group (P<0.05 or P<0.01). The tissue HSC positive rates of type I collagen, α-SMA and TIMP-1 mRNA, which represented the active phenotype of HSCs in tissues, remained very high from wk 6 to the end of model making in control group. While in prophylaxis group, they were at a relatively low level. In treatment group, there was a gradual decreasing trend. Time- and dose-dependent effects of anti-lipid peroxidation on isolated mitochondria, cell proliferation and apoptosis in cultured HSCs were also observed during the study. CONCLUSION: Yi-gan-kang can effectively inhibit or inverse the course of liver fibrogenesis in CCI4- and pig serum-induced rat models.     

Suppressive effects of 171β-estradiol on hepatic fibrosis in CCI4-induced rat model

AIM: To investigate the pathway via which 17β-estradio(β-Est) exerts suppressive effects on rat hepatic fibrosis.METHODS: In vivo study was done in CCI4-induced female hepatofibrotic rats. Fibrosis-suppressive effect of β-Est(20 μg/kg&#183;d) was evaluated in intact and ovariectomized rat models. Six weeks after the treatment, all the rats were sacrificed and specimens of serum or liver tissue were collected for the studies. Serum liver enzymes,fibrosis markers and estradiol levels were determined by standard enzymatic methods, ELISA and RIA, respectively.Degrees of fibrosis and areas of hepatic stellate cells(HSCs) positive for alpha-smooth muscle actin (a-SMA) in the liver were determined by van Gieson (VG) stain and immunohistochemistry. In vitro studies, HSCs were isolated by a combination of pronase-collagenase perfusion and density gradient centrifugation. First-passage HSCs were randomly divided into 10 groups, and different concentrations of β-Est, 2-hydroxyestradiol (2OHE) or 2-methoxyestradiol(2MeOE) were separately added to the cell groups. After incubation for 72 h, the degree of cell proliferation, collagen production, ~-SMA or estrogen receptor (ER) expression was determined by MTT assay, ELISA and immunohistochemistry,respectively.RESULTS: β-Est treatment reduced aspartate aminotransfer-ase (AST), alanine aminotransferase (ALT), hyaluronic acid(HA) and type IV collagen (C IV) in sera, suppressed hepatic collagen content, decreased the areas of HSCs positive for α-SMA significantly in both intact and ovariectomized female hepatofibrotic rats. There was a negative correlation between the percentage of fibrotic area of liver tissue and the serum estradiol level; the calculated correlation coefficient was -0.57 (P&lt;0.01). β-Est and its metabolites concentration-dependently (10^-9 mol/L-10^-7 mol/L) inhibited HSC proliferation and collagen synthesis. At the concentration of 10^-7 mol/L, they could inhibit α-SMA expression. The order of potency was 2MeOE&gt;2OHE&gt;β-Est.CONCLUSION: β-Est may suppress hepatic fibrosis probably via its biologically active metabolites.     

The regulatory role of AT 1 receptor on activated HSCs in hepatic fibrogenesis:effects of RAS inhibitors on hepatic fibrosis induced by CCl(4)

AIM:To assess the effect of ACE inhibitor and Ang II type 1 (AT1) receptor antagonist in preventing hepatic fibrosis caused by CCl(4) administration in rats;to investigate whether or not there are expression of AT 1 receptors on hepatic stellate cells; and to observe the effect of Ang II on proliferation and ECM synthesis of cultured HSCs.METHODS:Studies were conducted in male Sprague-Dawley rats. Except for the hepatofibrotic model group and the control group, in three treated groups, either enalapril (5mg/kg), or losartan (10mg/kg), or enalapril + losartan were given to the fibrotic rats by daily gavage, and saline vehicle was given to model and normal control rats. After 6 weeks, liver fibrosis was assessed directly by hepatic morphometric analysis, which has been considered the gold standard for the quantification of fibrosis. The expressions of AT 1 receptors and (alpha-mooth muscle actin,alpha-SMA) in liver tissue or isolated hepatic stellate cells (HSCs) were detected by immunohistochemical techniques. The effect of Ang II on HSC proliferation was determined by MTT method. Effect of Ang II on collagen synthesis of HSCs was determined by (3)H-proline incorporation.RESULTS:Contrasted to the fibrosis in rats of the model group, groups of rats treated with either enalapril or losartan, or a combination of two drugs showed a limited expansion of the interstitium (4.23 plus minus 3.70 vs 11.22 plus minus 4.79, P<0.05), but no difference was observed among three treated groups (5.38 plus minus3.43, 4.96 plus minus 2.96, 4.23 plus minus 2.70, P>0.05). Expression of AT 1 receptors was found in fibrotic interstitium of fibrotic rats, whereas in normal control rats they were limited to vasculature only to a very slight degree. AT 1 receptors were also expressed on activated HSCs in the culture. At concentrations from 10(-9) to 10(-5)mol/L, Ang II stimulated HSC proliferation in culture in a dose dependent manner. Increasing Ang II concentrations produced corresponding increases in (3)H-proline incorporation. Differences among groups were significant.CONCLUSION:Angiotensin converting enzyme inhibitors and AT 1 blocker may slow the progression of hepatic fibrosis;activated HSCs express AT 1 receptors, and Ang II can stimulate the proliferation and collagen synthesis of HSCs in a dose-dependent manner; and activation of RAS may be related to hepatic fibrogenesis induced by CCl(4).     

抗纤软肝颗粒对肝纤维化大鼠肝组织核因子-κB基因表达的影响

目的观察中药抗纤软肝颗粒对四氯化碳加乙醇诱导肝纤维化大鼠的肝组织核因子-κB（NF-κB）p65 mRNA表达的影响。方法 SD雄性大鼠40只,随机分为模型对照组（M）15只,抗纤软肝颗粒组（K）15只,正常对照组（N）10只。M组、K组分别以40%四氯化碳橄榄油液腹腔注射,每周2次,并以5%乙醇为唯一饮水。造模同时,K组即予抗纤软肝颗粒20 g.kg-1.d-1（中等剂量）灌胃,每天一次。7周末,各组取肝右叶分别作肝组织病理观察及检测肝组织NF-κB p65 mRNA表达。结果抗纤软肝颗粒组肝细胞变性坏死及肝纤维化水平较模型对照组明显减轻（P〈0.05）,肝组织NF-κB p65 mRNA表达较模型对照组明显减弱（P〈0.05）。结论抗纤软肝颗粒能下调肝纤维化大鼠肝组织NF-κB p65 mRNA表达,抑制肝纤维化的形成和发展,其机制可能是通过调控NF-κB p65 mRNA转录过程实现的。     

Cystamine ameliorates liver fibrosis induced by carbon tetrachloride via inhibition of tissue transglutaminase

AIM To investigate the anti-fibrosis effect of the tissue transglutaminase (tTG) specific inhibitor cystamine on liver fibrosis.METHODS Sixty-eight male Sprague Dawley rats were divided into three groups normal control, liver fibrosis control and cystamine-treated group. Liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl4), and Cystamine was administrated by intraperitoneal injection starting 2 d before the first administration of CCl4. Animals in each group were further divided into 2 subgroups according to two time points of 4 wk and 8 wk after treatment. Hepatic function, pathological evaluation (semi-quantitative scoring system, SSS) and liver hydroxyproline (Hyp)content were examined. Real-time PCR was used to detect the expression of tTG, smooth muscle alpha actin (α-SMA), tissue inhibitor of metalloproteinase 1 (TIMP-1)and collagen-1 mRNA. The expressions of tTG and α-SMA protein were detected by Western Blotting.RESULTS Eight weeks after treatment, the SSS score of liver was significantly less in the cystamine group than that in the fibrosis control group (P ＜ 0.01). The levels of alanine aminotransferase (ALT) and total bile acid (TBA)at the 4 wk and 8 wk time points were decreased in the cystamine group compared with those in fibrosis controls (P ＜ 0.01). Liver hydroxyproline content at the 4 wk and 8 wk time points showed a substantial reduction in the cystamine group compared to fibrosis controls (P ＜ 0.01).The expression of tTG, α-SMA, collagen-1, TIMP-1 mRNA and tTG, as well as α-SMA protein was downregulated in the cystamine group compared to fibrosis controls.CONCLUSION Cystamine can ameliorate CCl4 induced liver fibrosis and protect hepatic function. The possible mechanism is related to the reduced synthesis of the extracellular matrix (ECM) caused by the inhibition of hepatic stellate cell activation and decreased expression of TIMP-1.     

Effect of WeiJia on carbon tetrachloride induced chronic liver injury

AIM:To study the effect of WeiJia on chronic liver injuryusing carbon tetrachloride(CCl_4)induced liver injuryanimal model.METHODS:Wistar rats weighing 180-220g were ran-domly divided into three groups:normal control group(Group A),CCl_4 induced liver injury control group(GroupB)and CCl_4 induction with WeiJia treatment group(GroupC).Each group consisted of 14 rats.Liver damage andfibrosis was induced by subcutaneous injection with 40?l_4 in olive oil at 3 mL/kg body weight twice a week foreight weeks for Groups B and C rats whereas olive oilwas used for Group A rats.Starting from the third week,Group C rats also received daily intraperitoneal injectionof WeiJia at a dose of 1.25 μg/kg body weight.Animalswere sacrificed at the fifth week(4 male,3 female),andeighth week(4 male,3 female)respectively.Degree offibrosis were measured and serological markers for liverfibrosis and function including hyaluronic acid(HA),typeIV collagen(CIV),γ-glutamyl transferase(γ-GT),alanineaminotransferase(ALT)and aspartate aminotransferase(AST)were determined.Alpha smooth muscle actin (α-SMA)and proliferating cell nuclear antigen(PCNA)immunohistochemistry were also performed.RESULTS:CCl_4 induction led to the damage of liver anddevelopment of fibrosis in Group B and Group C ratswhen compared to Group A rats.The treatment of WeiJiain Group C rats could reduce the fibrosis condition sig-nificantly compared to Group B rats.The effect could beobserved after three weeks of treatment and was moreobvious after eight weeks of treatment.Serum HA,CIV,ALT,AST and γ-GT levels after eight weeks of treatmentfor Group C rats were 58±22 μg/L(P<0.01),57±21 μg/L(P<0.01),47±10 U/L(P<0.01),139±13 U/L(P<0.05)and 52±21 U/L(P>0.05)respectively,similar to normalcontrol group(Group A),but significantly different fromCCl_4 induced liver injury control group(Group B).An in-crease in PCNA and decrease in α-SMA expression levelwas also observed.CONCLUSION:WeiJia could improve liver function andreduce liver fibrosis which might be through the inhibi-tion of stellate cell activity.     

Effect of TL-10 on the expression of HSC growth factors in hepatic fibrosis rat

AIM: To study the effect of IL-10 on the expression of growth factors - transforming growth factor-β1 (TGF-β1),epidermal growth factor (EGF), hepatocyte growth factor (HGF)and platelet-derived growth factor (PDGF)of hepatic stellate cells (HSCs) of hepatic fibrosis rat and the anti-fibrogenic role of exogenous IL-10.METHODS: Hepatic fibrosis was induced by CCl4administration intra-peritoneally. Sixty clean male SpragueDawley (SD) rats were randomly divided into three groups:normal control group (GN, 8 rats), hepatic fibrosis model group (GC, 28 rats) and IL-10 treated group (GI, 24 rats).At the beginning of the 7th and 11th wk, rats in each group were routinely perfused with pronase E and type Ⅳ collagenase through a portal vein catheter and the suspension obtained from the liver was spun by centrifugation with 11% Nycodenz density gradient to isolate HSCs. Histological examination was used to determine the degree of hepatic fibrosis. RT-PCR was employed to analyze mRNA expression from freshly isolated cells. Tmmunocytochemistry was performed to detect protein expression in primary cultured HSCs.RESULTS: Rat hepatic fibrosis was developed with the increase of injection frequency of CCl4, and HSCs were successfully isolated. At the 7th and 11th wk, TGF-β1, EGF,and HGF mRNA in GC increased obviously compared with GN (P = 0.001/0.042, 0.001/0.001, 0.001/0.001) and GI (P= 0.001/0.007, 0.002/0.001, 0.001/0.001). For TGF-β1,no difference was observed between GI and GN. For EGF,mRNA level in GI increased compared with GN during the 7th wk (P= 0.005) and 11th wk (P= 0.049). For HGF, mRNA level in GI decreased compared with GN at the 7th wk (P = 0.001) and 11th wk (P = 0.021). Between these two time points, TGF-β1 expression at the 7th wk was higher than that of the 11th wk (P = 0.049), but for EGF, the former was lower than the latter (P = 0.022). As for PDGF mRNA, there was no significant difference between these groups, but difference seemed to exist in protein levels.Results by immunocytochemistry of TGF-β1 and EGF were paralleled with the above findings.CONCLUSION: The expression of TGF-β1, EGF and HGF increased in HSC of hepatic fibrosis rat and decreased after treatment with IL-10. IL-10 plays an anti-fibrogenic role by suppressing growth factors expression.     

Long-term administering low anticoagulant activity heparin can lessen rat hepatic fibrosis induced by either CCl(4) or porcine serum injection.

BACKGROUND/AIM: Heparin or heparin-derived molecules have been demonstrated to have a renoprotective activity in a number of experimental nephropathies and anti-fibrotic effect on pulmonary fibrosis induced by Bleomycin. However, the effectiveness of low anticoagulant activity heparin (LAAH) in the treatment of liver fibrosis has not been well defined. We are here demonstrating by both biochemical and morphological methods that long term LAAH administering can considerably decrease the hepatic fibrosis in rats elicited by carbon tetrachloride (CCl(4)) or injection of porcine serum. And its inhibition of fibrosis may be associated with the intervention of ERK signal transduction pathway and down-regulation of AP-1 activity in hepatic stellate cells. METHODS: LAAH were given to rats that subjected to CCl(4) or porcine serum injection induced liver fibrosis regimens up to 10 weeks. Immunohistochemical stainings for collagen types I and III plus image analysis as well as measurement of hydroxyproline content in the livers were carried out to evaluate the fibrosis of livers in rats. The activated HSCs (strong positive immunohistochemical staining for alpha-smooth actin) in the livers were counted under microscopy. The activities of extracellular signal-regulated kinase (ERK) in HSCs in vitro were estimated by the Western blot. Activator protein-1 (AP-1) activity in nuclear proteins of HSCs was measured by the electrophoresis motility shift assay (EMSA). RESULTS: The immunohistochemical staining plus image analysis showed that LAAH administration could reduce the contents of collagen types I and III to 16.48% and 27.94% of those in saline administration control in livers of rats treated with CCl(4) or 30.98% and 35.43% of those in saline administration control in livers of rats subjected to injection of porcine serum. Meanwhile, LAAH could decrease the content of hydroxyproline in the livers of rats treated with CCl(4) or injection of porcine serum by 32.83% or 32.41%, respectively. Long-term administration of LAAH can remarkably reduce the alpha-SMA positive cells only in the fibrotic livers induced by CCl(4). In vitro studies of HSCs showed that LAAH could inhibit the PDGF-BB augmented both expression and phosphorylation of ERK proteins and AP-1 activity in a dose-dependent manner. CONCLUSIONS: Long-term administering LAAH can suppress the hepatic fibrosis either induced by CCl(4) or injection of porcine serum. The mechanism of its anti-fibrotic effect may partly be related to the inference of ERK signal transduction pathway and AP-1 activity in activated hepatic stellate cells.     

内毒素受体在肝星状细胞的表达及其作用

目的了解内毒素受体在肝星状细胞活化中的变化和作用。方法分离正常大鼠的肝星状细胞，以逆转录聚合酶链反应法检测其在体外培养过程中内毒素受体（CD14和TLR4）mRNA的表达变化。以细胞免疫染色法检测肝星状细胞内毒素受体CD14的表达。制作肝纤维化和肝硬化的大鼠模型，免疫组织化学法动态检测肝组织内CD14和α平滑肌肌动蛋白的表达变化和定位。结果初分离的肝星状细胞表达低水平的CD14 mRNA，不表达TLR4 mRNA，培养活化的肝星状细胞内毒素受体的表达增强，内毒素可上调这种表达。体外培养10d的肝星状细胞表达CD14蛋白，内毒素作用后CD14表达更明显。在肝纤维化的发展过程中，肝组织内CD14阳性细胞增多，阳性细胞多分布于肝窦周围，晚期CD14阳性细胞聚集在纤维隔内，与α平滑肌肌动蛋白阳性细胞的分布一致。结论肝星状细胞在体内外的活化过程中内毒素受体的表达增强，因此，内毒素受体可能参与肝星状细胞在肝脏炎症和纤维化中的作用。     

Effect of IL-10 on the expression of HSC growth factors in hepatic fibrosis rat

AIM: To study the effect of IL-10 on the expression of growth factors - transforming growth factor-β1(TGF-β1), epidermal growth factor (EGF), hepatocyte growth factor (HGF)and platelet-derived growth factor (PDGF) of hepatic stellate cells (HSCs) of hepatic fibrosis rat and the anti-fibrogenic role of exogenous IL-10. METHODS: Hepatic fibrosis was induced by CCl4 administration intra-peritoneally. Sixty clean male Sprague-Dawley (SD) rats were randomly divided into three groups: normal control group (GN, 8 rats), hepatic fibrosis model group (GC, 28 rats) and IL-10 treated group (GI, 24 rats). At the beginning of the 7th and 11th wk, rats in each group were routinely perfused with pronase E and type IV collag-enase through a portal vein catheter and the suspension obtained from the liver was spun by centrifugation with 11% Nycodenz density gradient to isolate HSCs. Histological examination was used to determine the degree of hepatic fibrosis. RT-PCR was employed to analyze mRNA expression from freshly isolated cells. Immunocytochemistry was performed to detect protein expression in primary cultured HSCs. RESULTS: Rat hepatic fibrosis was developed with the increase of injection frequency of CCl4, and HSCs were successfully isolated. At the 7th and 11th wk, TGF-β1, EGF, and HGF mRNA in GC increased obviously compared with GN (P= 0.001/0.042, 0.001/0.001, 0.001/0.001) and GI (P=0.001/0.007, 0.002/0.001, 0.001/0.001). For TGF-β1, no difference was observed between GI and GN. For EGF, mRNA level in GI increased compared with GN during the 7th wk (P=0.005) and 11th wk (P=0.049). For HGF, mRNA level in GI decreased compared with GN at the 7th wk (P=0.001) and 11th wk (P=0.021). Between these two time points, TGF-β1 expression at the 7th wk was higher than that of the 11th wk (P=0.049), but for EGF, the former was lower than the latter (P=0.022). As for PDGF mRNA, there was no significant difference between these groups, but difference seemed to exist in protein levels. Results by immunocytochemistry of TGF-β1 and EGF were paralleled with the above findings. CONCLUSION: The expression of TGF-β1, EGF and HGF increased in HSC of hepatic fibrosis rat and decreased after treatment with IL-10. IL-10 plays an anti-fibrogenic role by suppressing growth factors expression.     

The role of Smad3 in mediating mouse hepatic stellate cell activation.

Transforming growth factor beta (TGF-beta) is the most potent profibrogenic mediator in liver fibrosis. Although Smad proteins have been identified as intracellular mediators in the TGF-beta signaling pathway, the function of individual Smad proteins remains poorly understood. The aim of this study was to explore the contribution of Smad3 in mediating TGF-beta responses in a model of acute liver injury in vivo and in culture-activated hepatic stellate cells (HSCs). Wild-type, Smad3 heterozygous or Smad3 homozygous knockout mice were treated with a single intragastric administration of CCl(4). After 72 hours, the induction of hepatic collagen alpha1(I) and alpha2(I) messenger RNA (mRNA) levels in Smad3 knockout mice was only 42% and 64%, respectively, of the levels induced in wild-type mice. However, smooth muscle alpha-actin (alpha-SMA) was expressed at a slightly higher level in livers from knockout mice compared with wild-type mice. In culture-activated HSCs from Smad3 knockout mice, collagen alpha1(I) mRNA was 73% of wild-type HSCs, but alpha-SMA expression was the same. HSCs from knockout mice showed a higher proliferation rate than wild-type HSCs. Smad3-deficient HSCs did not form TGF-beta1-induced Smad-containing DNA-binding complexes. In conclusion, (1) maximal expression of collagen type I in activated HSCs requires Smad3 in vivo and in culture; (2) Smad3 is not necessary for HSC activation as assessed by alpha-SMA expression; (3) Smad3 is necessary for inhibition of proliferation of HSCs, which might be TGF-beta-dependent; and (4) Smad3 is required for TGF-beta1-mediated Smad-containing DNA-binding complex formation in cultured HSCs.     

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.     

IGF-1在肝纤维化大鼠中的表达及IL-10的干预作用

目的：探讨胰岛素样生长因子-l(IGF-1)在实验性大鼠肝纤维化过程中的表达状况及白细胞介素—10(IL-10)对肝纤维化大鼠IGF—l表达的影响。方法：建立大鼠肝纤维化模型并行IL-l0干预实验。分批从肝纤维化进程中不同阶段的对照组、CCl4诱导肝纤维化模型组及IL-l0干预肝纤维化组的大鼠中取肝脏组织，采用S—P免疫组织化学方法分别检测分析不同组及同组不同阶段大鼠肝组织中IGF-l的表达状况。结果：经CCl4诱导成功建立大鼠肝纤维化模型。随着肝纤维化程度的进展，IGF-l在肝组织中阳性表达明显增强；经Ridit分析，对照组(38．1％)与肝硬化模型组(92．0％)组间比较IGF-l阳性表达均有显著性差弄(P＜0．01)；IL-l0干预肝纤维化组IGF-l阳性表达(71．4％)较肝纤维化模型组均明显减弱，经Ridit分析组间均有显著性差异(P＜0．05)，且随IL-l0干预时间的延长，IGF—l在肝组织中阳性表达均逐渐减弱。结论：IGF-l随着肝纤维化程度的进展阳性表达增强，外源性IL-l0可降低CCl4诱导大鼠肝纤维化中IGF-l的表达。