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.     

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.     

Effects of interleukin-10 on activation and apoptosis of hepatic stellate cells in fibrotic rat liver

AIM:To study the effects of interleukin-10(IL-10)on the expression of α-smooth muscle actin(α-SMA),nuclear factor-κB(NF-κB)and Fas/Fas ligand(FasL)inhepatic stellate cells of experimental rats with hepaticfibrosis.METHODS:Sixty clean SD rats were randomly dividedinto control group(group N),liver fibrotic group(groupC)and IL-10 treatment group(group I).Control groupreceived intraperitoneal injection of saline(2ml·kg~(-1)),twicea week.Fibrotic group was injected intraperitoneallywith 50% carbon tetrachloride(CCl_4)(2 ml·kg~(-1)),twicea week.IL-10 treatment group was given IL-10 at adose of 4 μg·kg~(-1)20 minutes before CCl_4 administrationfrom the third week.Hepatic stellate cells(HSCs)wereisolated from these rats at the seventh and eleventhweeks during the course of liver fibrosis,respectively.The expression of α-SMA and NF-κB in HSCs wasmeasured by S-P immunohistochemistry.The expressionof Fas and FasL mRNA was measured by RT-PCR.Furthermore,liver tissues were harvested from threegroups at the same time.RESULTS:The CCl_4- induced experimental rat hepaticfibrosis model was established successfully.The purityof extracted hepatic stellate cells was about 95% andthe yield of hepatic stellate cells was 1.2-2.3×10~6/g livertissue averagely.The positive expression of α-SMA andNF-κB was 36.5% and 28.5% respectively in group N.The positive levels of α-SMA and NF-κB were increasedsignificantly in group C compared to group N(P<0.01).The positive signals decreased significantly(P<0.05)ingroup I.In the 11~(th)week,the HSCs of group I becameround with visible pyknotic nuclei.The expression ofNF-μB in group C was significantly increased in a time-dependentmanner(P<0.01),but there was no difference in the α-SMA expression(P>0.05).The mRNA of Fasand FasL in group C was significantly increased in a time-dependent manner compared to that in control group.After treated with IL-10,the expression level of Fas andFasL was higher in group I than in group C.CONCLUSION:The positive expression of α-SMA andNF-κB in hepatic stellate cells is decreased by ectogenicIL-10 in liver fibrosis induced by CCl_4.The expression ofFas and FasL is increased in the course of liver fibrosis,and is further increased by IL-10.IL-10 could inhibitthe activation of HSCs and cause apoptosis of activatedHSCs.     

Expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 in hepatic stellate cells during rat hepatic fibrosis and its intervention by IL-10

AIM: To investigate the expression of matrix metallopr-oteinase-2 and tissue inhibitor of metalloproteinase-1 in hepatic fibrosis and the antifibrogenic role of exogenous interleukin-10 (IL-10). METHODS: Hepatic fibrosis was induced by CCI4 administration and 60 male Sprague-Dawley rats were randomly divided into normal control group (group N, 8 rats), CCI4-induced group (group C, 28 rats) and IL-10-treated group (group I, 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 portal vein catheter and the suspension was centrifuged by 11% Nycodenz density gradient to isolate hepatic stellate cells (HSCs). RT-PCR was used to analyze mRNA of MMP-2 and TIMP-1 from freshly isolated cells. Densitometric data were standardized with β-actin signals. Immunocytochemistry was performed to detect MMP-2 and TIMP-1 expression in HSC cultured for 72 h. RESULTS: Compared to group N in the 7th wk, MMP-2 and TIMP-1 mRNA increased in group C (P= 0.001/0.001) and group I (P= 0.001/0.009). The level of MMP-2 and TIMP-1 mRNA in group I was significantly lower than that in group C (P= 0.001/0.001). In the 11th wk, MMP-2 mRNA in group I was still lower than that in group C (P = 0.005), but both dropped compared with that in the 7th week (P = 0.001/0.004). TIMP-1 mRNA in group I was still lower than that in group C (P= 0.001), and increased in group C (P= 0.001) while decreased in group I (P = 0.042) compared with that in the 7th wk. Same results were found by immunocytochemistry. CONCLUSION: Expression of MMP-2 and TIMP-1 is increased in hepatic fibrosis. IL-10 exhibits an antifibrogenic effect by suppressing MMP-2 and TIMP-1 expression.     

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.     

Oral imatinib treatment reduces early fibrogenesis but does not prevent progression in the long term

BACKGROUND/AIMS: Transactivated hepatic stellate cells (HSCs) represent the key source of extra cellular matrix (ECM) in fibrotic liver. Imatinib, a potent inhibitor of the PDGF receptor tyrosine kinase, reduces HSC proliferation and fibrogenesis when treatment is initiated before fibrosis has developed. We tested the antifibrotic potential of imatinib in ongoing liver injury and in established fibrosis. METHODS: BDL-rats were gavage fed with 20 mg/kg/d imatinib either early (days 0-21) or late (days 22-35) after BDL. Untreated BDL-rats served as controls. ECM and activated HSCs were quantified by morphometry. Tissue activity of MMP-2 was determined by gelatin zymography. mRNA expression of TIMP-1 and procollagen alpha1(I) were measured by RT-PCR. Liver tissue concentration of imatinib was measured by tandem mass spectrometry. RESULTS: Early imatinib reduced ECM formation by 30% (P=0.0455) but left numbers of activated HSCs and procollagen I expression unchanged. MMP-2 activity and TIMP-1 expression were reduced by 50%. Late imatinib treatment did not alter histological or molecular markers of fibrogenesis despite high imatinib tissue levels. CONCLUSIONS: The antifibrotic effectiveness of imatinib is limited to the early phase of fibrogenesis. In ongoing liver injury other mediators most likely compensate for the inhibited PDGF effect.     

PDGF-BB对肝星状细胞表达MMP-2及TIMP-1的影响

目的：探讨血小板衍生生长因子－BB（PDGF－BB）对体外培养大鼠肝星状细胞（HSC）表达基质金属蛋白酶－2（MM－2）及金属蛋白酶组织抑制因子－1（TIMP－1）的影响。方法：原位灌流分离HSC，体外培养激活，用PDGF－BB干预，采用半定RT－PCR方法检测各组MMP－2、TIMP－1 mRNA的表达情况。结果：PDGF－BB干预组TIMP－1 mRNA的表达较对照组明显增强（P＜0．01），且随着干预时间的延长其表达逐渐增强（P＜0．01）；而PDGF－BB干预组MMP－2表达与对照组无明显差异（P＞0．05）。结论：PDGF－BB促进肝纤维化与其引起HSC表达TIMP－1增强有关。     

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.     

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.     

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.     

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.     

内毒素对实验性肝纤维化大鼠基质金属蛋白酶-2的影响

目的探讨内毒素对早期实验性肝纤维化大鼠基质金属蛋白酶-2（MMP-2）的影响及其在肝纤维形成过程中的作用机制。方法建立实验性肝纤维化大鼠模型,采用免疫组化染色法半定量测定MMP-2、α-SMA在肝纤维化过程中的时序动态表达,网状纤维法判定肝纤维化程度,同时采用ELISA法测定血浆内毒素和TNF-α的水平。结果有内毒素血症发生,不同时期（0、2、4、6周）血浆内毒素水平呈梯度增加,组间差异明显,血浆内毒素与TNF-α及肝组织MMP-2、α-SMA表达有较好的相关性。MMP-2表达的部位、时序、量的变化都与α-SMA的表达大致平行,以中央静脉周围、汇管区、纤维间隔内为明显,提示MMP-2主要由活化的肝星状细胞（HSC）合成。肝纤维化程度与α-SMA、MMP-2在肝脏的表达呈正相关。结论内毒素可能通过细胞因子TNF-α,介导肝组织MMP-2、α-SMA的表达,而后者又与肝纤维化的程度密切相关。     

脂联素对原代大鼠肝星状细胞表达MMP-2和MMP-13的影响

目的观察外源性脂联素对体外培养原代大鼠肝星状细胞（HSC）表达基质金属蛋白酶-2（MMP-2）和基质金属蛋白酶-13（MMP-13）的影响,探讨脂联素对肝纤维化的影响。方法改良的肝脏二步原位灌注法分离肝星状细胞,外源性脂联素（125μg/L、250μg/L、500μg/L）处理体外培养的原代肝星状细胞。Real-time PCR方法分析其对原代HSCMMP-2和MMP-13 mRNA表达的影响,ELISA方法检测原代HSC分泌MMP-2和MMP-13蛋白水平,酶谱法检测细胞外MMP-2的酶活性。结果脂联素促进MMP-2和MMP-13 mRNA表达,随脂联素浓度增加,作用更加明显。脂联素可促进HSC分泌MMP-2及MMP-13蛋白,提高MMP-2酶活性,但均无剂量依赖性。结论脂联素调节MMP-2表达和活性,促进MMP-13表达,可能对肝纤维化产生影响。     

慢性乙型肝炎肝组织中MMP-2、TIMP-1与肝纤维化关系的研究

为了研究慢性乙型肝炎患者肝纤维化与肝组织中基质金属蛋白酶-2(MMP-2)及其天然抑制物金属蛋白酶组织抑制剂-1(TIMP-1)的关系,对60例慢性乙型肝炎患者进行肝组织病理活检,其中S_110例,S_224例,S_312例,S_414例。采用苦叶酸天狼猩红染色检测胶原面积百分比,单克隆抗体(McAb)免疫组织化学染色检测MMP-2、TIMP-1阳性细胞的方法进行病理分析。结果显示：慢性乙型肝炎肝组织中胶原面积百分比与肝组织纤维化分期正相关(r=0．885,P=0．000);慢性乙型肝炎肝组织中MMP-2与肝纤维化的分期无关(r=0．034,P= 0．896);慢性乙型肝炎肝组织中TIMP-1和肝纤维化的分期正相关(r=0．760,P=0．000);慢性乙型肝炎肝组织中MMP-2/TIMP-1和肝纤维化分期负相关(r=-0．674,P=0．000)。总之,TIMP-1参与了慢性乙型肝炎肝纤维化纤维化的过程,而MMP-2/TIMP-1是诊断肝纤维化的比较合适的指标。     

四种中药单体的抗肝纤维化作用及其机制

目的 比较丹参素、黄芩甙、黄芪、三七总皂甙4种中药单体抗肝纤维化的疗效并探讨其作用机制.方法 以秋水仙碱为对照,将4种中药用于大鼠肝纤维化模型和大鼠肝星状细胞(HSC).观察肝组织形态变化;检测血清透明质酸、Ⅳ型胶原含量;检测肝组织羟脯氨酸、丙二醛(MDA)含量,超氧化物歧化酶(SOD)活性,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)、转化生长因子(TGF)β1的表达;观察HSC活化增殖周期、细胞凋亡及Ⅰ、Ⅲ型胶原蛋白的表达.结果 与模型组比较,中药处理组的肝组织形态显著改善,血清透明质酸、Ⅳ型胶原含量明显减少,肝组织MDA、羟脯氨酸含量及TIMP-1、TGF β1表达亦明显减少、SOD活性显著升高,丹参素组作用最强,黄芩甙组、黄芪组次之,各组比较,差异有统计学意义(P＜0.01或P＜0.05).四种中药都能抑制HSC活化增殖、促进细胞凋亡、抑制HSC的Ⅰ、Ⅲ型胶原蛋白表达,丹参素、黄芩甙、黄芪作用较强.结论 四种中药均能减缓实验性肝纤维化的发生.丹参素作用最强,黄芩甙和黄芪强于三七总皂甙.机制可能是这些中药能通过下调TGF β1抑制HSC活化增殖、促进细胞凋亡而抑制HSC的Ⅰ、Ⅲ型胶原蛋白表达;降低肝组织MDA含量,增加SOD活性;下调TIMP-1/MMP-1比值从而促进细胞外基质降解,减少其沉积.     

Effects of oxymatrine on experimental hepatic fibrosis and its mechanism in vivo

AIM: Hepatic fibrogenesis has close relation with hepatic stellate cells (HSC)and tissue inhibitors of metalloproteinase (TIMP). Oxymatrine (OM) is a kind of Chinese herb that is found to have some effects on liver fibrosis. We aimed to determine the effects of OM on hepatic fibrosis and explore the possible mechanism. METHODS: Thirty-two rats were randomly divided into four groups; 16 were used to develop hepatic fibrosis by carbon tetrachloride (CCI4) and treated with or without OM, and 16 were used as controls. The expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and α-smooth muscle actin (α-SMA) in the livers of rats was detected by immunohisto-chemical assay. Liver pathology was determined by H&E staining and reticulum staining. RESULTS: In CCl4-injured rats, the normal structure of lobules was destroyed, and pseudolobules were formed. Hyperplasia of fibers was observed surrounding the lobules. While the degree of fibrogenesis in liver tissues was significantly decreased in those rats with OM-treatment compared with those without OM treatment. The pseudolobules were surrounded by strong, multi-layer reticular fibers, which netted into pseudolobules in CCl4-injured rats, however, there was a significant decrease in reticular fibers in OM-treated rats. The expression of TIMP-1 in hepatic cells was weak in control groups, but strong in CCl4-injured groups, however, the expression of TIMP-1 was significantly inhibited by OM (F = 52.93, P<0.05). There was no significant change in the expression of α-SMA between CCl4-injured rats with or without OM treatment (F= 8.99, P>0.05). CONCLUSION: OM effectively inhibits CCl4-induced fibrogenesis in rat liver tissues, probably by reducing the expression level of TIMP-1.