复方红景天干预大鼠肝纤维化作用的实验研究

目的：从血清、组织、分子等水平探讨复方红景天抗大鼠肝纤维化的作用及其可能的机制，为抗肝纤维化研究提供新的选择。方法：用四氯化碳皮下注射法诱导SD大鼠肝纤维化模型，同时给予复方红景天颗粒口服进行干预性治疗，观察大鼠肝功能，血清纤维化相关酶活性ALT、AST、β-NAG、MAO，血清肝纤维化指标PCⅢ、CIV、HA以及肝组织羟脯氨酸含量和病理学变化，同时检测复方红景天对大鼠肝组织TGF β1、I型前胶原mRNA表达水平的影响。结果：口服复方红景天可改善大鼠肝功能，降低血清PCⅢ、CⅣ、HA水平，抑制β－NAG、MAO的活性，降低肝组织羟脯氨酸含量，明显改善大鼠肝组织病理变化，并降低大鼠肝组织TGFβ、I型前胶原mRNA的表达水平。结论：复方红景天可能通过抑制TGF β1、I型前胶原mRNA的表达从而减少胶原合成、保护肝细胞等机制有效干预CC14诱导的大鼠肝纤维化，值得深入研究。     

依达拉奉对实验性肝纤维化大鼠脂质过氧化的影响

目的探讨依达拉奉（EDA）对实验性肝纤维化大鼠脂质过氧化的影响。方法以四氯化碳诱导大鼠肝纤维化模型。30只Sprague—Dawley大鼠随机分为对照组（10只）、肝纤维化模型组（10只）和EDA防治组（10只）。检测各组大鼠血清谷丙转氨酶、谷草转氨酶水平及肝组织羟脯氨酸、丙二醛含量和超氧化物歧化酶活性。结果EDA防治大鼠血清谷丙转氨酶和谷草转氨酶水平分别为714．2±28．2U／L和766．0±11．0U／L,较模型组（1110．3±45．9U／L和1640．3±26．7U／L,P〈0．05和P〈0．01）明显降低;EDA组大鼠肝组织羟脯氨酸和丙二醛含量分别为0．4±0．1μg／mg和5．5±2．3nmol／gl,较模型组（0．8±0．1μg／mg和7．5±2．1nmol／gl,P均〈0．01）显著下降;EDA组大鼠超氧化物歧化酶活性为129．7±2．3u／g,较模型组（933±3．9u／g,P〈0．01）明显升高;EDA组和模型组大鼠肝组织纤维化评分分别为2．7±1．0和3．5±0．7,差异显著（P〈0．01）。结论EDA对大鼠肝纤维化有一定的防治作用,其机制很可能与抗脂质过氧化损伤有关。     

肝纤维化大鼠肝组织Smads基因表达状况及意义

目的:研究CCl4诱导的肝纤维化大鼠肝组织Smad基因表达的变化及其意义.方法:80只健康雄性SD大鼠分为2组:正常组(C组,n=40)和模型组(M组,n=40).以CCl4 sc 法诱导肝纤维化,HE和VG胶原染色观察肝脏胶原沉积情况,原位杂交法及免疫组化法检测肝组织Smads分子表达水平变化.结果:与C组比较,M组大鼠肝脏组织学积分显著增加(3.29±0.68 vs0,P<0.05),平均胶原面积显著增加(290.86±89.37 μm2 vs 56.12±21.45 μm2,P<0.01),肝组织Smad4蛋白表达率较C组明显增加(4.27%±0.43% vs 2.86%±0.86%,P<0.05),而Smad7蛋白表达虽然增加,但水平低下;Smad 3 mRNA表达A值明显增加(0.167±0.092 vs 0.010±0.002,P<0.05),Smad4 mRNA表达A值也明显增加(0.24 1±0.098 vs 0.021±0.004,P<0.05),Smad6、Smad7 mRNA虽然增加,但表达水平仍然低下.结论:实验性大鼠肝纤维化存在肝脏Smads分子表达水平的比例失调,TGF-Smad信号通路可能参与了肝纤维化的形成与发展.     

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.     

Black bean extract ameliorates liver fibrosis in rats with CCl4-induced injury

We assessed the anti-fibrotic effects of methanolic black bean extract antioxidants in a carbon tetrachloride (CCl4) liver injury model in rats. Experimentally intoxicated animals received CCl4 for eight weeks, the reference and test groups received daily intragastric quercetin or daily intragastric black bean extract. Liver fibrosis was assessed and quantified using morphometric analysis. Expression of fibrosis related genes was measured by real time RT-PCR. Qualitative and quantitative histological analysis showed that administration of 70 mg/kg b.w. of black bean extract reduced hepatic fibrosis index by 18% compared to positive controls (P 0.006), as a result of a decrease in type I (44.3% less, P 0.03) and type IV (68.9% less, P 0.049) collagen gene expression compared to CCl4-injured and Quercetin treated rats. In conclusion, we provide evidence that this methanol black bean extract ameliorates liver fibrosis and types I and IV collagen gene expression, in the animal model used. PRACTICAL APPLICATIONS: The compounds contained in this black bean extract exhibited strong antifibrotic effects in the CCl4 chronic liver injury model used; considering that this compounds are contained in a leguminous that has been used in human diet for a long time, their toxic potential should be very low, and this characteristic should favor their potential use in some other chronic or degenerative states that include an increase in inflammation and oxidative burst in their pathogenesis. Another possible application of this kind of extract could be its use as an antimicrobial or even antiparasitic therapeutic agent, although it is purely speculative.     

不同浓度CCl4注射联合饮用乙醇诱导大鼠肝硬化模型研究

目的 探索不同浓度CCl4联合饮用乙醇制备大鼠肝硬化模型,寻求最佳的药物和乙醇浓度。方法 取90只SD大鼠, 分为对照组10只和实验组A组、B组、C组和D组,每组20只,给予不同浓度的CCl4油溶液腹腔注射,同时以不同浓度的乙醇溶液为饮用水,制备肝硬化模型。结果 在实验12 w末,A组大鼠死亡10只, B组死亡8只,C组死亡4只,D组死亡5只;实验A组肝细胞走向紊乱,纤维组织增生,纤维间隔形成。有假小叶形成,肝细胞大小不一,呈点灶状坏死,肝细胞凋亡、再生,汇管区内炎细胞浸润;B组与A组变化相似;C组肝细胞走向紊乱,纤维间隔形成,但无假小叶形成,肝细胞脂肪变性,纤维组织增生,肝细胞凋亡,汇管区内炎细胞浸润;D组与C组表现类似。结论 适当浓度的CCl4油溶液结合乙醇溶液为饮用水诱导大鼠肝硬化模型可显著提高造模成功率。     

p38MAPK在大鼠实验性肝纤维化发生中的表达及其意义

目的:观察p38MAPK在实验性大鼠肝纤维化(hepatic fibrosis,HF)发生过程中表达量的变化及其定位,从而揭示p38MAPK信号传导通路与HF形成之间的关系.方法:采用CCl4 sc诱导大鼠HF模型,36R ♂sD大鼠(体质量在180-220 g)随机分为正常对照组(12只)和CCl4造模组(24只),造模3、6、9 wk结束时分别随机处死各组大鼠.取其肝脏观察HF形成过程中不同时间段各组大鼠肝组织病理变化,用RT-PCR技术检测p38MAPKmRNA在造模过程中肝组织中的表达变化,免疫组化方法检测p38MAPK在造模过程中肝组织中蛋白表达量的变化,及其在肝组织中的表达分布情况.结果:与正常对照组比较,CCl4诱导的实验性大鼠HF不同时间段纤维化程度明显加重,随着HF的形成,p38MAPK在mRNA水平和蛋白水平都表现出增加的趋势,并且主要表达于肝脏的间质细胞,肝细胞未见染色.结论:p38MAPK在HF的形成中持续上调,参与HF形成的病理过程,p38MAPK信号传导通路活化可能促进CCl4诱导的HF的形成.     

β受体阻断剂卡维地洛抗肝纤维化的作用

目的 研究非选择性β受体阻断荆和α-1受体阻滞剂卡维地洛对抗大鼠肝纤维化的作用及机理.方法 40只sD大鼠分为4组.分别为正常对照组、肝纤维化组、单用卡维地洛组、卡维地洛治疗组,分别测定各组的肝功能[丙氨酸氮基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、白蛋白/球蛋白(A/G)],肝纤维化血清指标(PC4、HA、CN),肝纤维化组织学指标(HE染色,Masson染色).SP免疫组织化学法测定肝组织α平滑肌肌动蛋白(α-SMA)和RT-PCR法测定肝组织转化生长因子β1 mRNA(TGF-β1 mRNA).结果 与造模组相比,卡维地洛改善了肝功能,血清肝纤维化指标和病理学检查指标都有所下降.结论 卡维地洛能够减轻CCl4致大鼠的肝纤维化.     

Protective effect of selenium-enriched Lactobacillus on CCl4-induced liver injury in mice and its possible mechanisms

AIM: To study the protective effects and mechanisms of Se-enriched lactobacillus on liver injury caused by carbon tetrachloride (CCl4) in mice. METHODS: Seventy-two ICR mice were randomly divided into four groups: normal group, CCl4-induced model group, low Se-enriched lactobacillus treatment group (L-Se group), and high Se-enriched lactobacillus treatment group (H-Se group). During a 3-wk experimental period, the common complete diet was orally provided daily for normal group and model group, and the mice in L-Se and H-Se groups were given a diet with 2 and 4 mg of organoselenium from Se-enriched lactobacillus per kg feed, respectively. From the 2nd wk of experiment, the model group, L-Se group, and H-Se group received abdominal cavity injection of olive oil solution containing 500 mL/L CCl4 (0.07 mL/100 g body mass) to induce liver injury, and the normal group was given olive oil on every other day for over 2 wk. In the first 2 wk post injection with CCl4, mice in each group were killed. The specimens of blood, liver tissue, and macrophages in abdominal cavity fluid were taken. Then the activities of the following liver tissue injury-associated enzymes including glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as well as malondialdehyde (MDA) content were assayed. Changes of phagocytic rate and phagocytic index in macrophages were observed with Wright-Giemsa stain. Plasma TNF-alpha level was measured by radioimmunoassay. The level of intracellular free Ca2+ ([Ca2+]i) in hepatocytes was detected under a laser scanning confocal microscope. RESULTS: During the entire experimental period, the AST and ALT activities in liver were greatly enhanced by CCl4 and completely blunted by both low and high doses of Se-enriched lactobacillus. The Se-enriched lactobacillus-protected liver homogenate GSH-Px and SOD activities were higher or significantly higher than those in model group and were close to those in normal group. CCl4 significantly increased MDA content in liver homogenates, while administration of Se-enriched lactobacillus prevented MDA elevation. Phagocytic rate and phagocytic index of macro-phages decreased after CCl4 treatment compared to those in normal control, but they were dramatically rescued by Se-enriched lactobacillus, showing a greatly higher phagocytic function compared to model group. CCl4 could significantly elevate plasma TNF-alpha and hepatocyte [Ca2+]i level, which were also obviously prevented by Se-enriched lactobacillus. CONCLUSION: Se-enriched lactobacillus can intervene in CCl4-induced liver injury in mice by enhancing macrophage function activity to keep normal and beneficial effects, elevating antioxidant-enzyme activities and reducing lipid peroxidation reaction, inhibiting excessive release of TNF-alpha, preventing the dramatic elevation of [Ca2+]i in hepatocytes.     

Effect of Oxymatrine on the TGFbeta-Smad signaling pathway in rats with CCl4-induced hepatic fibrosis

AIM： To explore the anti-fibrotic effect of Oxymatrine on CCl4-induced liver fibrosis in rats and its modulation on the TGFbeta-Smad signaling pathway. METHODS： One hundred healthy male SD rats were randomly divided into three groups： normal group （n = 20）, treatment group of Oxymatrine （n = 40） and CCh-induced fibrosis group （n = 40）. Experimental hepatic fibrosis was induced by subcutaneous injection of carbon tetrachloride （CCh soluted in liquid paraffin with the concentration of 300 g/L, the dosage of injection was 3 mL/kg, twice per week for 8 wk）. The treated rats received Oxymatrine via celiac injection at a dosage of 10 mg/kg twice a week at the same time. The deposition of collagen was observed with H＆E and Masson staining. The concentration of serum TGF-β1 was assayed with ELISA. The gene expression of Smads and CBP （CREB binding protein） was detected with in situ hybridization （ISH） and immunohistochemistry （IH）, respectively. All the experimental figures were scanned and analyzed with special figure-analysis software. RESULTS： A significant reduction of collagen deposition and rearrangement of the parenchyma was noted in the liver tissue of Oxymatrine-treated rats. The semi- quantitative histological scores （2.43 ± 0.47 μm^2 vs 3.76 ±0.68, P 〈 0.05） and average area of collagen/in those rats were significantly decreased when compared with hepatic cirrhosis model rats （94.41 ± 37.26 μm^2 vs 290.86 ± 89.37 μm^2, P 〈 0.05）. The gene expression of Smad 3 mRNA was considerably decreased in the treated animals. The A value of Smad 3 mRNA was lower in the treated rats than the model rats （0.034 ± 0.090 vs 0.167 ± 0.092, P 〈 0.05）. Contrarily, the A value of Smad 7 mRNA was increased considerably in the treated animals （0.175 ± 0.065 vs 0.074 vs 0.012, P 〈 0.05）. There was an obvious decrease in the expression of CBP mRNA in treated rats as illuminated by a reduction of its A value when compared with model rats （0.065±0.049 vs 0.235 ?     

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.     

四氯化碳诱导大鼠慢性肝损伤模型方法的探讨

目的研究大鼠慢性肝损伤模型的建立方法。方法以20％和50％四氯化碳植物油溶液给SD大鼠腹腔注射8周,制备大鼠慢性肝损伤模型,观察大鼠饮食、体重和血清ALT、AST水平的变化,采用TUNEL法观察肝细胞凋亡情况,以评价成模效果。结果实验组大鼠饮食量降低,体重增加缓慢。实验组ALT和AST分别为204．1±35．7U／L和307．5±54．1U／L,而对照组分别27．6±3．1U／L和50．5±9．0U／L。实验组动物出现肝细胞变性、凋亡、坏死及再生等病变。大剂量四氯化碳容易弓l起肝纤维化。结论应用20％～50％四氯化碳溶液在1．5ml·kg^-1 bw剂量下腹腔注射可诱导大鼠典型的肝损伤模型,病变稳定,操作简便,可供实验研究应用。     

Transplantation of bone marrow cells reduces CCl4-induced liver fibrosis in mice

We investigated the effect of bone marrow cell (BMC) transplantation on established liver fibrosis. BMCs of green fluorescent protein (GFP) mice were transplanted into 4-week carbon tetrachloride (CCl4)-treated C57BL6 mice through the tail vein, and the mice were treated for 4 more weeks with CCl4 (total, 8 weeks). Sirius red and GFP staining clearly indicated migrated BMCs existing along with fibers, with strong expression of matrix metalloproteinase (MMP)-9 shown by anti-MMP-9 antibodies and in situ hybridization. Double fluorescent immunohistochemistry showed the expression of MMP-9 on the GFP-positive cell surface. Film in situ zymographic analysis revealed strong gelatinolytic activity in the periportal area coinciding with the location of MMP-9-positive BMCs. Four weeks after BMC transplantation, mice had significantly reduced liver fibrosis, as assessed by hydroxyproline content of the livers, compared to that of mice treated with CCl4 alone. Subpopulation of Liv8-negative BMCs was responsible for this fibrolytic effect. In conclusion, mice with BMC transplants with continuous CCl4 injection had reduced liver fibrosis and a significantly improved survival rate after BMC transplantation compared with mice treated with CCl4 alone. This finding introduces a new concept for the therapy of liver fibrosis.