肝苏对人肝细胞和肝星状细胞增殖、氧应激以及细胞外基质表达的影响

背景：有研究显示肝苏对肝细胞具有保护、抗炎、抗氧化和抗肝纤维化的作用,但其作用机制未明。目的：探讨肝苏对人肝细胞和肝星状细胞（HSC）增殖、氧应激以及细胞外基质表达的影响。方法：分别用0．01～1．0mg／ml肝苏培养肝细胞和HSC,以M1丫r法检测肝苏对肝细胞和HSC增殖的影响：用次氮基三乙酸铁（Fe-NTa）和0．05—1．0mg／ml肝苏共同培养肝细胞和HSC,检测超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量;用1．5mg／ml和2．5mg／ml肝苏培养HSC,以酶联免疫吸附测定（ELISA）检测细胞外基质透明质酸（HA）、层黏连蛋白（LN）、I型胶原、Ⅲ型胶原和细胞因子转化生长因子（TGF）-β1含量。结果：在0．05。1．0mg／ml浓度范围内,肝苏可促进肝细胞增殖,但各浓度肝苏对HSC增殖无明显影响。肝苏可增高肝细胞SOD活性,降低肝细胞和HSCMDA含量,但对HSCSOD活性无明显影响。同时肝苏可抑制HSC细胞外基质HA、LN和细胞因子TGF-β1的表达,而I型胶原、Ⅲ型胶原无明显差异。结论：肝苏可促进肝细胞增殖,保护肝细胞和HSC免受氧应激损伤,抑制HSC分泌HA、LN和TGF-β1,提示其具有肝细胞保护、抗氧化和抗肝纤维化作用。     

三七总皂甙诱导大鼠肝星状细胞凋亡的研究

三七及其主要有效成分三七总皂甙（PNS）具有抗肝纤维化作用。采用体外培养的传代大鼠肝星状细胞（HSC），利用有关凋亡检测技术，观察PNS对LISC增殖、调亡，胶原和层黏连蛋白（LN）合成的影响，从细胞水平探讨PNS抗肝纤维化机制。     

抗血管生成药苏拉明对大鼠肝星状细胞的影响

目的 探讨抗血管生成药苏拉明(Suramin)对体外培养肝星状细胞(HSC)的影响及其抗纤维化的作用.方法 将大鼠HSC-T6体外培养,用MTT法初测细胞增殖趋势、流式细胞仪定性及定量检测HSC的细胞周期和增殖指数;免疫组织化学检测α-平滑肌肌动蛋白(α-SMA);ELISA法测定细胞上清液中Ⅲ型胶原含量.结果 与对照组相比,不同浓度Suramin组的HSC增殖均受抑制,阻抑在细胞周期G1期,呈时间和剂量依赖性(P＜0.05).各浓度组α-SMA灰度值亦低于对照组(P＜0.05).不同浓度Suramin组的上清液中Ⅲ型胶原均低于对照组,TGF-β1,+Suramin组的含量介于TGF-β1组和Suramin组之间.结论 抗血管生成药Suramin抑制HSC增殖和活化,从而减轻Ⅲ型胶原沉积,起到抗肝纤维化作用,即Suramin在临床应用上有多靶点抗肝纤维化的优势,但其具体机制有待进一步研究.     

丹酚酸B对大鼠肝星状细胞增殖周期的抑制作用

丹酚酸B(SAB)是丹参的主要水溶性成分，具有良好的抗D-半乳糖胺急性肝损伤和抗四氯化碳诱导的肝纤维化作用，还可抑制肝星状细胞(HSC)增殖及胶原合成，对脂质过氧化有很强的抑制作用，临床上已初步显示具有一定的抗肝纤维化作用。肝纤维化发生过程中，细胞外基质的主要来源是活化的HSC。我们试图在细胞增殖的整体水平上探讨SAB抗肝纤维化的作用机制。     

川芎嗪对内毒素刺激大鼠肝星状细胞增殖及瘦素和胶原表达的影响

目的观察川芎嗪对内毒素刺激肝星状细胞（HSC）增殖及瘦素表达的影响，并观察Ⅰ型胶原表达的变化。研究川芎嗪抗肝纤维化过程中的机制。方法内毒素刺激大鼠肝星状细胞的基础上，加川芎嗪分组培养。MTT法检测其吸光度，ELISA法检测其上清液瘦素水平和Ⅰ型胶原。结果内毒素刺激HSC增殖，川芎嗪抑制内毒素诱导的HSC增殖，并抑制Ⅰ型胶原表达和瘦素的表达。结论川芎嗪抑制内毒素诱导的HSC活化和Ⅰ型胶原表达。川芎嗪有可能通过抑制HSC增殖和抑制瘦素的分泌发挥抗纤维化作用。     

实验性大鼠肝纤维化模型中细胞外基质成份的变化

应用大鼠肝纤维化模型,通过光镜、电镜观察肝组织学改变,并应用放免法测定肝匀浆中Ⅲ型前胶原(PCⅢ)、透明质酸(HA)、层粘连蛋白(LN)水平和测定肝组织羟脯氨酸(Hyp)含量,结合计算机图像分析研究肝纤维化中细胞外基质成分的动态变化。结果发现,随着肝纤维化向肝硬变的发展,PCⅢ、HA、LN逐渐沉积于肝实质内,在肝纤维化早、中期以Ⅲ型胶原增长为主,在肝纤维化高峰期及肝硬变期则以Ⅰ型胶原增生为主。研究提示细胞外基质在肝纤维化发生发展中起重要作用。     

黄芪注射液对肝纤维化抑制作用的实验研究

目的探讨黄芪注射液对大鼠肝星状细胞(HSC)和肝纤维化的作用。方法体外细胞实验: 用不同浓度黄芪注射液(0、25、50、100、200、400 mg/ml)作用HSC不同时间(24、48、72h)后,采用四甲基偶氮唑盐法检测其活化增殖;流式细胞术检测HSC增殖周期;溴乙锭/吖啶橙荧光染色和流式细胞术检测HSC凋亡。动物实验:用40%四氯化碳和5%乙醇制备大鼠肝纤维化动物模型,实验分为正常对照组、模型组和黄芪注射液组。黄芪注射液组和模型组在模型制备的同时分别给予黄芪注射液(800 mg·kg-1·d-1)和等渗盐水腹腔注射,第8周时测定血清透明质酸(HA),层黏连蛋白(LN)水平及肝组织中超氧化物歧化酶(SOD)活性,丙二醛(MDA)含量,免疫组织化学方法观察肝组织LN的表达,苏木素-伊红、苦味酸-酸性品红染色观察肝组织病理改变。结果在体外细胞实验中,与0 mg/ml组比较,黄芪注射液其它浓度组明显抑制了HSC增殖,并呈剂量和时间依赖性;HSC增殖周期被抑制在G2-M期;黄芪注射液各浓度组荧光染色法和流式细胞术均未检测到HSC凋亡。在体内实验中,血清HA、LN含量:模型组分别为(114.3±25.6)μg/L和(78.8±11.7)μg/L,黄芪注射液组分别为(85.6±37.3)μg/L和(66.8±17.6)μg/L,P <0.05;肝组织SOD活性黄芪注射液组为(75.9±5.9)NU/mg,模型组为(49.6±5.7)NU/mg,P< 0.01;而MDA含量黄芪注射液组为(2.4±0.2)μmol/g,模型组为(3.7±0.4)μmol/g,P<0.01。显微镜下黄芪注射液组肝纤维化程度明显轻于模型组,免疫组织化学结果黄芪注射液组肝组织LN表达明显减少。结论黄芪注射液可延缓肝纤维化的发生,其机制除可直接抑制HSC增殖外,还有抗氧化、抗脂质过氧化、减少LN产生,防止肝窦毛细血管化等作用。     

龙血素B对肝星状细胞增殖及细胞外基质分泌的影响

目的观察龙血素B对大鼠肝星状细胞(HSC)增殖及细胞外基质分泌的影响。方法用不同浓度的龙血素B处理大鼠HSC;MTT法计算药物的抑制率;放射免疫法测定细胞上清中透明质酸(HA)、层粘连蛋白(LN)和Ⅳ型胶原(Ⅳ-C)。结果随药物浓度的升高,龙血素B对HSC-T6增殖的抑制作用逐渐增强,有明显的量效关系。细胞上清中HA、LN和Ⅳ-C的含量与对照组相比降低,其中龙血素B浓度为0.300μg/μL时降低最明显。结论龙血素B可抑制HSC-T6增殖,并不同程度抑制HA、LN和Ⅳ-C的分泌。     

扶正化瘀胶囊对肝星状细胞激活的干预

目的：观察扶正化瘀胶囊(319方)对肝星状细胞(HSC)激活的抑制作用。方法：①制备319方含药血清，温育原代HSC，并以正常血清为对照，观察HSC的增殖与I型胶原分泌。②用含药血清分别添加于损伤肝枯否细胞与传代HSC中培养，制备含药血清作用过的损伤肝枯否细胞条件培养液与传代HSC条件培养液，并以添加正常血清的条件培养液作为对照，温育HSC，观察HSC的增殖与I型胶原分泌。③测定枯否细胞条件培养液中转化生长因子-β(TGF-β)、血小板衍生生长因子(PDGF)、表皮生长因子(VEGF)及传代HSC条件培养液中VEGF。结果：①含药血清能明显抑制原代HSC的增殖与I型胶原分泌。②损伤肝枯否细胞与传代HSC可明显促进原代HSC的增殖与I型胶原分泌。含药血清可抑制这一促进作用。③损伤肝枯否细胞FGF-β、PDGF、VEGF与传代HSC中VEGF分泌明显增加，含药血清可抑制损伤枯否氏细胞TGF-β、PDGF及传代HSC中VEGF的分泌。结论：319方不仅可直接抑制HSC的增殖与I型胶原的分泌，并可抑制TGF-β1、PDGF、VEGF等细胞因子，抑制HSC激活的旁分泌与自分泌途径，这一作用可能是该药抗肝纤维化的机制之一。     

木犀草素抑制肝星状细胞增殖及其胶原合成

目的 研究木犀草素对体外培养的肝星状细胞(hepatic stellate cells,HSC)增殖及其胶原表达、合成的影响。方法 从Wistar大鼠肝脏分离培养HSC,并用~3H-TdR和~3H-pro同位素掺入实验,基因探针原位杂交等技术研究了木犀草素对HSC增殖、胶原基因表达合成的影响。结果 当木犀草素的浓度分别达到10 μmol/L和20 μmol/L 时抑制HSC增殖(t=2.542,P＜0.05)和胶原合成(t=3.650,P＜0.01),其作用具有剂量依赖关系;25 μmol/L木犀草素使Ⅰ、Ⅲ型前胶原mRNA的表达降低,其中Ⅰ型前胶原基因表达降低具有统计学差异(x~2=6.850,P＜0.01)。结论 木犀草素在体外抑制HSC增殖和胶原表达合成,在体内可能会具有预防或冶疗肝纤维化的作用。     

Effects of Ginkgo biloba extract on cell proliferation, cytokines and extracellular matrix of hepatic stellate cells.

BACKGROUND/AIMS: Hepatic fibrosis is the common wound-healing response to chronic liver injury. Ginkgo biloba extract (GbE) has been indicated to reverse hepatic fibrosis and exhibit therapeutic effects both in vitro and in vivo. This study aimed to investigate the underlying mechanism of GbE using HSC-T6 cells, a subline of hepatic stellate cells (HSC) as a model. METHODS: HSC-T6 cells were seeded into six-well plates and allowed to attach overnight. After exposure to different concentrations of GbE761 for 24 or 48 h, cell cycle analysis, semiquantitative RT-PCR, Western blotting analysis and analysis of ECM secretion were performed. RESULTS: It was revealed that GbE (1, 10, 100, 500 mg/l) suppressed HSC proliferation and caused G0/G1 phase arrest in a concentration-dependent manner. RT-PCR and Western blot assays were applied to detect the decline of transforming growth factor beta1(TGF-beta1) and connective tissue growth factor (CTGF) in both mRNA and protein levels after GbE treatment in HSC-T6 cells for 24 or 48 h. Meanwhile, GbE inhibited the synthesis of type I and type III collagens. Secretion of some extracellular matrix (ECM) proteins, such as type III procollagen (PC III), type IV collagen (collagen IV), laminin (LN), hyaluronic acid (HA), were all decreased in supernatant of GbE treated HSC cells. CONCLUSIONS: Our results suggest that GbE confers its anti-fibrosis effects through inhibiting HSC proliferation, reducing TGF-beta1 and CTGF expression and consequently suppressing the collagen production and ECM secretion.     

Effect of emodin on pancreatic fibrosis in rats

AIM： To establish the rats model of chronic fibrosing pancreatitis and to prove the anti-fibrotic effect of emodin in chronic pancreatitis with fibrosis.
METHODS： Fifty rats were randomly divided into five groups, 10 rats in each group. Trinitrobenzene sulfonic acid （TNBS） was infused into the pancreatic duct to induce chronic pancreatitis in rats （except for normal group）. Emodin-treated rats were fed with different doses of emodin （20, 40 and 80 mg/kg body weight） for 28 d, while normal group and control group received 0.9% sodium chloride solution. Serum levels of hyaluronic acid （HA） and laminin （LN） were determined by radioimmunoassay. Histopathological alterations were studied by optical microscopy. Expression of collagen was also examined while transforming growth factor- beta-1 （TGF-131） was localized by immunochemistry. RESULTS： In emodin-treated rats, the serum levels of HA and LN were decreased significantly （HA, 62.2 ± 19.3 μg/L vs 112.7 ± 26.5μg/L, P 〈 0.05; LN 44.3 ± 10.4 μg/L vs 86.2 ± 16.5 μg/L, P 〈 0.05）; the degree of fibrosis was ameliorated observably; the expression of collagen in pancreatic tissue was reduced especially in high-dose emodin-treated group （36% ± 5% vs 42% ± 6%, P 〈 0.05）; with the increased doses of emodin, the expression of TGF-β1 was declined, compared with those in control group.
CONCLUSION： Emodin has an anti-fibrotic effect on pancreatic fibrosis in rats. Because of its anti-fibrotic effect, it could be a potential herb for the treatment of chronic pancreatitis.     

促肝细胞生长素及其类似物的生物活性研究

目的 研究促肝细胞生长素(pHGF)对肝细胞及肝星状细胞生物活性的影响。方法 以人肝细胞株及大鼠肝星状细胞株为研究靶细胞,采用MTT法研究pHGF对细胞增殖活性的影响,放免法检测透明质酸(HA),细胞基因转染结合报告基因测活检测Ⅰ型胶原基因转录活性的变化,SDS-PAGE分析pHGF主要成分。结果 pHGF具有明确促进肝细胞增生及抑制肝纤维化主要形成细胞肝星状细胞系增殖的作用,能抑制细胞外间质成分HA的产生,抑制Ⅰ型胶原基因启动子样活性。研究的12种pHGF中仅1种在15％SDS-PAGE中具有明确的蛋白条带。结论 pHGF具有促进肝细胞再生及抗纤维化作用,结构-功能关系需进一步研究。     

Effects of Fuzhenghuayu decoction on collagen synthesis of cultured hepatic stellate cells, hepatocytes and fibroblasts in rats

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Differential modulation of rat hepatic stellate phenotype by natural and synthetic retinoids

Activation of hepatic stellate cells (HSC) is a central event in the pathogenesis of liver fibrosis during chronic liver injury. We examined the expression of retinoic acid (RAR) and retinoid X receptors (RXR) during HSC activation and evaluated the influence of natural and synthetic retinoic acids (RA) on the phenotype of culture-activated HSC. The expression of the major RAR/RXR subtypes and isoforms was analyzed by Northern hybridization. Presence of functional receptor proteins was established by gel shift analysis. Retinoic acids, RAR, and RXR selective agonists and an RAR antagonist were used to evaluate the effects of retinoid signalling on matrix synthesis by Northern blotting and immunoprecipitation, and on cell proliferation by BrdU incorporation. The 9-cisRA and synthetic RXR agonists reduced HSC proliferation and synthesis of collagen I and fibronectin. All-trans RA and RAR agonists both reduced the synthesis of collagen I, collagen III, and fibronectin, but showed a different effect on cell proliferation. Synthetic RAR agonists did not affect HSC proliferation, indicating that ATRA inhibits cell growth independent of its interaction with RARs. In contrast, RAR specific antagonists enhance HSC proliferation and demonstrate that RARs control proliferation in a negative way. In conclusion, natural RAs and synthetic RAR or RXR specific ligands exert differential effects on activated HSC. Our observations may explain prior divergent results obtained following retinoid administration to cultured stellate cells or to animals subjected to fibrogenic stimuli.     

Morphological and serum hyaluronic acid, laminin and type IV collagen changes in dimethylnitrosamine-induced hepatic fibrosis of rats

AIM: To study the morphological and serum hyaluronic acid (HA), laminin (LN), and type IV collagen changes in hepatic fibrosis of rats induced by dimethylnitrosamine (DMN). METHODS: The rat model of liver fibrosis was induced by DMN. Serum HA, type IV collagen, and LN were measured by ELISA. The liver/weight index and morphological changes were examined under electron microscope on d 7, 14, 21, and 28 by immunohistochemical alpha smooth muscle actin alpha-SMA staining as well as Sirius-red and HE staining. RESULTS: The levels of serum HA, type IV collagen and LN significantly increased from d 7 to d 28 (P = 0.043). The liver/weight index increased on d 7 and decreased on d 28. In the model group, the rat liver stained with HE and Sirius-red showed evident hemorrhage and necrosis in the central vein of hepatic 10 lobules on d 7. Thin fibrotic septa were formed joining central areas of the liver on d 14. The number of alpha-SMA positive cells was markedly increased in the model group. Transitional hepatic stellate cells were observed under electron microscope. All rats in the model group showed micronodular fibrosis in the hepatic parenchyma and a network of alpha-SMA positive cells. Typical myofibroblasts were embedded in the core of a fibrous septum. Compared to the control group, the area-density percentage of collagen fibrosis and pathologic grading were significantly different in the model group (P<0.05) on different d (7, 14, and 28). The area-density percentage of collagen fibrosis in hepatic tissue had a positive correlation with the levels of serum HA, LN, and type IV collagen. CONCLUSION: The morphological and serum HA, type IV collagen, and LN are changed in DMN-induced liver fibrosis in rats.     

Morphological and serum hyaluronic acid, laminin and type Ⅳ collagen changes in dimethylnitrosamine-induced hepatic fibrosis of rats

AIM: To study the morphological and serum hyaluronic acid (HA), laminin (LN), and type Ⅳ collagen changes in hepatic fibrosis of rats induced by dimethylnitrosamine (DMN).METHODS: The rat model of liver fibrosis was induced by DMN. Serum HA, type Ⅳ collagen, and LN were measured by ELISA. The liver/weight index and morphological changes were examined under electron microscope on d 7, 14, 21, and 28 by immunohistochemical alpha smooth muscle actin α-SMA staining as well as Sirius-red and HE staining.RESUJLTS: The levels of serum HA, type Ⅳ collagen and LN significantly increased from d 7 to d 28 (P = 0.043).The liver/weight index increased on d 7 and decreased on d 28. In the model group, the rat liver stained with HE and Sirius-red showed evident hemorrhage and necrosis in the central vein of hepatic 10 lobules on d 7. Thin fibrotic septa were formed joining central areas of the liver on d 14. The number of α-SMA positive cells was markedly increased in the model group. Transitional hepatic stellate cells were observed under electron microscope.All rats in the model group showed micronodular fibrosis in the hepatic parenchyma and a network of α-SMA positive cells. Typical myofibroblasts were embedded in the core of a fibrous septum. Compared to the control group, the area-density percentage of collagen fibrosis and pathologic grading were significantly different in the model group (P＜0.05) on different d (7, 14, and 28). The area-density percentage of collagen fibrosis in hepatic tissue had a positive correlation with the levels of serum HA, LN, and type Ⅳ collagen.CONCLUSION: The morphological and serum HA, type Ⅳ collagen, and LN are changed in DMN-induced liver fibrosis in rats.