肝细胞凋亡模型的建立

目的　建立体内、外肝细胞凋亡模型 ,为进一步研究药物对肝细胞凋亡的调控作用奠定基础。方法　 (1)采用D GalN +LPS小鼠腹腔注射建立体内肝细胞损伤模型 ;(2 )采用H2 O2 (0 0 5～ 0 4mmol·L-1)与原代大鼠肝细胞共培养建立体外肝细胞损伤模型 ,分别用形态学、DNA电泳和流式细胞术检测等方法。结果　 (1)D GalN(70 0mg·kg-1,ip) +LPS(1μg·kg-1,ip)可明显升高小鼠血中TNF水平和MDA含量 ,使肝线粒体Mn SOD活性降低 ,小鼠肝细胞皱缩变小、核染色质凝聚和DNA片段化 (DNA梯状条带 ) ;(2 )H2 O2(0 1mmol·L-1,1h)可致大鼠肝细胞增殖受抑、肝细胞MDA含量升高 ,肝细胞DNA的AO荧光染色变亮 ,DNA电泳呈片段化 ,流式细胞术肝细胞DNA亚G1峰 (即凋亡峰 )明显升高。结论　采用D GalN +LPS与低浓度H2 O2 可分别建立较理想的体内、外肝细胞凋亡模型     

黄芪总提物对肝细胞凋亡的抑制作用

为研究黄芪总提物 (TEA)对肝细胞凋亡的保护作用及其机理 ,分别用D 氨基半乳糖 (D GalN ,70 0mg·kg- 1,ip) +脂多糖 (LPS ,1μg·kg- 1,ip)诱导小鼠在体肝细胞凋亡和H2 O2 (0 .1mmol·L- 1,1h)诱导原代培养大鼠肝细胞凋亡 ,采用形态学观察 ,DNA凝胶电泳和流式细胞术等方法检测细胞凋亡 .结果表明 :①TEA (40mg·kg- 1,ig× 2 ,5h)使D GalN +LPS升高的小鼠血中肿瘤坏死因子 (TNF)水平和肝脏丙二醛 (MDA)含量降低 ,以及使降低的肝线粒体锰 超氧化物歧化酶 (Mn SOD)活性升高 ;TEA可明显抑制D GalN +LPS引起的小鼠肝细胞皱缩变小 ,核染色质凝聚和DNA片段化 .②TEA (2 0mg·L- 1)可恢复或减轻由H2 O2 所致肝细胞增殖受抑和肝细胞MDA含量升高 ;TEA (40mg·L- 1)可使H2 O2 致DNA较强的AO荧光染色变淡 ,TEA(2 0mg·L- 1)对H2 O2 所致的DNA片段化有抑制作用 ,使H2 O2 升高的大鼠肝细胞DNA亚G1峰 (即凋亡峰 )明显降低 ,经DNA软件分析 ,TEA可使H2 O2 升高的细胞凋亡率从 6 3.7%降至 4 .2 % .提示 ,TEA对体内外肝细胞凋亡均有保护作用 ,其抗肝损伤作用可能与其抗凋亡和抗氧化作用有关     

W3D固体分散体的制备及其抗对乙酰氨基酚诱导的急性肝损伤的保护作用

目的 将4-(5’-二甲氨基)-萘磺酰氧基苯并噁唑酮(W3D)制成固体分散体，并研究其对对乙酰氨基酚(N-acetyl-para-aminophenol，APAP)诱导的急性肝损伤(acute liver injury, ALI)的保护作用。方法 采用溶剂法将原料药W3D及辅料PVPk30按1:7反应得W3D固体分散体。采用差示扫描量热(DSC)和X-粉末射线衍射(XRD)手段表征固体分散体的物相变化，同时对其饱和溶解度、溶出重现性进行考察。建立对乙酰氨基酚诱导的ALI模型，分别灌胃给予CMC-Na、阳性药N-乙酰半胱氨酸(NAC 12.5 mg·kg-1)、W3D原料药(12.5 mg·kg-1)、W3D固体分散体(12.5、6.25和3.125 mg·kg-1)，24 h后处死小鼠，取出肝脏，计算肝脏指数；HE染色观察肝组织病理变化，微孔板法检测血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)的含量及肝组织中超氧化物歧化酶(SOD)活力、微量还原型谷胱甘肽(GSH)含量，TBA法检测丙二醛(MDA)的含量，ELISA法检测血清中肿瘤坏死因子(TNF-α)、白介素6(IL-6)的含量，免疫组化检测肝组织MD2蛋白的表达。结果 W3D固体分散体为无定形形态，溶出度10 min即可达70 %；W3D可降低APAP诱导的ALI小鼠肝脏指数，减少肝组织血管周围细胞胞核固缩、碎裂或溶解等现象，剂量依赖性地减少血清中ALT、AST、TNF-α、IL-6的含量，升高肝组织中GSH含量及SOD活力，降低MDA含量而呈现出优于临床用药NAC的肝保护活性；W3D亦可降低ALI小鼠肝组织中MD2蛋白的含量。结论 W3D固体分散体可通过抑制氧化应激和炎症而发挥抗APAP诱导的ALI作用，其作用机制可能与降低MD2蛋白表达有关。     

人胎盘提取物促进大鼠肝再生活性

为了研究开发新的的抗肝炎药物,探讨了人胎盘提取物（HPE）对ANIT（α－naphthylisothiocyanate)肝病态大鼠的肝保护,肝再生作用的影响。体内实验表明,HPE静脉给药,可使肝病态大鼠肝标记指数（Labeling index)提高16．5倍。初代培养大鼠肝细胞实验发现,HPE可使肝细胞DNA合成活性增高6倍,HPE可明显降低肝病态大鼠血清中胆红素以及转氨酶等值,加热后的HPE丧失其肝再生促进效果但仍保持其肝保护作用,利用肝素亲和力柱层析将HPE的有效成分分离后发现,HPE的肝再生促进效果主要与肝素亲和性成分有关,而肝素非亲和性成分可促进肝素亲和性成分的肝再生效果。结论：1．HPE可促进肝病态大鼠的肝再生效果。2．HPE中肝再生促进因子和肝保护因子分别与热稳定和热不稳定成分有关。3．HPE中肝素亲和性和非亲和性成分以协同作用促进肝细胞再生。     

牛肝提取物对四氯化碳诱发小鼠急性肝损伤的保护作用

牛肝提取物以乳牛肝为主要原料 ,经物理方法提取的小分子蛋白质 ,主要含肝细胞生长刺激因子及钙、锌、铁等人体必需微量元素、维生素等。为了评价该胶囊是否有保肝作用 ,我们对其进行了下列检测。1　材料与方法1 1　受试物　牛肝提取物呈浅黄色颗粒状 ,用蒸馏水配制成所需浓度进行试验 ,由福建省某公司提供。1 2　实验动物　ＩＣＲ雄性小鼠 ,体重 2 0～ 2 5ｇ ,由上海西普尔 必凯实验动物有限公司提供。1 3　实验方法1 3 1　小鼠急性肝损伤模型建立 :用精致玉米油将ＣＣｌ4 配制成 2 0、2 5、3 0、3 5、4 0、80、160ｍｇ ｋｇ等剂量 ,动物…     

氯丙嗪对撤除苯巴比妥钠诱导的小鼠肝细胞凋亡有抑制作用(英文)

目的:研究氯丙嗪(Chl)、维拉帕米和阿斯匹林对撤除苯巴比妥钠(Phe)引起的小鼠肝细胞凋亡有无抑制作用及其时效关系。方法:以肝DNA含量、肝/体重比、DNA片段含量、DNA电泳、原位DNA末端标记试验(TUNEL)及形态学改变为指标,研究Phe 75mg·kg~(-1)·d~(-1)ip诱导小鼠肝细胞增殖及撤药后增殖肝回落的动态过程,并观察Chl25mg·kg~(-1)、维拉帕米50mg·kg~(-1)或阿司匹林60mg·kg~(-1)ip对小鼠肝细胞凋亡的影响及其时程效应。结果:Phe诱导小鼠肝细胞增殖及停药后增殖肝的回落由增殖期、平台期、快速回落期和缓慢回落期四个时相组成,快速回落期是一个典型的凋亡过程,其早期能被钙调蛋白拮抗剂Chl阻断。结论:钙调蛋白在撤除Phe引起的小鼠肝细胞凋亡中起重要作用。     

中药抗癌I号对S_(180)腹水瘤和实体瘤的抑制作用

中药抗癌I号系中药组成的复方制剂 ,具有抗自由基和免疫调节等作用 ,为探索其对肿瘤细胞的抑制作用 ,本试验进行了中药抗癌I号抗S180 腹水瘤和实体瘤的体内研究。一、实验材料1 试药　中药抗癌I号 ,由豆苷等组成 ,批号 :2 0 0 2 10 15。2 动物　昆明种小鼠 ,体重 2 0± 2 g ,由山东鲁抗医药集团动物中心提供 ,合格证号 :鲁动质字 :D2 0 0 2 0 92 1。二、方法1 S180 腹水瘤试验　健康昆明种小鼠 ,腹腔接种S180 肉瘤细胞混悬液 5～ 10 5个 /鼠 ,次日腹腔注射给药 ,剂量分别相当于豆苷 5 0、2 5、12 5mg·kg- 1,对照组同法给予生理盐水 0 …     

中药“男宝”的药理作用研究(二)

中药“男宝”能增加小鼠肝、脾DNA的合成,对全血和白细胞DNA含量无明显影响。对小鼠无耐缺氧和抗疲劳作用。小鼠口服LD_(50)为7克/公斤以上。     

肝再生的研究进展

肝脏是机体具有强大防御功能和再生能力的重要器官之一，相对于细胞增殖来说，成人或动物的肝脏是一个相对静止的器官。在正常肝组织中仅有0.0012％-0.1％的肝细胞进行有丝分裂[1]，而大部分肝细胞处于相对静止的G0期。但在肝脏被部分切除、受到药物（如D2氨基半乳糖胺，CCl4）毒害后，残余的或未受到损害的肝细胞可以通过DNA合成和有丝分裂增殖，重建与机体大小相适应的体积。而肝细胞再生停止是由肝脏的重量与个体体重的比例来决定的，当肝脏再生后其重量与整个体重达到一定比例时，肝脏的再生将自动停止[2]。肝脏的这种再生并不是被切除的肝叶重新长出，而是残余肝细胞的增生，称之为肝再生（liver regeneration，LR），肝再生是一个多基因参与和多步骤协同的复杂过程[3]。     

口服二磷酸果糖对小鼠急性肝损伤的保护作用

目的研究二磷酸果糖钠 (FDP)对CCl4 和D 氨基半乳糖两种模型引起小鼠肝损伤的保护作用 ,并分析其对肝微粒体细胞色素P45 0酶的活性影响。方法在对小鼠连续灌胃FDP 4d后 ,采用ipCCl4 和D 氨基半乳糖两种化学物质造成小鼠急性肝损害 ,研究其有无保肝作用 ;并采用Omura法测定了FDP对正常小鼠的肝细胞色素P45 0酶活力的影响。结果FDP 0 .15g/kg和 0 .30g/kg灌胃剂量下对肝损伤模型小鼠有一定的保护作用 ,并有提高正常小鼠肝微粒体P45 0酶活性的作用。结论FDP能防止化学物质造成的肝损伤 ,保护肝功能     

Leukotriene-mediated liver injury

The pathogenic mechanism of fulminant hepatitis induced by 700 mg/kg D-galactosamine plus 33 micrograms/kg endotoxin was investigated in male NMRI mice. The extent of liver injury was assessed by measurement of serum transaminases and sorbitol dehydrogenase activities 9 hr after intoxication, as well as by histopathological evaluation. When the hepatic glutathione content of galactosamine endotoxin-treated animals had been decreased by more than 90% following administration of 250 mg/kg phorone or 400 mg/kg diethyl maleate given three times, no signs of liver injury were observed. Since different agents interfering with the leukotriene synthesis pathway also prevented galactosamine/endotoxin-induced hepatitis, we suspected that a glutathione-derived peptidoleukotriene may be the pathogenic metabolite. In vivo inhibition of the catabolism of leukotriene C4 by administration of 50 mg/kg of the glutamyl transpeptidase inhibitor AT 125 (Acivicin) also protected the animals against liver injury. In order to elucidate which metabolite of leukotriene C4 was responsible for the observed hepatotoxicity we intravenously injected leukotrienes into animals that had received only galactosamine. Injection of 50 micrograms/kg leukotriene E4 1 hr after galactosamine had no effect. The same dose of leukotriene D4 led to a fulminant hepatitis which was prevented when the leukotriene D4 antagonist FPL 55712 had been given before. In contrast, lipoxygenase inhibitors or AT 125 did not protect against galactosamine + LTD4. Galactosamine/endotoxin-induced and galactosamine/leukotriene D4-induced hepatitis resulted in similarly localized histopathological changes, i.e. diffuse necrosis in the organ. We conclude from our results that galactosamine/endotoxin-induced hepatitis is mediated by a leukotriene D4-dependent mechanism.     

人工发酵虫草菌粉及其提取物保肝作用的研究

目的 观察人工发酵虫草菌粉及其提取物对实验性肝损伤的保护作用。方法 分别采用D-氨基半乳糖(D—G1aN)和四氯化碳(CCl4)致小鼠急性实验性肝损伤，测定血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)的含量及肝组织切片病理观察。结果 对D—G1aN致小鼠急性肝损伤，人工发酵虫草菌粉及其提取物可使升高的ALT降低(P＜0．01)，病理检查显示有明显保肝作用，对AST作用不明显；而对CCl4致小鼠急性肝损伤，仅菌粉和其提取物高剂量组有一定的降ALT作用。结论 人工发酵虫草菌粉及其提取物有一定保肝作用。     

Glycyrrhizin inhibits TNF-induced, but not Fas-mediated, apoptosis in the human hepatoblastoma line HepG2.

To determine the transaminase-lowering action of glycyrrhizin (GL) immunologically, the effect of GL on tumor necrosis factor (TNF)-alpha- and Fas-mediated apoptosis was assessed using a human hepatoblastoma line, HepG2 cells. The HepG2 cells were resistant to TNF-alpha and anti-Fas antibody, but were rendered susceptible to TNF-alpha and anti-Fas antibody in the presence of actinomycin D (Act D), an inhibitor of RNA synthesis. The cytotoxicity induced by TNF-alpha/Act D or anti-Fas/Act D was accompanied by DNA fragmentation, indicating apoptotic death of HepG2 cells. GL partially prevented the apoptosis of HepG2 cells induced by TNF-alpha/Act D in a GL-dose dependent fashion. However, this protective effect of GL was not observed in the cytotoxicity of HepG2 caused by anti-Fas/Act D. Although the protection mechanism of GL, observed in a limited fashion against TNF-alpha-mediated apoptosis, is unclear, the present results provide an immunological explanation for the transaminase-lowering action of GL in the GL treatment of chronic liver diseases involving apoptotic hepatocyte death in their pathogenesis.     

Effect of HD-03--a herbal formulation in galactosamine-induced hepatopathy in rats

The effect of HD-03 a herbal preparation was studied on galactosamine (400 mg/kg b.wt., i.p.) induced hepatotoxicity in rats. Animals were pre-treated for 14 days with HD-03 and compared against untreated group for SGPT, SGOT, serum bilirubin and liver glycogen. Histopathology of liver lobes was considered to evaluate the extent of hepatic injury induced by galactosamine. These were reversed by HD-03 pre-treatment. HD-03 provided convincing evidence of hepatoprotection against galactosamine induced hapatotoxicity.     

Protective activity of silipide on liver damage in rodents.

The activity of silipide, a silybin-phosphatidylcholine complex (IdB 1016), was tested in different models of liver damage in rodents. After oral administration, silipide exhibited a significant and dose-related protective effect against the hepatotoxicity induced by CCl4, praseodymium, ethanol and galactosamine. The ED50 values for inhibition of the rise in ASAT and ALAT levels caused by CCl4 and praseodymium and for antagonism of the increase in liver triglycerides caused by ethanol ranged from 93 to 156 mg/kg (as silybin). At a dose of 400 mg/kg (as silybin), silipide was also active in protecting against paracetamol-induced hepatotoxicity. Silybin and phosphatidylcholine at doses equivalent to those contained in the active doses of silipide failed to show any significant protective activity in these models. The liver protective effect of silipide is probably related to its antioxidant activities and to a stimulating effect on the hepatic synthesis of RNA and proteins.     

肝细胞生长因子的部分纯化与活性研究

报告新生牛肝细胞生长因子的提取,Sephadex G-50过柱分离、SDS—凝胶电泳及高效液相色谱分析,结果表明,新生牛肝脏中提取的肝细胞生长因子含有四种组分,分子量(u)分别为21000(Ⅰ),14400(Ⅱ),10000(Ⅲ)及10000以下(Ⅳ)含量各占8.01%,76.09%,15.72%,o.14%。生物活性测定表明,四组分中以分子量14400u(Ⅱ)者刺激肝细胞DNA合成活性最强,其次为Ⅲ,而Ⅰ,Ⅳ组分无或仅有微弱活性。     

Function and regulation of production of hepatocyte growth factor (HGF)

Hepatocyte growth factor (HGF) was purified as a potent mitogen for rat hepatocytes in primary culture and is believed to be the most physiological hepatotrophic factor that triggers liver regeneration. HGF is one of the largest disulfide-linked cytokines, consisting of a 60-kDa heavy chain and a 35-kDa light chain. Human HGF is synthesized as a single polypeptide chain precursor of 728 amino acid residues that has an appreciable homology with plasminogen, and it is processed proteolytically to release an N-terminal signal peptide of 31 amino acids and to generate an active heterodimer after secretion. The novel serine protease HGF activator and urokinase-type plasminogen activator (u-PA) are responsible for the latter extracellular processing. HGF stimulates the proliferation of rat hepatocytes in primary culture at concentrations as low as 10 pM. It also stimulates the growth of various epithelial cells, endothelial cells, and some kinds of mesenchymal cells. HGF inhibits the proliferation of several tumor cell lines and induces apoptosis of some of them. It also has motogenic, morphogenic, anti-apoptotic, angiogenic, and immunoregulatory activities. The receptor of HGF is the product of c-met proto-oncogene with tyrosine kinase activity that mediates the transduction of multiple biological signals of HGF. During liver regeneration, HGF gene expression in the liver, spleen, and lung and HGF levels in the blood and liver increase prior to the induction of liver DNA synthesis. Liver regeneration is markedly inhibited by continuous administration of a neutralizing anti-HGF antibody. HGF production in cultured cells is induced by PKC-activating agents, cAMP-elevating agents, PKA-activating agents, growth factors, and inflammatory cytokines; and it is inhibited by TGF-beta, glucocorticoids, 1,25-dihydroxyvitamin D3, and retinoic acid. There are many reports on potential application of HGF as a therapeutic agent for organ diseases that are difficult to cure such as liver cirrhosis, chronic renal failure, pulmonary fibrosis, myocardial infarction, and arteriosclerosis obliterans utilizing its potent growth-stimulating activity for a wide variety of cells. ELISA kits for assays of serum and plasma HGF levels are clinically used to prognosticate the development of fulminant hepatic failure.     

Free-radical scavenging action of medicinal herbs from Ghana: Thonningia sanguinea on experimentally-induced liver injuries.

The antioxidant action of medicinal herbs used in Ghana for treating various ailments was evaluated in vitro and in vivo. Five plants, Desmodium adscendens, Indigofera arrecta, Trema occidentalis, Caparis erythrocarpus, and Thonningia sanguinea were tested for their free radical scavenging action by their interaction with 1,1-diphenyl-2-picrylhydrazyl (DPPH). Of these five plants, only Thonningia sanguinea was found to scavenge the DPPH radical. Lipid peroxidation in liver microsomes induced by H2O2 was also inhibited by T. sanguinea. The hepatoprotective effect of T. sanguinea was studied on acute hepatitis induced in rats by a single dose of galactosamine (GalN, 400 mg/kg, IP) and in mice by carbon tetrachloride (CCl4, 25 microl/kg, IP). GalN induced hepatotoxicity in rats as evidenced by an increase in alanine aminotransferase (ALT) and glutathione (GSH) S-transferase activities in serum was significantly inhibited when T. sanguinea extract (5 ml/kg, IP) was given to rats 12 hr and 1 hr before GalN treatment. The activity of liver microsomal GSH S-transferase, which is known to be activated by oxidative stress, was increased by the GaIN treatment and this increase was blocked by T. sanguinea pretreatment. Similarly, T. sanguinea pretreatment also inhibited CCl4-induced hepatotoxicity in mice. These data indicate that T. sanguinea is a potent antioxidant and can offer protection against GalN- or CCl4-induced hepatotoxicity.     

S-adenosylmethionine but not glutathione protects against galactosamine-induced cytotoxicity in rat hepatocyte cultures

A gradual but extensive depletion of hepatic GSH has long been known to accompany development of galactosamine-induced hepatotoxicity in rats, and some protection from liver injury has been observed after administration of sulfhydryl-donating compounds. Although these observations support a key role for GSH in the underlying mechanism, the impact of GSH depletion and repletion on the hepatotoxic response to galactosamine is unclear. To investigate the role of GSH in galactosamine-induced liver injury, we examined the effect of modulating GSH content on galactosamine toxicity in rat primary hepatocyte cultures. Galactosamine (4 mM) cytotoxicity was assessed by release of lactate dehydrogenase into the culture medium, and hepatocellular GSH content was measured by HPLC with electrochemical detection. The data indicated that prior depletion of GSH with either diethyl maleate or buthionine sulfoximine significantly enhanced galactosamine toxicity; however, addition of GSH-ester or alternate sulfur nucleophiles at various times during the incubation did not abrogate toxicity. In contrast, co-addition of S-adenosylmethionine (SAMe) with galactosamine exerted a marked protective effect without significantly altering hepatocyte GSH content. These data suggest that GSH depletion is not directly involved in the sequelae for galactosamine-induced hepatotoxicity, and raise the possibility that SAMe may have hepatoprotective effects that are not dependent on its ability to enhance GSH synthesis.     

巯基氧化剂对急性肝损伤时库普弗细胞功能的影响

目的　观察巯基氧化剂在急性肝损伤时对库普弗细胞功能的影响。方法　采用半乳糖胺 /脂多糖 (Galn/LPS)所致急性肝损伤模型 ,雄性昆明种小鼠于注射Galn/LPS(每只 4kg/g)前 1h ,腹腔注射巯基氧化剂马来酸二乙酯 (DEM)每周 5mmol/kg ,并分别于Galn/LPS注射后 6h ,测定血浆丙氨酸转氨酸 (ALT)活性、肿瘤坏死因子 (TNF) α水平、肝脏谷胱甘肽 (GSH)含量和库普弗细胞的吞噬功能。结果　DEM预处理可抑制Galn/LPS导致的血浆ALT活性、TNF α水平、库普弗细胞的吞噬功能升高。结论　巯基氧化剂DEM可通过改变肝脏GSH含量的变化 ,影响细胞氧化还原状态 ,抑制LPS导致的库普弗细胞活化 ,从而减轻肝损伤的发生