肝再生刺激因子对小鼠实验性急性肝损伤的保护作用

本工作采用雄性初断乳 SD 大鼠肝按 LaBrecque 法提取肝再生刺激因子(HSS),用四氯化碳和半乳糖胺分别损伤小鼠肝来研究 HSS 的保肝作用。结果如后:(1)HSS 可使 CCl_4致肝损伤小鼠的血清 GPT 和 GOT 升高幅度降低,并呈量效关系。(2)肝组织切片表明 HSS 可使 CCl_4损伤肝组织的程度减轻。(3)肝组织化学法表明 HSS 可使 CCl_4损害肝细胞线粒体琥珀酸脱氢酶的活性恢复。(4)胰岛素-胰高糖素可降低半乳糖胺所致的小鼠死亡率,减弱对肝组织的损害和刺激肝细胞增殖,这项实验可作为 HSS 具有保肝作用的证据。     

肝细胞生长因子对四氯化碳损伤肝细胞的保护作用及相关基因表达

利用无血清原代培养大鼠肝细胞,观察重组人肝细胞生长因子(rhHGF)对CCl4染毒肝细胞的保护作用. 结果表明: (1) rhHGF (5 ng/ml)预处理后可显著提高CCl4 (15 mmol/L)染毒肝细胞存活率,降低细胞内丙氨酸氨基转移酶(ALT)、 K+的漏出;(2) 表皮生长因子(EGF,50ng/ml)和rhHGF (5 ng/ml)合用预处理肝细胞,CCl4染毒后细胞内ALT、 K+漏出较rhHGF和EGF单独保护组进一步降低;(3)大鼠肝部分切除和CCl4 (50%,2.5 ml/kg bw)染毒后,再生肝内HGF基因及其受体基因/c-met的表达分别较假手术和盐水对照组显著升高. 结果提示,rhHGF对CCl4染毒大鼠肝细胞具有保护作用;EGF和rhHGF有协同保护作用;HGF及其受体的表达在肝脏再生及修复中可能有重要作用.     

肝再生刺激因子对小鼠实验性急性肝损伤的保护机制

我们前文证明肝再生刺激因子(HSS)对小鼠实验性肝损伤有保护作用。本文进一步探讨其机制并获得如下结果:(1)HSS显著提高由CCl_4所降低肝细胞膜、线粒体膜和微粒体膜的流动性,使其上升到对照水平。(2)HSS使CCl_4所致的肝组织丙二醛升高幅度降低。(3)HSS使CCl_4所致的肝组织谷胱甘肽降低的含量回升。(4)HSS能刺激受CCl_4损伤的肝再生,促进肝细胞合成DNA和~3H-TdR掺入肝细胞DNA。这些结果提示,HSS具有抗氧化作用,能抗CCl_4所产生的自由基对膜脂质的过氧化。此外还加强肝细胞本身抗氧化能力和促进受损肝脏再生。这些保肝机制可能相互联系。     

田基黄提取物抗CCl_4诱导大鼠肝纤维化的作用及其机制研究

探讨田基黄提取物(HJT)抗四氯化碳(CCl_4)诱导的大鼠肝纤维化(HF)的作用及其机制。将60只雄性SD大鼠随机分为正常组、模型组、秋水仙碱(0. 12 mg/kg)组和HJT(16 000、8 000、4 000 mg/kg)剂量组,每组10只。除正常组外,其余各组大鼠均腹腔注射40%的CCl_4橄榄油溶液(1 mL/kg),2次/周,建立HF模型;同时各给药组灌胃相应剂量药物,正常组和模型组给予等体积蒸馏水,连续6周。末次给药后,采用HE染色和Masson染色观察大鼠肝组织病理学改变;生化法检测血清中ALT、AST活性;生化法检测肝组织中SOD、GSH-PX活性和MDA含量;ELISA法检测血清中HA、LN、PCⅢ、Ⅳ-C含量;ELISA法检测肝组织IL-1β、IL-6和TNF-α的含量;Western blot法检测大鼠肝组织中TGF-β1和α-SMA表达水平。结果显示,HJT(16 000、8 000、4 000 mg/kg)剂量组和秋水仙碱组能显著降低HF大鼠血清中ALT、AST、HA、LN、PC-Ⅲ、Ⅳ-C活性或含量,降低肝组织MDA含量,增加SOD和GSH-PX活性,同时减少肝组织IL-1β、IL-6及TNF-α含量,抑制肝组织TGF-β1和α-SMA蛋白表达;病理组织切片结果显示:HJT各剂量组和秋水仙碱组大鼠肝组织炎症坏死和HF病变程度明显减轻。本研究结果表明,HJT对CCl_4诱导的HF大鼠有显著保护作用,其机制可能与抗炎、抗氧化和抑制TGF-β1蛋白表达有关。     

水杨柳根D_(101)树脂提取物保肝降酶作用的实验研究

目的研究水杨柳根D_(101)树脂提取物(HRE)对肝损伤模型动物的保护作用。方法取小鼠行连续灌胃给予50%白酒30天制造酒精性肝损伤模型,于小鼠腹腔注射CCl_4制造急性肝损伤模型,每周2次于大鼠腹腔连续注射CCl_430天制造慢性肝损伤模型,然后分别测定各动物血清ALT和AST水平。结果在酒精性和CCl_4慢性肝损伤动物模型中,HRE高、中剂量组(小鼠为2.7g/kg,1.8g/kg;大鼠为3.9g/kg,2.6g/kg)均能显著降低血清ALT和AST水平(P0.01或P0.05);在CCl_4急性肝损模型小鼠中,HRE高、中剂量组(2.7g/kg,1.8g/kg)也能显著降低血清ALT和AST水平(P0.01或P0.05)。结论水杨柳根的D_(101)树脂提取物的主要成分为黄酮、多糖和皂苷,具有保肝降酶作用,且随剂量增加而增强。     

川芎嗪促进大鼠部分肝切除后修复性再生机制的研究

目的:探讨大鼠部分肝切除后肝功能的变化及川芎嗪对肝修复性再生能力的影响。方法:在相同月龄的动物中按体重均衡的原则随机分组。实验动物共分为5组:设正常对照组(对照组)、青年假手术对照组(假术组)、青年肝切除组(青切组)、中年肝切除组(中切组)和中年肝切除治疗组(切治组),每组动物10只,常规饲养,自由饮水。参照Higgins and Aderson给大鼠施行肝脏70%切除手术,中切组大鼠术前以川芎嗪(200 mg/kg/d)腹腔注射7d,其余组注射生理盐水。假术组大鼠以同样的手术程序打开腹腔但不施行肝部分切除术。各组施行手术动物在切除术后24 h沿腹中线切开动物腹腔,于腹主动脉两髂分支处取血分离血清,切取所有肝脏,待测。采用试剂盒法分别测定各组血清中丙氨转氨酶(ALT)、谷草转氨酶(AST)含量;肝脏匀浆后,采用八木国夫法检测肝组织中丙二醛(MDA)含量,采用western blot法测定肝组织中核增殖抗原(PCNA)和铜锌超氧化物歧化酶(SOD1)、锰超氧化物歧化酶(SOD2)的蛋白表达。结果:与中切组相比手术动物相比,切治组组大鼠血清中ALT、AST水平显著降低(P<0.05),肝细胞中PCNA表达显著升高(P<0.05),肝组织中MDA含量显著降低(P<0.05);肝组织中SOD1、SOD2表达显著增加。结论:肝脏切除70%后,肝功受损,氧化应激增加,但核增殖能力增强。川芎嗪可以抑制肝切手术导致的氧化应激损伤,促进SOD的表达,抑制MDA的升高,降低ALT、AST水平,提高PCNA的表达。提示中年大鼠肝切除后肝功能受损与氧化应激相关,给予抗氧化药物能够促进肝再生修复能力,青年肝切除手术大鼠肝的修复能力强于中年动物。     

芍药苷减轻四氯化碳诱导的急性肝损伤

为研究芍药苷对四氯化碳(CCl_4)诱导的急性肝损伤的保护作用及相关机制。将36只健康雄性C57BL/J小鼠随机分成6组:空白组,芍药苷对照组,CCl_4模型组(0.1%,20 mL/kg),高、中、低剂量芍药苷+CCl_4组(10 mg/kg,30 mg/kg,100 mg/kg),每组6只。24 h后眼球取血收集血清,测定丙氨酸氨基转移酶(ALT),天门冬氨酸氨基转移酶(AST)活性。用苏木精-伊红(HE)染色,观察肝脏的组织学改变;试剂盒测定肝组织中SOD、GSH-PX、CAT的活性及MDA和GSH含量;ELISA法检测血清中TNF-α、IL-6含量;试剂盒检测小鼠肝组织中Caspase-3的活性;q RT-PCR检测肝组织中HO-1 mRNA的表达。实验发现PAE降低小鼠血清ALT、AST水平,改善肝脏的病理形态;芍药苷抑制CCl_4诱导氧化应激,升高肝组织HO-1 mRNA水平;降低TNF-α、IL-6含量;芍药苷减低肝组织中Caspase-3活性,减少肝细胞凋亡。由此可知,芍药苷可保护CCl_4诱导的急性肝细胞损伤,该保护作用可能与抑制脂质过氧化,减少促炎细胞因子产生,减少肝细胞凋亡,促进抗氧化蛋白表达有关。     

小鼠肝再生过程中ROS及线粒体代谢变化规律的研究

目的:肝脏是维持人体发挥功能的重要器官,同时肝脏再生能力十分强大。本文通过部分肝切除术后小鼠肝再生模型,观察肝再生过程中氧化应激及线粒体代谢变化规律,以期为将来的调控肝再生提供新的干预靶点。方法:选择雄性健康体重均匀的Balb/c小鼠,采用经典70%肝切除模型,随机分为假手术对照组(Sham组)以及70%肝切除组(70%PH组)。肝切除术后6 h、1d、2 d、3 d、5 d、7 d不同时间点取肝组织,制备冰冻切片检测活性氧(ROS)水平,Western blot分别检测细胞增殖相关蛋白PCNA、Cyclin D1;氧化应激相关蛋白SOD1、SOD2、CAT、GPX1;以及线粒体代谢相关蛋白PGC-1α、Nrf1、TFAM、Drp1、Fis1、Mfn1、Mfn2、OPA1的表达并分析其变化规律。结果:70%肝切除术后小鼠肝脏增长迅速,细胞增殖关键蛋白PCNA和Cyclin D1表达显著增加;在此过程中细胞ROS水平呈现先升高后降低的变化,细胞主要抗氧化酶SOD1、SOD2、CAT、Gpx1与ROS相一致出现先升高后降低的变化。线粒体生物合成调控因子PGC-1α、Nrf1、TFAM呈现先降低后升高的趋势,而线粒体分裂蛋白Drp1和Fis1呈现先降低后显著升高的趋势,线粒体融合相关蛋白Mfn1、Mfn2和OPA1总体为先降低后恢复至正常水平。结论:在小鼠70%肝切除再生过程中,存在着明显的氧化应激,线粒体生物合成增加,线粒体分裂/融合平衡偏向分裂,并且这些变化呈现具有一定的时间变化规律,这些变化及规律很可能作为将来调控肝再生的重要的潜在干预靶点。     

大鼠再生肝刺激因子抗四氯化碳损伤的研究

我们以往的研究证明,大鼠再生肝具有抗四氯化碳损伤的能力。本工作进一步研究其机制,首先从部分（６８％）肝切除后不同的再生肝的取肝刺激因子,用＾３Ｈ胸腺嘧喧核苷测定ｒＨＳＳ的生物活性,结果表明部分切除肝后７２ｈ的ｒＨＳＳ活性较对照组约增加７．７倍。然后将ｒＨＳＳ注射给小鼠,观察其抗ＣＣｌ４损伤肝的效应,具体表现如下：ｒＨＳＳ能减少ＣＣｌ４中毒小鼠的死亡率和降低ＣＣｌ４所增高的血清谷丙转氨酶和谷草转氨酶     

四氯化碳中毒对大鼠离体再生肝细胞钾离子外漏的影响

本工作用三种剂量四氯化碳(CCl_4,10,15和20mmol/L)损伤正常大鼠离体肝细胞,分别在5,10,15和20min测定细胞内K~+和GPT漏出量。实验观察到细胞内K~+和GPT漏出量与CCl_4染毒的剂量和时间有明显关系,而且K~+漏出量较GPT更能灵敏地反映细胞的损伤程度;用中等剂量CCl_4(15mmol/L)损伤离体再生肝细胞20min后,细胞内K~+漏出的变化百分数明显低于正常肝细胞。这些结果表明,大鼠离体再生肝细胞具有较强的抗CCl_4损伤作用,其机制可能与再生肝细胞膜稳定性较强有关。     

Effect of realimentation after several days' pure carbohydrate intake on DNA synthesis in regenerating rat liver

The authors studied the effect of realimentation after several days' isolated glucose or fructose intake on DNA synthesis in liver regenerating after partial hepatectomy (PH) (65-70%) or after carbon tetrachloride (CCl4) poisoning 1.5 ml/kg. Two days before PH or the administration of CCl4 and two days after, the experimental rats were given glucose (50% solution) of fructose (50% solution) as the only source of energy. Rats with PH were then fed for one day on a standard laboratory diet (25 cal% protein) or a high protein diet (81 cal% protein). Rats with CCl4 liver damage were fed for one day on the standard laboratory diet only. In the rats given glucose, liver DNA synthesis and the total amount of these nucleic acids in the liver 48 hours after CCl4 administration was lower than in the controls or the rats given fructose. In all the experimental groups (PH and CCl4), stimulation of liver DNA synthesis was observed after one day's realimentation. The total DNA content of the liver of rats with PH rose markedly during realimentation. The experiments indicate that the regenerative activity of damaged liver can be influenced by the nutritional regimen.     

Impairment of liver regeneration during inhibition of mitochondrial protein synthesis by oxytetracycline

Under standard conditions, liver regeneration is not impaired if mitochondrial protein synthesis is completely blocked. By treating rats with oxytetracycline for various periods of time directly prior to partial hepatectomy, livers were led to a condition of relative deficiency in cytochrome c oxidase and ATP synthetase. To this end, oxytetracycline was administered by means of continuous intravenous infusion up to concentrations of 20 μg/ml serum, giving a gradual decrease in cytochrome c oxidase activity. This activity was used as a marker for functionally capable mitochondria and as a tool to monitor the efficiency of inhibition of mitochondrial protein synthesis. It is shown that liver regeneration is strongly impaired after a period of pretreatment of 22 days or more and continuation of oxytetracycline treatment during regeneration. The mitochondrial respiratory capacity is reduced to 14% of the control value under these conditions. To obtain inhibitory levels within the regenerating liver, it was necessary to raise the serum levels slightly above 20 μg/ml. This measure is most likely required because of the poor vascularization of the regenerating liver. The serum levels were kept, however, far below those known to inhibit cytoplasmic protein synthesis. The results show that in normal liver the respiratory capacity must be reduced drastically before energy-requiring processes become affected. In Zajdela hepatoma cells, similar effects are found after reduction of the cytochrome c oxidase activity to 38%. This difference in sensitivity is probably based on the different mitochondrial content of liver cells and the liver-derived Zajdela cells.     

Liver lipase and high-density lipoprotein. Lipoprotein changes after incubation of human serum with rat liver lipase

Under standard conditions, liver regeneration is impaired if mitochondrial protein synthesis is completely blocked. By treating rats with oxytetracycline for various periods of time directly prior to partial hepatectomy, livers were led to a condition of relative deficiency in cytochrome c oxidase and ATP synthetase. To this end, oxytetracycline was administered by means of continuous intravenous infusion up to concentrations of 20 micrograms/ml serum, giving a gradual decrease in cytochrome c oxidase activity. This activity was used as a marker for functionally capable mitochondria and as a tool to monitor the efficiency of inhibition of mitochondrial protein synthesis. It is shown that liver regeneration is strongly impaired after a period of pretreatment of 22 days or more and continuation of oxytetracycline treatment during regeneration. The mitochondrial respiratory capacity is reduced to 14% of the control value under these conditions. To obtain inhibitory levels within the regenerating liver, it was necessary to raise the serum levels slightly above 20 micrograms/ml. This measure is most likely required because of the poor vascularization of the regenerating liver. The serum levels were kept, however, far below those known to inhibit cytoplasmic protein synthesis. The results show that in normal liver the respiratory capacity must be reduced drastically before energy-requiring processes become affected. In Zajdela hepatoma cells, similar effects are found after reduction of the cytochrome c oxidase activity to 38%. This difference in sensitivity is probably based on the different mitochondrial content of liver cells and the liver-derived Zajdela cells.     

Hepatotoxic effects of acetaminophen. Protective properties of tryptophan derivatives

Rat intoxication with acetaminophen (APAP) (500–1500 mg/kg body weight, intragastrically) caused a considerable dose-dependent decrease in reduced glutathione (GSH) level in both liver cell cytoplasm and mitochondria (at the dose 1500 mg/kg body weight by 60% and 33%, respectively). The decrease in cytoplasmic GSH level was more pronounced than in mitochondria. Despite of significant mitochondrial GSH depletion we did not observe any inactivation of the mitochondrial enzymes: succinate dehydrogenase, α-ketoglutarate dehydrogenase, glutathione peroxidase, and also any decrease in the respiratory activity of liver mitochondria isolated from APAP-intoxicated rats. We have investigated hepatoprotector properties of tryptophan derivatives, melatonin and N-acetyl-nitrosotryptophan (a nitric oxide donor). The pineal gland hormone, melatonin, a known antioxidant (10 mg/kg body weight), did not prevent intramitochondrial GSH, but decreased the APAP hepatotoxicity evaluated as the decrease in the activity of marker enzymes of hepatic damage, ALT and AST and total bilirubin content in blood plasma of intoxicated rats, whereas NNT did not exhibit any hepatoprotective effects.     

Effect of dimethyl sulphoxide on mitochondrial biogenesis in regenerating rat liver and Saccharomyces cerevisiae

Effect of dimethyl sulphoxide (DMSO) on mitochondrial biogenesis in regenerating rat liver and cells of Saccharomyces cerevisiae during aerobiosis has been studied by monitoring the cytochrome oxidase activity. A single dose of DMSO (275 mg/100-125 g body wt) to normal rats stimulated cytochrome oxidase activity in liver mitochondria while the same dose to partial hepatectomized rats inhibited the enzyme activity. Administration of low dose of DMSO (92 mg/100-125 g body wt) to partial hepatectomized rats did not alter the enzyme activity. Anaerobic cells of S. cerevisiae on aerobiosis for 2 hr attained cytochrome oxidase activity level on par with aerobic cells. Inclusion of DMSO (275 mg/100 ml) in the growth medium of S. cerevisiae during respiratory adaptation exerted partial inhibitory effect on the formation of cytochrome oxidase at 2 hr period, while the 10-fold concentration inhibited the enzyme formation completely. However, the inhibitory effect of DMSO on enzyme formation was abolished on prolonged growth (18 hr and above), while these doses had no influence on cytochrome oxidase in aerobic cells of S. cerevisiae. The results imply that DMSO may be exerting its effect on the assembly of subunits into active enzyme complex during mitochondrial biogenesis.     

Tumor necrosis factor-alpha increases hepatic DNA and RNA and hepatocyte mitosis.

Tumor necrosis factor-alpha (TNF) has been reported to increase DNA synthesis in normal rat liver. Therefore, we examined the effects of TNF on rat liver regeneration. TNF, 1.5 micrograms ip every 4 h for 5 d, significantly increased hepatic DNA and RNA contents of regenerating and sham operated livers by up to 45%. Mitotic figures in sham operated liver, usually rare, were increased substantially by TNF. ODC mRNA content and enzyme activity were increased in regenerating liver, and were further increased by TNF. These data indicate that TNF, although not specific for regenerating liver, is a potent stimulus for hepatocyte DNA synthesis and mitosis.     

Mitochondrial functional changes during hepatic hyperplasia and azo dye carcinogenesis.

Resting and active-state respiratory velocities, respiratory control, high amplitude volume changes, and latent ATPase activities were examined in hepatic mitochondria from rats fed 3'-methyl-4-dimethylaminoazobenzene (3'MeDAB) for production of liver tumors and from rats in three phases of liver regeneration subsequent to subtotal hepatectomies. Tetrabutylammonium bromide, a lipophilic probe capable of selectively inhibiting phosphorylating oxidation or uncoupling oxidation from phosphorylation, was used to detect subtle alterations in lipophilicity characteristics of the organelles and it was concluded that mitochondria from pre-hyperplastic, hyperplastic, and neoplastic tissues had a higher than normal degree of membrane lipophilicity at specific functional sites. Control of respiration by ADP was markedly augmented in all experimental groups; this behavior, plus depressed sensitivity to swelling agents and energized contraction, were similar in mitochondria from hepatomas and from 3-day regenerating livers. These mitochondrial functions were even more pronounced, however, in cells in pre-hyperplastic states (6 and 16 h subsequent to partial hepatectomy). Many forms of liver damage result in mitochondrial alterations which elevate the capacity for oxidative phosphorylation. Such changes associated with induction of azo dye oncogenesis are mimicked by the degree of hyperplasia in the tissue following the first mitotic wave of regeneration; implications relevant to hepatocarcinogenesis are discussed.