Lipid peroxidation in thioacetamide-induced macronodular rat liver cirrhosis

Microsomes and isolated hepatocytes from thioacetamide (TAA)-induced macronodularly cirrhotic rat livers were analysed for their susceptibility to unstimulated and stimulated lipid peroxidation measured as malondialdehyde (MDA) formation. In microsomes from TAA-induced macronodularly cirrhotic livers the MDA production stimulated either by ascorbate-iron or by ADP-iron in a NADPH-regenerating system was decreased. Hepatic microsomes from TAA-treated rats exhibited a reduced cytochrome P450 content and lowered activities of ethylmorphine N-demethylase, ethoxycoumarin O-deethylase and epoxide hydrolase. Besides this, the microsomal fatty acid pattern of phosphatidylcholine and phosphatidylethanolamine was significantly changed after 6 months of TAA administration. The 182/204 ratio of phospholipid fatty acids was markedly increased. In contrast to the microsomes, in isolated hepatocytes from macronodularly cirrhotic livers the iron- and ascorbate-iron-stimulated MDA formation was increased. The hepatocellular GSH content was unaffected by TAA pretreatment, whereas the GSSG content exhibited a significant increase, thus leading to a pronounced reduction of the GSH/GSSG ratio. The calcium channel blocker verapamil (200 M), known to be able to scavenge OH radicals produced by the Fenton reaction, revealed an inhibitory effect on ascorbate-iron- and ADP-iron-stimulated lipid peroxidation in hepatocytes from normal as well as TAA-treated livers which is attributed to its antioxidative properties. In summary, lipid peroxidation is altered in TAA-induced macronodularly cirrhotic rat livers. Furthermore, the data clearly show that isolated microsomes and parenchymal cells prepared from cirrhotic livers react differently to prooxidant stimuli.     

内毒素血症在大鼠肝硬化发生发展中的作用

目的:探讨内毒素血症在大鼠肝硬化发生发展过程中的作用。方法:采用饮水中加入硫代乙酰胺(TAA),另一组同时小剂量注射脂多糖(LPS),以观察其对肝硬化形成的影响。结果:TAA可使动物血浆内毒素水平与肝脏胶原蛋白含量明显增高,且二者存在直线相关;血浆和肝组织中肿瘤坏死因子α(TNFα)、内皮素-1(ET-1)、一氧化氮(NO)、丙二醛(MDA)含量均增加,而且血浆中各成分含量均与内毒素血症呈正相关;在TAA+LPS组动物唯有TNFα浓度和胶原蛋白含量明显高于TAA组。结论:在肝硬化形成过程中内毒素可能主要通过激活枯否细胞分泌TNFα发挥其作用,但ET-1、NO和自由基也参与其中。     

Low-dose oral rapamycin treatment reduces fibrogenesis, improves liver function, and prolongs survival in rats with established liver cirrhosis

BACKGROUND/AIMS: Mammalian target of rapamycin (mTOR) signalling is central in the activation of hepatic stellate cells (HSCs), the key source of extracellular matrix (ECM) in fibrotic liver. We tested the therapeutic potential of the mTOR inhibitor rapamycin in advanced cirrhosis. METHODS: Cirrhosis was induced by bile duct-ligation (BDL) or thioacetamide injections (TAA). Rats received oral rapamycin (0.5 mg/kg/day) for either 14 or 28 days. Untreated BDL and TAA-rats served as controls. Liver function was quantified by aminopyrine breath test. ECM and ECM-producing cells were quantified by morphometry. MMP-2 activity was measured by zymography. mRNA expression of procollagen-alpha1, transforming growth factor-beta1 (TGF-beta1) and beta2 was quantified by RT-PCR. RESULTS: Fourteen days of rapamycin improved liver function. Accumulation of ECM was decreased together with numbers of activated HSCs and MMP-2 activity in both animal models. TGF-beta1 mRNA was downregulated in TAA, TGF-beta2 mRNA was downregulated in BDL. 28 days of rapamycin treatment entailed a survival advantage of long-term treated BDL-rats. CONCLUSIONS: Low-dose rapamycin treatment is effectively antifibrotic and attenuates disease progression in advanced fibrosis. Our results warrant the clinical evaluation of rapamycin as an antifibrotic drug.     

Co-administration of cyclosporine an alleviates thioacetamide-induced liver injury

AIM: To investigate the effects of cyclosporine A (CsA) on thioacetamide (TAA)-induced liver injury.METHODS: CsA was co-administrated (7.5 μg/kg body weight per day, i.p.) into rat to investigate the role of CsA on TAA-(200 mg/kg body weight per 3 d for 30 d, i.p.)induced liver injury.RESULTS: The data show that TAA caused liver fibrosis in rat after 30 d of treatment. CsA alleviates the morphological changes of TAA-induced fibrosis in rat liver. The blood glutamyl oxaloacetic transaminase (GOT)/glutamyl pyruvic transaminase (GPT) in the TAA-injury group is elevated compared to that of the normal rat. Compared with the TAA-injury group, the blood GOT/GPT and TGFβ1 (by RT-PCR analysis) are reduced in the CsA plus TAA-treated rat. The level of the transforming growth factor receptor I (TGFβ-R1) in the CsA plus TAA-treated group showsh igher than that in the TAA only group, but shows a lower level of the fibroblast growth factor receptor 4 (FGFR4) in the CsA plus TAA-treated group, when using the Western blot analysis. After immunostaining of the frozen section,TGFβ-R1 and FGFR4 are more concentrated in rat liverafter CsA plus TAA injury.CONCLUSION: This result suggests that CsA has an alleviated effect on TAA-induced liver injury by increasing the multidrug resistance P-glycoprotein and could be through the regulation of TGFβ-R1 and FGFR4.     

Depletion of Kupffer cell function by gadolinium chloride attenuates thioacetamide-induced hepatotoxicity. Expression of metallothionein and HSP70

Kupffer cell function plays an important role in drug-induced liver injury. Thus, gadolinium chloride (GD), by selectively inactivating Kupffer cells, can alleviate drug-induced hepatotoxicity. The effect of GD was studied in reference to metallothionein and heat shock proteins expression in an in vivo model of liver necrosis induced by thioacetamide. Rats, pre-treated or not with GD (0.1 mmol/kg), were intraperitoneally injected with thioacetamide (6.6 mmol/kg), and samples of blood and liver were obtained at 0, 12, 24, 48, 72 and 96 hr. Parameters related to liver damage, Kupffer cell function, microsomal FAD monooxygenase activity, oxidative stress, and the expression of metallothionein and HSP70 were determined. GD significantly reduced serum myeloperoxidase activity and serum concentration of TNF alpha and IL-6, increased by thioacetamide. The extent of necrosis, the degree of oxidative stress and lipoperoxidation and microsomal FAD monooxygenase activity were significantly diminished by GD. The effect of GD induced noticeable changes in the expression of both metallothionein and HSP70, compared to those induced by thioacetamide. We conclude that GD pre-treatment reduces thioacetamide-induced liver injury and enhances the expression of metallothionein and HSP70. This effect, parallel to reduced levels of serum cytokines and myeloperoxidase activity, demonstrates that Kupffer cells are involved in thioacetamide-induced liver injury, the degree of contribution being approximately 50%.     

猪再生肝中肝细胞生长因子对药物性肝细胞损伤的影响

本实验以体外培养大鼠肝细胞硫代乙酰胺（TAA）损伤为模型，采用四甲基偶氮唑盐（MTT）显色法，观察比较了成年小型猪部分肝切除后，取正在再生肝脏制得的肝细胞生长因子（PRHGF）和市场销售的乳猪肝肝细胞生长因子（PHGF）对肝细胞损伤保护作用的大小。结果表明，PRHGF和PHGF对TAA引起的肝细胞损伤均有保护作用，但PRHGF的最大效应高于PHGF组，提示PRHGF是一种有效的抗TAA肝损伤和刺激损伤后肝细胞再生的药物。     

Single cell analysis in toxicity testing: the mitogenic activity of thioacetamide in cultured rat hepatocytes analyzed by DNA/protein flow cytometry

Rat hepatocytes were cultured at 4% O₂ and 13% O₂ and exposed to the nongenotoxic rodent carcinogen thioacetamide (TA) from 24 to 72 h after isolation at exposure levels between 0.01 and 0.33 mM. Hepatocytes and isolated nuclei were analyzed by DNA-protein flow cytometry. An aggregate correction procedure was applied and the proportion of S-phase, diploid, tetraploid or octoploid hepatocytes as well as binucleated cells, were measured or calculated. The proportion of S-phase cells within the diploid hepatocytes increased with increasing concentration of TA up to 3.9-fold, whereas the corresponding increase in S-phase mononucleated tetraploid cells was only 1.8-fold. S-phase binucleate tetraploid cells showed no increase. In the tetraploid hepatocytes, the mitogenic stimuli was detectable only in cultures maintained at 4% O₂. The relative contribution of binuclear cells was increased 1.5-fold in the octoploid cells. It is concluded that the mitogenic activity of TA initiates DNA synthesis in diploid hepatocytes in the G1 and in the following G2 cell-cycle phase, omitting karyogenesis. The cellular protein content is not affected which indicates that the mitogenic activity of the chemical is not necessarily associated with an increase in cellular protein content. The results obtained correspond well with data of in vivo studies. The method applied therefore allows the mitogenic activity of nongenotoxic carcinogens to be detected in vitro within 48 h and their mode of action to be elucidated.Dedicated to Professor Dr. Gerhard Zbinden on the occasion of his retirement     

Retinal gliopathy accompanying thioacetamide-induced liver insufficiency: light and electron microscopic observations

A recent examination of retinae of patients who had died with symptoms of liver insufficiency (LI) including hepatic encephalopathy (HE) revealed morphological changes in retinal Müller glia similar to the astrocytic changes normally accompanying HE, and the term “hepatic retinopathy” (HR) was coined to define these changes. In the present study, the immunomorphology and ultrastructure of Müller cells were examined in rats in which LI with accompanying HE was induced with a hepatotoxin, thioacetamide (TAA). Light microscopically, retinae of rats with LI were characterized by swelling of the Müller cell cytoplasm. Immunostaining for glia-specific marker proteins in Müller cells from LI rats revealed a strongly enhanced expression of glial fibrillary acidic protein, and a considerable increase in glutamine synthetase immunoreactivity, as compared to control animals. Ultrastructurally, the Müller cells of LI rats showed swelling and vacuolization of cell processes. In particular, the endfeet contained many swollen mitochondria. By contrast, LI produced no morphologically demonstrable changes in retinal neurons and photoreceptor cells. Thus, the retinal changes induced by TAA in the rats strongly resembled those described in human HR, rendering the present rat model suitable for more detailed investigations of the pathomechanism(s) of HR. Received: 4 August 1997 / Revised, accepted: 12 January 1998     

丁酸钠对肝性脑病大鼠血氨浓度的影响初探

目的：观察不同浓度丁酸钠对肝性脑病（HE）大鼠血氨浓度的影响以及对HE的防治效果。方法采用腹腔注射硫代乙酰胺（TAA）诱导急性肝性脑病模型。饲喂两周后造模两天,观察大鼠一般情况,并进行神经反射评级,血浆TBil、DBil、AST、ALT、血氨和肠道pH值等指标的测定。结果丁酸钠各组HE大鼠TBil、DBil、ALT、AST均有所下降,其中丁酸钠A组的肠道pH值较模型组差异有统计学意义（P ＜0.05）,丁酸钠A、B两组的血氨浓度较模型组明显降低（P均＜0.001）。丁酸钠处理可改善大鼠的神经反射,降低大鼠肝性脑病的分期。结论丁酸钠通过酸化肠道,降低HE大鼠的血氨浓度,进而改善HE大鼠所表现出来的精神症状。     

穿心莲内酯调控硫代乙酰胺诱发小鼠肝纤维化的作用机制

目的 利用硫代乙酰胺(TAA)诱发小鼠肝纤维化的动物模型,探讨穿心莲内酯(AG)减缓肝纤维化的作用及其分子机制.方法 每周给予小鼠2次100 mg· kg-TAA,分别于第1、4周取样,并同时喂食高(100 mg· kg-1)、低(20 mg·kg-1)剂量AG,于第4周后取肝脏组织做组织切片、免疫组织化学染色、双重免疫荧光染色,收集血清做丙氨酸氨基转移酶分析.结果 AG可降低TAA所诱发肝内炎性物质与过氧化反应的表达,降低肝组织中Neutrophil、CD11b、F4/80的表达以及NF-κB、COX-2、p-cPLA2、Nrf2蛋白质等的表达量,并能调控肝内脂质过氧化作用,上调SMP30蛋白质水平;组织切片染色显示:给予TAA4周后的小鼠肝脏有明显纤维化,而随着给予AG剂量的增加有显著改善作用;随着AG剂量的增加减少了肝脏内d-SMA、TGF-βR1蛋白质的表达量;使用双重免疫荧光染色法发现AG减缓肝纤维化的作用与调控星状细胞活化相关.结论 AG能调控肝内炎性病变进而减缓TAA所引起的肝脏纤维化.