Quantitative and qualitative changes of serum proteolytic activity in rats with liver damage induced by galactosamine

Serum proteolytic activity was determined in galactosamine-treated rats and in controls. Injection of the hepatotoxin at a dose of 400 mg/kg resulted in a 3.4-fold elevation in the serum proteolytic activity, while AST (aspartate aminotransferase), ALT (alanine aminotransferase) and bilirubin were increased by factors of 3.9, 8.8 and 4.5, respectively. Studies with proteinase inhibitors revealed that the serum proteolytic activity was partially metal-dependent as well as puromycin and antipain sensitive. Differences in susceptibility to a combination of N-ethylmaleimide and antipain indicated presence of different proteolytic systems in the sera of liver damaged and control rats. Separation of serum proteinases by gel filtration showed that the galactosamine-intoxicated rat serum contained activity which did not appear in the control serum. This activity was partially metal dependent, antipain and N-ethylmaleimide sensitive, and was more susceptible to dithiothreitol than the control activity. These findings demonstrate that hepatocellular damage induced by galactosamine caused not only an increase in serum proteinases, but was also associated with the appearance of enzymes not normally released by the liver of untreated animals.Abbreviations AP alkaline phosphatase - TBil total bilirubin - AST aspartate aminotransferase - ALT alanine aminotransferase - GGT gamma-glutamyltranspeptidase - BiAc bile acids - PrAm primary amines - ProAc proteolytic activity     

复方黄芪提取物抗急性肝损伤作用的研究

目的：研究复方黄芪提取物（ERAC）的抗急性肝损伤作用。方法： 分别采用D-氨基半乳糖（D-Galn）和四氯化碳（CCl4）造成急性肝损伤模型，检测血清中丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）的活性及胆红素的含量，并进行病理学检查。结果： ERAC对肝损伤引起的ALT和AST活性增加以及胆红素增高均有明显的降低作用（P<0.05）。病理组织学检查：ERAC对肝损伤引起的肝细胞变性、坏死等病变有改善作用。结论： ERAC能减轻D-Galn和CCl4所引起的肝损伤，改善肝功能。     

Oncotic necrosis and caspase-dependent apoptosis during galactosamine-induced liver injury in rats

The mode of cell death during galactosamine (Gal)-induced liver injury was originally thought to be oncotic necrosis but recently it was suggested to be apoptosis. Thus, the objective was to assess whether apoptosis and oncosis are sequential or independent events in the pathophysiology. In addition, the role of caspases in Gal-induced apoptotic signaling was investigated. A dose of 500 mg/kg Gal caused a time-dependent increase in plasma alanine transaminase (ALT) levels (24 h: 430 +/- 122 U/L) in female Sprague-Dawley rats. This was accompanied by processing of procaspase-3 and significant increases in hepatic and plasma caspase-3 activities. Using morphology and TUNEL staining, apoptotic and oncotic cells were quantitated. The number of apoptotic hepatocytes increased from 0.14% in controls to 5.4 +/- 1.0% 24 h after Gal treatment. In addition, the number of cells with oncotic morphology increased from 0 to 6.9% of total hepatocytes. Treatment with the pan-caspase inhibitor IDN-7314 (10 mg/kg) or pretreatment with uridine (1 g/kg), reduced all parameters of apoptosis to baseline. However, IDN-7314 administration did not affect plasma ALT activities and the number of oncotic cells at 6 h and only modestly reduced these parameters at 24 h. Uridine, on the other hand, prevented the increase of plasma ALT levels and reduced the number of apoptotic and oncotic cells by >80%. In conclusion, galactosamine-induced hepatocellular apoptosis in rats is caspase dependent. Although some of the apoptotic cells may undergo secondary necrosis, a significant number of hepatocytes die through oncotic necrosis as an independent mechanism of cell death.     

Hydrogen peroxide derived from amine oxidation mediates the interaction between aminosugars and semicarbazide-sensitive amine oxidase

Summary Semicarbazide-sensitive amine oxidase (SSAO) also functions as a vascular-adhesion protein (VAP-1). The nature of the target site on lymphocytes to which endothelial-cell SSAO/VAP-1 binds is unknown. We have shown that amino sugars (galactosamine, glucosamine and mannosamine), which are not SSAO substrates, can bind to the enzyme as reversible inhibitors. Thus, they serve as a model system in which to study the interaction process. Binding occurred during substrate (benzylamine) oxidation but not when the amino sugar was incubated, for extended periods, with SSAO alone. These results suggest that one, or more of the products of the SSAO-catalysed amine oxidation might be necessary for the inhibitory process to occur. Two of the reaction products of benzylamine oxidation, benzaldehyde and ammonia were found to have no effect on the inhibition of SSAO by galactosamine. Preincubation of the enzyme with galactosamine plus H₂O₂ was, however, found to result in time-dependent inhibition. This is not a result of the non-enzymic reaction between H₂O₂ and the amino sugar, since preincubation of galactosamine with H₂O₂ alone, for extended periods, did not give rise to an inhibitory species. The amount of exogenously added H₂O₂ necessary for inhibition was very much greater than that formed during substrate oxidation. These results suggest that the H₂O₂ formed as a product of the SSAO-catalysed oxidation reaction is more efective in promoting the binding of amino sugars.     

Cholesteryl hemisuccinate treatment protects rodents from the toxic effects of acetaminophen, adriamycin, carbon tetrachloride, chloroform and galactosamine

In addition to its use as a stabilizer/rigidifier of membranes, cholesteryl hemisuccinate, tris salt (CS) administration has also been shown to protect rats from the hepatotoxic effects of carbon tetrachloride (CCl₄). To further our understanding of the mechanism of CS cytoprotection, we examined in rats and mice the protective abilities of CS and the non-hydrolyzable ether form of CS, γ-cholesteryloxybutyric acid, tris salt (CSE) against acetaminophen-, adriamycin-, carbon tetrachloride-, chloroform-, and galactosamine-induced toxicity. The results of these studies demonstrated that CS-mediated protection is not selective for a particular species, organ system or toxic chemical. A 24-h pretreatment of both rats and mice with a single dose of CS (100mg/kg, i.p.), resulted in significant protection against the hepatotoxic effects of CCl₄, CHCl₃, acetaminophen and galactosamine and against the lethal (and presumably cardiotoxic) effect of adriamycin administration. Maximal CS-mediated protection was observed in experimental animals pretreated 24 h prior to the toxic insult. These data suggest that CS intervenes in a critical cellular event that is an important common pathway to toxic cell death. The mechanism of CS protection does not appear to be dependent on the inhibition of chemical bioactivation to a toxic reactive intermediate (in light of the protection observed against galactosamine hepatotoxicity). However, based on the data presented, we can not exclude the possibility that CS administration inhibits chemical bioactivation. Our findings do suggest that CS-mediated protection is dependent on the action of the intact anionic CS molecule (non-hydrolyzable CSE was as protective as CS), whose mechanism has yet to be defined.     

Hepatotoxin-induced hypertyrosinemia and its toxicological significance

A ¹H Nuclear Magnetic Resonance (NMR) spectroscopic investigation of the effects of single doses of four model hepatotoxins on male Sprague–Dawley rats showed that hypertyrosinemia was induced by three of the treatments (ethionine 300 mg/kg, galactosamine hydrochloride 800 mg/kg and isoniazid 400 mg/kg) but not by the fourth (thioacetamide 200 mg/kg). Concomitant histopathological and clinical chemistry analyses showed that hypertyrosinemia could occur with or without substantial hepatic damage and that substantial hepatic damage could occur without hypertyrosinemia. However, in the rats dosed with galactosamine hydrochloride, which showed highly variable amounts of liver damage at ca. 24 h after dosing, a clear relationship was found between the degree of hypertyrosinemia and the extent of the hepatic necrosis induced. In line with the cause of clinically observed Type II Tyrosinemia, we consider that the critical event in the onset of hepatotoxin-induced hypertyrosinemia is likely to be a reduction in hepatic tyrosine aminotransferase (TAT) activity. We discuss mechanisms by which TAT activity could be lost with special consideration given to pyridoxal 5′-phosphate (P5P) depletion and to the inhibition of protein synthesis. This analysis may have implications for the interpretation of clinical measures of liver status such as Fischer’s ratio and the branched-chain tyrosine ratio (BTR).     

半乳糖胺盐致肝损害与肾上腺皮质激素水平的关系

半乳糖胺盐(GaiN)一次和多次染毒后引起大鼠肝损害和肾上腺抗坏血酸含量下降,而血浆皮质酮、尿17-羟类固醇和17-羟类固酮无明显改变;肝△~4还原酶活性受抑制,而未见血浆肾上腺皮质激素水平增加和尿皮质类固醇排出减少。组织学上可见肾上腺皮质束状带细胞部分肿胀和脂质脱失。肾上腺中抗坏血酸含量下降可能是GalN直接引起的,而不是肝损害的后果。机体在皮质激素高水平的情况下可增加GalN肝毒作用。     

保肝康对急性肝损伤大鼠肝细胞凋亡的影响

目的：探讨保肝康抗肝损伤的作用机理。方法：采用D－GlaN所致大鼠急性肝损伤模型，观察保肝康对此模型肝细胞凋亡及相关基因表达的影响。结果：（1）保肝康有抑制肝细胞凋亡的作用。（2）Fas/FasL的表达率与肝细胞凋亡呈平行相关，结论：（1）保肝康抑制肝细胞凋亡的作用是其抗肝损伤的作用机理之一。（2）保肝康抑制细胞凋亡作用机理可能是下调Fas/FasL的表达。     

Fructose 1,6-bisphosphate reduced TNF-α-induced apoptosis in galactosamine sensitized rat hepatocytes through activation of nitric oxide and cGMP production

Fructose 1,6-P2 (F1,6BP) protects rat liver against experimental hepatitis induced by galactosamine (GalN) by means of two parallel effects: prevention of inflammation, and reduction of hepatocyte sensitization to tumour necrosis factor-alpha (TNF-α). In a previous paper we reported the underlying mechanism involved in the prevention of inflammation. In the present study, we examined the intracellular mechanisms involved in the F1,6BP inhibition of the apoptosis induced by TNF-α in parenchyma cells of GalN-sensitized rat liver. We hypothesized that the increased nitric oxide (NO) production in livers of F1,6BP-treated rats mediates the antiapoptotic effect. This hypothesis was evaluated in cultured primary rat hepatocytes challenged by GalN plus tumour necrosis factor-alpha (GalN + TNF-α), to reproduce in vitro the injury associated with experimental hepatitis. Our results show a reduction in apoptosis concomitant with an increase in NO production and with a reduction in oxidative stress. In such conditions, guanylyl cyclase is activated and the increase in cGMP reduces the TNF-α-induced apoptosis in hepatocytes. These results provide new insights in the protective mechanism activated by F1,6BP and confirm its interest as a hepatoprotective agent.     

混合核苷酸对小鼠急性化学性肝损伤的保护作用

目的：研究了混合核苷酸对急性化学性肝损伤的保护作用及其对免疫系统的作用。方法：采用四氯化碳、D－氨基半乳糖化学性肝损伤模型及碳粒廓清和血清溶血素等免疫学模型的动物实验。结果：混合核苷酸由D－氨半乳糖引起的小鼠急性肝损伤及由四氯化碳引起的小鼠急性肝损伤具有明显的保护作用。事核苷酸1000,500mg/kg能降低小鼠血清AST及ALT水平,混合核苷酸组与模型对照组比较有显著性差异,与肌苷、联苯双酯组比较无明显差异。对混合核苷酸免疫调节作用的研究结果显示,混合核苷酸对正常动物免疫系统作用不明显,但能增强免疫低下动物的免疫,使之恢复正常水平。结论：混合核苷酸可治疗急、慢性肝炎及肝损伤,可能是通过对免疫系统的作用而发挥作用的。