暴发性肝功能衰竭大鼠肺血管壁通透性、血气的变化及其与内毒素血症的关系

本实验对硫代乙酰胺所致暴发性肝损伤大鼠肺血管壁通透性及血气的变化进行了观察。结果表明,肝损伤大鼠出现暴发性肝功能衰竭症状时,其肺血管对伊文思蓝的通透性增加(P<0.05),血浆内毒素水平亦较正常动物为高(P<0.05)。血气分析表现为动脉血CO_2分压增高(P<0.05);pH降低,且其水平与血浆内毒素浓度呈负相关(r=-0.730,P<0.05),70%大鼠出现混合性酸中毒,但动脉血氧分压及血氧饱和度无变化。     

Murine model to study brain,behavior and immunity during hepatic encephalopathy

AIM:To propose an alternative model of hepatic encephalopathy(HE) in mice,resembling the human features of the disease.METHODS:Mice received two consecutive intraperitoneal injections of thioacetamide(TAA) at low dosage(300 mg/kg).Liver injury was assessed by serum transaminase levels(ALT) and liver histology(hematoxylin and eosin).Neutrophil infiltration was estimated by confocal liver intravital microscopy.Coagulopathy was evaluated using prolonged prothrombin and partial thromboplastin time.Hemodynamic parameters were measured through tail cuff.Ammonia levels were quantified in serum and brain samples.Electroencephalography(EEG) and psychomotor activity score were performed to show brain function.Brain edema was evaluated using magnetic resonance imaging.RESULTS:Mice submitted to the TAA regime developed massive liver injury,as shown by elevation of serum ALT levels and a high degree of liver necrosis.An intense hepatic neutrophil accumulation occurred in response to TAA-induced liver injury.This led to mice mortality and weight loss,which was associated with severe coagulopathy.Furthermore,TAA-treated mice presented with increased serum and cerebral levels of ammonia,in parallel with alterations in EEG spectrum and discrete brain edema,as shown by magnetic resonance imaging.In agreement with this,neuropsychomotor abnormalities ensued 36 h after TAA,fulfilling several HE features observed in humans.In this context of liver injury and neurological dysfunction,we observed lung inflammation and alterations in blood pressure and heart rate that were indicative of multiple organ dysfunction syndrome.CONCLUSION:In summary,we describe a new murine model of hepatic encephalopathy comprising multiple features of the disease in humans,which may provide new insights for treatment.     

硫代乙酰胺诱导的急性肝损伤大鼠肝组织Norrin/Frizzled-4表达的变化*

目的 建立硫代乙酰胺（TAA）诱导的大鼠急性肝损伤模型,探讨Norrin/Frizzled-4在肝损伤间质重建中的作用。 方法 随机将32只SD大鼠分为正常对照组（N组）8只和TAA诱导组（M组）24只。建立TAA诱导的肝损伤大鼠模型。采用ELISA法检测血清CD105和血管内皮细胞生长因子（VEGF）水平,采用Western blot法检测原代肝星状细胞和肝组织Norrin/Frizzled-4信号通路中细胞外配体蛋白Norrin和细胞膜特异性受体蛋白Frizzled-4的表达。 结果 24只TAA诱导组大鼠死亡11只（45.8%）;病理学检查显示急性肝损伤大鼠模型建立成功;对照组血清CD105水平为（2.18±0.05） ng/mL,显著低于TAA处理1 w组的（2.36±0.07） ng/mL或2 w组的（2.42±0.03） ng/mL,差异均有显著性（P<0.05）;对照组血清VEGF为（61.48±0.39） pg/mL,显著低于模型组的（64.52±0.25） pg/mL,差异均有显著性（P<0.01）;原代肝星状细胞和正常肝组织不表达Norrin蛋白或Frizzled-4蛋白,或表达量很低,急性肝损伤大鼠肝组织Norrin/Frizzled-4表达显著高于正常对照组,差异均有显著性（P<0.01）。 结论 急性肝损伤大鼠肝组织Norrin/Frizzled-4蛋白表达上调,可能与急性肝损伤初期维持血管网络形态及肝间质重建有关。     

Involvement of P53 and Bax/Bad triggering apoptosis in thioacetamide-induced hepatic epithelial cells

AIM: Thioacetamide (TAA) has been used in studying liver fibrosis and cirrhosis, however, the mechanisms of TAA-induced apoptosis in liver are still unclear. The hepatic epithelial cell line clone 9 was cultured and treated with TAA to investigate the causes of cell death. METHODS: The cell viability of TAA-induced clone 9 cells was determined using MTT assay. Total cellular GSH in TAA-induced clone 9 cells was measured using a slight modification of the Tietze assay. The activity of caspase 3 in TAA-induced clone 9 cells was monitored by the cleavage of DEVD-p-nitroanaline. TUNEL assay and flow cytometry were applied for the determination of DNA fragmentation and the proportion of apoptosis in TAA-induced clone 9 cells, respectively. The alterations of caspase 3, Bad, Bax and Phospho-P53 contents in TAA-induced clone 9 cells were measured by Western blot. RESULTS: The experimental data indicated that TAA caused rat hepatic epithelial cell line clone 9 cell death in a dose-and time-dependent manner; 60% of the cells died (MTT assay) within 24 h after 100 mg/L TAA was applied. Apoptotic cell percentage (TUNEL assay) and caspase 3 activities were highest after 100 mg/L TAA was added for 8 h. The release of GSH and the elevation in caspase content after TAA treatment resulted in clone 9 cell apoptosis via oxidative stress and a caspase-dependent mechanism. The phospho-p53, Bax and Bad protein expressions in clone 9 cells were increased after TAA treatment. CONCLUSION: These results reveal that TAA activates p53, increases caspase 3, Bax and Bad protein contents, perhaps causing the release of cytochrome c from mitochondria and the disintegration of membranes, leading to apoptosis of cells.     

Inhibitory effect of Solanum nigrum on thioacetamide-induced liver fibrosis in mice

AIM: Solanum nigrum (Solanaceae) has been used in traditional folk medicine for its hepatoprotective agent. The purpose of this study was to investigate the effects of Solanum nigrum extract (SNE) on thioacetamide (TAA)-induced liver fibrosis in mice. MATERIALS AND METHODS: Hepatic fibrosis was produced by TAA (0.2g/kg, i.p.) three times a week for 12 weeks. Mice in the three TAA groups were treated daily with distilled water and SNE (0.2 or 1.0g/kg) via gastrogavage throughout the experimental period. RESULTS: SNE reduced the hepatic hydroxyproline and alpha-smooth muscle actin protein levels of TAA-treated mice. SNE inhibited TAA-induced collagene (alpha1)(I) and transforming growth factor-beta1 (TGF-beta1) mRNA levels in the liver. Histological examination also confirmed that SNE reduced the degree of fibrosis caused by TAA treatment. CONCLUSION: Oral administration of SNE significantly reduces TAA-induced hepatic fibrosis in mice, probably through the reduction of TGF-beta1 secretion.     

Caffeine mitigates experimental nonalcoholic steatohepatitis and the progression of thioacetamide-induced liver fibrosis by blocking the MAPK and TGF-β/Smad3 signaling pathways

Introduction and objectivesCaffeine consumption is associated with beneficial effects on hepatic disorders. The objectives of this study were to evaluate the antifibrotic effects of caffeine on experimental nonalcoholic steatohepatitis (NASH) induced with a high-fat, high-sucrose, high-cholesterol diet (HFSCD), as well as to evaluate the ability of caffeine to prevent the progression of experimental liver fibrosis induced by the administration of thioacetamide (TAA) in rats and explore the mechanisms of action.MethodsNASH and fibrosis were induced in rats by the administration of an HFSCD for 15 weeks, and liver fibrosis was induced by intraperitoneal administration of 200 mg/kg TAA 3 times per week, for 6 weeks. Caffeine was administered at a dose of 50 mg/kg body weight. The effects of diet, TAA, and caffeine on fibrosis were evaluated by biochemical and histological examinations. The profibrotic pathways were analyzed by western blotting and immunohistochemistry.ResultsRats exhibited liver fibrosis after HFSCD feeding and the administration of TAA. Caffeine could reduce the hepatic level of collagen and the fibrotic area in the liver. Caffeine prevented the progression of liver fibrosis by decreasing transforming growth factor-beta (TGF-β), connective tissue growth factor (CTGF), and alpha-smooth muscle actin (α-SMA) expression and by inhibiting the activation of mitogen-activated protein kinases (MAPKs) and Smad3 phosphorylation.ConclusionsCaffeine attenuates NASH and the progression of liver fibrosis due to its antifibrotic effects and modulating the MAPK and TGF-β pathways. Therefore, caffeine could be a suitable candidate for treating liver diseases associated with fibrosis.     

The role of vitamin D3 upregulated protein 1 in thioacetamide-induced mouse hepatotoxicity

Thioacetamide (TA) is a commonly used drug that can trigger acute hepatic failure (AHF) through generation of oxidative stress. Vitamin D3 upregulated protein 1 (VDUP1) is an endogenous inhibitor of thioredoxin, a ubiquitous thiol oxidoreductase, that regulates cellular redox status. In this study, we investigated the role of VDUP1 in AHF using a TA-induced liver injury model. VDUP1 knockout (KO) and wild-type (WT) mice were subjected to a single intraperitoneal TA injection, and various parameters of hepatic injury were assessed. VDUP1 KO mice displayed a significantly higher survival rate, lower serum alanine aminotransferase and aspartate aminotransferase levels, and less hepatic damage, compared to WT mice. In addition, induction of apoptosis was decreased in VDUP1 KO mice, with the alteration of caspase-3 and -9 activities, Bax-to-Bcl-2 expression ratios, and mitogen activated protein kinase (MAPK) signaling pathway. Importantly, analysis of TA bioactivation revealed lower plasma clearance of TA and covalent binding of [¹⁴C]TA to liver macromolecules in VDUP1 KO mice. Furthermore, the level of oxidative stress was significantly less in VDUP1 KO mice than in their WT counterparts, as evident from lipid peroxidation assay. These results collectively indicate that VDUP1 deficiency protects against TA-induced acute liver injury via lower bioactivation of TA and antioxidant effects.     

Attenuation of thioacetamide-induced hepatocellular injury by short-term repeated injections associated with down-regulation of metabolic enzymes and relationship with MHC class II-presenting cells

The liver is the primary organ participating in the metabolism of xenobiotics and is therefore an important target in the safety assessment of drugs, chemicals and environmental toxins. Drug-induced liver injury (DILI) has recently become widely recognized in human medicine as an adverse event. The progression of DILI often involves “damage-associated molecular patterns” (DAMPs) of gene and protein expression such as high-mobility group boxes (HMGBs), S100 proteins and heat shock proteins (Hsp). DAMPs are released from injured or necrotic cells and are bound to Toll-like receptors (TLRs) and modulate inflammatory reactions. Previously, in thioacetamide (TAA; 300 mg/kg body weight, single injection)-induced rat liver, we demonstrated that the expressions of DAMPs, TLR4 and major histocompatibility complex (MHC) class II were simultaneously increased, accompanied with progression of hepatocellular injury and inflammation. Here we investigated the association of DILI and DAMPs, TLRs and MHC class II by using rat livers repeated injections with TAA (100 mg/kg body weight, once, three times). Two days after TAA single injection, centrilobular hepatocellular necrosis with infiltration of mononuclear cells was observed, being paralleled with increase in serum levels of aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP). However, two days after duplicate and triplicate injections, only mild degenerative change of hepatocytes and slight infiltration of mononuclear cells were seen in the affected centrilobular area. Serum levels of AST, ALT and ALP were also decreased to the same levels of control. mRNA expressions of DAMPs (HMGBs, S100A4 and Hsp 70-2), TLR4 and MHC class II tended to be increased only on single injection, although the number of MHC class II-positive cells in the centrilobular area was still increased on each examination point. The analysis of enzymes (CYP2E1 and Flavin monooxygenase (FMO) 3), which metabolize TAA in hepatocytes, showed a significant decrease in FMO3 on the duplicate and triplicate injections. Autophagy and regulatory T cells were not significantly changed for the attenuation of hepatocyte injury. Collectively, these results suggest that hepatocytes may adapt accumulation of the toxicant by changing their enzyme functions; furthermore, MHC class II cells, which still showed increased number in the duplicate and triplicate injections, may be related with protection from the toxicant.     

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.     

丹参素和肝细胞生长因子对药物性肝细胞损伤的影响

目的:观察丹参素、猪再生肝细胞生长因子(PRHGF)和猪促肝细胞生长素(PHGF)对药物性肝损伤的影响。方法:以硫代乙酰胺(TAA)复制体外培养大鼠肝细胞损伤模型,通过检测培养液中LDH选出量和四甲基偶氮唑盐(MTT)显色情况了解肝细胞的存活和增殖情况,并同时检测细胞内、外SOD含量。结果:培养介质中加入TAA后,致LDH逸出明显增加,肝细胞增生指数下降,上清液和细胞内SOD活性显著降低。TAA和丹参素或PRHGF合用,则肝细胞逸出的LDH明显减少,增生指数上升并超过正常对照组,上清液和细胞内SOD活性显著升高。结论:丹参素、PRHGF和PHGF均能阻止肝细胞TAA损伤和促进肝细胞增生的作用。