Characterization of glial fibrillary acidic protein (GFAP)-expressing hepatic stellate cells and myofibroblasts in thioacetamide (TAA)-induced rat liver injury

Hepatic stellate cells (HSCs), which can express glial fibrillary acidic protein (GFAP) in normal rat livers, play important roles in hepatic fibrogenesis through the conversion into myofibroblasts (MFs). Cellular properties and possible derivation of GFAP-expressing MFs were investigated in thioacetamide (TAA)-induced rat liver injury and subsequent fibrosis. Seven-week-old male F344 rats were injected with TAA (300 mg/kg BW, once, intraperitoneally), and were examined on post single injection (PSI) days 1–10 by the single and double immunolabeling with MF and stem cell marker antibodies. After hepatocyte injury in the perivenular areas on PSI days 1 and 2, the fibrotic lesion consisting of MF developed at a peak on PSI day 3, and then recovered gradually by PSI day 10. MFs expressed GFAP, and also showed co-expressions such cytoskeletons (MF markers) as vimentin, desmin and α-SMA in varying degrees. Besides MFs co-expressing vimentin/desmin, desmin/α-SMA or α-SMA/vimentin, some GFAP positive MFs co-expressed with nestin or A3 (both, stem cell markers), and there were also MFs co-expressing nestin/A3. However, there were no GFAP positive MFs co-expressing RECA-1 (endothelial marker) or Thy-1 (immature mesenchymal cell marker). GFAP positive MFs showed the proliferating activity, but they did not undergo apoptosis. However, α-SMA positive MFs underwent apoptosis. These findings indicate that HSCs can proliferate and then convert into MFs with co-expressing various cytoskeletons for MF markers, and that the converted MFs may be derived partly from the stem cell lineage. Additionally, well-differentiated MFs expressing α-SMA may disappear by apoptosis for healing. These findings shed some light on the pathogenesis of chemically induced hepatic fibrosis.     

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。     

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。     

表皮生长因子对多器官功能障碍小鼠的作用

目的 研究重组人表皮生长因子(recombinant human epidermal growth factor,rhEGF)对多脏器功能障碍综合征小鼠预后的影响.方法 江西省人民医院动物实验室清洁级雄性昆明种小鼠120只,SPF级,随机(随机数字法)分为生理盐水(normal saline,NS)空白对照组15只、MODS模型对照组15只,采用ME Caballero方法用硫代乙酰胺(thioacetamide,TAA) 2000 mg/kg单次腹腔注射建立单相速发小鼠多脏器功能障碍综合征模型.rhEGF MODS治疗组90只,其中随机(随机数字法)分为两种给药方式:腹腔注射组45只,皮下注射组45只;每组随机分为3个剂量组:10 μg/kg,30 μg/kg和50 μg/kg,每个剂量组15只.观察各组小鼠24 h内呼吸、体质量、饮食等一般情况的变化.24h后,计算各组病死率;颈椎脱臼处死动物后立即收集肝脏、肾脏、心脏、脑、肺、脾脏、胰腺、肠、胃,行苏木素-伊红染色光镜下观察组织学变化.实验数据以均数±标准差((x-)±s)表示,体质量自身变化比较采用t检验,不同给药方式及不同剂量的rhECF给药对MODS小鼠体质量变化影响采用Dunnett法分析;不同给药方式对MODS小鼠病死率的比较采用Fisher精确概率法,以P＜0.05为差异具有统计学意义.结果 rhEGF皮下给药组与MODS模型对照组的病死率比较差异无统计学意义(P＞0.05),但hrEGF MODS腹腔给药组病死率明显低于MODS模型对照组病死率73.3％(P＜0.01),其中腹腔给药50 μg/kg和30 μg/kg剂量组的病死率(6.7％、20.0％)较MODS模型对照组降低(P =0.000,0.009);而且腹腔给药50 μg/kg的病死率(6.7％)比10 μg/kg更低(P=0.014).同时,rhEGF MODS (50 μg/kg)腹腔给药组的病死率(6.7％)较皮下给药组的病死率(40.0％)降低更明显(P=0.031).在组织病理学上rhEGFMODS治疗组能改善MODS小鼠肝肺组织的损伤,随rhEGF腹腔给药剂量增加,肝淤血程度减轻,肝细胞凋亡减少,肝细胞浊肿和空泡变减少.同时随rhEGF MODS治疗组给药剂量增加肺间质充血减轻,炎细胞减少,偶见凋亡小体,支气管柱状纤毛上皮较少脱落.结论 rhEGF对TAA诱导的MODS小鼠模型的肝肺等脏器的组织损伤有修复作用,且腹腔注射较皮下注射能更有效地降低MODS小鼠的24 h病死率.     

体重监测下使用硫代乙酰胺诱导大鼠肝硬化模型

目的探索根据体重变化监测肝硬化诱导过程中大鼠对硫代乙酰胺 (thioacetamide,TAA)反应的个体差异 ,以提高肝硬化形成率和质量。方法雄性Wistar大鼠 4 6只 (2 0 0～ 2 30 g) ,随机分为 3组。 1组 (2 0只 )以 0 0 3% (w/v)TAA作为饮用水 ,共 12周。 2组 (2 0只 )以 0 0 3%TAA作为初始浓度 ,然后根据每周体重变化调整饮用水中TAA浓度。 3组 (6只 )给予饮用水 ,作为对照组。结果 1组大鼠病死率为 30 % (6 / 2 0 ) ,肝硬化形成率仅 4 5 % (9/ 2 0 )。 2组病死率为 0 ,肝硬化形成率为 90 % (18/ 2 0 )。结论肝硬化诱导过程中大鼠对TAA反应的个体差异可根据每周体重变化加以监测。该法可降低大鼠病死率至 0 ,同时显著提高肝硬化形成率和质量。     

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.     

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%.     

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