Simvastatin for rats with thioacetamide-induced liver failure and encephalopathy

Background and Aim:  Nitric oxide (NO) inhibition aggravates hepatic damage and encephalopathy and increases mortality in rats with thioacetamide (TAA)-induced acute liver failure. Statins enhance NO synthase expression beyond their lipid-lowering capability, but the impact on encephalopathy remains unexplored. The aim of this study was to assess the effects of simvastatin on rats with TAA-induced acute liver damage and hepatic encephalopathy.
Methods:  Sprague–Dawley rats received TAA (350 mg/kg/day) or normal saline (NS) by intraperitoneal injection for 3 consecutive days. Two days before injections, each group was divided into three subgroups, taking (i) distilled water; (ii) simvastatin (20 mg/kg/day); or (iii) simvastatin plus N ^G-nitro- l -arginine methyl ester (L-NAME, 25 mg/kg/day) by oral gavage for 5 days. On the fifth day, severity of encephalopathy was assessed and plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin and ammonia were measured.
Results:  The TAA subgroups showed higher ALT, AST, bilirubin and ammonia levels and lower motor activity counts as compared with the NS subgroups. Among the TAA-treated subgroups, rats with simvastatin treatment exerted higher motor activity counts and survival rate ( P  = 0.043), and a trend of lower ALT, AST, bilirubin and ammonia levels than those receiving saline. All rats that underwent simvastatin plus L-NAME treatment died during or after TAA injections.
Conclusions:  Simvastatin improved encephalopathy and survival in TAA-administered rats. The beneficial effect was offset by L-NAME, suggesting the role of NO in liver damage and encephalopathy.     

硫代乙酰胺诱导大鼠急性肝性脑病模型建立的用药时间研究

目的通过硫代乙酰胺（TAA,300mg·kg^-1·d^-1）不同用药时间诱导A型肝性脑病,比较大鼠的行为学、生物化学以及组织学改变,探讨造模最适时间。方法将大鼠分为A、B、C、D四组,其中A组为正常对照组;B、C、Di组用TAA（300mg·kg^-1·d^-1）分别连续灌胃2d、3d、4d,A组用相等量生理盐水灌胃4d。比较各组大鼠行为学变化、脑功能评分、AHE的诱导率和致死率,并分析各组给药结束24h后血氨、ALT、AST、TBIL的差异。结果C、D组比B组大鼠脑功能评分高,差异有统计学意义（P〈0．0083）;C、D组比B组诱导率高（P〈0．0083）,而D组比B、C组的大鼠致死率高,差异有统计学意义（P〈0．0083）;C、D组血氨及ALT、AST、TBIL肝功能指标比B组高,差异有统计学意义（P〈0．0083）;C、D组TAA用药后肝组织学观察炎症浸润、坏死、纤维化等损害最明显。结论300mg·kg^-1·d^-1的TAA连续灌胃3d,行为学改变显著．致死率较低．血氨较高．肝功能损害明显．为TAA诱导大鼠急性肝性脑病适宜时间。     

Hepatic stimulator substance administration ameliorates liver regeneration in an animal model of fulminant hepatic failure and encephalopathy

Abstract: Aims/Background: Hepatic stimulator substance (HSS) is a liver‐specific growth factor implicated in hepatocellular proliferation and hepatoprotection in models of acute liver injury. In the present study, we examined the effect of exogenous HSS administration on liver proliferating capacity and survival outcome in an experimental animal model of fulminant hepatic failure (FHF) and encephalopathy, induced by repeated injections of thioacetamide (TAA) in rats. Methods: Fulminant hepatic failure was induced in adult male Wistar rats by three consecutive intraperitoneal injections of TAA (400 mg/kg of body weight), at 24 h time intervals. The animals received intraperitoneally either a saline solution or HSS (50 mg protein/kg of body weight), 2 h after the second and third TAA injections. The animals were killed at 6, 12 and 18 h post the last injection of TAA. Results: Levels of liver enzymes and urea in serum, blood ammonia values, liver histology, stage of hepatic encephalopathy and survival were statistically significantly improved in TAA‐intoxicated and HSS‐treated rats compared to TAA‐intoxicated and saline‐treated ones. Furthermore, HSS ameliorated liver regenerative indices – DNA biosynthesis, thymidine kinase activity and hepatocyte mitotic activity – in a statistically significant manner. Conclusions: Our data suggest the beneficial effect of HSS administration in this animal model of FHF and encephalopathy, supporting evidence for a possible use of HSS as supportive therapy, by increasing hepatocellular proliferation, in management of FHF.     

丹参对暴发性肝衰竭大鼠肝损伤及肝再生的影响

目的：探讨丹参对暴发性肝衰竭（FHF）大鼠肝损伤及肝再生的影响。方法：Wistar大鼠随机分为4组：正常对照组、FHF组、丹参组、促肝细胞生长素（PHGF）组。FHF、丹参组和PHGF组动物模型采用TAA皮下注射,剂量按每kg体重600mg,2次,每次间隔24h,复制FHF动物模型。丹参组和PHGF组动物除皮下注射TAA外,于实验前3天至实验结束分别于皮下注射丹参注射液1ml/100g和PHGF1ml/100g,正常对照组和FHF组大鼠同时皮下注射生理盐水1ml/100g。FHF组、丹参组和PHGF组,于第2次注射TAA后24h,随机取大鼠各8只,腹主动脉取血测肝功能。迅速取肝组织,用10%甲醛液固定,制成石蜡切片,检测肝细胞有丝分裂指数（MI）和增殖细胞核抗原（PCNA）。结果：丹参具有降低FHF大鼠ALT、AST及TBil,显著提高肝细胞MI和PCNA的作用（P〈0.05,P〈0.01）。结论：丹参具有改善FHF大鼠肝功能和促进肝细胞增殖及再生的作用。     

急性肝衰竭大鼠肝组织组蛋白去乙酰化酶2的表达

目的观察D-氨基半乳糖盐酸盐诱导急性肝衰竭模型大鼠在丙酮酸乙酯保护下,组蛋白去乙酰化酶2(HDAC2)的表达情况。方法将78只雄性Wistar大鼠随机分成正常组、模型组、EP干预组和EP治疗组。采用免疫组织化学法检测肝组织HDAC2表达;采用ELISA法检测肝组织匀浆HDAC2水平。结果与正常组比,模型组HDAC2的表达量减少(P0.05),其中造模12h和24h肝组织HDAC2表达的IOD/Area值分别为0.0817±0.0118和0.0726±0.0111,明显低于正常组(0.1125±0.0047,P0.01);EP干预组和治疗组与模型组间HDAC2的表达无明显差异(P0.05)。结论 EP虽然对肝脏有保护作用,但不能影响HDAC2的表达。HDAC2的减少在急性肝衰竭的进展过程中,可能发挥重要的作用。     

Pathogenesis of hepatic encephalopathy in acute liver failure

Hepatic encephalopathy (HE) in acute liver injury signifies a serious prognosis. Brain edema and intracranial hypertension are major causes of death in this syndrome. Comparison of HE in acute liver failure (ALF) with that of cirrhosis allows recognition of important differences and similarities. A key role for ammonia in the pathogenesis of both HE and brain edema is now firmly supported by clinical and experimental data. Additional factors, such as infection, products of the necrotic liver, and synergistic toxins, may contribute to an altered mental state. A low plasma osmolarity, high temperature, and both high and low arterial pressure may affect brain water content. A combined derangement of cellular osmolarity coupled with cerebral hyperemia can explain the development of brain edema in ALF. Increasingly, study of the mechanisms responsible for brain swelling provides critical information for understanding the pathogenesis of HE.     

丹黄方对硫代乙酰胺所致大鼠急性肝衰竭的防治作用

目的:探讨丹黄方抗大鼠急性肝衰竭的作用和可能机制。方法:按600mg/kg的剂量经皮下注射硫代乙酰胺(TAA)1次,24小时后按相同剂量重复注射1次,造成大鼠急性肝衰竭模型,于实验前3天至实验结束分别给予各组大鼠生理盐水,不同剂量丹黄方及促肝细胞生长素,观察造模后48小时内大鼠病死率,并于造模48小时后经腹主动脉采血测定ALT、AST、TBil、TNF-α、IL-6,取肝组织作病检。结果:丹黄方能明显降低急性肝衰竭大鼠死亡率、降低ALT、AST、TBil及TNF-α、IL-6水平,改善肝组织病变,与模型组及促进肝细胞生长素阳性对照组比较,P<0.05或P<0.01,差异有显著性意义。结论:丹黄方具有抗TAA大鼠急性肝衰竭作用,可能与降低大鼠急性肝衰竭的重要介质TFN-α、IL-6有关。     

Prostacyclin inhibition by indomethacin aggravates hepatic damage and encephalopathy in rats with thioacetamide-induced fulminant hepatic failure

AIM: Vasodilatation and increased capillary permeability have been proposed to be involved in the pathogenesis of acute and chronic form of hepatic encephalopathy. Prostacyclin (PGI2) and nitric oxide (NO) are important contributors to hyperdynamic circulation in portal hypertensive states. Our previous study showed that chronic inhibition of NO had detrimental effects on the severity of encephalopathy in thioacetamide (TAA)-treated rats due to aggravation of liver damage. To date, there are no detailed data concerning the effects of PGI2 inhibition on the severity of hepatic encephalopathy during fulminant hepatic failure. METHODS: Male Sprague-Dawley rats weighing 300-350 g were used. Fulminant hepatic failure was induced by intraperitoneal injection of TAA (350 mg/(kg·d) for 3 d. Rats were divided into two groups to receive intraperitoneal injection of indomethacin (5 mg/(kg·d), n = 20) or normal saline (N/S, n = 20) for 5 d, starting 2 d before TAA administration. Severity of encephalopathy was assessed by the counts of motor activity measured with Opto-Varimex animal activity meter. Plasma tumor necrosis factor-α (TNF-α, an index of liver injury) and 6-keto-PGF1α (a metabolite of PGI2) levels were measured by enzyme-linked immunosorbent assay. RESULTS: As compared with N/S-treated rats, the mortality rate was significantly higher in rats receiving indomethacin (20% vs5%, P<0.01). Inhibition of PGI2 created detrimental effects on total movement counts (indomethacin vs N/S: 438±102 vs 841±145 counts/30 min, P<0.05). Rats treated with indomethacin had significant higher plasma levels of TNPa (indomethacin vs N/S: 22±5 vs 10±1 pg/mL, P<0.05) and lower plasma levels of 6-keto-PGF1α (P<0.001), but not total bilirubin or creatinine (P>0.05), as compared with rats treated with N/S. CONCLUSION: Chronic indomethacin administration has detrimental effects on the severity of encephalopathy in TAA-treated rats and this phenomenon may be attributed to the aggravation of liver injury. This study suggests that PGI2 may provide a protective role in the development of fulminant hepatic failure.     

大黄对暴发性肝衰竭大鼠肝损伤及肝再生的影响

[目的]探讨大黄对暴发性肝衰竭(FHF)大鼠肝损伤及肝再生的影响.[方法]Wistar大鼠随机分4组:正常对照(对照)组、模型(FHF)组、大黄组、促肝细胞生长素(PHGF)组.FHF、大黄组和PHGF组动物模型采用皮下注射(sc)硫代乙酰胺(TAA)600 mg/kg体重,2次,每次间隔24 h,复制FHF动物模型.大黄组和PHGF组动物除予TAA外,于实验前3天至实验结束分别sc大黄注射液1 ml/100 g和PHGF 1 ml/100g,对照组和FHF组同时sc 0.85%氯化钠液1 ml/100g.FHF组、大黄组和PHGF组,于第2次注射TAA后24 h,随机各取8只,腹主动脉取血测肝功能.迅速取肝组织,用10%甲醛液固定,石蜡切片,检测肝细胞有丝分裂指数(MI)和增殖细胞核抗原(PCNA).[结果]大黄具有降低FHF大鼠丙氨酸氨基转移酶、天冬氨酸转氨酶及总胆红素,并能显著提高MI和PCNA(P＜0.05,＜0.01).[结论]大黄具有改善FHF大鼠肝功能和促进肝细胞增殖及再生的作用.     

Infection and the progression of hepatic encephalopathy in acute liver failure

BACKGROUND & AIMS: Progression of hepatic encephalopathy (HE) is a major determinant of outcome in acute liver failure (ALF). Our aim was to identify predictive factors of worsening HE, including the relation of encephalopathy with the systemic inflammatory response (SIRS) and infection. METHODS: We included 227 consecutive patients with stage I-II HE prospectively enrolled in the U.S. Acute Liver Failure Study. Univariate and multivariate analysis of 27 variables at admission were performed separately for acetaminophen (n = 96) and nonacetaminophen (n = 131) etiologies. RESULTS: On multivariate analysis, acquisition of infection during stage I-II HE (P < 0.01), increased leukocyte levels at admission (P < 0.01), and decreased platelet count (P < 0.05) were predictive factors of worsening HE in the acetaminophen group. By contrast, only increased pulse rate (P < 0.05) and AST levels (P < 0.05) at admission were predictors in nonacetaminophen patients. In patients who progressed to deep HE, the first confirmed infection preceded progression in 15 of 19 acetaminophen patients compared with 12 of 23 nonacetaminophen patients. In patients who did not demonstrate positive microbiologic cultures, a higher number of components of SIRS at admission was associated with more frequent worsening of HE (25% vs. 35% vs. 50% for 0, 1, and >or=2 components of SIRS, P < 0.05). CONCLUSIONA: This prospective evaluation points to infection and/or the resulting systemic inflammatory response as important factors contributing to worsening HE in ALF, mainly in patients with acetaminophen- induced ALF. The use of prophylactic antibiotics in these patients and the mechanisms by which infection triggers hepatic encephalopathy require further investigation.     

Evidence against a role for endotoxin in the hepatic encephalopathy of rats with thioacetamide-induced fulminant hepatic failure

BACKGROUND AND AIMS: Endotoxin has been proposed to participate in the development of hepatic encephalopathy. However, there is no published data concerning the effects of endotoxin neutralization on the degree of hepatic encephalopathy. The present study investigated the effect of chronic intraperitoneal injection of polymyxin B, a neutralizing antagonist of endotoxin, on hepatic encephalopathy in rats with thioacetamide (TAA)-induced fulminant hepatic failure. METHODS: Male Sprague-Dawley rats weighing 300-350 g were used. Fulminant hepatic failure was induced by intraperitoneal injection of TAA (350 mg/kg/day) for 3 days. Two series of rats were designed to compare the effects of low dose (0.1 mg) or high dose (0.2 mg) intraperitoneal polymyxin B administration versus normal saline (NS) on hepatic encephalopathy. The injection was twice daily started from 2 days prior to TAA administration and lasted for 5 days. Severity of encephalopathy was assessed by the counts of motor activity in an Opto-Varimex animal activity meter. Plasma levels of endotoxin and tumor necrosis factor-alpha (an index of liver injury) were measured by Limulus assay and the ELISA method, respectively. RESULTS: Neutralization of endotoxin by either low dose or high dose polymyxin B administration did not significantly alleviate the degree of hepatic encephalopathy, as represented by the counts of motor activities (P > 0.05). Plasma levels of endotoxin and tumor necrosis factor-alpha were comparable between rats treated with polymyxin B or NS (P > 0.05). CONCLUSION: Our findings do not support the notion that endotoxin plays a major role in the pathogenesis of hepatic encephalopathy in rats with TAA-induced fulminant hepatic failure.     

Hepatic stimulator substance activity in animal model of fulminant hepatic failure and encephalopathy

Hepatic stimulator substance (HSS) is a known liver-specific but species-nonspecific growth factor. In the present study we examined the activity of the endogenously produced HSS in an established experimental model of fulminant hepatic failure (FHF) and encephalopathy, induced by repeated injections of thioacetamide (TAA). FHF was induced by three consecutive intraperitoneal injections of TAA (400 mg/kg body weight) in rats, at time intervals of 24 hr. The animals were killed at 0, 6, 12, or 18 hr following the last injection of TAA. The rate of tritiated thymidine incorporation into hepatic DNA, the enzymatic activity of liver thymidine kinase (EC 2.7.1.21), and the assessment of mitotic index in hepatocytes were used to estimate liver regeneration. HSS extract obtained from the livers of TAA-treated rats, sacrificed at the above-mentioned time points was tested for its activity. Increased HSS activity was noted in all TAA-treated animals, presenting a peak at 12 hr following the third TAA dose, suggesting active participation of this growth factor in hepatocyte replication in this animal model of FHF and encephalopathy. It may also be suggested that up-regulation of HSS activity could be used in future as a therapeutic approach in FHF.     

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。     

Autoimmune acute liver failure: an emerging etiology for intractable acute liver failure

Diagnosis of acute onset autoimmune hepatitis (AIH) is the most challenging task because of atypical clinicopathological features. We examined the nature of acute onset AIH consisting of nonsevere, severe, and fulminant AIH based on our published data and other published papers, and propose how to diagnose and treat this intractable hepatitis. We analyzed clinical, biochemical, immunological, radiological, and histological features of acute onset AIH. Thirty percent of fulminant hepatitis was due to AIH and autoimmune acute liver failure (ALF) was not rare. The important characteristic of acute onset AIH is its histological, radiological, and clinical heterogeneity. Sometimes acute onset AIH develops into ALF in a sub-acute clinical course without appropriate diagnosis and treatment, and becomes resistant to immunosuppressive therapy and has poor prognosis. Unenhanced computed tomography (CT) often shows heterogeneous hypoattenuation in autoimmune ALF. The revised original scoring system (1999) performed better in patients with acute onset AIH than the simplified scoring system (2008). Liver regeneration from periportal progenitor cells to mature hepatocytes was impaired in ALF, resulting in resistance to immunosuppressive therapy. Precise histological evaluation (the presence of centrilobular necrosis/collapse) along with the revised original scoring system and CT findings of heterogeneous hypoattenuation after systematic exclusion of other causes 36 plays an important role in the diagnosis. The most important strategy for autoimmune ALF is to diagnose and treat acute onset AIH before its development into ALF. Liver transplantation should be considered before the occurrence of infectious complications in the case of fulminant liver failure.     

Dynamic tracking of stem cells in an acute liver failure model

AIM:To investigate a dual labeling technique,which would enable real-time monitoring of transplanted embryonic stem cell(ESC) kinetics,as well as long-term tracking.METHODS:Liver damage was induced in C57/BL6 male mice(n = 40) by acetaminophen(APAP) 300 mg/kg administered intraperitoneally.Green fluorescence protein(GFP) positive C57/BL6 mouse ESCs were stained with the near-infrared fluorescent lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide(DiR) immediately before transplantationinto the spleen.Each of the animals in the cell therapy group(n = 20) received 5 × 10 6 ESCs 4 h following treatment with APAP.The control group(n = 20) received the vehicle only.The distribution and dynamics of the cells were monitored in real-time with the IVIS Lumina-2 at 30 min post transplantation,then at 3,12,24,48 and 72 h,and after one and 2 wk.Immunohistochemical examination of liver tissue was used to identify expression of GFP and albumin.Plasma alanine aminotransferase(ALT) was measured as an indication of liver damage.RESULTS:DiR-stained ESCs were easily tracked with the IVIS using the indocyanine green filter due to its high background passband with minimal background autofluorescence.The transplanted cells were confined inside the spleen at 30 min post-transplantation,gradually moved into the splenic vein,and were detectable in parts of the liver at the 3 h time-point.Within 24 h of transplantation,homing of almost 90% of cells was confirmed in the liver.On day three,however,the DiR signal started to fade out,and ex vivo IVIS imaging of different organs allowed signal detection at time-points when the signal could not be detected by in vivo imaging,and confirmed that the highest photon emission was in the liver(P 0.0001).At 2 wk,the DiRsignal was no longer detectable in vivo ;however,immunohistochemistry analysis of constitutively-expressed GFP was used to provide an insight into the distribution of the cells.GFP +ve cells were detected in tissue sections resembling hepatocytes and were dispersed throughout the hepatic parenchyma,with the presence of a larger number of GFP +ve cells incorporated within the sinusoidal endothelial lining.Very faint albumin expression was detected in the transplanted GFP +ve cells at 72 h;however at 2 wk,few cells that were positive for GFP were also strongly positive for albumin.There was a significant improvement in serum levels of ALT,albumin and bilirubin in both groups at 2 wk when compared with the 72 h time-point.In the cell therapy group,serum ALT was significantly(P = 0.016) lower and albumin(P = 0.009) was significantly higher when compared with the control group at the 2 wk time-point;however there was no difference in mortality between the two groups.CONCLUSION:Dual labeling is an easy to use and cheap method for longitudinal monitoring of distribution,survival and engraftment of transplanted cells,and could be used for cell therapy models.     

急性肝衰竭大鼠胃肠动力的变化

急性肝衰竭患者临床常见,病死率高,是危害人类健康的重要疾病.急性肝衰竭发病机制复杂,一直是研究的难点.临床上肝衰竭患者常发生恶心、呕吐、腹胀等现象,提示可能存在胃肠动力障碍.目前,关于急性肝衰竭胃肠动力改变及其机制的研究探讨甚少.本研究通过动物实验了解急性肝衰竭大鼠胃肠动力的变化、胃肠激素及内毒素水平的改变情况,进一步探讨急性肝衰竭的发病机制.     

急性肝衰竭大鼠胃肠动力的变化

急性肝衰竭患者临床常见,病死率高,是危害人类健康的重要疾病.急性肝衰竭发病机制复杂,一直是研究的难点.临床上肝衰竭患者常发生恶心、呕吐、腹胀等现象,提示可能存在胃肠动力障碍.目前,关于急性肝衰竭胃肠动力改变及其机制的研究探讨甚少.本研究通过动物实验了解急性肝衰竭大鼠胃肠动力的变化、胃肠激素及内毒素水平的改变情况,进一步探讨急性肝衰竭的发病机制.