Effects of putrescine on D-galactosamine-induced acute liver failure in rats

We studied the effect of putrescine on acute liver failure caused in rats by two injections of 1 gm/kg D-galactosamine. The hepatic polyamine level rose only slightly in the D-galactosamine-injected rats treated with glucagon and insulin, and [3H]thymidine incorporation into DNA increased little; these hormones did not improve the survival rate. When D-galactosamine-injected rats were given putrescine, the putrescine concentration in the liver increased and the survival rate of the rats was significantly higher than that of control rats given only D-galactosamine. Putrescine administration tended to lower the serum level of alanine aminotransferase in rats injected with D-galactosamine, so the polyamine might have a protective effect on hepatocytes. Putrescine significantly increased [3H]thymidine incorporation in the liver; thus it accelerated liver regeneration. Difluoromethylornithine decreased the level of putrescine in the liver, decreasing both [3H]thymidine uptake and the survival rate. In the rats treated with D-galactosamine, in which liver damage was so severe that treatment with glucagon and insulin was ineffective, the intraperitoneal administration of putrescine increased the survival rate in acute liver failure. This probably resulted mainly from activation of liver regeneration and possibly from a protective effect of putrescine on the liver.     

Hybrid bioartificial liver support in cynomolgus monkeys with D-galactosamine-induced acute liver failure

AIM: To evaluate a hybrid bioartificial liver support system (HBALSS) in cynomolgus monkeys with acute liver failure.METHODS: To establish a model of acute liver failure, 0.3 g/kg of D-galactosamine was injected intravenously into cynomolgus monkeys. Chinese human liver cells were introduced into a perfusion bioreactor to carry out hybrid bioartificial liver support treatment. Forty-eight hours after the injection, one group of cynomolgus monkeys received HBALSS care, and a second experimental group received no treatment. Clinical manifestations of all animals, survival time, liver and kidney functions and serum biochemistry changes were recorded. Simultaneous detection of the number, viability and function of hepatocytes in the hybrid bioartificial liver were also performed.RESULTS: Forty-eight hours after the injection of D-galactosamine, serum biochemistry levels were significantly increased, whereas albumin levels and the Fischer index were significantly reduced compared to baseline (all Ps < 0.05). Of the ten monkeys in the HBALSS treatment group, five survived, with an average duration of survival of 128 ± 3 h. All cynomolgus monkeys in the control group died, with a duration of survival of 112 ± 2 h. Survival time was significantly longer with HBALSS treatment (P < 0.05). Moreover, the number, viability and function of hepatocytes were maintained at a high level with HBALSS.CONCLUSION: The novel hybrid bioartificial liver plays a significant role in liver support by significantly reducing serum biochemistry levels and extending animal survival time.     

Effects of intraperitoneal transplantation of microcarrier-attached hepatocytes on D-galactosamine-induced acute liver failure in rats.

A study was conducted to investigate morphologic as well as metabolic characteristics of microcarrier-attached hepatocytes in culture, and also to evaluate the effect of intraperitoneal transplantation of the microcarrier-attached hepatocytes on acute hepatic failure in rats induced by D-galactosamine (GalN). Rat hepatocytes were isolated by collagenase perfusion, and cultured on collagen-coated microcarriers. Protein synthesis estimated by [14C] leucine incorporation was four-fold higher in microcarrier culture than in cell suspension. The rates of albumin, transthyretin and bile acid syntheses in hepatocytes cultured on microcarriers were similar to those in monolayer culture. When microcarrier-attached hepatocytes were intraperitoneally transplanted into rats with Galn-induced acute liver failure, a marked improvement in survival rate was observed as compared with control rats which received injections of microcarriers alone (80% vs 0% beyond 6 days of transplantation). Mean serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), methionine and glucose levels were similar in both groups, while serum bilirubin and ammonia levels were lower (P less than 0.1, P less than 0.05) in rats transplanted with the microcarrier-attached hepatocytes. Immunohistochemical examinations revealed that the transplanted hepatocytes around microcarriers had albumin synthesis activity, whereas almost no albumin synthesis was demonstrated in recipient liver. In conclusion, intraperitoneal transplantation of the microcarrier-attached hepatocytes will provide sufficient metabolic support, representing detoxication of ammonia (and presumably bilirubin) and synthesis of albumin, to allow GalN-damaged liver function to restore. Microcarrier culture of isolated hepatocytes seems to be one of the most appropriate tools for an artificial liver support.     

Novel D-galactosamine-induced cynomolgus monkey model of acute liver failure

AIM To establish a simplified, reproducible D-galactosamineinduced cynomolgus monkey model of acute liver failure having an appropriate treatment window. METHODS Sixteen cynomolgus monkeys were randomly dividedinto four groups(A, B, C and D) after intracranial pressure(ICP) sensor implantation. D-galactosamine at 0.3, 0.25, 0.20 + 0.05(24 h interval), and 0.20 g/kg body weight, respectively, was injected via the small saphenous vein. Vital signs, ICP, biochemical indices, and inflammatory factors were recorded at 0, 12, 24, 36, 48, 72, 96, and 120 h after D-galactosamine administration. Progression of clinical manifestations, survival times, and results of HE staining, TUNEL, and Masson staining were recorded. RESULTS Cynomolgus monkeys developed different degrees of debilitation, loss of appetite, and jaundice after D-galactosamine administration. Survival times of groups A, B, and C were 56 ± 8.7 h, 95 ± 5.5 h, and 99 ± 2.2 h, respectively, and in group D all monkeys survived the 144-h observation period except for one, which died at 136 h. Blood levels of ALT, AST, CK, LDH, TBi L, Cr, BUN, and ammonia, prothrombin time, ICP, endotoxin, and inflammatory markers [(tumor necrosis factor(TNF)-α, interleukin(IL)-1β, and IL-6)] significantly increased compared with baseline values in different groups(P 0.05). Pathological results showed obvious liver cell necrosis that was positively correlated with the dose of D-galactosamine.CONCLUSION We successfully established a simplified, reproducible D-galactosamine-induced cynomolgus monkey model of acute liver failure, and the single or divided dosage of 0.25 g/kg is optimal for creating this model.     

Protective effect of pretreatment with low-dose lipopolysaccharide on D-galactosamine-induced acute liver failure

BACKGROUND AND AIMS: The effect of low-dose lipopolysaccharide (LPS) induced nitric oxide (NO) on liver damage and survival in rats with acute liver failure caused by a lethal dose of D-galactosamine (D-gal) was studied. RESULTS: Ninety percent of control animals died within 4 days after D-gal injection, but pretreatment with low-dose LPS significantly decreased mortality to 5%. There was marked elevation in serum aspartate aminotransferase and alanine aminotransferase levels 24 h after D-gal injection. These aminotransferases were significantly improved in low-dose LPS pretreated rats 24 h after the administration of D-gal. NG-Nitro-L-arginine-methyl ester, but not NG-nitro-D-arginine-methyl ester, reversed this cytoprotection. CONCLUSION: Pretreatment with low-dose LPS prevents experimental liver failure induced by D-gal through activation of endogenous NO synthesis.     

小剂量内毒素导致非酒精性脂肪性肝炎大鼠急性肝功能衰竭

通过持续24周的高脂饮食建立了大鼠肥胖、高脂血症、脂肪性肝炎大鼠模型，观察了小剂量内毒素处理后模型大鼠肝功能衰竭的发生情况。     

急性肝衰竭大鼠血浆D-乳酸、二胺氧化酶和内毒素的变化及其意义

目的探讨血浆D-乳酸、二胺氧化酶（DAO）和内毒素水平在急性肝衰竭大鼠的变化及其意义。方法选取健康雌性SD大鼠40只,随机分为对照组（15只）和实验组（25只）,采用分光光度法检测血浆中D-乳酸、DAO和内毒素水平,分别用HE染色及电镜观察大鼠回肠的组织形态和超微结构。结果 实验组大鼠血浆D-乳酸、DAO和内毒素水平均明显高于对照组（P〈0.05）;实验组大鼠回肠黏膜明显萎缩,部分绒毛断裂、脱落。结论急性肝衰竭大鼠肠黏膜屏障出现明显障碍。     

Immunohistochemical study of proteoglycans in D-galactosamine-induced acute liver injury in rats

In this study, we carried out an immunohistochemical investigation of time-dependent alterations in the distribution of proteoglycans, and the proliferation profiles of hepatocytes and fat-storing cells (FSCs) in the livers of rats intoxicated withd-galactosamine (GalN). The proliferative cells were analyzed by proliferative cell nuclear antigen (PCNA) staining. In untreated rats, heparan sulfate, dermatan sulfate, and chondroitin/chondroitin sulfate were detected within the portal spaces and the central veins, and, with the exception of chondroitin, also within the reticular fibers. After administration of GalN, the number of PCNA-positive cells (FSCs and hepatocytes) and FSCs increased, reaching maximal on the 2nd and 3rd days, respectively. Heparan sulfate showed complicated changes. Dermatan sulfate decreased in portal spaces from the 2nd to the 3rd day, and in reticular fibers from 12 h to the 6th day. Chondroitin/chondroitin sulfate staining was observed from 2 h to the 6th day in the sinusoidal endothelia, which suggests that the sinusoidal endothelia may produce chondroitin/chondroitin sulfate transiently during liver damage as part of the mechanism of regeneration. Heparan sulfate and chondroitin/chondroitin sulfate were detected in necrotic regions, but dermatan sulfate was not. These observations suggest that heparan sulfate and chondroitin/chondroitin sulfate are involved in cell proliferation or morphogenesis and that the dermatan sulfate plays a role in the differentiation or functional maintenance of cells in liver regeneration.     

急性肝衰竭大鼠肠黏膜屏障功能变化特点分析

目的探讨急性肝衰竭(ALF)大鼠肠黏膜屏障功能变化特点,为ALF的诊治提供新思路。方法将120只SD大鼠随机分为模型组及对照组各60只。模型组腹腔注射硫代乙酰胺复制ALF模型;对照组腹腔注射等量生理盐水。两组分别于制模后24、36、48 h取血检测血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、总胆红素(TBil)、肠脂肪酸结合蛋白(iFABP)、内毒素水平及肠系膜淋巴结和门静脉血细菌移位率,并观察回肠病理变化。结果与对照组比较,模型组制模后24、36、48 h血清ALT、AST、TBil含量均明显升高;24、36 h血清iFABP水平明显升高(P<0.05或P<0.01);Pearson相关性分析显示,模型组血清iFABP水平与肝功能指标ALT、AST均呈显著正相关。早期内毒素水平有明显升高,48 h达到峰值(P<0.01)。肠系膜淋巴结细菌移位在制模24 h后明显升高,48 h达到30%(P=0.013),门静脉血细菌移位48 h达到30%(P=0.013)。模型组回肠病理可见黏膜下充血、水肿、固层中性粒细胞浸润,部分绒毛尖端脱落。结论 ALF早期即可出现肠黏膜屏障功能受损,引发肠道细菌和内毒素移位;iFABP升高可能是ALF早期肠黏膜屏障功能受损的预警指标。     

Ingestion of resistant starch protects endotoxin influx from the intestinal tract and reduces D-galactosamine-induced liver injury in rats

BACKGROUND AND AIMS: The aim of the present study was to examine the protective effect of a dietary high-amylose cornstarch (HAS) against D-galactosamine (D-GalN)-induced liver injury, focusing specifically on intestinal endotoxin translocation. METHODS: Male Wistar rats fed a HAS-free basal diet or a 30% HAS-supplemented diet were injected intraperitoneally with D-GalN. Serum transaminase activities, serum concentrations of tumor necrosis factor (TNF)-alpha, and portal venous endotoxin concentrations were determined at various time points. Ileal mucosal proliferation, small intestinal immunoglobulin (Ig)A and mucin, and the size of the cecal short-chain fatty acids (SCFA) pool were also determined. RESULTS: High-amylose cornstarch ingestion significantly reduced the increase in serum transaminase activities at 22 h after the injection of D-GalN. Rats fed the HAS diet showed a greater cecal SCFA production as measured by pool size than those fed the basal diet. Luminal IgA and mucin content were significantly greater in rats fed the HAS diet. Protein, DNA and RNA contents in the ileal mucosa were also higher in rats fed the 30% HAS diet. In a further experiment, portal venous endotoxin concentrations in rats fed the basal diet reached 72 ng/L at 4 h after D-GalN administration, but endotoxin was not detected in rats fed the HAS diet. At this time, portal endotoxin concentrations were significantly and positively correlated with the serum concentrations of TNF-alpha and serum alanine aminotransferase activities. CONCLUSION: These data support the view that HAS ingestion may reduce D-GalN-induced liver injury as a result of an inhibitory effect on endotoxin influx from the intestinal tract, at least in part as a result of alterations in the mucosal barrier functions.     

急性肝衰竭大鼠肠道菌群和内毒素的动态研究

目的 研究急性肝衰竭大鼠肠道菌群及内毒素的动态变化。 方法 腹腔注射半乳糖胺建立急性肝衰竭大鼠模型。40只SD大鼠随机分为A组(对照组)10只;B组12只,C组18只(均为肝衰竭大鼠)。实验开始时(A组)、造模后24 h(B组)和48 h(C组)分别处死各组大鼠并检测肝功能,定性、定量分析空肠、回肠及结肠菌群,定量测定门静脉、右心室血,回肠及结肠内毒素。 结果 肝功能检测显示:B组大鼠的肝脏损伤最为严重;与B组相比,C组大鼠的肝功能开始好转。肠道菌群分析显示:B组大鼠肠道内肠杆菌科细菌显著增加(空肠、回肠间,P<0.01;结肠间,P<0.05)、乳酸杆菌下降(P<0.01);与B组相比,C组空肠和结肠内的肠杆菌科细菌出现下降(P<0.05)、乳酸杆菌增加,以空肠为显著(P<0.05)。内毒素测定表明B组回肠内毒素增加(P<0.05);C组空肠和回肠内毒素继续增高与对照组差异有显著性(P<0.01);门静脉内内毒素在B组最高,与A、C两组比较差异有显著性(P<0.01)。 结论 急性肝衰竭大鼠肠道存在菌群失调、肠杆菌科细菌过度生长,菌群失调程度与肝损伤程度有关;肠道菌群失调伴有回肠和结肠内内毒素升高;门静脉内毒素的增加与肠道菌群失调有关     

Changes in cerebral oxidative metabolism in patients with acute liver failure

Acute liver failure patients with a persistence of hyperammonemia are at an increased risk of intracranial hypertension due to development of brain oedema. In vitro studies of brain tissue and cell cultures that indicates that exposure to ammonium inhibits enzymatic activity in the tricarboxylic acid cycle, induces substrate depletion through marked glutamate utilization for glutamine synthesis and leads to mitochondrial dysfunction. In patients with acute liver failure cerebral microdialysis studies show a linear correlation between the lactate to pyruvate ratio and the glutamine concentration, as well as to some of the adenosine triphosphate degradation products. However, clinical observations of cerebral exchange rates of oxygen, glucose, lactate and amino acids challenge the interpretation of these findings. In this review the conflicting data of cerebral metabolism during acute liver failure is discussed.     

Intestinal expressions of eNOSmRNA and iNOSmRNA in rats with acute liver failure

AIM To observe the gene expression change ofeNOSmRNA and iNOSmRNA in the small and largeintestines with acute liver failure(ALF),and to reveal thebiological function of NO on the pathogenesis of ALF andmultiple organs dysfunction at the molecular level.METHODS Sixty male Wistar rats were selected,weighing from 250g to 350g,and divided into 5 groupsrandomly:SO,ALF(6h,12h),L-Arg,L-NAME,L-Arg andL-NAME,each group with 10 rats.The dose of L-Arg was300mg.kg~(-1),and L-NAME was 30mg.kg~(-1),the reagentsdiluted by normal saline were injected through tail vein 30minutes pre-and post-operation.The rats in the ALFgroup were respectively sacrificed postoperatively at 6h,12h,and the rats in the other groups were sacrificedpostoperatively at 6h.The tissues of small and largeIntestines were harvested in 4% psraforaldehydecontaining the reagent of DEPC and fixed at 6h,embeddedin paraffin,and 4μm section was cut.The expression ofeNOSmRNA and INOSmRNA in these tissues wasdetermined with in situ hybridization,and analyzed withthe imaging analysis system of CMM-3 and SPSSstatistical software.RESULTS The expression of eNOSmRNA in the largeIntestine and INOSmRNA in the small and large intestinesIncreased significantly at 6h after ALF,but the expressionof iNOSmRNA in the small and large intestines reducednotably at 12h after ALF(P<0.05);the expression ofeNOSmRNA in the large intestine and iNOSmRNA in thesmall and large intestines decreased significantly with thereagents of L-Arg at 6h ALF,but the expression ofeNOSmRNA and iNOSmRNA in the small and largeintestines decreased totally with the reagents of L-NAMEor association with L-Arg 6h ALF.CONCLUSION The expression of eNOSmRNA in the largeintestine increased notably at the early stage of ALF,NOinduced by the enzyme of eNOS from the transplantationof eNOSmRNA can protect the function of the largeintestine,the high expression of iNOSmRNA is involved inthe damaged function of the small and large intestines.NOprecursor can reduce the expression of iNOSmRNA in the small and large intestines and the damage to intestines;NOS inhibitor or association with NO precursor can totallylower the expression of eNOSmRNA and iNOSmRNA in thesmall and large intestines,it cannot notably influence theNOS inhibitor in the gene expression of eNOSmRNA andiNOSmRNA to supply the additional NO precursor.     

Protective action of glutamine in rats with severe acute liver failure

BACKGROUND Severe acute liver failure(SALF) is a rare, but high-mortality, rapidly evolving syndrome that leads to hepatocyte degeneration with impaired liver function.Thioacetamide(TAA) is a known xenobiotic, which promotes the increase of the formation of reactive oxygen species. Erythroid 2-related factor 2(Nrf2) activates the antioxidant protection of cells. Studies have evidenced the involvement of inflammatory mediators in conditions of oxidative stress.AIM To evaluate the antioxidant effects of glutamine on Nrf2 activation and NFκBmediated inflammation in rats with TAA-induced IHAG.METHODS Male Wistar rats(n = 28) were divided into four groups: control,control+glutamine, TAA, and TAA + glutamine. Two TAA doses(400 mg/kg)were administered intraperitoneally, 8 h apart. Glutamine(25 mg/kg) was administered at 30 min, 24 h, and 36 h. At 48 h, blood was collected for liver integrity analysis [aspartate aminotransferase(AST), alanine aminotransferase(ALT), and alkaline phosphatase(ALP)]. The liver was harvested for histology and assessment of oxidative stress [thiobarbituric acid-reactive substances(TBARS), catalase(CAT), glutathione peroxidase(GPx), glutathione S-transferase(GST), glutathione(GSH), Nrf2, Kelch-like ECH-associated protein 1(Keap1),NADPH quinone oxidoreductase1(NQO1), superoxide dismutase(SOD)] and inflammatory process.RESULTS TAA caused disruption of the hepatic parenchyma, with inflammatoryinfiltration, massive necrosis, and ballooning degeneration. Glutamine mitigated this tissue damage, with visible regeneration of hepatic parenchyma; decreased TBARS(P 0.001), GSH(P 0.01), IL-1β, IL6, and TNFα levels(P 0.01) in hepatic tissue; and decreased blood levels of AST, ALT, and ALP(P 0.05). In addition, CAT, GPx, and GST activities were restored in the glutamine group(P0.01, P 0.01, and P 0.001, respectively vs TAA alone). Glutamine increased expression of Nrf2(P 0.05), NQO1, and SOD(P 0.01), as well as levels of IL-10(P 0.001), while decreasing expression of Keap1, TLR4, NFκB(P 0.001), COX-2 and iNOS,(P 0.01), and reducing NO_2 and NO_3 levels(P 0.05).CONCLUSION In the TAA experimental model of IHAG, glutamine activated the Nrf2 pathway,thus promoting antioxidant protection, and blunted the NFκB-mediated pathway, reducing inflammation.     

Changes of neurotensin and endotoxin in rats with intestinal ischemia

AIM: To investigate the changes of neurotensin (NT) and endotoxin in rats with segmental intestinal ischemia. METHODS: The distal ileal mesenteric arteries in rats were ligated to make segmental intestinal ischemia models. At the 2^nd, 6^th and 12^th hours after intestinal ischemia, endotoxin levels in portal blood were tested by limulus lysate test and NT levels in plasma from the heart and in intestine tissues (ischemia and peri-ischemia areas) were assayed by radioimmunoassay. Histological changes of the mucosa were examined under light and electron microscopes. RESULTS: NT levels decreased significantly in intestinal ischemia and peri-ischemia areas (34.07 ± 5.93 vs 40.14 ± 5.38, P < 0.05; 7.47 ± 1.38 vs 40.14 ± 5.38, P < 0.01), especially lower in ischemia area (34.07 ± 5.93 vs 7.47 ± 1.38, P < 0.05. However, NT level increased obviously in plasma (0.76 ± 0.16 vs 0.47 ± 0.10, P < 0.05). Levels of endotoxin elevated obviously in portal blood (389.0 ± 105.0 vs 55.1 ± 6.7, P < 0.01), and the mucosa was injured both in ischemia and peri-ischemia areas. CONCLUSION: Intestinal ischemia injures intestinal mucosa and leads to decrease of intestinal NT level, which is accelerated by endotoxemia and increase of blood NT level.     

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.     

急性肝功能衰竭大鼠肝组织内巨噬细胞形态变化特点

目的探讨肝组织内巨噬细胞在急性肝功能衰竭（acuteliverfailure,ALF）大鼠模型中的形态变化及其在肝脏炎性损伤中的意义。方法Wistar大鼠分为两组：实验对照组6只,模型组24只。通过腹腔注射D-氨基半乳糖（D-GalN）及脂多糖（LPS）建立大鼠急性肝功能衰竭模型,动态观察各时间点（4、8、16、24h）大鼠肝组织的病理变化以及ALT、AST、TBil和TNF-a的含量变化,通过透射电镜观察各时间点大鼠肝组织中巨噬细胞的形态变化。结果D-GalN／LPS联合注射成功诱导大鼠急性肝功能衰竭模型,表现为建立模型后ALT、AST及TBil水平显著上升,于造模后24h表达最高（P〈O．01）,病理变化符合急性肝功能衰竭的病理特点。以细胞体积增大、形状不规则、胞质伸出较多微绒毛和伪足者为激活的巨噬细胞判断标准。建立模型后,在同样放大倍数下,肝组织中巨噬细胞形态发生变化,以8h模型组的大鼠肝组织中巨噬细胞的形态变化最为明显,随着炎性反应的进一步发展,其形态变化反而减弱。与此同时,TNF-a建立模型后其含量逐渐升高,以8h模型组的大鼠血清内含量最高,此后逐渐下降。结论肝组织中巨噬细胞参与急性肝功能衰竭的病理生理过程,其在急性肝功能衰竭的初始阶段及促进炎性反应发展中发挥了重要的作用。