Disruption of mitochondrial calcium homeostasis after chronic alpha-naphthylisothiocyanate administration: relevance for cholestasis.

BACKGROUND: Hepatocyte dysfunction caused by impaired mitochondrial function has been pointed out as a probable leading cause of cholestatic liver injury. The aim of this study was to evaluate liver mitochondrial bioenergetics that followed repeated in vivo administration of alpha-naphthylisothiocyanate, a known cholestatic agent. METHODS: Serum markers of liver injury and endogenous adenine nucleotides were measured in alpha-naphthylisothiocyanate-treated rats (intraperitoneally, 100 mg/Kg/wk x 6 wk). Changes in membrane potential, mitochondrial respiration, as well as alterations in mitochondrial calcium homeostasis were monitored. RESULTS: In rats injected with alpha-naphthylisothiocyanate, liver injury with cholestasis developed within 48 hours, as indicated by both serum enzyme activities and total bilirubin concentration. However, 1 week after the last injection, serum enzyme activity returned to control levels. In addition, after chronic alpha-naphthylisothiocyanate administration, no alterations in mitochondrial respiratory function and membrane potential were observed. Associated with these parameters, mitochondria from treated animals exhibited increased susceptibility to disruption of mitochondrial calcium homeostasis by calcium phosphate and by bile acids, which was probably caused by induction of permeability transition pore. CONCLUSIONS: Our data suggest that chronic cholestasis in rats leads to impaired mitochondrial function due to the disruption of mitochondrial calcium homeostasis. The initiating event is the induction of a cyclosporine A-sensitive release of calcium. This event may be an important determinant of the progression of cholestatic liver injury and associated liver cirrhosis. In addition, in the present study we observed that impairment of mitochondrial function is potentiated by chenodeoxycholate, a bile acid that is known to be toxic. Ursodeoxycholate (the beta- epimer of chenodeoxycholate) is approved for the treatment of chronic cholestatic liver disease. Interestingly, we show that the susceptibility to the cyclosporine A-sensitive release of calcium was increased by the combination of both bile acids. These results indicate that the reported improvement of biochemical parameters in cholestatic patients treated with ursodeoxycholate would not prevent the associated mitochondrial dysfunction. This may explain the progression of the histological stage and the maintenance of symptoms during cholestasis.     

An antibody to neutrophils attenuates alpha-naphthylisothiocyanate-induced liver injury.

alpha-Naphthylisothiocyanate (ANIT) causes cholestasis and injury to bile duct epithelium and hepatic parenchymal cells in rats. The mechanism of toxicity is unknown. Neutrophils (PMNs) infiltrate periportal regions of the liver after ANIT intoxication. Because PMNs play a causal role in other extrahepatic models of tissue injury, we determined whether PMNs might be involved in ANIT-induced liver injury in rats by reducing circulating PMN numbers with a polyclonal antibody (antineutrophil serum). ANIT treatment caused cholestasis and elevations in serum of total bilirubin concentration, total bile acid concentration, aspartate amino-transferase activity, gamma-glutamyltransferase activity and histologic lesions consistent with acute, neutrophilic cholangiohepatitis. Cotreatment of rats with antineutrophil serum reduced circulating PMN numbers, prevented ANIT-induced cholestasis and attenuated other markers of liver injury elevated by ANIT. In addition, antineutrophil serum treatment attenuated the severity of histologic lesions within the liver and reduced the number of PMNs in periportal regions. Numbers of PMNs in liver sections correlated positively with markers of liver injury, histologic evidence of cholangiohepatitis and numbers of circulating PMNs in peripheral blood. The protection afforded by antineutrophil serum appeared to result from a specific reduction of PMNs and not lymphocytes, because administration of an antilymphocyte serum reduced circulating lymphocyte numbers without offering protection. Inasmuch as ANIT stimulates PMNs in vitro to release O2- and since PMN-derived oxygen species may cause tissue injury, we determined whether administration of agents which degrade oxygen radicals afforded protection against the liver injury caused by ANIT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma alkaline phosphodiesterase I in intrahepatic cholestasis induced by alpha-naphthylisothiocyanate in rats

1. Administration of alpha-naphthylisothiocyanate (ANIT) to rats produced dose-dependent increases in plasma bile acid and bilirubin concentrations. Similar increases in plasma bile acid and bilirubin concentrations were evident in bile duct ligated rats, indicating that the severity of cholestasis is almost identical in both models. 2. Plasma alkaline phosphodiesterase I was increased by only 50-80% while alkaline phosphatase was increased more than threefold after ANIT administration. This is in contrast to an earlier study [S. R. Simpson, K. Rahman & D. Billington (1984) Clinical Science 67, 647-652] where, after bile duct ligation, serum alkaline phosphodiesterase I was elevated sixfold before any increase in alkaline phosphatase activity became apparent. Thus, plasma alkaline phosphodiesterase I does not offer as sensitive a marker of intrahepatic cholestasis (induced by ANIT) as it does of extrahepatic cholestasis (induced by bile duct ligation). 3. Hepatic alkaline phosphodiesterase I was unaffected by ANIT pretreatment while hepatic alkaline phosphatase was increased up to seven times. It is suggested that raised plasma alkaline phosphodiesterase I is due to regurgitation of the biliary enzyme rather than overspill of the enzyme from liver into blood. 4. Gel filtration showed that 24 h and 96 h after ANIT administration, rat serum contained a high molecular weight form of alkaline phosphodiesterase I, suggesting a different isoenzyme profile.     

The Effect of Prolonged Administration of Ethanol on Cardiac Metabolism and Performance in the Dog

The effect of prolonged administration of alcohol on mitochondrial function and high-energy phosphate (ATP) of heart muscle was investigated in dogs. Animals were divided into two groups, a control group and a group that received alcohol. In the experimental series, dogs received 400 ml of a 25% solution of alcohol added to the food and drinking water. Measurements were carried out after ethanol had been withheld for 2 days. Total myocardial blood flow, cardiac output, and myocardial O₂ consumption remained at control levels. Measurement of cardiac contractility using the maximal rate of left ventricular pressure rise (dP/dt_max) showed no change in animals exposed to alcohol. When the afterload of the heart was increased with angiotensin, a slight but not significant decline in cardiac contractility was observed. Activities of various intramitochondrial and extramitochondrial enzymes were measured in both groups. After alcohol administration, the primarily intramitochondrial isocitrate dehydrogenase diminished. ATP in heart muscle of dogs exposed to alcohol declined, and mitochondrial oxygen consumption and respiratory control indices diminished. These observations suggest that the primary lesion leading to alteration of myocardial performance is a biochemical malfunction of the mitochondria, which at this early stage is not reflected in changes in myocardial contractility.     

Saikokeishito Extract Exerts a Therapeutic Effect on alpha-Naphthylisothiocyanate-Induced Liver Injury in Rats through Attenuation of Enhanced Neutrophil Infiltration and Oxidative Stress in the Liver Tissue

We examined whether Saikokeishito extract (TJ-10), a traditional Japanese herbal medicine, exerts a therapeutic effect on alpha-naphthylisothiocyanate (ANIT)-induced liver injury in rats through attenuation of enhanced neutrophil infiltration and oxidative stress in the liver tissue. In rats treated once with ANIT (75 mg/kg, i.p.), liver injury with cholestasis occurred 24 h after treatment and progressed at 48 h. When ANIT-treated rats orally received TJ-10 (0.26, 1.3 or 2.6 g/kg) at 24 h after the treatment, progressive liver injury with cholestasis was significantly attenuated at 48 h after the treatment at the dose of 1.3 or 2.6 g/kg. At 24 h after ANIT treatment, increases in hepatic lipid peroxide and reduced glutathione contents and myeloperoxidase activity occurred with decreases in hepatic superoxide dismutase and glutathione reductase activities. At 48 h after ANIT treatment, these changes except for reduced glutathione were enhanced with decreases in catalase, Se-glutathione peroxidase, and glucose-6-phosphate dehydrogenase activities. TJ-10 (1.3 or 2.6 g/kg) post-administered to ANIT-treated rats attenuated these changes found at 48 h after the treatment significantly. These results indicate that TJ-10 exerts a therapeutic effect on ANIT-induced liver injury in rats possibly through attenuation of enhanced neutrophil infiltration and oxidative stress in the liver tissue.     

清肝灵煎剂对大鼠急性肝损伤的保护作用

目的:观察清肝灵煎剂(清肝灵)对α-萘异硫氰酸酯(Alpha-naphthylisothiocyanate,ANIT)所致急性肝损伤的保护作用。方法:SD大鼠48只,随机分为6组:正常对照组、ANIT模型组、茵栀黄注射液(菌栀黄)治疗组、清肝灵预保护组、清肝灵低剂量组以及清肝灵高剂量组。除正常对照组外,其它5组均用ANIT 100mg·kg~1体重灌胃造成急性肝损伤模型。除清肝灵预保护组于ANIT攻毒前3d起作预保护给药外,所有实验组应用ANIT后分别给予相应药物治疗。观察各组大鼠一般状况的变化并采用全自动生化分析仪检测各组动物ANIT中毒后24、48、72h的血清ALT、AST和TBil等肝功能指标。结果:经ANIT灌胃后各实验组大鼠的ALT、AST、TBil的含量均较正常组明显升高(P<0.01)。与模型组比较,在ANIT攻毒后24、48、72h茵栀黄治疗组和各清肝灵治疗组的ALT、AST、TBil水平均明显降低(P<0.01);各清肝灵治疗组的上述指标明显优于茵栀黄治疗组(P<0.01或P<0.05)且以预保护组为最佳。结论:清肝灵能明显减轻ANIT所致的大鼠急性肝损伤,其保肝降酶退黄效果明显优于茵栀黄注射液。     

ANIT诱导的肝内胆汁淤积大鼠肝胆系统AQP1的表达

[目的]探讨水通道蛋白1（AQP1）在α-萘异硫氰酸（ANIT）诱导的肝内胆汁淤积大鼠肝胆系统的表达情况.[方法]将健康雄性Wistar成年大鼠16只随机分为ANIT组和对照组, 每组8只.ANIT组由10 g/L ANIT花生油溶液一次性灌胃诱导肝损伤.对照组仅灌花生油.检测两组AQP1的表达.平均光密度（OD）值.[结果]两组大鼠肝胆系统均有AQP1表达.对照组OD值为（0.38±0.08）明显高于ANIT组（0.10±0.03）（P〈0.01）.[结论]ANIT诱导的胆汁淤积大鼠肝胆系AQP1的蛋白表达下调,提示AQP1参与胆汁淤积的病理机制.     

Protective effects of ranolazine and propranolol,alone or combined,on the structural and functional alterations of cardiomyocyte mitochondria in a pig model of ischemia/reperfusion

Preventing the consequences of ischemia/reperfusion (I/R)‐induced lesions in the clinic requires the administration of pharmacological agents prior to restoring coronary vascularization. The aim of this study was to evaluate the effects of ranolazine and propranolol when administered either alone or combined prior to I/R induction in a pig model. Thirty domestic pigs were randomly assigned to five groups of six animals including (i) sham animals; (ii) untreated animals with 45‐min ischemia and 1‐min reperfusion; animals administered intravenously with (iii) ranolazine, or (iv) propranolol, or (v) both combined, prior to 45‐min ischemia and 1‐min reperfusion. The heart rate (HR), duration of monophasic action potentials (dMAP), and peak of the time derivative of left ventricular pressure (LV dP/dt max) were measured during ischemia and after 1 min of reperfusion. Structural and functional parameters of mitochondria were analyzed in tissue samples taken from the left ventricle ischemic area at the end of the experiment. I/R induced expected effects, namely accelerated HR, decreased dMAP and LV dP/dt max, and altered mitochondrial structural and functional parameters including decreased oxygen consumption, increased reactive oxygen species (ROS) production, and reduced calcium retention capacity resulting in the opening of mitochondrial permeability transition pores (mPTP). Ranolazine and propranolol administered either alone or combined prior to I/R significantly decreased all of these deleterious consequences. The protective effects of ranolazine and propranolol are seemingly due to the prevention of calcium overload and resulting lesions in mitochondria.     

Sickle cell disease,extreme hyperbilirubinemia,and pericardial tamponade: case report and review of the literature

OBJECTIVE: Sickle cell disease is a relatively common disease seen predominantly in the African-American population with numerous important sequelae that require critical care management. We report a patient who presented with intrahepatic cholestasis, a rare and potentially lethal complication of sickle cell disease. DESIGN: Individual case report and review of the literature. SETTING: Medical intensive care unit of a tertiary care hospital. PATIENT: A 37-yr-old African-American male, with known sickle cell disease, who developed fulminant hepatic failure with subsequent extreme hyperbilirubinemia, coagulopathy, and pericardial tamponade. Additional organ dysfunction included renal insufficiency, respiratory failure, and cardiac dysrhythmias. INTERVENTIONS: The patient underwent serial measurements of complete blood count, hepatic profile, coagulation profiles, and hemoglobin electrophoresis. The patient received exchange transfusion, and his hemoglobin S concentration gradually decreased. Coagulopathy and anemia were corrected with transfusion of fresh frozen plasma and packed red blood cells. Serum bilirubin and other hepatic variables gradually improved. Pericardial tamponade was suggested by right heart catheterization measurements and diagnosed by echocardiographic findings. The tamponade resolved after pericardiocentesis and was managed by drainage through the pericardial catheter. Pulmonary, renal, and cardiac sequelae resolved with intensive supportive care that included intubation, mechanical ventilation, pulmonary artery catheterization, continuous renal replacement therapy, and permanent cardiac pacemaker. The patient was discharged home on hospital day 23 and has not required further hospitalization. CONCLUSION: Sickle cell intrahepatic cholestasis, a potentially fatal complication of sickle cell disease, can present with abdominal pain, acute hepatomegaly, coagulopathy, hyperbilirubinemia, and fulminant hepatic failure. Prompt recognition and early intervention with exchange transfusion and intensive supportive care of multiple organ dysfunction can result in a favorable outcome.     

Heat shock pretreatment prevents cardiac mitochondrial dysfunction during sepsis

The present study was designed to investigate the effect of previous heat shock treatment on the mitochondria function of the heart during a cecal ligation and puncture (CLP)-induced sepsis model. Rats of the heated group were heated by whole-body hyperthermia 24 h before the CLP operation. Cardiac mitochondria were freshly collected 9 and 18 h after CLP, indicating early and late sepsis, respectively. The expressions of heat shock protein 72 (Hsp72), glucose-regulated protein 75 (Grp75), and mitochondrial complexes I, II, III, and IV were evaluated by Western blot and immunochemical analysis. Enzyme activities of NADH cytochrome c reductase (NCCR), succinate cytochrome c reductase (SCCR), and cytochrome c oxidase (CCO) were measured after the reduction or oxidation of cytochrome c using a spectrophotometer. The results showed that the ATP content in the heart significantly declined during late sepsis, whereas heat shock treatment reversed this declination. The enzyme activities of NCCR, SCCR, and CCO were apparently suppressed during late stage of sepsis. The protein expressions of mitochondrial complex II and complex IV and Grp75 were also down-regulated during sepsis. Previously treated by heat shock, late-sepsis rats emerged with a high preservation of mitochondrial respiratory chain enzymes, both the protein amount and enzyme activity. Aspects of morphology were observed by electron microscopy, while heat shock treatment revealed the attenuation of cardiac mitochondrial damage induced by sepsis. In conclusion, structural deformity and the decrease of respiratory chain enzyme activity in mitochondria and its leading to a decline of ATP content are highly correlated with the deterioration of cardiac function during sepsis, and heat shock can reverse adverse effects, thus achieving a protective goal.     

The evaluation of altered redox status in plasma and mitochondria of acute and chronic diabetic rats

OBJECTIVES: An increase in plasma oxidative stress and decreased mitochondrial lipid hydroperoxides may contribute to the imbalance in the redox status between intramitochondrial and extramitochondrial milieu in chronic experimental diabetic rats. DESIGN AND METHODS: To determine the effect of hyperglycemia in promoting redox imbalance, we determined lipid hydroperoxides (LHP), protein carbonyl (PCO), total antioxidant activity (ferric reducing/antioxidant power; FRAP) and albumin as markers of redox status of plasma, and mitochondrial lipid hydroperoxide levels as a marker of lipid peroxidation in liver, pancreas and kidney tissue of acute and chronic diabetic male Sprague-Dawley rats and their controls. The levels of the studied markers were determined by colorimetric methods. RESULTS: Plasma and mitochondrial oxidative stress parameter levels of acute diabetic rats were not significantly different from their controls. Plasma LHP and PCO levels of chronic diabetic rats were increased significantly as compared to those of both acute diabetic rats and the controls. Plasma FRAP levels of chronic diabetic animals were decreased significantly as compared to those of the controls. On the other hand, LHP levels in liver, pancreas and kidney mitochondria of chronic diabetic rats were decreased significantly as compared to those of both acute diabetic rats and the controls. We observed a negative correlation between LHP levels in liver mitochondria of chronic diabetic rats, and PCO and fructosamine levels in plasma of chronic diabetic rats were correlated. LHP levels in the pancreatic mitochondria of chronic diabetic rats and plasma oxidative stress parameters of chronic diabetic rats were not significantly correlated. LHP levels in kidney mitochondria of chronic diabetic rats were significantly correlated with serum albumin. There was no correlation between LHP levels in kidney mitochondria and other plasma oxidative stress parameters in chronic diabetic rats. CONCLUSIONS: Our data suggest that redox imbalance between plasma and liver mitochondria might become a major threat to chronic diabetic rats.     

Defective membrane systems in dystrophic skeletal muscle of the UM-X7.1 strain of genetically myopathic hamster.

1. The function of mitochondria, sarcotubular membranes (heavy microsomes), sarcolemma and myofibrils from the hind-leg skeletal muscle of about 60- and 150-day-old normal and myopathic (UM-X7.1) hamsters was examined. 2. The mitochondrial calcium uptake as well as mitochondrial phosphorylation and respiratory rates were lower in 60-day-old myopathic skeletal muscle, unlike 150-day-old myopathic animals, when pyruvate-malate and glutamate-malate were used as substrates. However, mitochondria from 150-day-old myopathic animals showed depressed glutamate-dependent respiratory and phosphorylation rates and succinate-supported initial rate of calcium uptake. 3. The microsomal calcium-uptake, but not calcium-binding, and Ca2+-stimulated adenosine triphosphatase (ATPase) activity of the 150-day-old myopathic skeletal muscle were lower than the control values. Although microsomal calcium-binding, calcium-uptake and ATPase activities of the 60-day-old myopathic muscle were not depressed significantly, the initial rate of calcium uptake was less than the control. 4. The sarcolemmal Ca2+-ATPase, but not Mg2+-ATPase or Na+ +K+-ATPase, activity was higher in 60-day-old myopathic muscle whereas the activities of all these enzymes from 150-day-old myopathic animals were higher than the control. On the other hand, the Na+ +K+-ATPase activities from 60- and 150-day-old myopathic animals were inhibited by ouabain to a lesser extent in comparison with the respective control values. 5. The myofibrillar Ca2+-ATPase and Mg2+-ATPase activities as well as inhibition of Mg2+-ATPase due to Na+ and K+ in myopathic muscle were no different from the control values. 6. The results reported here give further support to the view that different membrane systems of the dystrophic muscle are defective.     

有氧运动对心肌梗死大鼠心肌能量代谢及线粒体呼吸功能的影响

目的 观察有氧运动对心肌梗死致慢性心力衰竭大鼠心脏能量代谢及线粒体呼吸功能的影响。方法 采用随机数字表法将45只SD大鼠分为假手术组、心衰对照组及心衰运动组。采用冠状动脉结扎术将心衰对照组及心衰运动组大鼠制成心肌梗死模型,术后4周时心衰运动组大鼠给予为期8周的跑台有氧运动。于运动干预结束后采用超声心动图检测各组大鼠心功能,采用递增负荷跑台实验测定大鼠运动能力,采用磁共振波谱法测定大鼠心肌磷酸肌酸（PCr）及三磷酸腺苷（ATP）含量,采用细胞呼吸测量仪评估大鼠心肌线粒体呼吸功能。结果 心衰对照组PCr含量、PCr/ATP比值、线粒体呼吸链复合体Ⅰ和Ⅱ的耗氧量、左心室缩短分数（FS）、射血分数（EF）以及递增负荷实验最高跑速、力竭距离和力竭时间等均不及假手术组水平（P<0.05）;心衰运动组ATP含量、复合体Ⅰ耗氧量、左心室FS和EF、递增负荷实验最高跑速、力竭距离和力竭时间均显著优于心衰对照组水平（P<0.05）,PCr/ATP比值组间差异无统计学意义（P>0.05）。结论 规律有氧运动能改善慢性心力衰竭大鼠心脏做功能力,表现为心功能及运动能力增强,其作用机制可能与上调心肌ATP水平及改善线粒体复合体Ⅰ功能有关;另外PCr/ATP比值可能不适合作为评估运动训练对心脏有益影响的生物标志物。     

Drug-induced hepatitis

AIM: To improve diagnosis and treatment of drug-induced hepatitis: to specify drugs with hepatotoxic effect, clinical variants of drug-induced hepatic diseases, management of drug-induced hepatitis (DIH). MATERIALS AND METHODS: Of 2300 examinees with hepatic lesions, 62 were diagnosed to have DIH. Measurements were made of serum levels of bilirubin and its fractions, total protein, some protein fractions, markers of hepatitis A, B, C, activity of aminotransferases (AlAT and AsAT), alkaline phosphatase (AP), glutamate dehydrogenase (GlDG), sorbitol dehydrogenase (SDG), gamma-glutamate transpeptidase (GGTP), choline esterase (CE). Timolveronal test was made. On demand, tests were made for CEA, AFP; device examinations were made: ultrasound investigation, computed tomography of the abdominal organs, endoscopic retrograde pancreatography, laparoscopy with hepatic biopsy, transcutaneous hepatic biopsy, laparotomy with cholecystocholangiography. RESULTS: A direct relationship of hepatitis with drugs intake was revealed in all the patients. Development of drug-induced intrahepatic cholestasis in patients on long-term treatment with anticancer drugs was accompanied with affection of cellular organellas and progressive destruction of small interlobular ducts provoking intrahepatic cholestasis and depletion of natural glutathione. CONCLUSION: Correction of intrahepatic cholestasis and deficiency of endogenic glutathione is successfully carried out with heptral.     

Biochemical and morphometric properties of mitochondrial populations in human muscle fibres

Two mitochondrial subpopulations were evaluated with biochemical and morphological techniques in human gastrocnemius muscle of 10 patients with peripheral arterial insufficiency and 12 control individuals. The subsarcolemmal mitochondria were released by gentle homogenization, with a recovery of 32-37%, and the intermyofibrillar by enzymic digestion and further mechanical disintegration, recovery 18-21%. The subsarcolemmal mitochondria were morphologically defined as those located within 2 micron from the sarcolemma membrane and the intermyofibrillar mitochondria as those located in the rest of the fibre. In the controls the intermyofibrillar mitochondria had a lower respiratory ratio than the subsarcolemmal, owing to a higher state II respiration. The subsarcolemmal space, which contained 25% of the mitochondria, had a mitochondrial volume density two- to three-fold that of the intermyofibrillar space in the controls. The patients, who had a 48-64% higher oxidative enzyme capacity in their muscle tissue, had higher respiratory rate and respiratory control index with similar ADP/O ratio in the subsarcolemmal fraction in comparison with the controls. The citrate synthase activity was higher in both mitochondrial fractions of the patients. The volume densities of mitochondria, total as well as for both subpopulations, were also higher in the patients, which was further reflected in higher yields of mitochondrial protein. The results demonstrate that both subpopulations of muscle mitochondria are able to adapt quantitatively and/or qualitatively. Furthermore, they show that the increased oxidative enzyme capacity of the patients is associated with an increased quantity of both mitochondrial populations and a qualitative improvement of the respiratory activity of the subsarcolemmal mitochondria.     

全脑缺血再灌注后脑线粒体功能变化及山莨菪碱的保护作用

目的：探讨全脑缺血再灌注后脑线粒体功能变化及山莨菪碱的保护作用。方法：采用家兔全脑缺血再灌注损伤模型，缺血20min、再灌注2h观察脑线粒体呼吸功能、呼吸链氧化酶活性、线粒体内Ca^2 和MDA含量的变化。结果:脑缺血再灌注后，脑线粒体呼吸控制率、磷氧比、氧化磷酸化效率及烟酰胺腺喋呤二核苷酸氧化酶、琥珀酸氧化酶、细胞色素C氧化酶活性明显降低(P＜0、01)，而线粒体Ca^2 、MDA含量明显升高(P＜0．01)；再灌注早期给予山莨菪碱治疗后，上述线粒体损伤性改变明显减轻。结论：山莨菪碱对脑缺血再灌注后线粒体损伤具有一定的保护作用，其机制可能与Ca^2 拮抗、抑制脂质过氧化及保护呼吸链酶活性有关。     

Effects of chronic nitric oxide activation or inhibition on early hepatic fibrosis in rats with bile duct ligation

Hepatic fibrosis or increased liver collagen contents drive functional abnormalities that, when extensive, may be life threatening. The purpose of this study was to assess the effects of the chronic stimulation or inhibition of nitric oxide synthesis in rats with hepatic fibrosis induced by permanent common bile duct ligation (3 weeks) and the role of expression of the different nitric oxide synthase isoforms. Bile duct ligation led to an important accumulation of collagen in the hepatic parenchyma, as shown both histologically and by the hydroxyproline contents of livers. Bilirubin and serum enzyme activities (measured as markers of cholestasis) increased several-fold after bile duct ligation. The area of fibrotic tissue, liver hydroxyproline content and serum markers of cholestasis were clearly related in obstructed rats. The absence of modifications in haemodynamic parameters excludes circulatory changes from being responsible for the development of liver alterations. In animals treated with NG-nitro-L-arginine methyl ester (L-NAME) the area of fibrosis was similar to that of untreated animals, the signs of cholestasis and cellular injury being more evident. In rats treated with L-arginine the area of fibrosis was almost three times larger than that found in bile duct ligated rats and in L-NAME-treated bile duct ligated rats, although the observed biochemical changes were similar to those seen in rats treated with L-NAME. Our results with inducible nitric oxide synthase, obtained by Western blots and immunohistochemistry, indicate a greater expression of the inducible enzyme in bile duct ligated and L-arginine-treated animals and a lower expression in the L-NAME and control groups. Constitutive nitric oxide synthase expression, obtained by Western blots, was very similar in all groups, except for the L-arginine-treated rats in which it was lower. These results suggest that nitric oxide production may be a key factor in the development of fibrosis in bile duct ligated rats. They also support the hypothesis of a dual role for nitric oxide; one beneficial, mediated by its circulatory effects, and the second negative, through its local toxic effects.     

“717复方”制剂对内毒素损伤大鼠肝细胞线粒体的保护作用

目的：观察“７１７复方”制剂对内毒素休克大鼠肝细胞线粒体的保护作用。方法：采用静脉注射内毒素制备大鼠内毒素休克模型，观察“７１７复方”对休克大鼠肝细胞线粒体功能的影响。结果：模型组大鼠肝细胞线粒体呼吸控制率（ＲＣＲ）明显下降，与正常对照组比较（４．５８±０．３１比５．７３±０．３５）有显著性差异（Ｐ＜０．０５）；电镜下，内毒素休克模型线粒体明显肿胀，嵴减少、模糊不清。“７１７复方”制剂组大鼠肝细胞线粒体ＲＣＲ（５．６８±０．４１）则接近正常，与模型组比较有极显著性差异（Ｐ＜０．０１）；其电镜下超微结构病变也较轻。体外实验测定肝细胞线粒体膜通透性（膜吸光度下降百分比），“７１７复方”制剂组明显低于内毒素组（Ｐ＜０．０１）。结论：“７１７复方”制剂具有拮抗内毒素损伤线粒体的作用     

The bile acid taurocholate impairs rat cardiomyocyte function: a proposed mechanism for intra-uterine fetal death in obstetric cholestasis

Obstetric cholestasis is a liver disease of pregnancy that can be complicated by sudden, hitherto unexplained, intra-uterine fetal death. Because intra-uterine death occurs suddenly, and because fetal heart rate abnormalities have been reported in obstetric cholestasis, we hypothesized that intra-uterine death is caused by impaired fetal cardiomyocyte function, resulting in fetal cardiac arrest. Obstetric cholestasis is associated with raised levels of maternal and fetal serum bile acids, and we propose that these may alter cardiomyocyte function. It was not possible to investigate the effects of bile acids on the intact human fetal heart at a cellular level. Therefore we used the closest available model of fetal myocardium at term: a primary culture of neonatal rat cardiomyocytes in which cells beat synchronously and develop pacemaker activity. The effect of the primary bile acid taurocholate (0.3 mM and 3 mM) on cultures of single cardiomyocytes, each with its own independent rate of contraction, was a reversible decrease in the rate of contraction and in the proportion of beating cells (P < 0.001). Addition of taurocholate to a network of synchronously beating cells caused a similar decrease in the rate of contraction. Furthermore, the integrity of the network was destroyed, and cells ceased to beat synchronously. Taurocholate also resulted in altered calcium dynamics and loss of synchronous beating. These data suggest that raised levels of the bile acid taurocholate in the fetal serum in obstetric cholestasis may result in the development of a fetal dysrhythmia and in sudden intra-uterine death.     

Cardiac troponins and creatine kinase content of striated muscle in common laboratory animals

Animal models are important for the investigation of human heart pathology, novel treatments, and medical or surgical interventions for disease. Serum markers of myocardial damage may also be important tools within this field of research. In order to assess the cardiac specificity of widely utilised serum markers, we measured the cardiac troponins and creatine kinase (CK) isoenzymes in cardiac and skeletal muscle samples taken from dog, monkey, pig and rat. These samples were also analysed by immunoblotting for cardiac troponin I (cTnI) and cardiac troponin T (cTnT). The content of cTnI and cTnT in skeletal muscle was below 0.6% of that found in heart for all animal species studied. This low immunoreactivity in skeletal muscle was confirmed by immunoblot analysis. The content of CK was higher in skeletal muscle than in heart muscle for all species. The CK-MB/total CK ratio was lower in skeletal muscle than in cardiac muscle for all species. The differences in CK-MB content of skeletal muscle and heart muscle were much less pronounced than the tissue differences in the amounts of the cardiac troponins. The cardiac troponins are potentially useful serum markers of myocardial damage, with high specificity for myocardial muscle in these common laboratory animals. Creatine kinase-MB is much less cardiac-specific.