Circadian Rhythm of Liver Parameters (Cellular Structures, Mitotic Activity, Glycogen and Lipids in Liver and Serum) During Three Consecutive Cycles in Phenobarbital-Treated Rats

The circadian rhythm of gastric content, serum alkaline phosphatase (alk.P.), serum lipids, body weight (wt), relative (rel.) liver wt, cellular structures (by light- and electron-microscopy), mitotic activity of hepatocytes, glycogen content, protein and lipids in liver was studied in 180 male Sprague-Dawley rats orally treated at 0830-1030 with 50 mg/kg phenobarbital (PB) for 7 days. Thereafter, five PB-treated males and five controls each were studied at 4-hr intervals at 0600, 1000, 1400, 1800, 2200 and 0200 on 3 consecutive days. The lighting schedule in the colony was 12:12 = light/dark (light from 0600 to 1800). Following the rhythm of gastric emptying, the rel. liver wt showed a clear circadian rhythm with a peak at 0800. The rel. liver wt was raised in PB-treated rats at all times of the day. The circadian rhythm of cellular structures was closely related to the hepatic glycogen content which exhibited a clear rhythm with the peak also at 0800, but lowered values were found in PB-treated rats. The mitotic activity of hepatocytes was significantly increased in PB-treated rats but displayed the same circadian rhythm as controls with peaks at noon and troughs at midnight. The well-known hypertrophy of the smooth endoplasmic reticulum in PB-treated rats was not found at 0600, but was fully developed at 1400 and 2200. PB-treatment increased significantly the liver content of cholesterol, triglycerides and phospholipids. Liver cholesterol showed a clear circadian rhythm with peaks at 1800. No rhythm of liver protein, triglycerides and phospholipids was observed. In serum, levels of cholesterol were significantly elevated, those of triglycerides and alk.P. significantly lowered, while those of phospholipids were not affected by the treatment. The three serum lipids, alk.P. and beta-lipoprotein exhibited a clear circadian rhythm, while serum glucose and non-esterified fatty acids did not.     

Circadian Rhythm of Liver Parameters (Cellular Structures,Mitotic Activity,Glycogen and Lipids in Liver and Serum) During Three Consecutive Cycles in Phenobarbital-Treated Rats

The circadian rhythm of gastric content, serum alkaline phosphatase (alk.P.), serum lipids, body weight (wt), relative (rel.) liver wt, cellular structures (by light- and electron-microscopy), mitotic activity of hepatocytes, glycogen content, protein and lipids in liver was studied in 180 male Sprague-Dawley rats orally treated at 0830-1030 with 50 mg/kg phenobarbital (PB) for 7 days. Thereafter, five PB-treated males and five controls each were studied at 4-hr intervals at 0600, 1000, 1400, 1800, 2200 and 0200 on 3 consecutive days. The lighting schedule in the colony was 12:12 = light/dark (light from 0600 to 1800). Following the rhythm of gastric emptying, the rel. liver wt showed a clear circadian rhythm with a peak at 0800. The rel. liver wt was raised in PB-treated rats at all times of the day. The circadian rhythm of cellular structures was closely related to the hepatic glycogen content which exhibited a clear rhythm with the peak also at 0800, but lowered values were found in PB-treated rats. The mitotic activity of hepatocytes was significantly increased in PB-treated rats but displayed the same circadian rhythm as controls with peaks at noon and troughs at midnight. The well-known hypertrophy of the smooth endoplasmic reticulum in PB-treated rats was not found at 0600, but was fully developed at 1400 and 2200. PB-treatment increased significantly the liver content of cholesterol, triglycerides and phospholipids. Liver cholesterol showed a clear circadian rhythm with peaks at 1800. No rhythm of liver protein, triglycerides and phospholipids was observed. In serum, levels of cholesterol were significantly elevated, those of triglycerides and alk.P. significantly lowered, while those of phospholipids were not affected by the treatment. The three serum lipids, alk.P. and beta-lipoprotein exhibited a clear circadian rhythm, while serum glucose and non-esterified fatty acids did not.     

Opposite effects of endotoxin on mitochondrial and endoplasmic reticulum functions

In this study, we determined functional integrity and reactive oxygen species generation in mitochondria and endoplasmic reticulum in liver of rats subjected to endotoxic shock to clarify whether intracellular reactive oxygen species (ROS) destabilize cellular integrity causing necrosis in rats challenged with lipopolysaccharide (LPS). LPS caused drastically increased plasma levels of alanine aminotransferase, suggesting damage to plasma membranes of liver cells. Liver necrosis was confirmed by histological examination. LPS induced a significant increase in ROS production in rat liver mitochondria (RLM), but did not impair mitochondrial function. In contrast to mitochondria, enzymatic activity and ROS production of cytochrome P450 were lower in microsomal fraction obtained from LPS-treated animals, suggesting the dysfunction of endoplasmic reticulum. Protein patterns obtained from RLM by two-dimensional electrophoresis showed significant upregulation of mitochondrial superoxide dismutase by LPS. We hypothesize that upregulation of this enzyme protects mitochondria against mitochondrial ROS, but does not protect other cellular compartments such as endoplasmic reticulum and plasma membrane causing necrosis.     

Ultrastructural changes in the liver of the sand lamprey,Lampetra reissneri,during sexual maturation

Ultrastructural changes of hepatocytes were examined in the sand lamprey,Lampetra reissneri, during various phases of the life cycle. In hepatocytes of ammocoetes, the rough endoplasmic reticulum was composed of short cisternae and the Golgi apparatus were scarcely developed, showing no sexual differences at this stage of life cycle. In hepatocytes of female lampreys at the metamorphic stages 4 to 5, the rough endoplasmic reticulum was developed to form long parallel cisternae and the Golgi apparatus were well-developed. The rough endoplasmic reticulum developed further to form stacks of long parallel cisternae extending over the cytoplasm in hepatocytes of females at the young adult stage, and became composed of both long parallel and vesicular cisternae in the cells of females at the adult stage. The Golgi apparatus were invariably welldeveloped in hepatocytes of young adult and adult females. No consipcuous development was observed in profiles of the rough endoplasmic reticulum and the Golgi apparatus in hepatocytes of males during and after metamorphosis. The ultrastructural changes of the rough endoplasmic reticulum and the Golgi apparatus observed in hepatocytes of female sand lampreys are considered to have an intimate relation to the activity of vitellogenin synthesis in the liver, and it is suggested that the hepatocytes begin to rapidly synthesize vitellogenin in the sand lamprey at the metamorphic stages 4 to 5.     

黄芪多糖预防2 型糖尿病大鼠肝损伤的实验研究

目的:研究分析黄芪多糖(Astragalus polysaecharides,APS)在预防2型糖尿病大鼠肝损伤的作用。方法:Wistar大鼠72只,其中48只以小剂量链脲佐菌素(Streptozotocin,STZ)建立2型糖尿病大鼠模型,并随机分为模型对照组(B组)和APS组(C组),余24只大鼠为正常对照组(A组)。C组大鼠给予APS 400 mg/kg/d灌胃,A、B组分别给予等量生理盐水。分别于第0、2、4、6周检测3组大鼠血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、超氧化物歧化酶(SOD)以及丙二醛(MDA)水平,透射电镜下观察3组大鼠肝细胞超微结构变化。结果:第0、2周时B、C组大鼠ALT、AST、SOD以及MDA水平比较无显著差异(P0.05)。第4、6周时,C组大鼠ALT、AST以及MDA水平均高于A组,但低于B组;而SOD水平低于A组,但高于B组;差异均有统计学意义(P0.05)。电镜下观察,A组肝细胞内大量线粒体、内质网,结构完好,未见损伤;B组细胞器数量减少,线粒体及内质网形态异常,膜结构破损,细胞空化;C组细胞器也存在损伤,但较B组明显减轻。结论:APS可增加肝细胞抗氧化酶的活性,促进氧自由基的清除,减少肝细胞损伤,对预防2型糖尿病引起的肝功能损害有一定的价值。     

Destructive and restorative processes of the liver in rats under intensive physical loading

The ultrastructure of rat liver cells after running exercise was investigated. When rats were trained for a month and sacrificed immediately after the last exercise it was revealed that the number of liver cells mitochondria increased, but many of them had alterations: mitochondria became swollen, had lucid matrix. There were some variations in degree of alterations between different mitochondria: a) in the same hepatocyte, b) in different hepatocytes of the same animal, that was connected with individual sensitivity of organelles on the levels of the cell and of the organ. Rough endoplasmic reticulum bore few ribosomes. Glycogen was absent. There were abundant vesicles of smooth endoplasmic reticulum, autophagic vacuoles and peroxisomes in the liver cell cytoplasm. Adaptation of rat liver to the exercise programme becomes evident by 1.5 month of exercise. Mitochondria and rough endoplasmic reticulum were numerous and of normal structure. There were many peroxisomes and glycogen granules in the cytoplasm of hepatocyte. The presence of large autophagic vacuoles in the cytoplasm of some hepatocytes were obviously connected with more rapid destruction of some organelles, than in control.     

Some histopathological effects of Aroclor 1254 on the liver and gonads of rainbow trout, Salmo gairdneri and carp, Cyprinus carpio

Sexually mature ♂ and ♀ rainbow trout ( Salmo gairdneri Richardson) and carp ( Cyprinus carpio L.) were injected intraperitoneally with Aroclor 1254 (25 mg kg⁻¹ body weight in 1 ml of arachis oil) once weekly for four weeks. Arachis oil was used as the control. At the end of the fourth week, samples of the liver, testis and ovary were taken from treated and control fish and examined by light and electron microscopy. The light microscope study indicated that there was general vacuolation of the hepatocytes of trout but not carp; fragmentation of the developing oocytes but no observable changes in the testis. Electron microscopy study showed that there was enlargement of the rough endoplasmic reticulum of the hepatocytes, some damage to spermatozoa and proliferation of the smooth endo- plasmic reticulum of the oocytes.     

CHOP deficiency attenuates cholestasis-induced liver fibrosis by reduction of hepatocyte injury

CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is a key component in endoplasmic reticulum (ER) stress-mediated apoptosis. The goal of the study was to investigate the role of CHOP in cholestatic liver injury. Acute liver injury and liver fibrosis were assessed in wild-type (WT) and CHOP-deficient mice following bile duct ligation (BDL). In WT livers, BDL induced overexpression of CHOP and Bax, a downstream target in the CHOP-mediated ER stress pathway. Liver fibrosis was attenuated in CHOP-knockout mice. Expression levels of alpha-smooth muscle actin and transforming growth factor-beta1 were reduced, and apoptotic and necrotic hepatocyte death were both attenuated in CHOP-deficient mice. Hepatocytes were isolated from WT and CHOP-deficient mice and treated with 400 microM glycochenodeoxycholic acid (GCDCA) for 8 h to examine bile acid-induced apoptosis and necrosis. GCDCA induced overexpression of CHOP and Bax in isolated WT hepatocytes, whereas CHOP-deficient hepatocytes had reduced cleaved caspase-3 expression and a lower propidium iodide index after GCDCA treatment. In conclusion, cholestasis induces CHOP-mediated ER stress and triggers hepatocyte cell death, and CHOP deficiency attenuates this cell death and subsequent liver fibrosis. The results demonstrate an essential role of CHOP in development of liver fibrosis due to cholestatic liver damage.     

Hepatocytic ultrastructure in splenectomized rats treated with pregnenolone-16alpha-carbonitrile, a microsomal enzyme inducer.

Pregnenolone-16alpha-carbonitrile (PCN), given orally at the dose of 6.8 mg/100 g body wt, twice daily for 3 or 6 days, increased liver weight in intact rats, and reduced zoxazolamine paralysis in both unoperated and splenectomized animals. The steroid induced smooth endoplasmic reticulum proliferation in the hepatocytes of intact and splenectomized rats, while splenectomy alone caused rough endoplasmic reticulum fragmentation and vesiculation. It appears that the spleen does not influence the hepatic action of PCN.     

Immunoperoxidase localization of albumin and fibrinogen in rat liver fixed by perfusion or immersion: effect of saponin on the intracellular penetration of labeled antibodies

Immunoperoxidase localization of albumin and fibrinogen in rat liver was tested with perfusion or immersion fixation and saponin as a membrane permeabilizing agent. The distribution of albumin- or fibrinogen-containing hepatocytes was examined by light microscopy. Labeled antibody penetration was assessed by electron microscopy on transversely cut cryostat sections. Paraformaldehyde liver fixation by perfusion, followed by incubation of the sections with labeled antibodies together with saponin, demonstrated that albumin and fibrinogen were present in all hepatocytes; mainly in the Golgi apparatus and rarely in the endoplasmic reticulum, the ultrathin sections being labeled throughout their entire thickness. A constant labeling of the endoplasmic reticulum was obtained when saponin was added from the beginning of fixation. In the absence of saponin, albumin was seen in most of the hepatocytes but only at the periphery of the transverse sections, in a few Golgi apparatus, and in some parts of the endoplasmic reticulum; under this condition, fibrinogen was not visualized in the hepatocytes. Paraformaldehyde liver fixation by immersion showed the presence of albumin or fibrinogen in a few hepatocytes only, with irregular labeled antibody penetration. The use of saponin did not improve albumin and fibrinogen localization, except when the liver was poorly fixed. These results show that liver fixation by perfusion gives a homogeneous labeling of all the hepatocytes, whereas fixation by immersion leads to a heterogeneous labeling. Satisfactory results are obtained with saponin, which must be used to improve the penetration of labeled antibodies when the liver is fixed by perfusion. Saponin does not work when immersion is employed, at least under the conditions tested.     

Structural and functional changes after the administration of tetrachlormethane in the liver of rats fed on diets with different protein contents

Morphological and biochemical changes characterizing the degree of liver damage and the development of liver repair were studied in rats fed 21 days on a low protein diet (LPD), a standard diet (SLD) and a high protein diet (HPD) and then given a single i.p. injection of tetrachlormethane (CCl4) in a dose of 0.75 ml/kg body weight. The HPD was found to increase sensitivity to CCl4, but it also promoted the liver repair process, as seen from the increment in liver DNA synthesis and the total DNA content of the liver, increased ploidy of the hepatocytes and growth of the size of their nuclei and of the hepatocytes themselves. An increase in the total surface area of the membranes of the granular endoplasmic reticulum and the inner and outer membrane of the mitochondria, but a decrease in the surface area of the membranes of the smooth endoplasmic reticulum, were also observed after the administration of CCl4. The LPD raised liver resistance to CCl4, but the development of liver repair activity differed from the process after the SLD and HPD, since polyploidy of the hepatocytes (especially the growth of octaploid cells) predominated and there was also an increase in the number of binuclear hepatocytes. Cell hypertrophy was expressed less in rats fed on the LPD than in animals given the HPD. As far as liver repair was concerned, the HPD showed no explicit advantages over the SLD.     

Morphology of the liver of the brook lamprey, Lampetra lamottenii before and during infection with the nematode, Truttaedacnitis stelmioides, hepatocytes, sinusoids, and perisinusoidal cells.

Routine light microscopy and transmission and scanning electron microscopy were used to describe and compare the livers of larval lampreys, Lampetra lamottenii before and during infection of the bile ducts by the nematode, Truttaedacnitis stelmioides. The hepatocytes of uninfected animals differ from other lamprey species in that they contain abundant glycogen, smooth endoplasmic reticulum (SER), and lipoprotein indicating that the liver may be involved in glucose metabolism. Infestation of the biliary tree by T. stelmioides coincides with alterations to the hepatocytes. These changes include dilation of the bile canaliculi, smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), and Golgi apparatus, swollen mitochondria in cells showing a high degree of hypertrophy, and an abundance of dense bodies. Following infection, the sinusoidal lumina became dilated and contain a moderate electron-dense precipitate, an abundance of melanomacrophages, lipocytes, and mononuclear cells. There is also a widening of the fenestrae of the sinusoidal endothelium following infection. Many of the changes in hepatocytes and sinusoids following parasite infections closely resemble those observed in hepatocytes in various pathologies and following experimental bile duct ligation and, therefore, are likely a consequence of increased biliary pressure due to bile duct obstruction.     

中药对欧鳗实验性肝病保护作用的超微结构观察

目的探讨由丹参、五味子及甘草组成的中药煎剂对实验性肝病欧鳗肝组织超微结构的影响。方法将健康组欧鳗养于清水中,三个处理组先暴露于0.12 mg/L Cu2 溶液4 d,后4 d其中一组作为肝病未治疗组不予治疗,其余两组为肝病中药治疗组,即中药煎剂治疗低剂量组(200 mg/L)和中药煎剂治疗高剂量组(400 mg/L),实验结束时,剖检并取肝脏样本,进行电镜观察。结果与健康组相比,肝病未治疗组欧鳗肝细胞线粒体肿胀,嵴断裂、减少或结构模糊,有的消失呈空泡状;粗面内质网扩张、脱粒或出现扭曲现象;滑面内质网增生、扩张呈囊泡化;池内隔离体增多,自噬体及空泡多见;部分肝细胞核皱缩,染色质边集。各治疗实验组与肝病未治疗组相比,肝组织超微结构修复非常显著。结论研究表明该中药煎剂可显著促进铜离子中毒致肝损伤欧鳗病变肝细胞结构的修复,具有良好的抗肝损伤及修复肝损伤作用。     

Ultrastructural location of albumin and fibrinogen in primary cultures of new-born rat liver tissue

In primary cultures of new-born rat liver tissue, albumin and frbrinogen, two proteins normally synthesized by the liver and secreted into plasma were demonstrated by specific antibodies labelled with peroxidase in about 50 and 70% of the hepatocytes; these proteins were not demonstrated in the other types of cells, in particular fibroblasts, present in primary cultures. These two proteins were detected on the ribosomes of the rough endoplasmic reticulum and were also present in the lumina of the rough and smooth endoplasmic reticulum and in the Golgi apparatus. It is concluded that

1. 1. In primary cultures of liver tissue, only the hepatocytes synthesize albumin and fibrinogen.

2. 2. Proliferating cultured hepatocytes are able to synthesize albumin and fibrinogen.

3. 3. The presence of detectable albumin and fibrinogen in the lumina of the rough and smooth endoplasmic reticulum and in the Golgi apparatus in hepatocytes of primary cultures and their absence in the lumina of the rough and smooth endoplasmic reticulum and in the Golgi apparatus in the hepatocytes of adult rat liver might indicate an alteration in the translocation of albumin and fibrinogen through these organelles in cultured hepatocytes.

     

Dense line in cisterna of rough-surfaced endoplasmic reticulum of hepatocytes of the rat perfused with saponin solution

The effect of brief perfusion of saponin solution to the membrane of rough-surfaced endoplasmic reticulum (r-ER) was observed in hepatocytes at electron microscope level. The liver of rats was perfused with 1% saponin solution in 0.2m phosphate buffer (pH 7.0) for 3 min. then perfused again with 4% glutaraldehyde buffered with 0.1m sodiumcacodylate-HCl at pH 7.4. The most striking alteration of ultrastructure of hepatocytes was the dense intracisternal line of the r-ER. That dense line often had cross striation or "ladder structure". Although its functional significance remains to be cleared, it should reflect the denature of membrane structure of r-ER as one of effects of saponin.     

Interferon suppresses glutamine synthetase induction in chick embryonic neural retina.

Two different proteins precipitable with antiserum to albumin exist in liver. One is albumin, the other is precursor albumin. Liver cells in suspension contain mainly precursor, but secrete only albumin. In subcellular fractions isolated from liver homogenate, 95.3% of anti-albumin precipitable protein in the rough endoplasmic reticulum, 51.4% in the smooth endoplasmic reticulum, 33.5% in the Golgi apparatus and 0% in the supernatant fraction was precursor albumin. The results suggest that albumin precursor is synthesized in the rough endoplasmic reticulum and converted into albumin in the smooth endoplasmic reticulum and the Golgi apparatus.     

A comparative study of ribonuclease hydrolysis of rat brain-cortex and liver membrane-bound ribosomes

Because it has been proposed that the ribosome–membrane interaction is different in endoplasmic reticulum derived from a non-secretory and secretory cell we undertook a study to determine whether attachment of the ribosome to the membrane involved ribosomal RNA and if the rRNA in ribosomes derived from the two classes of cell possessed an altered susceptibility to RNAase (ribonuclease) hydrolysis. We found that brain ribosomes appeared to possess more regions accessible to nuclease attack, independent of whether a sequence-dependent RNAase (T₁) or a sterically hindered RNAase bound to Enzite polymer was employed. These results were independent of whether the ribosomes were membrane-bound or detached from the endoplasmic reticulum membranes, but at high RNAase concentration these differences became negligible. No conclusions, however, could be drawn as to whether ribosomal RNA is involved in the attachment of the ribosome to the endoplasmic reticulum membrane, because of the presence of endogeneous membrane-associated RNAases. Analysis of the rRNA fragments by polyacrylamide-gel electrophoresis suggests that the sites available for attack by low concentrations of nuclease in bound-ribosomes derived from brain cortex are different from those of liver.     

Inhibition by colchicine of fibrinogen translocation in hepatocytes

In the rat, 8 h after intraperitoneal administration of colchicine, fibrinogen (detected by antirat fibrinogen antibodies labeled with peroxidase) accumulated in the lumina of the rough endoplasmic reticulum of the hepatocytes; 16 and 24 h after colchicine administration, fibrinogen was detected, respectively, in the lumina of the smooth endoplasmic reticulum and in the Golgi apparatus. The effect of colchicine on the cytoplasmic translocation of fibrinogen could be due to a direct action of the drug on the membranes of the endoplasmic reticulum or could be the indirect result of the disruptive action of the drug on the microtubules.