Nitric oxide synthase inhibitors influence dynorphin A (1-17) immunoreactivity in the rat brain following hyperthermia

Summary.  The possibility that nitric oxide synthase (NOS) inhibitors influence dynorphin immunoreactivity following hyperthermia was examined in a rat model using a pharmacological approach. Previous reports from our laboratory show that hyperthermia induces an upregulation of NOS in several brain regions that seems to be instrumental in causing cell injury. Recent reports suggest that nitric oxide (NO) can influence dynorphin neurotransmission in the normal brain as well as in several pathological states. Since dynorphin is neurotoxic in different animal models of brain or spinal cord injury, it may be that the peptide will contribute to the cell injury in hyperthermia. The present investigation was carried out to determine whether hyperthermia can influence dynorphin immunoreactivity in the brain, and if so, whether inhibition of NOS will influence the peptide distribution in the brain following heat stress. Rats subjected to hyperthermia at 38°C for 4 h in a biological oxygen demand incubator (BOD) resulted in a marked upregulation of dynorphin immunoreactivity in several brain regions e.g., cerebral cortex, hippocampus, cerebellum and brain stem. Pretreatment of rats with two potent NOS inhibitors, L-NAME (30 mg/kg/day, i.p. for 7 days) or L-NMMA (35 mg/kg/day, i.p. for 7 days) significantly attenuated the dynorphin immunoreactivity in the brain. These drugs were also able to reduce hyperthermia induced blood-brain barrier (BBB) permeability, brain edema formation and cell injury. Taken together, our results suggest that (i) hyperthermia has the capacity to upregulate dynorphin immunoreactivity in the brain, (ii) inhibition of NOS considerably attenuates the dynorphin immunoreaction following heat stress and (iii) upregulation of dynorphin is somehow contributing to hyperthermia induced brain damage, not reported earlier. Received July 3, 2001 Accepted August 6, 2001 Published online July 31, 2002     

Cuprizone Administration Alters the Iron Metabolism in the Mouse Model of Multiple Sclerosis

Cuprizone (CZ) is a widely used copper chelating agent to develop non-autoimmune animal model of multiple sclerosis, characterized by demyelination of the corpus callosum (CC) and other brain regions. The exact mechanisms of CZ action are still arguable, but it seems that the only affected cells are the mature oligodendrocytes, possibly via metabolic disturbances caused by copper deficiency. During the pathogenesis of multiple sclerosis, high amount of deposited iron can be found throughout the demyelinated areas of the brain in the form of extracellular iron deposits and intracellularly accumulated iron in microglia. In the present study, we used the accepted experimental model of 0.2% CZ-containing diet with standard iron concentration to induce demyelination in the brain of C57BL/6 mice. Our aim was to examine the changes of iron homeostasis in the CC and as a part of the systemic iron regulation, in the liver. Our data showed that CZ treatment changed the iron metabolism of both tissues; however, it had more impact on the liver. Besides the alterations in the expressions of iron storage and import proteins, we detected reduced serum iron concentration and iron stores in the liver, together with elevated hepcidin levels and feasible disturbances in the Fe–S cluster biosynthesis. Our results revealed that the CZ-containing diet influences the systemic iron metabolism in mice, particularly the iron homeostasis of the liver. This inadequate systemic iron regulation may affect the iron homeostasis of the brain, eventually indicating a relationship among CZ treatment, iron metabolism, and neurodegeneration.     

Dietary Folate and Biogenic Amines in the CNS

Abnormal biogenic amine biosynthesis has been observed in humans and animals with endogenous and exogenous disturbances in folate metabolism. In an attempt to study this interaction biochemically, rats were depleted or repleted with folate for 10 weeks. Folate levels in depleted animals in serum and CSF correlated with stores in liver and brain, respectively. In depleted or repleted animals, there was no significant effect on biogenic amine metabolism in the CNS, as determined by quantitation of biogenic amines in brain and their respective metabolites in brain and CSF. These results are contrary to studies by other investigators. We suspect, however, that specific genetic defects in folate metabolism do result in impaired biogenic amine metabolism and probably at the level of disturbed biopterin cofactor functions.     

Th1/Th2类细胞因子的变化对急性免疫性肝损伤的影响研究

目的探讨Th1/Th2类细胞因子的变化对ConA诱导的急性免疫性肝损伤的机制,以及脾脏对急性免疫性肝损伤的影响作用。方法将Balb/c小鼠随机分为两组：正常对照组,肝损伤组。正常对照组尾静脉注射等量生理盐水,肝损伤组尾静脉注射12.5mg/Kg ConA一次。各组分别于ConA注射后8h,24h,72h取材,进行下列研究：①HE染色观察各组小鼠肝脏病理学改变。②经眼球取血,收集血清测ALT和AST。③收集各组小鼠血清及新鲜肝、脾组织（各100mg）,获取肝、脾组织裂解液。用多参数细胞因子检测技术即FlowCytomix技术,通过流氏细胞仪对荧光素PE信号强度的检测,实现对各组小鼠血清、肝组织、脾组织内多种Th1/Th2类因子的定性定量分析。结果①HE染色：正常对照组肝组织结构正常。肝损伤组8h时表现为急性肝损伤表现,24h时可见大片坏死灶,72h时肝损伤缓解。②血清ALT和AST检测：正常对照组3个时间点内无明显升高,肝损伤组3个时间段内ALT和AST均高于正常对照组,有显著性差异。③Th1/Th2细胞因子检测结果：肝脏：肝损伤组8h时Th1和Th2类细胞因子均明显升高,与正常对照组比较有显著性差异,24h后开始下降,降至正常水平或正常水平以下,呈明显下降趋势。血清：肝损伤组Th1,Th2类细胞因子8h均升高,24h后逐步降低。脾脏：肝损伤组Th1,Th2类细胞因子8h时均升高,与正常对照组比较,有显著性差异,24h时明显降低。结论①ConA诱导的急性免疫性肝损伤主要是由Th1类细胞、巨噬细胞和Th2类细胞分泌的炎性因子所造成。②脾脏通过Th1/Th2类细胞因子的分泌对急性免疫性肝损伤起到免疫调控作用。     

The thioacetamide-poisoned rat as an animal experimental model for endocrinological studies of estrogen metabolism in chronic liver injury)]

Liver microsomes of rats poisoned with thioacetamide show a significant reduction of cytochrome P-450. Consequently, oxidative reactions of drug metabolism and the estrogen 2-hydroxylase are diminished. Enhancement of microsomal transformation of estradiol to estrone and 16alpha-hydroxyestrone is observed after treatment of rats with thioacetamide, due to diminished metabolism of estradiol by the alternative oxidation at C-2. Estriol formation is reduced by thioacetamide pretreatment. These changes in estrogen breakdown closely correlate with those observed in humans suffering from cirrhosis of the liver. It is concluded that the thioacetamide poisoned rat should be an experimental model suitable for studying estrogen metabolism in liver injury.     

Effects of phytoestrogen-coumestrol on lipid and carbohydrate metabolism in young ovariectomized rats may be independent of its estrogenicity

Two groups of young ovariectomized female rats received one of two treatments. The first group was fed coumestrol in lab chow (200 microg of coumestrol per day) for 14 days; the second group received coumestrol (40 mg/L) via perfusion medium. There was a significant increase (78% compared with the control group) in the uterine weight after coumestrol treatment, which supports the estrogen-like activity of coumestrol. Phytoestrogen diminished the liver and skeletal muscle glycogen contents by 18% and 29%, respectively, and increased the blood glucose level by 24%. Glycogenolytic activity of coumestrol was observed when it acted directly on the liver areas. Although phytoestrogen did not influence insulin and glucagon blood level, liver and to some degree muscle susceptibility to insulin (measured as hormone binding by insulin receptors) was decreased. Coumestrol increased the content of triglycerides in muscle by 113% and enhanced the liver lipid synthesis from glucose by 179%. Liver cholesterol concentration was increased both after coumestrol feeding (by 12%) and when it acted directly on the liver (by 16%). These observations suggest that coumestrol is in general anabolic with regard to lipid and catabolic within-carbohydrate metabolism of young ovariectomized female rats. Based on the results of this study, it is concluded that influence of coumestrol on lipid and carbohydrate metabolism of ovariectomized rats is in part not related to its estrogenic action.     

Role of nitric oxide in the oxidant stress during ischemia/reperfusion injury of the liver.

The potential role of nitric oxide (NO) and its reaction product with superoxide, peroxynitrite, was investigated in a model of hepatic ischemia-reperfusion injury in male Fischer rats in vivo. Pretreatment with the NO synthase inhibitor nitro-L-arginine (10 mg/kg) did neither affect the post-ischemic oxidant stress and liver injury during the initial reperfusion phase nor the subsequent infiltration of neutrophils into the liver and the later, neutrophil-induced injury phase. Furthermore, no evidence was found for a postischemic increase of the urinary excretion of nitrite, a stable oxidation metabolite of NO. In contrast, the administration of Salmonella enteritidis endotoxin (1 mg/kg) induced a significant diuresis in Fischer rats and an 800-fold enhancement of the urinary nitrite excretion. Nitro-L-arginine pretreatment inhibited the endotoxin-induced nitrite formation by 97%. Hepatic cGMP levels, as index of NO formation in the liver, were only increased significantly after endotoxin administration but not after ischemia and reperfusion. Our results provide no evidence for any enhanced generation of NO or peroxynitrite either systemically or locally during reperfusion and therefore it is unlikely that any of these metabolites are involved in the oxidant stress and liver injury during reperfusion after hepatic ischemia.     

The estimation of the nitric oxide role in the aggravation of combined radiation-thermal injuries

The influence of specific inhibitor of inducible NO synthase S-ethil-isothiourea (as "Difetur" preparation) on liver NO production level, and 30-days survival, mean survival time and probability of mortality within animals under combined radiation/thermal injury (CRTI) were evaluated. Experiments were carrying out on mice (whole body gamma-irradiation at the dose of 7 Gy + 10% body surface full-thickness thermal burn). It was shown, that CRTI induce 2-fold statistical significant increase of NO production in liver of experimental animals. Mice pretreatment with Difetur preparation lead to practically full inhibition of NO production. In the group of animals, with Difetur administration during first two days after CRTI 60% mice survived as compared 15% survive in control group. In pair with data on probability of mortality it was suggested that growth of NO production in the early period of CRTI increase sensitivity of animals to pathological processes leading to death on 10-12 days.     

Short-term effects of thalidomide analogs on hepatic glycogen and nitric oxide in endotoxin-challenged rats

Hepatic glycogen metabolism is altered by nitric oxide (NO) during endotoxic shock. Thalidomide analogs immunomodulate the endotoxin-induced cytokines which regulate the NO release. We analyzed the short-term effects of some thalidomide analogs on the hepatic glycogen store and on the plasma and hepatic NO in an acute model of endotoxic challenge in rat. An endotoxin dose selection was performed. Rats received vehicle, thalidomide or analogs orally and, two hours after last dose, they were injected with endotoxin (5 mg/kg). Animals were sacrificed 2 h after challenge. Liver glycogen was quantified by the anthrone technique. Plasma and hepatic NO was determined by Griess reagent and HPLC. Hepatic interferon-gamma, a NO co-inducer, was measured by ELISA. Endotoxin caused inverse dose-dependent effects on plasma NO and on glycogen.Thalidomide analogs showed short-term regulatory effects on glycogen, some of them increased it. Plasma NO was almost unaffected by analogs but hepatic NO was strikingly modulated. Analogs slightly up-regulated the liver interferon-gamma and two of them increased it significantly. Thalidomide analogs may be used as a pharmacological tool due to their short-term regulatory effects on glycogen and NO during endotoxic shock. Drugs that increase glycogen may improve liver injury in early sepsis.     

Level of nitrated proteins in the plasma,platelets and liver of patients with liver cirrhosis

Abstract

Over-expression of nitric oxide synthase (NOS) and nitric oxide (NO) formation are associated with the pathogenesis of liver cirrhosis. NO-related stress alters the functions of biomolecules, especially proteins, probably as a result of nitration. The aim of this study was to assess the level of protein nitration and its correlation with the severity of the disease. Liver cirrhosis patients with different grades of severity (grades A, B, and C according to the Child–Pugh classification) were enrolled in this study. Nitroprotein content, arginine, citrulline, NO in terms of total nitrite, nitrosothiol (RSNO) and protein carbonyls were measured in blood. Immunohistochemical detection of nitroprotein was carried out in liver sections of cirrhosis patients. A significant elevation in the levels of serum and platelet arginine, arginase, citrulline, plasma, and platelet nitroproteins, RSNO, total nitrite, protein carbonyls and also a significant amount of nitrated proteins by immunohistochemical detection in tissue were observed in cirrhosis patients. The alterations were highly significant in grade C patients with bleeding complications when compared to those of grade B and A patients. In platelets, both cytosolic and cytoskeletal proteins were found to be nitrated significantly. The level of nitrite seems to have positive correlation with the level of nitroproteins in different grades of cirrhosis. The level of nitroproteins in plasma, platelets and liver tissue can be correlated with the severity of liver cirrhosis.     

具有保肝作用的天然药物开发进展

肝脏是机体代谢的最主要场所,也是机体最容易遭受到损伤的脏器之一,各种因素引起的肝损伤已成为威胁人类健康的重要疾病之一。肝损伤机制主要与线粒体损伤、自由基脂质过氧化、炎症细胞因子分泌和细胞膜损伤有关。目前已报道很多天然药物具有显著保肝作用,且具有疗效稳定、副作用低、多途径作用、作用温和持久等优势,已广泛用于肝脏疾病的防治。本文对肝损伤的生理机制以及具有保肝作用的天然药物开发进展进行了综述,提出了目前存在的一些问题并进行了展望。     

Acceleration of metabolism of stress-related substances by L-carnosine]

Liver dysfunction was produced in the rat by injecting CCl4 subcutaneously in the back twice a week, and the effects of L-carnosine (CAR) on the resulting liver injury were examined. When CCl4 was administered to 6-week-old rats for 9 weeks, GOT and GPT values increased, but these changes were suppressed in the group concomitantly treated with CAR, indicating a protective effect of the agent on liver function. No such preventive effects of CAR was observed in 40-week-old rats, but when the CCl4 administration was discontinued after 4 weeks, GOT and GPT decreased to normal levels within 1 week of discontinuation, indicating a therapeutic effect of CAR on hepatopathy. Based on these findings, we determined the cortisone beta-reductase activity in the rat liver. The increase in this enzyme activity in the group treated with CAR indicated acceleration of cortisone metabolism. Changes of blood cortisol level and cerebral and blood noradrenaline (NA) levels were studied by exposing 6-week-old rats to electric shocks at 30 V. Cortisol released into the circulation after the stress was quickly metabolized in the CAR group and the blood level normalized after 3 hours. Following the release of NA from the brain into the circulation, the NA concentration rapidly returned to the normal level both in the brain and the blood. CAR enhanced the liver function and accelerated the metabolism of stress-related substances also in aged animals. CAR, moreover, restored the RNA contents of the mouse spleen and the immunological abilities represented by PFC reaction, which are reduced by stresses such as forced immersion, fasting, and administration of MMC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical and functional changes of rat liver spheroids during spheroid formation and maintenance in culture: II. nitric oxide synthesis and related changes

Liver cells isolated from intact tissue can reaggregate to form three-dimensional, multicellular spheroids in vitro. During this process, cells undergo a histological and environmental change. How cells respond biochemically to this change has not been studied in detail previously. We have investigated some biochemical changes in rat liver cells during the formation and maintenance of spheroids. Liver cells were isolated from male Sprague rats and spheroids cultured by a gyrotatory-mediated method. Liver cells were shown to respond to the isolation procedure and the formation of spheroids triggered histological environmental changes that increased arginine uptake, nitric oxide (NO) and urea syntheses, as well as raised levels of GSH, GSSG, glutamic acid and aspartic acid secretion within the first couple of days after cell isolation. Levels were maintained at a relatively stable level in the mature spheroids (>5 days) over the 3 week period of observation. P450 1A1 activity was lost in the first 2 days and gradually recovered thereafter. This study, for the first time, shows that liver cells after isolation and during spheroid formation actively uptake arginine and increase NO and urea syntheses. A high level of NO is likely to play an important role in modulating a series of biochemical changes in liver cells. It is considered that liver cells actively respond to the 'challenge' induced by the isolation procedure and subsequent histological environmental changes, and biochemical modulation and instability result. The stable cell-cell contacts and histological environment in mature spheroids permit and support functional recovery and maintenance in vitro. This period of stability permits the use of spheroids in toxicity studies to establish acute and chronic paradigms.     

脑室内注入TRH对大鼠肝脏无机磷代谢影响的核磁共振分析

采用在大鼠脑室内注入促甲状腺激素释放激素（Ｔｈｙｒｏｔｒｏｐｉｎｒｅｌｅａｓｉｎｇｈｏｒｍｏｎｅ,ＴＲＨ）,并利用３１Ｐ－核磁共振法测定活体大鼠肝脏中的含磷化学物质,并观察ＴＲＨ对肝脏无机磷代谢的影响,研究证明,ＴＲＨ通过中枢神经影响肝脏中无机磷的代谢。由侧脑室注入ＴＲＨ,使肝脏无机磷含量发生显著增加,此作用由于副交感神经的阻断剂阿托品的加入而消失,由此可以认为,ＴＲＨ是经由副交感神经而影响肝脏的代谢。     

Sensitivity to in vitro lipid peroxidation in liver and brain of aged rats.

Lipid peroxidation in rat liver and brain has been studied to see if it increases with old age. No significant differences in the level of endogenous, nonstimulated lipid peroxidation (TBA-RS) is found between 9 month-old (mature adults) and 28 month-old animals in liver or cerebral cortex. Liver homogenates subjected in vitro to an oxidative stress (ascorbate-Fe++), show a clearly slower peroxidation rate in old than in young animals. On the other hand, the in vitro peroxidation rate of cerebral homogenates was similar in young and old animals. The in vitro peroxidation rate was much higher in brain than in liver tissue. These results do not support the view that old rats liver and brain are more susceptible to free radical oxidative damage than those of young ones.     

Melatonin exerts a more potent effect than S-adenosyl-l-methionine against iron metabolism disturbances, oxidative stress and tissue injury induced by obstructive jaundice in rats

Melatonin and S-adenosyl-l-methionine (SAMe) prevent oxidative stress and tissue dysfunction in obstructive jaundice (OJ). Lipid peroxidation is exacerbated in the presence of trace amounts of iron (Fe). The study investigated the regulation by melatonin and SAMe the induction of oxidative stress, iron metabolism disturbances and tissue injury in an experimental model of OJ. Different parameters of lipid peroxidation, antioxidant status, tissue injury and Fe metabolism were determined in liver and blood. OJ induced Fe accumulation in liver, and increased transferrin (Tf) saturation and loosely bound Fe content in blood. Melatonin, and SAMe at lesser extent, enhanced protein Tf content in liver and blood, that reduced loosely bound Fe content in blood. Melatonin and SAMe did not affect ferritin (FT) and Tf mRNA expression, but reduced Tf receptor (TfR) mRNA expression in liver. In conclusion, the effect of melatonin and SAMe on Fe metabolism may be included in the beneficial properties of these agents on lipid peroxidation and tissue injury induced by OJ.     

Developmental patterns of copper and zinc concentrations in mouse liver and brain: evidence that the gene crinkled (cr) is associated with an abnormality in copper metabolism

An abnormality in copper metabolism during both the prenatal and postnatal (preweaning) periods was found to be associated with the autosomal recessive gene ”crinkled“ (cr) in mice. Liver copper concentration was significantly lower in crinkled mice (cr/cr) than in littermate controls (+/?) from 18 days of gestation to 20 days after birth. Crinkled mice older than 20 days of age had liver copper concentrations similar to those of littermate controls. Liver zinc and brain copper and zinc were similar in crinkled and noncrinkled mice at all times tested. In both crinkled and noncrinkled mice, brain copper concentration increased during the suckling period, and liver copper concentration decreased.     

Nucleoside mono- and nucleoside diphosphate kinase activities in rat liver and brain in radiation sickness

Activity of nucleoside di- and nucleoside triphosphates metabolism enzymes in tissues of rats gamma-irradiated by a dose of 30 Gy was studied 0.5, 1, 3, 6 and 24 hours after the radiation effect. It is shown that the nucleoside monophosphate kinase activity of the liver and brain is enhanced almost at all stages of the studies and the nucleoside diphosphate kinase activity is inhibited. A significant but reversible decrease of the nucleoside monophosphate kinase activity is observed in the liver 3 h later. By an end of the first day after irradiation the nucleoside mono- and nucleoside diphosphate kinase activities increase significantly both in the liver and brain.