Safety evaluation of auraptene in rats in acute and subacute toxicity studies.

Auraptene (AUR) is a natural, bioactive, monoterpene coumarin ether. It has anti-inflammatory, anti-carcinogenic, anti-bacterial, neuroprotective, and hepatoprotective properties. The aim of the present study was to assess the acute and subacute toxicity of oral administration of AUR in rats by evaluating clinical signs, haematology, biochemical factors, pathological changes and immune-toxicity. Acute administration of AUR in doses of 125, 250, 500, 1000 and 2000 mg/kg body weight had no mortality or clinical signs in a period of two days. To evaluate subacute toxicity, AUR was administrated for 28 days by oral gavage in doses of 125 and 250 mg/kg. There were significant differences in the haematological and biochemical data of the treated and untreated groups. However, almost all haematological differences were within normal reference ranges. Subacute administration of AUR showed no toxic histopathological effects on organ tissue. Evaluation of immune-toxicity also revealed no significant differences between treatment and untreated groups.

Anti-angiogenic effects of ethanolic extract of Artemisia sieberi compared to its active substance, artemisinin

Abstract Angiogenesis plays a key role in tumor growth, invasion and metastasis of cancer diseases and therefore, the inhibition of angiogenesis can provide an important therapeutic approach in cancer diseases. This study was designed to compare the anti-angiogenic activities of the ethanolic extract of Artemisia sieberi Besser, Asteraceae, and its active substance, artemisinin in both in vitro and in vivo models. To compare cytotoxicity level of ethanolic extract of A. sieberi with artemisinin, different concentrations (1–100 µg/ml) were tested using MTT assay on human umbilical vein endothelial cells. The anti-angiogenic properties of serial concentrations of ethanolic extract of A. sieberi and artemisinin were examined on human umbilical vein endothelial cells using a three-dimensional angiogenesis assay (in vitro model) and in the chick chorioallantoic membrane assay as in vivo model. The effects of ethanolic extract of A. sieberi and artemisinin were also tested on the expression of VEGFR-1, VEGFR-2 and CD34 genes using real-time PCR. Ethanolic extract of A. sieberi and artemisinin significantly (p < 0.001) inhibited the angiogenesis in the human umbilical vein endothelial cells culture whilst the ethanolic extract of A. sieberi showed higher effect in a concentration-dependent fashion (p < 0.001). The chick chorioallantoic membrane angiogenesis was also completely inhibited by ethanolic extract of A. sieberi at concentration of 33 ng/100 µl/egg. The gene expression analysis showed that the ethanolic extract of A. sieberi and artemisinin reduced the transcription of VEGFR-1, VEGFR-2 and CD34 genes in a concentration-dependent manner. This study demonstrated that the ethanolic extract of A. sieberi is strongly able to inhibit the angiogenesis in human umbilical vein endothelial cells and chick chorioallantoic membrane models compared to the artemisinin.

Liver ischemia preconditions the heart against ischemia-reperfusion arrhythmias.

OBJECTIVES: This study aimed to examine the hypothesis that an antiarrhythmic effect might be obtained by ischemic preconditioning of the liver, and also to characterize the potential underlying mechanisms. MATERIALS AND METHODS: Male Wistar rats were anesthetized by thiopental sodium (50 mg/kg, IP) followed by IV injection of heparin (250 IU). Remote ischemic preconditioning (RIPC) was induced by 3 cycles of 5 min liver ischemia followed by 5 min of reperfusion. The hearts were excised within 5 min after the final cycle of preconditioning and perfused using Langendorff's system. The isolated perfused hearts were subjected to 30 min global ischemia followed by 90 min reperfusion. The myocardial arrhythmias induced by ischemia- reperfusion (I/R) were determined in accordance with the guidelines of Lambeth Conventions. The potential role of KATP channels on RIPC was assessed by injection of glibenclamide (nonselective KATP blocker) or 5-hydroxydecanoate (mitochondrial KATP blocker) on rats 30 and 15 min before induction of RIPC in the liver, respectively. RESULTS: Hepatic remote preconditioning of the heart significantly (P<0.0001) prevented the incidence of myocardial arrhythmias induced by I/R in the perfused hearts (5.33±1.54 vs. 32.33±6.44,). However, the protective effects of remote preconditioning was significantly (P<0.01) abolished by the KATP blocker, glibenclamide (25.5±4.9 vs. 5.33±1.54,). CONCLUSION: Hepatic RIPC may prevent the arrhythmias induced by I/R in the isolated perfused hearts via KATP channels.

Free radical scavengers improve liver function but not morphological changes induced by reperfusion injury.

OBJECTIVE: Reperfusion injury (RI) is associated with high generation of reactive oxygen species (ROS), but the extent of involvement of these agents in the injury remains controversial. The present study aimed to examine the effectiveness of ROS scavengers against hepatic reperfusion injury in the rat. METHODS: The RI was induced in the liver using an isolated slow-flow, reflow perfused rat liver in both anterograde and retrograde perfusion. The effects of gentisic acid, N-acetyl cysteine, and trolox C on the superoxide production, liver function, and morphological changes were examined using different biochemical and histological assays. RESULTS: The hepatic RI caused a significant (p < 0.05) increase in superoxide production and enzyme releases and a decrease in bile flow in both directions. Histological changes induced by RI include apoptosis, necrosis, pale cytoplasm, cell vacuolation, and attenuation of cell cords. Although the production of superoxide in retrograde direction was significantly less than the anterograde, the extent of the injury in the retrograde was greater than the anterograde direction. Pretreatment of the livers with each of the test compounds significantly reduced the release of lactate dehydrogenase and aspartate aminotransferase and improved bile flow in the liver exposed to hypoxia/reperfusion. However, they failed to protect the liver against the structural alterations induced by RI. CONCLUSION: ROS scavengers can reduce superoxide-induced damage and improve the liver function, but they are not able to prevent the structural changes. It shows that ROS are not the sole causative mechanism of hepatic RI and other mechanisms and mediators may be involved.

Effects of nitric oxide modulating activities on development of enteric nervous system mediated gut motility in chick embryo model.

The enteric nervous system (ENS) arises from the enteric neural crest-derived cells (ENCCs), and many molecules and biochemical processes may be involved in its development. This study examined the effects of modulating embryonic nitric oxide (NO) activity on the intestinal motility induced by ENS. One-hundred-and-twenty fertilized chicken eggs were assigned to three main groups and incubated at 37 degrees Centigrade and 60 percent humidity. The eggs were treated with NG-nitro-Larginine methyl ester (L-NAME), sodium nitroprusside (SNP), L-arginine (L-Arg) or vehicle from days 3 (1st group), 7 (2nd group) and 10 (3rd group) of incubation and continued up to day 18. On day 19, the embryos were sacrificed, the jejunal and colorectal segments were taken and the intestinal motility was assessed using isolated organ system. The intestinal motility was recorded normally and following cholinergic, adrenergic and non-adrenergic non-cholinergic (NANC) stimulations. The ENS structure was assessed by immunohistochemistry (IHC) using glial fibrillary acidic protein (GFAP). Rhythmic intestinal contractions were seen in all treatment groups, but inhibition of NO in the LNAME- treated embryos caused significant decrease (p less than 0.01) in the frequency and amplitude of the contraction. The responsiveness to adrenergic, cholinergic and NANC stimulations was also significantly decreased (p less than 0.05). The GFAP expression was significantly (p less than 0.05) reduced in the L-NAME-treated embryos. This study showed that the inhibition of NO caused a deficient development of the ENS, leading to a decrease in the frequency and amplitude of the intestinal contractions and reduced the responsiveness to adrenergic, cholinergic and NANC signalling.

Allometric scaling relationship between frequency of intestinal contraction and body size in rodents and rabbits.

This study aimed to establish an allometric scaling relationship between the frequency of intestinal contractions and body mass of different mammalian species. The frequency of intestinal contractions of rabbit, guinea pig, rat and mouse were measured using an isolated organ system. The isolated rings were prepared from proximal segments of jejunums and the frequency of contractions was recorded by an isometric force procedure. The coefficients of the obtained allometric equation were ascertained by computation of least squares after logarithmic transformation of both body mass and frequency. Significant differences (p less than 0.001) were shown in the frequency of contractions between different species. The highest frequency that corresponded to the mice was 57.7 min-1 and the 95 percent confidence interval (CI) ranged from 45.4 to 70, while rabbits showed the lowest frequency (12.71 min-1, CI: 8.6-16.8). Logarithms of frequency were statistically proportional to logarithms of body mass (r00.99; p less than 0.001). The data fitted an equation F 1/4 18:51B 0:31 and the 95 percent confidence interval of the exponent ranged from -0.30 to -0.32. The results of this study suggest that it is probably possible to extrapolate the intestinal contraction frequency of other mammalian species by the means of allometry scaling.

A comparison of hepatic ischemia/hypoxia-reperfusion injury models.

INTRODUCTION: A number of hepatic ischemia/hypoxia-reperfusion models have been described. This study characterised the functional and structural changes induced by the most commonly used in vivo and in situ models for hypoxia/ischemia-reperfusion in the rat liver. METHODS: A range of no-flow, slow-flow and lobar ischemia and reperfusion models were established in the rat liver. Changes following reperfusion were monitored using physiological, biochemical, histological and pharmacological assessments, including bile production, oxygen consumption, lignocaine extraction, enzyme release, and disposition of exogenous markers. RESULTS: Short periods of hepatic ischemia/hypoxia-reperfusion led to minimal changes in liver function whereas long periods of ischemia-reperfusion led to substantial liver injury. The most severe injury was found with the slow flow, reflow model. The formation of cell vacuoles, blebs and focal hepatitis were the most important liver morphological changes observed as a consequence of ischemia/hypoxia. The major liver histological findings after reperfusion were dispersed apoptosis and local necrosis. Hepatic ischemia/hypoxia-reperfusion was also associated with significant changes in the hepatic extracellular and intracellular spaces. DISCUSSION: The morphology and function of the liver associated with a range of hepatic ischemia/hypoxia-reperfusion models varies with the duration of the insult and between models. The choice of model is therefore an important consideration in seeking to resolve any particular hypothesis associated with hepatic ischemia/hypoxia-reperfusion.

Alteration of renal functional, oxidative stress and inflammatory indices following hepatic ischemia-reperfusion.

Liver ischemia/reperfusion (IR) injury is a complex phenomenon that may cause local as well as remote organ injuries. Reactive oxygen species (ROS) along with many pro- and anti- inflammatory cytokines are implicated in the development of organ injury. The renal functional, histological, oxidative stress and inflammatory indices were studied during a short and a longer period of liver IR. Rats were subjected to either sham operation or 90 min partial liver ischemia followed by 4 or 24 h of reperfusion. Serum ALT, AST, ALK and LDH levels, BUN and creatinine, renal MDA level, SOD and catalase activities were evaluated as well as serum IL-6 and IL-10 concentrations along with renal histological evaluation. Ninety minutes liver ischemia /4 h reperfusion caused an increase in BUN and renal MDA levels and a decrease in SOD and catalase activities. It also caused an increase in serum IL-6 and IL-10 levels. 24 h liver reperfusion resulted in a reduction in BUN levels and lower oxidative damages demonstrated by a decrease in renal MDA levels and an increase in renal SOD and catalase activities comparing to 4 h reperfusion group. Evaluations indicated improvement in histology such as less cytoplasmic vacuolation and lower tubular debris. Serum inflammatory indices (IL-6 and IL-10 levels) were also reduced. This study showed that liver IR damage causes renal injury including functional, inflammatory and oxidative status changes. The remote kidney damage was then improved by continuing reperfusion from 4 to 24 h.

Histological and biochemical alterations in early-stage lobar ischemia-reperfusion in rat liver.

AIM: To investigate the structural and biochemical changes in the early stage of reperfusion in the rat livers exposed to lobar ischemia-reperfusion (IR). METHODS: The median and left lobes of the liver were subjected to 60 min ischemia followed by 5, 10, 30, 45, 60 and 120 min reperfusion. Blood samples were taken at different time intervals to test enzyme activities and biochemical alterations induced by reperfusion. At the end of each reperfusion period, the animals were killed by euthanasia and tissue samples were taken for histological examination and immunohistochemistry. RESULTS: Cell vacuolation, bleb formation and focal hepatitis were the most important changes occur during ischemia. While some changes including bleb formation were removed during reperfusion, other alterations including portal hepatitis, inflammation and the induction of apoptosis were seen during this stage. The occurrence of apoptosis, as demonstrated by apoptotic cells and bodies, was the most important histological change during reperfusion. The severity of apoptosis was dependent on the time of reperfusion, and by increasing the time of reperfusion, the numbers of apoptotic bodies was significantly enhanced. The amounts of lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, creatinine and urea were significantly increased in serum obtained from animals exposed to hepatic IR. CONCLUSION: Inflammation and subsequent apoptotic cell death were the most important changes in early-stage hepatic reperfusion injury, and the number of apoptotic bodies increased with time of reperfusion.

Dual lentivirus infection potentiates neuroinflammation and neurodegeneration: viral copassage enhances neurovirulence.

Infection by multiple lentiviral strains is recognized as a major driving force in the human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic, but the neuropathogenic consequences of multivirus infections remain uncertain. Herein, we investigated the neurovirulence and underlying mechanisms of dual lentivirus infections with distinct viral strains. Experimental feline immunodeficiency virus (FIV) infections were performed using cultured cells and an in vivo model of AIDS neuropathogenesis. Dual infections were comprised of two FIV strains (FIV-Ch and FIV-PPR) as copassaged or superinfected viruses, with subsequent outcome analyses of host immune responses, viral load, neuropathological features, and neurobehavioral performance. Dual infections of feline macrophages resulted in greater IL-1beta (interleukin-1beta), TNF-alpha (tumor necrosis factor alpha), and IDO (indoleamine 2,3-dioxygenase) expression and associated neurotoxic properties. FIV coinfection and sequential superinfection in vivo also induced greater IL-1beta, TNF-alpha, and IDO expression in the basal ganglia (BG) and cortex (CTX), compared to the monovirus- and mock-infected groups, although viral loads were similar in single virus- and dual virus-infected animals. Immunoblot analyses disclosed lower synaptophysin immunoreactivity in the CTX resulting from FIV super- and coinfections. Cholinergic and GABAergic neuronal injury was evident in the CTX of animals with dual FIV infections. With increased glial activation and neuronal loss in dual FIV-infected brains, immunohistochemical analysis also revealed elevated detection of cleaved caspase-3 in dysmorphic neurons, which was associated with worsened neurobehavioral abnormalities among animals infected with the copassaged viruses. Dual lentivirus infections caused an escalation in neuroinflammation and ensuing neurodegeneration, underscoring the contribution of infection by multiple viruses to neuropathogenesis.

Proteinase-activated receptor-2 exerts protective and pathogenic cell type-specific effects in Alzheimer's disease.

The proteinase-activated receptors (PARs) are a novel family of G protein-coupled receptors, and their effects in neurodegenerative diseases remain uncertain. Alzheimer's disease (AD) is a neurodegenerative disorder defined by misfolded protein accumulation with concurrent neuroinflammation and neuronal death. We report suppression of proteinase-activated receptor-2 (PAR2) expression in neurons of brains from AD patients, whereas PAR2 expression was increased in proximate glial cells, together with up-regulation of proinflammatory cytokines and chemokines and reduced IL-4 expression (p < 0.05). Glial PAR2 activation increased expression of formyl peptide receptor-2 (p < 0.01), a cognate receptor for a fibrillar 42-aa form of beta-amyloid (Abeta(1-42)), enhanced microglia-mediated proinflammatory responses, and suppressed astrocytic IL-4 expression, resulting in neuronal death (p < 0.05). Conversely, neuronal PAR2 activation protected human neurons against the toxic effects of Abeta(1-42) (p < 0.05), a key component of AD neuropathogenesis. Amyloid precursor protein-transgenic mice, displayed glial fibrillary acidic protein and IL-4 induction (p < 0.05) in the absence of proinflammatory gene up-regulation and neuronal injury, whereas PAR2 was up-regulated at this early stage of disease progression. PAR2-deficient mice, after hippocampal Abeta(1-42) implantation, exhibited enhanced IL-4 induction and less neuroinflammation (p < 0.05), together with improved neurobehavioral outcomes (p < 0.05). Thus, PAR2 exerted protective properties in neurons, but its activation in glia was pathogenic with secretion of neurotoxic factors and suppression of astrocytic anti-inflammatory mechanisms contributing to Abeta(1-42)-mediated neurodegeneration.