Optimization of primary hepatocyte isolation for the pharmacological characterization of metabotropic glutamate receptor (mGluR) s ubtype 5: A study on Reduction

To minimize the number of animals used during experiments, it is important to choose the suitable enzyme according to the final goal. In our work, we demonstrated the superiority of collagenase IV in the maintenance of functional transmembrane receptor, thus the pharmacological activity, in isolated rat hepatocytes

Liver plays a central role in asymmetric dimethylarginine-mediated organ injury

Asymmetric-dimethylarginine (ADMA) competes with L-arginine for each of the three isoforms of nitric oxide synthase: endothelial; neuronal; inducible. ADMA is synthesized by protein methyltransferases followed by proteolytic degradation. ADMA is metabolized to citrulline and dimethylamine, by dimethylarginine dimethylaminohydrolase (DDAH) and enters cells through cationic amino-acid transporters extensively expressed in the liver. The liver plays a crucial role in ADMA metabolism by DDAH-1 and, as has been recently demonstrated, it is also responsible for ADMA biliary excretion. A correlation has been demonstrated between plasma ADMA levels and the degree of hepatic dysfunction in patients suffering from liver diseases with varying aetiologies: plasma ADMA levels are increased in patients with liver cirrhosis, alcoholic hepatitis and acute liver failure. The mechanism by which liver dysfunction results in raised ADMA concentrations is probably due to impaired activity of DDAH due to severe inflammation, oxidative stress, and direct damage to DDAH. High plasma ADMA levels are also relevant as they are associated with the onset of multi-organ failure (MOF). Increased plasma concentration of ADMA was identified as an independent risk factor for MOF in critically-ill patients causing enhanced Intensive Care Unit mortality: a significant reduction in nitric oxide synthesis, leading to malperfusion in various organs, eventually culminating in multi organs dysfunction

Detailed Molecular Mechanisms Involved in Drug-Induced Non-Alcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis: An Update

: Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are some of the biggest public health challenges due to their spread and increasing incidence around the world. NAFLD is characterized by intrahepatic lipid deposition, accompanied by dyslipidemia, hypertension, and insulin resistance, leading to more serious complications. Among the various causes, drug administration for the treatment of numerous kinds of diseases, such as antiarrhythmic and antihypertensive drugs, promotes the onset and progression of steatosis, causing drug-induced hepatic steatosis (DIHS). Here, we reviewed in detail the major classes of drugs that cause DIHS and the specific molecular mechanisms involved in these processes. Eight classes of drugs, among the most used for the treatment of common pathologies, were considered. The most diffused mechanism whereby drugs can induce NAFLD/NASH is interfering with mitochondrial activity, inhibiting fatty acid oxidation, but other pathways involved in lipid homeostasis are also affected. PubMed research was performed to obtain significant papers published up to November 2021. The key words included the class of drugs, or the specific compound, combined with steatosis, nonalcoholic steatohepatitis, fibrosis, fatty liver and hepatic lipid deposition. Additional information was found in the citations listed in other papers, when they were not displayed in the original search

Changes in Glutathione Content in Liver Diseases: An Update

Glutathione (GSH), a tripeptide particularly concentrated in the liver, is the most important thiol reducing agent involved in the modulation of redox processes. It has also been demonstrated that GSH cannot be considered only as a mere free radical scavenger but that it takes part in the network governing the choice between survival, necrosis and apoptosis as well as in altering the function of signal transduction and transcription factor molecules. The purpose of the present review is to provide an overview on the molecular biology of the GSH system; therefore, GSH synthesis, metabolism and regulation will be reviewed. The multiple GSH functions will be described, as well as the importance of GSH compartmentalization into distinct subcellular pools and inter-organ transfer. Furthermore, we will highlight the close relationship existing between GSH content and the pathogenesis of liver disease, such as non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), chronic cholestatic injury, ischemia/reperfusion damage, hepatitis C virus (HCV), hepatitis B virus (HBV) and hepatocellular carcinoma. Finally, the potential therapeutic benefits of GSH and GSH-related medications, will be described for each liver disorder taken into account

Localization and role of metabotropic glutamate receptors subtype 5 in the gastrointestinal tract

Metabotropic glutamate receptor subtype 5 (mGluR5) is a Group I mGlu subfamily of receptors coupled to the inositol trisphosphate/diacylglycerol pathway. Like other mGluR subtypes, mGluR5s contain a phylogenetically conserved, extracellular orthosteric binding site and a more variable allosteric binding site, located on the heptahelical transmembrane domain. The mGluR5 receptor has proved to be a key pharmacological target in conditions affecting the central nervous system (CNS) but its presence outside the CNS underscores its potential role in pathologies affecting peripheral organs such as the gastrointestinal (GI) tract and accessory digestive organs such as the tongue, liver and pancreas. Following identification of mGluR5s in the mouth, various studies have subsequently demonstrated its involvement in mechanical allodynia, inflammation, pain and oral cancer. mGluR5 expression has also been identified in gastroesophageal vagal pathways. Indeed, experimental and human studies have demonstrated that mGluR5 blockade reduces transient lower sphincter relaxation and reflux episodes. In the intestine, mGluR5s have been shown to be involved in the control of intestinal inflammation, visceral pain and the epithelial barrier function. In the liver, mGluR5s have a permissive role in the onset of ischemic injury in rat and mice hepatocytes. Conversely, livers from mice treated with selective negative allosteric modulators and mGluR5 knockout mice are protected against ischemic injury. Similar results have been observed in experimental models of free-radical injury and in vivo mouse models of acetaminophen intoxication. Finally, mGluR5s in the pancreas are associated with insulin secretion control. The picture is, however, far from complete as the review attempts to establish in particular as regards identifying specific targets and innovative therapeutic approaches for the treatment of GI disorders

Changes in ADMA/DDAH Pathway after Hepatic Ischemia/Reperfusion Injury in Rats: The Role of Bile

We investigated the effects of hepatic ischemia/reperfusion (I/R) injury on asymmetric dimethylarginine (ADMA, a nitric oxide synthase inhibitor), protein methyltransferase (PRMT) and dimethylarginine dimethylaminohydrolase (DDAH) (involved, resp., in ADMA synthesis and degradation), and the cationic transporter (CAT). Male Wistar rats were subjected to 30 or 60 min hepatic ischemia followed by 60 min reperfusion. ADMA levels in serum and bile were determined. Tissue ADMA, DDAH activity, DDAH-1 and CAT-2 protein, DDAH-1 and PRMT-1 mRNA expression, GSH/GSSG, ROS production, and lipid peroxidation were detected. ADMA was found in bile. I/R increased serum and bile ADMA levels while an intracellular decrease was detected after 60 min ischemia. Decreased DDAH activity, mRNA, and protein expression were observed at the end of reperfusion. No significant difference was observed in GSH/GSSG, ROS, lipid peroxidation, and CAT-2; a decrease in PRMT-1 mRNA expression was found after I/R. Liver is responsible for the biliary excretion of ADMA, as documented here for the first time, and I/R injury is associated with an oxidative stress-independent alteration in DDAH activity. These data are a step forward in the understanding of the pathways that regulate serum, tissue, and biliary levels of ADMA in which DDAH enzyme plays a crucial role

Associations between serum trace elements and inflammation in two animal models of nonalcoholic fatty liver disease.

BackgroundThe comparison of hepatic steatosis animal models has allowed the understanding of mechanisms involved in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) and the progression to nonalcoholic steatohepatitis (NASH). We investigated the changes in serum levels of trace elements and inflammation markers in fatty livers using two rat models of NAFLD, the methionine and choline deficient (MCD) diet model and Obese-Zucker rats.Material and methodsNAFLD was induced in male Wistar rats by 3-week MCD diet administration, after which, blood samples were collected. 12-week old Obese (fa/fa) and Lean (fa/-) male Zucker rats were also used. Serum levels of hepatic enzymes, Urea, Uric acid, Ca2+, Cl, Fe, K, Na, Mg and Zn were quantified, as well as the inflammation markers TNF-alpha, IL-1beta and IL-6.ResultsIn MCD rats, a serum increase in Cl, Mg and Na and a decrease in Ca2+, Zn were detected in comparison with control rats. An increase in only serum Ca2+ was found in Obese-Zucker rats. In MCD rat serum, Zn was inversely correlated with IL-1beta, IL-6, TNF-alpha, Urea and Uric Acid; Ca2+ was inversely correlated with IL-1beta, IL-6 and Urea; Cl and Mg were directly correlated with Uric Acid and Urea, respectively. In Obese-Zucker rats, Cl and IL-1beta were inversely correlated, whereas Ca2+ and Urea where directly correlated, as well Fe and TNF-alpha.ConclusionsThe serum concentrations of trace elements change significantly only in MCD rats, which spontaneously progress to NASH. The causes of these changes may be a result of defense strategies of the organism, which is regulated by immunoregulatory cytokines. These results might suggest that the impairment of trace element status should be taken into account when the effectiveness of a pharmacological treatment is under evaluation

Fluorescence excitation properties of bilirubin in solution and in serum

The bichromophore nature of bilirubin explains the presence of at least two partially overlaying bands in both absorption and fluorescence emission spectra, and accounts for interchromophore exciton transfer events responsible for the emission sensitivity to the molecular environment and excitation wavelength. These concepts were likely responsible for the previously reported good yield of the unexpected remarkable bilirubin fluorescence emission under excitation at 366 nm, at which bilirubin absorption is very low. In this connection, aim of this work is to further investigate bilirubin spectral excitation properties and their diagnostic potential, until now poorly considered. Fluorescence excitation spectra of pure bilirubin in solution with solubilizing agents observed at 520 and 570 nm showed a wide region in the 430-510 nm range, similar to the absorption profile. In addition, an excitation band centered at about 400 nm was detected. Comparable excitation features were detected in rat serum. The 430-510 nm excitation region was well separated from a main band at shorter wavelength, ascribable to other endogenous fluorophores, with a shoulder at about 400 nm which was also easily discriminated by spectral fitting analysis. The bands ascribable to bilirubin showed changes of their relative contribution to the overall spectral region after liver ischemia/reperfusion, comparable to bilirubin biochemical data. Excitation spectra proved to discriminate the fluorescence of serum bilirubin at levels much lower than emission spectra, opening promising perspectives to improve the real time fluorescence analysis of crude serum in the absence of any exogenous labelling agent, and advance the diagnostic application of optical-biopsy in experimental hepatology and biomedicine

Spectrofluorometric Analysis of Autofluorescing Components of Crude Serum from a Rat Liver Model of Ischemia and Reperfusion

Autofluorescence (AF) of crude serum was investigated with reference to the potential of its intrinsic AF biomarkers for the noninvasive diagnosis of liver injury. Spectral parameters of pure compounds representing retinol (vitamin A) and fluorescing free fatty acids were characterized by spectrofluorometry, to assess spectral parameters for the subsequent AF analysis of serum, collected from rats undergoing liver ischemia/reperfusion (I/R). Differences in AF spectral profiles detected between control and I/R were due to the increase in the AF components representing fatty acids in I/R serum samples. No significant changes occurred for retinol levels, consistently with the literature reporting that constant retinol levels are commonly observed in the blood, except for malnutrition or chronic severe liver disease. Conversely, fatty acids, in particular arachidonic and linoleic acid and their derivatives, act as modulating agents in inflammation, representing both a protective and damaging response to stress stimuli. The biometabolic and pathophysiological meaning of serum components and the possibility of their direct detection by AF spectrofluorometry open up interesting perspectives for the development of AF serum analysis, as a direct, cost effective, supportive tool to assess liver injury and related systemic metabolic alterations, for applications in experimental biomedicine and foreseen translation to the clinics