Background & Aims Alcohol abuse is a major cause of liver injury. The pathologic features of alcoholic liver disease develop over prolonged periods, yet the cellular defense mechanisms against the ...detrimental effects of alcohol are not well understood. We investigated whether macroautophagy, an evolutionarily conserved cellular mechanism that is commonly activated in response to stress, could protect liver cells from ethanol toxicity. Methods Mice were acutely given ethanol by gavage. The effects of ethanol on primary hepatocytes and hepatic cell lines were also studied in vitro. Results Ethanol-induced macroautophagy in the livers of mice and cultured cells required ethanol metabolism, generation of reactive oxygen species, and inhibition of mammalian target of rapamycin signaling. Suppression of macroautophagy with pharmacologic agents or small interfering RNAs significantly increased hepatocyte apoptosis and liver injury; macroautophagy therefore protected cells from the toxic effects of ethanol. Macroautophagy induced by ethanol seemed to be selective for damaged mitochondria and accumulated lipid droplets, but not long-lived proteins, which could account for its protective effects. Increasing macroautophagy pharmacologically reduced hepatotoxicity and steatosis associated with acute ethanol exposure. Conclusions Macroautophagy protects against ethanol-induced toxicity in livers of mice. Reagents that modify macroautophagy might be developed as therapeutics for patients with alcoholic liver disease.
Chronic inflammation plays a critical role in the pathogenesis of atherosclerosis. Currently, the mechanism(s) by which inflammation contributes to this disease are not entirely understood. ...Inflammation is known to induce oxidative stress, which can lead to lipid peroxidation. Lipid peroxidation can result in the production of reactive by-products that can oxidatively modify macromolecules including DNA, proteins, and lipoproteins. A major reactive by-product of lipid peroxidation is malondialdehyde (MDA). MDA can subsequently break down to form acetaldehyde (AA). These two aldehydes can covalently interact with the epsilon (ε)-amino group of lysines within proteins and lipoproteins leading to the formation of extremely stable, highly immunogenic malondialdehyde/acetaldehyde adducts (MAA-adducts). The aim of this study was to investigate the inflammatory response to MAA-modified human serum albumin (HSA-MAA) and low-density lipoprotein (LDL-MAA). We found that animals injected with LDL-MAA generate antibodies specific to MAA-adducts. The level of anti-MAA antibodies were further increased in an animal model of atherosclerosis fed a Western diet. An animal model that combined both high fat diet and immunization of MAA-modified protein resulted in a dramatic increase in antibodies to MAA-adducts and vascular fat accumulation compared with controls. In vitro exposure of endothelial cells and macrophages to MAA-modified proteins resulted in increased fat accumulation as well as increased expression of adhesion molecules and pro-inflammatory cytokines. The expression of cytokines varied between the different cell lines and was unique to the individual modified proteins. The results of these studies demonstrate that different MAA-modified proteins elicit unique responses in different cell types. Additionally, the presence of MAA-modified proteins appears to modulate cellular metabolism leading to increased accumulation of triglycerides and further progression of the inflammatory response.
Novel Antioxidant Properties of Doxycycline Clemens, Dahn L; Duryee, Michael J; Sarmiento, Cleofes ...
International journal of molecular sciences,
12/2018, Letnik:
19, Številka:
12
Journal Article
Recenzirano
Odprti dostop
Doxycycline (DOX), a derivative of tetracycline, is a broad-spectrum antibiotic that exhibits a number of therapeutic activities in addition to its antibacterial properties. For example, DOX has been ...used in the management of a number of diseases characterized by chronic inflammation. One potential mechanism by which DOX inhibits the progression of these diseases is by reducing oxidative stress, thereby inhibiting subsequent lipid peroxidation and inflammatory responses. Herein, we tested the hypothesis that DOX directly scavenges reactive oxygen species (ROS) and inhibits the formation of redox-mediated malondialdehyde-acetaldehyde (MAA) protein adducts. Using a cell-free system, we demonstrated that DOX scavenged reactive oxygen species (ROS) produced during the formation of MAA-adducts and inhibits the formation of MAA-protein adducts. To determine whether DOX scavenges specific ROS, we examined the ability of DOX to directly scavenge superoxide and hydrogen peroxide. Using electron paramagnetic resonance (EPR) spectroscopy, we found that DOX directly scavenged superoxide, but not hydrogen peroxide. Additionally, we found that DOX inhibits MAA-induced activation of Nrf2, a redox-sensitive transcription factor. Together, these findings demonstrate the under-recognized direct antioxidant property of DOX that may help to explain its therapeutic potential in the treatment of conditions characterized by chronic inflammation and increased oxidative stress.
Acute and chronic ethanol administration increase autophagic vacuole (i.e., autophagosome; AV) content in liver cells. This enhancement depends on ethanol oxidation. Here, we used parental ...(nonmetabolizing) and recombinant (ethanol-metabolizing) Hep G2 cells to identify the ethanol metabolite that causes AV enhancement by quantifying AVs or their marker protein, microtubule-associated protein 1
l
ight
c
hain
3-II
(LC3-II). The ethanol-elicited rise in LC3-II was dependent on ethanol dose, was seen only in cells that expressed alcohol dehydrogenase (ADH) and was augmented in cells that coexpressed cytochrome CYP2E1 (P450 2E1). Furthermore, the rise in LC3-II was inversely related to a decline in proteasome activity. AV flux measurements and colocalization of AVs with lysosomes or their marker protein
L
ysosomal-
A
ssociated
M
embrane
P
rotein 1 (LAMP1) in ethanol-metabolizing VL-17A cells (ADH
+
/CYP2E1
+
) revealed that ethanol exposure not only enhanced LC3-II synthesis but also decreased its degradation. Ethanol-induced accumulation of LC3-II in these cells was similar to that induced by the microtubule inhibitor, nocodazole. After we treated cells with either 4-methylpyrazole to block ethanol oxidation or GSH-EE to scavenge reactive species, there was no enhancement of LC3-II by ethanol. Furthermore, regardless of their ethanol-metabolizing capacity, direct exposure of cells to acetaldehyde enhanced LC3-II content. We conclude that both ADH-generated acetaldehyde and CYP2E1-generated primary and secondary oxidants caused LC3-II accumulation, which rose not only from enhanced AV biogenesis, but also from decreased LC3 degradation by the proteasome and by lysosomes.
Ethanol metabolism leads to the formation of acetaldehyde and malondialdehyde. Acetaldehyde and malondialdehyde can together form malondialdehyde–acetaldehyde (MAA) adducts. The role of alcohol ...dehydrogenase (ADH) and cytochrome P4502E1 (CYP2E1) in the formation of MAA-adducts in liver cells has been investigated.
Chronic ethanol treated VL-17A cells over-expressing ADH and CYP2E1 were pretreated with the specific CYP2E1 inhibitor — diallyl sulfide or ADH inhibitor — pyrazole or ADH and CYP2E1 inhibitor — 4-methyl pyrazole. Malondialdehyde, acetaldehyde or MAA-adduct formation was measured along with assays for viability, oxidative stress and apoptosis.
Inhibition of CYP2E1 with 10μM diallyl sulfide or ADH with 2mM pyrazole or ADH and CYP2E1 with 5mM 4-methyl pyrazole led to decreased oxidative stress and toxicity in chronic ethanol (100mM) treated VL-17A cells. In vitro incubation of VL-17A cell lysates with acetaldehyde and malondialdehyde generated through ethanol led to increased acetaldehyde (AA)-, malondialdehyde (MDA)-, and MAA-adduct formation. Specific inhibition of CYP2E1 or ADH and the combined inhibition of ADH and CYP2E1 greatly decreased the formation of the protein aldehyde adducts. Specific inhibition of CYP2E1 led to the greatest decrease in oxidative stress, toxicity and protein aldehyde adduct formation, implicating that CYP2E1 accelerates the formation of protein aldehyde adducts which can be an important mechanism for alcohol mediated liver injury.
CYP2E1-mediated metabolism of ethanol leads to increased AA-, MDA-, and MAA-adduct formation in liver cells which may aggravate liver injury.
Methotrexate (MTX) is an immunosuppressant commonly used for the treatment of autoimmune diseases. Recent observations have shown that patients treated with MTX also exhibit a reduced risk for the ...development of cardiovascular disease (CVD). Although MTX reduces systemic inflammation and tissue damage, the mechanisms by which MTX exerts these beneficial effects are not entirely known. We have previously demonstrated that protein adducts formed by the interaction of malondialdehyde (MDA) and acetaldehyde (AA), known as MAA-protein adducts, are present in diseased tissues of individuals with rheumatoid arthritis (RA) or CVD. In previously reported studies, MAA-adducts were shown to be highly immunogenic, supporting the concept that MAA-adducts not only serve as markers of oxidative stress but may have a direct role in the pathogenesis of inflammatory diseases. Because MAA-adducts are commonly detected in diseased tissues and are proposed to mitigate disease progression in both RA and CVD, we tested the hypothesis that MTX inhibits the generation of MAA-protein adducts by scavenging reactive oxygen species. Using a cell free system, we found that MTX reduces MAA-adduct formation by approximately 6-fold, and scavenges free radicals produced during MAA-adduct formation. Further investigation revealed that MTX directly scavenges superoxide, but not hydrogen peroxide. Additionally, using the Nrf2/ARE luciferase reporter cell line, which responds to intracellular redox changes, we observed that MTX inhibits the activation of Nrf2 in cells treated with MDA and AA. These studies define previously unrecognized mechanisms by which MTX can reduce inflammation and subsequent tissue damage, namely, scavenging free radicals, reducing oxidative stress, and inhibiting MAA-adduct formation.
The production of reactive oxygen species (ROS) can result in lipid peroxidation. Lipid peroxidation can, in turn, result in the formation of malondialdehyde (MDA), which can spontaneously breakdown forming acetaldehyde (AA). Malondialdehyde and acetaldehyde can interact to form the stable hybrid malondialdehyde-acetaldehyde adduct (MAA) on proteins and other macromolecules. Binding of MAA-adducted proteins to cells can lead to expression of pro-inflammatory cytokines, resulting in inflammation. MAA-adducted proteins are also themselves immunogenic and can initiate an inflammatory response. Importantly, methotrexate can scavenge free radicals. This may ameliorate the formation of lipid peroxides and the resulting formation of MDA and AA. Additionally, methotrexate can directly inhibit the formation of MAA-protein adducts. Thus, methotrexate can scavenge free radicals and inhibit the formation of MAA-adducts. These are previously undescribed mechanisms by which methotrexate may reduce inflammation and the tissue damage associated with chronic inflammatory diseases. Display omitted
•MTX is commonly used to treat RA and is being tested in CVD patients.•MDA and AA are produced during lipidperoxidation and can interact to form MAA-adducts.•MAA-adducts are found in atheromas and in diseased synovial tissue of RA patients.•MTX scavenges the free radical O2− and prevents the formation of MAA-adducts.•Scavenging O2− may be a mechanism by which MTX reduces inflammation and disease.
Persistent infection of hepatitis C virus (HCV) is one of the leading causes of end‐stage liver disease (ESLD), such as decompensated cirrhosis and liver cancer. Of particular note, nearly half of ...HCV‐infected people in the United States are reported to be heavy drinkers. This particular group of patients is known to rapidly progress to the ESLD. Although accelerated disease progression among alcohol abusers infected with HCV is clinically well recognized, the molecular pathophysiology behind this manifestation has not been well elucidated. Hepatocytes metabolize ethanol (EtOH) primarily through two steps of oxidative catabolism in which alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) play central roles. The ADH‐ALDH pathway also governs the metabolism of retinol (vitamin A) to its transcriptionally active metabolite, retinoic acid (RA). In this study, we defined that the ADH‐ALDH pathway serves as a potent antiviral host factor in hepatocytes, which regulates the expression of interferon (IFN)‐stimulated genes (ISGs) by biogenesis of RA. ISGs constitute over 300 antiviral effectors, which cooperatively govern intracellular antiviral innate immunity. Our study revealed that intracellular RA levels greatly influence ISG expression under basal conditions. Moreover, RA augments ISG induction in response to viral infection or exposure to IFN in a gene‐specific manner. Lastly, our results demonstrated that EtOH attenuates the antiviral function of the ADH‐ALDH pathway, which suggests the possibility that EtOH‐retinol metabolic competition is one of the molecular mechanisms for the synergism between HCV and alcohol abuse in liver disease progression. Conclusions: RA plays a critical role in the regulation of intracellular antiviral innate immunity in hepatocytes. (Hepatology 2016;63:1783‐1795)
The hepatic asialoglycoprotein receptor (ASGP-R) is posttranslationally modified in the Golgi en route to the plasma membrane, where it mediates clearance of desialylated serum glycoproteins. It is ...known that content of plasma membrane-associated ASGP-R is decreased after ethanol exposure, although the mechanisms remain elusive. Previously, we found that formation of compact Golgi requires dimerization of the largest Golgi matrix protein giantin. We hypothesize that ethanol-impaired giantin function may be related to altered trafficking of ASGP-R. Here we report that in HepG2 cells expressing alcohol dehydrogenase and hepatocytes of ethanol-fed rats, ethanol metabolism results in Golgi disorganization. This process is initiated by dysfunction of SAR1A GTPase followed by altered COPII vesicle formation and impaired Golgi delivery of the protein disulfide isomerase A3 (PDIA3), an enzyme that catalyzes giantin dimerization. Additionally, we show that SAR1A gene silencing in hepatocytes mimics the effect of ethanol: dedimerization of giantin, arresting PDIA3 in the endoplasmic reticulum (ER) and large-scale alterations in Golgi architecture. Ethanol-induced Golgi fission has no effect on ER-to-Golgi transportation of ASGP-R, however, it results in its deposition in cis-medial-, but not trans-Golgi. Thus, alcohol-induced deficiency in COPII vesicle formation predetermines Golgi fragmentation which, in turn, compromises the Golgi-to-plasma membrane transportation of ASGP-R.
Many medications exhibit clinical benefits that are unrelated to their primary therapeutic uses. In many cases, the mechanisms underpinning these pleotropic effects are unknown. Two commonly ...prescribed medications that exhibit pleotropic benefits in cardiovascular disease and other diseases associated with chronic inflammation are methotrexate (MTX) and doxycycline (DOX). The vast majority of cardiovascular disease is associated with atherosclerosis. Because atherosclerosis is a chronic inflammatory disease, possible mechanisms by which MTX and DOX reduce inflammation have been investigated. Interestingly, the primary structure of both of these medications contain aromatic phenolic rings, which resemble polyphenols that are known to possess antioxidant activity. Inflammation and oxidative stress are intimately related. Inflammation promotes oxidative stress, which in turn leads to further inflammation; in this way, oxidative stress and inflammation can establish a self-perpetuating cycle. It has been shown that MTX and DOX act as antioxidants and are capable of scavenging free radicals and the reactive oxygen species (ROS) superoxide (O2−). Furthermore, both MTX and DOX inhibit the formation of malondialdehyde acetaldehyde (MAA) adducts, products of oxidative stress and lipid peroxidation. Importantly, MAA-adducts are highly immunogenic and initiate inflammatory responses; thereby, fueling the cycle of inflammation and oxidative stress that results in chronic inflammation. Thus, reducing the formation of MAA-adducts may ameliorate inflammation that leads to ROS production and in this way, break the self-sustaining cycle of oxidative stress and inflammation. It is possible that the under-recognized antioxidant properties of these medications may be a mechanism by which they and other medications provide pleotropic benefit in the treatment of chronic inflammatory disease.
The association between alcohol consumption and pancreatitis has been recognized for over 100 years. Despite the fact that this association is well recognized, the mechanisms by which alcohol abuse ...leads to pancreatic tissue damage are not entirely clear. Alcohol abuse is the major factor associated with pancreatitis in the Western world. Interestingly, although most cases of chronic pancreatitis and many cases of acute pancreatitis are associated with alcohol abuse, only a small percentage of individuals w...
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