ABSTRACT
In recent years, the impact of lipotoxicity on male fertility has received extensive attention, especially on Sertoli cells (SCs). In SCs, energy metabolism is important as disorders of energy metabolism result in infertility eventually. However, the underlying mechanism of lipotoxicity on energy metabolism in SCs remains unknown. Advances in high-throughput metabolomics and lipidomics measurement platforms provide powerful tools to gain insights into complex biological systems. Here, we aimed to explore the potential molecular mechanisms of palmitic acid (PA) regulating energy metabolism in SCs based on metabolomics and lipidomics. The results showed that glucose metabolism-related metabolites were not significantly changed, which suggested that PA treatment had little effect on glucose metabolism and may not influence the normal energy supply from SCs to germ cells. However, fatty acid ß-oxidation was inhibited according to accumulation of medium- and long-chain acylcarnitines in cells. In addition, the pool of amino acids and the levels of most individual amino acids involved in the tricarboxylic acid (TCA) cycle were not changed after PA treatment in SCs. Moreover, PA treatment of SCs significantly altered the lipidome, including significant decreases in cardiolipin and glycolipids as well as remarkable increases in ceramide and lysophospholipids, which indicated that mitochondrial function was affected and apoptosis was triggered. The increased apoptosis rate of SCs was verified by elevated protein expression levels of Cleaved Caspase-3 and Bax as well as decreased Bcl-2 protein expression level. Together, these findings indicated that PA may result in mitochondrial dysfunction and increased apoptosis by inhibiting fatty acid ß-oxidation of SCs.
Subject(s)
Palmitic Acid , Sertoli Cells , Amino Acids/metabolism , Apoptosis , Cardiolipins/metabolism , Cardiolipins/pharmacology , Caspase 3/metabolism , Caspase 3/pharmacology , Ceramides/metabolism , Glucose/metabolism , Glycolipids/metabolism , Humans , Lysophospholipids/metabolism , Lysophospholipids/pharmacology , Male , Mitochondria/metabolism , Palmitic Acid/pharmacology , Tricarboxylic Acids/metabolism , Tricarboxylic Acids/pharmacology , bcl-2-Associated X Protein/metabolism , bcl-2-Associated X Protein/pharmacologyABSTRACT
Mitochondria are recognized as an important target organelle for the toxicity of nanomaterials. Although the toxic effects of silver nanoparticles (AgNPs) on mitochondria have been widely reported, the mechanism behind the toxicity remains unclear. In this study, the effects of two forms of silver (AgNPs and AgNO3) on carp gill mitochondria were investigated by analyzing the mitochondrial ultrastructure, physicochemical properties of mitochondrial membrane, and mitochondrial proteomics. After exposure of common carp to AgNPs (0.75 mg/L) and AgNO3 (0.05 mg/L) for 96 h, both forms of silver were shown to cause gill mitochondrial lesions, including irregular shape, loss of mitochondrial cristae, and increased mitochondrial membrane permeability. Proteomics results revealed that AgNPs and AgNO3 induced 362 and 297 differentially expressed proteins (DEPs) in gill mitochondria, respectively. Among the DEPs, 244 were shared between AgNPs and AgNO3 treatments. These shared proteins were mainly distributed in the mitochondrial membrane and matrix, and were significantly enriched in the tricarboxylic acid (TCA) cycle and oxidative phosphorylation pathway. The functional annotation of DEPs induced by both silver forms was mainly involved in energy production and conversion. These results indicated that the toxic mechanism of AgNPs and AgNO3 on gill mitochondria were comparable and the two forms of silver caused mitochondrial dysfunction in fish gills by inhibiting the TCA cycle and disrupting the electron transport chain.
Subject(s)
Carps , Metal Nanoparticles , Water Pollutants, Chemical , Animals , Silver Nitrate/toxicity , Gills , Metal Nanoparticles/chemistry , Proteomics , Water Pollutants, Chemical/toxicity , Silver/toxicity , Silver/metabolism , Coloring Agents/metabolism , Coloring Agents/pharmacology , Tricarboxylic Acids/metabolism , Tricarboxylic Acids/pharmacologyABSTRACT
Herbacetin (HBN) is a glycosylated flavonoid, which possesses numerous pharmacological properties. Cyclophosphamide (CYC) is a chemotherapeutic drug that adversely affects the kidneys. The present investigation aimed to evaluate the curative potential of HBN against CYC-induced nephrotoxicity. Sprague Dawley rats (n = 48) were randomly divided into four groups: control (0.1% DMSO + food), CYC (150 mg/kg b.wt.), CYC+HBN (150 + 40 mg/kg b.wt.), and HBN (40mg/kg b.wt.). CYC treatment significantly decreased the activities of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GSR) while elevating the concentration of reactive oxygen species (ROS) and malondialdehyde (MDA). Treatment with HBN significantly recovered the activity of CAT, SOD, GPx, and GSR while reducing the concentrations of ROS and MDA. Moreover, an increase in the level of renal functional markers, including Urea, creatinine, kidney injury molecule-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL), and a decrease in creatinine clearance after CYC administration was recovered to control values by HBN treatment. Furthermore, HBN treatment normalized the increased levels of inflammatory markers such as nuclear factor kappa-B (NF-κB), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) after CYC administration. Besides, HBN administration increased the expression of anti-apoptotic markers (Bcl-2) while decreasing the apoptotic markers (Bax and Caspase-3). Furthermore, HBN decreased the activities of tricarboxylic acid (TCA) cycle enzymes (ICDH, αKGDH, SDH, and MDH) as well as renal mitochondrial respiratory-chain complexes (I-IV) and repolarized mitochondrial membrane potential (ΔΨm). Additionally, HBN administration significantly protected against renal histological damage induced by CYC. In conclusion, CYC-induced toxicity was effectively ameliorated by the HBN administration. These results indicate that HBN might be considered as a potential protective agent against nephrotoxicity. The observed protection may be due to its antioxidant, anti-inflammatory, and anti-apoptotic potential.
Subject(s)
NF-kappa B , Tumor Necrosis Factor-alpha , Animals , Anti-Inflammatory Agents/pharmacology , Antioxidants/metabolism , Antioxidants/pharmacology , Antioxidants/therapeutic use , Apoptosis , Caspase 3/metabolism , Catalase/metabolism , Creatinine/metabolism , Cyclooxygenase 2/metabolism , Cyclophosphamide/therapeutic use , Cyclophosphamide/toxicity , Dimethyl Sulfoxide/metabolism , Dimethyl Sulfoxide/pharmacology , Dimethyl Sulfoxide/therapeutic use , Flavonoids/pharmacology , Glutathione Peroxidase/metabolism , Glutathione Reductase/metabolism , Inflammation/chemically induced , Inflammation/drug therapy , Inflammation/metabolism , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Kidney , Lipocalin-2 , Malondialdehyde/metabolism , Mitochondria/metabolism , NF-kappa B/metabolism , Nitric Oxide Synthase Type II/metabolism , Oxidative Stress , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Superoxide Dismutase/metabolism , Tricarboxylic Acids/metabolism , Tricarboxylic Acids/pharmacology , Tricarboxylic Acids/therapeutic use , Tumor Necrosis Factor-alpha/metabolism , Urea , bcl-2-Associated X Protein/metabolismABSTRACT
This study aimed to evaluate the in vitro effect of the novel bioactive adhesive monomer CMET, a calcium salt of 4-methacryloxyethyl trimellitate acid (4-MET), on human dental pulp stem cells (hDPSCs) and its capacity to induce tertiary dentin formation in a rat pulp injury model. Aqueous solutions of four tested materials [4-MET, CMET, Ca(OH)2, and mineral trioxide aggregate (MTA)] were added to the culture medium upon confluence, and solvent (dH2O) was used as a control. Cell proliferation was assessed using the Cell Counting Kit-8 assay, and cell differentiation was evaluated by real-time quantitative reverse transcription-polymerase chain reaction. The mineralization-inducing capacity was evaluated using alizarin red S staining and an alkaline phosphatase activity assay. For an in vivo experiment, a mechanical pulp exposure model was prepared on Wistar rats; damaged pulp was capped with Ca(OH)2 or CMET. Cavities were sealed with composite resin, and specimens were assessed after 14 and 28 days. The in vitro results showed that CMET exhibited the lowest cytotoxicity and highest odontogenic differentiation capacity among all tested materials. The favorable outcome on cell mineralization after treatment with CMET involved p38 and c-Jun N-terminal kinases signaling. The nuclear factor kappa B pathway was involved in the CMET-induced mRNA expression of odontogenic markers. Similar to Ca(OH)2, CMET produced a continuous hard tissue bridge at the pulp exposure site, but treatment with only CMET produced a regular dentinal tubule pattern. The findings suggest that (1) the evaluated novel bioactive adhesive monomer provides favorable biocompatibility and odontogenic induction capacity and that (2) CMET might be a very promising adjunctive for pulp-capping materials.
Subject(s)
Dental Pulp/cytology , Dentin/cytology , Methacrylates/pharmacology , Odontoblasts/cytology , Odontogenesis , Regeneration , Stem Cells/cytology , Tricarboxylic Acids/pharmacology , Adhesives , Animals , Cell Differentiation , Cell Proliferation , Cells, Cultured , Dental Pulp/drug effects , Dental Pulp/metabolism , Dentin/drug effects , Dentin/metabolism , Male , Odontoblasts/drug effects , Odontoblasts/metabolism , Rats , Rats, Wistar , Signal Transduction , Stem Cells/drug effects , Stem Cells/metabolismABSTRACT
The squalene synthase inhibitor squalestatin 1 (Squal1) is a potent and efficacious inducer of CYP2B expression in primary cultured rat hepatocytes and rat liver. To determine whether Squal1 is also an inducer of human CYP2B, the effects of Squal1 treatment were evaluated in primary cultured human hepatocytes, differentiated HepaRG cells, and humanized mouse livers. Squal1 treatment did not increase CYP2B6 mRNA levels in human hepatocytes or HepaRG cells and only slightly and inconsistently increased CYP2B6 mRNA content in humanized mouse liver. However, treatment with farnesol, which mediates Squal1's effect on rat CYP2B expression, increased CYP2B6 mRNA levels in HepaRG cells expressing the constitutive androstane receptor (CAR), but not in cells with knocked-down CAR. To determine the impact of cholesterol biosynthesis inhibition on CAR activation, the effects of pravastatin (Prava) were determined on CITCO-mediated gene expression in primary cultured human hepatocytes. Prava treatment abolished CITCO-inducible CYP2B6 expression, but had less effect on rifampicin-mediated CYP3A4 induction, and CITCO treatment did not affect Prava-inducible HMG-CoA reductase (HMGCR) expression. Treatment with inhibitors of different steps of cholesterol biosynthesis attenuated CITCO-mediated CYP2B6 induction in HepaRG cells, and Prava treatment increased HMGCR expression and inhibited CYP2B6 induction with comparable potency. Transfection of HepG2 cells with transcriptionally active sterol regulatory element binding proteins (SREBPs) reduced CAR-mediated transactivation, and inducible expression of transcriptionally active SREBP2 attenuated CITCO-inducible CYP2B6 expression in HepaRG cells. These findings suggest that Squal1 does not induce CYP2B6 in human hepatocytes because Squal1's inhibitory effect on cholesterol biosynthesis interferes with CAR activation. SIGNIFICANCE STATEMENT: The cholesterol biosynthesis inhibitor squalestatin 1 induces rat hepatic CYP2B expression indirectly by causing accumulation of an endogenous isoprenoid that activates the constitutive androstane receptor (CAR). This study demonstrates that squalestatin 1 does not similarly induce CYP2B6 expression in human hepatocytes. Rather, inhibition of cholesterol biosynthesis interferes with CAR activity, likely by activating sterol regulatory element binding proteins. These findings increase our understanding of the endogenous processes that modulate human drug-metabolizing gene expression.
Subject(s)
Anticholesteremic Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cholesterol/biosynthesis , Constitutive Androstane Receptor/metabolism , Sterol Regulatory Element Binding Proteins/metabolism , Tricarboxylic Acids/pharmacology , Animals , Cell Line , Cytochrome P-450 CYP2B6/biosynthesis , Cytochrome P-450 CYP2D6/biosynthesis , Cytochrome P-450 CYP2D6/genetics , Farnesol/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Gene Knockdown Techniques , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Liver/drug effects , Liver/enzymology , Mice , Pravastatin/pharmacology , RatsABSTRACT
A stable preparation of agaricinic acid nanoparticles was obtained. The mean hydrodynamic size of nanoparticles according to photon correlation spectroscopy was 200 nm and zeta potential was -57 mV. Cytotoxic activity of agaricinic acid nanoparticles against human HepG2 hepatoma cells was evaluated. Nanoparticles with a low concentration of agaricinic acid stimulated and with high concentration - suppressed metabolic activity and viability of hepatoma cells. The EC50 for the stimulating effect was 32.8 µg/ml, and the IC50=602.1 mg/ml. The preparation of agaricinic acid nanoparticles can be used in medicine as a potential antitumor agent.
Subject(s)
Alkanes/pharmacology , Antineoplastic Agents/pharmacology , Coriolaceae/chemistry , Nanoparticles/chemistry , Tricarboxylic Acids/pharmacology , Alkanes/isolation & purification , Antineoplastic Agents/isolation & purification , Cell Survival/drug effects , Dose-Response Relationship, Drug , Fruiting Bodies, Fungal/chemistry , Hep G2 Cells , Humans , Particle Size , Tricarboxylic Acids/isolation & purificationABSTRACT
Caffeoylquinic acids are well known for their prominent antiviral activities. Beyond our expectations, we initially found 3,4,5-Tri-O-caffeoylquinic acid methyl ester (3,4,5-CQME) from L. japonica can facilitate HBV DNA and antigens secretion. This study aimed to investigate its underlying molecular mechanism. The results indicate that 3,4,5-CQME signally increased intracellular and secreted HBsAg levels by more than two times in HepG2.2.15 cells and HepAD38 cells. Furthermore, levels of HBeAg, HBV DNA and RNA were significantly enhanced by 3-day 3,4,5-CQME treatment; it didn't directly affect intracellular cccDNA amount, although it slightly increased cccDNA accumulation as a HBV DNA replication feedback. In addition, treatment with 3,4,5-CQME significantly induced HBx protein expression for viral replication. We utilized a phospho-antibody assay to profile the signal transduction change by 3,4,5-CQME to illuminate its molecular mechanism. The results indicate that treatment with 3,4,5-CQME activated AKT/mTOR, MAPK and NF-κB pathways verified by immunoblot. Moreover, 3,4,5-CQME upregulated the expression of nuclear transcriptional factors PGC1α and PPARα. In short, 3,4,5-CQME promotes HBV transcription and replication by upregulating HBx expression and activating HBV transcriptional regulation-related signals. As caffeoylquinic acids are widely present in traditional Chinese medicines, the risk of intaking caffeoylquinic acids-containing herbs for hepatitis B treatment requires more evaluation and further research.
Subject(s)
Hepatitis B virus/drug effects , Lonicera/chemistry , Quinic Acid/analogs & derivatives , Tricarboxylic Acids/pharmacology , Virus Replication/drug effects , Cell Line , Cell Survival/drug effects , DNA, Viral/metabolism , Flowers/chemistry , Hep G2 Cells , Hepatitis B/virology , Hepatitis B Antigens/metabolism , Hepatitis B e Antigens/metabolism , Humans , Mitogen-Activated Protein Kinases/metabolism , Monosaccharides/chemistry , Monosaccharides/isolation & purification , Monosaccharides/pharmacology , Plant Extracts/chemistry , Plant Extracts/pharmacology , Protein Serine-Threonine Kinases , Quinic Acid/chemistry , Quinic Acid/pharmacology , Signal Transduction/drug effects , Tricarboxylic Acids/isolation & purification , Up-Regulation/drug effectsABSTRACT
Invasive aspergillosis (IA) is a life-threatening disease impacting immunocompromised individuals. Standard treatments of IA, including polyenes and azoles, suffer from high toxicity and emerging resistance, leading to the need to develop new antifungal agents with novel mechanisms of action. Ergosterol biosynthesis is a classic target for antifungals, and squalene synthase (SQS) catalyzes the first committed step in ergosterol biosynthesis in Aspergillus spp. making SQS of interest in the context of antifungal development. Here, we cloned, expressed, purified and characterized SQS from the pathogen Aspergillus flavus (AfSQS), confirming that it produced squalene. To identify potential leads targeting AfSQS, we tested known squalene synthase inhibitors, zaragozic acid and the phosphonosulfonate BPH-652, finding that they were potent inhibitors. We then screened a library of 744 compounds from the National Cancer Institute (NCI) Diversity Set V for inhibition activity. 20 hits were identified and IC50 values were determined using dose-response curves. 14 compounds that interfered with the assay were excluded and the remaining 6 compounds were analyzed for drug-likeness, resulting in one compound, celastrol, which had an AfSQS IC50 value of 830â¯nM. Enzyme inhibition kinetics revealed that celastrol binds to AfSQS in a noncompetitive manner, but did not bind covalently. Since celastrol is also known to inhibit growth of the highly virulent Aspergillus fumigatus by inhibiting flavin-dependent monooxygenase siderophore A (SidA, under iron starvation conditions), it may be a promising multi-target lead for antifungal development.
Subject(s)
Antifungal Agents/pharmacology , Aspergillus flavus/enzymology , Enzyme Inhibitors/pharmacology , Farnesyl-Diphosphate Farnesyltransferase/antagonists & inhibitors , Farnesyl-Diphosphate Farnesyltransferase/metabolism , Aspergillosis/drug therapy , Aspergillosis/microbiology , Aspergillus flavus/genetics , Aspergillus flavus/metabolism , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cloning, Molecular , Farnesyl-Diphosphate Farnesyltransferase/genetics , Humans , Models, Molecular , Molecular Targeted Therapy , Pentacyclic Triterpenes , Tricarboxylic Acids/pharmacology , Triterpenes/pharmacologyABSTRACT
Geranylgeranoic acid (GGA) has been reported to induce autophagic cell death via upregulation of lipid-induced unfolded protein response in several human hepatoma-derived cell lines, and its 4,5-didehydro derivative has been developed as a preventive agent against second primary hepatoma in clinical trials. We have previously reported that GGA is a natural diterpenoid synthesized in several medicinal herbs. Here, we provide unequivocal evidence for de novo GGA biosynthesis in mammals. First, with normal male Wistar rats, the levels of GGA in liver were found to be far greater than those in other organs analyzed. Second, we demonstrated the metabolic GGA labeling from the 13C-labeled mevalonolactone in the human hepatoma-derived cell line, HuH-7. Isotopomer spectral analysis revealed that approximately 80% of the cellular GGA was newly synthesized from mevalonate (MVA) in 12 h and the acid picked up preexisting farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), suggesting that GGA is derived from FPP and GGPP through the MVA pathway. Third, zaragozic acid A, a squalene synthase inhibitor, induced dose-dependent upregulation of endogenous GGA content in HuH-7 cells and their concomitant cell death. These results strongly suggest that a cancer-preventive GGA is biosynthesized via the MVA pathway in mammals.
Subject(s)
Diterpenes/metabolism , Mevalonic Acid/metabolism , Animals , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Death/drug effects , Cell Line, Tumor , Male , Rats , Rats, Wistar , Tricarboxylic Acids/pharmacologyABSTRACT
Our previous studies have shown that ACPT-I [(1S, 3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid], a blood-brain barrier permeable agonist of group III metabotropic glutamate (mGlu) receptors, was neuroprotective against middle cerebral artery occlusion/reperfusion (MCAO/R) in normotensive rats. Preclinical studies are typically performed on healthy animals, whereas stroke patients predominately exhibit comorbidities, such as hypertension; therefore, in the present study, we investigated the effect of ACPT-I in spontaneously hypertensive rats (SHR) after MCAO/R. We examined the potential neuroprotective action of ACPT-I (30â¯mg/kg) when administered during occlusion or reperfusion via the assessment of not only the brain infarction volume but also motor (CatWalk gait analysis and open field test) and sensorimotor (vibrissae-evoked forelimb-placing test) functions following MCAO/R. We determined that ACPT-I not only reduced the cortico-striatal infarction but also improved several gait parameters (run speed, run and stand durations, swing speed and stride length) and mobility when administered 30â¯min after the start of the occlusion or 30â¯min after the start of reperfusion. Moreover, the sensorimotor function was improved in hypertensive rats treated with ACPT-I during occlusion. In conclusion, the current findings provide further evidence for the neuroprotective effects of ACPT-I against ischemic damage. These findings may have clinical implications because hypertension is an important risk factor for ischemic stroke.
Subject(s)
Brain Ischemia/drug therapy , Cyclopentanes/pharmacology , Essential Hypertension/drug therapy , Neuroprotective Agents/pharmacology , Stroke/drug therapy , Tricarboxylic Acids/pharmacology , Animals , Brain/drug effects , Brain/pathology , Brain Ischemia/complications , Brain Ischemia/pathology , Essential Hypertension/complications , Essential Hypertension/pathology , Gait/drug effects , Male , Random Allocation , Rats, Inbred SHR , Receptors, Metabotropic Glutamate/agonists , Sensation/drug effects , Stroke/complications , Stroke/pathologyABSTRACT
Lichens are a source of secondary metabolites which possess important biological activities, including antioxidant, antibacterial, anti-inflammatory, and cytotoxic effects. The anticancer activity of lichens was shown in many types of tumors, including colorectal cancers (CRC). Several studies revealed that the application of lichen extracts diminished the proliferation of CRC cells and induced apoptosis. Colon carcinogenesis is associated with aberrations in Wnt signaling. Elevated transcriptional activity of ß-catenin induces cell survival, proliferation, and migration. Thus, the inhibition of Wnt signaling is a promising therapeutic strategy in colorectal cancer. The aim of this study was the evaluation of the effects of lichen-derived depsides (atranorin, lecanoric acid, squamatic acid) and depsidones (physodic acid, salazinic acid) and a poly-carboxylic fatty acid-caperatic acid, on Wnt signaling in HCT116 and DLD-1 colorectal cancer cell lines. HCT116 cells were more sensitive to the modulatory effects of the compounds. PKF118-310, which was used as a reference ß-catenin inhibitor, dose-dependently reduced the expression of the classical ß-catenin target gene-Axin2 in both cell lines. Lecanoric acid slightly reduced Axin2 expression in HCT116 cells while caperatic acid tended to reduce Axin2 expression in both cell lines. Physodic acid much more potently decreased Axin2 expression in HCT116 cells than in DLD-1 cells. Physodic acid and caperatic acid also diminished the expression of survivin and MMP7 in a cell line and time-dependent manner. None of the compounds affected the nuclear translocation of ß-catenin. This is the first report showing the ability of caperatic acid and physodic acid to modulate ß-catenin-dependent transcription.
Subject(s)
Colorectal Neoplasms/metabolism , Dibenzoxepins/pharmacology , Lichens/chemistry , Tricarboxylic Acids/pharmacology , Wnt Signaling Pathway/drug effects , Axin Protein/metabolism , Cell Line, Tumor , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Dibenzoxepins/chemistry , Humans , Neoplasm Proteins/metabolism , Tricarboxylic Acids/chemistryABSTRACT
The production of amyloid-ß (Aß) is the key factor driving pathogenesis in Alzheimer's disease (AD). Increasing concentrations of Aß within the brain cause synapse degeneration and the dementia that is characteristic of AD. Here the factors that affect the release of disease-relevant forms Aß were studied in a cell model. 7PA2 cells expressing the human amyloid precursor protein released soluble Aß oligomers that caused synapse damage in cultured neurons. Supernatants from 7PA2 cells treated with the cholesterol synthesis inhibitor squalestatin contained similar concentrations of Aß42 to control cells but did not cause synapse damage in neuronal cultures. These supernatants contained reduced concentrations of Aß42 oligomers and increased concentrations of Aß42 monomers. Treatment of 7PA2 cells with platelet-activating factor (PAF) antagonists had similar effects; it reduced concentrations of Aß42 oligomers and increased concentrations of Aß42 monomers in cell supernatants. PAF activated cholesterol ester hydrolases (CEH), enzymes that released cholesterol from stores of cholesterol esters. Inhibition of CEH also reduced concentrations of Aß42 oligomers and increased concentrations of Aß42 monomers in cell supernatants. The Aß monomers produced by treated cells protected neurons against Aß oligomer-induced synapse damage. These studies indicate that pharmacological manipulation of cells can alter the ratio of Aß monomer:oligomer released and consequently their effects on synapses.
Subject(s)
Amyloid beta-Peptides/metabolism , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Enzyme Inhibitors/pharmacology , Sterol Esterase/antagonists & inhibitors , Synapses/drug effects , Tricarboxylic Acids/pharmacology , Animals , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , Down-Regulation/drug effects , Embryo, Mammalian , Humans , Mice , Neurons/drug effects , Neurons/metabolism , Polymers/metabolism , Synapses/metabolismABSTRACT
1,2,3,4-Butane tetracarboxylic acid (BTCA) has been considered as one of the most promising crosslinking agent to replace dimethylol dihydroxy ethylene urea (DMDHEU) for anti-wrinkle finishing on cotton fabrics. However, it could cause significant strength loss of the treated fabrics. In this study, a 5-(carbonyloxy succinic)-benzene-1,2,4-tricarboxylic acid (BSTA) was synthesized and applied as an effective crosslinking agent. The results show that fabrics treated with BSTA present the same or even better anti-wrinkle properties as that with BTCA. FTIR was employed to analyze ester bond formation process on cellulose. Finishing conditions such as agent concentration, curing temperature, and bath pH were also discussed to evaluate crosslinking effect of cellulose. BSTA, as a derivative of photo-active conjugated compound, can absorb ultraviolet lights and offer ultraviolet (UV) protective property on treated materials. The treated fabrics showed excellent UV protection performance due to the addition of aromatic conjugated system on fabrics.
Subject(s)
Cotton Fiber , Sunscreening Agents/pharmacology , Tricarboxylic Acids/chemistry , Tricarboxylic Acids/pharmacology , Chemistry Techniques, Synthetic , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Spectroscopy, Fourier Transform Infrared , Temperature , Ultraviolet Rays/adverse effectsABSTRACT
Squalene synthase inhibitors (SSIs), such as squalestatin 1 (SQ1), reduce cholesterol biosynthesis but cause the accumulation of isoprenoids derived from farnesyl pyrophosphate (FPP), which can modulate the activity of nuclear receptors, including the constitutive androstane receptor (CAR), farnesoid X receptor, and peroxisome proliferator-activated receptors (PPARs). In comparison, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (e.g., pravastatin) inhibit production of both cholesterol and nonsterol isoprenoids. To characterize the effects of isoprenoids on hepatocellular physiology, microarrays were used to compare orthologous gene expression from primary cultured mouse and rat hepatocytes that were treated with either SQ1 or pravastatin. Compared with controls, 47 orthologs were affected by both inhibitors, 90 were affected only by SQ1, and 51 were unique to pravastatin treatment (P < 0.05, ≥1.5-fold change). When the effects of SQ1 and pravastatin were compared directly, 162 orthologs were found to be differentially coregulated between the two treatments. Genes involved in cholesterol and unsaturated fatty acid biosynthesis were up-regulated by both inhibitors, consistent with cholesterol depletion; however, the extent of induction was greater in rat than in mouse hepatocytes. SQ1 induced several orthologs associated with microsomal, peroxisomal, and mitochondrial fatty acid oxidation and repressed orthologs involved in cell cycle regulation. By comparison, pravastatin repressed the expression of orthologs involved in retinol and xenobiotic metabolism. Several of the metabolic genes altered by isoprenoids were inducible by a PPARα agonist, whereas cytochrome P450 isoform 2B was inducible by activators of CAR. Our findings indicate that SSIs uniquely influence cellular lipid metabolism and cell cycle regulation, probably due to FPP catabolism through the farnesol pathway.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cholesterol/biosynthesis , Gene Expression Regulation/drug effects , Hepatocytes/drug effects , Hepatocytes/metabolism , Pravastatin/pharmacology , Terpenes/metabolism , Tricarboxylic Acids/pharmacology , Animals , Drug Synergism , Female , Male , Mice , Rats , Sequence Homology, Nucleic AcidABSTRACT
Skin produces cholesterol and a wide array of sterols and non-sterol mevalonate metabolites, including isoprenoid derivative farnesyl pyrophosphate (FPP). To characterize FPP action in epidermis, we generated transcriptional profiles of primary human keratinocytes treated with zaragozic acid (ZGA), a squalene synthase inhibitor that blocks conversion of FPP to squalene resulting in endogenous accumulation of FPP. The elevated levels of intracellular FPP resulted in regulation of epidermal differentiation and adherens junction signaling, insulin growth factor (IGF) signaling, oxidative stress response and interferon (IFN) signaling. Immunosuppressive properties of FPP were evidenced by STAT-1 downregulation and prominent suppression of its nuclear translocation by IFNγ. Furthermore, FPP profoundly downregulated genes involved in epidermal differentiation of keratinocytes in vitro and in human skin ex vivo. Elevated levels of FPP resulted in induction of cytoprotective transcriptional factor Nrf2 and its target genes. We have previously shown that FPP functions as ligand for the glucocorticoid receptor (GR), one of the major regulator of epidermal homeostasis. Comparative microarray analyses show significant but not complete overlap between FPP and glucocorticoid regulated genes, suggesting that FPP may have wider transcriptional impact. This was further supported by co-transfection and chromatin immunoprecipitation experiments where we show that upon binding to GR, FPP recruits ß-catenin and, unlike glucocorticoids, recruits co-repressor GRIP1 to suppress keratin 6 gene. These findings have many clinical implications related to epidermal lipid metabolism, response to glucocorticoid therapy as well as pleiotropic effects of cholesterol lowering therapeutics, statins. J. Cell. Physiol. 231: 2452-2463, 2016. © 2016 Wiley Periodicals, Inc.
Subject(s)
Cell Movement/drug effects , Epidermis/pathology , Inflammation/pathology , Oxidative Stress/drug effects , Polyisoprenyl Phosphates/pharmacology , Sesquiterpenes/pharmacology , Skin/metabolism , Adherens Junctions/metabolism , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Carrier Proteins/metabolism , Cell Differentiation/drug effects , Cell Movement/genetics , Cells, Cultured , Dexamethasone/pharmacology , Gene Expression Regulation/drug effects , Humans , Inflammation/genetics , Insulin-Like Growth Factor I/metabolism , Interferons/metabolism , Keratin-6/genetics , Keratin-6/metabolism , Keratinocytes/drug effects , Keratinocytes/metabolism , Models, Biological , NF-E2-Related Factor 2/metabolism , Nerve Tissue Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Oxidative Stress/genetics , Promoter Regions, Genetic/genetics , Signal Transduction/drug effects , Transcription, Genetic/drug effects , Tricarboxylic Acids/pharmacology , Wound Healing/drug effects , beta Catenin/metabolismABSTRACT
In the present study, we investigated the effect of ACPT-I [(1S, 3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid], a blood-brain-barrier permeable agonist of group III mGlu receptor, against oxygen-glucose deprivation (OGD)-evoked neuronal cell death in primary neuronal cell cultures and in the model of transient middle cerebral artery occlusion (MCAO) in rats. We found that ACPT-I (1-200 µM) in a concentration- and time-dependent way attenuated the OGD-induced neuronal cell damage, being also effective after a delayed application (30 min after OGD). The neuroprotective effects of ACPT-I were blocked by the group III mGlu receptor antagonist, (RS)-alpha-cyclopropyl-4-phosphonophenyl glycine (CPPG), and by the activator of cAMP-dependent PKA, 8-Bromo-cAMP, but not by an inhibitor of PI-3-K signaling pathway. Moreover, ACPT-I attenuated the OGD-induced calpain activity and glutamate release. In the in vitro study, we also demonstrated the neuroprotective potential of mGluR4 positive allosteric modulators (PAMs), PHCCC (30 µM) and VU0155041 (10 and 30 µM) and synergism in neuroprotective action of low concentrations of ACPT-I and mGluR4 PAMs suggesting an important role of mGluR4 activation in prevention of ischemic neuronal cell death. In the rat MCAO model, we demonstrated that ACPT-I (30 mg/kg) injected intraperitoneally either 30 min after starting MCAO or 30 min after beginning reperfusion not only diminished the infarction volume by about 30%, but also improved selected gait parameters (CatWalk analysis) and the mobility of animals in the open field test. In conclusion, our results indicate that ACPT-I may be not only neuroprotective against ischemic neuronal damage but may also diminish the postischemic functional deficits.
Subject(s)
Brain Ischemia/drug therapy , Cyclopentanes/therapeutic use , Excitatory Amino Acid Agonists/therapeutic use , Neuroprotective Agents/therapeutic use , Receptors, Metabotropic Glutamate/agonists , Stroke/drug therapy , Tricarboxylic Acids/therapeutic use , Animals , Brain Ischemia/metabolism , Cells, Cultured , Cyclopentanes/pharmacology , Disease Models, Animal , Excitatory Amino Acid Agonists/pharmacology , L-Lactate Dehydrogenase/metabolism , Male , Mice , Neurons/drug effects , Neurons/metabolism , Neuroprotective Agents/pharmacology , Rats , Rats, Sprague-Dawley , Stroke/metabolism , Tricarboxylic Acids/pharmacologyABSTRACT
Farnesyl pyrophosphate (FPP) is a branch-point intermediate in the mevalonate pathway that is normally converted mainly to squalene by squalene synthase in the first committed step of sterol biosynthesis. Treatment with the squalene synthase inhibitor squalestatin 1 (SQ1) causes accumulation of FPP, its dephosphorylated metabolite farnesol, and several oxidized farnesol-derived metabolites. In addition, SQ1 treatment of primary cultured rat hepatocytes increases CYP2B expression through a mechanism that requires FPP synthesis and activation of the constitutive androstane receptor (CAR). Because direct farnesol treatment also increases CYP2B expression, it seems likely that SQ1-mediated CAR activation requires FPP dephosphorylation to farnesol. The lipid phosphatase, phosphatidic acid phosphatase domain containing 2 (PPAPDC2), was recently reported to catalyze FPP dephosphorylation. We therefore determined the effect of overexpressing or knocking down PPAPDC2 on SQ1-mediated CAR activation in primary cultured rat hepatocytes. Cotransfection of rat hepatocytes with a plasmid expressing rat or human PPAPDC2 enhanced SQ1-mediated activation of a CAR-responsive reporter by 1.7- or 2.4-fold over the SQ1-mediated activation that was produced when hepatocytes were cotransfected with empty expression plasmid. Similarly, transduction of rat hepatocytes with a recombinant adenovirus expressing PPAPDC2 enhanced SQ1-mediated CYP2B1 mRNA induction by 1.4-fold over the induction that was seen in hepatocytes transduced with control adenovirus. Cotransfection with a short hairpin RNA targeting PPAPDC2 reduced SQ1-mediated CAR activation by approximately 80% relative to the activation that occurred in hepatocytes transfected with nontargeting short hairpin RNA. These results indicate that PPAPDC2 plays an important role in SQ1-mediated CAR activation, most likely by catalyzing the conversion of FPP to farnesol.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Hepatocytes/drug effects , Hepatocytes/metabolism , Phosphatidate Phosphatase/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Tricarboxylic Acids/pharmacology , Animals , Cells, Cultured , Constitutive Androstane Receptor , Cytochrome P-450 CYP2B1/metabolism , Humans , Male , Polyisoprenyl Phosphates/metabolism , Protein Structure, Tertiary , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Sesquiterpenes/metabolism , Transfection/methodsABSTRACT
Cytosolic sulfotransferase 1C2 (SULT1C2) is expressed in the kidney, stomach, and liver of rats; however, the mechanisms regulating expression of this enzyme are not known. We evaluated transcriptional regulation of SULT1C2 by mevalonate (MVA)-derived intermediates in primary cultured rat hepatocytes using several cholesterol synthesis inhibitors. Blocking production of mevalonate with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin (30 µM), reduced SULT1C2 mRNA content by â¼40% whereas the squalene synthase inhibitor squalestatin (SQ1, 0.1 µM), which causes accumulation of nonsterol isoprenoids, increased mRNA content by 4-fold. Treatment with MVA (10 mM) strongly induced SULT1C2 mRNA by 12-fold, and this effect was blocked by inhibiting squalene epoxidase but not by more distal cholesterol inhibitors, indicating the effects of MVA are mediated by postsqualene metabolites. Using rapid amplification of cDNA ends (RACE), we characterized the 5' end of SULT1C2 mRNA and used this information to generate constructs for promoter analysis. SQ1 and MVA increased reporter activity by â¼1.6- and 3-fold, respectively, from a construct beginning 49 base pairs (bp) upstream from the longest 5'-RACE product (-3140:-49). Sequence deletions from this construct revealed a hepatocyte nuclear factor 1 (HNF1) element (-2558), and mutation of this element reduced basal (75%) and MVA-induced (30%) reporter activity and attenuated promoter activation following overexpression of HNF1α or 1ß. However, the effects of SQ1 were localized to a more proximal promoter region (-281:-49). Collectively, our findings demonstrate that cholesterol biosynthetic intermediates influence SULT1C2 expression in rat primary hepatocytes. Further, HNF1 appears to play an important role in mediating basal and MVA-induced SULT1C2 transcription.
Subject(s)
Cholesterol/biosynthesis , Gene Expression Regulation, Enzymologic/physiology , Hepatocytes/enzymology , Sulfotransferases/biosynthesis , Sulfotransferases/genetics , Animals , Anticholesteremic Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Genes, Reporter , Hepatocyte Nuclear Factor 1/genetics , Hepatocyte Nuclear Factor 1/metabolism , Hepatocytes/drug effects , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Male , Mevalonic Acid/pharmacology , Primary Cell Culture , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Squalene Monooxygenase/antagonists & inhibitors , Sulfotransferases/drug effects , Transfection , Tricarboxylic Acids/pharmacologyABSTRACT
Previous studies have shown that fosmidomycin, risedronate, and nerolidol exert antimalarial activity in vitro. We included squalestatin, an inhibitor of the isoprenoid metabolism in Erwinia uredovora, and found that combinations of compounds which act on different targets of the plasmodial isoprenoid pathway possess important supra-additivity effects.
Subject(s)
Antimalarials/pharmacology , Biosynthetic Pathways/drug effects , Plasmodium falciparum/drug effects , Terpenes/metabolism , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Drug Interactions , Fosfomycin/analogs & derivatives , Fosfomycin/pharmacology , Malaria/drug therapy , Parasitic Sensitivity Tests , Plasmodium falciparum/metabolism , Risedronic Acid/pharmacology , Sesquiterpenes/pharmacology , Tricarboxylic Acids/pharmacologyABSTRACT
Constitutive androstane receptor (CAR) is one of the principal regulators of hepatic cytochrome P450s (CYPs) 3A (CYP3A). cDNA-mediated expression of a mature rat CAR (rCAR) into rat hepatoma cells induced CYP3A1 and CYP2B mRNAs. Aberrant rCAR failed in these inductions. Three important human CYP3A4 regulatory elements (REs), proximal ER6 (proER6), xenobiotic responsive enhancer module (XREM) and constitutive liver enhancer module (CLEM), support constitutive and inducible expression of CYP3As mediated by CAR and pregnane X receptor (PXR). NHR-scan software predicted proER6, XREM and CLEM at -255 b, -8 kb and -11.5 kb, respectively of CYP3A4, but neither XREM nor CLEM was predicted in rat CYP3A. A luciferase reporter construct carrying a 5'-flanking sequence of CYP3A1 (-31,739 to -31,585 from its transcription initiation site) revealed important for the rCAR-dependent transactivation of CYP3A1. This region includes two putative binding motifs of nuclear receptors (DR4 and DR2), a putative hepatocyte nuclear factor-1 binding motif (HNF1), nuclear factor-kappa B binding motif (NFκB), activator protein 1 binding motif (AP-1), and ecotropic viral integration site 1 binding motif (Evi1). We hereby conclude DR4 and/or DR2 motifs being primarily responsible and HNF1 being synergistically functioning elements for the rCAR-mediated transcription of CYP3A1.