Intracellular Adenosine Research Articles

Blue Whiting (Micromesistius poutassou) Protein Hydrolysates Increase GLP-1 Secretion and Proglucagon Production in STC-1 Cells Whilst Maintaining Caco-2/HT29-MTX Co-Culture Integrity.

Inducing the feeling of fullness via the regulation of satiety hormones presents an effective method for reducing excess energy intake and, in turn, preventing the development of obesity. In this study, the ability of blue whiting soluble protein hydrolysates (BWSPHs) and simulated gastrointestinal digested (SGID) BWSPHs, to modulate the secretion and/or production of satiety hormones, such as glucagon-like peptide-1 (GLP-1), cholecystokinin (CCK) and peptide YY (PYY), was assessed in murine enteroendocrine STC-1 cells. All BWSPHs (BW-SPH-A to BW-SPH-F) (1.0% w/v dw) increased active GLP-1 secretion and proglucagon production in STC-1 cells compared to the basal control (Krebs–Ringer buffer) (p < 0.05). The signaling pathway activated for GLP-1 secretion was also assessed. A significant increase in intracellular calcium levels was observed after incubation with all BWSPHs (p < 0.05) compared with the control, although none of the BWSPHs altered intracellular cyclic adenosine monophosphate (cAMP) concentrations. The secretagogue effect of the leading hydrolysate was diminished after SGID. Neither pre- nor post-SGID hydrolysates affected epithelial barrier integrity or stimulated interleukin (IL)-6 secretion in differentiated Caco-2/HT-29MTX co-cultured cells. These results suggest a role for BWSPH-derived peptides in satiety activity; however, these peptides may need to be protected by some means to avoid loss of activity during gastrointestinal transit.

Antibacterial Effect of Oregano Essential Oil against Vibrio vulnificus and Its Mechanism.

Oregano essential oil (OEO) is an effective natural antibacterial agent, but its antibacterial activity against Vibrio vulnificus has not been widely studied. The aim of this study was to investigate the inhibitory effect and germicidal activity of OEO on V. vulnificus and its possible inhibition mechanism. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of OEO against four V. vulnificus strains (ATCC 27562 and three isolates from seafoods) were from 0.06 to 0.15 μL/mL. Compared with untreated bacteria, OEO reduced the concentration of intracellular adenosine triphosphate (ATP), hyperpolarized the cell membrane, increased the level of reactive oxygen species (ROS), and increased the concentration of intracellular malondialdehyde (MDA), but there was no obvious DNA damage at the OEO test concentration. It was indicated that OEO inactivated V. vulnificus by generating ROS which caused lipid peroxidation of cell membranes, thereby reducing the permeability and integrity of cell membranes and causing morphological changes to cells, but there was no obvious damage to DNA. In addition, OEO could effectively kill V. vulnificus in oysters at 25 °C, and the number of bacteria decreased by 48.2% after 0.09% OEO treatment for 10 h. The good inhibitory effect and bactericidal activity of OEO showed in this study, and the economy and security of OEO make it possible to apply OEO to control V. vulnificus contamination in oysters and other seafoods.

Increase in Ribosomal Fidelity Benefits Salmonella upon Bile Salt Exposure.

Translational fidelity is maintained by multiple quality control steps in all three domains of life. Increased translational errors (mistranslation) occur due to genetic mutations and external stresses. Severe mistranslation is generally harmful, but moderate levels of mistranslation may be favored under certain conditions. To date, little is known about the link between translational fidelity and host–pathogen interactions. Salmonella enterica can survive in the gall bladder during systemic or chronic infections due to bile resistance. Here we show that increased translational fidelity contributes to the fitness of Salmonella upon bile salt exposure, and the improved fitness depends on an increased level of intracellular adenosine triphosphate (ATP). Our work thus reveals a previously unknown linkage between translational fidelity and bacterial fitness under bile stress.

Agmatine mitigates palmitate (PA)-induced mitochondrial and metabolic dysfunction in microvascular endothelial cells.

Agmatine is an arginine metabolite that has neuroprotective capacity. Recently, it has been found to ameliorate atherosclerosis progression in rabbits. However, further molecular mechanisms of its anti-atherosclerotic properties remain unclear. High plasma levels of free fatty acids (FFAs) are an important risk factor for atherosclerosis due to their detrimental effects on vascular endothelial cells (ECs). Here, we used palmitate (PA), a kind of FFA, to induce endothelial dysfunction in human microvascular endothelial cells (HMECs) to determine the possible biological functions of agmatine. We found that PA caused ECs dysfunction in HMEC-1 cells, decreased cell viability, and elevated lactate dehydrogenase (LDH) release which could be reversed by agmatine treatment. Agmatine also improved the nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) activity in PA-induced HMEC-1 cells. The PA-caused mitochondrial dysfunction of HMEC-1 cells was diminished after agmatine treatment, as proven by the increased intracellular Adenosine Triphosphate (ATP) level, decreased mitochondrial reactive oxygen species (ROS) level, and increased mitochondrial oxygen consumption rate (OCR). Further, agmatine could alleviate PA-caused lipid accumulation with increased levels of Triglyceride (TG) and total cholesterol (TC) in HMEC-1 cells. Furthermore, Western blot analysis revealed that agmatine administration markedly decreased the expression levels of phosphorylated-AMP-activated protein kinase α (p-AMPKα), p-protein kinase B (p-AKT), and p-eNOS in PA-induced HMEC-1 cells. Inhibition of AMPK by compound C reversed the protective effects of agmatine on PA-induced HMEC-1 cells. Taken together, we hypothesize that agmatine mitigated PA-induced HMEC-1 cell dysfunction by alleviating mitochondrial and metabolic dysfunction via the AMPK/PI3K/Akt/eNOS signaling pathway.

Evaluation of the membrane damage mechanism of thymol against Bacillus cereus and its application in the preservation of skim milk

The goal of this study was to elucidate the mechanism by which thymol caused membrane damages in Bacillus cereus cells and the feasibility of its application in the preservation of skim milk. The minimum inhibitory concentration (MIC) of thymol was determined using the broth microdilution method. The membrane potentials, intracellular adenosine triphosphate (ATP) concentrations, and pH gradients across the membrane were characterized using fluorescence spectroscopy. The degree of membrane damage and membrane structural integrity were observed under an electron microscope. The quantity of B. cereus in skim milk at different temperatures and concentrations of thymol was calculated using the plate counting method. As a result, the MIC of thymol against B. cereus BNCC 103930 was 0.625 mg/mL. Thymol significantly induced B. cereus cell dysfunction, membrane potential hyperpolarization, pH gradient dissipation, the blockade of ATP synthesis, and intracellular ATP depletion. Confocal laser scanning microscopy (CLSM) and field emission gun scanning electron microscope (FEGSEM) assays revealed pronounced membrane damages of B. cereus after thymol administration. Of note, low thymol concentrations, together with low-temperature storage, have the potential to control B. cereus in skim milk. These results indicated that thymol could be used as a food additive preventing B. cereus food poisoning. • The antimicrobial mechanism of thymol against Bacillus cereus was described. • Cell dysfunction induced by thymol was characterized using fluorescent probes. • Thymol causes morphological damages in Bacillus cereus. • Thymol controls the growth of Bacillus cereus in skim milk stored at low temperature.

Novel anti-adipogenic effect of CF3-allylated indole in 3T3-L1 cells

Indole derivatives from various plants are known to have health benefits because of their anti-cancer, anti-oxidant, anti-inflammatory, and anti-tubercular effects. However, their effects on adipogenesis have not been fully elucidated yet. Herein, we show that a newly synthesized indole derivative, CF3-allylated indole, [(E)-1-(pyrimidin- 2-yl)-2-(4,4,4- trifluorobut-2-enyl)-1H-indole], effectively inhibits adipogenesis. We found that CF3-allylated indole inhibited lipid accumulation and suppressed the expression of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator activated receptor γ (PPARγ) in 3T3-L1 cells. The inhibitory effect of CF3-allylated indole primarily occurred at the early phase of adipocyte differentiation by increasing intracellular cyclic adenosine monophosphate (cAMP) levels and enhancing protein kinase A (PKA) and adenosine monophosphate-activated protein kinase (AMPK) signaling. Conversely, depletion of PKA or treatment with a protein kinase A inhibitor (H89) reversed such inhibitory effects of CF3-allylated indole on adipogenesis and PPARγ expression. These results suggest that CF3-allylated indole inhibits early stages of adipogenesis by increasing phosphorylation of PKA/AMPK, leading to decreased expression of adipogenic genes in 3T3-L1 cells. These results indicate that CF3-allylated indole has potential for controlling initial adipocyte differentiation in metabolic disorders such as obesity.

The Cell Protective Effect of Adenine on Hypoxia-Reoxygenation Injury through PPAR Delta Activation.

Ischemia followed by blood supply reperfusion in cardiomyocytes leads to an overproduction of free radicals and a rapid decrease of adenosine triphosphate concentration. The cardioprotective effect of a potential drug, adenine, was evaluated using H9c2 rat cardiomyoblasts. After hypoxia–reoxygenation (HR) treatment consisting of hypoxia for 21 h followed by reoxygenation for 6 h, it was revealed that pretreatment with 200 µM adenine for 2 h effectively prevented HR-induced cell death. Adenine also significantly decreased the production of reactive oxygen species and reduced cell apoptosis after HR injury. The antioxidant effect of adenine was also revealed in this study. Adenine pretreatment significantly reduced the expression of activating transcription factor 4 (ATF4) and glucose-regulated protein 78 (GRP78) proteins, and protein disulfide isomerase induced a protective effect on mitochondria after HR stimulation. Intracellular adenosine monophosphate-activated protein kinase, peroxisome proliferator-activated receptor delta (PPARδ), and perilipin levels were increased by adenine after HR stimulation. Adenine had a protective effect in HR-damaged H9c2 cells. It may be used in multiple preventive medicines in the future.

Bacteriostatic effects of high-intensity ultrasonic treatment on Bacillus subtilis vegetative cells

The bacteriostatic effects of high-intensity ultrasonic treatment (HIU) on Bacillus subtilis vegetative cells were evaluated, and the related mechanisms were explored using quantitative proteomics. The bacteriostatic effect of HIU on B. subtilis was proportional to the ultrasound treatment time and power, and the number of cultivable B. subtilis cells was decreased by approximately one log (at 270 W for 15 min) or half log (at 90 W for 25 min or 360 W for 5 min). Scanning electron microscopy images and gel electrophoresis results showed that HIU caused the destruction of the cell structure and intracellular protein leakage. In addition, HIU treatment at 270 W for 15 min resulted in the greatest decrease (84.22%) in intracellular adenosine triphosphate (ATP) content. The quantitative proteomic analysis showed that B. subtilis resisted the stress of HIU treatment by regulating the key proteins in physiological activities related to membrane transport (ATP-binding cassette [ABC] transporter), signal transduction (the two-component system), and energy metabolism (the tricarboxylic acid [TCA] cycle). HIU-induced physical damage, stress, and metabolic disorders were the main causes of the bacteriostatic effects on B. subtilis. These findings provide a foundation for the subsequent optimization and potential applications of HIU inactivation of B. subtilis.

Melanogenic effect of dersimelagon (MT-7117), a novel oral melanocortin 1 receptor agonist.

BackgroundThe activation of melanocortin 1 receptor (MC1R) on melanocytes stimulates the production of eumelanin. A tridecapeptide α melanocyte‐stimulating hormone (αMSH) is known to induce skin pigmentation.ObjectivesWe characterised the properties of a novel oral MC1R agonist dersimelagon (MT‐7117) with respect to its specific binding to MC1R, downstream signalling and eumelanin production in experimental models.MethodsThe competitive binding and production of intracellular cyclic adenosine 3′, 5′‐monophosphate in cells expressing recombinant melanocortin receptors were examined. A mouse melanoma cell line B16F1 was used for the evaluation of in vitro melanin production. The in vitro activity of MT‐7117 was determined with αMSH and [Nle4, D‐Phe7]‐αMSH (NDP‐αMSH) as reference comparators. The change of coat colour and skin pigmentation were evaluated after repeat administration of MT‐7117 by oral gavage to C57BL/6J‐Ay/+ mice and cynomolgus monkeys, respectively.ResultsMT‐7117 showed the highest affinity for human MC1R compared to the other melanocortin receptors evaluated and agonistic activity for human, cynomolgus monkey and mouse MC1R, with EC50 values in the nanomolar range. In B16F1 cells, MT‐7117 increased melanin production in a concentration‐dependent manner. In vivo, MT‐7117 (≥0.3 mg/kg/day p.o.) significantly induced coat colour darkening in mice. MT‐7117 (≥1 mg/kg/day p.o.) induced significant skin pigmentation in monkeys and complete reversibility was observed after cessation of its administration.ConclusionsMT‐7117 is a novel oral MC1R agonist that induces melanogenesis in vitro and in vivo, suggesting its potential application for the prevention of phototoxic reactions in patients with photodermatoses, such as erythropoietic protoporphyria and X‐linked protoporphyria.

PDGF Promotes Dermal Fibroblast Activation via a Novel Mechanism Mediated by Signaling Through MCHR1.

Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy and excessive fibrosis of the skin and internal organs. To this day, no effective treatments to prevent the progression of fibrosis exist, and SSc patients have disabilities and reduced life expectancy. The need to better understand pathways that drive SSc and to find therapeutic targets is urgent. RNA sequencing data from SSc dermal fibroblasts suggested that melanin-concentrating hormone receptor 1 (MCHR1), one of the G protein-coupled receptors regulating emotion and energy metabolism, is abnormally deregulated in SSc. Platelet-derived growth factor (PDGF)-BB stimulation upregulated MCHR1 mRNA and protein levels in normal human dermal fibroblasts (NHDF), and MCHR1 silencing prevented the PDGF-BB-induced expression of the profibrotic factors transforming growth factor beta 1 (TGFβ1) and connective tissue growth factor (CTGF). PDGF-BB bound MCHR1 in membrane fractions of NHDF, and the binding was confirmed using surface plasmon resonance (SPR). MCHR1 inhibition blocked PDGF-BB modulation of intracellular cyclic adenosine monophosphate (cAMP). MCHR1 silencing in NHDF reduced PDGF-BB signaling. In summary, MCHR1 promoted the fibrotic response in NHDF through modulation of TGFβ1 and CTGF production, intracellular cAMP levels, and PDGF-BB-induced signaling pathways, suggesting that MCHR1 plays an important role in mediating the response to PDGF-BB and in the pathogenesis of SSc. Inhibition of MCHR1 should be considered as a novel therapeutic strategy in SSc-associated fibrosis.

Clinical Value of Serum Mitochondria-Inhibiting Substances in Assessing Renal Hazards: A Community-Based Prospective Study in Korea.

BackgroundMitochondrial dysfunction is strongly associated with several kidney diseases. However, no studies have evaluated the potential renal hazards of serum mitochondria-inhibiting substance (MIS) and aryl hydrocarbon receptor ligand (AhRL) levels.MethodsWe used serum level of MIS and AhRL and clinical renal outcomes from 1,511 participants of a prospective community-based cohort in Ansung. MIS was evaluated based on intracellular adenosine triphosphate (MIS-ATP) or reactive oxygen species (MIS-ROS) generation measured using cell-based assays.ResultsDuring a mean 6.9-year follow-up, 84 participants (5.6%) developed a rapid decline in kidney function. In the lowest quartile group of MIS-ATP, patients were older and had metabolically deleterious parameters. In multivariate logistic regression analysis, higher MIS-ATP was associated with decreased odds for rapid decline: the odds ratio (OR) of 1% increase was 0.977 (95% confidence interval [CI], 0.957 to 0.998; P=0.031), while higher MIS-ROS was marginally associated with increased odds for rapid decline (OR, 1.014; 95% CI, 0.999 to 1.028; P=0.055). However, serum AhRL was not associated with the rapid decline in kidney function. In subgroup analysis, the renal hazard of MIS was particularly evident in people with hypertension and low baseline kidney function.ConclusionSerum MIS was independently associated with a rapid decline in kidney function, while serum AhRL was not. The clinical implication of renal hazard on serum MIS requires further evaluation in future studies.

Evaluation of the Membrane Damage Mechanism of Chlorogenic Acid against Yersinia enterocolitica and Enterobacter sakazakii and Its Application in the Preservation of Raw Pork and Skim Milk.

Plant-derived antimicrobial agents have adequate antimicrobial effects on food-borne pathogens, which can be used as food preservatives. The purpose of this study was to evaluate the antibacterial mechanism of chlorogenic acid (CA) against Yersinia enterocolitica and Enterobacter sakazakii. The minimum inhibitory concentration (MIC) of CA was determined by employing the broth microdilution method. Then, the cell function and morphological changes of Y. enterocolitica and E. sakazakii treated with CA were characterized. Finally, the growth inhibition models of Y. enterocolitica in raw pork and E. sakazakii in skim milk were constructed through the response surface methodology. The results demonstrated that CA has a satisfactory inhibitory effect against Y. enterocolitica and E. sakazakii with a MIC of 2.5 mg/mL. In addition, CA inhibited the growth of Y. enterocolitica and E. sakazakii via cell membrane damage, such as depolarization of the cell membrane, reduction in intracellular adenosine triphosphate (ATP) and pH levels, and destruction of cell morphology. Moreover, CA reduced two log cycles of Y. enterocolitica in raw pork and E. sakazakii in skim milk at a certain temperature. According to the corresponding findings, CA has the potential to be developed as an effective preservative to control Y. enterocolitica and E. sakazakii-associated foodborne diseases.

Structural and functional basis of the selectivity filter as a gate in human TRPM2 channel.

Transient receptor potential melastatin 2 (TRPM2), a Ca2+-permeable cation channel, is gated by intracellular adenosine diphosphate ribose (ADPR), Ca2+, warm temperature, and oxidative stress. It is critically involved in physiological and pathological processes ranging from inflammation to stroke to neurodegeneration. At present, the channel's gating and ion permeation mechanisms, such as the location and identity of the selectivity filter, remain ambiguous. Here, we report the cryo-electron microscopy (cryo-EM) structure of human TRPM2 in nanodisc in the ligand-free state. Cryo-EM map-guided computational modeling and patch-clamp recording further identify a quadruple-residue motif as the ion selectivity filter, which adopts a restrictive conformation in the closed state and acts as a gate, profoundly contrasting with its widely open conformation in the Nematostella vectensis TRPM2. Our study reveals the gating of human TRPM2 by the filter and demonstrates the feasibility of using cryo-EM in conjunction with computational modeling and functional studies to garner structural information for intrinsically dynamic but functionally important domains.

Palmatine sensitizes chemoresistant triple negative breast cancer cells via efflux inhibition of multidrug resistant protein 1.

Chemoresistance develops in triple negative breast cancer cells during prolonged therapy with chemotherapeutic drugs, diminishing the benefits of these drugs in breast cancer patients. Palmatine, a natural alkaloid extracted from several medicinal plants in West Africa, has been studied in vitro and found to be a promising anticancer agent for treating triple negative breast cancer. This study seeks to investigate the therapeutic potential of palmatine in sensitizing resistant 4T1 triple negative breast cancer cells to doxorubicin therapy. Cell viability, Hoechst dye membrane transport, intracellular doxorubicin accumulation and Adenosine triphosphatase (ATPase) assays were performed. From the results, the intracellular concentrations of the Hoechst dye and doxorubicin were substantially increased following treatment with palmatine. In addition, palmatine inhibited the doxorubicin efflux activity of the Multidrug resistance protein 1 (MDR1) by stimulating the activity of the ATPase enzymes. A combination of low concentrations of palmatine and verapamil best achieved these results. This research highlights the use of palmatine as a chemosensitizer to aid the treatment of triple negative breast cancer disease.

Identification of surrogates for rapid monitoring of microbial inactivation by ozone for water reuse: A pilot-scale study

The complex contaminants in reclaimed water sources and delayed feedback of microbial detection have brought tremendous challenges to disinfection process control. The identification of sensitive and online surrogates for indicating microbial inactivation efficacy is vital to evaluate and optimize the disinfection technologies and processes. This study analyzes the inactivation of microbial indicators during ozone disinfection at a pilot-scale study over 5 months. It is identified that total fluorescence (TF) intensity, ultraviolet absorbance at 254 nm (UV254) and intracellular adenosine triphosphate (cATP) concentration can act as surrogates in predicting microbial inactivation by ozone. Particularly, the empirical linear correlations for log removal values (LRV) of TF, UV254 and cATP concentration are developed for the inactivation of four widely applied microbial indicators, namely the total coliforms, fecal coliforms, Escherichia coli (E. coli) and heterotrophic plate count (HPC) (R2 = 0.86–0.96). Validation analyses are further conducted to verify the robustness and effectiveness of empirical models. Notably, TF is considered as the most efficient surrogate due to its high sensitivity, accuracy and reliability, whereas cATP concentration is an efficient supplement to directly reflect total microbial counts. The study is important to provide a rapid and reliable approach for ozone disinfection efficiency evaluation and prediction.

Potentiation of long-acting \u03b22-agonist and glucocorticoid responses in human airway epithelial cells by modulation of intracellular cAMP

IntroductionOver 300 million people in the world live with asthma, resulting in 500,000 annual global deaths with future increases expected. It is estimated that around 50–80% of asthma exacerbations are due to viral infections. Currently, a combination of long-acting beta agonists (LABA) for bronchodilation and glucocorticoids (GCS) to control lung inflammation represent the dominant strategy for the management of asthma, however, it is still sub-optimal in 35–50% of moderate-severe asthmatics resulting in persistent lung inflammation, impairment of lung function, and risk of mortality. Mechanistically, LABA/GCS combination therapy results in synergistic efficacy mediated by intracellular cyclic adenosine monophosphate (cAMP).HypothesisIncreasing intracellular cAMP during LABA/GCS combination therapy via inhibiting phosphodiesterase 4 (PDE4) and/or blocking the export of cAMP by ATP Binding Cassette Transporter C4 (ABCC4), will potentiate anti-inflammatory responses of mainstay LABA/GCS therapy.MethodsExpression and localization experiments were performed using in situ hybridization and immunohistochemistry in human lung tissue from healthy subjects, while confirmatory transcript and protein expression analyses were performed in primary human airway epithelial cells and cell lines. Intervention experiments were performed on the human airway epithelial cell line, HBEC-6KT, by pre-treatment with combinations of LABA/GCS with PDE4 and/or ABCC4 inhibitors followed by Poly I:C or imiquimod challenge as a model for viral stimuli. Cytokine readouts for IL-6, IL-8, CXCL10/IP-10, and CCL5/RANTES were quantified by ELISA.ResultsUsing archived human lung and human airway epithelial cells, ABCC4 gene and protein expression were confirmed in vitro and in situ. LABA/GCS attenuation of Poly I:C or imiquimod-induced IL-6 and IL-8 were potentiated with ABCC4 and PDE4 inhibition, which was greater when ABCC4 and PDE4 inhibition was combined. Modulation of cAMP levels had no impact on LABA/GCS modulation of Poly I:C-induced CXCL10/IP-10 or CCL5/RANTES.ConclusionModulation of intracellular cAMP levels by PDE4 or ABCC4 inhibition potentiates LABA/GCS efficacy in human airway epithelial cells challenged with viral stimuli. The data suggest further exploration of the value of adding cAMP modulators to mainstay LABA/GCS therapy in asthma for potentiated anti-inflammatory efficacy.

The protective effect of the PDE-4 inhibitor rolipram on intracerebral haemorrhage is associated with the cAMP/AMPK/SIRT1 pathway

Rolipram specifically inhibits phosphodiesterase (PDE) 4, thereby preventing inactivation of the intracellular second messenger cyclic adenosine monophosphate (cAMP). Rolipram has been shown to play a neuroprotective role in some central nervous system (CNS) diseases. However, the role of PDE4 and the potential protective effect of rolipram on the pathophysiological process of intracerebral haemorrhage (ICH) are still not entirely clear. In this study, a mouse model of ICH was established by the collagenase method. Rolipram reduced brain oedema, blood–brain barrier (BBB) leakage, neuronal apoptosis and inflammatory cytokine release and improved neurological function in our mouse model of ICH. Moreover, rolipram increased the levels of cAMP and silent information regulator 1 (SIRT1) and upregulated the phosphorylation of AMP-activated protein kinase (AMPK). Furthermore, these effects of rolipram could be reversed by the SIRT1 inhibitor sirtinol. In conclusion, rolipram can play a neuroprotective role in the pathological process of ICH by activating the cAMP/AMPK/SIRT1 pathway.

Research progress of phosphodiesterase inhibitors in inflammatory bowel disease treatment.

Inflammatory bowel disease is a recurrent chronic intestinal inflammatory disease with unknown etiology and no effective treatment. Phosphodiesterase (PDE) regulates a variety of physiological and pathophysiological processes by mediating the hydrolysis of intracellular second messengers cyclic adenosine monophosphate and cyclic guanosine monophosphate. In recent years, a series of researches suggest that PDE inhibitors such as several PDE4 inhibitors, PDE5 inhibitors (sildenafil, tadalafil and vardenafil), PDE3 inhibitors (cilostazol), PDE9 inhibitor (PF-04447943) and PDE3/PDE4 double inhibitor (pumafentrine) have ameliorating effect on experimental colitis in animals. In clinical trials, PDE4 inhibitor apremilast showed more therapeutic advantage than tetomilast. This article reviews the recent research progress of PDE inhibitors in treatment of inflammatory bowel disease.

Protease-Activated Receptor 2 (PAR-2) Antagonist AZ3451 Mitigates Oxidized Low-Density Lipoprotein (Ox-LDL)-Induced Damage and Endothelial Inflammation.

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial dysfunction plays an important role in the initiation and development of cardiovascular diseases, especially atherosclerosis (AS). Protease-activated receptor 2 (PAR-2) is a receptor for inflammatory proteases. However, the biological function of PAR-2 in endothelial cells and the pathophysiological process of AS are still unknown. In the current study, we found that treatment with ox-LDL increased the gene and protein expressions of PAR-2 in EA.hy926 endothelial cells. Interestingly, we found that antagonism of PAR-2 with its specific antagonist AZ3451 could ameliorate ox-LDL-induced lactate dehydrogenase (LDH) release. Treatment with AZ3451 considerably improved the mitochondrial function by restoring the mitochondrial membrane potential and increasing the levels of intracellular adenosine triphosphate (ATP). Also, we found that AZ3451 attenuated ox-LDL-induced expression and production of pro-inflammatory cytokines such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-8 (IL-8). Treatment with AZ3451 also mitigated the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). Notably, our results demonstrated that the presence of AZ3451 alleviated ox-LDL-induced expression of the endothelial cell adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1). Mechanistically, we found that AZ3451 attenuated ox-LDL-induced activation of nuclear factor-κB (NF-κB) by reducing the levels of intracellular NF-κB p65 and the luciferase activity of NF-κB promoter. Based on these findings, we conclude that PAR-2 might become a novel therapeutic target for the treatment of AS.

Assessing the Biocompatibility of Multi-Anchored Glycoconjugate Functionalized Iron Oxide Nanoparticles in a Normal Human Colon Cell Line CCD-18Co.

We have previously demonstrated that iron oxide nanoparticles with dopamine-anchored heterobifunctional polyethylene oxide (PEO) polymer, namely PEO-IONPs, and bio-functionalized with sialic-acid specific glycoconjugate moiety (Neu5Ac(α2-3)Gal(β1-4)-Glcβ-sp), namely GM3-IONPs, can be effectively used as antibacterial agents against target Escherichia coli. In this study, we evaluated the biocompatibility of PEO-IONPs and GM3-IONPs in a normal human colon cell line CCD-18Co via measuring cell proliferation, membrane integrity, and intracellular adenosine triphosphate (ATP), glutathione GSH, dihydrorhodamine (DHR) 123, and caspase 3/7 levels. PEO-IONPs caused a significant decrease in cell viability at concentrations above 100 μg/mL whereas GM3-IONPs did not cause a significant decrease in cell viability even at the highest dose of 500 μg/mL. The ATP synthase activity of CCD-18Co was significantly diminished in the presence of PEO-IONPs but not GM3-IONPs. PEO-IONPs also compromised the membrane integrity of CCD-18Co. In contrast, cells exposed to GM3-IONPs showed significantly different cell morphology, but with no apparent membrane damage. The interaction of PEO-IONPs or GM3-IONPs with CCD-18Co resulted in a substantial decrease in the intracellular GSH levels in a time- and concentration-dependent manner. Conversely, levels of DHR-123 increased with IONP concentrations. Levels of caspase 3/7 proteins were found to be significantly elevated in cells exposed to PEO-IONPs. Based on the results, we assume GM3-IONPs to be biocompatible with CCD-18Co and could be further evaluated for selective killing of pathogens in vivo.