Intracellular Oxidation Research Articles

Protective effects of dexamethasone combined with valsartan on chronic obstructive pulmonary disease in mice and its mechanism

Objective: To investigate the possible protective effects of combined dexamethasone and valsartan against cigarette induced chronic obstructive pulmonary disease (COPD) in mice. Methods: Forty C57BL/6 mice were randomly divided into control group, COPD group, dexamethasone treated group, valsartan treated group and dexamethasone + valsartan combined treatment group, with 8 mice in each group. Mice in COPD group were exposed to cigarette for 8 weeks. On the basis of cigarette exposure, mice in dexamethasone treated group were intraperitoneally injected with dexamethasone (2 mg / kg) before cigarette exposure for 5-8 weeks. Mice in valsartan treated group were intraperitoneally injected with valsartan (30 mg/kg) before cigarette exposure for 1-8 weeks. Dexamethasone (2 mg/kg) and valsartan (30 mg/kg) were injected intraperitoneally into mice in the dexamethasone + valsartan combined treatment group. After 8 weeks, the lung tissues and bronchoalveolar lavage fluid (BALF) of mice in each group were collected. The pathological score of lung tissue was evaluated. The activities of superoxide dismutase(SOD) and matrix metalloproteinase-9 (MMP-9), and the contents of malondialdehyde (MDA), intracellular adhesion molecule-1 (ICAM-1), C-reactive protein (CRP) and nitric oxide (NO) in BALF were determined. Results: Compared with the control group, COPD mice had emphysema and alveolar congestion, the levels of MDA, ICAM-1, MMP-9, CRP and lymphocytes in BALF were increased, while the levels of SOD, macrophages and NO were decreased (all P＜0.05). Compared with COPD group, there was no significant improvement in emphysema and alveolar congestion, the levels of SOD and NO in BALF were increased, and the levels of MDA, lymphocytes and macrophages were decreased in dexamethasone or valsartan group (all P＜0.05). Compared with dexamethasone or valsartan group, the dexamethasone + valsartan combined treatment was more effective in preventing pulmonary emphysema and alveolar congestion caused by cigarette smoke. The levels of MDA, ICAM-1, MMP-9, CRP and lymphocyte in BALF were decreased, while the levels of SOD, macrophage and NO were increased (all P＜0.05). Conclusion: Compared with dexamethasone or valsartan, dexamethasone combined with valsartan has a more effective protective effect in COPD mice by inhibiting oxidative stress and inflammation.

Selective Fluorescence Imaging of Cancer Cells Based on ROS-Triggered Intracellular Cross-Linking of Artificial Enzyme.

Inside living cells, regulation of catalytic activity of artificial enzymes remains challenging due to issues such as biocompatibility, efficiency, and stability of the catalyst, by which the practical applications of artificial enzymes have been severely hindered. Here, an artificial enzyme, PTT-SGH, with responsiveness to reactive oxygen species (ROS), was obtained by introducing a catalytic histidine residue to pentaerythritol tetra(3-mercaptopropionate) (PTT). The artificial enzyme formed large aggregates in cells via the intracellular ROS-mediated oxidation of thiol groups. The process was significantly facilitated in tumor cells because of the higher ROS concentration in the tumor microenvironment. The catalytic activity of this artificial enzyme was intensively enhanced through deprotonation of cross-linked PTT-SGH, which showed typical esterase activities. Selective fluorescence imaging of tumor cells was achieved using the artificial enzyme to trigger the cleavage of the ester bond of the caged fluorophore inside living cells.

Inhibition of interferon-gamma-stimulated melanoma progression by targeting neuronal nitric oxide synthase (nNOS)

Interferon-gamma (IFN-γ) is shown to stimulate melanoma development and progression. However, the underlying mechanism has not been completely defined. Our study aimed to determine the role of neuronal nitric oxide synthase (nNOS)-mediated signaling in IFN-γ-stimulated melanoma progression and the anti-melanoma effects of novel nNOS inhibitors. Our study shows that IFN-γ markedly induced the expression levels of nNOS in melanoma cells associated with increased intracellular nitric oxide (NO) levels. Co-treatment with novel nNOS inhibitors effectively alleviated IFN-γ-activated STAT1/3. Further, reverse phase protein array (RPPA) analysis demonstrated that IFN-γ induced the expression of HIF1α, c-Myc, and programmed death-ligand 1 (PD-L1), in contrast to IFN-α. Blocking the nNOS-mediated signaling pathway using nNOS-selective inhibitors was shown to effectively diminish IFN-γ-induced PD-L1 expression in melanoma cells. Using a human melanoma xenograft mouse model, the in vivo studies revealed that IFN-γ increased tumor growth compared to control, which was inhibited by the co-administration of nNOS inhibitor MAC-3-190. Another nNOS inhibitor, HH044, was shown to effectively inhibit in vivo tumor growth and was associated with reduced PD-L1 expression levels in melanoma xenografts. Our study demonstrates the important role of nNOS-mediated NO signaling in IFN-γ-stimulated melanoma progression. Targeting nNOS using highly selective small molecular inhibitors is a unique and effective strategy to improve melanoma treatment.

Characterizing oxiodoreductase Pyrox-D1

PYROX-D1 (pyridine oxidoreductase domain 1) is a human enzyme involved with intracellular oxidation management. Natural mutations in this protein have been identified and implicated with causing a newly defined form of limb-girdle muscular dystrophy. Pedigree analysis reveals an autosomal recessive inheritance pattern (requiring a mutated allele from both parents). The disease manifests with bulk muscle wasting, atypical connective tissue elasticity, progressive proximal and distal weakness, nasal speech, elevated creatine kinase, flat feet, and ambulation issues usually beginning between 20-30 years of age.

The toxicity of cooking oil fumes on human bronchial epithelial cells through ROS-mediated MAPK, NF-κB signaling pathways and NLRP3 inflammasome.

Cooking oil fumes (COFs) are the main pollutants in kitchen and indoor air, which threaten human health. Exposure to COFs may lead to respiratory diseases and impair pulmonary function. To investigate the toxicity of COFs on human bronchial epithelial cells (Beas-2B) and explore the underlying mechanisms, MTT assay was conducted to detect the viability of Beas-2B. Intracellular reactive oxygen species (ROS) levels and nitric oxide (NO) levels were determined with DCFH-DA assay and DAF-FM assay. The expression of genes involved in inflammation were measured with quantitative real-time PCR (qRT-PCR). The phosphorylation and the expression of proteins related to Mitogen-activated protein kinase (MAPK), NF-κB signaling pathways were measured with western blot. Our results revealed that COFs decreased cell viability, increased the ROS levels and NO levels and induced apoptosis in Beas-2B cells. The results of qRT-PCR and western blot showed that the expression of NLRP3, p65, iNOS, IL-1β, and the factors related to oxidative stress and inflammation increased, NF-κB signaling pathway and MAPK signaling pathway were activated. This study provided some useful information to evaluate the toxicity of COFs and revealed the possible mechanism for the damage on respiratory system induced by COFs.

Antioxidant activity assessment of Yingjisha sweet almond oil

SummarySweet almond is one of the main cash crops in Yingjisha, Xinjiang Autonomous Region, China. The composition of sweet almond oil (SAO) was analysed by gas chromatography‐mass spectrometry (GC‐MS). SAO’s antioxidant activities were investigated by measuring its scavenging ability with 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH), 2,2′‐Azino‐bis (3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS), hydroxyl radical scavenging assay and 6‐hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylic acid (Trolox) equivalent antioxidant capacity (TEAC) assay. Furthermore, in vivo antioxidant capacity was assessed using Saccharomyces cerevisiae model stimulated by hydrogen peroxide (H2O2) and carbon tetrachloride (CCl4). The results showed that SAO was mainly composed of methyl oleate (21.28%), methyl stearate (18.55%) and methyl palmitate (14.1%). In vitro antioxidant test results showed that compared with the scavenging three free radicals capacity of 1 mmol L−1 Trolox, the SAO needed to reach 5 mg mL−1, 20 mg mL−1 and less than 20 mg mL−1, respectively. In vivo experiments showed that SAO rescued the survival and inhibited intracellular oxidation and lipid peroxidation of Saccharomyces cerevisiae exposed to H2O2 and CCl4. Additionally, our results showed that the catalase encoded by the Gsh1 gene may be involved in the mechanism of action of SAO against intracellular oxidation. These findings suggest that SAO possesses antioxidant capacities, and it is recommended to develop dietary antioxidants based on SAO.

Thiol-Based Antioxidants and the Epithelial/Mesenchymal Transition in Cancer

Significance: The epithelial/mesenchymal transition (EMT) is commonly associated with tumor metastasis. Oxidative and nitrosative stress is maintained in cancer cells and is involved in the EMT. Cancer cells are endowed with high levels of enzymatic and nonenzymatic antioxidants, which counteract the effects of oxidative and nitrosative stress. Thiol-based antioxidant systems such as the thioredoxin/thioredoxin reductase (Trx/TrxR) and glutathione/glutaredoxin (GSH/Grx) are continually active in cancer cells, while the thioredoxin-interacting protein (Txnip), the negative regulator of the Trx/TrxR system, is downregulated. Recent Advances: Trx/TrxR and GSH/Grx systems play a major role in maintaining EMT signaling and cancer cell progression. Critical Issues: Enhanced stress conditions stimulated in cancer cells inhibit EMT signaling. The elevated expression levels of the Trx/TrxR and GSH/Grx systems in these cells provide the antioxidant protection necessary to guarantee the occurrence of the EMT. Future Directions: Elevation of the intracellular reactive oxygen species and nitric oxide concentrations in cancer cells has been viewed as a promising strategy for elimination of these cells. The development of inhibitors of GSH synthesis and of the Trx/TrxR system together with genetic-based strategies to enhance Txnip levels may provide the necessary means to achieve this goal. Antioxid. Redox Signal. 36, 1037-1050.

Trans‐ferulic acid attenuates hyperglycemia‐induced oxidative stress and modulates glucose metabolism by activating AMPK signaling pathway in vitro

Adenosine monophosphate-activated protein kinase (AMPK) is a potent metabolic regulator and an attractive target for antidiabetic activators. Here we report for the first that, trans-ferulic acid (TFA) is a potent dietary bioactive molecule of hydroxycinnamic acid derivative for the activation of AMPK with a maximum increase in phosphorylation (2.71/2.67±0.10; p<.001 vs. high glucose [HG] control) in hyperglycemia-induced human liver cells (HepG2) and rat skeletal muscle cells (L6), where HG suppresses the AMPK pathway. It was also observed that TFA increased activation of AMPK in a dose- and time-dependent manner and also increased the phosphorylation of acetyl-CoA carboxylase (ACC), suggesting that it may promotes fatty acid oxidation; however, pretreatment with compound C reversed the effect. In addition, TFA reduced the level of intracellular reactive oxygen species (ROS) and nitric oxide (NO) induced by hyperglycemia and subsequently increased the level of glutathione. Interestingly, TFA also upregulated the glucose transporters, GLUT2 and GLUT4, and inhibited c-Jun N-terminal protein kinase (JNK1/2) by decreasing the phosphorylation level in tested cells under HG condition. Our studies provide critical insights into the mechanism of action of TFA as a potential natural activator of AMPK under hyperglycemia. PRACTICAL APPLICATIONS: Hydroxycinnamic acid derivatives possess various pharmacological properties and are found to be one of the most ubiquitous groups of plant metabolites in almost all dietary sources. However, the tissue-specific role and its mechanism under hyperglycemic condition remain largely unknown. The present study showed that TFA is a potent activator of AMPK under HG condition and it could be used as a therapeutic agent against hyperglycemia in type 2 diabetes.

Impact of small fractions of abnormal erythrocytes on blood rheology.

Red blood cell (RBC) populations are inherently heterogeneous, given mature RBC lack the transcriptional machinery to re-synthesize proteins affected during in vivo aging. Clearance of older, less functional cells thus aids in maintaining consistent hemorheological properties. Scenarios occur, however, where portions of mechanically impaired RBC are re-introduced into blood (e.g., damaged from circulatory support, blood transfusion) and may alter whole blood fluid behavior. Given such perturbations are associated with poor clinical outcomes, determining the tolerable level of abnormal RBC in blood is valuable. Thus, the current study aimed to define the critical threshold of blood fluid properties to re-infused physically-impaired RBC. Cell mechanics of RBC were impaired through membrane cross-linking (glutaraldehyde) or intracellular oxidation (phenazine methosulfate). Mechanically impaired RBC were progressively re-introduced into the native cell population. Negative alterations of cellular deformability and high shear blood viscosity were observed following additions of only 1-5% rigidified RBC. Low-shear blood viscosity was conversely decreased following addition of glutaraldehyde-treated cells; high-resolution microscopy of these mixed cell populations revealed decreased capacity to form reversible aggregates and decreased aggregate size. Mixed RBC populations, when exposed to supraphysiological shear, presented with compounded mechanical impairment. Collectively, key determinants of blood flow behavior are sensitive to mechanical perturbations in RBC, even when only 1-5% of the cell population is affected. Given this fraction is well-below the volume of rigidified RBC introduced during circulatory support or transfusion practice, it is plausible that some adverse events following surgery and/or transfusion may be related to impaired blood fluidity.

Piracetam-induced neuroprotection in lipopolysaccharides-challenged EOC-20 cells and mouse brain via attenuating oxidative stress

Therapeutically, piracetam has been used for decades as a cognitive enhancer for memory- related neuronal disorders. The present study aimed to investigate the neuroprotective potential of piracetam on lipopolysaccharides (LPS)-induced neuronal deficit using both in-vitro and in-vivo experimental models. For the in-vitro analysis, EOC-20 murine microglial cells were induced with a neuronal toxicity of 100 µg/ml of LPS, and the formation of intracellular reactive oxygen species (ROS) and nitric oxide (NO) productions were determined. For in-vivo neuroprotective analysis, groups of mice were treated orally with two doses of piracetam (200 and 400 mg/kg) for 30 days. Neuronal toxicity was induced by four intraperitoneal injections of LPS (250 µg/kg/day). The malondialdehyde (MDA) level was measured for oxidative stress, and catalase reduced glutathione (GSH), glutathione reductase (GRD), and superoxide dismutase (SOD) levels were determined as the antioxidant parameters. The result of the cell viability study was that pre-treatment with piracetam significantly protected the LPS-induced cell loss, and attenuated the ROS generation and NO production in LPS-induced EOC-20 cells. Moreover, the treatment of piracetam significantly reduced the MDA levels and improved catalase, GSH, GRD, and SOD activities in LPS-induced mice brains. The overall results from this study supported the neuroprotective effects of piracetam against LPS-induced neuronal toxicity.

Sb(III) resistance mechanism and oxidation characteristics of Klebsiella aerogenes X

Resistant bacteria are potential natural materials for the bioremediation of soil metalloid pollution. A strain isolated from farmland soil chronically exposed to Sb was identified as K. aerogenes X with high antimonite [Sb(III)] tolerance and oxidation ability. The resistance mechanism of K. aerogenes X and its extracellular polymeric substances (EPS), antioxidant enzymes, and oxidation characteristics in Sb(III) stress were investigated in this study by stress incubation experiments and FTIR. The biotoxicity of Sb was limited by the binding of the organic compounds in EPS, and the anionic functional groups (e.g., amino, carboxyl and hydroxyl groups, etc.) present in the cell envelope were the components primarily responsible for the metalloid-binding capability of K. aerogenes X. The K. aerogenes X can oxidize Sb(III), and its metabolites induce changes in reactive oxygen species (ROS), catalase (CAT), total superoxide dismutase (SOD) and glutathione s-transferase (GSH-S) activity, indicating that the resistance mechanisms of K. aerogenes X are mediated by oxidative stress, EPS restriction and cell damage. Oxidation of Sb(III) is driven by interactions in intracellular oxidation, cell electron transport, extracellular metabolism including proteins and low molecular weight components (LMWs). LMWs (molecular weight <3 kDa) are the main driving factor of Sb(III) oxidation. In addition, Sb resistance genes arsA, arsB, arsC, arsD and acr3 and potential oxidation gene arsH were identified in K. aerogenes X. Owing to its natural origin, high tolerance and oxidation ability, K. aerogenes X could serve as a potential bioremediation material for the mitigation of Sb(III) in contaminated areas.

Rheum tanguticum Alleviates Cognitive Impairment in APP/PS1 Mice by Regulating Drug-Responsive Bacteria and Their Corresponding Microbial Metabolites.

Drugs targeting intestinal bacteria have shown great efficacy for alleviating symptoms of Alzheimer’s disease (AD), and microbial metabolites are important messengers. Our previous work indicated that Rheum tanguticum effectively improved cognitive function and reshaped the gut microbial homeostasis in AD rats. However, its therapeutic mechanisms remain unclear. Herein, this study aimed to elaborate the mechanisms of rhubarb for the treatment of AD by identifying effective metabolites associated with rhubarb-responsive bacteria. The results found that rhubarb reduced hippocampal inflammation and neuronal damage in APP/PS1 transgenic (Tg) mice. 16S rRNA sequencing and metabolomic analysis revealed that gut microbiota and their metabolism in Tg mice were disturbed in an age-dependent manner. Rhubarb-responsive bacteria were further identified by real-time polymerase chain reaction (RT-PCR) sequencing. Four different metabolites reversed by rhubarb were found in the position of the important nodes on rhubarb-responsive bacteria and their corresponding metabolites combined with pathological indicators co-network. Furthermore, in vitro experiments demonstrated o-tyrosine not only inhibited the viabilities of primary neurons as well as BV-2 cells, but also increased the levels of intracellular reactive oxygen species and nitric oxide. In the end, the results suggest that rhubarb ameliorates cognitive impairment in Tg mice through decreasing the abundance of o-tyrosine in the gut owing to the regulation of rhubarb-responsive bacteria. Our study provides a promising strategy for elaborating therapeutic mechanisms of bacteria-targeted drugs for AD.

Genetically encoded biosensor G-geNOp for detection and quantification of intracellular nitric oxide in isolated skeletal muscle fibres

Genetically encoded biosensor G-geNOp for detection and quantification of intracellular nitric oxide in isolated skeletal muscle fibres

Association of 5-aminolevulinic acid with intraoperative hypotension in malignant glioma surgery

BackgroundUse of 5-aminolevulinic acid for photodynamic malignant tumor diagnosis reportedly causes intraoperative hypotension (systolic blood pressure < 70 mmHg) during urologic surgery. However, its association with intraoperative hypotension in malignant glioma surgery and underlying mechanisms has not yet been elucidated.. This study aimed to investigate whether 5-aminolevulinic acid administration is associated with intraoperative hypotension in malignant glioma surgery and explore the mechanisms of 5-aminolevulinic acid-induced hypotension in vitro. MethodsIn this retrospective multicenter cohort study, we investigated intracellular nitric oxide as a candidate mediator of hypotension in response to 5-aminolevulinic acid in vitro in human umbilical vein endothelial cell cultures. ResultsOf 142 patients, 94 underwent 5-aminolevulinic acid-guided surgery. Systolic blood pressure was significantly lower throughout surgery with 5-aminolevulinic acid administration. 5-Aminolevulinic acid administration was an independent risk factor for intraoperative hypotension according to multivariable logistic regression analysis (89% vs. 56%; odds ratio = 6.72, 95% confidence interval [2.05–22.1], P = 002). In subgroup analysis of the 5-aminolevulinic acid group, increasing age and use of renin-angiotensin system inhibitors had a synergistic effect with 5-aminolevulinic acid on decreased blood pressure. In the vascular endothelial cell culture study, 5-aminolevulinic acid induced a significant increase in intracellular nitric oxide generation. Conclusions5-Aminolevulinic acid administration was associated with intraoperative hypotension in malignant glioma surgery, with increasing age and use of renin-angiotensin system inhibitors boosting the blood pressure-lowering effect of 5-aminolevulinic acid. According to in vitro results, the low blood pressure induced by 5-aminolevulinic acid may be mediated by a nitric oxide increase in vascular endothelial cells.

Cationic amino acid transporters and their modulation by nitric oxide in cardiac muscle cells.

Cationic amino acid transporters (CATs) play a central role in the supply of the substrate L-arginine to intracellular nitric oxide synthases (NOS), the enzymes responsible for the synthesis of nitric oxide (NO). In heart, NO produced by cardiac myocytes has diverse and even opposite effects on myocardial contractility depending on the subcellular location of its production. Approximately a decade ago, using a combination of biophysical and biochemical approaches, we discovered and characterized high- and low-affinity CATs that function simultaneously in the cardiac myocyte plasma membrane. Later on, we reported a negative feedback regulation of NO on the activity of cardiac CATs. In this way, NO was found to modulate its own biosynthesis by regulating the amount of L-arginine that becomes available as NOS substrate. We have recently solved the molecular determinants for this NO regulation on the low-affinity high-capacity CAT-2A. This review highlights some biophysical and biochemical features of L-arginine transporters and their potential relation to cardiac muscle physiology and pathology.

Bai-Hu-Tang regulates endothelin-1 and its signalling pathway in vascular endothelial cells

Ethnopharmacological relevanceBai-Hu-Tang (BHT) is traditionally used to treat human and animal fever syndrome with four symptoms: large and vigorous pulse, large thirst, high sweat, and high heat. Aim of the studyTo investigate the mechanism of vasodilation regulation of Bai-Hu-Tang in primary vascular endothelial cells stimulated by lipopolysaccharide (LPS). Materials and methodsA hydrophilic concentrate of BHT was prepared, and the main components of mangiferin and timosaponin BⅡ were determined by HLPC analysis. The rabbit fever model was constructed by intravenous injection of LPS (15 μg/kg body weight), and BHT was gavaged to treat febrile rabbits. After treatment for 6 h, animal peripheral blood was collected, and serum was isolated for endothelin-1 (ET-1) and nitric oxide (NO) assays. Rabbit vascular endothelial cells (RVECs) were isolated and stimulated with 1 μg/mL LPS, and then inflammatory cells were treated with 125 or 250 μg/mL BHT for 24 h. The supernatant cytokines TNF-ɑ, IL-1β, IL-6, and ET-1 were detected by ELISA kits. Gene expression levels of endothelin receptor type B (ETB receptor) were analysed by real-time polymerase chain reaction (RT-PCR), and protein expression levels of PI3K and Akt were detected by Western blot. A nitrite assay was used to measure intracellular nitric oxide (NO) production, and nitric oxide synthase (NOS) was measured by the T-NOS colorimetric method. ResultsAnimal experiments demonstrated that BHT significantly restored ET-1 and NO in animal peripheral blood, which were disordered in LPS-induced fever rabbits. Moreover, a cytotoxicity assay demonstrated that BHT ≤700 μg/mL is innoxious to RVECs. BHT significantly repressed cellular TNF-α, IL-1β, and ET-1, which were originally elevated by LPS in RVECs. Meanwhile, BHT elevated the gene expression level of the ETB receptor and promoted NOS and NO production in RVECs induced by LPS. ConclusionBHT can inhibit excessive ET-1 secretion induced by LPS in vascular endothelial cells and activate the classic ET-1 signalling pathway to promote NO production, which may facilitate vasodilation of smooth muscle cells.

Thrombin-Induced Oxidative Phosphorylation in Platelets Is Repressed By Dengue Virus through Inhibition of the PI3K/Akt Signaling Pathway

Background:There are many very important events that condition the progression to severe dengue, these include cytokine storm and a profound thrombocytopenia. Still, the molecular mechanisms underlying these events are not fully understood. Increasing evidence supports the fact that platelet dysfunction, in patients with dengue, could be due to direct interaction between platelets and dengue virus. This event induces changes in platelet function and contributes to pathogenesis. Recent results from our laboratory have shown that thrombin induces oxygen consumption through activation of glycoprotein Ib and subsequent phosphorylation of PI3K and Akt enzymes. Here we want to evaluate changes in these signalling pathways as well as their impact on mitochondrial function.Aims: To evaluate DENV2 effects on platelet function, PI3K/Akt signaling, oxidative phosphorylation and GPIb expression.Methods:Blood samples from healthy volunteers were collected and platelet fraction was obtained. Purified DENV2 particles were incubated with platelets for 2 h at 37ºC. Then, morphological changes, activation and aggregation, mitochondrial function and surface markers expression were tested in platelets.Results:We found that DENV2 induces conspicuous morphological changes on platelets not induced by other arboviruses; increases CD41 (p<0.05), CD62P (8979 ± 1189 vs1723 ± 160 MFI; p<0.05) and reduces CD42b (p<0.5) expressions on platelet surface; reduces agonist-induced platelet aggregation (30 ± 10 vs 71 ± 9%; p<0.05) through increases intracellular NO production (0.4 ± 0.2 vs 0.07 ± 0.01; p<0.05); inhibits thrombin-induced oxidative phosphorylation (45 ± 6.69 vs 348 ± 26.9 ngatomO/min/10 9 platelets; p<0.0001) through PI3K/Akt signaling pathways inhibition (p<0.001). Surprisingly, rDC-SIGN treatment reverts DENV2 effects on platelet activation (9693 ± 1033 vs 2379 ± 390 MFI; p<0.01).Conclusions:DENV2 inhibits thrombin-induced oxidative phosphorylation through PI3K/Akt signaling pathway inhibition. Dengue virus inhibits thrombin-induced phosphorylation at different levels in the platelet. First, by reducing the expression of the GPIb molecule on the platelet surface. This molecule is important for thrombin-induced intracellular signalling. Second, dengue virus inhibits the PI3K/Akat signalling pathway, which is important for the induction of oxidative phosphorylation in mitochondria. And third, dengue virus increases intracellular nitric oxide levels and promotes its excretion into the extracellular milieu, this mechanism could be involved in the inhibition of platelet aggregation processes.These changes induce by short-term interaction of platelets with DENV2 has a direct effect over platelet function and may be correlated with the clinical manifestations observed during severe dengue stage, such as thrombocytopenia and hemorrhages. The overall impact of these changes in signaling pathways related to immune activity remains to be determinate, as does the potential to develop new pharmacological treatments. DisclosuresNo relevant conflicts of interest to declare.

Exploiting the Anti-Inflammatory Potential of White Capsicum Extract by the Nanoformulation in Phospholipid Vesicles.

The peppers of the Capsicum species are exploited in many fields, as flavoring agents in food industry, or as decorative and therapeutic plants. Peppers show a diversified phytochemical content responsible for different biological activities. Synergic activity exerted by high levels of antioxidant compounds is responsible for their important anti-inflammatory property. A methanolic extract was obtained from a new pepper genotype and tested for anti-inflammatory activity. The extract was incorporated into phospholipid vesicles to increase the bioavailability of its bioactive components. Two types of phospholipid vesicles were produced, conventional liposomes and Penetration Enhancer containing Vesicles (PEVs). They were tested in human monoblastic leukemia U937 cell line, showing no cytotoxic effect. The intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels were measured to value the in vitro efficacy of the vesicles in regulating inflammatory responses. Liposomal incorporation significantly reduced ROS levels in extract-treated LPS-activated cells. Furthermore, LC-MS/MS analyses demonstrated that liposomes facilitated the transport of the extract components across the cell membrane and their accumulation into the cytoplasm.

Nicotinamide Attenuates UV-Induced Stress Damage in Human Primary Keratinocytes from Cancerization Fields

UVB radiation directly damages DNA, increases ROS and nitric oxide (NO) release, and promotes inflammation leading to genomic instability and cell death. Nicotinamide (NAM) is the precursor of NAM adenine dinucleotide, essential for cell energy production and DNA damage repair. NAM protects HaCat cells from UV-induced impairment; however, little is known about its effects on human primary keratinocytes and those isolated from field cancerization (i.e., field cancerization human primary keratinocytes [FC-HPKs]). We examined the role of NAM against UV-induced oxidative stress damages in FC-HPKs, isolated from precancerous lesions and skin cancers, and in normal human epidermal keratinocytes. Cells were treated for 18, 24, and 48 hours with NAM (5, 25, and 50 μM, respectively) before UVB irradiation. FC-HPK showed four-fold higher basal ROS levels than normal human epidermal keratinocytes; NAM downregulated ROS production only in irradiated FC-HPKs, which showed a greater sensibility to UV rays. UV exposure increased OGG1, inducible nitric oxide synthase, and IL-1β expression, an effect counteracted by NAM pretreatment. Intracellular nitric oxide production and DNA damages were inhibited by NAM exposure before irradiation. Collectively, our findings indicate that pretreatment with 25 μM NAM 24 hours before UVB irradiation effectively prevents oxidative stress formation, DNA damage, and inflammation in both normal human epidermal keratinocytes and FC-HPKs.

Mechanisms of TLR4-Mediated Autophagy and Nitroxidative Stress.

Pathogenic infections have badly affected public health and the development of the breeding industry. Billions of dollars are spent every year fighting against these pathogens. The immune cells of a host produce reactive oxygen species and reactive nitrogen species which promote the clearance of these microbes. In addition, autophagy, which is considered an effective method to promote the destruction of pathogens, is involved in pathological processes. As research continues, the interplay between autophagy and nitroxidative stress has become apparent. Autophagy is always intertwined with nitroxidative stress. Autophagy regulates nitroxidative stress to maintain homeostasis within an appropriate range. Intracellular oxidation, in turn, is a strong inducer of autophagy. Toll-like receptor 4 (TLR4) is a pattern recognition receptor mainly involved in the regulation of inflammation during infectious diseases. Several studies have suggested that TLR4 is also a key regulator of autophagy and nitroxidative stress. In this review, we describe the role of TLR4 in autophagy and oxidation, and focus on its function in influencing autophagy-nitroxidative stress interactions.