Nitric Oxide Synthase Protein Research Articles

Briavioids E–G, Newly Isolated Briarane-Diterpenoids from a Cultured Octocoral Briareum violaceum

The chemical screening of a cultured soft coral, Briareum violaceum, led to the isolation of eight natural, briarane-related diterpenoids, including three unreported metabolites, briavioids E-G (1-3), and five known briaranes, briacavatolides B (4) and C (5), briaexcavatin L (6), briaexcavatolide U (7) and briarenol K (8). The structures of briaranes 1-8 were established using spectroscopic methods. The absolute configuration of briavioid A (9), obtained in a previous study, was reported for the first time in this study by a single-crystal X-ray diffraction analysis using a copper radiation source. The anti-inflammatory activity of briaranes 1 and 2 and briaranes 4-8 was evaluated by screening their inhibitory ability against the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins in lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells.

Antidiabetic drug sitagliptin blocks cyclophosphamide cerebral neurotoxicity by activating Nrf2 and suppressing redox cycle imbalance, inflammatory iNOS/NO/NF-κB response and caspase-3/Bax activation in rats

Cyclophosphamide (CYP) is a classic DNA-interacting anticancer agent with broad application in chemotherapy. However, CYP cerebral neurotoxicity is a worrisome side effect for clinicians and patients. Strategies to mitigate the underlying oxidative inflammatory cascades and neuroapoptosis induced by CYP are urgently needed. Herein, we have repurposed an antidiabetic drug, sitagliptin (STG), for a possible abrogation of CYP-induced cerebral neurotoxicity in rats. Healthy rats were administered STG (20 mg/kg body weight) for 5 days prior to neurotoxicity induced by CYP (200 mg/kg body weight, ip) on day 5 only, and rats were sacrificed after 24 h post-CYP injection. CYP caused profound increases in the cerebral levels of nitric oxide (NO), acetylcholinesterase (AChE), malondialdehyde (MDA), interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), nuclear factor-kappaB (NF-κB), inducible nitric oxide synthase (iNOS), caspase-3 and Bax protein compared to the control. Furthermore, CYP markedly depressed the activities of glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD), along with levels of reduced glutathione (GSH) and nuclear factor erythroid 2-related factor2 (Nrf2) compared to the control (p < 0.05). Interestingly, STG pretreatment inhibited the CYP-induced alterations in caspase-3, Bax, pro-inflammatory cytokines, NO, iNOS, AChE, NF-κB, and restored the cerebral antioxidant apparatus, including the Nrf2 and histopathological abrasions. Therefore, these findings show that STG could be repurposed to prevent CYP-induced cerebral toxicity in the brain.

Adrenomedullin in paraventricular nucleus attenuates adipose afferent reflex and sympathoexcitation via receptors mediated nitric oxide-gamma-aminobutyric acid A type receptor pathway in rats with obesity-related hypertension.

Hypothalamic paraventricular nucleus (PVN) is an important central site for the control of the adipose afferent reflex (AAR) that increases sympathetic outflow and blood pressure in obesity-related hypertension (OH). In this study, we investigated the effects of nitric oxide (NO) and cardiovascular bioactive polypeptide adrenomedullin (ADM) in the PVN on AAR and sympathetic nerve activity (SNA) in OH rats induced by a high-fat diet. The results showed that ADM, total neuronal NO synthase (nNOS) and phosphorylated-nNOS protein expression levels in the PVN of the OH rats were down-regulated compared to the control rats. The enhanced AAR in OH rats was attenuated by PVN acute application of NO donor sodium nitroprusside (SNP), but was strengthened by the nNOS inhibitor nNOS-I, guanylyl cyclase inhibitor (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one, ODQ) and gamma-aminobutyric acid A type receptor (GABAA) antagonist Bicuculline. Moreover, PVN ADM microinjection not only decreased basal SNA but also attenuated the enhanced AAR in OH rats, which were effectively inhibited by ADM receptor antagonist ADM22-52, nNOS-I, ODQ or Bicuculline pretreatment. Bilateral PVN acute microinjection of ADM also caused greater increases in NO and cyclic guanosine monophosphate (cGMP) levels, and nNOS phosphorylation. Adeno-associated virus vectors encoding ADM (AAV-ADM) transfection in the PVN of OH rats not only decreased the elevated AAR, basal SNA and blood pressure (BP), but also increased the expression and activation of nNOS. Furthermore, AAV-ADM transfection improved vascular remodeling in OH rats. Taken together, our data highlight the roles of ADM in improving sympathetic overactivation, enhanced AAR and hypertension, and its related mechanisms associated with receptors mediated NO-cGMP-GABAA pathway in OH condition.

Abstract WP234: Mitochondria Therapy Mitigates Neuroinflammation And Provides Neuroprotection Against Ischemic Stroke In Mice

Background: Ischemic stroke is the second prevalent life-threat problem associated with long-lasting neurological disabilities and a high mortality rate worldwide. Despite this, therapeutic options remain limited. Recently, mitochondria transplantation therapy has emerged as a potential approach for increasing brain protection. This study aimed to investigate the protective role of mitochondria transplantation against ischemic stroke-induced behavioral deficits and molecular alterations in a photothrombotic stroke model. Methods: Ischemic stroke was induced by a Rose Bengal photothrombosis method in young adult C57BL/6 mice. Repeated doses of isolated liver mitochondria (100μg/mouse) were administrated intranasally at 1 h, 3 h, 24 h, and 48 h post-stroke. After 72 h, mice were tested for behavioral outcomes and euthanized, and tissues were harvested to measure infarct volume, brain edema, and molecular analysis. Results: Mitochondria-treated mice showed a significant improvement in behavioral outcomes, reduction in infarct size, and brain edema compared to the vehicle-treated group. Additionally, mitochondria significantly inhibited the protein expression of NOD-like receptor protein-3, phospho-nuclear factor kappa-B, cleaved-interleukin-1β, apoptosis-associated speck-like protein, glial fibrillary acidic protein, and ionized calcium-binding adapter molecule-1 proteins. Furthermore, mitochondria treatment showed a lower expression of nitrotyrosine, and 4-hydroxynonenal and higher expression of endothelial nitric oxide synthase and sulfotransferase-4A1 proteins, oxidative stress markers ( Table 1 ). Conclusion: Taken together, these data indicate that mitochondria reveal neuroprotection through the attenuation of inflammation against stroke. Further investigations into the role of neuroprotection of mitochondria transplantation are needed to determine whether it can be an effective therapy for stroke patients.

Attenuation of Lipopolysaccharide-Induced Inflammatory Responses through Inhibition of the NF-κB Pathway and the Increased NRF2 Level by a Flavonol-Enriched n-Butanol Fraction from Uvaria alba.

Pathologic hyperreactive inflammatory responses occur when there is excessive activation of a proinflammatory NF-κB pathway and a reduced cytoprotective NRF2 cascade. The noncytotoxic, highly selective COX-2 inhibitory flavonol-enriched butanol fraction (UaB) from Uvaria alba (U. alba) was investigated for its inflammatory modulating potential by targeting NF-κB activation and NRF2 activity. Enzyme-linked immunosorbent assay was initially performed to measure levels of proinflammatory mediators [nitric oxide (NO), prostaglandin E2, and reactive oxygen species (ROS)] and cytokines [tumor necrosis factor-alpha (TNF-α), IL-1β, and IL-6], followed by reverse transcription-polymerase chain reaction and western blotting to determine mRNA and protein expression, respectively. Using immunofluorescence staining combined with western blot analysis, the activation of NF-κB was further investigated. NRF2 activity was also measured using a luciferase reporter assay. UaB abrogated protein and mRNA expressions of inducible nitric oxide synthase (iNOS), COX-2, TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophages, thereby suppressing the production of proinflammatory mediators and cytokines. This was further validated when a concentration-dependent decrease in NO and ROS production was observed in zebrafish (Danio rerio) larvae. UaB also increased NRF2 activity in HaCaT/ARE cell line and attenuated NF-κB activation by inhibiting the nuclear translocation of transcription factor p65 in RAW 264.7 macrophages. Nontargeted LC-MS analysis of UaB revealed the presence of the flavonols quercitrin (1), quercetin (2), rutin (3), kaempferol (4), and kaempferol 3-O-rutinoside (5). Molecular docking indicates that major flavonol aglycones have high affinity toward COX-2 NSAID-binding sites, TNF-α, and TNF-α converting enzyme, while the glycosylated flavonoids showed strong binding toward iNOS and IKK-all possessing dynamic stability when performing molecular dynamics simulations at 140 ns. This is the first report to have elucidated the mechanistic anti-inflammatory potential of the Philippine endemic plant U. alba.

Melatonin Mitigates iNOS-Related Effects of HEMA and Camphorquinone in Human Dental Pulp Cells: Relevance for Postoperative Sensitivity Mechanism in Type 2 Diabetes.

High elution and diffusion of 2-hydroxylethyl methacrylate (HEMA) and camphorquinone (CQ) through dentinal tubules may induce pulp injury and postoperative sensitivity. We aimed to investigate the melatonin protective effect in HEMA- and CQ-treated human dental pulp cells (hDPCs) as well as its relevance in a mechanism for postoperative sensitivity in diabetic patients. hDPCs were exposed to HEMA (5 mM) and/or CQ (1 mM) in the absence and presence of melatonin (MEL) (0.1 mM and 1 mM). Heme oxygenase-1 (HMOX1), NADPH oxidase-4 (NOX4), BCL-2-associated X-protein (BAX), B-cell lymphoma-2 (BCL-2) and caspase-3 (CASP3) gene expression levels, and superoxide dismutase (SOD) activity were measured in hDPCs while inducible nitric oxide synthase (iNOS) and melatonin protein expression were measured in human dental pulp as well, by RT-PCR, by ELISA, and spectrophotometrically. Bioinformatic analyses were performed by using the ShinyGO (v.0.75) application. Type 2 diabetic patients showed a higher incidence of postoperative sensitivity and lower melatonin and higher iNOS content in dental pulp tissue compared with non-diabetic patients. Melatonin, when co-added in hDPC culture, reverses HEMA and CQ cytotoxic effects via anti-apoptotic and anti-inflammatory/antioxidant iNOS-related effects. Enrichment analyses showed that genes/proteins, altered by HEMA and CQ and normalized by melatonin, are the most prominently overrepresented in type 2 diabetes mellitus pathways and that they share subcellular localization in different oligomeric protein complexes consisting of anti- and pro-apoptotic regulators. This is the first evidence of the ability of melatonin to counteract iNOS-mediated inflammatory and stress effects in HEMA- and CQ-treated hDPCs, which could be of significance for the modulation of presently observed immediate postoperative sensitivity after composite restoration in type 2 diabetic patients.

α7 nicotinic acetylcholine receptor agonist improved brain injury and impaired glucose metabolism in a rat model of ischemic stroke.

Vagus nerve stimulation through the action of acetylcholine can modulate inflammatory responses and metabolism. α7 Nicotinic Acetylcholine Receptor (α7nAChR) is a key component in the biological functions of acetylcholine. To further explore the health benefits of vagus nerve stimulation, this study aimed to investigate whether α7nAChR agonists offer beneficial effects against poststroke inflammatory and metabolic changes and to identify the underlying mechanisms in a rat model of stroke established by permanent cerebral ischemia. We found evidence showing that pretreatment with α7nAChR agonist, GTS-21, improved poststroke brain infarction size, impaired motor coordination, brain apoptotic caspase 3 activation, dysregulated glucose metabolism, and glutathione reduction. In ischemic cortical tissues and gastrocnemius muscles with GTS-21 pretreatment, macrophages/microglia M1 polarization-associated Tumor Necrosis Factor-α (TNF-α) mRNA, Cluster of Differentiation 68 (CD68) protein, and Inducible Nitric Oxide Synthase (iNOS) protein expression were reduced, while expression of anti-inflammatory cytokine IL-4 mRNA, and levels of M2 polarization-associated CD163 mRNA and protein were increased. In the gastrocnemius muscles, stroke rats showed a reduction in both glutathione content and Akt Serine 473 phosphorylation, as well as an elevation in Insulin Receptor Substrate-1 Serine 307 phosphorylation and Dynamin-Related Protein 1 Serine 616 phosphorylation. GTS-21 reversed poststroke changes in the gastrocnemius muscles. Overall, our findings, provide further evidence supporting the neuroprotective benefits of α7nAChR agonists, and indicate that they may potentially exert anti-inflammatory and metabolic effects peripherally in the skeletal muscle in an acute ischemic stroke animal model.

NEDD4 attenuates oxidized low‑density lipoprotein‑induced inflammation and dysfunction in vascular endothelial cells via regulating APEX1 expression.

Atherosclerosis chiefly results from inflammation as well as vascular endothelial cell dysfunction. Methylation levels of neuronally expressed developmentally downregulated 4 (NEDD4) were found to be fortified in atherosclerosis patients and NEDD4 deficiency enhanced vascular calcification. However, the exact function of NEDD4 in inflammation and vascular endothelial dysfunction remains to be elucidated. In the present study, CCK-8 assay was used to estimate cell viability. Reverse transcription-quantitative PCR was adopted to examine the expression of NEDD4, inflammation-associated enzymes and apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1). Western blotting was used to test NEDD4, endothelial nitric oxide synthase, inducible nitric oxide synthase and APEX1 protein levels. Cytotoxicity was detected by a lactate dehydrogenase (LDH) kit. Reactive oxygen species level was tested by a corresponding kit. Vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 contents were examined with ELISA. Cell adhesion assays evaluated the adhesion of endothelial cells. Co-immunoprecipitation assay was used to test the relationship between NEDD4 and APEX1. The data revealed that NEDD4 expression rapidly declined in oxidized low density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs). Following NEDD4 overexpression, the active damage, inflammatory release and endothelial cell dysfunction in ox-LDL-induced HUVECs were attenuated. After co-transfection of APEX1 interference plasmids and NEDD4 overexpression plasmids, cell damage, inflammatory release and endothelial cell dysfunction in ox-LDL-induced HUVECs were improved again. Taken together, NEDD4 attenuated ox-LDL-induced inflammation and endothelial dysfunction by regulating APEX1 expression.

Sucrose enhances the chromogenic ability ofStaphylococcus xylosusby improving nitric oxide synthase activity

AbstractIn this paper, the effect of different concentrations of sucrose stress on color formation of the Staphylococcus xylosus was investigated. The results showed that the highest a* value and the best coloring effect similar to those of nitrite were observed after the addition of 0.05 g/mL sucrose to stress the S. xylosus. UV-Vis and electron spin resonance spectra analysis showed that production of coloring product Mb-NO was significantly enhanced after 0.05 g/mL sucrose stress. The growth curve, reactive oxygen content, cell cycle, nitric oxide synthase (NOS) activity, zeta potential, cell size, and protein composition of S. xylosus were investigated to reveal the mechanism of sucrose stress to enhance the coloring effect of the strain. The result showed that sucrose inhibited the growth of S. xylosus, which changed the physiological state by activating the oxidative stress response. The stress altered the rate of intracellular metabolism of S. xylosus by delaying the cell cycle and increasing cell surface zeta potential and cell particle size. These changes altered the protein composition of the cells and significantly enhanced the activity of intracellular NOS, which could improve the chromogenic ability of S. xylosus. This study will provide theoretical support for sucrose stress on S. xylosus to enhance its coloring effect, and sucrose stress for S. xylosus might be a promising biological alternative to nitrite in meat products.

A new terpenoid from Olea europaea L. leaves and potential aptitude of different leaf extracts as anti-inflammatory agents

Background The olive tree (Olea europaea L.) has played a significant role in medical history, being used to relieve a wide range of illnesses in traditional medicine. The value of olive leaves came from their medicinal and nutritional properties. In recent times, it has been proven that there is a potential role of leaves in cancer prevention and inflammatory illnesses.Objective The purpose of this study was to look at the chemical makeup of methylene chloride fraction and also to evaluate the chemopreventive properties and the anti-inflammatory activity of different olive leave fractions using hepa1c1c7 cells and RAW264.7 macrophages activated by lipopolysaccharides, respectively.Materials and methods Air-dried powdered olive leaves were defatted with hexane, and the marc was then soaked in 80% methanol and successively extracted with CH2Cl2, EtOAc, and n-BuOH. Phytochemical investigation of CH2Cl2 fraction was done. The chemopreventive effect was determined in vitro using a hepa1c1c7 human carcinoma cell line, and the anti-inflammatory was carried out using RAW264.7 macrophages.Results and conclusion One novel compound, identified and given the name oleuropeinone (1), was obtained together with two known compounds, blumenol A (2) and megaritolactonol (3). Spectroscopic data such as 1H and 13C NMR were used to identify their chemical structures. The CH2Cl2 fraction is the most effective fraction to inhibit inflammatory markers inducible nitric oxide synthase and COX-2 protein expression in RAW264.7 macrophages. Moreover, it has a potent effect in inducing the chemopreventive marker NQO1 protein expression.

Antioxidant and Anti-inflammatory Activity of Sea Cucumber (Holothuria scabra) Active Compounds against KEAP1 and iNOS Protein.

Oxidative stress and inflammation have a role in the development of various diseases. Oxidative stress and inflammation are associated with many proteins, including Kelch ECH associating protein 1 (KEAP1) and inducible nitric oxide synthase (iNOS) proteins. The active compounds contained in Holothuria scabra have antioxidant and anti-inflammatory properties. This study aimed to evaluate the antioxidant and anti-inflammatory activity of sea cucumber's active compounds by targeting KEAP1 and iNOS proteins. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) scavenging activity of H. scabra extract were measured spectrophotometrically. The 3-dimensional (3D) structures of sea cucumber's active compounds and proteins were obtained from the PubChem and Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) databases. Molecular docking was performed using AutoDock Vina software. Molecular dynamics simulations were carried out using Yet Another Scientific Artificial Reality Application (YASARA) software with environmental parameters according to the cell's physiological conditions. The membrane permeability test was performed using the PerMM web server. The methanol extract of H. scabra had a weak antioxidant activity against DPPH and strong activity against NO radical. Scabraside and holothurinoside G had the most negative binding affinity values when interacting with the active site of KEAP1 and iNOS proteins. Molecular dynamics simulations also showed that both compounds were stable when interacting with KEAP1 and iNOS. However, scabraside and holothurinoside G were difficult to penetrate the cell plasma membrane, which is seen from the high energy transfer value in the lipid acyl chain region of phospholipids. Scabraside and holothurinoside G are predicted to act as antioxidants and anti-inflammations, but in their implementation to in vitro and in vivo study, it is necessary to have liposomes or nanoparticles, or other delivery methods to help these 2 compounds enter the cell.

Effects of Cavitation from Extracorporeal Shock Wave Combined with Sulfur Hexafluoride Microbubble on Myocardial Ultrastructure in Rats.

In the present study, the effects of extracorporeal cardiac shock waves combined with different concentrations of sulfur hexafluoride ultrasound microbubbles on myocardial ultrastructure in rats were observed. Thirty-six rats were randomly divided into 6 groups: control group (N), extracorporeal cardiac shock wave group, and combined group, i.e., extracorporeal cardiac shock wave combined with different concentrations of sulfur hexafluoride microbubble (0.225 mL/kg/min, 0.45 mL/kg/min, 0.9 mL/kg/min, 1.8 mL/kg/min). The combination of extracorporeal cardiac shock wave combined with sulfur hexafluoride microbubbles of different concentrations had no significant effect on hemodynamic indexes and left ventricular function in rats. There were significant differences in cardiac troponin I (cTnI) and nitricoxide among different groups. Histopathology showed that inflammatory cells infiltrated in the shock wave+microbubble 0.9 and shock wave+microbubble 1.8 groups. The myocardial ultrastructural injury score of shock wave+microbubble1.8 group was significantly higher than that of the N group, shock wave group, shock wave+microbubble 0.225 group, and shock wave+microbubble 0.45 group. The score of shock wave+microbubble 0.9 group was higher than that of the control group (P=.009). Western blot results showed that the expression of vascular endothelial growth factor and endothelial nitricoxide synthase (eNOS) protein in the rats treated with extracorporeal cardiac shock wave combined with sulfur hexafluoride microbubbles of different concentrations was higher than that in the N group and shock wave group, with shock wave+microbubble 0.45 group having the strongest expression. Myocardial ultrastructure damage occurs when high concentrations of sulfur hexafluoride microbubbles are present, but a proper concentration of sulfur hexafluoride microbubbles could promote the cavitation effect of extracorporeal cardiac shock waves. Thus combination therapy may become a new paradigm in coronary heart disease, especially contributing to the treatment of refractory angina. Combination therapy may change coronary heart disease treatment, especially for refractory angina.

Investigation of the neuroprotective effect of kefir in experimental spinal cord injury

In this study, the antioxidant, anti-inflammatory, and neuroprotective effects of kefir were investigated in spinal cord injury that was experimentally created on rats with a compression trauma model. A total of 56 Wistar-Albino male rats were used in the study. Daily freshly prepared 18 ml/kg/day of kefir was given by oral gavage to animals 7 days before and during the trauma and during the trauma. Spinal cord injury was created according to the weight drop method. On the 1st and 7th days before euthanasia, intracardiac blood was collected for analysis, and then they were sacrificed. The damaged spinal cord segments were examined biochemically, immunohistochemically, and histopathologically. When compared to the sham groups, kefir had a positive effect in the preconditioning and treatment groups by decreasing spinal cord bleeding, edema, myelin sheath damage, liquefactive necrosis, neuronal necrosis, selectivity of canalis centralis, and gitter cell levels significantly. When compared to the sham groups, kefir was found to have a positive effect in the treatment groups by decreasing the neuron specific enolase (NSE), ionized calcium binding adapter molecule 1 (IBA-1), inducible nitric oxide synthase (INOS), cyclooxygenase 2 (COX-2) and myelin basic protein (MBP) levels significantly on the 1st and 7th days, and by increasing the glial fibrillary acidic protein (GFAP) level significantly. As a result, it was demonstrated that kefir had a protective and therapeutic effect on spinal cord injury.

Antiliver Fibrosis Formula of Fuzheng Huayu Alleviates Inflammatory Response.

Fuzheng Huayu's (FZHY) formula ameliorated liver fibrosis in clinical and experimental practice. Based on the close link between fibrosis and inflammation, its anti-inflammatory effect and related mechanisms were explored in this present study. With the aid of the inflammatory macrophage model, FZHY significantly blocked nitrite accumulation without observable cytotoxicity due to its suppression of inducible nitric oxide synthase (iNOS) gene and protein expressions in a concentration-depended manner. Proinflammatory mediators including IL-6, CD86, and CD40 were also restrained by FZHY. Interestingly, FZHY induced anti-inflammatory mediators heme oxygenase 1 (HO-1) and peroxisome proliferator-activated receptor γ (PPAR-γ) expressions simultaneously. Downregulation of iNOS and miR-155 and upregulation of PPAR-γ were also observed in CCl4-induced liver fibrosis mice upon FZHY administration. Mechanically, FZHY strikingly eliminated the phosphorylation of STAT1 and MAPK. Taken together, FZYH regulated the balance of proinflammatory and anti-inflammatory mediators partially via modulating STAT1/MAPK pathways and the miR-155/PPAR-γ axis.

Abstract 13147: Formate Stimulates S-Nitrosylation and Mitochondrial Activity to Induce Cardioprotection

Formate (FM) is a substrate in one-carbon metabolism, and recent studies implicate its function in both health and disease, but a role in the heart remains unexplored. Research from our group demonstrates that the loss of formaldehyde dehydrogenase, which oxidizes formaldehyde into FM, ameliorates sex-dependent cardioprotection against ischemia-reperfusion (I/R) injury in the female mouse heart. Considering that FM depletion may be detrimental, we tested the hypothesis that FM yields cardioprotective benefit in a model of I/R injury. Hearts from male and female mice (n=5-10/group) were subjected to ex vivo I/R injury with or without FM via Langendorff perfusion. FM significantly enhanced post-ischemic functional recovery in male hearts (27.1%, 95% CI 10.8 to 45.9, p=0.0023; females: 9.5%, 95% CI -6.9 to 27.8, p=0.2020) and decreased infarct size (males: -24.6%, 95% CI -35.1 to -12.2, p&lt;0.0001; females: -3.7%, 95% CI -9.6 to -0.3, p=0.1128), indicating that FM protects the myocardium against I/R injury. Although FM had a greater protective effect in male hearts than in female hearts, plasma FM levels were greater in female mice, suggesting that FM-mediated cardioprotection may already exist near steady state in females. Mechanistically, cardiomyoblasts stimulated with FM exhibited a significant increase in total protein S-nitrosylation (SNO) without changing nitric oxide synthase (NOS) phosphorylation or protein expression; this was suppressed by NOS inhibition, suggesting that FM contributes to NOS-dependent NO cycling in the heart. FM-treated cardiomyocytes and cardiomyoblasts also exhibited significant increases in mitochondrial marker protein expression, metabolic activity, and maximal oxygen consumption, demonstrating an enhanced respiratory capacity to respond to increased energetic demand. Furthermore, FM treatment significantly increased phospho-activation of the energy regulator AMPKα and the protein expression of PGC-1α targets, indicating a potential upregulation in mitochondrial biogenesis. Taken together, these data suggest that FM may stimulate protein SNO and mitochondrial activity to protect the heart against I/R injury.

Semen Sojae Praeparatum improves anxiety in mice by inhibiting HPA axis hyperactivity and modulating gut microbiota

• SSP increased the desire of anxious mice to explore in elevated plus maze and the open field. • SSP exerts improved anxiety effects in mice by inhibiting HPA axis hyperactivity. • SSP decreased the levels of IL-1β, IL-6 and TNF-α in anxious mice. • SSP modulated the unbalanced gut microbiota in anxious mice. Semen Sojae Praeparatum (SSP) is a functional food made from fermented black soybeans used in folk medicine to treat psychiatric diseases. This study aimed to evaluate whether SSP affects anxiety and explain the improved anxiety mechanism of SSP. We found that SSP increased the exploratory desire of anxiety anxious mice in behavioral experiments, reduced cytokine (IL-1β, IL-6, and TNF-α) release in serum, dose-dependently decreased the protein and mRNA levels of neuronal nitric oxide synthase (nNOS), corticotropin-releasing factor (CRF), and decreased corticosterone concentrations downstream of the HPA axis. Additionally, SSP ameliorated intestinal disorders in mice by reducing the Firmicutes/Bacteroidetes ratio and increasing the abundance of Bacteroidota , Muribaculaceae , and Bacillus . These results demonstrate that SSP improved anxiety effects linked to regulation of HPA axis hyperactivity and gut microbiota composition.

Chronic E-Cigarette Use Impairs Endothelial Function on the Physiological and Cellular Levels.

The harmful vascular effects of smoking are well established, but the effects of chronic use of electronic cigarettes (e-cigarettes) on endothelial function are less understood. We hypothesized that e-cigarette use causes changes in blood milieu that impair endothelial function. Endothelial function was measured in chronic e-cigarette users, chronic cigarette smokers, and nonusers. We measured effects of participants' sera, or e-cigarette aerosol condensate, on NO and H2O2 release and cell permeability in cultured endothelial cells (ECs). E-cigarette users and smokers had lower flow-mediated dilation (FMD) than nonusers. Sera from e-cigarette users and smokers reduced VEGF (vascular endothelial growth factor)-induced NO secretion by ECs relative to nonuser sera, without significant reduction in endothelial NO synthase mRNA or protein levels. E-cigarette user sera caused increased endothelial release of H2O2, and more permeability than nonuser sera. E-cigarette users and smokers exhibited changes in circulating biomarkers of inflammation, thrombosis, and cell adhesion relative to nonusers, but with distinct profiles. E-cigarette user sera had higher concentrations of the receptor for advanced glycation end products (RAGE) ligands S100A8 and HMGB1 (high mobility group box 1) than smoker and nonuser sera, and receptor for advanced glycation end product inhibition reduced permeability induced by e-cigarette user sera but did not affect NO production. Chronic vaping and smoking both impair FMD and cause changes in the blood that inhibit endothelial NO release. Vaping, but not smoking, causes changes in the blood that increase microvascular endothelial permeability and may have a vaping-specific effect on intracellular oxidative state. Our results suggest a role for RAGE in e-cigarette-induced changes in endothelial function.

Identification of TLR2/4-mediated phagocytosis and immune response activation pathways by vacuoles isolated from Saccharomyces cerevisiae.

The vacuoles of the yeast Saccharomycescerevisiae are closely related to mammalian lysosomes and play a role in macromolecular degradation due to the hydrolytic enzymes present inside. The vacuoles also regulate osmotic pressure and control cellular homeostasis. In previous results, vacuoles were shown to activate the immune response of macrophages by promoting the production of immune-mediated transporters nitric oxide (NO), reactive oxygen species (ROS), and pro-inflammatory cytokines. In this study, the effects of vacuoles on the phagocytosis activity of RAW264.7 cells and their potential as immune enhancers were evaluated, and receptors capable of recognizing vacuoles were examined. An investigation using the phagocytes assay showed that phagocytosis activity increased by the vacuole. Besides, after treatment with TLR2/4 inhibitor, the expression of pro-inflammatory cytokines by vacuoles was significantly reduced and the inducible nitric oxide synthase (iNOS) protein was also significantly reduced. However, treatment with a TLR2 inhibitor did not reduce the production of interleukin-6 (IL)-6, a pro-inflammatory cytokine. As a result of confirming the activation of TLR2/4 using Western blot and immunofluorescence (IF), the TLR2/4 protein expression and fluorescence intensity increased depending on the concentration of vacuoles. Yeast vacuoles significantly upregulate protein expression of p-p65/p-p38 MAPKs. In summary, the vacuoles isolated from S. cerevisiae in macrophages have increased phagocytic ability at a concentration of 20 (µg/ml) and can function as immune-enhancing agent suggesting that TLR2/4 mediated the p38 MAPK/nuclear factor kappa Bsignaling pathway.

Resveratrol inhibits Toxoplasma gondii-induced lung injury, inflammatory cascade and evidences of its mechanism of action

BackgroundToxoplasma gondii is an opportunistic protozoan that can infect host to cause toxoplasmosis. We have previously reported that resveratrol (RSV) has protective effects against liver damage in T. gondii infected mice. However, the effect of RSV on lung injury caused by T. gondii infection and its mechanism of action remain unclear. PurposeIn this work, we studied the protective effects of RSV on lung injury caused by T. gondii infection and explored the underlying mechanism. MethodsMolecular docking and localized surface plasmon resonance assay were used to detect the molecular interactions between RSV and target proteins. In vitro, the anti-T. gondii effects and potential anti-inflammatory mechanisms of RSV were investigated by quantitative competitive-PCR, RT-PCR, ELISA, Western blotting and immunofluorescence using RAW 264.7 cells infected with tachyzoites of T. gondii RH strain. In vivo, the effects of RSV on lung injury caused by T. gondii infection were assessed by observing pathological changes and the expression of inflammatory factors of lung. ResultsRSV inhibited T. gondii loads and T. gondii-derived heat shock protein 70 (T.g.HSP70) expression in RAW 264.7 cells and lung tissues. Moreover, RSV interacts with T.g.HSP70 and toll-like receptor 4 (TLR4), respectively, and interferes with the interaction between T.g.HSP70 and TLR4. It also inhibited the overproduction of inducible nitric oxide synthase, TNF-α and high mobility group protein 1 (HMGB1) by down-regulating TLR4/nuclear factor kappa B (NF-κB) signaling pathway, which is consistent with the effect of TLR4 inhibitor CLI-095. In vivo, RSV improved the pathological lung damage produced by T. gondii infection, as well as decreased the number of inflammatory cells in bronchoalveolar lavage fluid and the release of HMGB1 and TNF-α. ConclusionThese findings indicate that RSV can inhibit the proliferation of T. gondii and T.g.HSP70 expression both in vitro and in vivo. RSV can inhibit excessive inflammatory response by intervening T.g.HSP70 and HMGB1 mediated TLR4/NF-κB signaling pathway activation, thereby ameliorating lung injury caused by T. gondii infection. The present study provides new data that may be useful for the development of RSV as a new agent for the treatment of lung damage caused by T. gondii infection.

2-Methoxyestradiol Damages DNA in Glioblastoma Cells by Regulating nNOS and Heat Shock Proteins

Gliomas are the most prevalent primary tumors of the central nervous system (CNS), accounting for over fifty percent of all primary intracranial neoplasms. Glioblastoma (GBM) is the most prevalent form of malignant glioma and is often incurable. The main distinguishing trait of GBM is the presence of hypoxic regions accompanied by enhanced angiogenesis. 2-Methoxyestradiol (2-ME) is a well-established antiangiogenic and antiproliferative drug. In current clinical studies, 2-ME, known as Panzem, was examined for breast, ovarian, prostate, and multiple myeloma. The SW1088 grade III glioma cell line was treated with pharmacological and physiological doses of 2-ME. The induction of apoptosis and necrosis, oxidative stress, cell cycle arrest, and mitochondrial membrane potential were established by flow cytometry. Confocal microscopy was used to detect DNA damage. The Western blot technique determined the level of nitric oxide synthase and heat shock proteins. Here, for the first time, 2-ME is shown to induce nitro-oxidative stress with the concomitant modulation of heat shock proteins (HSPs) in the SW1088 grade III glioma cell line. Crucial therapeutic strategies for GMB should address both cell proliferation and angiogenesis, and due to the above, 2-ME seems to be a perfect candidate for GBM therapy.