Inducible Nitric Oxide Research Articles

Nitro-oxidative stress impairs single cardiomyocyte adrenergic reserve in a murine model of HFpEF

Abstract Background Exercise intolerance is the central symptom of patients with heart failure and preserved ejection fraction (HFpEF). Underlying reduced cardiac functional reserve in response to adrenergic stimuli (stress testing) has been suggested but the molecular mechanisms are insufficiently understood. In cardiomyocytes, nitric oxide (NO) modifies contractility and is required to achieve a full adrenergic response. Recently, dysregulation of NO release has been described to contribute to HFpEF. In a murine model of HFpEF, we investigated cardiomyocyte's adrenergic functional reserve and the role of NO in cardiomyocyte contractility, calcium handling and adrenergic reserve. Methods Firstly, the effects of NO on sarcomere shortening and calcium handling (Fura-2) were studied in isolated, adult ventricular cardiomyocytes (AMVMs) from 8–10 weeks old, male C57BL/6J mice. Secondly, male C57BL/6J mice (12w) were fed regular CHOW (Sham) or a high fat diet (D12492, Research Diet) and L-NAME (1g/l, via the drinking water) for 15 weeks to induce HFpEF. At week 27, mice underwent echocardiography and exercise testing (treadmill). In AMVMs isolated from HFpEF and Sham mice, we quantified sarcomere shortening, calcium handling (Fura-2), release of NO (CuFL2) and reactive oxygen species (DCF) before and after the addition of isoproterenol (ISO, 1μM), in the absence and presence (40 min preincubation) of an inhibitor of inducible NO Synthase (1400W) or a denitrosylating agent (glutathione). Results In AMVMs, addition of the NO donor SNAP (10μM) increased calcium transient amplitude and accelerated relaxation time. Inhibition of NO synthesis with L-NAME (100μM) impaired adrenergic response upon exposure to ISO. HFpEF mice (evident by diastolic dysfunction (e/e' ratio) and lung edema (wet lung weight / TL)) exhibited significantly reduced exercise capacity (running distance). In AMVMs isolated from HFpEF mice, NO and ROS release were increased at baseline, associated with an increased sarcomere shortening amplitude and faster relaxation and calcium removal as compared to Sham. Strikingly, after the addition of ISO, the adrenergic functional reserve (cellular inotropy and lusitropy) was significantly lower in HFpEF vs. Sham AMVMs. Preincubation with the iNOS inhibitor 1400W or glutathione restored adrenergic inotropic and lusitropic reserve in HFpEF AMVMs. Conclusion In HFpEF, adrenergic reserve is impaired on a single cardiomyocyte level. This is at least partially related to an increase in NO release. Pharmacologic inhibition of iNOS improved adrenergic reserve in HFpEF. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG

Nitric Oxide Production and Effects in Group B Streptococcus Chorioamnionitis.

Intrauterine infection, or chorioamnionitis, due to group B Streptococcus (GBS) is a common cause of miscarriage and preterm birth. To cause chorioamnionitis, GBS must bypass maternal-fetal innate immune defenses including nitric oxide (NO), a microbicidal gas produced by nitric oxide synthases (NOS). This study examined placental NO production and its role in host-pathogen interactions in GBS chorioamnionitis. In a murine model of ascending GBS chorioamnionitis, placental NOS isoform expression quantified by RT-qPCR revealed a four-fold expression increase in inducible NOS, no significant change in expression of endothelial NOS, and decreased expression of neuronal NOS. These NOS expression results were recapitulated ex vivo in freshly collected human placental samples that were co-incubated with GBS. Immunohistochemistry of wild type C57BL/6 murine placentas with GBS chorioamnionitis demonstrated diffuse inducible NOS expression with high-expression foci in the junctional zone and areas of abscess. Pregnancy outcomes between wild type and inducible NOS-deficient mice did not differ significantly although wild type dams had a trend toward more frequent preterm delivery. We also identified possible molecular mechanisms that GBS uses to survive in a NO-rich environment. In vitro exposure of GBS to NO resulted in dose-dependent growth inhibition that varied by serovar. RNA-seq on two GBS strains with distinct NO resistance phenotypes revealed that both GBS strains shared several detoxification pathways that were differentially expressed during NO exposure. These results demonstrate that the placental immune response to GBS chorioamnionitis includes induced NO production and indicate that GBS activates conserved stress pathways in response to NO exposure.

Omarigliptin Mitigates 6-Hydroxydopamine- or Rotenone-Induced Oxidative Toxicity in PC12 Cells by Antioxidant, Anti-Inflammatory, and Anti-Apoptotic Actions.

Dipeptidyl peptidase-4 (DPP-4) inhibitors are reported to exhibit promising effects on several pathological processes associated with Parkinson’s disease (PD). To explore its repositioning potential as an antiparkinsonian agent, we evaluated the effects of omarigliptin (OMG), a DPP-4 inhibitor recently approved as a hypoglycemic drug, on neurotoxin-induced toxicity, using PC12 cells as a cellular model of PD. The molecular mechanism(s) underlying its protective activity was also investigated. OMG alleviated oxidative toxicity and the production of reactive oxygen species induced by 6-hydroxydopamine (6-OHDA) or rotenone. It also partially attenuated the formation of DPPH radicals and lipid peroxidation, demonstrating the antioxidant properties of OMG. OMG upregulated Nrf2 and heme oxygenase-1 (HO-1). Notably, treatment with a selective HO-1 inhibitor and Nrf2 knockdown by siRNA abolished the beneficial effects of OMG, indicating that the activated Nrf2/HO-1 signaling was responsible for the protective activity. Moreover, OMG exhibited anti-inflammatory activity, blocking inflammatory molecules, such as nitric oxide (NO) and inducible NO synthase, through inhibition of IκBα phosphorylation and NF-κB activation in an Akt-dependent fashion. Finally, OMG decreased the levels of cleaved caspase-3 and Bax and increased the level of Bcl-2, indicating its anti-apoptotic properties. Collectively, these results demonstrate that OMG alleviates the neurotoxin-induced oxidative toxicity through Nrf2/HO-1-mediated antioxidant, NF-κB-mediated anti-inflammatory, and anti-apoptotic mechanisms in PC12 cells. Our findings elucidating multiple mechanisms of antiparkinsonian activity strongly support the therapeutic potential of OMG in the treatment of PD.

Sonidegib Suppresses Production of Inflammatory Mediators and Cell Migration in BV2 Microglial Cells and Mice Treated with Lipopolysaccharide via JNK and NF-κB Inhibition.

Our structure-based virtual screening of the FDA-approved drug library has revealed that sonidegib, a smoothened antagonist clinically used to treat basal cell carcinoma, is a potential c-Jun N-terminal kinase 3 (JNK3) inhibitor. This study investigated the binding of sonidegib to JNK3 via 19F NMR and its inhibitory effect on JNK phosphorylation in BV2 cells. Pharmacological properties of sonidegib to exert anti-inflammatory and anti-migratory effects were also characterized. We found that sonidegib bound to the ATP binding site of JNK3 and inhibited JNK phosphorylation in BV2 cells, confirming our virtual screening results. Sonidegib also inhibited the phosphorylation of MKK4 and c-Jun, the upstream and downstream signals of JNK, respectively. It reduced the lipopolysaccharide (LPS)-induced production of pro-inflammatory factors, including interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), and nitric oxide (NO), and the expression of inducible NO synthase and cyclooxygenase-2. The LPS-induced cell migration was suppressed by sonidegib. Sonidegib inhibited the LPS-induced IκBα phosphorylation, thereby blocking NF-κB nuclear translocation. Consistent with these findings, orally administered sonidegib attenuated IL-6 and TNF-α levels in the brains of LPS-treated mice. Collectively, our results indicate that sonidegib suppresses inflammation and cell migration in LPS-treated BV2 cells and mice by inhibiting JNK and NF-κB signaling. Therefore, sonidegib may be implicated for drug repurposing to alleviate neuroinflammation associated with microglial activation.

Murine macrophages do not support the proliferation of Leishmania (Viannia) braziliensis amastigotes even in absence of nitric oxide and presence of high arginase activity.

Leishmania (Viannia) braziliensis is the main species responsible for American tegumentary leishmaniasis in Brazil. Nevertheless, the use of this parasite species to study Leishmania infection in the murine model has been less conducted when compared with other Leishmania species. The control of murine infection with Leishmania has been associated with nitric oxide (NO) produced by inducible NO synthase (iNOS) from M1 macrophages, while arginase expressed by M2 macrophages is related to Leishmania proliferation. Here we use three different strains of L. (V.) braziliensis and one strain of L. (L.) major to study a 9-day infection of macrophages in vitro. Wild-type bone marrow-derived macrophages (BMDM) supported the proliferation of L. (L) major amastigotes from the 3rd day after infection, while all strains of L. (V.) braziliensis did not proliferate even inside IL-4-treated or iNOS knockout (KO) macrophages. The arginase activity was higher in iNOS KO than IL-4-treated macrophage showing that the absence of proliferation is independent of arginase. Importantly, L. (V.) braziliensis was able to cause uncontrolled disease in iNOS KO mice in vivo demonstrating that murine macrophages present at the site of infection have additional changes beyond inhibition of NO production or stimulation of arginase activity to support parasite proliferation.

Luteolin alleviates vascular dysfunctions in CLP-induced polymicrobial sepsis in mice.

Luteolin, a naturally occurring flavonoid, is thought to have health-promoting properties as a part of human diet and has been reported to possess a wide range of pharmacological activities. Therefore, the present study was undertaken to evaluate the effect of luteolin pre-treatment on vascular dysfunctions in sepsis induced by caecal ligation and puncture (CLP) in the mouse model. Mice were divided into four groups: sham, luteolin plus sham, CLP, and luteolin plus CLP. Luteolin was administered (0.2mg/kg body weight) intraperitoneally one hour (h) before CLP surgery in mice. 20 ± 2h post CLP surgery, the isolated thoracic aorta of mice was assessed for its vascular reactivity to noradrenaline (NA) and acetylcholine (ACh). To explore the underlying mechanism, aortic mRNA expressions of α1D adrenoceptors, eNOS and iNOS were investigated. In mice with CLP-induced sepsis luteolin pre-treatment markedly increased the survival time and attenuated serum lactate level. The CLP group manifested the reduced vascular reactivity to NA and this deficit was restored by luteolin pre-treatment. However, luteolin pre-treatment did not improve α1D adrenoceptors down-regulation observed in septic mice aorta. In the presence of 1400W, the NA contractile response was significantly restored in CLP mice aortic tissue in comparison with the respective control of septic mice and further enhanced in the presence of luteolin. Luteolin reduced the iNOS mRNA expression and iNOS-derived nitrite production. Pre-treatment with luteolin restored the endothelial dysfunction in septic mice aorta by improving eNOS mRNA expression and enhanced eNOS-derived nitric oxide (NO) production in septic mice aorta and aortic iNOS gene expression and inducible NO production. The present study suggests that the vasoplegic state to NA in aorta was restored through the iNOS pathway and endothelial dysfunction was reversed via eNOS and NO production pathway.

Nitrooleic acid inhibits macrophage activation induced by lipopolysaccharide from Prevotella intermedia

The hypothesis of the present study was that nitro-fatty acids (NO2-FAs) would suppress inflammation associated with periodontal disease. To test this hypothesis, we investigated the influence of nitrooleic acid, a prototypical NO2-FA, on the inflammatory response of murine macrophages activated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen associated the etiology of different types of periodontal diseases. LPS was prepared from P. intermedia cells by using phenol-water protocol. Culture supernatants were assayed for nitric oxide (NO), interleukin-1β (IL-1β), and IL-6. Real-time polymerase chain reaction and immunoblotting analyses were performed to quantify messenger RNA and protein expression, respectively. The secreted embryonic alkaline phosphatase reporter assay was performed to measure NF-κB activation. The transcription factor assay kit was used to measure DNA-binding of NF-κB subunits. Findings obtained from the present study revealed that nitrooleic acid suppresses the generation and messenger RNA expression of inducible NO synthase-derived NO, IL-1β, and IL-6 in RAW264.7 cells activated with P. intermedia LPS and promotes macrophage polarization toward anti-inflammatory M2 phenotype. We also found that nitrooleic acid exerts its effect via heme oxygenase-1 induction and suppression of NF-κB signaling. The inhibition of NO and proinflammatory cytokine production by nitrooleic acid was independent from PPAR-γ, JNK, p38, and STAT1/3. Nitrooleic acid may represent a novel class of agent as a host modulator which has therapeutic benefit in periodontal disease, though more work is needed to confirm this.

Detoxification Response of Pseudomonas fluorescens MFAF76a to Gaseous Pollutants NO2 and NO.

Bacteria are often exposed to nitrosative stress from their environment, from atmospheric pollution or from the defense mechanisms of other organisms. Reactive nitrogen species (RNS), which mediate nitrosative stress, are notably involved in the mammalian immune response through the production of nitric oxide (NO) by the inducible NO synthase iNOS. RNS are highly reactive and can alter various biomolecules such as lipids, proteins and DNA, making them toxic for biological organisms. Resistance to RNS is therefore important for the survival of bacteria in various environments, and notably to successfully infect their host. The fuel combustion processes used in industries and transports are responsible for the emission of important quantities of two major RNS, NO and the more toxic nitrogen dioxide (NO2). Human exposure to NO2 is notably linked to increases in lung infections. While the response of bacteria to NO in liquid medium is well-studied, few data are available on their exposure to gaseous NO and NO2. This study showed that NO2 is much more toxic than NO at similar concentrations for the airborne bacterial strain Pseudomonas fluorescens MFAF76a. The response to NO2 involves a wide array of effectors, while the response to NO seemingly focuses on the Hmp flavohemoprotein. Results showed that NO2 induces the production of other RNS, unlike NO, which could explain the differences between the effects of these two molecules.

Loganin attenuates interleukin-1β-induced chondrocyte inflammation, cartilage degeneration, and rat synovial inflammation by regulating TLR4/MyD88/NF-κB.

ObjectiveInflammation plays a crucial part in osteoarthritis (OA) development. This work aimed to explore loganin’s role and molecular mechanism in inflammation and clarify its anti-inflammatory effects in OA treatment.MethodsChondrocytes were stimulated using interleukin (IL)-1β and loganin at two concentrations (1 μM and 10 μM). Nitric oxide (NO) and prostaglandin E2 (PGE2) expression was assessed. Real-time polymerase chain reaction was used to evaluate inducible NO synthase (iNOS), cyclooxygenase (COX)-2, IL-6, and tumor necrosis factor (TNF)-α mRNA levels. Western blot was used to investigate TLR4, MyD88, p-p65, and IκB-α expression. p65 nuclear translocation, synovial inflammatory response, and cartilage degeneration were also assessed.ResultsLoganin significantly reduced IL-1β-mediated PGE2, NO, iNOS, and COX-2 expression compared with that of the IL-1β stimulation group. The TLR4/MyD88/NF-κB pathway was suppressed by loganin, which decreased inflammatory cytokine (TNF-α and IL-6) levels compared with those of the IL-1β stimulation group. Loganin inhibited IL-1β-mediated NF-κB p65 nuclear translocation compared with that of the IL-1β stimulation group. Loganin partially suppressed cartilage degeneration and the synovial inflammatory response in vivo.ConclusionThis work demonstrated that loganin inhibited IL-1β-mediated inflammation in rat chondrocytes through TLR4/MyD88/NF-κB pathway regulation, thereby reducing rat cartilage degeneration and the synovial inflammatory response.

Citri Reticulatae Pericarpium extract and flavonoids reduce inflammation in RAW 264.7 macrophages by inactivation of MAPK and NF‐κB pathways

AbstractCitri reticulatae Pericarpium (CRP) is widely consumed as a food and herbal medicine in various countries and has many biological activities, including anti‐inflammatory effects, but the molecular mechanism is not completely known. This study aims to investigate whether CRP extract and its major flavonoids hesperidin (Hsd) and hesperetin (Hst) can reduce inflammation in macrophages and the underlying mechanism. The chemical composition of CRP was detected by ultra‐performance liquid chromatography‒mass spectrometry (UPLC‒MS) analysis. The inflammatory cell model was induced by stimulating RAW 264.7 cells with lipopolysaccharide (LPS, 1 μg/ml). Levels of nitric oxide (NO) and cytokines were determined by Griess assay and enzyme‐linked immunosorbent assay kits, respectively. Protein expressions were examined by western blotting. CRP extract dose‐dependently suppressed LPS‐induced production of NO, interleukin‐6 (IL‐6), and tumor necrosis factor‐α. Elevated expressions of inducible NO synthase (iNOS) and cyclooxygenase‐2 (COX‐2) and phosphorylation of c‐Jun NH2‐terminal kinase, extracellular signal‐regulated kinase, p38, and p65 in LPS‐stimulated cells were prevented by CRP extract. Hsd and Hst are flavonoids present in CRP, as confirmed by UPLC‒MS analysis. Both Hsd and Hst ameliorated LPS‐triggered inflammation to reduce NO release, IL‐6 generation, and protein expressions of iNOS and COX‐2. These results reveal that CRP extract possesses anti‐inflammatory activity by inactivating the mitogen‐activated protein kinase and nuclear factor kappa‐light chain‐enhancer of activated B cells pathways, where Hsd and Hst are bioactive constituents, implying the potential application of CRP as dietary supplements against inflammatory diseases.

Hydrogen sulfide potently promotes neuronal differentiation of adipose tissue-derived stem cells involving nitric oxide-mediated signaling cascade with the aid of cAMP-elevating agents

Neuronal differentiation of adipose tissue-derived stem cells (ASCs) is potently promoted by valproic acid (VPA) through a gaseous signaling molecule, nitric oxide (NO). Here, we investigated the involvement of hydrogen sulfide (H2S), another gaseous signaling molecule, in neuronal differentiation of ASCs. VPA-promoted neuronal differentiation of ASCs was accompanied by increased intracellular H2S and sulfane sulfur with increased mRNA expression of enzymes synthesizing sulfane sulfur including cystathionine β-synthase (CBS), of which inhibition reduced the differentiation efficiency. H2S donors, GYY4137 (GYY) or NaHS, potently promoted neuronal differentiation of ASCs when cAMP-elevating agents, dibutyryl cyclic adenosine monophosphate and isobutyl methyl-xanthine, were added as neuronal induction medium (NIM). Neuronal differentiation of ASCs promoted by NaHS or GYY was accompanied by Ca2+ entry and increased mRNA expression of voltage-gated Ca2+ channels. NaHS or GYY also increased mRNA expression of enzymes of the NO-citrulline cycle including inducible NO synthase (iNOS). It was concluded from these results that H2S potently promoted differentiation of ASCs into neuronal cells expressing functional voltage-gated Ca2+ channels with the aid of cAMP-elevating agents, involving NO-mediated signaling cascade. These effects of H2S were also considered as a partial mechanism for the VPA-promoted neuronal differentiation of ASCs.

Sulindac acetohydrazide derivative attenuates against cisplatin induced organ damage by modulation of antioxidant and inflammatory signaling pathways

This study aimed to explore the mechanisms of action of a sulindac acetohydrazide derivative, N'-(4-dimethylaminobenzylidene)-2-1-(4-(methylsulfinyl) benzylidene)-5-fluoro-2-methyl-1H-inden-3-yl) acetohydrazide, against anticancer drug cisplatin induced organ damage. Using a rodent model, various markers of organ function and signaling pathways were examined and validated by molecular docking studies. The study involves five groups of animals: control, DMSO, CDDP, CDDP + DMFM, and DMFM. Biochemical enzyme activity, histopathology, tissue antioxidant, and oxidative stress markers were examined. RT-PCR and western blot analyses were conducted for the expression of inducible cyclooxygenase enzyme (COX-2), nuclear factor kappa beta (NF-κB), p65, IL-1, TNF-α, and inducible nitric oxide synthase (iNOS). Flow cytometry analysis of CD4 + TNF-α, CD4 + COX-2, and CD4 + STAT-3 cells in whole blood was performed. Structural and dynamic behavior of DMFM upon binding with receptor molecule molecular docking and dynamic simulations were performed using bioinformatics tools and software. Treatment with DMFM reversed cisplatin-induced malondialdehyde (MDA) and nitric oxide (NO) induction, whereas the activity of glutathione peroxidase (GPx), and superoxide dismutase (SOD) in the kidney, heart, liver, and brain tissues were increased. DMFM administration normalized plasma levels of biochemical enzymes. We observed a marked decline in CD4 + STAT3, TNF-α, and COX2 cell populations in whole blood after treatment with DMFM. DMFM downregulated the expression factors related to inflammation at the mRNA and protein levels, i.e., IL-1, TNF-α, iNOS, NF-κB, STAT-3, and COX-2. Dynamic simulations and in silico docking data supports the experimental findings. Our experimental and in silico results illustrated that DMFM may affect protective action against cisplatin-induced brain, heart, liver, and kidney damage via reduction of inflammation and ROS.

SIRT3 attenuates coronary atherosclerosis in diabetic patients by regulating endothelial cell function.

BackgroundThis study aimed to explore the relationship between the Sirtuin 3 (SIRT3) gene and endothelial cell dysfunction, contributing to the progression of coronary atherosclerosis driven by hyperglycemia.MethodsWe measured serum SIRT3 levels using enzyme‐linked immunosorbent assay in 95 patients with type 2 diabetes mellitus (T2DM) who underwent diagnostic coronary angiography. The patients were divided into two groups according to the presence (n = 45) or absence (n = 50) of coronary artery disease (CAD). Human aortic endothelial cells (HAECs) grown in vitro in a medium with various concentrations of glucose (5.5, 11, 16.5, 22, 27.5, 33, and 38.5 mM) for 24 h were assessed for protein expression of SIRT3, peroxisome proliferator‐activated receptor alpha (PPAR‐α), endothelial nitric oxide (NO) synthase (eNOS), and inducible NO synthase (iNOS) using Western blot analysis. HAECs were subjected to SIRT3 overexpression or inhibition through SIRT3 adenovirus and siRNA transfection.ResultsSerum SIRT3 levels were significantly lower in T2DM patients with CAD than in those without CAD (p = 0.048). The in vitro results showed that HG significantly increased SIRT3, PPAR‐α, and eNOS protein expression in a concentration‐dependent manner. Moreover, iNOS expression was decreased in HAECs in response to HG. Reduced PPAR‐α and eNOS levels and increased iNOS levels were observed in SIRT3 silenced HAECs cells. In contrast, SIRT3 overexpression significantly improved PPAR‐α and eNOS expression and suppressed iNOS expression.ConclusionSIRT3 was associated with the progression of atherosclerosis in T2DM patients through upregulation of PPAR‐α and eNOS and downregulation of iNOS, which are involved in endothelial dysfunction under hyperglycemic conditions.

Effects of Compounds Isolated from Lindera erythrocarpa on Anti-Inflammatory and Anti-Neuroinflammatory Action in BV2 Microglia and RAW264.7 Macrophage.

Lindera erythrocarpa contains various constituents such as cyclopentenedione-, flavonoid-, and chalcone-type components. In this study, a novel bi-linderone derivative and 17 known compounds were isolated from the leaves of L. erythrocarpa by using various chromatographic methods. The structures of the components were determined from nuclear magnetic resonance and mass spectrometry data. All isolated compounds were tested for anti-inflammatory and anti-neuroinflammatory activities in lipopolysaccharide (LPS)-induced BV2 and RAW264.7 cells. Some of these compounds showed anti-inflammatory effects by inhibiting the nitric oxide (NO) produced by LPS. In particular, linderaspirone A (16), bi-linderone (17) and novel compound demethoxy-bi-linderone (18) showed significant inhibitory effects on the production of prostaglandin E2 (PGE2), tumor necrosis factor-α, and interleukin-6. The three compounds also inhibited the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), which are pro-inflammatory proteins, and the activation of nuclear factor κB (NF-κB). Therefore, linderaspirone A (16), bi-linderone (17), and demethoxy-bi-linderone (18) isolated from the leaves of L. erythrocarpa have therapeutic potential in neuroinflammatory diseases.

Anti-inflammatory effects of anthraquinones of Polygonum multiflorum roots

Background: The roots of Polygonum multiflorum Thunberg (Polygonaceae) are a crude drug known as Kashu and are thought to improve blood deficiency as a Kampo concept. Kashu is added to Kampo formulas, including Tokiinshi, which is used to treat eczema and dermatitis with itchiness by facilitating blood circulation in the skin. Although anti- inflammatory effects were expected, the reports on anti-inflammatory constituents of Polygonum roots are very few. Objective: To purify pharmacologically active constituents from Polygonum roots and compare their anti- inflammatory potency using hepatocytes. Methods: Polygonum roots were extracted and fractionated based on hydrophobicity to three fractions, the ethyl acetate (EtOAc)-soluble, n-butanol-soluble, and water-soluble fractions. Constituents were purified from the EtOAc- soluble fraction, and its contents were measured using liquid chromatography-triple quadrupole mass spectrometer. The interleukin (IL)-1β-induced production of nitric oxide (NO), a proinflammatory mediator, in rat hepatocytes was monitored to assess anti-inflammatory activity. Results: The EtOAc-soluble fraction inhibited IL-1β-induced NO production, whereas the other fractions did not. Six constituents were isolated from the EtOAc-soluble fraction and identified as (E)-2,3,5,4'-tetrahydroxystilbene-2-O-β- D-glucoside, emodin, emodin-8-O-β-D-glucopyranoside, physcion, physcion-8-O-β-D-glucopyranoside, and catechin. Emodin and emodin-8-O-β-D-glucopyranoside significantly inhibited NO production in hepatocytes, and the others showed less potency in suppressing NO production. These two anthraquinones decreased the protein and mRNA levels of inducible NO synthase, as well as the mRNA levels of tumor necrosis factor α. In a search of the literature to date, the comparison of pharmacological activity of the constituents of Polygonum roots was not reported.Conclusion: These results suggest that hydrophobic constituents in the EtOAc-soluble fraction, especially anthraquinones, may exert the anti-inflammatory effects by treatment with Polygonum roots.Keywords: anthraquinone, nitric oxide, inflammation, Kampo medicine, LC–MS/MS.

Diammonium glycyrrhizinate ameliorates portal hypertension by regulating portal macrophage oxidation and superoxide dismutase 3

Portal hypertension (PHT) is a complication of liver diseases. Increased intrahepatic vascular resistance is attributed to reduced bioavailability of vasodilator substances. The macrophage activation and superoxide dismutase 3 (SOD3) involve in the pathogenesis of PHT. Diammonium glycyrrhizinate (DG) is the salt form of glycyrrhizin derived from Radix glycyrrhizae, exerting anti-oxidant activities and be beneficial for liver injury. Here, we aimed to investigate effects of DG on PHT and explore its underlying mechanisms on regulation of macrophages and SOD3. The carbon tetrachloride induced PHT rats received administration of liposome-encapsulated clodronate for hepatic macrophage depletion, or PBS liposomes for matched control. DG (25 mg/kg) or vehicle was gavaged. Portal pressure in vivo, and serum biomarkers of macrophage activation were measured. The nitric oxide (NO) and prostacyclin (PGI2) bioavailability was evaluated in the isolated portal perfused rat livers. Liver tissues were collected to evaluate cirrhosis, macrophage oxidation, and SOD3 activity. Depletion of hepatic macrophages decreased portal pressure, increased bioavailability of NO and PGI2, and restored SOD3 activity. DG effectively decreased portal pressure, relieved cirrhosis, inhibited macrophage activation. DG increased bioavailability of NO and PGI2 to relax portal veins. DG relieved portal macrophage oxidation through decreasing nicotinamide adenine dinucleotide phosphate oxidase 2 and inducible NO synthase expressions, elevated SOD3 activities and increased SOD3 expressions at portal triads. These findings indicated that DG restored SOD3 activity, against portal macrophage oxidation, protected bioavailability of NO and PGI2, thereby reduced portal pressure. It suggested a potential use of DG for PHT treatment.

Abstract 3133: Glucose is important for macrophage metabolism and response to immunotherapy in a pancreatic cancer model

Abstract Introduction: Pancreatic cancer (PC) is resistant to systemic therapies. This is at least in part due to the characteristically harsh tumor microenvironment which limits the delivery of systemic therapies and is not conducive to anti-tumor immune cell function. Unfortunately, development of novel therapeutics is arduous. Thus, identification of methods to augment the efficacy of existing therapeutics, such as immunotherapies, represents an attractive option. Herein, we attempt to sensitize PC to a colony stimulating factor 1 receptor (CSF1R) inhibitor by augmenting peripheral glucose levels. Methods Murine PC cells (KPC) and bone marrow derived macrophages were cultured in hypoglycemic (≤2.5mM) or hyperglycemic (25mM) conditions. Phenotypic (western blot, flow cytometry), metabolic (seahorse, LC-MS metabolomics), and cell survival (clonogenic assay) assays were performed. KPC cells were orthotopically injected into the pancreas of immunocompetent mice for survival studies. Mice were randomized to pexidartinib (a CSF1R inhibitor) or vehicle and received either normal water of 30% dextrose (D30). Results: In a co-culture experiment, tumor-supporting M2 macrophages increased KPC cell growth by 30%, whereas tumor-fighting M1 macrophages decreased KPC growth by 95%. M1 macrophages were poorly suited to survive in hypoglycemic conditions as compared to hyperglycemic conditions (relative survival: 38% vs 100%, p<0.01). M2 macrophage survival was equivalent (93% vs 100%, p=0.39). When cultured in hypoglycemic conditions, M1 macrophages adopted an M2-like metabolic profile (i.e., increased TCA cycle metabolites and decreased glycolytic metabolites) assessed by both seahorse and LC-MS metabolomics. As glucose concentrations decreased in culture media over time, protein levels of an M1 marker (inducible nitric oxide, iNOS) decreased and an M2 marker (arginase) appeared, suggesting a phenotypic switch. Pexidartinib augmented M1 iNOS protein levels and simultaneously decreased M2 arginase levels. On an in vitro flow cytometric analysis, the percentage of M2 macrophages (CD206+, CD301+, F480+, CD11b+) decreased as glucose concentrations were increased (33.7% vs 38.0%), and decreased further when pexidartinib was combined with hyperglycemia (20.5%). Mice treated with pexidartinib and D30 had improved median survival relative to mice receiving pexidartinib alone (median survival: 42 (IQR: 38, 52) vs 34 (IQR: 32, 38) days, p<0.05) in two independent experiments. Of note, pexidartinib alone did not improve survival as compared to vehicle or D30. Conclusion: Higher glucose conditions promote M1 macrophage survival and function, while preventing the switch to an M2 phenotype. Also, higher peripheral glucose levels appear to sensitize PC to a CSF1R inhibitor. We plan to validate these findings using other immunotherapeutics (e.g., checkpoint inhibitors). Citation Format: Jonathan J. Hue, Mehrdad Zarei, Hallie J. Graor, Erryk S. Katayama, Omid Hajihassani, Alexander W. Loftus, Luke D. Rothermel, Ali Vaziri-Gohar, Jordan M. Winter. Glucose is important for macrophage metabolism and response to immunotherapy in a pancreatic cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3133.

γ-Glutamylcysteine attenuates amyloid-β oligomers-induced neuroinflammation in microglia via blocking NF-κB signaling pathway

Alzheimer's disease (AD) is the most prevalent neurogenerative disease, characterized by progressive memory loss and cognitive deficits. Intracellular neurofibrillary tangles (NFTs) and amyloid-β (Aβ)-formed neuritic plaques are major pathological features of AD. Aβ evokes activation of microglia to release inflammatory mediators and ROS to induce neurotoxicity, leading to neurodegeneration. γ-Glutamylcysteine (γ-GC), an intermediate dipeptide of the GSH-synthesis pathway with anti-inflammatory and anti-oxidative properties, represents a relatively unexplored option for AD treatment. In the present study, we investigated the anti-inflammatory effect of γ-GC on Aβ oligomer (AβO)-induced neuroinflammation and the associated molecular mechanism in microglia. The results showed that γ-GC reduced AβO-induced release of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and nitric oxide (NO), and the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2). γ-GC decreased ROS and MDA production and increased the GSH level, GSH/GSSG ratio, and SOD activity in AβO-treated microglia. Mechanistically, γ-GC inhibited activation of nuclear factor kappa B (NF-κB), and upregulated the nuclear receptor-related 1 (Nurr1) protein expression to suppress the transcriptional effect of NF-κB on the inflammatory genes. Besides, γ-GC suppressed the AβO-induced neuroinflammation in mice. These findings suggested that γ-GC might represent a potential therapeutic agent for anti-neuroinflammation.

Extract of Pinus densiflora needles suppresses acute inflammation by regulating inflammatory mediators in RAW264.7 macrophages and mice

Context Pinus densiflora Siebold & Zucc. (Pinaceae) needle extracts ameliorate oxidative stress, but research into their anti-inflammatory effects is limited. Objective To investigate antioxidant and anti-inflammatory effects of a Pinus densiflora needles (PINE) ethanol extract in vitro and in vivo. Materials and methods We measured levels of reactive oxygen species (ROS), superoxide dismutase (SOD) and inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW264.7 cells at various PINE concentrations (25, 50 and 100 μg/mL; but 6.25, 12.5 and 25 μg/mL for interleukin-1β and prostaglandin E2 (PGE2)). Thirty ICR mice were randomized to six groups: vehicle, control, PINE pre-treatment (0.1, 0.3 and 1 mg/left ear for 10 min followed by arachidonic acid treatment for 30 min) and dexamethasone. The posttreatment ear thickness and myeloperoxidase (MPO) activity were measured. Results PINE 100 μg/mL significantly decreased ROS (IC50, 70.93 μg/mL, p < 0.01), SOD (IC50, 30.99 μg/mL, p < 0.05), malondialdehyde (p < 0.01), nitric oxide (NO) (IC50, 27.44 μg/mL, p < 0.01) and tumour necrosis factor-alpha (p < 0.05) levels. Interleukin-1β (p < 0.05) and PGE2 (p < 0.01) release decreased significantly with 25 μg/mL PINE. PINE 1 mg/ear inhibited LPS-stimulated expression of cyclooxygenase-2 and inducible NO synthase in RAW264.7 macrophages and significantly inhibited ear oedema (36.73–15.04% compared to the control, p < 0.01) and MPO activity (167.94–105.59%, p < 0.05). Discussion and conclusions PINE exerts antioxidant and anti-inflammatory effects by inhibiting the production of inflammatory mediators. Identified flavonoids such as taxifolin and quercetin glucoside can be attributed to effect of PINE.

Focused ultrasound-induced targeted delivery of miR-155-5p inhibitor employing biomimetic microbubbles reduces NADPH oxidase expression and inflammation in atherosclerotic ApoE-/- mice

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): UEFISCDI Background MicroRNAs are important biomarkers and potential therapeutic targets in atherosclerosis. MiR-155-5p amplifies inflammation in atherosclerosis by targeting negative regulators of NF-kB signaling pathway. Inhibition of pro-atherogenic miRNAs or mimicking the function of selected anti-atherogenic miRNAs have emerged as prospective oligonucleotide-base therapeutic interventions in order to correct the expression of key genes that are mechanistically linked to atherosclerotic lesion formation. Purpose The aim of this study was to develop a strategy to reduce oxidative stress and inflammation in atherosclerotic mice employing sterically stabilized biomimetic microbubbles (MB) as focused ultrasound-responsive targeted delivery systems of miR-155-5p inhibitor. Methods ApoE-/- mice were fed a normal (control) or a high-fat, cholesterol-rich diet for 14 weeks to accelerate de development of aortic atherosclerotic lesions. FAM-labelled miRNA negative control inhibitor, miRNA negative control inhibitor or miR-155-5p inhibitor were charge-coupled to VCAM1-targeted MB. Targeted MB were delivered to mice in a single dose via retro-orbital injection. Ultrasound imaging interrogation of atherosclerotic lesions in ApoE-/- mice was performed using a conventional 30 MHz transducer (Vevo 2100 System). The Sonidel SP100 sonoporator was used for ultrasound-induced targeted release of miRNA inhibitors in mice. The biodistribution of MB was monitored by high-resolution fluorescence imaging. Atherosclerotic lesions were assessed by Oil Red O staining. Real time PCR and Western blot were employed to determine the expression of NADPH oxidase (Nox) subtypes, a major source of oxidative stress, and markers of oxidative stress inflammation (4-HNE-protein adducts, inducible nitric oxide synthase/NOS2, CD68). Results Epi-fluorescence analysis demonstrated the enhanced uptake of FAM-labelled miRNA control inhibitor by lungs and the atherosclerotic aorta of ApoE-/- mice following ultrasound wave exposure (1 MHz, 2.0 w/cm2, 50% duty cycles, 15 minutes). The mRNA/protein levels of Nox1-4 subtypes, 4-HNE-protein adducts, NOS2, and macrophage infiltration marker CD68 were found significantly elevated in the atherosclerotic aorta of ApoE-/- mice. The expression levels of Nox isoforms, 4-HNE-protein adducts, NOS2, and CD68 were significantly decreased in the atherosclerotic aorta of ApoE-/- mice 1-week after administration of targeted MB carrying miR-155-5p inhibitor as compared with atherosclerotic ApoE-/- mice treated with targeted MB carrying miRNA negative control inhibitor. Conclusion Systemic administration of miR-155-5p inhibitor conjugated with VCAM1-targeted MB reduces the expression of oxidative stress and inflammation-related markers in experimental atherosclerosis. Ultrasound-induced targeted delivery of miRNA-oriented oligonucleotides could become an important theranostic strategy combining imaging and treatment procedures in clinical atherosclerosis.