Nitric Oxide Synthase-2 Research Articles

Lycium barbarum polysaccharide alleviates dextran sodium sulfate-induced inflammatory bowel disease by regulating M1/M2 macrophage polarization via the STAT1 and STAT6 pathways.

Disruption of colonic homeostasis caused by aberrant M1/M2 macrophage polarization contributes to the development of inflammatory bowel disease (IBD). Lycium barbarum polysaccharide (LBP) is the primary active constituent of traditional Chinese herbal Lycium barbarum L., which has been widely demonstrated to have important functions in regulating immune activity and anti-inflammatory. Thus, LBP may protect against IBD. To test this hypothesis, the DSS-induced colitis model was established in mice, then the mice were treated with LBP. The results indicated that LBP attenuated the weight loss, colon shortening, disease activity index (DAI), and histopathological scores of colon tissues in colitis mice, suggesting that LBP could protect against IBD. Besides, LBP decreased the number of M1 macrophages and the protein level of Nitric oxide synthase 2(NOS2) as a marker of M1 macrophages and enhanced the number of M2 macrophages and the protein level of Arginase 1(Arg-1) as a marker of M2 macrophages in colon tissues from mice with colitis, suggesting that LBP may protect against IBD by regulating macrophage polarization. Next, the mechanistic studies in RAW264.7 cells showed that LBP inhibited M1-like phenotype by inhibiting the phosphorylation of STAT1, and promoted M2-like phenotype by promoting the phosphorylation of STAT6. Finally, immunofluorescence double-staining results of colon tissues showed that LBP regulated STAT1 and STAT6 pathways in vivo. The results in the study demonstrated that LBP could protect against IBD by regulating macrophage polarization through the STAT1 and STAT6 pathways.

Abstract 3588: Multiplex proteomics profiling with the imaging mass cytometry (IMC) identifies immune cell phenotypes associated with high NOS2/COX2 expression in ER- negative and TNBC patients

Abstract Elevated expression of Inducible Nitric oxide Synthase-2 (NOS2) occurs during acute and chronic inflammation and wound healing processes however, it is also a predictor of bad prognosis in aggressive ER-negative tumors. Overexpression of NOS2 correlates with other prognostic biomarkers making it a unique multifunctional oncoprotein. Cyclooxygenase-2 (COX2) is another enzyme associated with cancer inflammation and is overexpressed in >50% of breast cancers. In our previous studies, we have shown that NOS2/COX2 co-expression is a strong predictor of poor clinical outcome and promotes CD8+ T cell exclusion from tumor epithelia in Triple-negative breast cancers (TNBC). This observation was further explored using multiplexing proteomics technologies. Imaging Mass Cytometry (IMC) has revolutionized tumor microenvironment research by allowing the interrogation of over 35 protein markers simultaneously for the elucidation of the spatial distribution of immune and tumor cells at sub-cellular resolution. Herein, we used multiplex capability IMC to explore the interactions of immune and tumor cell counterparts in ER-negative and TNBC tumors. We employed Formalin-fixed paraffin-embedded (FFPE) tissue from 26 ER-negative and TNBC samples from a clinical cohort of African American breast cancer patients in the Baltimore MD area. We developed a panel of 15 protein metal-labeled antibody markers including antibodies targeting NOS2, COX2, T cells (CD45, CD3, CD4, CD8,) endothelium (CD31), monocytes (CD14), macrophages (CD68), basal markers (α-SMA), cancer stem cells (CD44v6), cancer cells (Pan-CK, B7H4, Cadherin), and serine proteases (granzyme B) secreted by T cells or Natural Killer (NK) cells. Single section slides of 4 µm size were stained with the cocktail of metal-labeled antibodies. Multiple regions of interest (ROIs) representing the tumor and immune cells compartments and tumor-facing stromal cell areas were ablated with the Hyperion Imaging system. The data analysis was performed by the Bodenmiller lab pipeline and VisioPharm software packages. Our preliminary data suggest that patient tumors with high or low NOS2/COX2 expression showed variable levels of CD68 positive macrophage population and T cells. We are currently analyzing the remaining data to understand the cell phenotypes associated with the immunosuppressive and immune-rich clinical samples and performing neighborhood analysis of tumor cells to immune cells and vessels. Citation Format: Milind Pore, Lisa Ridnour, Paul Mallory, Ashley Cardamone, Stephen Lockett, William Bocik, David Wink. Multiplex proteomics profiling with the imaging mass cytometry (IMC) identifies immune cell phenotypes associated with high NOS2/COX2 expression in ER- negative and TNBC patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3588.

Elevations of Extracellular Vesicles and Inflammatory Biomarkers in Closed Circuit SCUBA Divers.

Blood-borne extracellular vesicles and inflammatory mediators were evaluated in divers using a closed circuit rebreathing apparatus and custom-mixed gases to diminish some diving risks. "Deep" divers (n = 8) dove once to mean (±SD) 102.5 ± 1.2 m of sea water (msw) for 167.3 ± 11.5 min. "Shallow" divers (n = 6) dove 3 times on day 1, and then repetitively over 7 days to 16.4 ± 3.7 msw, for 49.9 ± 11.9 min. There were statistically significant elevations of microparticles (MPs) in deep divers (day 1) and shallow divers at day 7 that expressed proteins specific to microglia, neutrophils, platelets, and endothelial cells, as well as thrombospondin (TSP)-1 and filamentous (F-) actin. Intra-MP IL-1β increased by 7.5-fold (p < 0.001) after day 1 and 41-fold (p = 0.003) at day 7. Intra-MP nitric oxide synthase-2 (NOS2) increased 17-fold (p < 0.001) after day 1 and 19-fold (p = 0.002) at day 7. Plasma gelsolin (pGSN) levels decreased by 73% (p < 0.001) in deep divers (day 1) and 37% in shallow divers by day 7. Plasma samples containing exosomes and other lipophilic particles increased from 186% to 490% among the divers but contained no IL-1β or NOS2. We conclude that diving triggers inflammatory events, even when controlling for hyperoxia, and many are not proportional to the depth of diving.

Monomeric CRP regulates inflammatory responses in human intervertebral disc cells.

CRP is an acute-phase protein that is used as a biomarker to follow severity and progression in infectious and inflammatory diseases. Its pathophysiological mechanisms of action are still poorly defined. CRP in its pentameric form exhibits weak anti-inflammatory activity. The monomeric isoform (mCRP) exerts potent proinflammatory properties in chondrocytes, endothelial cells, and leucocytes. No data exist regarding mCRP effects in human intervertebral disc (IVD) cells. This work aimed to verify the pathophysiological relevance of mCRP in the aetiology and/or progression of IVD degeneration. We investigated the effects of mCRP and the signalling pathways that are involved in cultured human primary annulus fibrosus (AF) cells and in the human nucleus pulposus (NP) immortalized cell line HNPSV-1. We determined messenger RNA (mRNA) and protein levels of relevant factors involved in inflammatory responses, by quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. We also studied the presence of mCRP in human AF and NP tissues by immunohistochemistry. We demonstrated that mCRP increases nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and Lipocalin 2 (LCN2) expression in human AF and NP cells. We also showed that nuclear factor-κβ (NF-κβ), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signalling of mCRP. Finally, we demonstrated the presence of mCRP in human AF and NP tissues. Our results indicate, for the first time, that mCRP can be localized in IVD tissues, where it triggers a proinflammatory and catabolic state in degenerative and healthy IVD cells, and that NF-κβ signalling may be implicated in the mediation of this mCRP-induced state.

Neurotherapeutic Effects of Quercetin and Its Metabolite Compounds on Cognitive Impairment and Parkinson's Disease: An In Silico Study.

Little is known about the metabolomic profile of quercetin and its biological effects. This study aimed to determine the biological activities of quercetin and its metabolite products, as well as the molecular mechanisms of quercetin in cognitive impairment (CI) and Parkinson's disease (PD). Key methods used were MetaTox, PASS Online, ADMETlab 2.0, SwissADME, CTD MicroRNA MIENTURNE, AutoDock, and Cytoscape. A total of 28 quercetin metabolite compounds were identified by phase I reactions (hydroxylation and hydrogenation reactions) and phase II reactions (methylation, O-glucuronidation, and O-sulfation reactions). Quercetin and its metabolites were found to inhibit cytochrome P450 (CYP) 1A, CYP1A1, and CYP1A2. The studied compounds demonstrated significant gastrointestinal absorption and satisfied Lipinsky's criterion. Due to their high blood-brain barrier permeability, P-glycoprotein inhibition, anticancer, anti-inflammatory, and antioxidant capabilities, quercetin and its metabolite products have been proposed as promising molecular targets for the therapy of CI and PD. By regulating the expression of crucial signaling pathways [mitogen-activated protein kinase (MAPK) signaling pathway, and neuroinflammation and glutamatergic signaling], genes [brain derived neurotrophic factor (BDNF), human insulin gene (INS), and dopamine receptor D2 (DRD2), miRNAs (hsa-miR-16-5p, hsa-miR-26b-5p, hsa-miR-30a-5p, hsa-miR-125b-5p, hsa-miR-203a-3p, and hsa-miR-335-5p], and transcription factors [specificity protein 1 (SP1), v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), and nuclear factor Kappa B subunit 1 (NFKB1)], quercetin exhibited its neurotherapeutic effects in CI and PD. In addition to inhibiting β-N-acetylhexosaminidase, quercetin also showed robust interactions and binding affinities with heme oxygenase 1 (HMOX1), superoxide dismutase 2 (SOD2), tumor necrosis factor (TNF), nitric oxide synthase 2 (NOS2), brain-derived neurotrophic factor (BDNF), INS, DRD2, and γ-aminobutyric acid type A (GABAa). This study identified 28 quercetin metabolite products. The metabolites have similar characteristics to quercetin such as physicochemical properties, absorption, distribution, metabolism, and excretion (ADME), and biological activities. More research, especially clinical trials, is needed to find out how quercetin and its metabolites protect against CI and PD.

Mucosal Genes Expression in Inflammatory Bowel Disease Patients: New Insights.

Individual differences in IBD illness severity, behavior, progression, and therapy response are evident. Since a break in the intestinal epithelial barrier causes IBD to begin, mucosal gene expression in IBD is crucial. Due to its high sensitivity and dynamic nature, molecular analysis of biomarkers in intestinal biopsies is feasible and provides a reliable means of evaluating localized inflammation. The goal of this investigation was to discover alterations in gene expression in the inflamed mucosa of IBD patients undergoing treatment with 5-amino salicylic acid (5ASA) (N = 39) or anti-TNF drugs (N = 22). The mucosal expression of numerous IBD-related genes was evaluated using qPCR. We discovered that the levels of the proteins Lipocalin-2 (LCN2), Nitric Oxide Synthase 2 (NOS2), Mucin 2 (MUC2), Mucin 5AC (MUC5AC), and Trefoil factor 1 (TFF1), which are overexpressed in untreated IBD patients compared to non-IBD subjects, are decreased by both therapy regimens. On the other hand, anti-TNF medicine helped the levels of ABCB1 and E-cadherin return to normal in IBD patients who were not receiving treatment.

Lnc-17Rik promotes the immunosuppressive function of Myeloid-Derived suppressive cells in esophageal cancer

Myeloid-derived suppressor cells (MDSCs) are a population of immature bone marrow cells that accumulate in large numbers in the spleen, peripheral blood, bone marrow, lymph nodes, and local and metastatic foci of tumors. C/EBP homologous protein (CHOP) and CCAAT/enhancer binding protein β (C/EBPβ) play key roles in regulating the immunosuppressive function and differentiation of MDSCs. Our study revealed that the long noncoding RNA Lnc-17Rik was able to promote immunosuppression in tumors by facilitating the activation and expression of key genes involved in MDSC differentiation. Lnc-17Rik was shown to directly interact with CHOP and C/EBPβ LIP to facilitate their dissociation from the transcriptional repressor complex involving C/EBP LAP/LIP/CHOP. Moreover, Lnc-17Rik increased the association of WD repeat-containing protein 5 (WDR5) with C/EBP LAP, promoting H3K4me3 enrichment in the promoter regions of arginase 1 (Arg-1), cyclooxygenase 2 (COX2), nitric oxide synthase 2 (NOS2) and NADPH oxidase 2 (NOX2) to enhance the expression of these genes. Furthermore, using a CD45 chimeric model we confirmed that Lnc-17Rik promoted the differentiation of monocytic (M)-MDSCs in vivo with the introduction of Lnc-17Rik-overexpressing MDSCs shown to promote tumor growth as a result of enhancing their immunosuppressive function. Notably, human Lnc-17Rik is highly homologous to mouse Lnc-17Rik and fulfills similar functions in human MDSC-like cells. In addition, we also found a high level of Lnc-17Rik in peripheral blood MDSC of patients with esophageal cancer. These findings suggest that Lnc-17Rik plays an important role in controlling the immunosuppressive function of MDSCs in the tumor environment and may further serve as a potential therapeutic target for patients with esophageal cancer.

The Chemical Biology of NO that Regulates Oncogenic Signaling and Metabolism: NOS2 and Its Role in Inflammatory Disease.

Nitric oxide (NO) and the enzyme that synthesizes it, nitric oxide synthase 2 (NOS2), have emerged as key players in inflammation and cancer. Expression of NOS2 in tumors has been correlated both with positive outcomes and with poor prognoses. The chemistry of NO is the major determinate to the biological outcome and the concentration of NO, which can range over five orders of magnitude, is critical in determining which pathways are activated. It is the activation of specific oncogenic and immunological mechanisms that shape the outcome. The kinetics of specific reactions determine the mechanisms of action. In this review, the relevant reactions of NO and related species are discussed with respect to these oncogenic and immunological signals.

Lectroacupuncture stimulation at CV4 sites prevents breast cancer-induced osteoporosis by activating the Wnt/β-catenin signaling pathway

Osteoporosis is a common condition among breast cancer patients, characterized by decreased bone density due to chemotherapy and hormone therapy. However, the application of electroacupuncture (EA) stimulation at Guanyuan (CV4) sites has been found to be beneficial in enhancing bone mineral density. In this study, we aimed to investigate the efficacy of EA stimulation at CV4 sites in breast cancer-induced osteoporosis and elucidate the potential underlying mechanisms. A breast cancer model in mice was established, EA stimulation at CV4 sites was performed, and hematoxylin-eosin (HE) stain was conducted to assess the bone mineral density and trabecular bone volume of femur tissues. Additionally, Tartrate-resistant acid phosphatase (TRAP) staining was performed to identify and visualize osteoclasts. Furthermore, Western blots were used to evaluate the expression levels of various proteins, including c-src, Cathepsin K (CtSK), matrix metalloproteinase 9 (MMP9), Collagen 1A1 (COL1A1), Osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), Wnt3a and β-catenin, and Enzyme Linked Immunosorbent Assay (ELISA) to measure the concentration of Alkaline phosphatase (ALP), NO synthase 2 (NOS2), glutathione reductase (GSR), superoxide dismutase (SOD), reactive oxygen species (ROS) and lactate dehydrogenase (LDH) in the serum. The results revealed that EA stimulation in the breast cancer model resulted in higher bone mineral density and trabecular bone volume, along with a reduction in the number of osteoclasts. EA stimulation at CV4 site significantly decreased the protein levels of c-src, CtSK and MMP9, while also reducing ROS and LDH production. Conversely, it promoted the expression of COL1A1, OPN, RUNX2, Wnt3a and β-catenin. Additionally, it elevated the concentration of ALP, NOS2, GSR and SOD in the serum. Overall, EA stimulation at the CV4 site may inhibit bone loss and promote bone differentiation by activating the Wnt/β-catenin pathway, indicating its potential use for breast cancer-induced osteoporosis treatment.

"Component-target-efficacy" network analysis and experimental verification of Qingkailing Oral Preparation

This study aims to explore the mechanism of Qingkailing(QKL) Oral Preparation's heat-clearing, detoxifying, mind-tranquilizing effects based on &quot;component-target-efficacy&quot; network. To be specific, the potential targets of the 23 major components in QKL Oral Preparation were predicted by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and SwissTargetPrediction. The target genes were obtained based on UniProt. OmicsBean and STRING 10 were used for Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of the targets. Cytoscape 3.8.2 was employed for visualization and construction of &quot;component-target-pathway-pharmacological effect-efficacy&quot; network, followed by molecular docking between the 23 main active components and 15 key targets. Finally, the lipopolysaccharide(LPS)-induced RAW264.7 cells were adopted to verify the anti-inflammatory effect of six monomer components in QKL Oral Preparation. It was found that the 23 compounds affected 33 key signaling pathways through 236 related targets, such as arachidonic acid metabolism, tumor necrosis factor α(TNF-α) signaling pathway, inflammatory mediator regulation of TRP channels, cAMP signaling pathway, cGMP-PKG signaling pathway, Th17 cell differentiation, interleukin-17(IL-17) signaling pathway, neuroactive ligand-receptor intera-ction, calcium signaling pathway, and GABAergic synapse. They were involved in the anti-inflammation, immune regulation, antipyretic effect, and anti-convulsion of the prescription. The &quot;component-target-pathway-pharmacological effect-efficacy&quot; network of QKL Oral Preparation was constructed. Molecular docking showed that the main active components had high binding affinity to the key targets. In vitro cell experiment indicated that the six components in the prescription(hyodeoxycholic acid, baicalin, chlorogenic acid, isochlorogenic acid C, epigoitrin, geniposide) can reduce the expression of nitric oxide(NO), TNF-α, and interleukin-6(IL-6) in cell supernatant(P&lt;0.05). Thus, the above six components may be the key pharmacodynamic substances of QKL Oral Preparation. The major components in QKL Oral Prescription, including hyodeoxycholic acid, baicalin, chlorogenic acid, isochlorogenic acid C, epigoitrin, geniposide, cholic acid, isochlorogenic acid A, and γ-aminobutyric acid, may interfere with multiple biological processes related to inflammation, immune regulation, fever, and convulsion by acting on the key protein targets such as IL-6, TNF, prostaglandin-endoperoxide synthase 2(PTGS2), arachidonate 5-lipoxygenase(ALOX5), vascular cell adhesion molecule 1(VCAM1), nitric oxide synthase 2(NOS2), prostaglandin E2 receptor EP2 subtype(PTGER2), gamma-aminobutyric acid receptor subunit alpha(GABRA), gamma-aminobutyric acid type B receptor subunit 1(GABBR1), and 4-aminobutyrate aminotransferase(ABAT). This study reveals the effective components and mechanism of QKL Oral Prescription.

ЕКСПРЕСІЯ ГЕНІВ PTGS2 ТА NOS2 У СИНОВІАЛЬНІЙ РІДИНІ ХВОРИХ НА ОСТЕОАРТРИТ ПІСЛЯ SARS-CoV2-ІНФЕКЦІЇ

Background. The emergence of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has created a serious global threat to the health of people in various countries. The 2019 coronavirus disease (Coronavirus disease 2019, COVID-19) has caused the development of many complications in the population of most countries of the world. Currently, many people have felt the unpleasant consequences of the coronavirus infection. Among them, a special group consists of patients with chronic diseases, particularly osteoarthritis. The development of inflammation and intensification of free radical processes is a leading link in the pathogenesis of osteoarthritis. Inflammatory and degenerative processes that develop in osteoarthritis cause modification of the molecular and cellular composition of the synovial fluid. Methods. The aim of the work was to determine the expression of (Prostaglandin-endoperoxide synthase 2) and NOS2 (Nitric Oxide Synthase 2) genes in synovial fluid cells of patients with osteoarthritis after SARS-CoV2 infection. All study participants were divided into two groups. The first group (n = 22) is patients with osteoarthritis of the knee joints II-III degree. The second group (n = 14) is patients with osteoarthritis of the knee joints II–III degree, who suffered a mild and moderate form of COVID-19 6–9 months ago. Expression of PTGS2 and NOS2 genes was determined in nuclear cells of synovial fluid. RNA was obtained by the Chomczynski method. Synthesis of cDNA and quantitative polymerase chain reaction in real time (Real-time PCR, qPCR) using the commercial kit "Thermo Scientific Verso SYBR Green 1-Step qRT-PCR ROX Mix" ("Thermo Scientific, Lithuania). Processing of research results was carried out using generally accepted methods of variational statistics. Results. In patients with osteoarthritis of the knee joints after SARS-CoV2 infection, the expression of PTGS2 and NOS2 genes increases in nuclear cells of the synovial fluid compared to patients diagnosed with osteoarthritis. Conclusions. Our results indicate that patients with osteoarthritis may develop a more severe course of synovial inflammation after infection with COVID-19.

Total rupture of Achilles tendon induces inflammatory response and glial activation on the spinal cord of mice.

Rupture of Achilles tendon is a common accident affecting professional and recreational athletes. Acute and chronic pain are symptoms commonly observed in patients with rupture. However, few studies have investigated whether Achilles tendon rupture is able to promote disorders in the central nervous system (CNS). Therefore, the current study aimed to evaluate nociceptive alterations and inflammatory response in the L5 lumbar segment of Balb/c mice spinal cord after Achilles tendon rupture. We found increased algesia in the paw of the ruptured group on the 7th and 14th days post-tenotomy compared with the control group. This phenomenon was accompanied by overexpression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase-2 (NOS-2) as well as hyperactivation of astrocytes and microglia in nociceptive areas of L5 spinal cord as evidenced by intense GFAP and IBA-1 immunostaining, respectively. Biochemical studies also demonstrated increased levels of nitrite in the L5 spinal cord of tenotomized animals compared with the control group. Thus, we have demonstrated for the first time that total rupture of the Achilles tendon induced inflammatory response and nitrergic and glial activation in the CNS in the L5 spinal cord region.

FTIR Characterization of Sulfated Polysaccharides Obtained from Macrocystis integrifolia Algae and Verification of Their Antiangiogenic and Immunomodulatory Potency In Vitro and In Vivo.

The aim of this study was to evaluate the antiangiogenic and immunomodulatory potential of sulfated polysaccharides from the marine algae Macrocystis integrifolia characterized by FTIR. The cytotoxicity of sulfated polysaccharides was evaluated using the 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay. Antiangiogenic activity was evaluated using the chicken chorioallantoic membrane (CAM) assay. Immunomodulatory activity was determined on macrophage functionality and allergic response. The results showed that sulfated polysaccharides significantly decreased angiogenesis in chicken chorioallantoic membranes (p &lt; 0.05). Likewise, they inhibited in vivo chemotaxis and in vitro phagocytosis, the transcription process of genes that code the enzymes cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and nitric oxide synthase-2 (NOS-2) and the nuclear factor kappa-light chain enhancer of activated B cells (NF-κB), showing immunomodulatory properties on the allergic response, as well as an in vivo inhibitory effect in the ovalbumin-induced inflammatory allergy model (OVA) and inhibited lymphocyte proliferation specific to the OVA antigen in immunized mice. Finally, these compounds inhibited the histamine-induced skin reaction in rats, the production of immunoglobulin E (IgE) in mice, and the passive response to skin anaphylaxis in rats. Therefore, the results of this research showed the potential of these compounds to be a promising source for the development of antiangiogenic and immunomodulatory drugs.

Manganese-induced PINK1 S-nitrosylation exacerbates nerve cell damage by promoting ZNF746 repression of mitochondrial biogenesis

Occupational exposure and non-occupational exposure to excessive levels of manganese (Mn) result in neuronal cell damage through mitochondrial dysfunction. The functional integrity of mitochondria is maintained by mitophagy and mitochondrial biogenesis. Although Mn-induced S-nitrosylation of PTEN-induced putative kinase 1 (PINK1) can interfere with mitophagy, its effect on mitochondrial biogenesis remains unclear. In this study, we established a rat model of Mn poisoning or “manganism” to examine the relationship between PINK1 S-nitrosylation and impairment of mitochondrial biogenesis, and found that treatment with 60 mg/kg Mn induced marked neurobehavioral abnormalities in rats and significantly increased the S-nitrosylation level of PINK1. We also found that the nuclear-encoded peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A)-mediated mitochondrial biogenesis was significantly upregulated in rats treated with 15 and 30 mg/kg Mn, and downregulated in rats treated with 60 mg/kg Mn. We further investigated the role of S-nitrosylated PINK1 and its molecular mechanism in the high-dose Mn-mediated impairment of mitochondrial biogenesis in primary cultured neurons treated with the nitric oxide synthase 2 (NOS2) inhibitor 1400 W. Our results revealed that the PPARGC1A-mediated mitochondrial biogenesis was upregulated in neurons treated with 100 μM, but downregulated in neurons treated with 200 μM Mn, which was similar to the in vivo results. However, treatment with 1400W could effectively prevent the 200 μM Mn-mediated impairment of mitochondrial biogenesis by suppressing nitric oxide (NO)-mediated PINK1 S-nitrosylation and rescuing Parkin-interacting substrate (PARIS, ZNF746) degradation, thereby upregulating mitochondrial biogenesis via PPARGC1A. These findings demonstrated that S-nitrosylation of PINK1 and subsequent prevention of ZNF746 degradation were crucial signaling processes involved in the Mn-mediated impairment of mitochondrial biogenesis, which might serve as an underlying mechanism of Mn-induced neurotoxicity. Furthermore, this study provided a reliable target for the prevention and treatment of manganism.

A combination of omega-3 and exercise reduces experimental Achilles tendinopathy induced with a type-1 collagenase in rats.

This study aimed to evaluate the effectiveness of omega-3 supplementation with exercise in acollagenase-induced Achilles tendinopathy (AT) ratmodel. Experimental groups (healthy control (HC), AT, exercise (Ex), omega-3 (W), and Ex+W) were randomly allocated. After a week of adaptation, oral omega-3 was initiated for 8 weeks (5 days/week). The exercise groups performed treadmill running for 30 min/day (5 days/week, 20 m/min, 8 weeks) following one week of adaptation (10 m/min, 15 min/day). Matrix metalloproteinase-13 (MMP-13), interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and total antioxidant-oxidant status (TAS) levels were determined in serum samples. Tendon samples were obtained for biomechanical, histopathological, and immunohistochemical assessments. Ultimate tensile force, yield force, stiffness values, collagen type-I alpha 1 expression, and serum TAS significantly decreased (P < 0.05) in AT vs. HC. These values and expression significantly increased in the Ex+W group vs. AT. Serum MMP-13, IL-1β, and TNF-α levels decreased in all treatment groups vs. AT. The most significant decrease was found in the Ex+W group (P < 0.01). Histopathologically, the improvement in degeneration was statistically significant in the Ex+W group (P < 0.05). Immunohistochemically, MMP-13, IL-1β, TNF-α, and nitric oxide synthase-2 expression was decreased in all treatment groups vs. AT. In conclusion, omega-3 and exercise might be recommended in AT patients.

NOS2/miR-493-5p Signaling Regulates in the LPS-Induced Inflammatory Response in the RAW264.7 Cells.

Tuberculosis (TB) is a fatal infectious disease; however, the molecular mechanisms underlying the pathogenicity of TB remain elusive. The present study aims to identify potential biomarkers associated with Mycobacterium tuberculosis (M.tb) infection by using integrated bioinformatics and in vitro validation studies. GSE50050, GSE78706, and GSE108844 data from the gene expression omnibus (GEO) database were downloaded to identify differentially expressed genes (DEGs). The functions of DEGs were further subjected to gene ontology (GO) and KEGG pathway analysis. The hub genes from the DEGs were determined based on the protein-protein interaction (PPI) network analysis. Finally, the hub genes were experimentally validated using the in vitro functional studies. A total of 26 common DEGs were identified among GSE50050, GSE78706, and GSE108844. The functional enrichment analysis showed that the common DEGs were associated with cytokines response and TB pathways. The PPI network analysis identified nine hub genes. Further in vitro studies showed that nitric oxide synthase 2 (NOS2) was up-regulated in RAW264.7 cells upon lipopolysaccharides (LPS) stimulation, which was accompanied by increased inflammatory cytokines release. Furthermore, NOS2 was found to be a target of miR-493-5p, which was confirmed by luciferase reporter assay. NOS2 was repressed by miR-493-5p overexpression and was up-regulated after miR-493-5p inhibition in RAW264.7 cells. The rescue experiments showed that LPS-induced increase in the inflammatory cytokines of the RAW264.7 cells was significantly attenuated by NOS2 knockdown and miR-493-5p overexpression. Collectively, our results for the first time demonstrated that NOS2/miR-493-5p signaling pathway may potentially involve in the inflammatory response during bacterial infection such as M. tb infection.

388 The impact of the susceptibility gene NOS2 and its bioactive molecule Nitric oxide in the pathogenesis of Psoriasis

Psoriasis is an immune-mediated systemic skin disorder with a strong genetic predisposition. The gene expression of the psoriasis susceptibility gene Nitric oxide synthase 2 (NOS2) is specifically upregulated in psoriatic involved skin. NOS2 catalyses the synthesis of nitric oxide (NO), a short-lived free radical and pivotal signaling molecule with both pro- and anti-inflammatory properties. In this study, we have investigated the functional relevance of NOS2 expression in psoriasis. Using qPCR, we found that NOS2 mRNA was strongly expressed in the epidermis of psoriatic involved skin compared to healthy skin.

Decreasing effects of protein kinase inhibitors on the expression of NOS2 and inflammatory cytokines and on phagocytosis in rat peritoneal macrophages is partly related to repolarization

The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) pathway plays a pivotal role in macrophage polarization, but other signaling routes may also be involved. The aim of this study was to reveal the relationship of activation between rat peritoneal macrophages and their polarization, to detect the signaling routes involved, and find selective protein kinase inhibitors decreasing the production of inflammatory proteins in activated peritoneal macrophages. Rat macrophages were elicited with i.p. casein injection. CD80 and CD206 markers, NOS2 (Nitric oxide synthase 2), arginase, cytokines and phagocytosis were investigated by ELISA (Enzyme Linked Immunosorbent Assay), Western Blot, fluorescent microscopic and flow cytometry. Statistical methods were ANOVA (Analysis Of Variance) and Student t-tests. Resident and elicited cells expressed both CD80 and CD206 polarization markers. The involvement of MAPK (mitogen-activated protein kinases) and JAK/STAT pathways in the polarization was evidenced by a phosphorylation array, supported by Western blotting, by cytokine markers and by the inhibitory effects of kinase inhibitors. The expression of NOS2 and inflammatory cytokines was higher in elicited cells suggesting their M1 polarization. This effect was reduced by the inhibitors of MAPK and JAK/STAT pathways. Phagocytosis was also higher in elicited macrophages and decreased by these inhibitors. Nevertheless, they cannot change macrophage polarization unambiguously, as levels of CD80 and CD206 markers were not changed. For comparison, human blood macrophages were also studied. Similar effects and several differences were observed between the two types of macrophages, suggesting the role of the previous differentiation in defining their characteristics. Selected anti-cancer protein kinase inhibitors of p38, MAPK and JAK/STAT pathways are possible candidates for the therapy of inflammatory diseases.

Effect of citronellol on oxidative stress, neuroinflammation and autophagy pathways in an in vivo model of Parkinson's disease

Citronellol, a monoterpene found in the essential oils of Cymbopogo plants has been reported to possess various biological properties. In the present study, we investigated the neuroprotective mechanisms of citronellol against rotenone induced neurodegeneration by using rat model of Parkinson's disease (PD). Our results demonstrated that oral administration of citronellol prevented rotenone induced reactive oxygen species production, lipid peroxidation and enhanced Nrf2 expression, catalase, glutathione peroxidase and superoxide dismutase levels in the brain. Enzyme-linked immunosorbent assays showed that citronellol reduced secretion of TNF-α, IL-1β, IL-6 and decreased MMP-9 expression levels. Further, citronellol prevented rotenone induced microglia (Iba-1 staining) and astrocyte (GFAP staining) activation. Western blot analysis showed that citronellol significantly decreased the expression of cyclooxygenase-2 and inducible nitric oxide synthase-2 that are key markers of neuroinflammation. We further evaluated the effect of citronellol on dopaminergic neurons in substantia nigra pars compacta (SNpc) and striatum (ST) which are key anatomical structures in PD. Tyrosine hydroxylase (TH) immunoreactivity showed that citronellol preserved Tyrosine hydroxylase (TH) positive dopaminergic neurons and enhanced TH striatal expression levels significantly compared to rotenone alone group. Further, to understand the effect of citronellol on apoptosis and proteotoxicity, we evaluated apoptotic markers (Bax, Bcl-2), growth regulator (mTOR) and α-synuclein expression. Citronellol attenuated rotenone induced expression of pro-apoptotic protein Bax, reduced α-synuclein expression and enhanced Bcl-2 and mTOR levels. In addition, citronellol modulated autophagy pathway by decreasing LC-3 (Microtubule-associated proteins) and p62 levels. Taken together, our results demonstrate that citronellol protected dopaminergic neurons through its antioxidant, anti-inflammatory, anti-apoptotic and autophagy modulating properties.

Dual functions of microRNA-17 in maintaining cartilage homeostasis and protection against osteoarthritis

Damaged hyaline cartilage has no capacity for self-healing, making osteoarthritis (OA) “difficult-to-treat”. Cartilage destruction is central to OA patho-etiology and is mediated by matrix degrading enzymes. Here we report decreased expression of miR-17 in osteoarthritic chondrocytes and its deficiency contributes to OA progression. Supplementation of exogenous miR-17 or its endogenous induction by growth differentiation factor 5, effectively prevented OA by simultaneously targeting pathological catabolic factors including matrix metallopeptidase-3/13 (MMP3/13), aggrecanase-2 (ADAMTS5), and nitric oxide synthase-2 (NOS2). Single-cell RNA sequencing of hyaline cartilage revealed two distinct superficial chondrocyte populations (C1/C2). C1 expressed physiological catabolic factors including MMP2, and C2 carries synovial features, together with C3 in the middle zone. MiR-17 is highly expressed in both superficial and middle chondrocytes under physiological conditions, and maintains the physiological catabolic and anabolic balance potentially by restricting HIF-1α signaling. Together, this study identified dual functions of miR-17 in maintaining cartilage homeostasis and prevention of OA.