Reduced iNOS Expression Research Articles

Inhibitor of serine peptidase 2 enhances Leishmania major survival in the skin through control of monocytes and monocyte-derived cells.

Leishmania major is the causative agent of the neglected tropical disease, cutaneous leishmaniasis. In the mouse, protective immunity to Leishmania is associated with inflammatory responses. Here, we assess the dynamics of the inflammatory responses at the lesion site during experimental long-term, low-dose intradermal infection of the ear, employing noninvasive imaging and genetically modified L. major. Significant infiltrates of neutrophils and monocytes occurred at 1–4 d and 2–4 wk, whereas dermal macrophage and dendritic cell (DC) numbers were only slightly elevated in the first days. Quantitative whole-body bioluminescence imaging of myeloperoxidase activity and the quantification of parasite loads indicated that the Leishmania virulence factor, inhibitor of serine peptidase 2 (ISP2), is required to modulate phagocyte activation and is important for parasite survival at the infection site. ISP2 played a role in the control of monocyte, monocyte-derived macrophage, and monocyte-derived DC (moDC) influx, and was required to reduce iNOS expression in monocytes, monocyte-derived cells, and dermal DCs; the expression of CD80 in moDCs; and levels of IFN-γ in situ. Our findings indicate that the increased survival of L. major in the dermis during acute infection is associated with the down-regulation of inflammatory monocytes and monocyte-derived cells via ISP2.—Goundry, A., Romano, A., Lima, A. P. C. A., Mottram, J. C., Myburgh, E. Inhibitor of serine peptidase 2 enhances Leishmania major survival in the skin through control of monocytes and monocyte-derived cells.

Thalidomide attenuates development of morphine dependence in mice by inhibiting PI3K/Akt and nitric oxide signaling pathways

Morphine dependence and the subsequent withdrawal syndrome restrict its clinical use in management of chronic pain. The precise mechanism for the development of dependence is still elusive. Thalidomide is a glutamic acid derivative, recently has been reconsidered for its clinical use due to elucidation of different clinical effects. Phosphoinositide 3-kinase (PI3K) is an intracellular transducer enzyme which activates Akt which in turns increases the level of nitric oxide. It is well established that elevated levels of nitric oxide has a pivotal role in the development of morphine dependence. In the present study, we aimed to explore the effect of thalidomide on the development of morphine dependence targeting PI3K/Akt (PKB) and nitric oxide (NO) pathways. Male NMRI mice and human glioblastoma T98G cell line were used to study the effect of thalidomide on morphine dependence. In both models the consequent effect of thalidomide on PI3K/Akt and/or NO signaling in morphine dependence was determined. Thalidomide alone or in combination with PI3K inhibitor, Akt inhibitor or nitric oxide synthase (NOS) inhibitors significantly reduced naloxone induced withdrawal signs in morphine dependent mice. Also, the levels of nitrite in hippocampus of morphine dependent mice were significantly reduced by thalidomide in compared to vehicle treated morphine dependent mice. In T98G human glioblastoma cells, thalidomide alone or in combination with PI3K and Akt inhibitors significantly reduced iNOS expression in comparison to the morphine treated cells. Also, morphine-induced p-Akt was suppressed when T98G cells were pretreated with thalidomide. Our results suggest that morphine induces Akt, which has a crucial role in the induction of NOS activity, leading to morphine dependence. Moreover, these data indicate that thalidomide attenuates the development of morphine dependence in vivo and in vitro by inhibition of PI3K/Akt and nitric oxide signaling pathways.

Comparison of Anti-Inflammatory Activities of Structurally Similar Triterpenoids Isolated from Bitter Melon

Momordica charantia L., or bitter melon, has been suggested to exhibit anti-inflammatory activity. In a previous study, three structurally similar triterpenes, namely 5β,19-epoxy-25-methoxycucurbita-6,23-diene-3β,19-diol (EMCD), 5β,19-epoxy-25-methoxycucurbita-6,23-dien-3β-ol (EMCO), and 5β,19-epoxy-19,25-dimethoxycucurbita-6,23-dien-3β-ol (EDMO), were isolated from bitter melon. EMCD has been shown to exhibit in vitro anti-inflammatory activity. In this study, the anti-inflammatory activities of EMCD, EMCO, and EDMO were compared. All three compounds were toxic to the RAW 264.7 macrophage cell line but not the FL83B cells. EMCD and EMCO inhibited tumor necrosis factor (TNF)-α-induced inducible nitric oxide synthase (iNOS) expression in FL83B cells, and the IC50 values were 19.8 and 25.7 μM, respectively. By contrast, EDMO did not effectively reduce iNOS expression. Furthermore, EMCD and EMCO suppressed other TNF-α-induced proinflammatory signals including the activation of inhibitor kappa B kinase complex, the phosphorylation of inhibitor of nuclear factor-κB, and the activation of c-Jun kinase. EMCD consistently exhibited a higher efficacy than did EMCO in these assays. Hence, the in vivo anti-inflammatory activity of EMCD was tested. EMCD clearly repressed 12- O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema in mice. In conclusion, differences in the functional group on carbon 19 do affect the anti-inflammatory activities of EMCD, EMCO, and EDMO. EMCD exhibited the highest anti-inflammatory activity among these molecules, and its in vivo anti-inflammatory activity was confirmed.

Carboxymethyl-chitosan attenuates inducible nitric oxide synthase and promotes interleukin-10 production in rat chondrocytes.

Osteoarthritis (OA) is a common age-related degenerative joint disease, which is caused by the breakdown of joint cartilage and the underlying bone. Carboxymethyl (CM)-chitosan is a soluble derivative of chitosan that has similar physicochemical properties to the extracellular proteoglycans identified in hyaline cartilage. Previous studies have demonstrated that CM-chitosan serves a protective role in a rabbit OA model. The aim of the present study was to investigate the effect of CM-chitosan on NO production and inflammation through its upregulation of interleukin (IL)-10, and the activation of the janus kinase (JAK)/signal transducer and activator of transcription (STAT)/suppressor of cytokine signaling (SOCS) signaling pathway. In the present study primary rat chondrocytes were induced to inflammation with 2 µg/ml lipopolysaccharide. The cells were subsequently subjected to increasing concentrations of CM-chitosan (50, 100 and 200 µg/ml) and the relative mRNA and protein expression of inducible nitric oxide synthase (iNOS), IL-10, JAK1, STAT3 and SOCS3 were measured by RT-qPCR and western blot analysis respectively. The results revealed that CM-chitosan attenuated inflammation by significantly reducing iNOS expression and upregulating the anti-inflammatory cytokine IL-10 in a dose-dependent manner (P<0.05). The expression of JAK1, STAT3 and SOCS3 were also significantly upregulated by CM-chitosan (all P<0.05). The protective role of CM-chitosan against NO production was due to its upregulation of IL-10 and its activation of the JAK/STAT/SOCS signaling pathway.

The role of insulin growth factor-1 on the vascular regenerative effect of MAA coated disks and macrophage-endothelial cell crosstalk.

The IGF-1 signaling pathway and IGF-1-dependent macrophage/endothelial cell crosstalk was found to be critical features of the vascular regenerative effect displayed by implanted methacrylic acid -co-isodecyl acrylate (MAA-co-IDA; 40% MAA) coated disks in CD1 mice. Inhibition of IGF-1 signaling using AG1024 an IGF1-R tyrosine kinase inhibitor abrogated vessel formation 14 days after disk implantation in a subcutaneous pocket. Explanted tissue had increased arginase 1 expression and reduced iNOS expression consistent with the greater shift from "M1" ("pro-inflammatory") macrophages to "M2" ("pro-angiogenic") macrophages for MAA coated disks relative to control MM (methyl methacrylate-co-IDA) disks; the latter did not generate a vascular response and the polarization shift was muted with AG1024. Invitro, medium conditioned by macrophages (both human dTHP1 cells and mouse bone marrow derived macrophages) had elevated IGF-1 mRNA and protein levels, while the cells had reduced IGF1-R but elevated IGFBP-3 mRNA levels. These cells also had reduced iNOS and elevated Arg1 expression, consistent with the invivo polarization results, including the inhibitory effects of AG1024. On the other hand, HUVEC exposed to dTHP1 conditioned medium migrated and proliferated faster suggesting that the primary target of the macrophage released IGF-1 was endothelial cells. Although further investigation is warranted, IGF-1 appears to be a key feature underpinning the observed vascularization. Why MAA based materials have this effect remains to be defined, however.

Krüppel-like factor 4 regulates the expression of inducible nitric oxide synthase induced by TNF-α in human fibroblast-like synoviocyte MH7A cells.

Krüppel-like factor 4 (KLF4), a zinc finger transcription factor, has been implicated in the inflammation mediated by macrophages and endothelial cells by regulating the expression of inflammatory mediators. Here, we investigated whether KLF4 affects the expression of inducible nitric oxide synthase (iNOS), an important inflammatory mediator, in the human RA fibroblast-like synovial cell line MH7A. A pcDNA3.1-KLF4 plasmid or short interfering RNA KLF4 was transfected into MH7A cells, and the iNOS expression and nitric oxide (NO) production were analyzed by quantitative PCR, immunoblotting, and nitrite measurement. The iNOS promoter activity was determined by luciferase assay. The results showed overexpression of KLF4 increased iNOS expression and NO production in the presence or absence of TNF-α. Conversely, KLF4 knockdown markedly reduced iNOS expression and NO production induced by TNF-α. KLF4 activated the transcription activity of iNOS promoter in MH7A cells stimulated by TNF-α. This study indicates that KLF4 is important for regulating the expression of iNOS by TNF-α in human synoviocytes.

Targeted siRNA delivery reduces nitric oxide mediated cell death after spinal cord injury

BackgroundTraumatic spinal cord injury (SCI) includes the primary insult as well as a sequela of biochemical and cellular cascades that amplifies the initial injury. This degenerative process, known as secondary injury, is often mediated by both reactive oxygen and nitrogen species released from damaged cells. Previous data suggests that dysregulated production of nitric oxide via inducible nitric oxide synthase (iNOS) is detrimental to spinal cord recovery. M1 macrophages have been implicated to overexpress iNOS post-SCI. In this work, we propose to inhibit iNOS expression through small interfering RNA (siRNA) complexed chitosan nanoparticles (NPs) that primarily target M1 macrophages.MethodssiRNA conjugated chitosan complexes were fabricated with and without an antibody (Ab) targeting moiety and screened for efficiency to reduce iNOS expression in vitro. Best formulations were subsequently applied in vivo following acute SCI in a rodent model. iNOS expression as well as Bax and Bcl-2 biomarkers were used to assess cell apoptosis within the lesion at 24 h post-injury.ResultsAb-siRNA conjugated chitosan NPs significantly reduced iNOS expression in vitro in M1 polarized macrophages. Results show high transfection efficiency with low cytotoxicity. Subsequent application of NPs in vivo after SCI demonstrated both a reduction in iNOS expression and cellular apoptosis.ConclusionProof of concept indicates that siRNA conjugated chitosan NPs can downregulate iNOS production and inhibit apoptosis following SCI. Our proposed gene silencing method putatively targets M1 macrophages as a means to attenuate secondary injury.

Difference in protective effects of GIP and GLP-1 on endothelial cells according to cyclic adenosine monophosphate response.

Receptors for glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are present in vascular endothelial cells. Previous studies investigating euglycemic status have demonstrated that GIP is directly involved in the physiology of blood vessels by controlling the blood flow rate of portal veins and that GLP-1 has a protective effect on blood vessels by acting on endothelial cells. However, to the best of our knowledge, the effects of GIP and GLP-1 on endothelial cells in patients with hyperglycemia remain unknown. Therefore, the present study investigated whether the effect of the incretin hormones GLP-1 and GIP differed with regards to the reversal of endothelial cell dysfunction caused by hyperglycemia. The production of nitric oxide (NO) was measured using the Griess reagent system kit and the expression of cyclic adenosine monophosphate (cAMP) in the cell was measured at a wavelength of 405 nm with the ELISA reader using the cyclic AMP EIA kit. Exposure of human umbilical vein endothelial cells (HUVEC) to a high glucose concentration decreased NO and endothelial nitric oxide synthase (eNOS) levels but increased inducible NOS (iNOS) levels. However, when HUVECs were pretreated with GLP-1, a reduction of iNOS expression was observed and the expression of eNOS and NO were increased, as opposed to pretreatment with GIP. The results differed according to the response of cAMP, the second messenger of incretin hormones: The GIP pretreatment group did not exhibit an increase in cAMP levels while the GLP-1 pretreatment group did. The results of the present study provide evidence that GLP-1, but not GIP, has a protective effect on endothelial function associated with cardiovascular disease, as it is associated with increased eNOS expression and the levels of NO. This effect may be due to an increase in the cAMP concentration during hyperglycemic events.

Effects of IFN-\u03b21a and IFN-\u03b21b treatment on the expression of cytokines, inducible NOS (NOS type II), and myelin proteins in animal model of multiple sclerosis

The aim of this study was to investigate the effects of interferon (IFN)-β1a and IFN-β1b treatment on inflammatory factors and myelin protein levels in the brain cortex of the Lewis rat experimental autoimmune encephalomyelitis (EAE), animal model of multiple sclerosis. To induce EAE, rat were immunized with inoculums containing spinal cord guinea pig homogenized in phosphate-buffered saline and emulsified in Freund’s complete adjuvant containing 110 µg of the appropriate antigen in 100 µl of an emulsion and additionally 4-mg/ml Mycobacterium tuberculosis (H37Ra). The rats were treated three times per week with subcutaneous applications of 300,000 units IFN-β1a or IFN-β1b. The treatments were started 8 days prior to immunization and continued until day 14 after immunization. The rats were killed on the 14th day of the experiment. EAE induced dramatic increase in interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-concentrations and inducible nitric oxide synthase (iNOS) expression in the brain, which closely corresponded to the course of neurological symptoms and the loss of weight. Both IFN-β1b and IFN-β1a treatments inhibited the pro-inflammatory cytokines (IL-6, IL-1β, TNF-α and IFN-γ), decreased the activation of astrocytes, increased the myelin protein level in the brain cortex, and improved the neurological status of EAE rats by different mechanisms; IFN-β1a reduced iNOS expression, at least in part, by the enhancement of IL-10, while IFN-β1b diminished IL-10 concentration and did not decrease EAE-induced iNOS expression.

Low-Level Laser Irradiation Improves Motor Recovery After Contusive Spinal Cord Injury in Rats.

This study investigated the therapeutic effects of low-level laser irradiation (LLLI) on the recovery of motor function and its underlying mechanisms in rats with spinal cord injury (SCI). The spinal cord was contused at the T11 level using a New York University impactor. Thirty-eight rats were randomly divided into four groups: LLLI with 0.08 J, 0.4 J, 0.8 J, and sham. We transcutaneously applied at the lesion site of the spinal contusive rats 5 min after injury and then daily for 21 days. The Basso, Beattie and Bresnahan (BBB) locomotor scale and combined behavioral score (CBS) were used to evaluate motor function. The spinal segments of rostral and caudal from the lesion site, the epicenter, and L4-5 were collected from normal and the all groups at 7 days after SCI. The expression of tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) was compared across groups in all regions. In the present study, LLLI with 0.4 J and 0.8 J led to a significant improvement in motor function compared to sham LLLI, which significantly decreased TNF-α expression at the lesion epicenter and reduced iNOS expression in the caudal segment for all LLLI groups and in the L4-5 segments for the 0.4 J and 0.8 J groups when compared to sham LLLI group. Our results demonstrate that transcutaneous LLLI modulate inflammatory mediators to enhance motor function recovery after SCI. Thus, LLLI in acute phase after SCI might have therapeutic potential for neuroprotection and restoration of motor function following SCI.

NS1 of H7N9 Influenza A Virus Induces NO-Mediated Cellular Senescence in Neuro2a Cells

Background/Aims: The novel avian H7N9 influenza A virus has been detected in brain tissues and associated with central nervous system (CNS) symptoms in infected human and mice. Roles of its virulence factor, NS1 protein in influenza virus infected neuron has yet to be explored. Methods: Nitric oxide (NO) release and inducible nitric oxide synthase (iNOS) expression in H7N9/NS1-expressed Neuro2a cells were detected by Griess test and western blotting. Cell proliferation rate of H7N9/NS1-expressing cells was recorded by Cell Counting Kit-8. Effects of H7N9/NS1 on cellular senescence were investigated by senescence-associated β-galactosidase (SA-β-gal) staining, immunofluorescent staining of phosphorylated heterochromatin protein 1γ (pHP1γ) and qPCR analysis of IL-6 and IL-8. Results: H7N9/NS1 in Neuro2a cells and primary cultured mouse cortical neurons increased the expression of iNOS and boosted NO release. Neuro2a cells constitutively expressing NS1 displayed a reduced proliferative ability, enhanced SA-β-gal staining, increased level of IL-6 and IL-8 and a typical punctuate structure of pHP1γ in nuclei. In addition, p38 MAPK was elevated in NS1-expressing Neuro2a cells. Reduced iNOS expression and subdued cellular senescence effect was found in p38 MAPK inhibitor-treated NS1-expressing Neuro2a cells. Conclusion: Our results suggest that H7N9/NS1 protein increases the iNOS expression and NO release in Neuro2a cells, which can induce cell growth arrest and cellular senescence.

Flavocoxid mitigates cadmium-induced toxicity: structural, immunohistochemical and molecular analysis in mice kidney

Background: Cadmium (Cd), a diffused environmental pollutant, has adverse effects on urinary apparatus [1]. The role of flavocoxid, a flavonoid with antioxidant activity [2], on the morphological and biochemical changes induced in vivo by Cd in mice kidneys was evaluated. Methods: C57 BL/6J mice received 0.9% NaCl alone, flavocoxid (20 mg/kg/day i.p.) alone, Cd chloride (CdCl2) (2 mg/kg i.p.) alone, or CdCl2 plus Flavocoxid (2 mg/kg i.p. plus 20 mg/kg/day i.p.) for 14 days. At the end of experiment, the mice were killed with an overdose of ketamine and xylazine and the kidneys were collected and processed for structural, immunohistochemical and biochemical analysis. Results: Cd treatment alone significantly increased iNOS, TNF-α and MMP-9 expression, induced structural damages in the glomeruli and in the proximal tubule epithelium, and reduced claudin-11, occludin and N-cadherin immunoreactivity. Flavocoxid administration reduced iNOS, TNF-α and MMP-9 expression, ameliorated glomerular and tubular changes and enhanced claudin-11, occludin and N-cadherin immunoreactivity. Conclusions: We demonstrated for the first time that flavocoxid has a protective role against Cd-induced damages in mice kidney. Therefore, flavocoxid may have a promising role against environmental Cd, in particular against its harmful effects on glomerular and tubular lesions.

Irgm1 promotes M1 but not M2 macrophage polarization in atherosclerosis pathogenesis and development

Background and aimsAtherosclerosis is a chronic inflammatory vascular disease related to macrophages uptake of low-density lipoprotein and their subsequent transformation into foam cells. M1 (inflammatory)/M2 (anti-inflammatory) balance was suggested to impact disease progression. In this study, we investigated whether the immunity related GTPase (Irgm1) regulates macrophage polarization during atherosclerosis development. MethodsWe used apolipoprotein E (ApoE) knockout and Irgm1 haplodeficient mice and induced atherosclerosis with high-cholesterol diet for the indicated months. Atherosclerotic arteries were collected from patients undergoing vascular surgery, to determine the lesional expression of Irgm1 and distribution of M1/M2 populations. ResultsOur results showed that IRGM/Irgm1 expression was increased in atherosclerotic artery samples (1.7-fold, p=0.0045) compared with non-atherosclerotic arteries, which was consistent with findings in the murine experimental atherosclerosis model (1.9-fold, p=0.0002). IRGM/Irgm1 expression was mostly found in lesional M1 macrophages. Haplodeficiency of Irgm1 in ApoE−/− mice resulted in reduced infiltrating M1 macrophages in atheroma (94%, p=0.0002) and delayed development of atherosclerotic plaques. In vitro experiments also confirmed that Irgm1 haplodeficiency reduced iNOS expression of polarized M1 macrophages (81%, p=0.0034), with negligible impact on the M2 phenotype. Moreover, we found that Irgm1 haplodeficiency in mice significantly reduced expression level of M1 function-related transcription factors, interferon regulatory factor (Irf) 5 and Irf8, but not Irf4, an M2-related transcription factor. ConclusionsThis study shows that Irgm1/IRGM participates in the polarization of M1 macrophage and promotes development of atheroma in murine experimental atherosclerosis.

Exercise does not activate the β3 adrenergic receptor–eNOS pathway, but reduces inducible NOS expression to protect the heart of obese diabetic mice

Obesity and diabetes are associated with higher cardiac vulnerability to ischemia-reperfusion (IR). The cardioprotective effect of regular exercise has been attributed to β3-adrenergic receptor (β3AR) stimulation and increased endothelial nitric oxide synthase (eNOS) activation. Here, we evaluated the role of the β3AR-eNOS pathway and NOS isoforms in exercise-induced cardioprotection of C57Bl6 mice fed with high fat and sucrose diet (HFS) for 12weeks and subjected or not to exercise training during the last 4 weeks (HFS-Ex). HFS animals were more sensitive to in vivo and ex vivo IR injuries than control (normal diet) and HFS-Ex mice. Cardioprotection in HFS-Ex mice was not associated with increased myocardial eNOS activation and NO metabolites storage, possibly due to the β3AR-eNOS pathway functional loss in their heart. Indeed, a selective β3AR agonist (BRL37344) increased eNOS activation and had a protective effect against IR in control, but not in HFS hearts. Moreover, iNOS expression, nitro-oxidative stress (protein s-nitrosylation and nitrotyrosination) and ROS production during early reperfusion were increased in HFS, but not in control mice. Exercise normalized iNOS level and reduced protein s-nitrosylation, nitrotyrosination and ROS production in HFS-Ex hearts during early reperfusion. The iNOS inhibitor 1400W reduced in vivo infarct size in HFS mice to control levels, supporting the potential role of iNOS normalization in the cardioprotective effects of exercise training in HFS-Ex mice. Although the β3AR-eNOS pathway is defective in the heart of HFS mice, regular exercise can protect their heart against IR by reducing iNOS expression and nitro-oxidative stress.

Chronic Exercise Reduces The Sirt1 S-nitrosation In The Liver Of Old Mice

The SIRT1 deacetylase protein plays a crucial role in cellular metabolism. It is known that during aging decreases in SIRT1 activity can contributes to development of metabolic disorders. PURPOSE: Determine if the reduction in iNOS genetically (knockout) or by exercise is related to increased activity of SIRT1 by S-nitrosation and/or the post-translational mechanisms responsible for this phenomenon. METHOD: “Young” (Y) (2 months), “sedentary-old” (O) (30 mo) “trained old” (TO) (30 mo) and “iNOS KO old” (iNOSKO) (30 mo) groups were submitted to a treadmill training for 4 weeks (5 days/week) at the intensity of 60% of maximum power (n=5). During the last experimental week, we performed a pyruvate tolerance test (PTT) and glucose samples were collected at rest, 30 and 60 minutes after pyruvate administration. 24 hours after the last exercise bout the animals were euthanized and the liver extracted for western blotting analysis. The database ´genenetwork.org` was used to perform bioinformatics analysis from data containing mRNA values from liver samples of bxd mices and humans, using mitochondrial biogenesis markers that were strongly correlated with the iNOS gene. Results were analyzed by One-Way ANOVA and Bonferroni post hoc test when necessary. RESULTS: “Y” and “TO” animal had reduced glucose production compared to “O” at minute 30 (Y=72.8±4.6 vs TO= 124.9±1.5 vs O= 261.9±32.2 mg/dl, p<0.05) during the PTT and area under glucose curve during PTT was increased in “O” animals compared to “Y” (Y= 5352.8±786.6 vs O=9738.7±1390.0 mg/dl.60min, p<0.05). Liver analysis showed reduced levels of gluconeogenesis enzymes G6Pase and PEPCK in “Y”, “TO” and “iNOSKO” compared to “O” animals. We found reduced iNOS expression, SIRT1 S-nitrosation and inproved mitochondrial complexes responsible for ATP synthesis and fatty acid oxidation (MTCO1 and Uqcrc1) in both “TO” and “iNOSKO” groups compared to “Y” and “O” groups. Bioinformatics analysis shows that bxd iNOS mRNA is negatively correlated to mitochondrial biogenesis markers. Such finding corroborates human analysis. CONCLUSION: The chronic physical exercise promotes attenuated expression of iNOS and S-nitrosation of SIRT1 contributing to increased activity of SIRT1 protein, improving liver function in aging mice. Supported by CNPq, Capes and FAPESP (2013/20293-2).

Selective inhibition of COX-2 improves cutaneous wound healing of pressure ulcers in mice through reduction of iNOS expression

AimsCyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) are involved in chronic inflammation observed in chronic lesions. Nonetheless, neither study demonstrated if decreased COX-2 activation could promote the wound healing of pressure ulcers. Therefore, this study investigated the effect of the administration of celecoxib (a selective COX-2 inhibitor) in wound healing of pressure ulcers. Materials and methodsMale mice were treated daily with celecoxib until euthanasia. One day after the beginning of treatment, two cycles of ischemia–reperfusion by external application of two magnetic plates were performed in skin to induce pressure ulcer formation. Key findingsCelecoxib administration reduced the protein expression of inducible nitric oxide synthase (iNOS), COX-2 and PGE2. The hydroperoxide levels, neutrophil and macrophage number, and protein elastase and matrix metalloproteinase-1 levels were reduced in celecoxib-treated group when compared to control group. Celecoxib administration increased myofibroblastic differentiation, re-epithelialization and wound contraction, and decreased the skin necrosis and angiogenesis. Celecoxib administration also stimulated the formation of a more organized and mature scar increasing collagen deposition and reducing tenascin-C expression. SignificanceCelecoxib administration improves the wound healing of pressure ulcers through decreased expression of iNOS and COX-2, which reduces wound inflammation and promotes dermal reconstruction and scar formation.

Adenosine kinase facilitated astrogliosis-induced cortical neuronal death in traumatic brain injury.

Adenosine kinase (ADK) plays a pivotal role in regulating brain function by regulating adenosine level, and ADK inhibition protects against neuronal damage in cerebral ischemia and epilepsy; however, the effects of ADK in traumatic brain injury (TBI) have not been investigated. For exploring its effects, we generated a blade-induced rat focal brain injury model. Western blot analysis, immunohistochemistry and immunofluorescent staining suggested that ADK was up-regulated after TBI, and it was temporally and spatially associated with astrogliosis. Terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling showed that neuronal apoptosis was paralleled with TBI-induced ADK up-regulation and astrogliosis. For further investigating the role of ADK in astrogliosis-induced neuronal death, primary cultured astrocytes and neurons were utilized, lipopolysaccharide (LPS) was employed to mediate astrogliosis, and condition medium (CM) of reactive astrocytes was used to treat neurons. The results showed that astrocytes increased iNOS expression and secreted pro-inflammatory cytokines after LPS treatment, and CM of reactive astrocytes resulted neuronal death. Additionally, ADK knock-down didn't ameliorate LPS-induced astrocyte proliferation, but it protected against neuronal death by reducing iNOS expression, tumor necrosis factor α and interleukin 1β secretion of reactive astrocytes. Taken together, ADK was associated with astrogliosis after TBI, its inhibition in reactive astrocytes ameliorated astrogliosis-induced neuronal death. Our findings extended the current knowledge on the role of ADK in astrogliosis, and also provided new evidence for the TBI treatment.

Involvement of inducible nitric oxide synthase in the loss of cardioprotection by ischemic postconditioning in hypothyroid rats

Cardioprotection by ischemic postconditioning (IPost) is negated in hypothyroidism; the underlying mechanisms however are unknown. This study aimed at determining whether changes in Bax, Bcl-2, eNOS, and iNOS gene expressions are involved in the negating effects of IPost against ischemia–reperfusion (IR) injury in hypothyroidism. The hearts from control and hypothyroid rats were perfused in Langendorff apparatus and exposed to 30min ischemia, followed by 120min reperfusion and IPost. In a subgroup of hypothyroid rats, ischemia duration was extended to 40min. Hemodynamic parameters, infarct size, and gene expressions were measured. Compared to controls, hypothyroid rats with 30min ischemia had higher recovery of post-ischemic LVDP and ±dp/dt, confirmed by decreased CK and LDH levels (187±16 vs. 485±41 and 191±9 vs. 702±48U/L, respectively; p<0.05), decreased infarct size (6.7±1.1 vs. 46.1±1.7%; p<0.05), and a reduced DNA laddering pattern. Recovery of post-ischemic LVDP and ±dp/dt decreased and infarct size increased following extension of ischemia period in hypothyroid rats. IPost increased eNOS and Bcl-2 expression by 3.2-fold and 3.7-fold and decreased Bax and iNOS expression by 79% and 38%, respectively; it also reduced IR-induced DNA laddering pattern in controls, whereas no change was observed in hypothyroid rats, regardless of the ischemia period. In conclusion, hearts from hypothyroid rats were resistant to IR injury, partly due to the lower expression of iNOS and subsequent reduction in apoptosis after IR. In hypothyroid rats, IPost was not associated with further reduction in iNOS expression and failed to provide additional cardioprotection against ischemia.

Unique immunomodulatory effect of paeoniflorin on type I and II macrophages activities

It has been widely accepted that macrophages are divided into M1 “pro-inflammatory” macrophages and M2 “anti-inflammatory” macrophages and an uncontrolled macrophage polarization plays an important role in the pathogenesis of different diseases. As the main substance of total glucosides of peony, paeoniflorin (PF), has been widely used to treat autoimmune and autoinflammatory diseases for years. Mechanistically, PF has been found to alter activities of many immune cells, which could further reduce inflammation and tissue damage. However, whether and how PF affects macrophages activities in vitro remains unknown. In current study, using M1 and M2 cells generated from mouse bone marrow precursors, we explored the role of PF in regulating M1/M2 cells activity in vitro. The results showed that PF inhibited LPS-induced M1 activity by reducing iNOS expression and NO production via decreasing LPS/NF-κB signaling pathway; whereas, PF enhanced IL-4-provoked M2 function by up-regulating Arg-1 production and activity via increasing IL-4/STAT6 signaling pathway. Our new finding indicates that PF can suppress M1 cells activity and enhance M2 cells function simultaneously, which could help to ameliorate autoimmune and autoinflammatory diseases in clinical treatment.

Abstract 10806: The Role of Neuronal Nitric Oxide Synthase on the Vascular Hyporeactivity and Multiple Organ Failure Associated With Hemorrhagic Shock

Introduction: Trauma can cause severe hemorrhage leading to a state of global ischemia and hemorrhagic shock (HS), resulting in multiple organ failure (MOF) and death. Several studies have shown the participation of nNOS-derived NO in the pathogenesis of the systemic inflammatory response and sepsis. However, the role of nNOS in HS is poorly understood. Hypothesis: The aim of this study is to investigate the role of nNOS-derived NO in the MOF associated with HS. Methods: Rats were subjected to HS under sodium thiopental anesthesia (120 mg/kg; i.p.). The blood pressure was reduced to 30 ± 2 mmHg for 90 min, followed by resuscitation with the shed blood. Rats were treated with 3 different nNOS inhbitors (S-methyl-L-thiocitrulline, ARL 17477 or 7 nitroindazol) or vehicle upon resuscitation. Four hours later, organ injury and dysfunction and the signaling events involved in the observed protective effects of nNOS inhibitors were investigated. Results: When compared to sham rats, HS resulted in a significant decrease in creatinine clearance and rises in serum creatinine, creatine kinase, lactate, aspartate and alanine aminotransferases, indicating the development of MOF. HS also resulted in vascular hyporeactivity to norepinephrine. Treatment of HS-rats with nNOS inhibitors protected animals against the MOF and vascular dysfunction induced by HS. When compared to HS control, analysis of kidney and liver tissue from HS rats treated with nNOS inhibitors revealed decreases in phosphorylation of IκB and p65 nuclear translocation of NF-κB, reduction in iNOS expression, NOx production and protein nitrosylation. nNOS inhibitors treatment also attenuated systemic pro-inflammatory cytokines formation. Conclusions: Our findings show that nNOS inhibition at the onset of resuscitation protects rats against the MOF and vascular dysfunction associated with HS thorough the attenuation of NF-κB activation and consequent reduction of NF-κB-dependent proteins (such as iNOS and pro-inflammatory cytokines), and thorough the attenuation of proteins nitrosylation by NO-derived species.