Decreased iNOS Expression Research Articles

Nicotine Modulates MyD88-Dependent Signaling Pathway in Macrophages during Mycobacterial Infection.

Recently, we reported that cigarette smoking, and especially nicotine, increases susceptibility to mycobacterial infection and exacerbates inflammation in patients with Crohn’s disease (CD). The macrophagic response to Mycobacterium avium subspecies paratuberculosis (MAP) in CD and Mycobacteria tuberculosis (MTB) continues to be under investigation. The role of toll-like-receptors (TLRs) and cytoplasmic adaptor protein (MyD88) in proinflammatory response during Mycobacterial infection has been suggested. However, the mechanism of how nicotine modulates macrophage response during infection in CD and exacerbates inflammatory response remain unclear. In this study, we elucidated the mechanistic role of nicotine in modulating MyD88-dependent/TLR pathway signaling in a macrophage system during mycobacterial infection. The data demonstrated that MAP infection in THP-1 derived macrophages was mediated through TLR2 and MyD88 leading to increase in IL-8 in expression and production. On the other hand, LPS-representing, Gram-negative bacteria mediated macrophage response through TLR4. Blocking TLR2 and TLR4 with antagonists voided the effect of MAP, and LPS, respectively in macrophages and reversed response with decrease in expression of iNOS, TNF-α and IL-8. Interestingly, nicotine in infected macrophages significantly (1) downregulated TLR2 and TLR4 expression, (2) activated MyD88, (3) increased M1/M2 ratio, and (4) increased expression and secretion of proinflammatory cytokines especially IL-8, as seen in CD smokers. We also discovered that blocking macrophages during MAP infection with MyD88 antagonist significantly decreased response which illustrates the key role for MyD88 during infection. Surprisingly, dual treatment of MAP-infected macrophages with MyD88 antagonist and nicotine absolutely impaired immune response and decreased MAP viability, which clearly validate the inflammatory role of nicotine in macrophages through TLR2/MyD88 pathway during infection. This is the first report to describe the mechanism by which nicotine modulates TLR2/MyDD88 and exacerbates inflammation in CD smokers associated with infection.

Evaluation of the modulation of nitric oxide synthase expression in the cerebellum of diabetic albino rats and the possible protective effect of ferulic acid

IntroductionDiabetes mellitus is a multisystem disease. Oxidative stress and nitric oxide isoforms are involved in diabetic pathogenesis. Ferulic acid is a natural substance that is distributed broadly in plants with strong potent properties. The aim of the researchThis research was designed to study the possible protective role of ferulic acid on oxidative stress and different Nitric oxide synthase isoforms (NOS) in the cerebellum of streptozotocin-induced diabetic rats. Materials and methodsTwenty-four albino male rats were randomly divided into equal four groups: control group, group 2 received ferulic acid orally (10 mg/kg), group 3 diabetic group, group 4 diabetic rats received ferulic acid. After 8 weeks, the left cerebellar hemisphere was taken for tissue homogenate for oxidative markers and real-time PCR for NOS isoforms. Paraffin sections of the right cerebellar hemisphere were stained with cresyl violet, Luxol fast blue and immnunohistochemically stained for neuronal NOS, inducible NOS and endothelial NOS. ResultsDegenerative changes were seen in the cerebella of the diabetic rats with significant elevation of Malondialdehyde, Nitric Oxide, and decrease of Superoxide dismutase levels. nNOS expression decreased and iNOS expression increased significantly. The ferulic acid-treated group showed a reduction of the degenerative changes in the cerebellum with significant improvement in oxidative stress marker, an increase of nNOS expression, and a decrease of iNOS expression. ConclusionsFerulic acid improves cerebellar functional and histopathological changes induced by diabetes which can be attributed mainly to its anti-oxidative effect and its ability to modulate NOS isoforms.

Baicalein inhibits non-small-cell lung cancer invasion and metastasis by reducing ezrin tension in inflammation microenvironment.

Baicalein, a flavonoid phytochemical, has been shown to be effective as an anti‐metastatic agent for various cancers, especially for non‐small‐cell lung cancer (NSCLC). However, the underlying mechanism of how baicalein targets cellular processes during NSCLC cell invasion and metastasis remains elusive. In this study, we found that non‐cytotoxic concentrations of baicalein still retained anti‐dissemination activity both in vitro and in vivo. Using a genetic encoding tension probe based on Förster resonance energy transfer (FRET) theory, baicalein was shown to significantly decrease ezrin tension by downregulating cellular ezrin S‐nitrosylation (SNO) levels in NSCLC cells in the inflammatory microenvironment. Decreased ezrin tension inhibited the formation of an aggressive phenotype of NSCLC cell and leader cell in collective migration, and subsequently suppressed NSCLC dissemination. Baicalein restrained SNO‐mediated ezrin tension by decreasing iNOS expression levels. Overall this study demonstrates the novel mechanism used by baicalein to suppress NSCLC invasion and metastasis from a mechanopharmacology perspective and illustrates a new direction for drug development.

Interventions of natural and synthetic agents in inflammatory bowel disease, modulation of nitric oxide pathways.

Inflammatory bowel disease (IBD) refers to a group of disorders characterized by chronic inflammation of the gastrointestinal (GI) tract. The elevated levels of nitric oxide (NO) in serum and affected tissues; mainly synthesized by the inducible nitric oxide synthase (iNOS) enzyme; can exacerbate GI inflammation and is one of the major biomarkers of GI inflammation. Various natural and synthetic agents are able to ameliorate GI inflammation and decrease iNOS expression to the extent comparable with some IBD drugs. Thereby, the purpose of this study was to gather a list of natural or synthetic mediators capable of modulating IBD through the NO pathway. Electronic databases including Google Scholar and PubMed were searched from 1980 to May 2018. We found that polyphenols and particularly flavonoids are able to markedly attenuate NO production and iNOS expression through the nuclear factor κB (NF-κB) and JAK/STAT signaling pathways. Prebiotics and probiotics can also alter the GI microbiota and reduce NO expression in IBD models through a broad array of mechanisms. A number of synthetic molecules have been found to suppress NO expression either dependent on the NF-κB signaling pathway (i.e., dexamethasone, pioglitazone, tropisetron) or independent from this pathway (i.e., nicotine, prednisolone, celecoxib, β-adrenoceptor antagonists). Co-administration of natural and synthetic agents can affect the tissue level of NO and may improve IBD symptoms mainly by modulating the Toll like receptor-4 and NF-κB signaling pathways.

IFN-γ-induced nitric oxide leads to inhibition of Th17 differentiation via induction of iNOS and nitration of ROR-γ T

Abstract Elucidating the molecular mechanisms involved in pleotropic cytokine signaling can lead to novel treatment approaches for autoimmune diseases that target proinflammatory functions while maintaining immune-regulatory functions. To that end, we sought to further investigate the immune-regulatory roles of the pleotropic cytokine IFN-γ. Previous studies demonstrated that IFN-γ prevents the differentiation of pathogenic Th17 cells. In addition, nitric oxide generated via inducible nitric oxide synthase (iNOS) prevents differentiation of Th17 cells by nitration of ROR-γT. However, it remains unresolved whether IFN-γ prevents Th17 differentiation directly via inducing nitric oxide (NO) and nitration of ROR-γT. In line with previous observations, we found an increase in the frequency of autoreactive Th17 cells and a decrease in iNOS expression and NO in the absence of IFN-γ in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Additionally, the increase in Th17 cells in the absence of IFN-γ was reversible both in vitro and in vivo with treatment with a NO donor. This indicates a role for IFN-γ in inhibiting the differentiation of Th17 cells via iNOS-derived NO. We are currently elucidating the underlying mechanisms of NO mediated suppression and the molecular link between ROR-γt-nitration and decreased Th17 differentiation by flow cytometry, single-cell western blotting of key transcription factors, and in vivo adoptive transfer studies. Our studies may lead to a better understanding of the role of IFN-g and NO/iNOS axis in Th17 cell differentiation and autoimmune diseases.

Vitamin D modulates innate immunity in pulmonary tuberculosis

Abstract Vitamin D and vitamin D receptor (VDR) may play role in altering host defence against Mtb via cathelicidin production and regulating nitric oxide (NO) production through inducible nitric oxide synthase (iNOS). 100 active pulmonary tuberculosis (PTB) patients, 75 household contacts and 70 healthy controls were recruited. Serum Vitamin D and NO levels were measured using CLIA and colorimetry based kit respectively. VDR and iNOS mRNA was studied in monocytes using qPCR. Serum VDR, cathelicidin and iNOS levels were measured using ELISA. Lower serum levels of vitamin D was found in PTB group compared to household contacts and healthy controls (P<0.001). VDR (both mRNA and protein) and iNOS mRNA levels were also lower in PTB group compared to household contacts and healthy controls (P<0.005)probably due to decreased levels of vitamin D. However, despite low serum vitamin D, serum cathelicidin levels were higher in PTB group compared to household contacts and healthy controls (p<0.01) which could be attributed to direct trigger via toll like receptors where Mtb is multiplying actively. Serum iNOS and NO levels were higher in PTB group compared to household contacts and healthy controls (p< 0.05), despite decreased iNOS expression which needs further evaluation. Increased VDR and iNOS expression in household contacts compared to both PTB and healthy controls could be due to optimum levels of vitamin D along with some exposure to Mtb as evident by increased levels of cathelicidin and NO compared to healthy controls. Adequate levels of vitamin D, VDR and iNOS that in turn promote production of antimicrobial cathelicidin and NO might have protective role against TB among household contacts and needs further investigation for its therapeutic potential.

Foxo3a aggravates inflammation and induces apoptosis in IL-1-treated rabbit chondrocytes via positively regulating tenascin-c

Osteoarthritis (OA) is the most common degenerative disease in middle-aged and elderly individuals that causes joint deformity and limb disability. Accumulating evidence has suggested that the pathogenesis of OA has been related to various mechanisms such as apoptosis, inflammation, oxidative stress and metabolic disorders. The aim of this study is to clarify the role of Foxo3a in the progress of OA in an in vitro model. The chondrocytes were derived from rabbit, and treated with IL-1β, which was used as an in vitro OA model. The over-expression and down-regulation of Foxo3a were achieved by transfection with overexpression vector or shRNA, respectively. The mRNA level of iNOS in chondrocytes was quantified by qPCR. Tenascin-c (Tnc) production was measured by ELISA and apoptosis-associated proteins were analyzed by Western blotting. The MTT assay was used to assess the viability of chondrocytes. Foxo3a and iNOS expression were upregulated in IL-1β-treated chondrocytes. Foxo3a silencing decreased iNOS expression, and inhibited apoptosis of IL-1β-treated chondrocytes. The production of Tnc was significantly increased in IL-1b-treated chondrocytes and was positively regulated by Foxo3. Importantly, extracellular addition of Tnc abrogated the protective effects of Foxo3a knockdown on IL-1β - treated chondrocytes. The present study indicated that down-regulation of Foxo3a protected IL-1β-treated chondrocytes by decreasing iNOS expression and suppressing chondrocytes' apoptosis via modulating tenascin-c, which could be regarded as a potent therapeutic target for the treatment of OA.

Modulation of microglial phenotypes improves sepsis-induced hippocampus-dependent cognitive impairments and decreases brain inflammation in an animal model of sepsis.

In order to modulate microglial phenotypes in vivo, M1 microglia were depleted by administration of gadolinium chloride and the expression of M2 microglia was induced by IL-4 administration in an animal model of sepsis to better characterize the role of microglial phenotypes in sepsis-induced brain dysfunction. Wistar rats were submitted to sham or cecal ligation and perforation (CLP) and treated with IL-4 or GdCl3. Animals were submitted to behavioral tests 10 days after surgery. In a separated cohort of animals at 24 h, 3 and 10 days after surgery, hippocampus was removed and cytokine levels, M1/M2 markers and CKIP-1 levels were determined. Modulation of microglia by IL-4 and GdCl3 was associated with an improvement in long-term cognitive impairment. When treated with IL-4 and GdCl3, the reduction of pro-inflammatory cytokines was apparent in almost all analyzed time points. Additionally, CD11b and iNOS were increased after CLP at all time points, and both IL-4 and GdCl3 treatments were able to reverse this. There was a significant decrease in CD11b gene expression in the CLP+GdCl3 group. IL-4 treatment was able to decrease iNOS expression after sepsis. Furthermore, there was an increase of CKIP-1 in the hippocampus of GdCl3 and IL-4 treated animals 10 days after CLP induction. GdCl3 and IL-4 are able to manipulate microglial phenotype in an animal models of sepsis, by increasing the polarization toward an M2 phenotype IL-4 and GdCl3 treatment was associated with decreased brain inflammation and functional recovery.

Could the Combination of Two Non-Psychotropic Cannabinoids Counteract Neuroinflammation? Effectiveness of Cannabidiol Associated with Cannabigerol.

Background and Objectives: Neuroinflammation is associated with many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). In this study, we investigate the anti-inflammatory, anti-oxidant, and anti-apoptotic properties of two non-psychoactive phytocannabinoids, cannabigerol (CBG) and cannabidiol (CBD). Materials and Methods: The motoneuron-like cell line NSC-34 differentiated by serum deprivation and with the additional treatment of all-trans retinoic acid (RA) is a valid model to investigate molecular events linked to neurodegeneration in ALS. Results: Pre-treatment with CBG (at 2.5 and 5 µM doses) alone and in combination with CBD (at 2.5 and 5 µM doses) was able to reduce neuroinflammation induced by a culture medium of LPS-stimulated macrophages. In particular, the pre-treatment with CBD at a 5 µM dose decreased TNF-α levels and increased IL10 and IL-37 expression. CBG–CBD association at a 5 µM dose also reduced NF-kB nuclear factor activation with low degradation of the inhibitor of kappaB alpha (IkBα). CBG and CBD co-administered at a 5 µM dose decreased iNOS expression and increased Nrf2 levels. Furthermore, the pre-treatment with the association of two non-psychoactive cannabinoids downregulated Bax protein expression and upregulated Bcl-2 expression. Our data show the anti-inflammatory, anti-oxidant, and anti-apoptotic effects PPARγ-mediated. Conclusions: Our results provide preliminary support on the potential therapeutic application of a CBG–CBD combination for further preclinical studies.

Aging results in accumulation of M1 and M2 hepatic macrophages and a differential response to gadolinium chloride.

Macrophages have vital roles in innate immunity by modulating the inflammatory response via their ability to alter their phenotype from pro-inflammatory (M1) to anti-inflammatory (M2). Aging increases activation of the innate immune system, and macrophage numbers increase in the aged liver. Since macrophages also produce free radical molecules, they are a potential source of age-related oxidative injury in the liver. This study evaluated macrophage phenotype in the aged liver and whether the increase in the number of macrophages with aging is associated with enhanced hepatic oxidative stress. Hepatic macrophage phenotype and oxidative stress were evaluated 2days after a single intraperitoneal injection of saline or gadolinium chloride (GdCl3, 10mg/kg) in young (6 months) and aged (24 months) Fischer 344 rats. GdCl3 has been shown to decrease the expression of macrophage-specific markers and impair macrophage phagocytosis in the liver. Saline-treated aged rats demonstrated greater numbers of both M1 (HO-1+/iNOS+) and M2 (HO-1+/CD163+) macrophages, without evidence of a phenotypic shift. GdCl3 did not alter levels of dihydroethidium fluorescence or malondialdehyde, suggesting that macrophages are not a major contributor to steady-state levels of oxidative stress. However, GdCl3 decreased M1 and M2 macrophage markers in both age groups, an effect that was attenuated in aged rats. In old animals, GdCl3 decreased iNOS expression to a greater extent than HO-1 or CD163. These results suggest a novel effect of aging on macrophage biology and that GdCl3 shifts hepatic macrophage polarization to the M2 phenotype in aged animals.

Inhibition of ovalbumin-induced allergic rhinitis by sumatriptan through the nitric oxide pathway in mice

AimsAllergic rhinitis is a global cause of disability, characterized by airway inflammation. Sumatriptan is a 5-hydroxytryptamine 1B/1D (5HT1B/1D) agonist used as a treatment for migraine headaches. Activation of 5HT1B/1D receptors can inhibit the release of neuropeptides and inhibit the inflammation cascades. This study investigated the effect of sumatriptan on ovalbumin-induced allergic rhinitis model in mice and the role of nitric oxide. MethodsFemale Balb/c mice were sensitized by intraperitoneal ovalbumin and challenged by intranasal ovalbumin. Mice received sumatriptan in doses 3, 10, 30 μg/kg intraperitoneally, 30 min before the last ovalbumin challenge. Key findingsIntraperitoneal injection of sumatriptan significantly decreased the nasal scratching, IL-4 and serum IgE levels of allergic mice, but it increased IFNγ levels. Histopathological analysis showed that the number of eosinophils was significantly elevated in nasal mucosa of ovalbumin-induced allergic mice, while sumatriptan treatment significantly reduced the number of eosinophils. GR-127935, a selective 5-HT1B/1D-receptor antagonist, reversed the anti-allergic effects of sumatriptan. Acute administration of l-NAME, a non-specific inhibitor of nitric oxide synthase, along with sumatriptan attenuated the anti-allergic effects of sumatriptan but chronic administration of l-NAME did not affect the influences of sumatriptan. Furthermore, sumatriptan decreased the inducible nitric oxide synthase (iNOS) protein expression in allergic mice, but it did not change the concentration of eNOS protein. SignificanceThis study shows that sumatriptan administration is associated with anti-allergic effects which are through 5HT1B/1D receptors. Decrease in iNOS expression and changes in T-helper 1&2 cytokines levels may indicate the involvement of inducible NOS and inflammation.

Role of Estrogen on Alveolar Macrophage Polarization in Response to Particulate Matter Exposure

BackgroundLung diseases affect more than 500 million people worldwide. Exposure to air pollutants such as particulate matter (PM) contributes significantly to lung disease, especially in women, indicating an intersection of air pollution and sex hormones in lung disease outcomes. Studies have been performed on the effects of estrogens on lung immunity, particularly in alveolar macrophages (AM). AMs provide one of the first lines of defense in the airways and are important mediators of inflammation. However, the mechanisms by which estrogens control the immune response to PM exposure remain unclear. Here, we hypothesized that PM exposure triggers AM polarization to the pro‐inflammatory (M1) macrophage phenotype in a sex‐dependent matter, and that this process is mediated by estrogens in female cells.MethodsWe tested this hypothesis by exposing male (RAW264.7, BALB/c) and female mouse AMs (J774A.1, BALB/c) to a synthetic PM preparation in the presence or absence of estrogen agonists/antagonists. Cell viability and cytotoxicity were measured at 8h to determine PM dose‐responses. Cells were then stimulated with LPS or IL‐4, which have been described as mediators of AM polarization. The polarization phenotype, determined by expression of specific markers (iNOS, Arg1), was evaluated by quantitative real‐time PCR.ResultsOur results demonstrate that estrogen treatment decreases iNOS expression and increases Arg1 expression, impairing the LPS‐induced pro‐inflammatory phase and triggering resolution of inflammation in RAW 264.7 cells. In addition, induction of AM polarization towards different phenotypes not only was altered by PM and estrogen treatment, but also depended on the sex of the cell. Interestingly, PM treatment led to decreased expression of estrogen receptors when compared to the non‐PM treated cells.ConclusionsWe conclude that estrogens regulate the immune response to PM in a sex‐dependent manner. These findings may be relevant to elucidate the transcriptional mechanisms involved in the sex‐specific inflammatory response and particularly for the identification of novel hormone receptor modulators efficacious in the prevention or mitigation of chronic inflammation in men and women.Support or Funding InformationThis research was supported by funding from NIH K01HL133520, the Center for Research on Women and Newborn Health and the American Physiological Society Porter Physiology Development Program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

THERAPEUTIC EFFICACY OF CHITOSAN NANOPARTICLES AND ALBENDAZOLE IN INTESTINAL MURINE TRICHINELLOSIS

Most of the drugs used for the treatment of trichinellosis showed a limited bioavailability and a high degree of resistance. Therefore, there is an urgent need to develop new agents to improve the bioavailability of these drugs. This work assessed the use Chitosan (CH) nanoparticles singly or combined with albendazole (ABZ) for the treatment of experimental trichinellosis. Fifty male albino mice were used. They were divided into six experimental groups. Chitosannanoparticles were used orally at a dose of 100mg/kg/day starting from 3rd dpi for three successive days for the treatment of the intestinal phase of infection either alone or loaded with full dose or half dose of ABZ. Results showed significant reduction in all treated groups with the highest reduction rate of adult 99.1%, improvement of the intestinal histopathological changes and a significant decrease in iNOS expression of the intestinal tissue were observed ingroups treated by CH loaded with full dose of ABZ compared to control group

THERAPEUTIC EFFICACY OF CHITOSAN NANOPARTICLES AND ALBENDAZOLE IN INTESTINAL MURINE TRICHINELLOSIS

Most of the drugs used for the treatment of trichinellosis showed a limited bioavailability and a high degree of resistance. Therefore, there is an urgent need to develop new agents to improve the bioavailability of these drugs. This work assessed the use Chitosan (CH) nanoparticles singly or combined with albendazole (ABZ) for the treatment of experimental trichinellosis. Fifty male albino mice were used. They were divided into six experimental groups. Chitosannanoparticles were used orally at a dose of 100mg/kg/day starting from 3rd dpi for three successive days for the treatment of the intestinal phase of infection either alone or loaded with full dose or half dose of ABZ. Results showed significant reduction in all treated groups with the highest reduction rate of adult 99.1%, improvement of the intestinal histopathological changes and a significant decrease in iNOS expression of the intestinal tissue were observed ingroups treated by CH loaded with full dose of ABZ compared to control group

Piceatannol pretreatment alleviates acute cardiac injury via regulating PI3K-Akt-eNOS signaling in H9c2 cells

Piceatannol (3,3′,4,5′-trans-trihydroxystilbene) is a natural polyphenols compound that occurs hydroxylated analogue of resveratrol showing widely biological activities. Previous studies have demonstrated its functions on anti-cancer, neuroprotection and cardioprotection. However, few studies have clarified the benefits of piceatannol on cardiomyocytes except its anti-oxidative effect based on the original property of polyphenols. Here we apply H9c2 cardiomyocytes to study the cardioprotective mechanisms of piceatannol in vitro. We firstly verify its anti-peroxidation effect by using H2O2-induced in vitro model. Then, flow cytometry results show piceatannol reduce cellular apoptosis by enhancing Bcl-2 expressions in immunoblot analysis. Meantime, piceatannol decreases H2O2-induced excessive ROS and calcium overloading, and prevents mitochondrial depolarization. Most importantly, piceatannol pretreatment can regulate PI3K-Akt-eNOS signaling pathway to alleviate peroxidative injury. Immunoblot analysis of PI3K, Akt, p-Akt and eNOS shows H2O2 significantly reduces expressions of these proteins. Pretreatment of piceatannol evidently increases their expressions and decreases iNOS expression, implying piceatannol can upregulate PI3K-Akt-eNOS signaling to protect cardiomyocytes from peroxidative injury.

Improved endogenous epoxyeicosatrienoic acid production mends heart function via increased PGC 1α-mitochondrial functions in metabolic syndrome

Metabolic syndrome (MS) is a combination of symptoms characterized by central obesity, hypertension, hyperglycemia, and hyperlipidemia, which together increase the risk of heart disease, stroke and diabetes. In our study, we hypothesized that an EET-agonist (AUDA) would increase expression of PGC 1α and improve mitochondrial and endothelial functions, resulting in improved heart function in a rat model of MS. To investigate this, rats were randomly divided into four groups: 1) Control; 2) MS + ABCT; 3) MS + AUDA; and 4) MS + AUDA + SnMP. MS rats were fed a high-fructose diet for 16 weeks and developed elevated inflammatory mediators, oxidative stress, and significant decreases in fractional shortening and hemodynamic parameters, indicating cardiac dysfunction. Histology revealed myocardial fibrosis and myocyte hypertrophy. AUDA improves mitochondrial function proven by increase in mt copy number and ATP production and significantly increased expression of PGC-1α and HO-1 in the rats and normalization of inflammatory cytokines, oxidative stress, and improves in cardiac function and myocardial fibrosis. These benefits were reversed by SnMP. Furthermore, AUDA increases eNOS but decreases iNOS expression which improved endothelial function. We therefore demonstrate that endogenous EET upregulation plays a novel role in protecting the heart from MS by regulating mitochondrial and endothelial function.

GPR30 Attenuates Myocardial Fibrosis in Diabetic Ovariectomized Female Rats: Role of iNOS Signaling

Premenopausal women have a reduced risk for cardiovascular disease. Estrogen deficiency augments cardiac inflammation and oxidative stress and, thereby, aggravates myocardial fibrosis (MF) and diastolic dysfunction in hypertensive female rats. However, estrogen replacement therapy has no effect on myocardial infarction and cardiac fibrosis in postmenopausal women. Further clinical studies showed that high blood glucose levels in patients with diabetes is an important cause of MF, but the underlying mechanism is unclear. To experimentally address this issue, diabetes mellitus (DM) was induced by injecting streptozotocin and administering a high-fat diet in ovariectomized (OVX) rats. High degrees of fibrosis and apoptosis were detected in the cardiac tissue of these rats, together with increased expression of iNOS. Further treatment with the G protein-coupled estrogen receptor 30 (GPR30) agonist G1 decreased iNOS expression and the apoptosis rate in cardiac tissue significantly and inhibited cardiac fibroblast (CF) proliferation. Similar trends were observed in cultured CFs treated with high concentrations of fat and glucose. In addition, treatment with the iNOS-specific inhibitor W1400 attenuated iNOS and vimentin expression, which is associated with a marked reduction in MF. These results suggest that GPR30 activation inhibits MF in diabetic OVX female rats by suppressing cardiac iNOS activity and consequently NO levels. Thus, GPR30 activation may provide novel cardioprotection strategies for postmenopausal women, especially those with DM.

Modulation of M1/M2 polarization by capsaicin contributes to the survival of dopaminergic neurons in the lipopolysaccharide-lesioned substantia nigra in vivo

The present study examined the neuroprotective effects of capsaicin (CAP) and explored their underlying mechanisms in a lipopolysaccharide (LPS)-lesioned inflammatory rat model of Parkinson’s dieases (PD). LPS was unilaterally injected into the substantia nigra (SN) in the absence or presence of CAP or capsazepine (CZP, a TRPV1 antagonist). The SN tissues were prepared for immunohistochemical staining, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, western blot analysis, blood–brain barrier (BBB) permeability evaluation, and reactive oxygen species (ROS) detection. We found that CAP prevented the degeneration of nigral dopamine neurons in a dose-dependent manner and inhibited the expression of proinflammatory mediators in the LPS-lesioned SN. CAP shifted the proinflammatory M1 microglia/macrophage population to an anti-inflammatory M2 state as demonstrated by decreased expression of M1 markers (i.e., inducible nitric oxide synthase; iNOS and interleukin-6) and elevated expression of M2 markers (i.e., arginase 1 and CD206) in the SN. RT-PCR, western blotting, and immunohistochemical analysis demonstrated decreased iNOS expression and increased arginase 1 expression in the CAP-treated LPS-lesioned SN. Peroxynitrate production, reactive oxygen species levels and oxidative damage were reduced in the CAP-treated LPS-lesioned SN. The beneficial effects of CAP were blocked by CZP, indicating TRPV1 involvement. The present data indicate that CAP regulated the M1 and M2 activation states of microglia/macrophage in the LPS-lesioned SN, which resulted in the survival of dopamine neurons. It is therefore likely that TRPV1 activation by CAP has therapeutic potential for treating neurodegenerative diseases, that are associated with neuroinflammation and oxidative stress, such as PD.

Analysis of NO-suppressing activity of Strawberry Wine supplemented with ball-milled achenes.

Inflammation is generally thought to be involved in the development of several chronical diseases, therefore, phytochemicals to modulate immune responses has attracted great interests. The objective of the present study was to evaluate the potential anti-inflammatory effects of wine supplemented using ball-milled achene on modulating NO production and inducible nitric oxide synthase (iNOS) expression. Ball-milled achenes were added in strawberry must prior to fermentation, and the wine samples were then concentrated and extracted with water and/or ethanol prior to analysis. Bioactivities of wine extracts were evaluated using the cell viability assay, cell cycle measurements, NO production and iNOS expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Treatments of achenes supplemented strawberry wine extract up to 100μg/mL inhibited the proliferation of LPS-stimulated RAW264.7 cell via affecting the progression of cell cycle. Moreover, no detectable cytotoxicity in RAW264.7 cells was observed. The supplemented wine extract suppressed the action of LPS and led to a decreased NO production in stimulated cells. The inhibitory effect of the wine extract on NO production was determined to be a 25-40% decrease in the level of 25-100μg/mL, in contrast to a 10% decrease for conventional wine samples. Additionally, an alcoholic wine extract (100μg/mL) led to a 40.31% decrease in iNOS expression in LPS-stimulated cells, which was more effective than the same dose of tocopherol. The results show that strawberry wine supplemented with ball-milled achenes causes a substantial inhibition of NO production, and this biofunction is exerted via the down-regulation of iNOS expression.

SKLB023 hinders renal interstitial fibrosis in obstructive nephropathy by interfering TGF-β1/Smad3 signaling.

Renal fibrosis is the principal process underlying the progression of chronic kidney disease to end-stage renal disease. It is a relatively uniform response involving glomerulosclerosis, tubulointerstitial fibrosis and changes in renal vasculature. A considerable number of studies have confirmed that inducible nitric oxide synthase (iNOS) was highly expressed in renal interstitial fibrosis and the overexpression of iNOS played a negative role in kidney disease progression. In our previous study, SKLB023 as a novel small-molecule inhibitor of iNOS, blocked joint inflammation and cartilage destruction in arthritis. However, the pharmacological role and function of SKLB023 in renal fibrosis remained poorly understood. In the study, oral administration of SKLB023 (25 and 50 mg per kg per day) for 7 day exhibited potent anti-fibrotic effects against the model UUO using the pathological assessment of H & E and Masson's trichrome staining. SKLB023 inhibited the expression of α-SMA, col I, col IV, fibronectin and further decreased iNOS expression as well as TGF-β1/Smad3 phosphorylation in the injured kidney tissues of UUO mice. Similarly, SKLB023 suppressed in vitro features of fibrosis in TGF-β1-induced NRK-49F by the inhibition of the corresponding fibrotic protein expression. These findings confirmed that SKLB023 hindered renal interstitial fibrosis by interfering with TGF-β1/Smad3 signaling, highlighting that SKLB023 has potential in therapeutic strategies.