Inducible Nitric Oxide Synthase Pathway Research Articles

Melissa officinalis Regulates Lipopolysaccharide-Induced BV2 Microglial Activation via MAPK and Nrf2 Signaling.

Neuroinflammation and microglial activation play critical roles in neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Modulating microglial activation may help prevent the progression of these disorders. This study aimed to investigate the effects and mechanisms of Melissa officinalis ethanol extract on lipopolysaccharide (LPS)-induced microglial activation in BV2 cells. Cell viability and nitric oxide (NO) production were assessed using MTT assay and Griess reagent, while inflammatory cytokine levels were measured by qPCR. Key inflammatory pathways, including MAPK, TLR4, and antioxidant biomarkers, were analyzed through western blot and immunofluorescence. Rosmarinic acid content in M. officinalis was determined using high-performance liquid chromatography (HPLC). The results demonstrated that M. officinalis ethanol extract significantly inhibited LPS-induced NO production and reduced inflammatory cytokine expression. Additionally, it downregulated inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TLR4, NF-κB, and MAPK signaling pathways (p38, JNK, ERK), while increasing the expression of antioxidant markers, including Nrf2, HO-1, catalase, and SOD2. In conclusion, M. officinalis ethanol extract exerts neuroprotective effects by modulating inflammation and enhancing antioxidant defenses, suggesting its potential in the prevention and treatment of inflammation-related neurodegenerative diseases.

Effect of a Silk Sericin and Methylsulfonylmethane (MSM) Blends on Inflammatory Response and Wound Healing

The natural product silk sericin has excellent biological properties, such as biocompatibility, biodegradability, and antioxidant activity, and it also facilitates cell proliferation, collagen type 1 production, and wound healing. Methylsulfonylmethane (MSM) is a natural organic sulfur compound known to relieve pain and inflammation. In this study, we formulated sericin and MSM blends in various ratios, and investigated their effects on the inflammatory response and subsequent healing of a wound in vitro using macrophages and fibroblasts. The experimental findings, derived from cell viability analyses and nuclear factor kappa B (NFκB)/cyclooxygenase-2 (COX-2)/inducible nitric oxide synthase (iNOS) pathway assays, demonstrated that the anti-inflammatory activity of MSM was more effective when it was blended with sericin. In contrast, MSM alone did not enhance wound healing but rather delayed it at higher concentrations. We confirmed that the blend of sericin and MSM exhibited a synergistic effect on suppressing inflammatory responses and promoting subsequent wound healing when the two materials were optimally blended.

COVID-19 Mimics Pulmonary Dysfunction in Muscular Dystrophy as a Post-Acute Syndrome in Patients

Although progressive wasting and weakness of respiratory muscles are the prominent hallmarks of Duchenne muscular dystrophy (DMD) and long-COVID (also referred as the post-acute sequelae of COVID-19 syndrome); however, the underlying mechanism(s) leading to respiratory failure in both conditions remain unclear. We put together the latest relevant literature to further understand the plausible mechanism(s) behind diaphragm malfunctioning in COVID-19 and DMD conditions. Previously, we have shown the role of matrix metalloproteinase-9 (MMP9) in skeletal muscle fibrosis via a substantial increase in the levels of tumor necrosis factor-α (TNF-α) employing a DMD mouse model that was crossed-bred with MMP9-knockout (MMP9-KO or MMP9-/-) strain. Interestingly, recent observations from clinical studies show a robust increase in neopterin (NPT) levels during COVID-19 which is often observed in patients having DMD. What seems to be common in both (DMD and COVID-19) is the involvement of neopterin (NPT). We know that NPT is generated by activated white blood cells (WBCs) especially the M1 macrophages in response to inducible nitric oxide synthase (iNOS), tetrahydrobiopterin (BH4), and tetrahydrofolate (FH4) pathways, i.e., folate one-carbon metabolism (FOCM) in conjunction with epigenetics underpinning as an immune surveillance protection. Studies from our laboratory, and others researching DMD and the genetically engineered humanized (hACE2) mice that were administered with the spike protein (SP) of SARS-CoV-2 revealed an increase in the levels of NPT, TNF-α, HDAC, IL-1β, CD147, and MMP9 in the lung tissue of the animals that were subsequently accompanied by fibrosis of the diaphragm depicting a decreased oscillation phenotype. Therefore, it is of interest to understand how regulatory processes such as epigenetics involvement affect DNMT, HDAC, MTHFS, and iNOS that help generate NPT in the long-COVID patients.

Regulation of microRNA-126 on the polarization of human macrophages stimulated by Porphyromonas gingivalis lipopolysaccharide

Objective: To study the effect of microRNA-126 (miR-126) on the polarization of human monocyte-derived macrophages stimulated by Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS). Methods: Macrophages derived from human myeloid leukemia mononuclear cells were stimulated by Pg-LPS (5 mg/L) and by Pg-LPS (5 mg/L) after 24 h-transfection of miR-126 mimic or negative control RNA for 48 h, respectively. Real-time quantitative-PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western blotting were conducted to detect the changes in miR-126, pro-inflammatory factor tumor necrosis factor-α (TNF-α), anti-inflammatory factors interleukin-10 (IL-10), inducible nitric oxide synthase (iNOS), arginase-1 (Arg-1) and M1 polarization-related pathways such as nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Results: Compared with non-LPS stimulation group (TNF-α: 1.000±0.020, iNOS: 1.125±0.064, miR-126: 1.004±0.113, IL-10: 1.003±0.053, Arg-1: 1.130±0.061), the mRNA levels of TNF-α (3.105±0.278) and iNOS (4.296±0.003) increased significantly (t=6.53, P=0.003; t=42.63, P<0.001, respectively), while miR-126, IL-10 and Arg-1 expressions (0.451±0.038, 0.545±0.004 and 0.253±0.017) decreased significantly (t=7.95, P=0.001; t=7.36, P=0.002; t=11.94, P<0.001, respectively) after Pg-LPS stimulated by human-derived macrophages for 48 h. The protein expression of iNOS, TNF-α, Arg-1 and IL-10 were consistent at mRNA levels. Meanwhile, the expressions of phospho-NF-κB p65 (p-p65), phospho-extracellular signal-regulated kinase (p-ERK) and phospho-p38 MAPK (p-p38) increased significantly, while the expression of Arg-1 decreased significantly. Compared with the negative controls (scramble RNA) (TNF-α: 1.141±0.197, iNOS: 1.173±0.115, IL-10: 1.032±0.138, Arg-1: 0.933±0.044), the mRNA levels of TNF-α (0.342±0.022) and iNOS (0.588±0.085) expressions significantly decreased (t=5.35, P=0.006; t=5.05, P=0.007), while IL-10 (1.786±0.221) and Arg-1 expressions (2.152±0.229) significantly increased (t=3.71, P=0.021; t=6.21, P=0.003) after Pg-LPS stimulation with miR-126 mimic transfection. The relative protein expressions of iNOS, p-p65, p-ERK and p-p38 significantly decreased (t=13.00, P<0.001; t=6.98, P=0.002; t=10.86, P<0.001; t=8.32, P=0.001), while the protein level of Arg-1 significantly increased (t=12.08, P<0.001). Conclusions: Pg-LPS could promote M1 polarization of macrophages. miR-126 might inhibit the effect of Pg-LPS on the M1 polarization of macrophages through down-regulating NF-κB and MAPK signaling pathways.

Hempseed (Cannabis sativa) Peptides WVSPLAGRT and IGFLIIWV Exert Anti-inflammatory Activity in the LPS-Stimulated Human Hepatic Cell Line.

WVSPLAGRT (H2) and IGFLIIWV (H3) are two transepithelial transported intestinal peptides obtained from the hydrolysis of hempseed protein with pepsin, which exert antioxidant activity in HepG2 cells. Notably, both peptides reduce the H2O2-induced reactive oxygen species, lipid peroxidation, and nitric oxide (NO) production levels in HepG2 cells via the modulation of the nuclear factor erythroid 2-related factor 2 and the inducible nitric oxide synthase (iNOS) pathways, respectively. Due to the close link between inflammation and oxidative stress and with the objective of fostering the multifunctional behavior of bioactive peptides, in this study, the molecular characterization of the anti-inflammatory and immunomodulatory properties of H2 and H3 was carried out in HepG2 cells. In fact, both peptides were shown to modulate the production of pro (IFN-γ: −33.0 ± 6.7% H2, p = 0.011; −13.1 ± 2.0% H3, p = <0.0001; TNF: −17.6 ± 1.7% H2, p = 0.0004; −20.3 ± 1.7% H3, p = <0.0001; and IL-6: −15.1 ± 6.5% H3, p = 0.010)- and anti (IL-10: +9.6 ± 3.1% H2, p = 0.010; +26.0 ± 2.3% H3, p = < 0.0001)-inflammatory cytokines and NO (−9.0 ± 0.7% H2, p = <0.0001; −7.2 ± 1.8% H3, p = <0.0001) through regulation of the NF-κB and iNOS pathways, respectively, in HepG2 cells stimulated by lipopolysaccharides.

Doxycycline hyclate stimulates inducible nitric oxide synthase and arginase imbalance, potentiating inflammatory and oxidative lung damage in schistosomiasis

BackgroundWe investigated the relationship between inducible nitric oxide synthase (iNOS) and arginase pathways, cytokines, macrophages, oxidative damage and lung granulomatous inflammation in S. mansoni-infected and doxycycline-treated mice. MethodsSwiss mice were randomized in four groups: (i) uninfected, (ii) infected with S. mansoni, (iii) infected + 200 mg/kg praziquantel (Pzt), (iv) and (v) infected + 5 and 50 mg/kg doxycycline. Pzt (reference drug) was administered in a single dose and doxycycline for 60 days. ResultsS. mansoni-infection determined extensive lung inflammation, marked recruitment of M2 macrophages, cytokines (IL-4, IL-5, IFN-γ, TNF-α) upregulation, intense eosinophil peroxidase (EPO) levels, arginase expression and activity, reduced iNOS expression and nitric oxide (NO) production. The higher dose of doxycycline aggravated lung granulomatous inflammation, downregulating IL-4 levels and M2 macrophages recruitment, and upregulating iNOS expression, EPO, NO, IFN-γ, TNF-α, M1 macrophages, protein carbonyl and malondialdehyde tissue levels. The number and size of granulomas in doxycycline-treated animals was higher than untreated and Pzt-treated mice. Exudative/productive granulomas were predominant in untreated and doxycycline-treated animals, while fibrotic/involutive granulomas were more frequent in Pzt-treated mice. The reference treatment with Pzt attenuated all these parameters. ConclusionOur findings indicated that doxycycline aggravated lung granulomatous inflammation in a dose-dependent way. Although Th1 effectors are protective against several intracellular pathogens, effective schistosomicidal responses are dependent of the Th2 phenotype. Thus, doxycycline contributes to the worsening of lung granulomatous inflammation by potentiating eosinophils influx and downregulating Th2 effectors, reinforcing lipid and protein oxidative damage in chronic S. mansoni infection.

The Inducible Nitric Oxide Synthase Pathway Promotes Osteoclastogenesis under Hypoxic Culture Conditions

Bone homeostasis depends on the balance between bone resorption by osteoclasts (OCs) and bone formation by osteoblasts. Bone resorption can become excessive under various pathologic conditions, including rheumatoid arthritis. Previous studies have shown that OC formation is promoted under hypoxia. However, the precise mechanisms behind OC formation under hypoxia have not been elucidated. The present study investigated the role of inducible nitric oxide synthase (iNOS) in OC differentiation under hypoxia. Primary bone marrow cells obtained from mice were stimulated with receptor activator of NF-κB ligand and macrophage colony-stimulating factor to induce OC differentiation. The number of OCs increased in culture under hypoxia (oxygen concentration, 5%) compared with that under normoxia (oxygen concentration, 20%). iNOS gene and protein expression increased in culture under hypoxia. Addition of an iNOS inhibitor under hypoxic conditions suppressed osteoclastogenesis. Addition of a nitric oxide donor to the normoxic culture promoted osteoclastogenesis. Furthermore, insulin-like growth factor 2 expression was significantly altered in both iNOS inhibition experiments and nitric oxide donor experiments. These data might provide clues to therapies for excessive osteoclastogenesis under several hypoxic pathologic conditions, including rheumatoid arthritis.

Myeloid-Derived Suppressor Cells in Immune-Related Bone Marrow Failure: Novel Therapeutic Implication

Immune-related bone marrow failure (IBMF) is characterized by pancytopenia and bone marrow (BM) hypoplasia. Abnormally activated CD8+ T cells are important in its pathogenesis and majority of the patients having this abnormity are diagnosed as aplastic anemia (AA). The exploration of intra-BM immune network will help to conduct novel treatments to this disease.BM mesenchymal stromal cells (MSCs), regulatory T cells (Tregs), natural killer (NK) cells, myeloid-derived suppressor cells (MDSCs) are all immune-regulators within BM microenvironment. Among them, lymphocytes including CD8+ T cells, Tregs, NKs were indicated relating to IBMF pathogenesis with either stimulating or suppressive function. However, the relationship within BM between activated CD8+ T cells and other immune-regulating component remains largely unknown. In this study, we investigated MDSCs' characterization in IBMF models and AA patients to look into the intra-BM immune response during IBMF occurrence and progression.IBMF mice models were produced by lymphocyte infusion as previously described (Chen J, et al. Blood. 2004). To determine the major T cell population responsible for attacking BM environment, we injected H2 b/b -CD3+CD4+ T cells or H2 b/b -CD3+CD8+ T cells in sublethally irradiated hybrid F1 recipients of H2 b/b and H2 d/d mice. The recipients infused with CD3+CD8+ T cells were manifested BMF as pancytopenia, promoted BM-MSC adipogenesis and shorter life span. To further clarify why BM is the major targeted organ of CD8+ T cell in IBMF models, we detected homing receptors and proliferative potential of CD8+ T cells in BM, spleen and lymph nodes. CXCR4, CXCR6, CD49d, TRAIL as well as CD25, EDU were increasingly expressed on intra-BM CD8+ T cells, suggesting that CD8+ T cells reside more sufficiently in BM than in other lymph organs.MDSCs, subgrouped as mMDSCs and gMDSCs by a panel of surface markers, are a heterogeneous population of immature myeloid cells with immunoregulatory function (Gabrilovich DI, et al. Nat Rev Immunol. 2009). The numbers of either circulating MDSCs (CD11b+CD33+HLA-DR-/low) in AA patients or intra-BM MDSCs (CD11b+Gr1+) in IBMF models were decreased compared to normal controls (p=0.0016 and 0.03, respectively), indicating that MDSCs are impacted in the presence of over-activated CD8+ T cells. In vitro co-cultivation of MDSC subpopulation with T cells showed that it was not gMDSCs (CD11b+Ly6G+Ly6Clow, p=0.112) but mMDSCs (CD11b+Ly6G-/lowLy6Chi, p=0.017) capable of reducing anti-CD3/CD28-stimulated T cell proliferation. A nitric oxide synthase (NOS) inhibitor, L-NG-Nitroarginine Methyl Ester (L-NAME), significantly reversed mMDSC-induced T cell hypoproliferation (38.7%±17.5% vs 72.3%±3.5%, p=0.031), suggesting that inducible NOS pathway is an important modulator in the interaction of mMDSC and activated CD8+ T cells.To further investigate whether mMDSCs exert immune regulating function in vivo, freshly sorted H2 b/b -mMDSCs were transfused in sublethally irradiated H2 b/d with CD3+ T cells or lately in IBMF models every three days. The earlier mMDSCs transfusion was able to reduce the severity of pancytopenia and intra-BM infiltration of CD8+ T cells. The overall survival (OS) of repeatedly transfusion group was significantly longer than that of non-mMDSCs transfusion controls (26 days vs 17days, p=0.025).Taking these together, mMDSCs serve as an important regulator with other immune suppressive parts on one side of the teeter-totter, the other side of which the overactivated CD8+ T cells are the major counterpart attacking hematopoietic components in IBMF. In BM microenvironment, immune balance together with orderly multi-differentiation of MSCs and hematopoietic stem cells are all essential to orchestrate normal hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

A Bacteria-Inspired Morphology Genetic Biomedical Material: Self-Propelled Artificial Microbots for Metastatic Triple Negative Breast Cancer Treatment.

Morphology genetic biomedical materials (MGBMs), referring to fabricating materials by learning from the genetic morphologies and strategies of natural species, hold great potential for biomedical applications. Inspired by the cargo-carrying-bacterial therapy (microbots) for cancer treatment, a MGBM (artificial microbots, AMBs) was constructed. Rather than the inherent bacterial properties (cancerous chemotaxis, tumor invasion, cytotoxicity), AMBs also possessed ingenious nitric oxide (NO) generation strategy. Mimicking the bacterial construction, the hyaluronic acid (HA) polysaccharide was induced as a coating capsule of AMBs to achieve long circulation in blood and specific tissue preference (tumor tropism). Covered under the capsule-like polysaccharide was the combinatorial agent, the self-assembly constructed by the amphiphilic dendrons with abundant l-arginine residues peripherally (as endogenous NO donor) and hydrophobic chemotherapeutic drugs at the core stacking on the surface of SWNTs (the photothermal agent) for a robust chemo-photothermal therapy (chemo-PTT) and the elicited immune therapy. Subsequently, the classic inducible nitric oxide synthase (iNOS) pathway aroused by immune response was revolutionarily utilized to oxidize the l-arginine substrates for NO production, the process for which could also be promoted by the high reactive oxygen species level generated by chemo-PTT. The NO generated by AMBs was intended to regulate vasodilation and cause a dramatic invasion (as the microbots) to disperse the therapeutic agents throughout the solid tumor for a much more enhanced curative effect, which we defined as "self-propulsion". The self-propelled AMBs exhibiting impressive primary tumor ablation, as well as the distant metastasis regression to conquer the metastatic triple negative breast cancer, provided pioneering potential therapeutic opportunities, and enlightened broad prospects in biomedical application.

Oxytocinergic system mediates the proconvulsant effects of sildenafil: The role of calcineurin

Sildenafil is a phosphodiesterase type 5 inhibitor used to treat male erectile dysfunction and pulmonary hypertension. A potential side effect of sildenafil is a noticeable decrease in seizure threshold. Oxytocin (OXT) secretion and the subsequent cAMP-responsive element-binding (CREB) phosphorylation are involved in proconvulsant effects of sildenafil in experimental models. The aim of the present study was to investigate the potential role of OXT receptors and their downstream calcineurin (CN)/inducible nitric oxide synthase (iNOS) pathways in proconvulsant effects of sildenafil. The pentylenetetrazole (PTZ)-induced seizure was used as a standard convulsion model in this study. Cortical CN activity, hippocampal nitrite levels, and proinflammatory cytokine content were measured. Our results indicated that following PTZ administration, sildenafil significantly increased CN activity at 40 mg/kg, respectively, in the control group. The combination of sildenafil and OXT receptor antagonist, atosiban (10 μg/kg, i.c.v) 30 min before sildenafil administration significantly reduced the CN activity. Also, the subeffective dose of CN inhibitor cyclosporine (5 mg/kg) 30 min before the administration of effective dose of sildenafil (40 mg/kg) reversed proconvulsant actions of sildenafil. This effect was iNOS-dependent because pretreatment of a low dose of aminoguanidine (20 mg/kg) 15 min before the administration of a low dose of cyclosporine (1 mg/kg) reversed the proconvulsant action of sildenafil (40 mg/kg). Finally, sildenafil induced the elevation of tumor necrosis factor alpha (TNF-α) and the nitrite level was blocked by the administration of cyclosporine in PTZ-treated mice.Collectively, our data provide insights into the role of OXT receptor/CN/iNOS pathway in the proconvulsant aspect of sildenafil.

TLR9/MyD88/TRIF signaling activates host immune inhibitory CD200 in Leishmania infection.

Virulent protozoans named Leishmania in tropical and subtropical areas produce devastating diseases by exploiting host immune responses. Amastigotes of Leishmania amazonensis stimulate macrophages to express CD200, an immunomodulatory ligand, which binds to its cognate receptor (CD200R) and inhibits the inducible nitric oxide synthase and nitric oxide (iNOS/NO) signaling pathways, thereby promoting intracellular survival. However, the mechanisms underlying CD200 induction in macrophages remain largely unknown. Here, we show that phagocytosis-mediated internalization of L. amazonensis amastigotes following activation of endosomal TLR9/MyD88/TRIF signaling is critical for inducing CD200 in infected macrophages. We also demonstrate that Leishmania microvesicles containing DNA fragments activate TLR9-dependent CD200 expression, which inhibits the iNOS/NO pathway and modulates the course of L. amazonensis infection in vivo. These findings demonstrate that Leishmania exploits TLR-signaling pathways not only to inhibit macrophage microbicidal function, but also to evade host systemic immune responses, which has many implications in the severity of the disease.

Modulation of inducible nitric oxide synthase pathway by eugenol and telmisartan in carbon tetrachloride-induced liver injury in rats

AimsInducible nitric oxide synthase (iNOS) pathway has been in the limelight since its discovery as a key mediator in the process of liver fibrogenesis. Therefore, the objective of the current study was to elucidate the in vivo molecular mechanism underlying the hepatic preventive relevance of eugenol (EUG) and telmisartan (TEL) through iNOS pathway modulation against carbon tetrachloride (CCl4)-induced hepatic injury. MethodsSixty healthy male albino rats were used in this study. Serum aminotransferases activities and NO levels were assessed. Hepatic malondialdehyde (MDA), total nitrite/nitrate content and reduced glutathione (GSH) concentration were estimated. Liver NF-kB, TNF-α, IL-6 and iNOS proteins expressions were investigated by western blot assay. Histopathological examination was done. Key findingsCCl4 resulted in damage to centrilobular regions of the liver, elevation of serum aminotransferases, rise in oxidative parameters level, and up-regulation of NF-kB, TNF-α, IL-6 as well as iNOS proteins expressions. Treatment of fibrotic rats with either EUG or TEL significantly alleviated CCl4-induced biochemical, inflammatory and histopathological changes. Moreover, the combined administration of EUG with TEL has an ameliorative effect which is greater than either of them alone. SignificanceIn conclusion, the combination therapy between EUG and TEL is more effective than either drug alone which is attributed to suppression of NO production and iNOS protein expression. The results support that use of EUG and TEL exerts beneficial effects in the attenuation of CCl4-induced liver fibrosis in rats.

Arsenic and/or copper caused inflammatory response via activation of inducible nitric oxide synthase pathway and triggered heat shock protein responses in testis tissues of chicken.

The aim of this study is to investigate the effects of arsenic (As) and copper (Cu) on the inflammatory response, and the protective roles of heat shock proteins (Hsps) in chicken testes. Seventy-two 1-day-old male Hy-line chickens were treated with 30mg/kg feed of arsenic trioxide (As2O3) and/or 300mg/kg feed of copper sulfate (CuSO4) for 4, 8, and 12weeks. The histological changes, inducible nitric oxide synthase (iNOS) activity, and the expressions of Hsps and inflammatory cytokines were detected. The results showed that slight histology changes were obvious in the testis tissue exposure to treatment groups. The activity and the protein level of iNOS were increased compared to the control group. The mRNA levels of proinflammatory cytokines and inflammatory factors were increased as a whole. However, anti-inflammatory cytokines were inhibited. The mRNA and protein levels of Hsp60, Hsp70, and Hsp90 were upregulated. These results suggested that sub-chronic exposure to As and/or Cu induced testicular poisoning in chickens. Increased Hsps tried to protect chicken testis tissues from tissues damage caused by inflammation. In conclusion, testicular poisoning induced by As and/or Cu caused inflammatory response and heat shock protein response in chicken testis tissues.

Lysophosphatidic acid-triggered pathways promote the acquisition of trophoblast endovascular phenotype in vitro.

Successful implantation and placentation requires that extravillous cytotrophoblast acquires an endovascular phenotype and remodels uterine spiral arteries. Defects in this mechanism correlate with severe obstetric complications as implantation failure and preeclampsia. Lysophosphatidic acid (LPA) participates in embryo implantation and contributes to vascular physiology in different biological systems. However, the role of LPA on trophoblast endovascular transformation has not been studied. Due to difficulties in studying human pregnancy in vivo, we adopted a pharmacological approach in vitro to investigate LPA action in various aspects of trophoblast endovascular response, such as the formation of endothelial capillary-like structures, migration, and proliferation. The HTR-8/SVneo cell line established from human first trimester cytotrophoblast was used to model the acquisition of the endovascular phenotype by the invading trophoblast. LPA increased HTR-8/SVneo tube formation, migration (wound healing assay and phalloidin staining) and proliferation (MTT assay). LPA G protein-coupled receptors, LPA1 and LPA3 , were expressed in HTR-8/SVneo. By using selective antagonists, we showed that enhanced tubulogenesis was mediated by LPA3 . In addition, cyclooxygenase-2 and inducible nitric oxide synthase pathways participated in LPA-stimulated tubulogenesis. Inducible nitric oxide synthase was activated downstream cyclooxygenase-2. Furthermore, prostaglandin E2 and a nitric oxide donor (SNAP) increased trophoblast tube formation in a concentration-dependent manner. Finally, we observed that cyclooxygenase-2 and inducible nitric oxide synthase were localized in the nucleus, and LPA did not modify their cellular distribution. Our results show that LPA-triggered regulatory pathways promote trophoblast endovascular response in vitro, suggesting a new role for LPA during spiral artery remodeling at the maternal-fetal interface.

Macrophages activate iNOS signaling in adventitial fibroblasts and contribute to adventitia fibrosis

A large amount of NO is generated through the inducible nitric oxide synthase (iNOS) pathway from the vascular adventitia in various vascular diseases. However, it is currently not fully understood how the iNOS signaling pathway is activated. In the present study, this question was addressed in the context of adventitial cellular interactions. A rat model of acute hypertension in the contralateral carotid arteries was established through transverse aortic constriction (TAC) surgery. In this model, activated macrophages were found surrounded by a large quantity of iNOS-expressing adventitial fibroblasts (AFs), suggesting a possible causal relationship between macrophages and iNOS activation of the neighboring AFs. In an in vitro model, a macrophage-like cell line RAW 264.7 was first activated by LPS treatment. The supernatant was then harvested and applied to treat primary rat AFs. iNOS in AFs was activated robustly by the supernatant treatment but not by LPS itself. Treating AFs with interleukin-1β (IL-1β) also activated iNOS signaling, suggesting that the IL-1β pathway might be a possible mediator. As a consequence of the iNOS activation, total protein nitration and S-nitrosylation significantly increased in those AFs. Additionally, increased deposition of type I and type III collagens was observed in both in vitro and in vivo models. The collagen deposition was partially restored by an iNOS inhibitor, 1400 W. These findings highlight the importance of iNOS signaling during vascular inflammation, and advance our understanding of its activation through a cellular interaction perspective.

Allicin protects traumatic spinal cord injury through regulating the HSP70/Akt/iNOS pathway in mice.

Allicin is a major component of garlic, extracted as an oily liquid. The present study was designed to investigate the beneficial effects of allicin on traumatic spinal cord injury (TSCI) in mice, and whether the effects are mediated via regulation of the heat shock protein 70 (HSP70), v-akt murine thymoma viral oncogene homolog 1 (Akt) and inducible nitric oxide synthase (iNOS) pathways. Adult BALB/c mice (30–40 g) received a laminectomy at the T9 vertebral level as a model of TSCI. In the present study, treatment of the TSCI mice with allicin significantly increased their Basso, Beattie and Bresnahan (BBB) scores (P<0.01) and reduced the spinal cord water content (P<0.01). This protective effect was associated with the inhibition of oxidative stress and inflammatory responses in TSCI mice. Western blot analysis demonstrated that allicin increased the protein levels of HSP70, increased the phosphorylation of Akt and reduced the iNOS protein expression levels in TSCI mice. Additionally, treatment with allicin significantly reduced the levels of ROS and enhanced the NADH levels in TSCI mice. Collectively, these data demonstrate that the effects of allicin on TSCI are mediated via regulation of the HSP70, Akt and iNOS pathways in mice.

Immunohistochemical study of inducible nitric oxide synthase and tumour necrosis factor alpha response in turbot (Scophthalmus maximus) experimentally infected with Aeromonas salmonicida subsp. salmonicida.

Aeromonas salmonicida subsp. salmonicida represents one of the major threats in aquaculture, especially in salmonid fish and turbot farming. In order to fight bacterial infections, fish have an immune system composed by innate and specific cellular and humoral elements analogous to those present in mammals. However, innate immunity plays a primordial role against bacterial infections in teleost fish. Among these non-specific mechanisms, the production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) pathway and the tumour necrosis factor-alpha (TNFα) produced by mononuclear phagocytes, are two of the main immune effectors to eliminate bacterial pathogens. In this study, the distribution and kinetic of iNOS and TNFα-producing cells of kidney and spleen of turbot experimentally inoculated with A.salmonicida was assessed by immunohistochemistry. In control and challenged fish, individual iNOS(+) and TNFα(+) cells, showing a similar pattern of distribution, were detected. In challenged fish, the number of immunoreactive cells was significantly increased in the evaluated organs, as well as the melanomacrophage centres showed variable positivity for both antigens. These results indicate that A.salmonicida induced an immune response in challenged turbot, which involved the increase of the activity of iNOS and TNFα in the leukocytic population from kidney and spleen.

Anti-Inflammatory Effect of Sedum takesimense Nakai Water Extract in RAW 264.7 Cells

Background: Sedum takesimense Nakai has been used as folk medicine in Korea. The present study aimed to determine the biological activity of S. takesimense by investigating the anti-inflammatory effects of S. takesimense water extract (SKLC) on the lipopolysaccharide-induced inflammatory response in RAW 264.7 cells. Methods and Results: Cytotoxicity of SKLC on RAW 264.7 cells was determinded by performing MTS assay was found to have no cytotoxic effect on RAW 264.7 cells at a concentration range of 62 - 500 ㎍/㎖. Further, pretreatment of SKLC inhibited lipopolysaccharide-induced nitric oxide (NO) production in a dose-dependent manner. To determined the inhibitory mechanisms of SKLC on inflammatory mediators, we assessed the inducible nitric oxide synthase (iNOS) and cyclooxygnease-2 (COX-2) pathways. The activities of these pathways were decreased in a dose-dependent manner by SKLC. The production of tumor necrosis factor- α (TNF-α), interleukin (IL)-1β‚ and IL-6 were also reduced. Conclusions: These results suggest that the down regulation of iNOS, COX-2, TNF-α, IL-1β‚ and IL-6 expression by SKLC are mediated by the down regulation of nuclear factor-κB (NF-κB) activity, a transcription factor necessary for pro-inflammatory mediators. This might be the mechanism underlying the anti-inflammatory effects of SKLC.

Topological control of nitric oxide secretion by tantalum oxide nanodot arrays.

BackgroundNitric oxide (NO) plays a very important role in the cardiovascular system as a major secondary messenger in signaling pathway. Its concentration regulates most of the important physiological indexes including the systemic blood pressure, blood flow, regional vascular tone and other cardiac functions. The effect of nanotopography on the NO secretion in cardiomyocytes has not been elucidated before. In this study, we report how the nanotopography can modulate the secretion profile of NO and attempt to elucidate the genetic pathways responsible for the same by using Tantalum Oxide nanodot arrays ranging from 10 to 200 nm. A series of nanodot arrays were fabricated with dot diameter ranging from 10 to 200 nm. Temporal NO release of cardiomyocytes was quantified when grown on different surfaces. Quantitative RT-PCR and Western blot were performed to verify the genetic pathways of NO release.ResultsAfter hours 24 of cell seeding, NO release was slowly enhanced by the increase of dot diameter from 10 nm up to 50 nm, mildly enhanced to a medium level at 100 nm, and increase rapidly to a high level at 200 nm. The temporal enhancement of NO release dropped dramatically on day 3. On day 5, a topology-dependent profile was established that maximized at 50 nm and dropped to control level at 200 nm. The NO releasing profile was closely associated with the expression patterns of genes associated with Endothelial nitric oxide synthase (eNOS) pathway [GPCR, PI3K, Akt, Bad, Bcl-2, NFκB(p65), eNOS], but less associated with Inducible nitric oxide synthase (iNOS) pathway (TNF-α, ILK, Akt, IκBα, NFκB, iNOS). Western blotting of Akt, eNOS, iNOS, and NFκB further validated that eNOS pathway was modulated by nanotopology.ConclusionsBased on the findings of the present study, 50, 100 nm can serve as the suitable nanotopography patterns for cardiac implant surface design. These two nanodot arrays promote NO secretion and can also promote the vascular smooth muscle relaxation. The results of this study can improve the heart stent design in the medical treatments.

Pinosylvin and monomethylpinosylvin, constituents of an extract from the knot of Pinus sylvestris, reduce inflammatory gene expression and inflammatory responses in vivo.

Scots pine (Pinus sylvestris) is known to be rich in phenolic compounds, which may have anti-inflammatory properties. The present study investigated the anti-inflammatory effects of a knot extract from P. sylvestris and two stilbenes, pinosylvin and monomethylpinosylvin, isolated from the extract. Inflammation is characterized by increased release of pro-inflammatory and regulatory mediators including nitric oxide (NO) produced by the inducible nitric oxide synthase (iNOS) pathway. The knot extract (EC50 values of 3 and 3 μg/mL) as well as two of its constituents, pinosylvin (EC50 values of 13 and 15 μM) and monomethylpinosylvin (EC50 values of 8 and 12 μM), reduced NO production and iNOS expression in activated macrophages. They also inhibited the production of inflammatory cytokines IL-6 and MCP-1. More importantly, pinosylvin and monomethylpinosylvin exerted a clear anti-inflammatory effect (80% inhibition at the dose of 100 mg/kg) in the standard in vivo model, carrageenan-induced paw inflammation in the mouse, with the effect being comparable to that of a known iNOS inhibitor L-NIL. The results reveal that the Scots pine stilbenes pinosylvin and monomethylpinosylvin are potential anti-inflammatory compounds.