iNOS mRNA Expression Research Articles

Lactobacillus plantarum KFY02 enhances the prevention of CCl4-induced liver injury by transforming geniposide into genipin to increase the antioxidant capacity of mice

This study investigated Lactobacillus plantarum KFY02 (KFY02) and its ability to enhance the antioxidant capacity of the body by converting geniposide to genipin, thereby decreasing the liver damage induced by CCl4 in mice. The experimental results showed that geniposide is partially converted into genipin by KFY02. In liver-injured mice, KFY02 and geniposide could decrease the liver index, the AST, ALT, TG, and MDA serum levels, and the levels of cytokines IL-6, IL-12, TNF-α, and IFN-γ while up-regulating SOD and GSH-Px levels. KFY02 and geniposide increased the mRNA and protein expression of Mn-SOD, Cu/Zn-SOD, CAT, GSH-Px, and IκB-α and decreased the mRNA and protein expression of COX-2, iNOS, and NF-κB in the liver tissue of liver-injured mice. In summary, the conversion of geniposide to genipin by L. plantarum KFY02 can effectively enhance the body's antioxidant capacity to prevent CCl4-induced liver damage in mice.

Possible contribution of the neprilysin/ACE pathway to sepsis in mice

AimOmapatrilat is an antagonist of angiotensin-converting (ACE) and neprilysin-neuropeptidase (NEP) enzymes. The aim of our study is to show that omapatrilat may have beneficial effects as a treatment for polymicrobial sepsis. Main methodsA cecal ligation and puncture (CLP) sepsis model was used to evaluate 10 and 20 mg/kg doses of omapatrilat in mice (n = 30) fasted for 12 h. The lungs were removed 12 h after CLP, and lung levels of cytokines (tumor necrosis factor-alpha [TNF-α], interleukin-6 [IL-6], NF-κB), iNOS and eNOS mRNA expression, GSH and MDA levels, and ACE and NEP activities were determined. Histopathological examinations were also performed. Key findingsOmapatrilat treatment provided a dose-dependent reduction in oxidative stress and inflammatory parameters in lung tissues. Omapatrilat administration decreased lung iNOS and eNOS mRNA levels at 20 mg/kg dose. Histopathological analysis revealed a decline in the thickening and edema areas in the alveolar septa in the Sepsis+OMA20 group. SignificanceOmapatrilat, a dual ACE and NEP inhibitor, protected lung tissue from sepsis damage by reducing ACE and NEP activities, by decreasing the mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-6, and NF-κB), by suppressing leukocyte infiltration and edema, by restoring iNOS and eNOS levels, and by restoring SOD activity and GSH and MDA levels, thereby reducing oxidative stress.

TRIM28/TIF1β and Fli-1 negatively regulate peroxynitrite generation via DUOX2 to decrease the shedding of membrane-bound fractalkine in human macrophages after exposure to substance P

The chemokine fractalkine is synthesized as a membrane-bound protein, but studies have shown that serum levels of soluble fractalkine are elevated in inflammatory and autoimmune diseases. Patients with autoimmune diseases also have increased serum levels of neuropeptide substance P (SP). The shedding activity of the ADAM family is induced by peroxynitrite, but that of SP is unclear. Treatment of human macrophages with SP upregulated levels of membrane-bound fractalkine. Interestingly, small interfering RNA (siRNA) for DUOX2 further increased membrane-bound fractalkine but decreased soluble fractalkine compared with cells treated with SP alone. SP induced nitric oxide 2/inducible nitric oxide synthase (NOS2/iNOS) mRNA and increased levels of nitrotyrosine, a biomarker of peroxynitrite, whereas transfection with DUOX2 siRNA blunted upregulation of nitrotyrosine. Most importantly, N(ω)-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor) decreased protein levels of nitrotyrosine and concomitantly increased expression of membrane-bound fractalkine after exposure to SP. As for the signaling pathway of TGFβ1 (an inhibitor of iNOS mRNA expression), silencing of RNA for TAK-1 upregulated membrane-bound fractalkine, but silencing of RNA for the Smad family did not. Interfering RNA of transcription factor specificity protein 1 (Sp1) upregulated protein levels of TGFβ1/LAP. Most importantly, double transfection with siRNA for Sp1 and TRIM28/TIF1βor Fli-1 led to a significant increase in TGFβ1/LAP levels and a corresponding reduction of NOS2/iNOS, which inhibited the shedding of membrane-bound fractalkine. In conclusion, TRIM28/TIF1β and Fli-1 negatively regulate TGFβ1 expression to upregulate the generation of peroxynitrite, leading to increased shedding of membrane-bound fractalkine induced by SP.

Loss of glutathione peroxidase 3 induces ROS and contributes to prostatic hyperplasia in Nkx3.1 knockout mice.

Glutathione peroxidase 3 (Gpx3) protects cells from oxidative stress, and its reduced expression in human prostate cancer has been reported. We hypothesized that Gpx3 might play an important role in the development of prostatic intraepithelial neoplasia (PIN), a pre-cancerous state of the prostate, and aimed to highlight the underlying molecular mechanism. The following double-knockout mice Nkx3.1-/-; Gpx3+/+, Nkx3.1-/-; Gpx3+/-, Nkx3.1-/-; Gpx3-/- were produced. Randomly divided animals were weighed, and their genitourinary tract (GUT) weights were determined after euthanasia at 4, 8, and 12months. The mRNA expression of the genes involved in oxidative stress and Wnt signaling was analyzed in the prostate. Histopathology, ROS, and superoxide dismutase (SOD) activities were also measured. Loss of Gpx3 did not affect body weight and GUT weight in Nkx3.1 knockout mice. The mRNA expression of SOD3, iNOS, Hmox, and CISD2, which are associated with oxidative stress, was increased in Nkx3.1-/-; Gpx3-/- mice at 4months but decreased at 8 and 12months. There was no change in β-catenin and its targets associated with Wnt signaling. Increased ROS and decreased SOD activity were observed in Nkx3.1-/-; Gpx3-/- mice at 12months of age. The histopathologic score and epithelium thickness were increased, and lumen area was decreased in Gpx3 knockout mice. Gpx3 loss increased the hyperplasia of PIN in the pre-cancerous stage of the prostate. Loss of Gpx3 induced oxidative stress. Histopathologically, no invasive carcinoma was identified, and Gpx3 loss did not increase Wnt/β-catenin signaling. Further research on the role of GPX3 in the transition of PIN to invasive carcinoma is needed. We show, for the first time, that the antioxidant enzyme GPX3 plays a vital role in inhibiting hyperplasia in the PIN stage of the prostate gland in vivo.

Oxidative stress, apoptosis and inflammatory responses involved in copper-induced pulmonary toxicity in mice.

At present, there are few studies focused on the relationship between copper (Cu) and oxidative stress, apoptosis, or inflammatory responses in animal and human lungs. This study was conducted to explore the effects of Cu on pulmonary oxidative stress, apoptosis and inflammatory responses in mice orally administered with 0 mg/kg (control), 10 mg/kg, 20 mg/kg, and 40 mg/kg of CuSO4 for 42 days. The results showed that CuSO4 increased ROS production, and MDA, 8-OHdG and NO contents as well as iNOS activities and mRNA expression levels. Meanwhile, CuSO4 reduced the activities and mRNA expression levels of antioxidant enzymes (GSH-Px, CAT, and SOD) and GSH contents, and ASA and AHR abilities. Also, CuSO4 induced apoptosis, which was accompanied by decreasing Bcl-2, Bcl-xL mRNA expression levels and protein expression levels, and increasing Bax, Bak, cleaved-caspase-3, cleaved-caspase-9 mRNA, and protein expression levels, and Bax/Bcl-2 ratio. Concurrently, CuSO4 caused inflammation by increasing MPO activities and activating the NF-κB signalling pathway, and down-regulating the mRNA and protein expression levels of anti-inflammatory cytokines (IL-2, IL-4, IL-10). In conclusion, the abovementioned findings demonstrated that over 10 mg/kg CuSO4 can cause oxidative stress, apoptosis, and inflammatory responses, which contribute to pulmonary lesions and dysfunction in mice.

Rhoifolin Alleviates Inflammation of Acute Inflammation Animal Models and LPS-Induced RAW264.7 Cells via IKKβ/NF-κB Signaling Pathway.

Rhoifolin (ROF) is a main effective component in Citrus grandis 'Tomentosa'. ROF has a potential anti-inflammatory activity, but its specific effects and mechanisms have not been studied. This study investigated the anti-inflammatory activity of ROF and searched for its possible molecular mechanisms. A mouse model of acute inflammation was induced by lipopolysaccharide, and the effects of ROF on pathological damages of the lung and liver were observed. Carrageenan-induced paw edema rat model was used to evaluate the effect of ROF on the volume of swelling paw. In LPS-induced RAW264.7 macrophages, the expression levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were measured using ELISA. Real-time PCR was used to measure the mRNA levels of iNOS and CCL2. Western blot was used to detect the activation of IκBα and IKKβ in NF-κB signaling pathways. The results showed that ROF accelerated the recoveries of liver and lung tissue damages in acute inflammation mice and inhibited carrageenan-induced paw edema in rats; in addition, ROF significantly suppressed the secretion of TNF-α, IL-1β, and IL-6 in the serum of rats and mouse model. In LPS-induced RAW264.7 cells, 100 μmol/L ROF enhanced cell viability and suppressed the production of TNF-α, IL-6, and IL-1β significantly. ROF also decreased the mRNA expression of iNOS and CCL2 and inhibited IκBα and IKKβ phosphorylation. In summary, ROF had a potential therapeutic value for inflammation. Our research provided experimental basis for the further development of ROF as an anti-inflammatory drug and for clarifying the anti-inflammatory substance basis of Citrus grandis 'Tomentosa'. Graphical Abstract.

Alveolar Macrophage Phenotype and Compartmentalization Drive Different Pulmonary Changes in Mouse Strains Exposed to Cigarette Smoke

COPD can manifest itself with different clinical phenotypes characterized by different disease progression and response to therapy. Although a remarkable number of studies have been carried out, little is known about the mechanisms underlying phenotypes that could guide the development of viable future therapies. Several murine strains mirror some human phenotypes after smoke exposure. It was of interest to investigate in these strains whether different pattern of activation of macrophages, and their distribution in lungs, is associated to changes characterizing different phenotypes. We chose C57Bl/6, and Lck deficient mice, which show significant emphysema, DBA/2 mice that develop changes similar to those of “pulmonary fibrosis/emphysema syndrome”, p66Shc ko mice that develop bronchiolitis with fibrosis but not emphysema, and finally ICR mice that do not develop changes at 7 months after smoke exposure. Unlike other strains, ICR mice show very few activated macrophages (Mac-3 positive) mostly negative to M1 or M2 markers. On the other hand, a large population of M1 macrophages predominates in the lung periphery of DBA/2, C57Bl/6 and in Lck deficient mice, where emphysema is more evident. M2 macrophages are mainly observed in subpleural and intraparenchymal areas of DBA/2 mice and around bronchioles of p66Shc ko mice where fibrotic changes are present. We observed slight but significant differences in mRNA expression of iNOS, ECF-L, arginase 1, IL-4, IL-13 and TGF-β between air- and smoke-exposed mice. These differences together with the different compartmentalization of macrophages may offer an explanation for the diversity of lesions and their distribution that we observed among the strains.

CD137 signaling promotes angiogenesis through regulating macrophage M1/M2 polarization

Objective: To investigate whether CD137 signaling can promote angiogenesis via regulating macrophage M1/M2 polarization. Methods: (1) The primary peritoneal macrophages in mice induced by 3% thiglycollate broth were divided into three groups: control group, CD137 signaling activated group and CD137 signaling inhibited group. Various specific markers of M1 and M2 macrophages were detected to observe the phenotype change of macrophages, and the macrophages protein expression of CD137, CD86 and CD206 was detected by flow cytometry (FCM). The protein and mRNA expression of induced nitric oxide synthase (iNOS), arginase Ⅰ(Arg-1) was determined by Western blot and RT-PCR, respectively. The secretion levels of IL-12 and IL-10 in culture supernatant of macrophages were detected by ELISA. (2) Macrophages were co-cultured with the endothelial cells (bEnd.3), and macrophages were implanted in the upper chamber, endothelial cells were implanted in stromal glue of the lower chamber. The experiment was divided into three groups: the control group, CD137 signaling activated group and peroxisome proliferator-activated receptor-γ (PPAR-γ) inhibited group, and tube formation ability of endothelial cells in each group was determined. Results: (1) The purity of primary peritoneal macrophages in mice was (97.93±1.31)%. The expression of CD137 on the surface of macrophages was (97.40±2.70)%. (2) Compared with control group, the mRNA and protein expression levels of Arg-1 were significantly increased and the mRNA and protein expression of iNOS were significantly decreased in CD137 signaling activated group (all P<0.05). Compared with CD137 signaling activated group, the mRNA and protein expression of Arg-1 were significantly lower and the mRNA and protein expression levels of iNOS were significantly higher in CD137 signaling inhibited group (all P<0.05). FCM results showed that the average fluorescence intensity of CD206 was higher, while the average fluorescence intensity of CD86 was lower in CD137 signaling activated group than in control group (P<0.05, P<0.01, respectively); the expression of CD206 was significantly lower, while the expression of CD86 was higher, in the CD137 signaling inhibited group than in CD137 signaling activated group (P<0.05, P<0.01, respectively). ELISA results showed that the secretion of IL-10 was higher, and the secretion level of IL-12 was significantly lower in CD137 signaling activated group than in control group (both P<0.01); the secretion of IL-10 was significantly lower and the secretion of IL-12 was significantly higher in CD137 signaling inhibited group than in CD137 signaling activated group (both P<0.05). (3) Values of the formation of tube length and branch number were both longer in CD137 signaling activated group than control group (P<0.05). The formation of the tube length and branch number were less in PPAR-γ inhibited group than in CD137 signaling activated group (P<0.05). Conclusion: CD137 signaling can promote angiogenesis by regulating macrophage M1/M2 polarization.

Structure-Dependent Effects of Bisphosphonates on Inflammatory Responses in Cultured Neonatal Mouse Calvaria.

Bisphosphonates (BPs) are classified into two groups, according to their side chain structures, as nitrogen-containing BPs (NBPs) and non-nitrogen-containing BPs (non-NBPs). In this study, we examined the effects of NBPs and non-NBPs on inflammatory responses, by quantifying the inflammatory mediators, prostaglandin E2 (PGE2) and nitric oxide (NO), in cultured neonatal mouse calvaria. All examined NBPs (pamidronate, alendronate, incadronate, risedronate, zoledronate) stimulated lipopolysaccharide (LPS)-induced PGE2 and NO production by upregulating COX-2 and iNOS mRNA expression, whereas non-NBPs (etidronate, clodronate, tiludronate) suppressed PGE2 and NO production, by downregulating gene expression. Additionally, [4-(methylthio) phenylthio] methane bisphosphonate (MPMBP), a novel non-NBP with an antioxidant methylthio phenylthio group in its side chain, exhibited the most potent anti-inflammatory activity among non-NBPs. Furthermore, results of immunohistochemistry showed that the nuclear translocation of NF-κB/p65 and tyrosine nitration of cytoplasmic protein were stimulated by zoledronate, while MPMBP inhibited these phenomena, by acting as a superoxide anion (O2−) scavenger. These findings indicate that MPMBP can act as an efficacious agent that causes fewer adverse effects in patients with inflammatory bone diseases, including periodontitis and rheumatoid arthritis.

P22077 inhibits LPS-induced inflammatory response by promoting K48-linked ubiquitination and degradation of TRAF6.

Inflammation is a biological process associated with multiple human disorders such as autoimmune diseases and metabolic diseases. Therefore, alleviation of inflammation is important for disease prevention or treatment. Recently, deubiquitinating enzymes (DUBs), especially ubiquitin specific protease-7 (USP7) attracts increasing attention as a potential drug target for inflammation. As an inhibitor of USP7, P22077 has been used to study the roles of USP7 in inflammatory response and neuroblastoma growth. However, the role and precise mechanism of P22077 in anti-inflammatory is still indistinct. In this study, we demonstrated that P22077 could attenuate the release of pro-inflammatory factors including TNF-α, IL-1β, IL-6 and NO, suppress mRNA expression of COX-2 and iNOS, and inhibit activation of NF-κB and MAPKs signaling pathways in Raw264.7 cells and mouse peritoneal macrophages after LPS stimulation. In vivo study showed that P22077 could relieve inflammatory response and reduce the lung injury in C57BL/6 mice with LPS-induced endotoxemia. Mechanically, P22077 might play an anti-inflammatory role by promoting tumor necrosis factor receptor-associated factor 6 (TRAF6) degradation via K48-linked polyubiquitination. These findings provide a rationale for the role of the P22077 in anti-inflammatory pathway and the promising clinical application of P22077 to treat inflammatory diseases.

Arbutin Improves Functional Recovery and Attenuates Glial Activation in Lysolecethin-Induced Demyelination Model in Rat Optic Chiasm.

Neuroinflammation, glial activation, and oxidative injury are the main pathological mechanisms of demyelination in multiple sclerosis (MS). Arbutin, a natural polyphenol compound, possesses antioxidant, anti-inflammatory, and neuroprotective properties whose therapeutic potential has not been studied in the experimental animal models of MS. In the present study, the efficiency of arbutin on lysolecthin (LPC)-induced local demyelination model was investigated. Demyelination was induced by micro-injection of 2μl LPC (1%) into the rat optic chiasm and the treated group received daily injection of arbutin (50mg/kg, i.p) during 2weeks. Visual-evoked potential (VEP) recordings were used to functionally assess the visual pathway. Gene expression analysis was done to evaluate the arbutin effect on the inflammatory, stress oxidative-related mediators, and myelin markers. The myelin-specific staining was performed to assess demyelination and GFAP staining as an astrocyte marker. We found that arbutin significantly reduced P1-latency of VEPs waves and demyelination at 7 and 14days post-demyelination. Arbutin decreased inflammatory cytokines (IL-1B, IL-17, TNF-α) and iNOS mRNA expression level. In addition, the expression level of anti-inflammatory cytokine (IL-10) and antioxidant mediators (Nrf-2 and HO-1) was enhanced by arbutin treatment. Arbutin increased MBP and Olig2 expression levels in demyelination context. Finally, arbutin attenuated GFAP as an astrocyte marker. Finally, this study demonstrates that arbutin improves functional recovery and myelin repair in the demyelinated optic chiasm through attenuation of inflammation, astrocyte activation, and oxidative stress. These findings might open new promising avenues for treating demyelinating disorders such as multiple sclerosis. Graphical abstract.

Protective effects of desipramine on alveolar bone in experimental periodontitis.

Desipramine is a tricyclic antidepressant with immune-modulatory activity, whose effects on ligature-induced periodontitis are yet to be investigated. Hence, its actions on alveolar bone resorption, gingival collagen content and key inflammatory mediators were herewith analyzed. A total of 60 male Wistar rats were randomly assigned into three groups: 1) control: rats without ligature treated with vehicle (saline); 2) ligature: rats with ligature-induced periodontitis treated with vehicle; 3) ligature + desipramine: rats with ligature-induced periodontitis treated with desipramine (20mg/kg/d in vehicle). Mandibles and gingival tissues were collected 3 or 15 days after ligature insertion (or no ligature insertion for controls) and treatments. Alveolar bone resorption and gingival collagen fibers were histologically analyzed using either HE or picrosirius red staining. Gingival mRNA expressions of interleukin (IL)-1β, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 were obtained through reverse transcription polymerase chain reaction. MMP-9 activity was analyzed by zymography. Alveolar bone loss was significantly reduced in the ligature + desipramine group (P<0.05), whereas gingival collagen degradation was like the ligature group (P>0.05). Desipramine administration downregulated mRNA expressions of IL-1β, iNOS, COX-2, and TIMP-1 when compared to vehicle alone in the ligature group (P<0.05). MMP-9 expression and MMP-9/TIMP-1 ratio were similar among rats with ligature-induced periodontitis (P>0.05); however, MMP-9 activity was lower in the group treated with desipramine (P<0.05). Desipramine administration reduced alveolar bone loss as histologically observed, and modulated key bone remodeling and inflammatory mediators in rats with ligature-induced periodontitis.

LP44 (4-[2-(methylthio)phenyl]-N-(1,2,3,4-tetrahydronaphthalen-1-yl)-1-piperazinehexanamide) exerts anti-ulcer effects via 5-hydroxytryptamine receptor7 activation on indomethacin-induced gastric ulcers in rats.

This study aimed to demonstrate the role of serotonin7 receptor (5-HT7) and the effects of 5-HT7 agonists and antagonists in an indomethacin-induced gastric ulcer. Male albino Wistar rats (n = 60) were used in the experiments. LP44 (5-HT7 agonist) and SB269970 (5-HT7 antagonist) were administered at 10mg/kg as a pre-treatment. One hour after the drug treatments, 25mg/kg of indomethacin (INDO) was administered to all groups except the healthy control group. Six hours after indomethacin administration, all the rats were euthanized. We analyzed the iNOS, eNOS, and 5-HT7 receptor mRNA levels in the stomach tissue of rats by real-time PCR. 5-HT7 mRNA expression was increased in the INDO group compared to the healthy group. LP44 administration exerted a significant upregulatory effect on eNOS mRNA expression and downregulatory effects on iNOS and 5-HT7 mRNA expression compared to the INDO group. However, antagonist (SB269970) administration did not result in such difference in gene expression, but even partially decreased the agonist's effect in combination. Famotidine and agonist exerted similar effects. Histopathological findings supported the beneficial effects of 5-HT7 agonist on gastric tissue. The study suggested that activation of 5-HT7 receptor showed a significant anti-ulcerogenic effect in the indomethacin-induced gastric ulcer model.

Inhibition of Skin Inflammation by Scytonemin, an Ultraviolet Sunscreen Pigment.

Scytonemin is a yellow-green ultraviolet sunscreen pigment present in different genera of aquatic and terrestrial blue-green algae, including marine cyanobacteria. In the present study, the anti-inflammatory activities of scytonemin were evaluated in vitro and in vivo. Topical application of scytonemin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear swelling in BALB/c mice. The expression of tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) was also suppressed by scytonemin treatment in the TPA-treated ear of BALB/c mice. In addition, scytonemin inhibited lipopolysaccharide (LPS)-induced production of TNF-α and nitric oxide (NO) in RAW 264.7 cells, a murine macrophage-like cell line, and the mRNA expressions of TNF-α and iNOS were also suppressed by scytonemin in LPS-stimulated RAW 264.7 cells. Further study demonstrated that LPS-induced NF-κB activity was significantly suppressed by scytonemin treatment in RAW 264.7 cells. Our results also showed that the degradation of IκBα and nuclear translocation of the p65 subunit were blocked by scytonemin in LPS-stimulated RAW 264.7 cells. Collectively, these results suggest that scytonemin inhibits skin inflammation by blocking the expression of inflammatory mediators, and the anti-inflammatory effect of scytonemin is mediated, at least in part, by down-regulation of NF-κB activity. Our results also suggest that scytonemin might be used as a multi-function skin care ingredient for UV protection and anti-inflammation.

Polysaccharide from Hizikia Fusiformis Enhances the Immunomodulatory Activity of Macrophages

Background and Objectives: &lt;i&gt;Hizikia fusiformis&lt;/i&gt; is widely used in oriental health food in Japan, China, and Korea, and is known for its anti-oxidation properties.Materials and Method: In this study, we investigated the anti-inflammatory and immune-modulatory effects and mechanisms of &lt;i&gt;Hizikia fusiformis&lt;/i&gt; (&lt;i&gt;H. fusiformis&lt;/i&gt;) extracts in lipopolysaccharide (LPS)-treated RAW 264.7 cells. RAW 264.7 cells were incubated in the presence of different concentrations of the viscozyme component of &lt;i&gt;H. fusiformis&lt;/i&gt; (1, 2, 5, and 10 μg/mL), and changes in expression of pro-inflammatory cytokines (GM-CSF, iNOS, VEGF, and COX-2) were evaluated by real-time PCR and immunoblotting. In addition, the associated signaling pathway including phospho (p)-pNF-κB 65, p-pIkBa, p-p38, and p-p44/42 was also evaluated.Results: The viscozyme component of &lt;i&gt;H. fusiformis&lt;/i&gt; downregulated the expression of GM-CSF, iNOS, VEGF, and COX-2 mRNA. The augmented NO and ROS production was decreased by administration of &lt;i&gt;H. fusiformis&lt;/i&gt;. The signal intensity of p-pNF-κB 65, p-pIkBa, p-p38, and p-p44/42 protein activated by LPS was ameliorated by administration of the viscozyme fraction in RAW 264.7 cells.Conclusion: These results suggest that &lt;i&gt;H. fusiformis&lt;/i&gt; has potential as a therapeutic agent for inflammatory diseases.

Changes in gene expression patterns in postmortem human myocardial infarction

In murine models, the expression of inducible nitric oxide synthase (iNOS) in myocardial infarction (MI) has been reported to be the result of tissue injury and inflammation. In the present study, mRNA expression of iNOS, hypoxia-inducible factor-1α (HIF-1α), and vascular endothelial growth factor (VEGF) was investigated in postmortem human infarction hearts. Since HIF-1α is the inducible subunit of the transcription factor HIF-1, which regulates transcription of iNOS and VEGF, the interrelation between the three genes was observed, to examine the molecular processes during the emergence of MI. iNOS and VEGF mRNAs were found to be significantly upregulated in the affected regions of MI hearts in comparison to healthy controls. Upregulation of HIF-1α was also present but not significant. Correlation analysis of the three genes indicated a stronger and significant correlation between HIF-1α and iNOS mRNAs than between HIF-1α and VEGF. The results of the study revealed differences in the expression patterns of HIF-1 downstream targets. The stronger transcription of iNOS by HIF-1 in the affected regions of MI hearts may represent a pathological process, since no correlation of iNOS and HIF-1α mRNA was found in non-affected areas of MI hearts. Oxidative stress is considered to cause molecular changes in MI, leading to increased iNOS expression. Therefore, it may also represent a forensic marker for detection of early changes in heart tissue.

The interaction of lead exposure and CCM3 defect plays an important role in regulating angiogenesis through eNOS/NO pathway

In this study, we aimed to explore the role of nitric oxide (NO) in regulating angiogenesis in cerebral cavernous malformations 3 gene (CCM3)-deficient mice exposed to lead during vascular development; further, we aimed to identify and study the potential mechanism involved as well. Angiogenesis was detected by whole mount immunofluorescent staining of retinal vessels in WT and CCM3+/− mice. Brain microvascular endothelial cells (BMECs) isolated from WT and CCM3+/− mice, primary HUVECs, and immortalized HUVECs (imHUVECs) (CCM3+/+ and CCM3-/-) were used and treated with lead acetate (PbAc). RT-PCR and Western blotting were used to detect the mRNA and protein expression of iNOS, eNOS, and VEGF genes. The results showed that both lead exposure and CCM3 gene deficiency adversely affected endothelial cell function, causing abnormal angiogenesis and vascular remodeling. The mRNA expression of eNOS and iNOS was significantly different in WT and CCM3+/− BMECs (0.04 ± 0.001 vs. 0.016 ± 0.002; 0.26 ± 0.002 vs. 0.306 ± 0.002, respectively), and the expression of eNOS and iNOS in imHUVECs (CCM3+/+ and CCM3-/-) also increased after PbAc exposure. In conclusion, CCM3 gene-deficient mice were more susceptible to abnormal vascular development after low-level lead exposure, probably due to the release of NO.

SUN-254 Angiotensin II Stimulates Microglia Cell Inflammatory Responses

Angiotensin II (AngII) is the principal effector molecule of the renin-angiotensin system (RAS). It’s effects on the cardiovascular and renal system are well-documented. AngII acts mainly via interaction with the AngII type-1 receptor (AT1R). Disordered levels of AngII lead to hypertension and cardiovascular disease. Increasing evidence suggests that AngII may also play a role in the pathophysiology of neurodegenerative diseases through unclear mechanisms. We investigated AngII, AT1R and AT2R levels in a mouse model of neurodegenerative disease, the experimentally induced autoimmune encephalomyelitis (EAE) mouse. In EAE mice, AngII and AT1R gene expression in brain tissue were significantly increased when compared to control mice (3.2 folds ±1.9, p<0.05, n=5; and 2.6 folds ±1.1, p<0.01, n=5 respectively). In addition, iNOS mRNA expression by qRT-PCR was likewise upregulated in EAE mice compared to control (3.4 ± 1.4 folds, p<0.01, n=5). We then studied the effects of AngII in human microglial cells (HMC3) -resident innate immune cells of the central nervous system (CNS). In HMC3 cells, treatment with AngII up-regulated the expression IL-6 (3.9 folds ± 1.2, p<0.01, n=4) and increased IL-6 concentration by 83% (p<0.05, n=4) by ELISA; effects that were blocked by the AT1R antagonist, Losartan. Also, AngII induced TNF-α production, increasing its concentration by 90% (p<0.05, n=4), an increase that was blocked by Losartan. We also quantified Nitric Oxide (NO) production by using Griess Reagent and reactive oxygen species (ROS) production by the MUSE Oxidative Stress assay. In these cells, NO and ROS production were significantly increased by AngII (p<0.05, n=4) and treatment with Losartan reduced their production (p<0.05, n=4). In addition, AngII treatment induced iNOS overexpression (2.5 folds ±0.8, p<0.05, n=4); results that are consistent with increases in the EAE mice. These data suggest that AngII can activate microglia cell inflammatory responses and as such may contribute to the pathophysiology of CNS inflammation and neurodegenerative diseases.

SUN-551 Impaired Vascular Relaxation and Altered eNOS Regulation in Boys with Hypospadias

Background: Sex hormones influence vascular function. Whether boys with hypospadias who have insufficient androgen exposure during the masculinisation programming window have altered vascular function is unknown. Objective: To investigate whether vascular function is impaired in boys with hypospadias and to explore the putative role of eNOS. Methods: Peripheral arteries from excess foreskin tissue obtained from boys undergoing hypospadias repair (cases) or circumcision (controls) were used. Vascular function was assessed by myography. mRNA expression was measured by qPCR in vascular smooth muscle cells (VSMCs). Nitric oxide (NO) was measured by DAF fluorescence assay and peroxynitrite levels measured via ELISA. Results: 23 boys with hypospadias and 34 age-matched controls were studied. There were 18 (52%) cases of distal, 7 (22%) of midshaft and 9 (26%) of proximal hypospadias and none of them had biochemical evidence of hypogonadism or a variant in AR. Clinical cardiometabolic parameters were similar between groups. Endothelium-dependent relaxation to acetylcholine (ACh) and endothelium-independent relaxation to sodium nitroprusside (SNP) were reduced in arteries from cases vs controls (Emax %U46619: 72.4 vs 1.2, p<0.0001 and Emax %U46619: (42.7 vs 11.8, p<0.01 respectively). Incubation with the NO synthase inhibitor, L-NAME (1x10-5 M) worsened endothelial-dependent relaxation in controls (Emax % U46619: 76.8 vs 1.2, p<0.0001) but had no effect in cases (Emax % U46619:60.6 vs 72.4, p=0.3). Testosterone (1x10-7 M) ameliorated vascular relaxation (p<0.05), whereas17[[Unsupported Character - Symbol Font 𝝱]];-estradiol stimulation (1x10-9 M) did not. In cultured VSMCs, mRNA expression of eNOS and iNOS was reduced whereas that of nNOS was increased in cases versus controls. Nitric oxide production was reduced in cases (5 fold, p<0.01), as was peroxynitrite production (0.5 fold, p<0.05). Testosterone increased expression of eNOS in VSMCs. There was no difference in mRNA expression of the AR and GPRC6A but cases had increased expression of ESR1 (2.71 fold), ESR2 (2.63 fold) and GPR30 (2.86 fold) (p<0.05). Conclusion: Arteries in eugonadal boys with hypospadias show vascular dysfunction which involves impaired NOS/NO regulation effects that are ameliorated with testosterone but not oestrogen. These processes may predispose to long-term cardiovascular disease.

Astronium fraxinifolium Schott Exerts Leishmanicidal Activity by Providing a Classically Polarized Profile in Infected Macrophages.

Leishmania braziliensis is prevalent in Latin American countries, including Brazil. It causes cutaneous and mucocutaneous leishmaniasis, leading to high morbidity, and has a low cure rate. Treatment is based on pentavalent antimonials; nonetheless, there are problems related to high toxicity, high cost, and parasitic resistance. Discovery of new leishmanicidal drugs without these limitations and that stimulate the cellular immune response is necessary. The present work evaluates whether Astronium fraxinifolium Schott exerts leishmanicidal activity against L. braziliensis by providing a classically polarized profile in infected macrophages. For the evaluation of the A. fraxinifolium Schott leishmanicidal activity, amastigote cell death was demonstrated in infected RAW 267.4 macrophages treated with an ethanolic extract from the plant sapwood (EEAF). For the evaluation of the EEAF capacity in providing a classically polarized profile in infected macrophages, the following analyses were done: detection of LAMP-1 protein by the baculovirus technology, measurement of superoxide anion by the NBT testing, quantification of TNF-α, IL-12p40, IL-10, IL-4, and TGF-β by sandwich-type enzyme immune assays, and iNOS and COX-2 expression by RT-PCR technique. The EEAF significantly reduced amastigote counts inside the cells. Vacuoles were visualized in infected and treated cells before and after May-Grünwald-Giemsa staining. A strong LAMP-1 protein fluorescence revealed phagosome maturation in infected cells treated with the EEAF. No production of superoxide was visualized in infected cells treated with the plant material. Nonetheless, high levels of TNF-α, IL-12p40, and IL-10 were found in cell supernatants, but reduced levels of TGF-β and no IL-4 production. We identified augmented mRNA expression for COX-2, but no expression of iNOS mRNA. Our results demonstrated that A. fraxinifolium induced a classically polarized profile in infected macrophages but also provided a less harmful environment by stimulating the production of certain anti-inflammatory mediators, such as IL-10.