mRNA Expression Levels Of iNOS Research Articles

Effects of octreotide on nitric oxide synthase expression in the small intestine of high fat diet-induced obese rats

To investigate whether obesity induced by high fat diet is associated with expression of neuronal, endothelial, and inducible nitric oxide synthase (nNOS, eNOS, and iNOS) in the intestine, and to test the effects of the somatostatin analog octreotide on this expression. The study included high fat diet-induced obese and normal control rats. The obese rats were further separated into an obese control group and an octreotide intervention group. Rats in the intervention group were injected with 40 μg/kg octreotide every 12 h for 8 days. Expressions of nNOS, eNOS, and iNOS in the small intestine were analyzed by RT-PCR and immunohistochemistry. The NO level of small intestinal homogenate was measured with an ELISA kit. The body weight; Lee's index; small intestinal eNOS and iNOS mRNA and protein expression levels; nNOS protein expression levels; and small intestinal homogenate NO levels were all significantly higher in the obese control group than in the normal controls (p < 0.01); nNOS mRNA expression was also higher in the obese control group, but not significantly so. Octreotide intervention significantly reduced the body weight and small intestinal homogenate NO level of the obese rats relative to the obese control group (p < 0.05). The mRNA and protein expression levels of eNOS and iNOS; the protein expression level of nNOS in the small intestine were also significantly lower in the octreotide intervention group than in the obese control group (p < 0.01), while nNOS mRNA expression was lower but not significantly so. High fat diet-induced obesity is associated with elevated small intestinal nNOS, eNOS, and iNOS expression levels. Octreotide treatment can inhibit nNOS, eNOS, and iNOS expression and lead to weight loss.

P3-09-05: Understanding the Role of Estrogen in Sex Differences in Adipocyte Biology for Cancer Prevention.

Abstract Background: Evidence shows that obesity increases the risk and mortality of many cancers, specifically breast cancer in post-menopausal women. Epidemiological studies demonstrate that males are at an increased risk for developing obesity, diabetes, and cancer compared to females, and after menopause, females mimic the males in their susceptibility to the above diseases. Furthermore, it has been established that obesity increases systemic and adipose tissue inflammation and oxidative stress, which is known to augment cancer progression. However, the role of estrogen in regulating these metabolic processes in adipose tissue is unclear; therefore, our objective is to determine the role of estrogen in adipose tissue morphology, inflammation and oxidative stress. Methods: To determine the role of estrogen in gender differences in the susceptibility to obesity we used C57BL/6J mice (15/group): 1) males 2) nonovariectomized females 3) ovariectomized females and 4) ovariectomized females supplemented with estrogen, which were randomized to the following diets: 30% calorie-restricted, low-fat or high-fat diet. We measured weight gain, percent body fat, abdominal adipose tissue, and adipocyte size. Additionally, we assessed markers of adipose tissue inflammation, DNA damage, and oxidative stress. Results: Male mice were more susceptible to obesity than female mice. Removal of the ovaries eliminated the protection to obesity and estrogen supplementation restored this protection in females. In the low-fat and high-fat diet groups, male and ovariectomized female mice gained more abdominal adipose tissue due to increased adipocyte size compared to nonovariectomized female mice and ovariectomized female mice supplemented with estrogen. In the mice consuming the high fat diet, the enlarged adipocytes observed in the male and ovariectomized female mice were accompanied with crownlike structures surrounding necrotic adipocytes and F480 positive macrophages, suggesting macrophage infiltration. To determine if there were sex differences in oxidative stress, we stained adipose tissue with γH2AX. Results suggest that nonovariectomized female mice and ovariectomized female mice supplemented with estrogen have less oxidative stress compared to males and ovariectomized females. Additionally, our results show that nonovariectomized female and ovariectomized female mice supplemented with estrogen had significantly lower CD68, TNFα and iNOS mRNA expression levels, but higher catalase mRNA expression levels. Conclusion: Male mice are more susceptible to the obesogenic effects of high fat diets compared to nonovariectomized female mice. In ovariectomized females, estrogen supplementation has a protective effect against obesity, adipose tissue inflammation and oxidative stress. Our future studies will determine the mechanisms by which estrogen protects female mice from adipose tissue inflammation and oxidative stress, whether it is a direct or indirect effect. Determining the role of estrogen on the above key metabolic processes related to obesity is necessary to develop effective strategies for cancer prevention specifically in post-menopausal females. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-09-05.

Toll-like receptors 1–9 are elevated in livers with fructose-induced hepatic steatosis

Studies in animals and human subjects indicate that gut-derived bacterial endotoxins may play a critical role in the development of non-alcoholic fatty liver disease (NAFLD). In the present study, we investigated if the liver is also sensitised by other microbial components during the onset of fructose-induced steatosis in a mouse model. C57BL/6 mice were either fed with 30 % fructose solution or tap water (control) with or without antibiotics for 8 weeks. Expression of toll-like receptors (TLR)1-9, TNF-α, inducible NO synthase (iNOS), myeloid differentiation factor 88 (MyD88) and number of F4/80 positive cells in the liver were assessed. Occludin protein, DNA of microbiota in the small and large intestine and retinol binding protein 4 (RBP4) in plasma were analysed using Western blot, DNA fingerprinting and ELISA, respectively. F4/80 positive cells were determined by immunohistochemistry. The accumulation of TAG found in the livers of fructose-fed mice was associated with a significant induction of TLR 1-4 and 6-8. Plasma RBP4 concentration and hepatic mRNA expression levels of TNF-α, iNOS, MyD88 and number of F4/80 positive cells of fructose-fed animals were significantly higher than those of controls; however, these effects of fructose were attenuated in antibiotic-treated mice. Whereas protein concentration of occludin was lower in the duodenum of fructose-treated mice, no systematic alterations of microbiota were found in this part of the intestine. Taken together, these data support the hypothesis that (1) an increased intestinal translocation of microbial components and (2) an increased number of F4/80 positive cells and induction of several TLR and dependent pathways (e.g. MyD88 and iNOS) may be involved in the onset of fructose-induced NAFLD.

Anti-inflammatory potential of an ethyl acetate fraction isolated from Justicia gendarussa roots through inhibition of iNOS and COX-2 expression via NF-κB pathway

Justicia gendarussa Burm.f. ( J. gendarussa) is a plant used as traditional medicine in different parts of India and China to treat inflammatory disorders like rheumatoid arthritis. But its mechanism of anti-inflammatory action is still unclear. Hence in this context, the objective of our study is to reveal the mechanism of anti-inflammatory activity of J. gendarussa which would form an additional proof to the traditional knowledge of this plant. The anti-inflammatory function and mechanism(s) of action was studied in an ethyl acetate fraction isolated from methanolic extract of J. gendarussa roots (EJG). Anti-inflammatory studies were conducted on rats using partitioned fractions isolated from methanolic extract of J. gendarussa roots. In carrageenan-induced rat paw edema, ethyl acetate fraction brought about 80% and 93% edema inhibition at 3rd and 5th hour at a dose of 50 mg/kg, when compared to other extracts and Voveran. We investigated whether EJG inhibits the release of cycloxygenase (COX), 5-lipoxygenase (5-LOX), interleukin-6 (IL-6) and nuclear factor kappa B (NF-κB) in LPS stimulated human peripheral blood mononuclear cells (hPBMCs). Results shows that EJG dose dependently inhibited LPS-activated COX, 5-LOX, IL-6, and NF-κB in hPBMCs. EJG also reduced LPS induced levels of iNOS and COX-2 mRNA expression in hPBMCs. This study provides an insight into the probable mechanism(s) underlying the anti-inflammatory activity of EJG and therefore, we report the first confirmation of the anti-inflammatory potential of this traditionally employed herbal medicine in vitro.

Effect of calcitonin gene‐related peptide on nitric oxide production in osteoblasts: an experimental study

The aim of this study was to investigate the in vitro effects and regulatory mechanism of CGRP (calcitonin gene-related peptide) on NO (nitric oxide) production in osteoblasts. MOB (primary human mandibular osteoblasts) and osteoblast-like cells (MG-63) were either cultured with CGRP or co-incubated with inhibitors targeting eNOS (endothelial nitric oxide synthase), iNOS (inducible nitric oxide synthase), nNOS (neuronal nitric oxide synthase) and [Ca2+]i (intracellular Ca2+). The NO concentration in cell culture supernatants was measured during the first 24 h using the Griess test; cellular NO was marked with the fluorescent marker DAF-FM, DA (3-amino, 4-aminomethyl-2',7'-difluorescein; diacetate) and measured by fluorescence microscopy from 1 to 4 h after treatment. eNOS and iNOS mRNA expression levels were measured by quantitative RT-PCR during the first 24 h after treatment. CGRP-induced NO production in the supernatants was high between 1 to 12 h, while cellular NO was highest between 1 to 2 h after treatment and returned to basal levels by 3 h. Both in MG-63 cells and MOBs, the most effective CGRP concentration was 10 nM with a peak time of 1 h. CGRP-induced NO production decreased when eNOS activity was inhibited or when voltage-dependent L-type Ca2+ channels were blocked at 4 h. CGRP was not able to induce changes in iNOS or eNOS mRNA levels and had no effect on the cytokine-induced increase of iNOS expression. Our results suggest that CGRP transiently induces NO production in osteoblasts by elevating intracellular Ca2+ to stimulate the activity of eNOS in vitro.

Effects of immunosuppressive therapy after experimental small bowel transplantation in rats

Significant improvements of graft and patient survival have been achieved over the past 20years in the field of intestinal transplantation. Tacrolimus monotherapy with corticosteroids, or in combination with sirolimus is the most commonly used immunosuppressive regimen. Early (24h) after experimental allogenic small bowel transplantation in rats, sirolimus reduces the cellular and molecular inflammatory response with subsequent graft dysmotility more efficiently than tacrolimus, with contrary effects at 7days after transplantation. This study evaluates three immunosuppressive strategies in the post-acute phase after intestinal transplantation – tacrolimus or sirolimus monotherapy and the combination therapy of tacrolimus with infliximab. After orthotopic intestinal transplantation between Brown Norway and Lewis rats, animals received 14days of immunosuppressive treatment. Histology, infiltration of neutrophils, monocytes and macrophages, cytokine and mediator mRNA expression (real time RT-PCR) and smooth muscle function in a standard organ bath were assessed at 14days after transplantation in all treatment groups and isogenic controls. Allogenic transplanted rats without immunosuppressive therapy and non-transplanted animals served as further control. Tacrolimus prevented acute rejection and graft dysmotility more effectively (p≥0.05) than sirolimus. Reduced mRNA expression levels of CD4, IFN-γ, IL-6, IL-10, iNOS, NFκB, TNF-α and MCP-1 (p≤0.05) were observed in tacrolimus treated animals compared to sirolimus. Additional infliximab application did not influence the cellular and molecular inflammatory response in the post-acute phase after transplantation.In conclusion, the severe cellular and molecular inflammatory response in allogenic transplanted grafts without immunosuppressive therapy is ameliorated by all three immunosuppressive regimens, but tacrolimus was found to be more efficient than sirolimus at 14days after transplantation. Our findings do not rule out the usage of sirolimus as single immunosuppressive therapy, but indicate and confirm the potency and effectivity of tacrolimus as basis immunosuppressant in the field of intestinal transplantation.

Gastrodin Inhibits Expression of Inducible NO Synthase, Cyclooxygenase-2 and Proinflammatory Cytokines in Cultured LPS-Stimulated Microglia via MAPK Pathways

BackgroundMicroglial activation plays an important role in neurodegenerative diseases by producing several proinflammatory enzymes and proinflammatory cytokines. The phenolic glucoside gastrodin, a main constituent of a Chinese herbal medicine, has been known to display anti-inflammatory properties. The current study investigates the potential mechanisms whereby gastrodin affects the expression of potentially pro-inflammatory proteins by cultured murine microglial BV-2 cells stimulated with lipopolysaccharide (LPS).Methodology/Principal FindingsBV-2 cells were pretreated with gastrodin (30, 40, and 60 µM) for 1 h and then stimulated with LPS (1 µg/ml) for another 4 h. The effects on proinflammatory enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and proinflammatory cytokines, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), are analysed by double-immunofluorescence labeling and RT-PCR assay. To reveal the mechanisms of action of gastrodin we investigated the involvement of mitogen-activated protein kinases (MAPKs) cascades and their downstream transcription factors, nuclear factor-κB (NF-κB) and cyclic AMP-responsive element (CRE)-binding protein (CREB). Gastrodin significantly reduced the LPS-induced protein and mRNA expression levels of iNOS, COX-2, TNF-α, IL-1β and NF-κB. LPS (1 µg/ml, 30 min)-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) and this was inhibited by pretreatment of BV-2 cells with different concentrations of gastrodin (30, 40, and 60 µM). In addition, gastrodin blocked LPS-induced phosphorylation of inhibitor κB-α (IκB-α) (and hence the activation of NF-κB) and of CREB, respectively.Conclusion and ImplicationsThis study indicates that gastrodin significantly attenuate levels of neurotoxic proinflammatory mediators and proinflammatory cytokines by inhibition of the NF-κB signaling pathway and phosphorylation of MAPKs in LPS-stimulated microglial cells. Arising from the above, we suggest that gastrodin has a potential as an anti-inflammatory drug candidate in neurodegenerative diseases.

Effects of Armepavine Against Hepatic Fibrosis Induced by Thioacetamide in Rats

The aim of this study was to investigate if armepavine (Arm, C₁₉H₂₃O₃N) could exert inhibitory effects against hepatic fibrosis in rats. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with tumour necrosis factor-α (TNF-α) to evaluate the inhibitory effects of Arm. Rats were injected with thioacetamide (TAA; 300 mg/kg, intraperitoneally) thrice a week for 4 weeks to induce hepatic fibrosis, with Arm (3 or 10 mg/kg) given by gavage twice a day. Liver sections were taken for western blotting, fibrosis scoring and immunofluorescence staining. Arm (1-10 µm) concentration-dependently attenuated TNF-α-stimulated: (i) protein expressions of α-smooth muscle actin (α-SMA), collagen type I and angiopoietin-1; (ii) H₂O₂ production; and (iii) NF-κB, JunD and C/EBPß (cytidine-cytidine-adenosine-adenosine-thymidine (CCAAT)/enhancer binding protein-ß (EBPß)) nuclear translocations in HSC-T6 cells. In vivo Arm treatment significantly reduced plasma aspartate transaminase and alanine transaminase levels, hepatic α-SMA expression and collagen contents, and fibrosis scores of TAA-injected rats. Moreover, Arm treatment decreased α-SMA- and NF-κB-positive cells in immunohistochemical staining, and mRNA expression levels of IL-6, TGF-ß1, TIMP-1, col1α2, iNOS and ICAM-1 genes, but up-regulated the metallothionein gene in the livers of TAA-injected rats. Our results indicated that Arm exerted both in vitro and in vivo antifibrotic effects in rats, with inhibition of NF-κB, JunD and C/EBPß pathways.

Phellinus baumii ethyl acetate extract inhibits lipopolysaccharide-induced iNOS, COX-2, and proinflammatory cytokine expression in RAW264.7 cells

Mushrooms are valuable sources of biologically active compounds possessing anticancer, antiplatelet, and anti-inflammatory properties. Phellinus baumii is a mushroom used in folk medicine for a variety of human diseases. However, its potential anti-inflammatory effect has remained unclear. Therefore, we studied the effect of P. baumii ethyl acetate extract (PBEAE) on inflammatory mediator and proinflammatory cytokine protein and/or mRNA expression levels using the nitric oxide (NO) assay, enzyme immunoassay (EIA), western blot, and reverse transcription polymerase chain reaction (RT-PCR) in lipopolysaccharide (LPS)-stimulated macrophage like RAW264.7 cells. PBEAE markedly inhibited NO generation and prostaglandin E(2) (PGE(2)) synthesis in a concentration-dependent pattern without any cytotoxic effect at the concentration range used. PBEAE also suppressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression. In addition, LPS-induced iNOS and COX-2 mRNA expression levels were dose-dependently inhibited by PBEAE pretreatment. Furthermore, PBEAE attenuated the mRNA expression levels of proinflammatory cytokines, specifically interleukin (IL)-1β, IL-6, and granulocyte macrophage colony-stimulating factor (GM-CSF), in a concentration-dependent fashion. Our study suggests that P. baumii might exhibit anti-inflammatory properties by downregulating proinflammatory mediators. Thus, further study on compounds isolated from PBEAE is warranted to investigate the associated molecular mechanisms and identify the potential therapeutic targets.

Adiponectin reduces proteinuria in streptozotocin-induced diabetic Wistar rats

The aim of the paper is to investigate the effects of adiponectin in diabetic nephropathy; we used an adenovirus to over-express adiponectin (Ad-Adipo) in streptozotocin (STZ)-induced diabetic rats. Animals were injected with either Ad-Adipo or control Ad-lacZ at 10 weeks after STZ treatment, and at two weeks postadenovirus injection, renal function was assessed. The degree of proteinuria was significantly reduced in Ad-Adipo rats compared with Ad-lacZ rats. Consistent with this, the mRNA expression levels of nephrin and transforming growth factor β (TGF-β) were significantly increased and decreased in the renal cortex of Ad-Adipo rats, respectively. Moreover, adiponectin over-expression in STZ rats decreased markers of endothelial dysfunction, a feature of diabetic nephropathy disease progression. Endothelin 1 (ET-1), plasminogen activator inhibitor 1 (PAI-1) and inducible nitric oxide synthase (iNOS) mRNA expression levels were significantly reduced in the renal cortex of Ad-Adipo rats, respectively. Concurrently, mRNA expression levels of endothelial nitric oxide synthase (eNOS), a positive regulator of endothelial function, were significantly increased in the renal cortex of Ad-Adipo rats. We have shown that chronic hyperadiponectinemia significantly alleviated the progression of proteinuria in early stage diabetic nephropathy. The mechanism whereby adiponectin decreases proteinuria involves an increase in nephrin expression, and an improvement of the endothelial dysfunction due to decreases in ET-1 and PAI-1, and an increase in eNOS expression in the renal cortex. Thus, over-expression of adiponectin has beneficial effects on early stage diabetic nephropathy.

Effects of Chinese herbal medicine Jinmaitong-medicated serum on inducible nitric oxide synthase and NADPH oxidase p22-phox subunit of rat Schwann cells cultured in high-glucose medium

To investigate the effects of medicated serum prepared by administration of Jinmaitong (JMT), a compound Chinese herbal medicine, on nicotinamide adenine dinucleotide phosphate (NADPH) oxidase p22-phox subunit and inducible nitric oxide synthase (iNOS) of rat Schwann cells cultured in high-glucose medium. Wistar rats were divided into normal control group (distilled water), JMT (JMT at dose of 1.31 g/(kg·d)) group and vitamin C (vitamin C at dose of 0.08 g/(kg·d)) group to prepare medicated serum. Bilateral sciatic nerves of new born Wistar rats were used to separate Schwann cells. Schwann cells cultured in high-glucose medium were divided into high glucose group (cultured with 50 mmol/L glucose medium), JMT group (cultured with JMT-medicated serum) and vitamin C (VC) group (cultured with VC-medicated serum). Schwann cells cultured in DMEM were used as the normal control. After 48 h culturing, the expression of iNOS was detected by immunofluorescence method and p22-phox mRNA was tested by real-time fluorescent quantitative polymerase chain reaction. The expression levels of iNOS and p22-phox mRNA in the high glucose group were higher than those in the normal control group (P<0.01). Compared with the high glucose group, expressions of iNOS protein and p22-phox mRNA in JMT group were significantly decreased (P<0.01) and JMT-medicated serum had better effect than VC (P<0.01). JMT-medicated serum can down-regulate the expressions of iNOS protein and NADPH oxidase p22-phox subunit mRNA of Schwann cells cultured in high-glucose medium.

Protective effect of Ulinastatin on intestinal motility in rats with bacterial peritonitis

Objective To explore the Protective effect and the mechanism of Ulinastatin (UTI) on intestinal motility in rats with bacterial peritonitis. Methods Sixty Wistar rats were divided randomly into control group, model group and UTI group (n=20 each), and the latter two groups were used to replicate bacterial peritonitis model. Rats in UTI group received UTI injection (50 000 U/kg) via tail vein, while rats in other groups were given equal volume saline. Gut transits in vivo and intestinal circular muscle contractions were measured in an organ bath. The contractile response of dispersed circular smooth muscle cells and the changes of[Ca2+]1 stimulated by aceylcholine were observed. Nuclear factor-κB (NF-κB) activity was examined by using electrophoretic mobility shift assay (EMSA). Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of inducible nitric oxide synthase (iNOS) mRNA within intestinal muscularis, and the level of nitric oxide (NO) in cell cultures was quantified. Results The gut transits in UTI group were improved obviously as compared with model group. In model group and UTI group, circular smooth muscle contractility stimulated by aceylcholine was (0. 75 ±0. 06 ) g/mm2· s - 1 and ( 1.87 ± O. 29 ) g/mm2· s - 1; contractile response of dispersed circular smooth muscle cells was ( 11.42 ± 4. 23 ) % and ( 37.22 ± 10. 08 ) %; changes of[Ca2+]1 was ( 119 ± 21 ) % and (205 ±49)%. In model group and UTI group, the activity of NF-κB was 303.25 ±47.18 and 170. 22 ±29.62; levels of iNOS mRNA expression were 1.38 ±0. 41 and 0. 58 ±0. 17; NO production was (98. 34± 19. 62) μmol/L and (32. 72 ±7.98) μmol/L respectively. Conclusion UTI has the ability to protect the intestinal motility in rats with bacterial peritonitis through inhibiting the activity of NF-κB and down-regulating the expression of iNOS mRNA within intestinal muscularis. Key words: Ulinastatin; Bacterial peritonitis; Intestinal; NF-κB; Nitric oxide

Stercurensin inhibits nuclear factor‐κB‐dependent inflammatory signals through attenuation of TAK1–TAB1 complex formation

We identified a chalcone, 2',4'-dihydroxy-6'-methoxy-3'-methylchalcone (stercurensin), as an active compound isolated from the leaves of Syzygium samarangense. In the present study, the anti-inflammatory effects and underlying mechanisms of stercurensin were examined using lipopolysaccharide (LPS)-stimulated RAW264.7 cells and mice. To determine the effects of stercurensin in vitro, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression were analyzed by RT-PCR and immunoblotting. Nuclear factor-κB (NF-κB) activation and its upstream signaling cascades were also investigated using a dual-luciferase reporter assay, electrophoretic mobility shift assay, immunoblotting, immunofluorescence, and immunoprecipitation. To verify the effects of stercurensin in vivo, the mRNA expression levels of iNOS and COX-2 were evaluated in isolated mouse peritoneal macrophages by quantitative real-time PCR, and the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β were assessed in serum samples from mice using a Luminex system. Pretreatment with stercurensin reduced LPS-induced iNOS and COX-2 expression, thereby inhibiting nitric oxide (NO) and prostaglandin E(2) production, respectively. In addition, an inhibitory effect of stercurensin on NF-κB activation was shown by the recovery of LPS-induced inhibitor of κB (I-κB) degradation after blocking the transforming growth factor-β-activated kinase 1 (TAK1)/I-κB kinase signaling pathway. In mouse models, stercurensin negatively regulated NF-κB-dependent pro-inflammatory mediators and cytokines. These results demonstrate that stercurensin modulates NF-κB-dependent inflammatory pathways through the attenuation of TAK1-TAB1 complex formation. Our findings demonstrating the anti-inflammatory effects of stercurensin in vitro and in vivo will aid in understanding the pharmacology and mode of action of stercurensin.

Inhibition of nitric oxide in LPS-stimulated macrophages of young and senescent mice by δ-tocotrienol and quercetin

BackgroundChanges in immune function believed to contribute to a variety of age-related diseases have been associated with increased production of nitric oxide (NO). We have recently reported that proteasome inhibitors (dexamethasone, mevinolin, quercetin, δ-tocotrienol, and riboflavin) can inhibit lipopolysaccharide (LPS)-induced NO production in vitro by RAW 264.7 cells and by thioglycolate-elicited peritoneal macrophages derived from four strains of mice (C57BL/6, BALB/c, LMP7/MECL-1-/- and PPAR-α-/- knockout mice). The present study was carried out in order to further explore the potential effects of diet supplementation with naturally-occurring inhibitors (δ-tocotrienol and quercetin) on LPS-stimulated production of NO, TNF-α, and other pro-inflammatory cytokines involved in the ageing process. Young (4-week-old) and senescent mice (42-week old) were fed control diet with or without quercetin (100 ppm), δ-tocotrienol (100 ppm), or dexamethasone (10 ppm; included as positive control for suppression of inflammation) for 4 weeks. At the end of feeding period, thioglycolate-elicited peritoneal macrophages were collected, stimulated with LPS, LPS plus interferon-β (IFN-β), or LPS plus interferon-γ (IFN-γ), and inflammatory responses assessed as measured by production of NO and TNF-α, mRNA reduction for TNF-α, and iNOS genes, and microarray analysis.ResultsThioglycolate-elicited peritoneal macrophages prepared after four weeks of feeding, and then challenged with LPS (10 ng or 100 ng) resulted in increases of 55% and 73%, respectively in the production of NO of 46-week-old compared to 8-week-old mice fed control diet alone (respective control groups), without affecting the secretion of TNF-α among these two groups. However, macrophages obtained after feeding with quercetin, δ-tocotrienol, and dexamethasone significantly inhibited (30% to 60%; P < 0.02) the LPS-stimulated NO production, compared to respective control groups. There was a 2-fold increase in the production of NO, when LPS-stimulated macrophages of quercetin, δ-tocotrienol, or dexamethasone were also treated with IFN-β or IFN-γ compared to respective control groups. We also demonstrated that NO levels and iNOS mRNA expression levels were significantly higher in LPS-stimulated macrophages from senescent (0.69 vs 0.41; P < 0.05), compared to young mice. In contrast, age did not appear to impact levels of TNF-α protein or mRNA expression levels (0.38 vs 0.35) in LPS-stimulated macrophages. The histological analyses of livers of control groups showed lesions of peliosis and microvesicular steatosis, and treated groups showed Councilman body, and small or large lymphoplasmacytic clusters.ConclusionsThe present results demonstrated that quercetin and δ-tocotrienols inhibit the LPS-induced NO production in vivo. The microarray DNA analyses, followed by pathway analyses indicated that quercetin or δ-tocotrienol inhibit several LPS-induced expression of several ageing and pro-inflammatory genes (IL-1β, IL-1α, IL-6, TNF-α, IL-12, iNOS, VCAM1, ICAM1, COX2, IL-1RA, TRAF1 and CD40). The NF-κB pathway regulates the production of NO and inhibits the pro-inflammatory cytokines involved in normal and ageing process. These ex vivo results confirmed the earlier in vitro findings. The present findings of inhibition of NO production by quercetin and δ-tocotrienol may be of clinical significance treating several inflammatory diseases, including ageing process.

Overexpressing autoimmune regulator regulates the expression of toll-like receptors by interacting with their promoters in RAW264.7 cells

Autoimmune regulator (Aire) is a transcriptional activator that regulates the ectopic expression of many tissue-restricted antigens in medullary thymic epithelial cells, and that has an important role in the negative selection of autoreactive T cells. However, the roles of Aire expression in peripheral lymphoid tissues and hematopoietic cells, especially monocytes/macrophages, remain poorly understood. In this study, we found that the mRNA and protein expression levels of toll-like receptor (TLR)1, TLR3, and TLR8 were notably up-regulated in a mouse macrophage-like cell line (RAW264.7) stably expressing Aire, while the expression of TLR2, TLR4, TLR5, TLR6, TLR7, and TLR9 were not significantly changed. In addition, the mRNA expression of TLR3 and TLR8 were significantly increased in primary peritoneal macrophages transiently transfected with Aire. Using chromatin immunoprecipitation and a luciferase activity assay, we also found that Aire interacted with the TLR1, TLR3, and TLR8 promoters and increased the luciferase transcriptional activity of these promoters in RAW264.7 cells. Moreover, after stimulation by Pam3CSK4, a TLR1 ligand, and poly(I:C), a TLR3 ligand, we found that the mRNA expression levels of IL-1α, TNFα, iNOS, and IFNα were increased in RAW264.7 cells stably expressing Aire. Together, these data suggest that Aire has a crucial role in the recognition of pathogenic microorganisms and peripheral immune tolerance in antigen-presenting cells (APCs) by regulating the expression of TLRs.

Anti-inflammatory/Anti-oxidative Stress Activities and Differential Regulation of Nrf2-Mediated Genes by Non-Polar Fractions of Tea Chrysanthemum zawadskii and Licorice Glycyrrhiza uralensis

Accumulating evidence from epidemiological studies indicates that chronic inflammation and oxidative stress play critical roles in neoplastic development. The aim of this study was to investigate the anti-inflammatory, anti-oxidative stress activities, and differential regulation of Nrf2-mediated genes by tea Chrysanthemum zawadskii (CZ) and licorice Glycyrrhiza uralensis (LE) extracts. The anti-inflammatory and anti-oxidative stress activities of hexane/ethanol extracts of CZ and LE were investigated using in vitro and in vivo approaches, including quantitative real-time PCR (qPCR) and microarray. Additionally, the role of the transcriptional factor Nrf2 (nuclear erythroid-related factor 2) signaling pathways was examined. Our results show that CZ and LE extracts exhibited potent anti-inflammatory activities by suppressing the mRNA and protein expression levels of pro-inflammatory biomarkers IL-1β, IL-6, COX-2 and iNOS in LPS-stimulated murine RAW 264.7 macrophage cells. CZ and LE also significantly suppressed the NO production of LPS-stimulated RAW 264.7 cells. Additionally, CZ and LE suppressed the NF-κB luciferase activity in human HT-29 colon cancer cells. Both extracts also showed strong Nrf2-mediated antioxidant/Phase II detoxifying enzymes induction. CZ and LE induced NQO1, Nrf2, and UGT and antioxidant response element (ARE)-luciferase activity in human hepatoma HepG2 C8 cells. Using Nrf2 knockout [Nrf2 (-/-)] and Nrf2 wild-type (+/+) mice, LE and CZ showed Nrf2-dependent transactivation of Nrf2-mediated antioxidant and phase II detoxifying genes. In summary, CZ and LE possess strong inhibitory effects against NF-κB-mediated inflammatory as well as strong activation of the Nrf2-ARE-anti-oxidative stress signaling pathways, which would contribute to their overall health promoting pharmacological effects against diseases including cancer.

Effect of topical application and intraperitoneal injection of oregonin on atopic dermatitis in NC/Nga mice

The diarylheptanoid, oregonin (ORE), which was isolated from the bark of Alnus japonica Steudel that grows natively in Korea, has been known to exert antioxidative, anti-inflammatory, anti-cancer and immune response inhibitory effects. The antioxidative effect of ORE was observed on the superoxide and 1,1-diphenyl-2-picrylhydrazyl radical, as well as on the expression of inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-treated RAW264.7 macrophages. The statistically significant inhibitory action of ORE against production of cytokines induced by bacterial products or by interleukin (IL)-1beta, free radicals and nitrogen species, and a corresponding increase in cellular calcium concentration because of ORE were confirmed in bone marrow and spleen dendritic cells that are known to play important functions in the development and advancement of atopic dermatitis (AD). It was thus expected that ORE would exert a beneficial effect in the treatment of AD. A study on the pharmaceutical benefits of ORE against AD has not yet been conducted in vivo. We therefore used an in vivo AD animal model, namely the NC/Nga mice, and by applying ORE onto the animals through skin application as well as intraperitoneal injection, we attempted to evaluate the benefits of ORE in this system. Evaluation of ORE was conducted by following the SCORE method to score the effect, as well as by measuring the Th2 cytokines IL-4, IL-5 and IL-13 levels from serum and lymphocytes, and IgE and eosinophil levels from serum. Additionally, the expression of mRNA and protein levels was estimated using real-time polymerase chain reaction and Western blotting analysis. The following categories of clinical evaluation, Th2 cytokines IL-4, IL-5 and IL-13 values, serum IgE levels, serum eosinophil levels, and mRNA and protein expression levels of iNOS and COX-2, were evaluated from topical application and intraperitoneal injection groups of ORE. The effects of ORE on AD in NC/Nga mice were confirmed as being similar to the positive control group, while a significant difference with the negative control group was observed. The results presented in this report suggest that ORE might be beneficial in the treatment of AD.

Differential uptake of amyloid-β soluble oligomers and fibrils by human microglia

b aggregates into non-b aggregates. Both oligomeric and filbrillar Ab can induce microglial activation, however the cellular signal ways are different due to the TNFa and iNOS mRNA expression levels, and NO metabolites activity are different when the BV2 cells were co-incubated with oligomeric and filbrillar Ab40, respectively.Ab-Cu(II) complexs play less roles on the activation of microglial than both oligomeric and fibrillar Ab. Conclusions: These results indicated that oligomeric and fibrillar Ab induce differentially activation of BV-2 cells and which were dramatically attenuated by Cu(II).

Effect of cardiomyopeptidin for injection on energy metabolism in isolated hearts of young rats after ischemia-reperfusion injury

To investigate the effect of cardiomyopeptidin for injection on energy metabolism in isolated hearts of young rats after ischemia-reperfusion injury. Fifty young healthy SD rats(aged 20 +/- 3 days and weighing 50-70 g) were randomly divided into 5 groups: a normal control group (NC group, n = 10 ): the isolated hearts were stable for 20 min, and then 150 min continuous perfusion; a normal + cardiomyopeptidin group (NCMP group, n = 10): the same as the normal control group, but K-H buffer solution was added with 50 mg/L cardiomyopeptidin, and 3 ischemia-reperfusion injury model groups, including a model control group (n = 10): the isolated rat hearts were perfused with K-H buffer and then arrested with cardioplegic solution; a CMP1 group (n = 10): the ST.Thomas'II cardioplegic solution was added with 100 mg/L cardiomyopeptidin; CMP2 group (n=10): K-H buffer and ST.Thomas'II cardioplegic solution was added with 50 mg/L and 100 mg/L cardiomyopeptidin respectively. The cardiac functional indexes were monitored, including heart rate, myocardial contractility and diastolic function, peak systolic and diastole myocardial velocities and coronary flow. In the 3 ischemia-reperfusion injury model groups, myocardial ultrastructure was observed through transmission electron microscopy; the creatine kinase isoenzyme (CK-MB) concentration was measured in the fluid outflow of coronary; the content of Na+-K+ ATPase, Ca2+-Mg2+ ATPase, total ATPase, superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide(NO), total nitric oxide synthase (TNOS), inducible nitric oxide synthase (iNOS) and aldosereductase were measured in the myocardium tissue; the relative expression levels of iNOS, eNOS, and Akr1b4 mRNA in the myocardial tissue were also detected by real-time fluorescence quantitative PCR. In the NC group, after prolonged perfusion, the cardiac function of isolated hearts had no significant change. Cardiomyopeptidin for injection had no significant effect on normal isolated hearts. Compared with the model control group, the cardiac function indexes and coronary flow in the groups treated with cardiomyopeptidin decreased much less. Cardiac myofibrillar fragmentation and mitochondrial swelling were observed in the control group, while in the CMP groups, the myocardial structure was nearly complete, and only mild mitochondria swelling and degeneration could be seen. After the reperfusion, the content of CK-MB was increased in the control group. Compared with the model control group, the CK-MB content was lower in the CMP1 and CMP2 groups. There was a slight decline in the contents of Na+-K+ ATPase, Ca2+-Mg2+ ATPase, and Total ATPase in the CMP1 and CMP2 groups, and an increase in SOD activity (P < 0.01 or P < 0.05). The concentration of NO and MDA produced after the ischemia-reperfusion injury was much lower in the CMP1 and CMP2 groups. The activity of iNOS and aldosereductase was inhibited, the expression levels of iNOS, and Akr1b4 mRNA were significantly down-regulated in the CMP1 and CMP2 groups. These changes were more prominent in the CMP2 group (P < 0.01 or P < 0.05). The eNOS mRNA levels in the CMP2 group was up-regulated (P < 0.05). Cardiomyopeptidin for injection may improve the energy metabolism, improve coronary blood flow and cardiac function after the reperfusion, thus protecting immature myocardial against ischemia-reperfusion injury in young rats. Administration of it in both K-H buffer and ST.Thomas'II cardioplegic solution is better than adding it in cardioplegic solution alone. The mechanism may be associated with the inhibition the mRNA expression of iNOS and Akr1b4 in cardiomyocytes, the inhibition activity of iNOS and aldosereductase, and the decrease of NO production.

Effects of progesterone on PMN-mediated inflammatory responses to gonococcal infection

Objective To evaluate the effects of progesterone on polymorphonuclear leukocyte (PMN)-mediated inflammatory responses to gonococcal infection. Methods Peripheral neutrophils were isolated from heparinized peripheral blood obtained from normal individuals, then divided into 4 groups: progesterone group (pretreated with progesterone only), gonococcus group (stimulated with gonococcal suspension), intervention group (pretreated with progesterone followed by stimuation with gonococcal suspension), and control group (receiving no pretreatment or stimulation). Real-time RT-PCR was conducted to detect the mRNA expression levels of inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β)in neutrophils from all groups at 0, 3, 8, 12 and 24 hours after the last treatment, and iNOS protein levels were measured by Western-blot in gonococcus group and intervention group. Results Real-time RT-PCR indicated that the expression levels of iNOS, TNF-α and IL-1β mRNA increased in gonococcus group and intervention group, and reached their peak at 8 hours in gonococcus group, while no significant changes were noted in the above parameters in progesterone group or control group. Also, the level of iNOS, TNF-α and IL-1β mRNA was lower in intervention group than that in the gonococcus group (P ＜ 0.05). Western blot showed an elevation in iNOS protein expression in both gonococcus group and intervention group, and the former group was higher than the latter group in the parameter (P ＜ 0.05). Conclusions Progesterone can downregulate the expressions of iNOS, TNF-α and IL-1 β by PMNs, inhibit the PMN-induced inflammatory responses induced by gonococcal infection, which is likely to be associated with the asymptomatic gonococcal infection in women. Key words: Neisseria gonorrhoeae; Progesterone; Granulocytes; Nitric oxide synthase; Tumor necrosis factor-alpha; Interleukin-1 beta