A novel Pleurotus nebrodensis polysaccharide (PN-S) was purified and characterized, and its immune-stimulating activity was evaluated in RAW264.7 macrophages. PN-S induced the proliferation of RAW264.7 cells in a dose-dependent manner, as determined by the MTT assay. After exposure to PN-S, the phagocytosis of the macrophages was significantly improved, with remarkable changes in morphology being observed. Flow cytometric analysis demonstrated that PN-S promoted RAW264.7 cells to progress through S and G2/M phases. PN-S treatment enhanced the productions of interleukin-6 (IL-6), nitric oxide (NO), interferon gamma (INF-γ), and tumor necrosis factor-α (TNF-α) in the macrophages, with up-regulation of mRNA expressions of interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), interferon gamma(INF-γ) and tumor necrosis factor-α (TNF-α) being observed in a dose-dependent manner, as measured by qRT-PCR. In conclusion, these results suggest that the purified PN-S can improve immunity by activating macrophages.
Animals ; Cell Cycle ; immunology ; Cell Line ; Cell Proliferation ; drug effects ; Fungal Polysaccharides ; pharmacology ; Immunity ; drug effects ; Interferon-gamma ; biosynthesis ; metabolism ; Interleukin-6 ; biosynthesis ; metabolism ; Macrophages ; immunology ; metabolism ; Mice ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; metabolism ; Pleurotus ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; biosynthesis ; metabolism ; Up-Regulation

The aim of this study was to determine the effect of dexamethasone (DEX) on renal ischemia/reperfusion injury (IRI). C57BL/6 mice were randomly divided into Sham group, IRI group and DEX group. The mice in IRI and DEX groups subjected to renal ischemia for 60 min, were treated with saline or DEX (4 mg/kg, i.p.) 60 min prior to I/R. After 24 h of reperfusion, the renal function, renal pathological changes, activation of extracellular signal-regulated kinase (ERK) and glucocorticoid receptor (GR), and the levels of iNOS and eNOS were detected. The results showed DEX significantly decreased the damage to renal function and pathological changes after renal IRI. Pre-treatment with DEX reduced ERK activation and down-regulated the level of iNOS, whereas up-regulated the level of eNOS after renal IRI. DEX could further promote the activation of GR. These findings indicated GR activation confers preconditioning-like protection against acute IRI partially by up-regulating the ratio of eNOS/iNOS.
Animals ; Dexamethasone ; pharmacology ; Gene Expression Regulation, Enzymologic ; drug effects ; Glucocorticoids ; pharmacology ; Male ; Mice ; Nitric Oxide Synthase Type II ; biosynthesis ; Nitric Oxide Synthase Type III ; biosynthesis ; Receptors, Glucocorticoid ; agonists ; Reperfusion Injury ; enzymology ; pathology ; Up-Regulation ; drug effects

OBJECTIVETo observe the analgesic effect of triptolide (TP) of high, middle and low doses on rats with adjuvant arthritis (AA), and the expressions of inducible nitric oxide synthase (iNOS) and substance P (SP) in spinal dorsal horn and dorsal root ganglion (DRG) of corresponding sections, in order to discuss the possible mechanism for the analgesic effect of TP on rats with adjuvant arthritis.

METHODFifty SD rats were selected and randomly divided into the normal group (group A), the model group (group B), and TP low (group C), middle (group D), high (group E) dose groups. Except for the group A, all of the remaining groups were injected with 0.1 mL of Freund's complete adjuvant through their right rear toes to establish the model. At 14 d after the model establishment, rats in C, D and E groups were intraperitoneally injected with different doses of TP (0.1 mg x kg(-1) for the group C, 0.2 mg x kg(-1) for the group D, 0.4 mg x kg(-1) for the group E) once a day for 9 days. Then the 50% mechanical withdraw threshold (MWT) was determined. And the expressions of iNOS and SP in lumbar5 (L5) spinal dorsal horn and DRG were detected with the immunohistochemical method.

RESULTThe 50% MWT of rats in the group B was significantly lower than that of the group A (P < 0.01). After being treated with TP, the Thermal withdrawal latencies of groups C, D and E were significantly higher than that of the group B (P < 0.01). TP could notably increase the MWT of AA rats, with a certain dose-effect relationship. The immunohistochemical results indicated that the iNOS and SP expressions significantly increased in the group B (P < 0.01), while the positive expression levels of iNOS and SP in groups C, D and E were significantly lower than that of the group B (P < 0.01), with a certain dose-effect relationship.

CONCLUSIONTP shows a good analgesic effect on AA, and could inhibit the iNOS and SP expressions in spinal dorsal horn and DRG in rats with adjuvant arthritis, which may be one of action mechanisms for the analgesic effect of TP.

Animals ; Anti-Inflammatory Agents, Non-Steroidal ; pharmacology ; Arthritis, Experimental ; drug therapy ; metabolism ; physiopathology ; Diterpenes ; pharmacology ; Dose-Response Relationship, Drug ; Epoxy Compounds ; pharmacology ; Female ; Ganglia, Spinal ; drug effects ; metabolism ; Immunohistochemistry ; Male ; Nitric Oxide Synthase Type II ; biosynthesis ; Pain Measurement ; methods ; Phenanthrenes ; pharmacology ; Phytotherapy ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; drug effects ; metabolism ; Substance P ; biosynthesis ; Time Factors ; Treatment Outcome ; Tripterygium ; chemistry

OBJECTIVETo observe the therapeutic effect of N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, on experimental autoimmune myocarditis (EAM) in Balb/C mice and discuss the therapeutic mechanism induced by apoptosis.

METHODSThirty male Balb/C mice were divided into normal control group, model control group and experimental group randomly (n = 10). Model control group and experimental group were created into EAM by injection of porcine cardiac myosin subcutaneously in double groin and axilla and pertussis toxin intraperitoneally on day 0 and 7 respectively. Model control group was intraperitoneally administered 5 mg/(kg x day) of physiological saline after infective myosin and pertussis toxin. Experimental group was intraperitoneally given 5 mg/(kg x day) of L-NAME on day 1-21. The hearts and blood were processed after sacrificed on day 21. Cardiac inflammation score was measured by HE staining. Heart weight / body weight (HW/BW), serum nitric oxide (NO) level, activity of induced nitric oxide synthase (iNOS) and mRNA expression of iNOS in heart were measured in each group. Degree of heart apoptosis were evaluated by cardiac apoptotic index through TUNEL, immunohistochemical examination and real time PCR of Caspase-3, Caspase-8 and Caspase-9.

RESULTSCompared with normal control group, cardiac inflammation score, HW/BW level of NO and activity of iNOS, mRNA expression of iNOS, the levels of mRNA and protein of Caspase-3, Caspase-8 and Caspase-9 and cardiac apoptotic index were significantly higher (P < 0.01) in model control group, and those of model control group were higher than those of experimental group (P < 0.01). HW/BW was only a little elevation in model control group compared with that in the experiment group (P < 0.05).

CONCLUSIONThe development of EAM is related with the NO catalyzed by iNOS. L-NAME protects cardiac myocyte via suppressing the activity of iNOS and further decreased production of NO in EAM. The mechanism might be that L-NAME alleviated myocardial inflammation through inhibited the apoptosis of cardiac myocyte.

Animals ; Apoptosis ; Autoimmune Diseases ; drug therapy ; metabolism ; pathology ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Disease Models, Animal ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; metabolism ; pathology ; Myocytes, Cardiac ; metabolism ; NG-Nitroarginine Methyl Ester ; therapeutic use ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; metabolism

OBJECTIVETo study possible immunobiological potential of Osmunda japonica Thunb.

METHODSImmunomodulatory effects of ethanol extracts prepared from rhizomes of O. japonica and phenolic compounds isolated from the extracts were investigated under the in vitro conditions using the rat peritoneal cells (2×10(6)/mL; 24 h culture). Biosynthesis of nitric oxide (NO) was assayed by Griess reagent, production of prostaglandin E2 (PGE2) and secretion of cytokines were determined by enzyme-linked immunoabsorbent assay.

RESULTSThe extracts activated dose dependently, with the onset at 2.5-5 μmol/L concentrations, the high output NO production, and secretion of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Mild enhancement of NO was produced by the aldehyde-type phenolics 4-hydroxybenzaldehyde and 3,4-hydroxybenzaldehyde. In contrasts, the acetone-type phenolics 4-hydroxybenzalacetone and 3,4-hydroxybenzalacetone inhibited production of immune mediators including cytokines (TNF-α, IL-1β, IL-6), NO, and PGE2. The 3,4-hydroxybenzalacetone was more effective than 4-hydroxybenzaldehyde. The IC50s estimates ranged within the interval of 5-10 μmol/L. No signs of cytotoxicity were observed up to the 50 μmol/L concentration of the compounds.

CONCLUSIONPhenolic compounds contained in medicinal herb Osmunda japonica possess distinct immunomodulatory activity.

Animals ; Cell Survival ; drug effects ; Cells, Cultured ; Dinoprostone ; biosynthesis ; Female ; Ferns ; chemistry ; Immunologic Factors ; pharmacology ; Interferon-gamma ; pharmacology ; Lipopolysaccharides ; pharmacology ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Peritoneum ; cytology ; drug effects ; Phenols ; chemistry ; isolation & purification ; pharmacology ; Plant Extracts ; chemistry ; isolation & purification ; pharmacology ; Polymyxin B ; pharmacology ; Proline ; analogs & derivatives ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Thiocarbamates ; pharmacology

OBJECTIVEBuckwheat has been considered as a potential source of nutraceutical components on the world market of probiotic foodstuffs. The purpose of this study was to evaluate the effects of tartary buckwheat (Fagopyrum tataricum) sprouts on oxidation and pro-inflammatory mediators.

METHODSThe anti-oxidant effects of buckwheat extract (BWE) and rutin were evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH)- and nitric oxide (NO)-scavenging activities, serum peroxidation and chelating assays. Lipopolysaccharide (LPS)-stimulated RAW264.7 cells were used to evaluate anti-inflammatory activities of buckwheat and rutin. NO production in LPS-stimulated RAW264.7 cells was determined by using Griess reagent. The expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF-κB) p65 subunit in cytosolic and nuclear portions were determined by Western blot analysis. Also, the production of inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was determined by enzyme-linked immunosorbent assay.

RESULTSInhibitory concentration 50 values for DPPH- and NO-scavenging activities of BWE were 24.97 and 72.54 μg/mL respectively. BWE inhibited serum oxidation and possessed chelating activity. Furthermore, BWE inhibited IL-6 and TNF-α production in LPS-stimulated RAW264.7 cells. Also, BWE inhibited iNOS and COX-2 expression and NF-κB p65 translocation.

CONCLUSIONBuckwheat sprouts possessed strong antioxidant activity and inhibited production of pro-inflammatory mediators in the applied model systems. Thus, buckwheat can be suggested to be beneficial in inflammatory diseases by inhibiting the free radicals and inflammatory mediators.

Animals ; Cells, Cultured ; Cyclooxygenase 2 ; analysis ; Fagopyrum ; Free Radical Scavengers ; pharmacology ; Inflammation Mediators ; antagonists & inhibitors ; Interleukin-6 ; biosynthesis ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; metabolism ; Mice ; NF-kappa B ; metabolism ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; analysis ; Plant Extracts ; pharmacology ; Tumor Necrosis Factor-alpha ; biosynthesis

Mitochondrial dysfunction and endoplasmic reticulum (ER) stress are considered the key determinants of insulin resistance. Impaired mitochondrial function in obese animals was shown to induce the ER stress response, resulting in reduced adiponectin synthesis in adipocytes. The expression of inducible nitric oxide synthase (iNOS) is increased in adipose tissues in genetic and dietary models of obesity. In this study, we examined whether activation of iNOS is responsible for palmitate-induced mitochondrial dysfunction, ER stress, and decreased adiponectin synthesis in 3T3L1 adipocytes. As expected, palmitate increased the expression levels of iNOS and ER stress response markers, and decreased mitochondrial contents. Treatment with iNOS inhibitor increased adiponectin synthesis and reversed the palmitate-induced ER stress response. However, the iNOS inhibitor did not affect the palmitate-induced decrease in mitochondrial contents. Chemicals that inhibit mitochondrial function increased iNOS expression and the ER stress response, whereas measures that increase mitochondrial biogenesis (rosiglitazone and adenoviral overexpression of nuclear respiratory factor-1) reversed them. Inhibition of mitochondrial biogenesis prevented the rosiglitazone-induced decrease in iNOS expression and increase in adiponectin synthesis. These results suggest that palmitate-induced mitochondrial dysfunction is the primary event that leads to iNOS induction, ER stress, and decreased adiponectin synthesis in cultured adipocytes.
3T3-L1 Cells ; *Adipocytes/drug effects/metabolism ; Adiponectin/biosynthesis ; Adipose Tissue/metabolism ; Animals ; Endoplasmic Reticulum Stress/drug effects ; Insulin Resistance/genetics ; Mice ; Mitochondria/drug effects/*metabolism/pathology ; Mitochondrial Turnover/drug effects/genetics ; *Nitric Oxide Synthase Type II/genetics/metabolism ; Nuclear Respiratory Factor 1 ; Obesity/genetics/metabolism ; Palmitic Acid/pharmacology ; Thiazolidinediones/pharmacology

PURPOSE: We investigated whether oxygen-induced retinopathy (OIR) results in changes in the protein expression of neuronal and inducible nitric oxide synthases (nNOS and iNOS, respectively) in rat model of OIR. In addition, we evaluated whether treatment of rats with triamcinolone acetonide (TA) prevents this response. METHODS: To promote OIR, Sprague-Dawley rats were exposed to hyperoxia from postnatal day 2 (P2) to P14. They were then returned to normoxia after P15. TA was injected into the right vitreous of P15 rats, while saline was injected into the left vitreous. At P18 the expression of nNOS and iNOS was determined using Western blotting and immunostaining techniques in retinas obtained from control rats. RESULTS: In P18 OIR rats, the abundance of nNOS and iNOS protein was significantly increased compared with controls. These increases were not observed in the retinas of rats treated with TA. The change in expression of nNOS and iNOS were specific to parvalbumin and glial fibrillary acidic protein-positive cells. Treatment with TA prevented the increased expression of nNOS and iNOS in all samples. CONCLUSIONS: Hypoxia upregulates expression of nNOS and iNOS in OIR rat retinas, which is can be prevented by treatment with TA.
Animals ; Animals, Newborn ; Anoxia/metabolism/pathology/*prevention & control ; Blotting, Western ; Disease Models, Animal ; Female ; Glucocorticoids/pharmacology ; Immunohistochemistry ; Neurons/metabolism ; Nitric Oxide Synthase Type II/*biosynthesis ; Oxygen/toxicity ; Pregnancy ; *Pregnancy, Animal ; Rats ; Rats, Sprague-Dawley ; Retina/*metabolism/pathology ; Retinal Diseases/chemically induced/pathology/*prevention & control ; Triamcinolone Acetonide/*pharmacology

Animals ; Animals, Newborn ; Hypoxia-Ischemia, Brain ; metabolism ; Neuroprotective Agents ; pharmacology ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; metabolism ; Progesterone ; pharmacology ; Rats ; Rats, Wistar

OBJECTIVETo explore the efficacy of inductible nitric oxide synthase (iNOS) inhibitor 1400W in vivo in blocking the death pathway of lipopolysaccharide (LPS)-induced activated-microglia to preoligodendrocytes (preOLs) in neonatal rats with infective-type periventricular leukomalacia (PVL) induced by LPS.

METHODSTwo-day-old neonatal rats were randomly divided into: a sham-operated group, an untreated PVL group, and four 1400W-treated PVL groups that were subcutaneously administrated with 20 mg/kg of 1400W at 0 h, 8 hrs, 16 hrs, and 24 hrs after LPS induction, respectively. The brain specimens were obtained 5 days after LPS induction. The pathological assessment of cerebral white matter was performed under a light microscope. Concentrations of nitric oxide (NO) were measured by nitric acid-deoxidize colorimetry. Synthesis of iNOS was determined by Western blot analysis. Peroxynitrite (ONOO(-)) level and the amount of preOLs were determined by immunocytochemistry. RETHODS: The obvious injuries of periventricular white matter, massive loss of positive O4-labelled preOLs, and increased levels of NO, ONOO(-) and iNOS were observed in neonatal rats with PVL. Compared to the untreated PVL group, the use of 1400W at 0 h, 8 hrs and 16 hrs after LPS induction significantly improved white matter injuries, reduced the levels of NO, ONOO(-) and iNOS, and increased the amount of O4-labelled preOLs. However, the use of 1400W at 24 hrs after LPS induction did not result in the improvements.

CONCLUSIONSiNOS inhibitor 1400W can effectively block the toxicity of LPS-activated microglia to preOLs and protect cerebral white matter through inhibiting iNOS and reducing the production of NO and ONOO(-). The use of 1400W within 16 hrs after LPS induction may provide cerebral protections in neonatal rats with PVL.

Amidines ; pharmacology ; Animals ; Apoptosis ; drug effects ; Benzylamines ; pharmacology ; Brain ; drug effects ; pathology ; Enzyme Inhibitors ; pharmacology ; Lipopolysaccharides ; toxicity ; Microglia ; cytology ; drug effects ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; antagonists & inhibitors ; Oligodendroglia ; cytology ; Peroxynitrous Acid ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology