Protein Expression Levels Of iNOS Research Articles

Anti-inflammatory Effect of Symplocos prunifolia Extract in an In Vitro Model of Acute Pneumonia.

Acute pneumonia is a respiratory disease characterized by inflammation within the lung tissue, exhibiting higher morbidity rates and mortality rates among immunocompromised children and older adults. Symplocos species have been traditionally used as herbal remedies for conditions like dysentery, skin ulcers, diarrhea, and dyspepsia. Contemporary research has employed various Symplocos species in the study of diverse diseases. However, the exact efficacy and mechanisms of action of Symplocos Prunifolia remain unknown. Therefore, this study investigated the anti-inflammatory mechanism of S. prunifolia extract (SPE) in A549 and RAW264.7 cells stimulated by lipopolysaccharide (LPS). SPE significantly reduced nitric oxide (NO) production and the protein expression levels of like inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 cells. Furthermore, it reduced the protein expression levels of iNOS, COX-2 and the levels of pro-inflammatory cytokines in LPS-stimulated A549 cells. The mechanism underlying the anti-inflammatory effect of SPE was associated with the inhibition of LPS stimulated the phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and Mitogen-activated protein kinase (MAPK) phosphorylation. Moreover, we confirmed that SPE decreased the nuclear translocation of nuclear factor-κB (NF-κB)/p65 stimulated by LPS. In conclusion, these results demonstrate that SPE alleviates inflammatory responses by deactivating the PI3K/Akt, MAPK, and NF-κB signaling pathways. Our findings suggest that SPE is a potential candidate for acute pneumonia prevention.

Acemannan과 aloesin의 혼합 비율에 따른 대식세포에서의 항염증 효과

This study aims to confirm the anti-inflammatory activities of acemannan and aloesin, which have been studied for various efficacies at various mixed sample ratios. The mixed samples were mixed at a ratio of 1:1 (AA-1), 1:2 (AA-2), 1:3 (AA-3), 2:1 (AA-4), and 3:1 (AA-5). Seven samples were evaluated for their cytotoxic ability on macrophages, and the results showed that all cell viability was over 90% at a concentration of 100 μg/mL. First, due to the NO production inhibitory activity, a better inhibitory effect was achieved when using a mixed sample rather than a single material. Afterward, the activity of inhibiting the production of PGE2, TNF-α, and IL-6 was confirmed using a mixed sample. It was confirmed that AA-2 had the best inhibitory activity on producing PGE2, TNF-α, and IL-6 rather than AA-1, AA-3, AA-4, and AA-5. For this reason, experiments were conducted using AA-2 to determine the protein expression levels of iNOS and COX-2, which are inflammation-related proteins. It was confirmed that AA-2 inhibited iNOS and COX-2 protein expression by 25.01% and 27.27%, respectively, compared to the LPS-alone treatment group. In conclusion, the mixed sample of acemannan and aloesin is judged to have anti-inflammatory activity and can potentially to be used as a functional material.

Anti-inflammatory effect and mechanism of active parts of Artemisia mongolica in LPS-induced Raw264.7 cells based on network pharmacology analysis.

Ethnopharmacological relevanceArtemisia mongolica is well known for its use in folk medicine, it is commonly used to alleviate a variety of diseases associated with inflammation, such as laryngitis, tonsillitis, headaches and hepatitis in northwest China. However, its anti-inflammatory mechanism is still unknown. Aim of the studyThe most potential anti-inflammatory part (AMPA) was identified by screening individual parts of A. Mongolica. After the network pharmacological analysis, the anti-inflammation effects and molecular mechanisms of AMPA were evaluated in RAW264.7 cells induced by LPS. Materials and methodsAMPA was chosen as the most anti-inflammatory of the A. Mongolica, as measured by the effect of each part of the A. Mongolica on NO and COX-2. The chemical composition of AMPA was identified using HPLC-Q-TOF-MS/MS, and targets of bioactive chemicals and targets related to inflammation were found using open-source databases. The “Compound-targets” network and PPI network were established by combining compounds and overlapped targets, and targets in the PPI networks were analyzed by GO and KEGG enrichment. The RAW26.7 cells induced by LPS were used as a model of inflammation examination. MTT assay was performed to assess the cytotoxicity of AMPA on LPS-induced RAW264.7 cells. The level of NO was measured by the Griess method while the inflammatory factors were detected by ELISA. The protein expression levels of iNOS, COX-2, MAPK, NF-κB signaling pathway and AMPK/Nrf2-related proteins were determined by Western blot. The results of nuclear translocation of p65 and Nrf2 were analyzed by immunofluorescence assay. ResultsA total of 18 compounds with potential bioactivity were identified, and after intersecting 640 compound-predicted targets and 1608 inflammation targets, the compounds and intersected targets were utilized to structure “compound-target” and PPI networks. Among AMPA, AM6, AM7, AM11, AM8 and AM1 compounds were essential in the “compound-targets” network, meanwhile, TNF, RELA, MAPK1, NOS2, PRKAG, and PTGS2 targets play important roles in the PPI network. The top 10 terms and pathways were obtained based on GO and KEGG. The cell experiments show that 50 μg/mL was the maximum concentration of AMPA without cytotoxicity in the LPS-induced RAW264.7 cell model. When compared with the LPS group, AMPA treatment not only effectively suppressed the generation of NO, TNF-α, IL-6, PGE2, IL-1β and MCP-1 in LPS-induced RAW264.7 cells, but also down-regulated the expression of COX-2, iNOS and the protein levels p-ERK, p-p38, p-IκB-α and p-p65, inhibited the nuclear translocation of p65. Furthermore, the expression levels of p-LKB1, p-AMPK, Nrf2 and HO-1 proteins were up-regulated and Nrf2 nuclear translocation was promoted. ConclusionAMPA should be considered an anti-inflammatory agent for the results of network pharmacology and in vitro, which could inhibit the MAPK pathway and NF-κB pathway and activate the AMPK/Nrf2 pathway in LPS-stimulated RAW264.7 cells.

Dim Blue Light at Night Induces Spatial Memory Impairment in Mice by Hippocampal Neuroinflammation and Oxidative Stress.

Light pollution is one of the most serious public problems, especially the night light. However, the effect of dim blue light at night (dLAN-BL) on cognitive function is unclear. In this study, we evaluated the effects of exposure to dLAN-BL in C57BL/6J mice for 4 consecutive weeks. Our results showed dLAN-BL significantly impaired spatial learning and memory and increased plasma corticosterone level in mice. Consistent with these changes, we observed dLAN-BL significantly increased the numbers and activation of microglia and the levels of oxidative stress product MDA in the hippocampus, decreased the levels of antioxidant enzymes Glutathione peroxidase (GSH-Px), Superoxide dismutase (SOD), Gluathione reductase (Gsr), total antioxidants (T-AOC) and the number of neurons in the hippocampus, up-regulated the mRNA expression levels of IL6, TNF-α and the protein expression levels of iNOS, COX2, TLR4, p-p65, Cleaved-Caspase3 and BAX, and down-regulated the mRNA expression levels of IL4, IL10, Psd95, Snap25, Sirt1, Dcx and the protein expression level of BCL2. In vitro results further showed corticosterone (10uM)-induced BV2 cell activation and up-regulated content of IL6, TNF-α in the cell supernatant and the protein expression levels of iNOS, COX2, p-p65 in BV2 cells. Our findings suggested dLAN-BL up-regulated plasma corticosterone level and hippocampal microglia activation, which in turn caused oxidative stress and neuroinflammation, leading to neuronal loss and synaptic dysfunction, ultimately leading to spatial learning and memory dysfunction in mice.

Mechanism of erythropoietin-induced M2 microglia polarization via Akt / Mtor / P70S6k signaling pathway in the treatment of brain injury in premature mice and its effect on biofilm

ABSTRACT We investigated the mechanism of erythropoietin (EPO) in brain injury in premature mice based on Akt/mTOR/p70S6K signaling pathway. The brain injury model group of premature mice was obtained by intraperitoneal injection of lipopolysaccharide during pregnancy. Normal mice were taken as the control group. The model mice were divided into low-dose EPO (1,000 IU/kg, L-EPO), medium-dose EPO (2,500 IU/kg, M-EPO), and high-dose EPO groups (5,000 IU/kg, H-EPO) by intraperitoneal injection. The levels of malondialdehyde (MDA) and total superoxide dismutase (T-SOD) were detected. TUNEL staining and Western blotting were used to detect the differences in neuronal apoptosis index (AI), microglial polarization marker protein, and Akt/mTOR/p70S6K-related protein expression levels in each group. Compared with the control group, the protein levels of AI, MDA, Bax, and iNOS in the model, L-EPO, and M-EPO groups were significantly increased, while the T-SOD level and Bcl-2, ARG1, p-Akt, p-mTOR, and p-70S6K protein levels were significantly decreased (P < 0.05). Compared with the model group, AI, MAD levels and Bax, iNOS protein expression levels in L-EPO, M-EPO, and H-EPO groups were significantly decreased, while T-SOD level and Bcl-2, ARG1, p-Akt, p-mTOR, and p-70S6K protein levels were significantly increased. The changes were dose-dependent. In summary, EPO can activate microglia transformation from M1 to M2 through Akt/mTOR/p70S6K signaling pathway.

Metformin-Loaded Alginate Nanoparticles Inhibits Mouse Atherosclerosis by Regulating Macrophage Differentiation by Activating the Adenosine Monophosphate-Activated Protein Kinase/Signal Transducer and Activator of Transcription 3 Pathway

Objective: By modulating macrophage phenotype and the adenylate-activated protein kinase/signal transducer and activator of transcription 3 (STAT3) signaling pathway, metformin-loaded alginate nanoparticles may prevent atherosclerosis (As). Methods: Flow cytometry was used to determine the percentage of macrophages with distinct phenotypes (CD86 and CD206). Analysis of protein expression levels of iNOS, arginase 1, AMPK, pAMPK, STAT3 and phosphorylated STAT3 were performed by Western Blot. To confirm the in vitro findings, ApoE−/− mice were employed. Results: AMPK activity and the fraction of M2 macrophages dramatically increased in cells treated with Met, but STAT3 activity was considerably reduced. It was also shown that the Met group had much shorter aortas and lower levels of lipid deposition than that of the control group; also, the fraction of M1 macrophages in the lipid plaques of the animals treated with Met was dramatically reduced by using immunofluorescence labeling. There was a considerable increase in AMPK activity in the Met group, but STAT3 activity was dramatically lowered. Conclusion: According to the results of this study, STAT3 activity is regulated by activation of AMPK and macrophage development in plaques is prevented in mice by metformin-loaded alginate nanoparticles.

Dracocephalum moldavica Ethanol Extract Suppresses LPS-Induced Inflammatory Responses through Inhibition of the JNK/ERK/NF-κB Signaling Pathway and IL-6 Production in RAW 264.7 Macrophages and in Endotoxic-Treated Mice.

The excessive synthesis of interleukin-6 (IL-6) is related to cytokine storm in COVID-19 patients. Moreover, blocking IL-6 has been suggested as a treatment strategy for inflammatory diseases such as sepsis. Sepsis is a severe systemic inflammatory response syndrome with high mortality. In the present study, we investigated the anti-inflammatory and anti-septic effects and the underlying mechanisms of Dracocephalum moldavica ethanol extract (DMEE) on lipopolysaccharide (LPS)-induced inflammatory stimulation in RAW 264.7 macrophages along with septic mouse models. We found that DMEE suppressed the release of inflammatory mediators NO and PGE2 and inhibited both the mRNA and protein expression levels of iNOS and COX-2, respectively. In addition, DMEE reduced the release of proinflammatory cytokines, mainly IL-6 and IL-1β, in RAW 264.7 cells by inhibiting the phosphorylation of JNK, ERK and p65. Furthermore, treatment with DMEE increased the survival rate and decreased the level of IL-6 in plasma in LPS-induced septic shock mice. Our findings suggest that DMEE elicits an anti-inflammatory effect in LPS-stimulated RAW 264.7 macrophages and an anti-septic effect on septic mouse model through the inhibition of the ERK/JNK/NF-κB signaling cascades and production of IL-6.

Sophora subprostrate polysaccharide regulates histone acetylation to inhibit inflammation in PCV2-infected murine splenic lymphocytes in vitro and in vivo

Porcine circovirus type 2 (PCV2) has caused large economic losses in the swine industry worldwide; therefore, research on relevant therapeutic medicines is still urgently needed. To define the relationship between histone acetylation and inflammation induced by PCV2, we investigated whether traditional Chinese medicinal polysaccharides could alleviate viral infection by regulating histone acetylation. In this study, Sophora subprostrate polysaccharide (SSP)-treated PCV2-infected murine splenic lymphocytes in vitro and murine spleen in vivo were used to explore the regulatory effects of SSP on inflammation and histone acetylation caused by PCV2. SSP at different concentrations significantly reduced the secretion levels of the proinflammatory cytokines TNF-α and IL-6, the activity of COX-2, the mRNA expression levels of TNF-α, IL-6, iNOS and COX-2 and the protein expression levels of iNOS and COX-2 but promoted the secretion and mRNA expression levels of IL-10. Furthermore, the different concentrations of SSP significantly regulated the activity of histone acetylase (HAT) and the mRNA expression of HAT1, increased the activity of histone deacetylase (HDAC) and the mRNA expression of HDAC1 and reduced the protein expression levels of Ac-H3 and Ac-H4. Overall, SSP inhibited inflammation in PCV2-infected murine splenic lymphocytes by regulating histone acetylation in vitro and in vivo, thus playing an important role in PCV2 infection.

Effects of simvastatin on iNOS and caspase‑3 levels and oxidative stress following smoke inhalation injury.

The overexpression of inducible nitric oxide synthase (iNOS) induces cell apoptosis through various signal transduction pathways and aggravates lung injury. Caspase-3 is an important protein in the apoptotic pathway and its activation can exacerbate apoptosis. Simvastatin, a hydroxymethyl glutaryl-A reductase inhibitor, protects against smoke inhalation injury by inhibiting the synthesis and release of inflammatory factors and decreasing cell apoptosis. Following the establishment of an animal model of smoke inhalation injury, lung tissue and serum were collected at different time points and the protein and mRNA expression of iNOS and caspase-3 in lung tissue by immunochemistry, western blot and reverse transcription-quantitative polymerase chain reaction, the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in lung tissue and serum were analyzed using thiobarbituric acid method and the WST-1 method. The results were statistically analyzed. The lung tissues of the rats in the saline group and the low-, middle- and high-dose groups exhibited clear edema and hemorrhage, and had significantly higher pathological scores at the various time points compared with the rats in the control group (P<0.05). Furthermore, lung tissue and serum samples obtained from these four groups had significantly higher mRNA and protein expression levels of iNOS and caspase-3 (P<0.05), significantly lower SOD activity and higher MDA content (P<0.05). Compared with the saline group, the low-, middle- and high-dose groups had significantly lower pathological scores (P<0.05), significantly lower mRNA and protein expression levels of iNOS, caspase-3 and MDA content in lung tissues (P<0.05) and significantly higher SOD activity in lung tissues and serum. The middle- and high-dose groups had significantly lower pathological scores (P<0.05), significantly decreased iNOS and caspase-3 mRNA and protein expression in lung tissues, significantly higher SOD activity in lung tissues and serum and a significantly lower MDA content (P<0.05) compared with the low-dose group. With the exception of SOD activity in lung tissues at 24 and 72 h and MDA content in serum at 48 h, no significant differences were observed between the middle- and high-dose groups. The present study demonstrated that there was an association between the therapeutic effect and dosage of simvastatin within a definitive range. In rats with smoke inhalation injury, simvastatin inhibited iNOS and caspase-3 expression in lung tissues and mitigated oxidative stress, thereby exerting a protective effect. In addition, the effect and dose were associated within a definitive range.

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.

Protective effect of avicularin on rheumatoid arthritis and its associated mechanisms.

The present study aimed to investigate the effect of avicularin on rheumatoid arthritis (RA) in vitro, and additionally explore the molecular mechanism. To perform this investigation, an in vitro model of RA was established by treatment of the human RA synovial MH7A cell line with tumor necrosis factor-α (TNF-α). MH7A cells were then treated with various concentrations (10, 30, 100 and 300 µM) of avicularin. Then, the levels of inflammatory factors [interleukin (IL)-1β, IL-6, IL-8, matrix metalloproteinase (MMP)-1 and MMP-13] were measured by ELISA. Cell viability and apoptosis were detected using an MTT assay and flow cytometry, respectively. In addition, the expression levels of genes and proteins were determined reverse transcription quantitative polymerase chain reaction and western blot analysis. The results of the present study indicated that avicularin significantly decreased the levels of inflammatory factors (IL-1β, IL-6, IL-8, MMP-1 and MMP-13), previously increased by TNF-α, in a dose-dependent manner. Concurrently, avicularin inhibited the mRNA and protein expression levels of iNOS and COX-2 increased by TNF-α. It was also identified that TNF-α administration significantly promoted MH7A cell viability and inhibited cell apoptosis, and avicularin treatment dose-dependently inhibited MH7A cell viability and induced cell apoptosis. In addition, these data suggested that avicularin prevented the activation of the mitogen-activated protein kinase kinase (MEK)/nuclear factor kappa light-chain-enhancer of activated B-cells (NF-κB) pathway activated by TNF-α. Taken together, these results demonstrated that avicularin may inhibit the inflammatory response, prevent cell viability and induce apoptosis in human RA synovial cells through preventing the activation of the MEK/NF-κB pathway.

Sanyang Xuedai enhances the radiosensitivity of human non-small cell lung cancer cells via increasing iNOS/NO production

ObjectiveIn this research, we aimed at finding out how San Yang Xue Dai (SYKT) promotes the radiosensitivity of non-small cell lung cancer (NSCLC) cell line NCI-H460. MethodsSurvival rate of NSCLC cells (A549, NCI-H460, NCI-H1650 and NCI-H1975) after the SYKT treatment or irradiation (IR) was calculated by the MTT assay. The radiosensitization of SYKT (0.5 g/mL and 1.0 g/mL) on cell line NCI-H460 and the radioresistant cell line NCI-H460R was studied by MTT assay and clone formation assay. The protein expression levels of iNOS, Cyclin B1 and CDC2 were determined by western blot, and the expression of NO was measured by Griess method. Finally, cell cycle and apoptotic rate of NSCLC cell line NCI-H460 were accessed by flow cytometry assay. BrdU staining was also applied to detect the cell proliferation after IR with or without SYKT treatment. ResultsThe IC10 value of SYKT for NCI-H460 cells was 1.03 g/mL. After 1.0 g/mL SYKT treatment, the radiosensitivity of NCI-H460R cells was enhanced. The level of iNOS in the cells was found decreased after IR. We also found that SYKT could enhance iNOS and NO expressions while inhibit cyclin B1 and CDC2 expressions in radiation resistant cells. Combining β-irradiation with SYKT caused cell cycle arrest in G2/M phase and increased cell apoptosis. ConclusionSYKT resensitized radioresistant NCI-H460R cells via increasing cell apoptosis and cell cycle arrest. This was due to an elevated NO level caused by accumulating iNOS and effects of SYKT on radiosensitization of NSCLC should be further investigated in clinical application.

L-Carnitine has a liver-protective effect through inhibition of inducible nitric oxide synthase induction in primary cultured rat hepatocytes

Background: L-Carnitine has protective effects on various injured organs. However, it has not been reported whether L-carnitine influences the induction of inducible nitric oxide synthase (iNOS) expression during inflammation. Nitric oxide (NO) produced by iNOS is an inflammatory indicator in organs which become inflamed, including the liver.Objective: This study aimed to examine whether L-carnitine influences the induction of iNOS gene expression in inflammatory cytokine-stimulated hepatocytes and the mechanisms involved in the action. Methods: L-Carnitine was added into the primary cultures of rat hepatocytes stimulated by interleukin-1β (an in vitro liver injury model). The production of NO and induction of iNOS and its signaling pathway were analyzed.Results: Transfection experiments with iNOS promoter-luciferase constructs revealed how L-carnitine inhibited iNOS mRNA synthesis activity and reduced its stability. In support of this observation, L-carnitine reduced iNOS mRNA and iNOS protein expression levels, resulting in reduced NO production. L-Carnitine blocked two essential pathways for iNOS induction: IκB kinase (IκB degradation/NF-κB activation) and phosphatidylinositol 3-kinase/Akt (type I IL-1 receptor upregulation).Conclusions: L-Carnitine inhibited the induction of inflammatory mediator iNOS, partially through inhibition of NF-κB activation, which demonstrated L-carnitine has protective effects in an in vitro liver injury model. L-Carnitine may have therapeutic potential for organ injuries, including the liver.Keywords: L-carnitine, hepatic encephalopathy, inducible nitric oxide synthase, liver injury, primary cultured hepatocytes, nuclear factor-κB, type I interleukin-1 receptor

Curcumin protects liver inflammation by suppressing expression of inducible nitric oxide synthase in primary cultured rat hepatocytes

Background: Curcumin has beneficial effects on organ metabolism. However, there is little evidence that curcumin affects inflammatory mediators, such as tumor necrosis factor (TNF)-α and nitric oxide (NO). In an inflamed liver, proinflammatory cytokines stimulate liver cells, followed by the induction of inducible NO synthase (iNOS). Excessive NO produced by iNOS is one of the factors in liver injury. Therefore, inhibiting iNOS induction for preventing liver injury is important.Objective: This study aimed to investigate liver protective effects of curcumin by examining interleukin (IL)-1β-stimulated hepatocytes.Methods: Primary cultured rat hepatocytes were treated with IL-1β in the presence or absence of curcumin. Induction of NO production and iNOS, and the signaling pathway of iNOS were analyzed.Results: Simultaneous addition of IL-1β and curcumin decreased expression levels of iNOS protein and mRNA, resulting in inhibition of NO production. Curcumin also reduced mRNA expression of TNF-α and IL-6. Curcumin inhibited two essential signaling pathways for iNOS induction, NF-κB activation and type I IL-1 receptor upregulation. Transfection experiments revealed that curcumin reduced iNOS mRNA levels at the promoter activation and mRNA stabilization steps. Delayed administration of curcumin after IL-1β addition also inhibited iNOS induction.Conclusions: Curcumin affects induction of inflammatory mediators, such as iNOS and TNF-α, in part through the inhibition of NF-κB activation in hepatocytes. Curcumin may have therapeutic potential for organ injuries, including the liver.Key words: curcumin, inducible nitric oxide synthase, liver injury, primary cultured hepatocytes, nuclear factor-κB, type I interleukin-1 receptor, tumor necrosis factor-α.

Role of ghrelin in small intestinal motility following pediatric intracerebral hemorrhage in mice

Small intestinal motility (SIM) disorder is a common complication following pediatric intracerebral hemorrhage(ICH), leading to a poor prognosis in patients. Previous studies have shown that ghrelin is involved in SIM in various diseases; however, the role of ghrelin in pediatric ICH‑induced SIM disorder remains to be elucidated. The present study was designed to investigate the association between ghrelin and SIM post‑ICH, and to examine the effect of exogenous ghrelin administration on SIM invivo. An ICH model was induced in mice by autologous blood infusion. Neurobehavioral deficits were evaluated using a Rotarod test, forelimb placing test, and corner turn test. Intestinal mucosal damage was examined using hematoxylin and eosin staining. SIM was measured using charcoal meal staining. An enzyme‑linked immunosorbent assay was used to evaluate serum levels of ghrelin and nitric oxide (NO). Reverse transcription‑quantitative polymerase chain reaction and western blot analyses were performed to determine the levels of inducible nitric oxide synthase (iNOS), neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) at the mRNA and protein levels. Nω‑nitro‑L‑arginine methyl ester hydrochloride (L‑NAME), L‑arginine, atropine, phentolamine and propranolol were used to manipulate the putative pathways induced by ghrelin. Neurological dysfunction was observed post‑ICH. ICH caused damage to the intestinal mucosa and delayed SIM. Serum levels of ghrelin increased between 3h and 3days, peaking at 12h, and showed a significant negative correlation with SIM post‑ICH. Ghrelin administration dose‑dependently attenua-ted ICH‑induced SIM disorder. Ghrelin also decreased NO levels by downregulating the mRNA and protein expression levels of iNOS, but not those of nNOS or eNOS, post‑ICH. Consistently, the effect was enhanced by L‑NAME and weakened by L‑arginine, respectively. The protective effect of ghrelin was eradicated by atropine, but not phentolamine or propranolol. These findings suggested that ghrelin ameliorated SIM disorder by downregulating iNOS/NO via the cholinergic pathway. Therefore, ghrelin may serve as a potential biomarker and useful target in ICH‑induced SIM disorder.

The Effect of Selenium on the Cd-Induced Apoptosis via NO-Mediated Mitochondrial Apoptosis Pathway in Chicken Liver

Cd-induced apoptosis and the protective effects of Se against Cd-induced injury have been reported in previous studies. However, little is known regarding the effects of Cd-induced apoptosis in hepatic cells and the antagonistic effects of Se on Cd in poultry. In the present study, 128 healthy 31-week-old laying hens were randomly divided into four groups, which were fed basic diets, with the addition of Se (Na2SeO3, 2mg/kg), Cd (CdCl2, 150mg/kg), or Se + Cd (150mg/kg of CdCl2 and 2mg/kg of Na2SeO3) for 90days. Ultrastructural changes, nitric oxide (NO) concentrations, inducible nitric oxide synthase (iNOS) activities, results of the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of apoptosis, and the expression of iNOS and apoptosis-related genes in livers were determined. It was observed that Cd treatment significantly increased the concentrations of NO and iNOS activity in chicken livers. The production of excessive NO initiated the mitochondrial apoptotic pathway. Exposure to Cd increased the mRNA and the protein expression levels of iNOS, caspase-3, Bax, p53, and Cyt-c. Furthermore, the ratio of Bax/Bcl-2 increased, while the expression of Bcl-2 decreased. Treatment with Se significantly alleviated Cd-induced apoptosis in chicken livers, as evidenced by a reduction in the production of NO, iNOS activity, the number of apoptotic cells, and mRNA and protein expression levels of iNOS, caspase-3, Bax, and Cyt-c. It indicated that Cd induced NO-mediated apoptosis through the mitochondrial apoptotic pathway and Se exerted antagonizing effects. The present study provides new insights as to how Se affects Cd-induced toxicity in the chicken liver.

Anti-inflammatory activity of a sulfated polysaccharide isolated from an enzymatic digest of brown seaweed Sargassum horneri in RAW 264.7 cells.

BACKGROUND/OBJECTIVESSargassum horneri is an edible brown alga that grows in the subtidal zone as an annual species along the coasts of South Korea, China, and Japan. Recently, an extreme amount of S. horneri moved into the coasts of Jeju Island from the east coast of China, which made huge economic and environmental loss to the Jeju Island. Thus, utilization of this biomass becomes a big issue with the local authorities. Therefore, the present study was performed to evaluate the anti-inflammatory potential of crude polysaccharides (CPs) extracted from S. horneri China strain in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells.MATERIALS/METHODSCPs were precipitated from S. horneri digests prepared by enzyme assistant extraction using four food-grade enzymes (AMG, Celluclast, Viscozyme, and Alcalase). The production levels of nitric oxide (NO) and pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-1β were measured by Griess assay and enzyme-linked immunosorbent assay, respectively. The levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), nuclear factor (NF)-κB, and mitogen-activated protein kinases (MAPKs) were measured by using western blot. The IR spectrums of the CPs were recorded using a fourier transform infrared spectroscopy (FT-IR) spectrometer.RESULTSThe polysaccharides from the Celluclast enzyme digest (CCP) showed the highest inhibition of NO production in LPS-stimulated RAW 264.7 cells (IC50 value: 95.7 µg/mL). Also, CCP dose-dependently down-regulated the protein expression levels of iNOS and COX-2 as well as the production of inflammatory cytokines, including TNF-α and IL-1β, compared to the only LPS-treated cells. In addition, CCP inhibited the activation of NF-κB p50 and p65 and the phosphorylation of MAPKs, including p38 and extracellular signal-regulated kinase, in LPS-stimulated RAW 264.7 cells. Furthermore, FT-IR analysis showed that the FT-IR spectrum of CCP is similar to that of commercial fucoidan.CONCLUSIONSOur results suggest that CCP has anti-inflammatory activities and is a potential candidate for the formulation of a functional food ingredient or/and drug to treat inflammatory diseases.

16α, 17α-epoxypregnenolone-20-oxime inhibits NO and IL-6 production in LPS-treated RAW264.7 cells.

It has previously been reported that 16α, 17α-epoxypregnenolone-20-oxime (EPREGO) exerts an inhibitory effect on nitric oxide (NO) production and inducible NO synthase (iNOS) expression in microglia. The present study aimed to investigate the effects of EPREGO on the lipopolysaccharide (LPS)‑induced inflammatory response in RAW264.7 macrophage cells, and to determine the underlying molecular mechanisms using western blot analysis, enzyme‑linked immunosorbent assays and fluorescence‑activated cell sorting. The present study demonstrated that LPS‑induced production of NO and interleukin (IL)-6, and the protein expression levels of iNOS, were reduced by EPREGO in a dose‑ and time‑dependent manner, whereas, EPREGO did not affect tumor necrosis factor‑α production. In addition, EPREGO suppressed LPS‑induced cellular reactive oxygen species production and phagocytosis. Furthermore, EPREGO significantly inhibited the LPS‑induced activation of mitogen‑activated protein kinases and inhibitor ofκBα degradation in LPS‑stimulated RAW264.7 cells, thus resulting in modulation of the production of NO and IL‑6. Taken together, these results suggest that EPREGO exhibits anti-inflammatory activity in macrophages, thus validating the hypothesis that EPREGO may be useful as a therapeutic agent for the treatment of macrophage-mediated inflammation.

LPS로 염증 유도된 RAW 264.7세포에 대한 참콩풍뎅이(Popillia flavosellata) 에탄올 추출물의 항염증 효과

The beetle Popillia flavosellata has been no reported its functional effects. In this study, we investigated the anti-inflammatory effect of P. flavosellata ethanol extract (PFE) on RAW 264.7 mouse macrophage cells treated with lipopolysaccharide (LPS) for the induction of inflammation. First, we examined the cytotoxicity of PFE in the RAW 264.7 cells at a concentration of 2,000 μg/ml or less. To evaluate the anti-inflammatory effects of PFE, we investigated the expression levels of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6, and proinflammatory enzymes, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-induced RAW 264.7 cells. In addition, we examined whether PFE inhibited the translocation of nuclear factor kappa B (NF-κB) p65 into the nucleus in the LPS-induced RAW 264.7 cells. We found that the protein levels of TNF-α and IL-6 were decreased in the LPS-induced RAW 264.7 cells after the treatment with PFE in a dose-dependent manner. In addition, we confirmed that PFE inhibited the translocation of NF-κB p65 into the nucleus, as well as the protein expression levels of iNOS and COX-2. Accordingly, we propose that PFE exerts an anti-inflammatory effect through the down-regulation of NF-κB p65, TNF-α, IL-6, iNOS, and COX-2 via the toll like receptor (TLR)-4 inflammatory signaling pathway.

Paeoniflorin ameliorates acute myocardial infarction of rats by inhibiting inflammation and inducible nitric oxide synthase signaling pathways.

Paeoniflorin (PF) is the main active component of the commonly used Traditional Chinese Medicine peony, Paeonia Suffruticosa. PF has diverse biological functions and exhibits anti‑oxidative, anti‑inflammatory and anti‑apoptotic activity. Inducible nitric oxide synthase (iNOS) is a catalyzing enzyme that is involved in the synthesis of nitric oxide (NO). NO has an important regulatory role in the cardiovascular, immune and nervous systems. PF has previously been demonstrated to inhibit the gene expression of iNOS. The present study aimed to identify a potentially novel cytoprotective function of PF, and to elucidate its effects against myocardial ischemic damage in a rat model of acute myocardial infarction (AMI). PF was able to significantly decrease the myocardial infarct size as well as the activities of creatine kinase (CK), the MB isoenzyme of CK, lactate dehydrogenase and cardiac troponin T. In addition, in the PF‑treated groups, the expression levels of tumor necrosis factor‑α, interleukin (IL)‑1β, IL‑6 and nuclear factor‑κB were markedly inhibited. Furthermore, treatment with PF inhibited the activities and protein expression levels of iNOS. Decreased caspase‑3 and caspase‑9 activities were also observed in the AMI rat model treated with various doses of PF. The results of the present study indicated that the cardioprotective effects of PF may be associated with the inhibition of inflammation and iNOS signaling pathways.