Effect of nitro-conjugated linoleic acid on the inflammatory response of murine macrophages activated with lipopolysaccharide derived from Prevotella intermedia.

Nitro-conjugated linoleic acid (NO2-CLA) has been observed to manifest salutary signaling responses, including anti-inflammatory and antioxidant properties. Here, the authors have explored the influence and underlying mechanisms of NO2-CLA on the proinflammatory reaction of murine macrophages that were challenged with lipopolysaccharide (LPS) derived from Prevotella intermedia, a putative periodontopathic bacterium. Treatment of LPS-activated RAW264.7 cells with NO2-CLA notably dampened the secretion of iNOS-derived NO, IL-1ß and IL-6 as well as their gene expressions and significantly enhanced the markers for M2 macrophage polarization. NO2-CLA promoted the HO-1 expression in cells challenged with LPS, and tin protoporphyrin IX, an HO-1 inhibitor, significantly reversed the NO2-CLA-mediated attenuation of NO secretion, but not IL-1ß or IL-6. We found that cells treated with NO2-CLA significantly increased mRNA expression of PPAR-γ compared to control cells, and NO2-CLA significantly reverted the decrease in PPAR-γ mRNA caused by LPS. Nonetheless, antagonists to PPAR-γ were unable to reverse the NO2-CLA-mediated suppression of inflammatory mediators. In addition, NO2-CLA did not alter the p38 and JNK activation elicited by LPS. Both NF-κB reporter activity and IκB-α degradation caused by LPS were notably diminished by NO2-CLA. NO2-CLA was observed to interrupt the nuclear translocation and DNA binding of p50 subunits caused by LPS with no obvious alterations in p65 subunits. Further, NO2-CLA attenuated the phosphorylation of STAT1/3 elicited in response to LPS. We propose that NO2-CLA could be considered as a possible strategy for the therapy of periodontal disease, although additional researches are certainly required to confirm this.

Mechanoregulation of Osteoclastogenesis-Inducing Potentials of Fibrosarcoma Cell Line by Substrate Stiffness.

A micro-physiological system is generally fabricated using soft materials, such as polydimethylsiloxane silicone (PDMS), and seeks an inflammatory osteolysis model for osteoimmunological research as one of the development needs. Microenvironmental stiffness regulates various cellular functions via mechanotransduction. Controlling culture substrate stiffness may help spatially coordinate the supply of osteoclastogenesis-inducing factors from immortalized cell lines, such as mouse fibrosarcoma L929 cells, within the system. Herein, we aimed to determine the effects of substrate stiffness on the osteoclastogenesis-inducing potential of L929 cells via cellular mechanotransduction. L929 cells showed increased expression of osteoclastogenesis-inducing factors when cultured on type I collagen-coated PDMS substrates with soft stiffness, approximating that of soft tissue sarcomas, regardless of the addition of lipopolysaccharide to augment proinflammatory reactions. Supernatants of L929 cells cultured on soft PDMS substrates promoted osteoclast differentiation of the mouse osteoclast precursor RAW 264.7 by stimulating the expression of osteoclastogenesis-related gene markers and tartrate-resistant acid phosphatase activity. The soft PDMS substrate inhibited the nuclear translocation of YES-associated proteins in L929 cells without reducing cell attachment. However, the hard PDMS substrate hardly affected the cellular response of the L929 cells. Our results showed that PDMS substrate stiffness tuned the osteoclastogenesis-inducing potential of L929 cells via cellular mechanotransduction.

Coadministration of Curcumin and Hydromorphone Hydrochloride Alleviates Postoperative Pain in Rats.

This study aimed to explore the effect of curcumin and hydromorphone hydrochloride (HH) cotreatment on postoperative pain in rats. An incision + formaldehyde-induced pain rat model was established. Rats were treated with vehicle, curcumin, HH, or curcumin + HH. Paw mechanical withdrawal threshold and thermal withdrawal latency were measured at 1 d before surgery as well as 1 , 2 h, 1 , 3 , and 7 d after surgery to assess pain sensitivity. The L4-6 region of the spinal cord was collected from each rat at 2 h, 1 , 3 , and 7 d after surgery. Western blot analysis and immunohistochemical staining were carried out to detect the protein expression of pain-related genes. Quantitative real-time PCR and enzyme-linked immunosorbent assay were conducted to measure the expression and production of proinflammatory mediators. Compared with other groups, Curcumin + HH significantly reduced pain sensitivity in the model rats. Mechanistically, curcumin + HH suppressed protein expression of stromal cell-derived factor-1 (SDF-1), CXC chemokine receptor 4 (CXCR4), p-Akt, and c-fos while enhancing protein expression of nerve growth factor (NGF) in the dorsal root ganglia (DRG) of model rats. Curcumin + HH inhibited the expression and production of interleukin 1ß (IL-1ß), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), and p65 nuclear factor kappa B (NF-κB) in the DRG. Coadministration of curcumin and HH alleviates incision + formaldehyde-induced pain in rats, possibly by suppressing the SDF-1/CXCR4 pathway and the production of proinflammatory mediators. Our results provide curcumin and HH cotreatment as a promising therapeutic strategy in the management of postoperative pain.

Immunologic characteristics of human gingival fibroblasts in response to oral bacteria.

BACKGROUND AND OBJECTIVE: There is ample evidence that gingival fibroblasts (GFs) participate in the immune response to oral bacteria and serve as immune-regulatory cells. The objective of this study was to investigate the innate immune response of GFs to oral bacteria. MATERIAL AND METHODS: Human GFs were cocultured with relatively less-pathogenic (Leptotrichia wadei, Fusobacterium nucleatum and Campylobacter gracilis) and pathogenic red-complex bacteria. The expression of mRNA for antimicrobial peptides [AMPs; namely human beta defensins (HBDs)], chemokines with antimicrobial activity [chemokine C-X-C motif (CXCL)10, CXCL11 and chemokine C-C motif ligand 20 (CCL20)] and proinflammatory mediators [interleukin (IL)6 and IL8] and the levels of CXCL11, CCL20, IL-6 and IL-8 accumulated in supernatants were analyzed using real-time PCR and ELISA, respectively. The proteolytic activities of CXCL11, CCL20, IL-6 and IL-8 produced by six species of bacteria were also determined. RESULTS: The relatively less-pathogenic bacteria strongly up-regulated the expression of antimicrobial chemokines and proinflammatory mediators, whereas the red-complex bacteria stimulated low levels, or often suppressed, expression of these factors. Regarding the regulation of AMPs, the inhibition of HBD3, HBD106 and HBD107 mRNAs by Porphyromonas gingivalis was noticeable; however, differences between the two bacterial groups were not conspicuous. Differential degradation of proteins by the six bacterial species was observed: P. gingivalis and Treponema denticola degraded proteins well, whereas the other species degraded proteins to a relatively lower degree. CONCLUSION: The invasion of red-complex bacteria into gingival connective tissue can suppress the immune response of GFs and can be a source of persistent infection in connective tissue.

Kinin B1 Receptor Inhibition With BI113823 Reduces Inflammatory Response, Mitigates Organ Injury, and Improves Survival Among Rats With Severe Sepsis.

BACKGROUND: This study examined the therapeutic effects of an orally active nonpeptide kinin B1 receptor antagonist, BI113823, in a clinically relevant experimental model of polymicrobial sepsis in rats. METHODS: Sepsis was induced by cecal ligation and puncture (CLP). Animals received treatment with either vehicle or BI113823. The experiment was terminated in the first set of animals 15 hours after CLP. Seven-day survival following CLP was determined in the second set of animals. RESULTS: Compared with vehicle treatment, administration of BI113823 reduced neutrophil and macrophage infiltration, reduced cytokine production, attenuated intestinal mucosal hyperpermeability, prevented hemodynamic derangement, and improved cardiac output. Furthermore, administration of BI113823 reduced inducible nitric oxide synthase expression and the injury score in the lung and attenuated nuclear factor ĸB activation and apoptosis in the liver. Treatment with BI113823 also reduced plasma levels of cardiac troponin, aspartate aminotransferase, alanine aminotransferase, urea, and lactate, as well as proteinuria. Finally, administration of BI113823 improved the 7-day survival rate following CLP in rats. CONCLUSIONS: Administration of BI113823 reduced systemic and tissue inflammatory responses, prevented hemodynamic derangement, attenuated multiorgan injury, and improved overall survival.

Sabiporide improves cardiovascular function and attenuates organ injury from severe sepsis.

BACKGROUND: The aim of the present study was to evaluate the efficacy of orally administered sabiporide, a selective Na(+)/H(+) exchanger inhibitor on whole body protection from severe sepsis in rats. METHODS: Series 1: Sepsis was induced by cecal ligation and puncture (CLP). Animals received treatment of vehicle or sabiporide (10 mg/kg, p.o.). The experiment was terminated 20 h after CLP. Series 2: At 20 h after CLP, the necrotic cecum was excised and the abdominal cavity was washed. The animals were then returned to their cages. The experiment was terminated 7 d after CLP. RESULTS: Series 1: Compared with vehicle treatment, administration of sabiporide prevented hemodynamic derangement and improved cardiac function as evidenced by improved arterial pressure, left ventricle systolic pressure, ±dp/dt max, ejection fraction and fractional shorting, attenuated left ventricle end-diastolic pressure elevation, and wall motion abnormality. Furthermore, administration of sabiporide attenuated intestinal mucosal hyperpermeability and reduced accumulation of abdominal ascites. In addition, treatment with sabiporide also reduced plasma levels of tumor necrosis factor-α, interleukin 6, interleukin 10, cardiac troponin, aspartate aminotransferase, alanine aminotransferase, urea, and lactate, and attenuated neutrophil infiltration in the liver and gut. Series 2: Administration of sabiporide improved the 7-day survival rate after CLP in rats (42% in vehicle group versus 75% in sabiporide group). CONCLUSIONS: Administration of sabiporide improved cardiovascular performance, lessened the inflammatory response, tissue hypoperfusion and multiorgan injury, and most importantly reduced mortality.

Nitrooleic acid inhibits macrophage activation induced by lipopolysaccharide from Prevotella intermedia.

The hypothesis of the present study was that nitro-fatty acids (NO2-FAs) would suppress inflammation associated with periodontal disease. To test this hypothesis, we investigated the influence of nitrooleic acid, a prototypical NO2-FA, on the inflammatory response of murine macrophages activated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen associated the etiology of different types of periodontal diseases. LPS was prepared from P. intermedia cells by using phenol-water protocol. Culture supernatants were assayed for nitric oxide (NO), interleukin-1ß (IL-1ß), and IL-6. Real-time polymerase chain reaction and immunoblotting analyses were performed to quantify messenger RNA and protein expression, respectively. The secreted embryonic alkaline phosphatase reporter assay was performed to measure NF-κB activation. The transcription factor assay kit was used to measure DNA-binding of NF-κB subunits. Findings obtained from the present study revealed that nitrooleic acid suppresses the generation and messenger RNA expression of inducible NO synthase-derived NO, IL-1ß, and IL-6 in RAW264.7 cells activated with P. intermedia LPS and promotes macrophage polarization toward anti-inflammatory M2 phenotype. We also found that nitrooleic acid exerts its effect via heme oxygenase-1 induction and suppression of NF-κB signaling. The inhibition of NO and proinflammatory cytokine production by nitrooleic acid was independent from PPAR-γ, JNK, p38, and STAT1/3. Nitrooleic acid may represent a novel class of agent as a host modulator which has therapeutic benefit in periodontal disease, though more work is needed to confirm this.

Excessive Expression of Microglia/Macrophage and Proinflammatory Mediators in Olfactory Bulb and Olfactory Dysfunction After Stroke.

BACKGROUND/AIM: Olfactory dysfunction can be caused by stroke but the pathogenesis is still unclear. Previous studies have proved that olfactory dysfunction could be caused by microglia activation in the olfactory bulb and that middle cerebral artery occlusion (MCAO) may induce ipsilateral olfactory bulb microglia activation. This study aimed to explore the possible pathogenesis of ischemic stroke-induced olfactory dysfunction. MATERIALS AND METHODS: We used a rat model of MCAO to simulate ischemic stroke. Olfactory function tests were performed using buried food test. The mRNA expression of olfactory marker protein (OMP), microglia/macrophage activation, and proinflammatory mediators were measured using reverse transcription-quantitative polymerase chain reaction. RESULTS: Following MCAO, rats had poorer olfactory performance. In the olfactory bulb of the rats, the mRNA expression of OMP decreased and the mRNA expression of microglia/macrophage activation and proinflammatory mediators increased. CONCLUSION: Ischemic stroke causes microglia/macrophage activation and promotes neuroinflammation in the olfactory bulb, causing olfactory dysfunction.

Adventitious root cultures of Oplopanax elatus inhibit LPS-induced inflammation via suppressing MAPK and NF-κB signaling pathways.

Bioreactor-cultured adventitious roots (ARs) of the endangered medicinal plant Oplopanax elatus Nakai is a novel alternative plant material. To utilize ARs in the product production, the present study investigated the anti-inflammatory effect of O. elatus ARs. In the in vivo experiment, lipopolysaccharide (LPS)-induced acute lung injury disease model was established and several inflammatory indexes were determined. For the LPS-stimulated mice, after pretreatment of AR crude extract (200 mg/kg), cell infiltration in lungs was decreased, the production of proinflammatory mediators, including nitric oxide (NO), tumor necrosis factor (TNF)-α, and interleukin (IL)-6, and 1ß in the bronchoalveolar lavage fluid was evidently reduced, which indicated that O. elatus ARs had an anti-inflammatory effect. In the in vitro experiment, ethyl acetate (EtOAc) fractions (12.5, 25, and 50 µg/mL) were used to treat LPS-induced peritoneal macrophages (PMs) of mice. The production of NO, prostaglandin E2, TNF-α, IL-6, and IL-1ß in LPS-stimulated PMs was obviously inhibited (p < 0.05) after pretreatment with EtOAc fractions, and the expression of the inducible nitric oxide synthase and cyclooxygenase were also suppressed. To clarify the anti-inflammatory mechanism, effects of EtOAc fraction on changes of proteins related to the pathways of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) were investigated. The phosphorylation of extracellular regulated protein kinases, c-jun n-terminal kinase, and p38 MAPK in LPS-induced PMs was inhibited after pretreatment of EtOAc fractions. In addition, EtOAc fractions enhanced inhibitor of nuclear factor-kappa B-α expression and decreased nuclear translocation of p65 NF-κB. Thus, EtOAc from O. elatus ARs is involved in regulating MAKP and NF-κB signaling pathways to inhibit LPS-induced inflammation.

Anti-Inflammatory Activity of Epimedium brevicornu Maxim Ethanol Extract.

Epimedium brevicornu Maxim has been used as a traditional herbal drug in China. In this study, the anti-inflammatory effects of E. brevicornu Maxim ethanol extract (EBME) were investigated in RAW264.7 macrophages and mice challenged with lipopolysaccharide (LPS). Results showed that EBME attenuated inflammation by decreasing the production of several proinflammatory mediators, such as nitric oxide (NO), prostaglandin (PG) E2, inducible nitric oxide synthase, and cyclooxygenase-2, in LPS-stimulated RAW264.7 macrophages. EBME increased the expression of heme oxygenase-1 (HO-1) and promoted the nuclear translocation of nuclear factor erythroid 2-related factor 2. The inhibitory effects of EBME on LPS-stimulated NO and PGE2 expression were partially reversed by HO-1 inhibitor. EBME also elicited an anti-inflammatory effect by inhibiting the production of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 in LPS-induced peritonitis. Therefore, EBME exhibited anti-inflammatory effects in vitro and in vivo.

[ATP-P2X7R signalling pathway and its effects in parasitic diseases].

ATP (Adenosine triphosphate) is an important endogenous damage - associated molecular pattern (DAMP). P2X7R is an ATP-gated cation channel. ATP-P2X7R plays a vital role in the pathophysiology of many diseases because P2X7R is distributed on various immune cells. ATP-P2X7R signal transduction pathway has been implicated to participate in the body's immune defense against pathogens. This paper reviews the recent progress regarding ATP-P2X7R and its effects on parasitic diseases.

Shiga Toxins Trigger the Secretion of Lysyl-tRNA Synthetase to Enhance Proinflammatory Responses.

Shiga toxins (Stxs) produced by Shiga toxin-producing Escherichia coli (STEC) strains are major virulence factors that cause fatal systemic complications, such as hemolytic uremic syndrome and disruption of the central nervous system. Although numerous studies report proinflammatory responses to Stx type 1 (Stx1) or Stx type 2 (Stx2) both in vivo and in vitro, none have examined dynamic immune regulation involving cytokines and/or unknown inflammatory mediators during intoxication. Here, we showed that enzymatically active Stxs trigger the dissociation of lysyl-tRNA synthetase (KRS) from the multi-aminoacyl-tRNA synthetase complex in human macrophage-like differentiated THP-1 cells and its subsequent secretion. The secreted KRS acted to increase the production of proinflammatory cytokines and chemokines. Thus, KRS may be one of the key factors that mediate transduction of inflammatory signals in the STEC-infected host.

ATP-P2X7R signalling pathway and its effects in parasitic diseases / 中国血吸虫病防治杂志

ATP (Adenosine triphosphate) is an important endogenous damage-associated molecular pattern (DAMP). P2X7R is an ATP-gated cation channel. ATP-P2X7R plays a vital role in the pathophysiology of many diseases because P2X7R is distributed on various immune cells. ATP-P2X7R signal transduction pathway has been implicated to participate in the body's im-mune defense against pathogens. This paper reviews the recent progress regarding ATP-P2X7R and its effects on parasitic diseas-es.

Generation of Proinflammatory Mediator of Intervertebral Disc Cells by Nicotine Stimulation

STUDY DESIGN: Experimental investigation in vitro. OBJECTIVES: To evaluate the relationship between the degeneration of intervertebral disc cells, and low back pain induced by degeneration of intervertebral disc cells and increases in use of proinflammatory mediators via nicotine stimulation. SUMMARY OF LITERATURE REVIEW: Smoking is a leading cause of degeneration of intervertebral disc cells and low back pain. According to the existing literature, nicotine, one of the main ingredients in cigarettes, causes the degeneration of intervertebral disk cells including decrease of glycoprotein through generation of carboxy-hemoglobin, vasoconstriction, and disability of fibrinolysis and changes of metabolism of nucleus pulposus cells. MATERIALS AND METHODS: Annulus fibrosus of intervertebral disc and knee joint cartilage were collected from pigs; these cells were acquired by gradual enzyme decomposition. Using Trypan blue, concentration and survival rate of cells were examined; cells were inserted on alginate beads for tertiary cultivation. Nicotine was then applied at 0, 50, 100, 200 and 300 nM, respectively, and the samples were cultivated for three, six and nine days, respectively. After collecting culture fluid, it was measured for interleukin(IL)-1beta, IL-6 and IL-8 with the ELISA Test. DNA of cells used for cultivation was quantitated and the amount of the resulting proinflammatory mediator was normalized. The results were then compared with the result of same study on cartilage of porcine knee joints. RESULTS: For changes of the inflammatory mediator based on the concentration of nicotine, in nicotine stimulation with low concentration of 50 nM and the control group, there was no significant change, while transient increases of inflammatory mediator showed in nicotine stimulation with concentrations of 100, 200 nM, respectively. There was not a significant increase of IL-1beta observed in all nicotine stimulation groups; these were the same results in porcine cartilage study. The level of IL-6 in 200, 300 nM nicotine concentration showed significant increases, respectively. The level of IL-8 in high dose nicotine stimulation groups also showed significant increases of DNA on the sixth day. And in porcine cartilage study group, significant changes were observed in 200, 300 nM, but the absolute value was lower than that of annulus fibrous cells group. CONCLUSION: Inflammatory mediators such as IL-6 and IL-8 increased as the result of tertiary cultivation of annulus fibrosus cells of porcine intervertebral disk and nicotine stimulation. It is believed that the cells of the disc annulus are more sensitive than articular chondrocytes to nicotine stimulation. This may be the focus of future long-term studies effects of nicotine other inflammatory cytokines.