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Ethnopharmacological relevance Eupafolin, a major bioactive compound found in Phyla nodiflora, has the anti-inflammatory property. Upregulation of cell adhesion molecules in the lung airway epithelium is associated with the epithelium–leukocyte interaction and plays a critical role in the pathogenesis of lung airway inflammatory disorders. To investigate the effects of eupafolin on tumor necrosis factor-α (TNF-α)-induced intercellular cell adhesion molecule-1 (ICAM-1) expression in A549 human lung airway epithelial cells and the underlying mechanisms. Materials and methods The effect of eupafolin on ICAM-1 expression in A549 cells were examined by Western blotting and immunofluorescent staining. The mice were injected intraperitoneally with or without eupafolin and then were left untreated or were injected intratracheally with TNF-α. To detect the effect of eupafolin on ICAM-1 expression, the lung tissues were also examined by Western blotting and immunohistochemical staining. Results Eupafolin pretreatment reduced the TNF-α-induced ICAM-1 expression and also the ERK1/2, JNK, p38, and AKT/PI3K phosphorylation. However, the increase in ICAM-1 expression with TNF-α treatment was unaffected by p38 and PI3K inhibitors. Eupafolin decreased the TNF-α-induced NF-κB p65 activation and its nuclear translocation. Furthermore, eupafolin reduced ICAM-1 expression in the lung tissues of TNF-α-treated mice. Conclusions Eupafolin exerts its anti-inflammatory activity by suppressing the TNF-α-induced ICAM-1 expression and subsequent monocyte adhesion via AKT/ERK1/2/JNK phosphorylation and nuclear translocation of NF-κB p65. These results suggest that eupafolin may represent a novel therapeutic agent targeting epithelial activation in lung inflammation.

International audience; Tumor necrosis factor-alpha (TNF-alpha) has been shown to induce the expression of adhesion molecules in airway resident cells and contribute to inflammatory responses. Here, the roles of mitogen-activated protein kinases (MAPKs) and NF-kappaB in TNF-alpha-induced expression of vascular cell adhesion molecule (VCAM)-1 were investigated in human tracheal smooth muscle cells (HTSMCs). TNF-alpha-enhanced expression of VCAM-1 protein and mRNA as well as phosphorylation of p42/p44 MAPK, p38, and JNK were significantly attenuated by inhibitors of MEK1/2 (U0126), p38 (SB202190), and JNK (SP600125). Transfection with dominant negative mutants of MEK1/2, ERK1, ERK2, p38, and JNK attenuated TNF-alpha-induced VCAM-1 expression. Furthermore, TNF-alpha-induced VCAM-1 expression was significantly blocked by a selective NF-kappaB inhibitor helenalin. TNF-alpha-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha was blocked by helenalin, but not by U0126, SB202190, or SP600125. VCAM-1 promoter activity was enhanced by TNF-alpha in HTSMCs transfected with VCAM-1-Luc, which was inhibited by helenalin, U0126, SB202190, and SP600125. Most surprisingly, VCAM-1 expression was also significantly blocked by a selective inhibitor of p300, curcumin. NF-kappaB transcription factor and p300 were associated with the VCAM-1 promoter, which was dynamically linked to histone H3 acetylation stimulated by TNF-alpha, as determined by chromatin immunoprecipitation assay. Moreover, the resultant enhancement of VCAM-1 expression increased the adhesion of polymorphonuclear cells (PMNs) to monolayer of HTSMCs, which was blocked by helenalin, U0126, SB202190, or SP600125. These results suggest that in HTSMCs, activation of MAPK pathways, NF-kappaB, and p300 is essential for TNF-alpha-induced VCAM-1 expression.

This study was to determine the mechanism of tumor necrosis factor-alpha (TNF-alpha)-enhanced cyclooxygenase (COX)-2 expression associated with prostaglandin E2 (PGE2) synthesis in human tracheal smooth muscle cells (HTSMCs). TNF-alpha markedly increased COX-2 expression and PGE2 synthesis in a time- and concentration-dependent manner, whereas COX-1 remained unaltered. Tyrosine kinase inhibitor (genistein), phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D-609) and PKC inhibitor (GF109203X) attenuated TNF-alpha-induced COX-2 expression and PGE2 synthesis in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis were also inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 and SB202190 (inhibitors of p38 MAPK), respectively, suggesting the involvement of p42/p44 and p38 MAPKs in these responses. This hypothesis was further supported by that TNF-alpha induced a transient activation of p42/p44 and p38 MAPKs in a time-and concentration-dependent manner. Furthermore, TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) reversely correlated with the degradation of IkappaB-alpha in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis was also inhibited by NF-kappaB inhibitor pyrrolidinedithiocarbamate (PDTC). These findings suggest that the increased expression of COX-2 correlates with the release of PGE2 from TNF-alpha-challenged HTSMCs, at least in part, mediated through p42/p44 and p38 MAPKs as well as NF-kappaB signaling pathways in HTSMCs.

Interleukin-1beta (IL-1beta) has been recognized as a potent stimulus for the synthesis of prostaglandin (PG), which has been implicated in inflammatory responses of the airways. However, the mechanisms underlying IL-1beta-induced cyclooxygenase (COX) expression and PGE(2) synthesis via activation of p42/p44 and p38 mitogen-activated protein kinases (MAPKs) in human tracheal smooth muscle cells (HTSMCs) are not completely understood. We found that IL-1beta increased COX-2 expression and PGE(2) synthesis in time- and concentration-dependent manners. Both specific phosphatidylcholine-phospholipase C inhibitor (D609) and protein kinase C inhibitor (GF109203X) attenuated IL-1beta-induced responses in HTSMCs. IL-1beta-induced COX-2 expression and PGE(2) synthesis were also inhibited by an inhibitor of MEK1/2 (PD98059) and inhibitors of p38 MAPK (SB203580 and SB202190), respectively, suggesting the involvement of p42/p44 and p38 MAPKs in these responses. This hypothesis was further supported by the transient activation of p42/p44 and p38 MAPKs induced by IL-1beta. Furthermore, IL-1beta-induced activation of nuclear factor-kappaB (NF-kappaB) was inversely correlated with the degradation of IkappaB-alpha in HTSMCs. IL-1beta-induced COX-2 expression and PGE(2) synthesis were inhibited by the NF-kappaB inhibitor pyrrolidinedithiocarbamate. These findings suggest that the expression of COX-2 is correlated with the release of PGE(2) from IL-1beta-challenged HTSMCs, which is mediated, at least in part, through p42/p44 and p38 MAPKs and NF-kappaB signaling pathways in HTSMCs.

Lipoteichoic acid (LTA), the principal component of the cell wall of gram-positive bacteria, triggers several inflammatory responses. However, the mechanisms underlying its action on human tracheal smooth muscle cells (HTSMCs) were largely unknown. This study was to investigate the mechanisms underlying LTA-stimulated p42/p44 mitogen-activated protein kinase (MAPK) using Western blotting assay. LTA stimulated phosphorylation of p42/p44 MAPK via a Toll-like receptor 2 (TLR2). Pretreatment with pertussis toxin attenuated the LTA-induced responses. LTA-stimulated phosphorylation of p42/p44 MAPK was attenuated by inhibitors of tyrosine kinase (genistein), phosphatidylcholine-phospholipase C (PLC; D609), phosphatidylinositol (PI)-PLC (U-73122), PKC (staurosporine, Gö-6976, rottlerin, or Ro-318220), MEK1/2 (U-0126), PI 3-kinase (LY-294002 and wortmannin), and an intracellular Ca2+chelator (BAPTA-AM). LTA directly evoked initial transient peak of [Ca2+]i, supporting the involvement of Ca2+mobilization in LTA-induced responses. These results suggest that in HTSMCs, LTA-stimulated p42/p44 MAPK phosphorylation is mediated through a TLR2 receptor and involves tyrosine kinase, PLC, PKC, Ca2+, MEK, and PI 3-kinase.

Background Ambient particulate matters (PMs) are known as inducers that adversely affect a variety of human organs. Objectives In this study, we aimed to evaluate the influence of PMs on the permeation of drugs and sunscreens via the skin. The role of skin-barrier properties such as the stratum corneum (SC) and tight junctions (TJs) during the delivery process was explored. Methods This work was conducted using both in vitro and in vivo experiments in pigs to check the responses of the skin to PMs. PMs primarily containing heavy metals (1648a) and polycyclic aromatic hydrocarbons (PAHs, 1649b) were employed to treat the skin. Results According to the transepidermal water loss (TEWL), 1649b but not 1648a significantly disrupted the SC integrity by 2-fold compared to the PBS control. The immunohistochemistry (IHC) of cytokeratin, filaggrin, and E-cadherin exhibited that 1649b mildly damaged TJs. The cytotoxicity of keratinocytes and skin fibroblasts caused by 1649b was stronger than that caused by 1648a. The 1649b elicited apoptosis via caspase-3 activation. The proteomic profiles showed that PMs upregulated Annexin A2 by >5-fold, which can be a biomarker of PM-induced barrier disruption. We found that the skin uptake of ascorbic acid, an extremely hydrophilic drug, was increased from 74 to 112 μg/g by 1649b treatment. The extremely lipophilic drug tretinoin also showed a 2.6-fold increase of skin accumulation. Oxybenzone and dextran absorption was not affected by PMs. The in vivo dye distribution visualized by fluorescence microscopy had indicated that 1649b intervention promoted permeant partitioning into SC. Conclusions Caution should be taken in exposing the skin to airborne dust due to its ability to reduce barrier function and increase the risk of drug overabsorption, although this effect was not very marked.

Extracts from natural plants have been used in traditional medicine for many centuries worldwide.Artocarpus communisis one such plant that has been used to treat liver cirrhosis, hypertension, and diabetes. To our knowledge, this study is the first to investigate the antihepatoma activity ofA. communistoward HepG2 and PLC/PRF/5 cells and the first to explore the relationship between antihepatoma activity and the active compound artocarpin content in different fractions ofA. communis.A. communismethanol extract and fractions induced dose-dependent reduction of tumor cell viability. DNA laddering analysis revealed thatA. communisextract and fractions did not induce apoptosis in HepG2 and PLC/PRF/5 cells. Instead, acridine orange staining revealed thatA. communistriggered autophagic cell death in a dose-dependent manner. The antihepatoma activity ofA. communisis attributable to artocarpin. The fractions with the highest artocarpin content were also the fractions with the highest antihepatoma activity in the following order: dichloromethane fraction > methanol extract > ethyl acetate fraction >n-butanol fraction >n-hexane fraction. Taken together,A. communisshowed antihepatoma activity through autophagic cell death. The effect was related to artocarpin content. Artocarpin could be considered an indicator of the anticancer potential ofA. communisextract.

Background An atypical pattern of neonatal sepsis, characterized by persistent positive blood culture despite effective antimicrobial therapy, has been correlated with adverse outcomes. However, previous studies focused only on coagulate-negative staphylococcus infection. Methods All episodes of persistent bloodstream infection (BSI), defined as 3 or more consecutive positive blood cultures with the same bacterial species, at least two of them 48 hours apart, during a single sepsis episode, were enrolled over an 8-year period in a tertiary level neonatal intensive care unit. These cases were compared with all non-persistent BSI during the same period. Results We identified 81 episodes of persistent BSI (8.5% of all neonatal late-onset sepsis) in 74 infants, caused by gram-positive pathogens (n=38, 46.9%), gram-negative pathogens (n=21, 25.9%), fungus (n=20, 24.7%) and polymicrobial bacteremia (n=2, 2.5%). Persistent BSI does not differ from non-persistent BSI in most clinical characteristics and patient demographics, but tends to have a prolonged septic course, longer duration of feeding intolerance and more frequent requirement of blood transfusions. No difference was observed for death attributable to infection (9.8% vs. 6.5%), but neonates with persistent BSI had significantly higher rates of infectious complications (29.6% vs. 9.2%, P < 0.001), death from all causes (21.6% vs. 11.7%, P = 0.025), and duration of hospitalization among survivors [median (interquartile range): 80.0 (52.5-117.5) vs. 64.0 (40.0-96.0) days, P = 0.005] than those without persistent BSI. Conclusions Although persistent BSI does not contribute directly to increased mortality, the associated morbidities, infectious complications and prolonged septic courses highlight the importance of aggressive treatment to optimize outcomes.

Phyla nodiflorais a creeping perennial herb, widely distributed in the most tropical and subtropical regions. It has been used as a folk medicine, herbal beverage, or folk cosmetic. For these usages, the development of a chemical quality control method of this plant is necessary. In the present study, ten compounds, namely, 3,7,4′,5′-tetrahydroxy-3′-methoxyflavone (1), nodifloretin (2), 4′-hydroxywogonin (3), onopordin (4), cirsiliol (5), 5,7,8,4′-tetrahydroxy-3′-methoxyflavone (6), eupafolin (7), hispidulin (8), larycitrin (9), andβ-sitosterol were isolated from the methanolic extract of the aerial part ofP. nodiflora(PNM) and their structures were identified by 1D-NMR comparing their spectra with the literature. The antioxidant activities of these compounds were evaluated by free radical scavenging activity and tyrosinase inhibitory effect in cell-free systems. Compounds4,5, and7showed strong antioxidant activity. To control the quality ofP. nodiflora, a simple and reliable method of high-performance liquid chromatography combined with ultraviolet detector (HPLC-UV) was established for both the fingerprint analysis and the quantitative determination of two selected active compounds, onopordin (4) and eupafolin (7). Statistical analysis of the obtained data demonstrated that our method achieved the desired linearity, precision, and accuracy. The results indicated that the developed method can be used as a quality evaluation method for PNM.

Background Polymicrobial bloodstream infections (PBSIs) have been associated with complex underlying medical conditions and a high incidence of specific microorganisms in several settings, but the relevant data are scarce in neonates. Methods Positive blood cultures from January 2004 to December 2011 in the neonatal intensive care unit (NICU) of Chang Gung Memorial Hospital (CGMH) were reviewed. Each neonate with PBSI (case episode) was matched to two episodes of monomicrobial BSI (control episode) by birth weight, gestational age and gender. Records were reviewed to compare their underlying medical conditions, organisms isolated, adequacy of therapy, clinical characteristics and outcomes. Results Forty-five episodes of PBSI (4.4% of all neonatal BSIs) were identified in 43 neonates. Gram-negative organisms constituted 59.8% of all PBSI pathogens, and 33 (73.3%) of PBSIs were caused by at least one Gram-negative organism. PBSIs were significantly more likely to be the recurrent episode and have endotracheal tube in place. No significant difference was found between PBSIs and controls in terms of demographics and most chronic conditions. PBSIs were significantly associated with a higher severity of illness, a longer duration of septic symptoms, and a higher rate of modification of antimicrobial regimens than monomicrobial BSIs. However, the sepsis-attributable mortality rates were comparable between these two groups. Conclusions In the NICU, PBSIs were more often caused by Gram-negative bacilli, and often occurred in neonates without any chronic conditions. The clinical significance of PBSIs included a more severe illness, longer duration of septic symptoms and a higher rate of modification of antimicrobial regimens.

Background Late-onset sepsis (LOS) is a common complication in the neonatal intensive care unit. We aimed to describe the epidemiology, clinical characteristics and risk factors for adverse outcome in neonates with LOS. Methods We conducted a cohort study of all neonates with LOS at the neonatal intensive care unit of a Tertiary Taiwan Medical Center from January 2004 through December 2011 and used multivariate logistic regression to identify risk factors for final adverse outcome. Results Among 5010 neonates over 253,644 neonate-days, 713 (14.2%) experienced a total of 942 episodes of LOS (incidence rate, 3.71 episodes per 1000 neonate-days). Although the rates of LOS were inversely proportional to birth weight and gestational age, the incidence rates were comparable among extremely preterm, late preterm and full term neonates. Fungemia was found to have significantly higher rate of infectious complication (30.8%), persistent bloodstream infection (19.2%) and sepsis attributable mortality (23.1%). The overall mortality rate was 12.6% (90/713), and sepsis attributable mortality rate was 7.2% (68/942 episodes). Independent predictors of in-hospital mortality were Pseudomonas LOS (adjusted odds ratio [OR], 14.31; 95% confidence interval [CI]: 3.87-53.0), fungemia (OR, 5.69; 95% CI: 2.48-13.01), presence of congenital anomalies (OR, 4.12; 95% CI: 1.60-10.60), neuromuscular comorbidities (OR, 3.34; 95% CI: 1.66-6.73) and secondary pulmonary hypertension with/without cor pulmonale (OR, 23.48; 95% CI: 5.96-92.49). Conclusions LOS predisposes hospitalized neonates to increased risk of mortality or morbidity, especially caused by Pseudomonas aeruginosa or Candida spp. More aggressive treatment strategy is worth consideration in neonates with presumed LOS, particularly those with certain underlying chronic conditions.

Upregulation of intercellular adhesion molecule-1 (ICAM-1) involves adhesions between both circulating and resident leukocytes and the human lung epithelial cells during lung inflammatory reactions. We have previously demonstrated that curcumin-loaded polyvinylpyrrolidone nanoparticles (CURN) improve the anti-inflammatory and anti-oxidative properties of curcumin in hepatocytes. In this study, we focused on the effects of CURN on the expression of ICAM-1 in TNF-α-treated lung epithelial cells and compared these to the effects of curcumin water preparation (CURH). TNF-αinduced ICAM-1 expression, ROS production, and cell-cell adhesion were significantly attenuated by the pretreatment with antioxidants (DPI, APO, or NAC) and CURN, but not by CURH, as revealed by western blot analysis, RT-PCR, promoter assay, and ROS detection and adhesion assay. In addition, treatment of TNF-α-treated cells with CURN and antioxidants also resulted in an inhibition of activation of p47 (phox) and phosphorylation of MAPKs, as compared to that using CURH. Our findings also suggest that phosphorylation of MAPKs may eventually lead to the activation of transcription factors. We also observed that the effects of TNF-α treatment for 30 min, which includes a significant increase in the binding activity of AP-1 and phosphorylation of c-jun and c-fos genes, were reduced by CURN treatment. In vivo studies have revealed that CURN improved the anti-inflammation activities of CURH in the lung epithelial cells of TNF-α-treated mice. Our results indicate that curcumin-loaded polyvinylpyrrolidone nanoparticles may potentially serve as an anti-inflammatory drug for the treatment of respiratory diseases.

Background We aimed to characterize the clinical manifestations and outcomes of patients with Acinetobacter baumannii bacteremia in the neonatal intensive care unit (NICU). Methods All patients with A baumannii bacteremia in our NICU from 2004 to 2010 were reviewed. A matched case-control study was performed by comparing each case of A baumannii to 2 uninfected controls and all cases of Escherichia coli and Klebsiella bacteremia, respectively. Results Thirty-seven cases with A baumannii bacteremia were identified. Multidrug-resistant isolate was noted in only 2 cases (5.4%), and the overall mortality rate was 8.1%. Compared with matched, uninfected controls, infants with A baumannii were more likely to have had a central vascular catheter (CVC) (P = .009), use of total parenteral nutrition (TPN) (P = .021), longer duration of ventilator use (P = .002), and hospitalization (P = .010). Compared with E coli or Klebsiella bacteremia, infants with A baumannii bacteremia had lower birth weight (median of 1,090 g vs 1,300 g, P = .044) and a higher rate of CVC and TPN use (both P Conclusion A baumannii bacteremia occurs endemically or sporadically in the NICU, primarily in low-birth-weight infants on TPN use and with CVC in situ. Although A baumannii does not often cause mortality, and multidrug-resistant A baumannii is uncommon, it contributes significantly to longer hospitalization.

Keloids are characterized by the vigorously continuous production of extracellular matrix protein and aberrant cytokine activity in the dermis. There is a growing body of evidence that thalidomide, α-N-phthalimidoglutarimide, has anti-fibrotic properties. The aims were to examine possible therapeutic effects of thalidomide on fibronectin expression in transforming growth factor-β1 (TGF-β1)-treated normal fibroblasts (NFs) and keloid-derived fibroblasts (KFs) and the underlying mechanism of action, especially the involvement of mitogen-activated protein kinase (MAPKs) and Sma- and Mad-related family (Smads) pathways. In surgically removed human tissues, TGF-β1 and fibronectin immunoreactivity was high in keloid tissue, but barely detectable in normal tissue. TGF-β1 induced significant fibronectin expression in NFs and KFs and the effect was inhibited by pretreatment with thalidomide. TGF-β1 also induced phosphorylation of MAPKs (ERK1/2, p38, and JNK) and Smad2/3 and pretreatment with PD98059 (an ERK1/2 inhibitor), SB203580 (a p38 inhibitor), or SP600125 (a JNK inhibitor) inhibited TGF-β1-induced fibronectin expression. Furthermore, pretreatment with thalidomide inhibited the TGF-β1-induced phosphorylation of p38 and Smad3, but not that of ERK1/2, JNK, and Smad2. In addition, thalidomide pretreatment inhibited the TGF-β-induced DNA binding activity of AP-1 and Smad3/4, caused fibronectin degradation by increasing the activity of matrix metalloproteinase 9, and decreased production of TGF-β1 and fibronectin and the number of fibroblasts in an in vivo keloid model. These results show that thalidomide has an antifibrotic effect on keloid fibroblasts that is caused by suppression of TGF-β1-induced p38 and Smad3 signaling. Our findings indicate that thalidomide may be a potential candidate drug for the treatment and prevention of keloids.

Resveratrol (RES), a well-known antioxidant and anti-inflammatory compound, is abundant in red wine and exerts numerous pharmacological effects, including hepatoprotection and cadioprotection. Unfortunately, RES is restricted in clinical application due to poor dissolution property and adsorption. In addition, red wine as a supplement for preventing disease is not recommended for patients with alcohol-related disorders. To address these limitations, we successfully developed a novel RES nanoparticle system (RESN) and demonstrated that RESN could circumvent the physicochemical drawbacks of raw RES with respect to dissolution, such as the reduction of particle size, amorphous transformation, and hydrogen-bond formation. In addition, we employed an animal model of CCl₄-induced hepatotoxicity to estimate the potential of the nanoparticle formulation to improve the hepatoprotective effect of orally administered RES. Our results demonstrated that RESN can diminish liver function markers (aspartate aminotransferase and alanine aminotransferase) by decreasing hepatocyte death due to CCl₄-induced hepatotoxicity in rats, when compared with RES administration. The effect was achieved by reducing oxidative stress (decreased reactive oxygen species and lipid peroxidation) and lowering inflammatory cytokines (decreased tumor necrosis factor-α and interleukin 1β) and protein expression (cyclooxygenase-2, inducible nitric oxide synthase, cytosolic phospholipase A2, and caspase-3). In conclusion, enhancement of the dissolution of RES through a nanoparticle engineering process can result in increased hepatoprotective effects mediated by antioxidant and anti-inflammatory activities. Consequently, we suggest that RESN deserves further study, perhaps in prophylaxis of chronic liver diseases.

Kaempferol (KAE) is a strong antioxidant flavonoid compound, but its clinical application is limited by quantity and poor dissolution property. However, the dissolution mechanism of a kaempferol nanoparticle formulation (KAEN) has not yet been elucidated. The aim of the present study was therefore to use a nanoparticle engineering process to resolve the dissolution problem. Our data indicated that KAEN effectively increased the dissolution percentage by particle size reduction, high encapsulation efficiency, amorphous transformation, and hydrogen-bond formation with excipients. In addition, we used several different antioxidant activity assays to evaluate KAE and KAEN. The data indicated that KAEN retained potent antioxidant activity after the nanoparticle engineering process and showed better antioxidant activity than KAE dissolved in water (P < 0.05). According to these findings, we concluded that KAEN could be a low-dose alternative to KAE in health food and future clinical research.

Cytosolic phospholipase A(2) (cPLA(2)) plays a pivotal role in mediating agonist-induced arachidonic acid (AA) release for prostaglandin (PG) synthesis during inflammation triggered by IL-1beta. However, the mechanisms underlying IL-1beta-induced cPLA(2) expression and PGE(2) synthesis in human tracheal smooth muscle cells (HTSMCs) remain unknown. IL-1beta-induced cPLA(2) protein and mRNA expression, PGE(2) production, or phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK1/2, which was attenuated by pretreatment with the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK1/2 (SP600125) or transfection with siRNAs of MEK1, p42, p38, and JNK2. IL-1beta-induced cPLA(2) expression was also inhibited by pretreatment with a NF-kappaB inhibitor, helenalin or transfection with siRNA of NIK, IKKalpha, or IKKbeta. IL-beta-induced NF-kappaB translocation was blocked by pretreatment with helenalin, but not U0126, SB202190, and SP600125. In addition, transfection with p300 siRNA blocked cPLA(2) expression induced by IL-1beta. Moreover, p300 was associated with the cPLA(2) promoter, which was dynamically linked to histone H4 acetylation stimulated by IL-1beta. These results suggest that in HTSMCs, activation of MAPKs, NF-kappaB, and p300 are essential for IL-1beta-induced cPLA(2) expression and PGE(2) secretion.

Histone acetylation regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) plays a critical role in the expression of inflammatory genes, such as vascular cell adhesion molecule-1 (VCAM-1). Oxidative processes have been shown to induce VCAM-1 expression. Here, we investigated the mechanisms underlying IL-1beta-induced VCAM-1 expression in human tracheal smooth muscle cells (HTSMCs). Our results showed that IL-1beta enhanced HTSMCs-monocyte adhesion through up-regulation of VCAM-1, which was inhibited by pretreatment with selective inhibitors of PKCalpha (Go6976), c-Src (PP1), NADPH oxidase [diphenylene iodonium (DPI) and apocynin (APO)], intracellular calcium chelator (BAPTA/AM), PI-PLC (U73122), CaM (calmidazolium chloride), CaM kinase II (KN62), p300 (garcinol), NF-kappaB (Bay11-7082), HDAC (trichostatin A), and ROS scavenger [N-acetyl-L-cysteine (NAC)] or transfection with siRNAs of MyD88, PKCalpha, Src, p47(phox), p300, and HDAC4. Moreover, IL-1beta stimulated NF-kappaB and CaMKII phosphorylation through MyD88-dependent PI-PLC/PKCalpha/c-Src/ROS and PI-PLC/Ca2+/CaM pathways, respectively. Activation of NF-kappaB and CaMKII may eventually lead to the acetylation of histone residues and phosphorylation of histone deacetylases. These findings suggested that IL-1beta induced VCAM-1 expression via these multiple signaling pathways in HTSMCs. Blockade of these pathways may reduce monocyte adhesion via VCAM-1 suppression and attenuation of the inflammatory responses in airway diseases.

Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid release for prostaglandin (PG) synthesis during stimulation with interleukin-1beta (IL-1beta). However, the mechanisms underlying IL-1beta-induced cPLA2 expression and PGE2 synthesis by canine tracheal smooth muscle cells (CTSMCs) have not been defined. IL-1beta induced cPLA2 protein and mRNA expression, PGE2 production, and phosphorylation of p42/p44 MAPK, p38 MAPK (ATF2), and JNK (c-Jun) in a time- and concentration-dependent manner, determined by Western blotting, RT-PCR, and ELISA, which was attenuated by the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK (SP600125), or transfection with dominant negative mutants of MEK1/2, p38, and JNK, respectively. Furthermore, IL-1beta-induced cPLA2 expression and PGE2 synthesis was inhibited by a selective NF-kappaB inhibitor (helenalin) or transfection with dominant negative mutants of NF-kappaB inducing kinase (NIK), IkappaB kinase (IKK)-alpha, and IKK-beta. Consistently, IL-1beta stimulated both IkappaB-alpha degradation and NF-kappaB translocation into nucleus in these cells. NF-kappaB translocation was blocked by helenalin, but not by U0126, SB202190, and SP600125. MAPKs together with NF-kappaB-activated p300 recruited to cPLA2 promoter thus facilitating the binding of NF-kappaB to cPLA2 promoter region and expression of cPLA2 mRNA. IL-1beta-induced cPLA2 expression and PGE2 production was inhibited by actinomycin D and cycloheximide, indicating the involvement of transcriptional and translational events in these responses. These results suggest that in CTSMCs, IL-1beta-induced cPLA2 expression and PGE2 synthesis was independently mediated through activation of MAPKs and NF-kappaB pathways and was connected to p300 recruitment and activation.

Up-regulation of vascular cell adhesion molecule-1 (VCAM-1) involves adhesions between both circulating and resident leukocytes and the human tracheal smooth muscle cells (HTSMCs) during airway inflammatory reaction. We have demonstrated previously that tumor necrosis factor (TNF)-alpha-induced VCAM-1 expression is regulated by mitogen-activated protein kinases, nuclear factor-kappaB, and p300 activation in HTSMCs. In addition to this pathway, phosphorylation of Akt and CaM kinase II has been implicated in histone acetyltransferase and histone deacetylase 4 (HDAC4) activation. Here, we investigated whether these different mechanisms participated in TNF-alpha-induced VCAM-1 expression and enhanced neutrophil adhesion. TNF-alpha significantly increased HTSMC-neutrophil adhesions, and this effect was associated with increased expression of VCAM-1 on the HTSMCs and was blocked by the selective inhibitors of Src [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1)], epidermal growth factor receptor [EGFR; 4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline, (AG1478)], phosphatidylinositol 3-kinase (PI3K) [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride(LY294002) and wortmannin],calcium[1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester; BAPTA-AM], phosphatidylinositol-phospholipase C (PLC) [1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122)], protein kinase C (PKC) [12-(2-cyanoethyl)-6,7,12, 13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö6976), rottlerin, and 3-1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl) maleimide (bisindolylmaleimide IX) (Ro 31-8220)], CaM (calmidazolium chloride), CaM kinase II [(8R(*),9S(*),11S(*))-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3,9, 10-tetrahydro-8,11-epoxy, 1H,8H, 11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinden-1-one (KT5926) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN62)], p300 (curcumin), and HDAC (trichostatin A) or transfection with short interfering RNAs for Src, Akt, PKCalpha, PKCmu, and HDAC4. At gene regulation level, reverse-transcriptase polymerase chain reaction and promoter assays revealed that expression of VCAM-1 was also attenuated by these signaling molecule inhibitors. Moreover, TNF-alpha induced Akt and CaM kinase II phosphorylation via cascades through Src/EGFR/PI3K and PLC/calcium/CaM, respectively. Finally, activation of Akt and CaM kinase II may eventually lead to the acetylation of histone residues and phosphorylation of histone deacetylase. These findings revealed that TNF-alpha induced VCAM-1 expression via multiple signaling pathways. Blockade of these pathways may be selectively targeted to reduce neutrophil adhesion via VCAM-1 suppression and attenuation of the inflammatory responses in airway diseases.

This study aimed to identify independent predictors of clinical and microbiological treatment failure and develop a predictive model for neonates with bloodstream infection (BSI). This study included 1087 episodes of BSIs in 793 neonates in a tertiary-level neonatal intensive care unit of northern Taiwan between 2004 and 2012. Patient demographics, underlying chronic comorbidities, clinical features, antimicrobial treatment and microbiological characteristics were evaluated. The presence of underlying congenital anomalies (odds ratio [OR] 2.12, 95% confidence interval [CI] 1.09 to 4.10) and pulmonary hypertension (OR 3.63, 95% CI 1.70 to 7.74), infections caused by multidrug-resistant gram-negative bacteria (OR 2.89, 95% CI 1.23 to 6.79), group B Streptococcus (OR 3.15, 95% CI 1.33 to 7.46), and fungi (OR 4.13, 95% CI 2.02 to 8.46), a Neonatal Therapeutic Intervention Scoring System score of ≥ 23 (OR 6.96, 95% CI 2.55 to 28.58), inappropriate antibiotics (OR 2.13, 95% CI 1.41 to 3.23), and concomitant meningitis (OR 4.25, 95% CI 2.08 to 8.69) and ventilator-associated pneumonia (OR 2.73, 95% CI 1.22 to 6.13) were identified as independent risk factors for 28-day treatment failure in neonatal BSI. A risk score model was created by adding the points for each independent risk factor, and had a c-statistic of 0.83. Patients with risk scores of 0, 4, 8, 12 and 15 had estimated 28-day treatment failure rates of approximately 3.5%, 17.0%, 53.5%, 86.6% and 95.9%, respectively. This predictive model, calculated after documentation of a BSI, reflects a spectrum of BSI severity and was associated with subsequent treatment failure through illness severity score and case mix variables. This simple score could prove useful in clinical and research settings, and practical in estimating the prognosis.

Lipopolysaccharide (LPS) has been shown to induce the expression of adhesion molecules on airway epithelial and smooth cells and contributes to inflammatory responses. Here, the roles of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) pathways for LPS-induced vascular cell adhesion molecule (VCAM)-1 expression were investigated in HTSMCs. LPS-induced expression of VCAM-1 protein and mRNA in a time-dependent manner, was significantly inhibited by inhibitors of MEK1/2 (U0126), p38 (SB202190), and c-Jun-N-terminal kinase (JNK; SP600125). The involvement of p42/p44 MAPK and p38 in these responses was further confirmed by that transfection with small interference RNAs (siRNA) direct against MEK, p42, and p38 significantly attenuated LPS-induced VCAM-1 expression. Consistently, LPS-stimulated phosphorylation of p42/p44 MAPK and p38 was attenuated by pretreatment with U0126 or SB202190, and transfection with these siRNAs, respectively. In addition, LPS-induced VCAM-1 expression was significantly blocked by a specific NF-kappaB inhibitor helenalin. LPS-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha was blocked by helenalin, U0126, SB202190, or SP600125. Moreover, the resultant enhancement of VCAM-1 expression increased the adhesion of polymorphonuclear cells to monolayer of HTSMCs which was blocked by pretreatment with helenalin, U0126, or SP600125 prior to LPS exposure. Taken together, these results suggest that in HTSMCs, activation of p42/p44 MAPK, p38, and JNK pathways, at least in part, mediated through NF-kappaB, is essential for LPS-induced VCAM-1 gene expression. These results provide new insight into the mechanisms of LPS action that bacterial toxins may promote inflammatory responses in the airway disease.

TNF-alpha has been shown to induce matrix metalloproteinase-9 (MMP-9) expression, which, in turn, degrades extracellular matrix in the inflammatory responses. However, the inductive mechanisms of the MMP-9 by TNF-alpha remain unclear. In human tracheal smooth muscle cells, TNF-alpha induced MMP-9 expression and Akt phosphorylation in a time-dependent manner, which was attenuated by the inhibitors of Src (PP1), epidermal growth factor receptor (AG1478), PDGFR (AG1296), and PI3K (LY294002), respectively, revealed by reporter gene assay, RT-PCR, zymographic, and Western blot analyses. Transfection with the dominant negative mutants of c-Src (KM, K295M [kinase inactive mutant]), p85, and Akt (KA, K179A) also reduced MMP-9 expression. These findings indicated that MMP-9 expression was regulated by PI3K/Akt via the transactivation of growth factor receptors. Furthermore, LY294002 or wortmannin inhibited Akt phosphorylation but had no effect on NF-kappaB translocation, which was blocked by helenalin. Mutated NF-kappaB DNA binding element in the MMP-9 promoter and helenalin also attenuated MMP-9 expression, suggesting that PI3K/Akt and NF-kappaB independently regulated MMP-9 expression. To support this notion, immunofluorescence staining and immunoprecipitation were applied to characterize the transcription factors involved in these responses. The results showed that LY294002 and curcumin blocked Akt translocation into nucleus. In contrast, p300, acetyl-histone (H3), and NF-kappaB p65 were found to be coimmunoprecipitated with the phosphorylated Akt, indicating that these components associated with the MMP-9 promoter are revealed by chromatin immunoprecipitation assay. Thus, our study provides a new insight into the molecular mechanisms that TNF-alpha-stimulated Akt phosphorylation mediated through transactivation of Src and growth factor receptors may stimulate the recruitment of p300, assemble transcription factor (p65), and then lead to MMP-9 expression.

Several factors have been shown to trigger the pathogenesis of asthma and airway inflammation mechanisms. Elevated levels of pro-inflammatory cytokines including tumor necrosis factor-alpha and interleukin-1beta in the bronchoalveolar lavage fluid have been detected in asthmatic patients. Cytokines exert as potent stimuli in inflammatory responses through up-regulation of many gene expressions, including cytokines, chemokines, cytosolic phospholipase A2, cyclooxygenase, adhesion molecules and matrix metalloproteinases. The extent of these gene expressions is correlated with the severity of inflammation. However, the intracellular signaling mechanisms underlying the expression of target proteins regulated by these factors are elusive. The mechanisms underlying actions by cytokines may be integrated to the signaling networks that augment airway inflammation by recruiting leukocytes and leading to airway remodeling. Although cytokines have been reported to activate mitogen-activated protein kinases including p42/p44 and p38, and c-Jun N-terminal kinase, the relationship between the activation of these pathways and expression of inflammatory genes remains unknown. Moreover, many genes regulated by mitogen-activated protein kinases are dependent on NF-kappaB for transcription. NF-kappaB has also been shown to be involved in target protein expression at the transcriptional level in various cell types. We review the mechanisms underlying the intracellular signaling involved in several target protein expressions induced by cytokines in airway resident cells.Increased understanding of signal transduction mechanisms underlying target protein gene expression will create opportunities for the development of anti-inflammation therapeutic strategies.

Interleukin-1beta (IL-1beta) has been shown to induce the expression of adhesion molecules on airway epithelial and smooth cells and contributes to inflammatory responses. Here, the roles of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) pathways for IL-1beta-induced vascular cell adhesion molecule (VCAM)-1 expression were investigated in human tracheal smooth muscle cells (HTSMC). IL-1beta induced expression of VCAM-1 protein and mRNA in a time-dependent manner, which was significantly inhibited by inhibitors of MEK1/2 (U0126 and PD-98059), p38 (SB-202190), and c-Jun NH(2)-terminal kinase (JNK; SP-600125). Consistently, IL-1beta-stimulated phosphorylation of p42/p44 MAPK, p38, and JNK was attenuated by pretreatment with U0126, SB-202190, or SP-600125, respectively. IL-1beta-induced VCAM-1 expression was significantly blocked by the specific NF-kappaB inhibitors helenalin and pyrrolidine dithiocarbamate. As expected, IL-1beta-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha were blocked by helenalin but not by U0126, SB-202190, or SP-600125. Moreover, the resultant enhancement of VCAM-1 expression increased the adhesion of polymorphonuclear cells to a monolayer of HTSMC, which was blocked by pretreatment with helenalin, U0126, SB-202190, or SP-600125 before IL-1beta exposure or by anti-VCAM-1 antibody. Together, these results suggest that in HTSMC, activation of p42/p44 MAPK, p38, JNK, and NF-kappaB pathways is essential for IL-1beta-induced VCAM-1 gene expression. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in airway disease.

Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid (AA) release for prostaglandins (PG) synthesis induced by bacterial lipopolysaccharide (LPS) and cytokines. However, the intracellular signaling pathways mediating LPS-induced cPLA2 expression and PGE2 synthesis in canine tracheal smooth muscle cells (TSMCs) remains unknown. LPS-induced expression of cPLA2 and release of PGE2 was attenuated by inhibitors of tyrosine kinase (genistein), phosphatidylcholine-phospholipase C (D609), phosphatidylinositol-phospholipase C (U73122), PKC (GF109203X and staurosporine), removal of Ca2+ by BAPTA/AM plus EDTA, MEK1/2 (PD98059), p38 (SB202190), JNK (SP600125), and phosphatidylinositol 3-kinase (PI3-K; LY294002 and wortmannin). The involvement of MPAKs in LPS-induced responses was further confirmed by transfection of TSMCs with dominant negative mutants of ERK2 and p38. LPS-induced cPLA2 expression and PGE2 synthesis was inhibited by a selective NF-kappaB inhibitor (helenalin) and transfection with dominant negative mutants of NF-kappaB inducing kinase (NIK), IkappaB kinase (IKK)-alpha, and IKK-beta, consistent with that LPS-stimulated both IkappaB-alpha degradation and NF-kappaB translocation into nucleus in these cells. LPS-stimulated cPLA2 phosphorylation was inhibited by PD98059, GF109203X, and staurosporine, indicating the regulation by p42/p44 MAPK and PKC. Moreover, LPS-induced up-regulation of cPLA2 and COX-2 linked to PGE2 synthesis was inhibited by AACOCF3 (a selective cPLA2 inhibitor), implying the involvement of cPLA2 in these responses. These findings suggest that phosphorylation and expression of cPLA2 correlates with the release of PGE2 from LPS-challenged TSMCs, at least in part, mediated through MAPKs and NF-kappaB signaling pathways. LPS-mediated responses were modulated by PLC, Ca2+, PKC, tyrosine kinase, and PI3-K in TSMCs.

BackgroundMany natural products used in preventive medicine have also been developed as cosmeceutical ingredients in skin care products, such asScutellaria baicalensisandGardenia jasminoides. Norartocarpetin is one of the antioxidant and antityrosinase activity compound inArtocarpus communis; however, the cytotoxicity, skin irritation and antimelanogenesis mechanisms of norartocarpetin have not been investigated yet.MethodsIn the present study, cell viabilityin vitroand skin irritationin vivoare used to determine the safety of norartocarpetin. The melanogenesis inhibition of norartocarpetin was determined by cellular melanin content and tyrosinase in B16F10 melanoma cell. Moreover, we examined the related-melanogenesis protein by western blot analysis for elucidating the antimelanogenesis mechanism of norartocarpin.ResultsThe result of the present study demonstrated that norartocarpetin not only present non-cytotoxic in B16F10 and human fibroblast cells but also non-skin irritation in mice. Moreover, our result also first found that norartocarpetin downregulated phospho-cAMP response element-binding (phospho-CREB) and microphthalmia-associated transcription factor (MITF) expression, which in turn decreased both synthesis of tyrosinases (TRP-1 and TRP-2) and cellular melanin content. This process is dependent on norartocarpetin phosphorylation by mitogen-activated protein kinases such as phospho-JNK and phospho-p38, and it results in decreased melanogenesis.ConclusionThe present study suggests that norartocarpetin could be used as a whitening agent in medicine and/or cosmetic industry and need further clinical study.