Ruey-Hseng Lin

Luteolin attenuates the pulmonary inflammatory response involves abilities of antioxidation and inhibition of MAPK and NFκB pathways in mice with endotoxin-induced acute lung injury.

Acute lung injury (ALI) in critically ill patients remains the leading cause of mortality and morbidity. Lipopolysaccharide (LPS) is a key mediator of lung injury. This study investigates the protective effects and mechanisms of luteolin in intratracheal instillation of LPS (100μg)-induced ALI in mice. Pretreatment of mice with 70μmol/kg luteolin significantly restores LPS-induced decrease in oxygen pressure and increase in carbon dioxide in arterial blood. The histopathological study established 70μmol/kg luteolin pretreatment markedly attenuates lung histopathological changes, such as haemorrhaging, interstitial edema, and infiltration of polymorphonuclear neutrophils (PMNs) into the lung parenchyma and alveolar spaces. Sufficient evidence for luteolin (35 and 70μmol/kg) suppresses activation and infiltration of PMNs is obtained in expression of surface marker CD11b and Ly6G on cells in bronchoalveolar lavage fluid (BALF) cells and myeloperoxidase activity in lung tissue. Furthermore, luteolin reduces the activity of catalase and superoxide dismutase, and the level of oxidative damage, and lipid peroxidation, in lung tissue. In addition, the secretion of TNF-α, KC, and ICAM-1 in the BALF after LPS challenge are also inhibited by luteolin. Moreover, luteolin reduced LPS-induced activation of MAPK and NFκB pathways. Therefore, luteolin is a potential protective antagonists for LPS-induced ALI in mice.

Protective effects of luteolin against lipopolysaccharide-induced acute lung injury involves inhibition of MEK/ERK and PI3K/Akt pathways in neutrophils

AbstractAim:To investigate whether luteolin, the major polyphenolic components of Lonicera japonica, has beneficial effects against lipopolysaccharide (LPS)-induced acute lung injury (ALI) and to determine whether the protective mechanism involves anti-inflammatory effects on neutrophils.Methods:ALI was induced with intratracheal instillation of LPS in mice. The level of ALI was determined by measuring the cell count and protein content in bronchoalveolar lavage (BAL) fluid. Neutrophils were stimulated with formyl-Met-Leu-Phe (fMLP) or LPS in vitro. Chemotaxis and superoxide anion generation were measured to evaluate neutrophil activation. The potential involvement of intracellular signaling molecules in regulating neutrophil activation was analyzed by using Western blot.Results:LPS induced ALI in mice, as evidenced with leukocyte infiltration and protein leakage into the lungs. Luteolin attenuated LPS-induced leukocyte infiltration and protein extravasation. In cell studies, luteolin attenuated the fMLP-induced neutrophil chemotaxis and respiratory burst (IC50 0.2±0.1 μmol/L and 2.2±0.8 μmol/L, respectively), but had a negligible effect on superoxide anion generation during phorbol myristate acetate stimulation. Furthermore luteolin effectively blocked MAPK/ERK kinase 1/2 (MEK), extracellular signal-regulated kinase (ERK), and Akt phosphorylation in fMLP- and LPS-stimulated neutrophils.Conclusion:These results indicate that luteolin has beneficial effects against LPS-induced ALI in mice, and the attenuation of neutrophil chemotaxis and respiratory burst by luteolin involves the blockade of MEK-, ERK-, and Akt-related signaling cascades.

Diet, vegetarian food and prostate carcinoma among men in Taiwan

In a case–control study in a veterans hospital in Taiwan, we compared 237 histology-confirmed prostate carcinoma cases with 481 controls, frequency matched by age, for their consumption of vegetarian food, namely soybean products, rice, wheat protein and other vegetables. The multivariable logistic regression analysis showed a significant association with such food (odds ratio (OR)=0.67, 95% confidence interval (CI)=0.47, 0.94). This beneficial effect presented for men with body mass index (BMI) ⩽25 kg m−2 (OR=0.50, 95% CI=0.32, 0.76) but not for men with greater BMI. The OR of prostate carcinoma for men with BMI ⩽25 kg m−2 was 1.74 (95% CI=1.21, 2.51), compared with men with higher BMI (>25 kg m−2). Other significant risk factors associated with the disease included higher income (OR=2.40, 95% CI=1.07, 5.42), physical activity (OR=1.75, 95% CI=1.08, 2.83), being married (OR=2.49, 95% CI=1.40, 4.43) and coffee consumption (OR=1.88, 95% CI=1.07, 3.30). Stratified analysis also showed that the consumption of fish/shellfish had an adverse association for men with higher BMI. This study suggests that the intake of the low fat local vegetarian food has a protective effect against prostate carcinoma for thin men in this study population.

Inhibition of nitric oxide production by the carbazole compound LCY-2-CHO via blockade of activator protein-1 and CCAAT/enhancer-binding protein activation in microglia

Excessive nitric oxide (NO) production by activated microglia plays a critical role in neurodegenerative disorders. In this study, we found that 9-(2-chlorobenyl)-9H-carbazole-3-carbaldehyde (LCY-2-CHO) suppressed the NO production in lipopolysaccharide (LPS)/interferon-gamma (IFNgamma)-stimulated murine microglial N9 and BV-2 cells and in LPS-stimulated N9 cells and rat primary microglia. LCY-2-CHO had no cytotoxic effect on microglia. In activated N9 cells, LCY-2-CHO abolished the expression of inducible nitric oxide synthase (iNOS) protein and mRNA but failed to alter the stability of expressed iNOS mRNA and the enzymatic activity of expressed iNOS protein. LCY-2-CHO did not block DNA-binding activity of nuclear factor-kappaB (NF-kappaB) or cyclic AMP response element-binding protein (CREB), but abolished that of activator protein-1 (AP-1), CCAAT/enhancer-binding protein (C/EBP) and nuclear factor IL6 (NF-IL6). LCY-2-CHO attenuated the nuclear levels of c-Jun and C/EBPbeta, but not those of p65, p50, C/EBPdelta, signal transducer and activator of transcription-1 (STAT-1) or the nuclear expression of IFN regulatory factor-1 (IRF-1). LCY-2-CHO had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), MAPK-activated protein kinase-2 (MAPKAPK-2), STAT-1, CREB or c-Jun in LPS/IFNgamma-stimulated N9 cells, whereas it attenuated the phosphorylation of C/EBPbeta at Ser105 and Thr235 residues, which occurred concomitantly with LCY-2-CHO inhibition of C/EBPbeta expression and phosphorylation. Taken together, these results suggest that LCY-2-CHO inhibits NO production in microglia through the blockade of AP-1 and C/EBP activation.

Distinct genotoxicity of phenylmercury acetate in human lymphocytes as compared with other mercury compounds

In the present study, the frequency of sister chromatid exchanges (SCEs) was assayed to evaluate the genotoxic effects of mercury nitrate (Hg2+), methylmercury chloride (CH3HgCl and phenylmercury acetate (PMA) on human lymphocytes. The free radical scavengers, catalase (CA) and superoxide dismutase (SOD) were tested for their antigenotoxic effects toward PMA. PMA (1-30 microM) increased SCE frequency in a concentration-dependent manner. However, CH3HgCl significantly increased SCE frequency only at a concentration of 20 microM, and all concentrations treated with Hg2+ did not induce a positive effect. On the other hand, we first reported that 30 microM Hg2+, 20 microM CH3HgCl and (3-30 microM) PMA significantly increased the frequency of endoreduplicated mitosis. PMA was about 3- or 5-fold more effective in inducing endoreduplication than CH3HgCl or Hg2+ at equivalent toxic concentrations, respectively. However, neither CA nor SOD in concentrations of 75 and 150 microg/ml showed antagonistic action on the genotoxic effects of PMA. The results suggest that the mechanism of PMA-induced genotoxicity is not mediated by superoxide anion nor H2O2. It is concluded that PMA, which was more effective in inducing the elevation of both SCEs and endoreduplication, may be especially hazardous of the three mercury compounds tested.

Inhibition of superoxide anion generation by CHS-111 via blockade of the p21-activated kinase, protein kinase B/Akt and protein kinase C signaling pathways in rat neutrophils

In formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils, 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111) inhibited superoxide anion (O(2)(-)) generation, which was not mediated by scavenging the generated O(2)(-) or by a cytotoxic effect, and attenuated migration. CHS-111 had no effect on the arachidonic acid-induced NADPH oxidase activation or the GTPgammaS-stimulated Rac2 membrane translocation in cell-free systems, whereas it effectively attenuated the membrane recruitment of p40(phox), p47(phox) and p67(phox), phosphorylation of Ser residues in p47(phox), association between p47(phox) and p22(phox), and Rac activation in fMLP-stimulated neutrophils. Moreover, the phosphorylation and membrane recruitment of p21-activated kinase (PAK), PAK kinase activity and the interaction of PAK with p47(phox) were inhibited by CHS-111. CHS-111 effectively reduced Akt kinase activity and the association between Akt and p47(phox), moderately inhibited the membrane recruitment of Akt and phospho-PDK1, and slightly attenuated Akt (Thr308) phosphorylation, whereas it had no effect on Akt (Ser473) phosphorylation or p110gamma membrane translocation. The membrane recruitment of protein kinase C (PKC)-alpha, -betaI, -betaII, -delta and -zeta, PKC phosphorylation and PKC kinase activity was attenuated by CHS-111, whereas CHS-111 did not affect the phosphorylation of p38 mitogen-activated protein kinase (MAPK) or downstream MAPK-activated protein kinase-2. Higher concentrations of CHS-111 were required to decrease fMLP-stimulated intracellular free Ca(2+) concentration ([Ca(2+)](i)) elevation in the presence but not in the absence of extracellular Ca(2+), and to reduce cellular cyclic AMP but slightly increase cyclic GMP levels. Taken together, these results suggest that CHS-111 inhibits fMLP-stimulated O(2)(-) generation in rat neutrophils through the blockade of PAK, Akt and PKC signaling pathways.

The in vivo effect of lipopolysaccharide on neuromuscular transmission in the mouse

The in vivo effect of lipopolysaccharide (endotoxin) on nerve-evoked muscle contractions and neuromuscular transmission was studied in the mouse phrenic nerve-diaphragm preparations. In lipopolysaccharide-treated mouse diaphragms it was observed that indirectly induced twitch tension was unchanged while tetanic tension significantly decreased. Neostigmine (50 nM) increased the amplitude of nerve evoked muscle contractions, while it caused partial fade of tetanic contractions (Wedensky inhibition) and accelerated the run-down of end-plate potentials (e.p.ps) evoked by repetitive nerve stimulation, in the diaphragm of saline-control mice, but not of lipopolysaccharide-treated mice. These effects of neostigmine could be abolished by ouabain (5 microM). Measurement of the quantal contents of e.p.ps revealed that ouabain (5 microM) significantly increased it in the diaphragm of saline-control mice to an extent similar to that in diaphragm of lipopolysaccharide-treated mice. Moreover, ouabain-sensitive Na+, K(+)-ATPase activity in the sciatic nerve of lipopolysaccharide-treated mice was markedly decreased. The alterations in neuromuscular transmission of the diaphragm during endotoxemia could be reversed by the administration of polymyxin B (a lipopolysaccharide neutralizer) and NG-nitro-L-arginine (a nitric oxide (NO) synthase inhibitor), suggesting that NO is involved in these lipopolysaccharide-induced alterations of neuromuscular transmission mediated by an impairment of ouabain-sensitive Na+, K(+)-ATPase activity in mouse motor nerves.

Artocarpol A stimulation of superoxide anion generation in neutrophils involved the activation of PLC, PKC and p38 mitogen-activated PK signaling pathways

1 Artocarpol A (ART), a natural phenolic compound isolated from Artocarpus rigida, stimulated a slow onset and long‐lasting superoxide anion generation in rat neutrophils, whereas only slightly activated the NADPH oxidase in a cell‐free system. 2 Pretreatment of neutrophils with pertussis toxin (1 μg ml−1), 50 μM 2′‐amino‐3′‐methoxyflavone (PD 98059), or 1 μM 1,4‐diamino‐2,3‐dicyano‐1,4‐bis(2‐aminophenylthio)butadiene (U0126) had no effect on ART‐stimulated superoxide anion generation. ART (30 μM) did not induce extracellular signal‐regulated kinase (ERK) phosphorylation. 3 4‐(4‐Fluorophenyl)‐2‐(4‐methylsulfinylphenyl)‐5‐(4‐pyridyl)‐1H‐imidazole (SB 203580) markedly attenuated the ART‐stimulated superoxide anion generation (IC50 value of 4.3±0.3 μM). Moreover, ART induced p38 mitogen‐activated PK (MAPK) phosphorylation and activation. 4 The superoxide anion generation in response to ART was also substantially inhibited in a Ca2+‐free medium, and by pretreatment with 1 μM 1‐[6‐((17β‐3‐methoxyestra‐1,3,5(10)‐trien‐17‐yl)amino)hexyl]‐1H‐pyrrole‐2,5‐dione (U‐73122) and 100 μM 2‐aminoethyldiphenyl borate (2‐APB). ART (30 μM) stimulated the [Ca2+]i elevation in the presence or absence of external Ca2+, and also increased the D‐myo‐inositol 1,4,5‐trisphosphate formation. 5 2‐[1‐(3‐Dimethylaminopropyl)‐1H‐indol‐3‐yl]‐3‐(1H‐indol‐3‐yl)‐maleimide (GF 109203X) greatly inhibited the ART‐stimulated superoxide anion generation (IC50 value of 7.8±1.0 nM). ART increased the recruitment of PKC‐α, ‐βI, and ‐βII to the plasma membrane of neutrophils, and stimulated Ca2+‐dependent PKC activation in the cytosol preparation. 6 ART induced the phosphorylation of p47phox, which was attenuated by GF 109203X. Moreover, ART evoked the membrane association of p47phox, which was inhibited by GF 109203X and SB 203580. 7 These results indicate that the ART stimulation of superoxide anion generation involved the activation of p38 MAPK, PLC/Ca2+, and PKC signaling pathways in rat neutrophils.

Effects of germanium oxide and other chemical compounds on phenylmercury acetate‐induced genotoxicity in cultured human lymphocytes

Phenylmercury acetate (PMA), which not only causes an elevation of sister chromatid exchanges (SCEs) but also induces high frequency of endore‐duplication in human lymphocytes, may be genotoxic to humans. The major aim of our study was to investigate the effects of germanium oxide (GeO2), D‐penicillamine (D‐PA), dimercaprol (BAL), and diltiazem (DTM) on PMA‐induced genotoxicity as quantified by SCEs. All concentrations of the four chemical compounds tested alone did not induce genotoxicity in cultured human lymphocytes. However, GeO2 significantly inhibited PMA‐induced genotoxicity in a concentration‐dependent manner. Similarly, D‐PA at concentrations of 3 μM and 10 μM, and BAL at a concentration of 30 μM produced the antigenotoxic effects. In addition, GeO2 (1.5 μM) significantly reversed an increase of endoreduplication frequency caused by PMA. In a cell cycle kinetic study, GeO2 (0.5–5.0 μM) reversed the inhibition of PMA on the proliferating rate index (PRI) of lymphocytes. On the contrary, both D‐PA and DTM at concentrations of 30–300 μM markedly potentiated PMA‐induced inhibition of PRI. These findings show that GeO2, D‐PA, and BAL could antagonize PMA‐induced genotoxicity, and GeO2 appears to be the most effective. Environ. Mol. Mutagen. 31:157–162, 1998

Signaling mechanisms of inhibition of phospholipase D activation by CHS-111 in formyl peptide-stimulated neutrophils

A selective phospholipase D (PLD) inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) inhibited the O(2)(-) generation and cell migration but not degranulation in formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils. A novel benzyl indazole compound 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111), which inhibited O(2)(-) generation and cell migration, also reduced the fMLP- but not phorbol ester-stimulated PLD activity (IC(50) 3.9±1.2μM). CHS-111 inhibited the interaction of PLD1 with ADP-ribosylation factor (Arf) 6 and Ras homology (Rho) A, and reduced the membrane recruitment of RhoA in fMLP-stimulated cells but not in GTPγS-stimulated cell-free system. CHS-111 reduced the cellular levels of GTP-bound RhoA, membrane recruitment of Rho-associated protein kinase 1 and the downstream myosin light chain 2 phosphorylation, and attenuated the interaction between phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and Arf6, whereas it only slightly inhibited the guanine nucleotide exchange activity of human Dbs (DH/PH) protein and did not affect the arfaptin binding to Arf6. CHS-111 inhibited the interaction of RhoA with Vav, the membrane association and the phosphorylation of Vav. CHS-111 had no effect on the phosphorylation of Src family kinases (SFK) but attenuated the interaction of Vav with Lck, Hck, Fgr and Lyn. CHS-111 also inhibited the interaction of PLD1 with protein kinase C (PKC) α, βI and βII isoenzymes, and the phosphorylation of PLD1. These results indicate that inhibition of fMLP-stimulated PLD activity by CHS-111 is attributable to the blockade of RhoA activation via the interference with SFK-mediated Vav activation, attenuation of the interaction of Arf6 with PLD1 and PIP5K, and the activation of Ca(2+)-dependent PKC in rat neutrophils.