Leng-Fang Wang

Inhibitory effects of a rice hull constituent on tumor necrosis factor α, prostaglandin E2, and cyclooxygenase-2 production in lipopolysaccharide-activated mouse macrophages

Abstract: Isovitexin, isolated from rice hull of Oryza sativa, has been characterized as a potent antioxidant. Its antioxidant activity, determined on the basis of inhibition of lipid peroxidation by the Fenton reaction, was comparable with that of α‐tocopherol, a well‐established antioxidant. Isovitexin was able to reduce the amount of hydrogen peroxide production induced by lipopolysaccharide (LPS) in mouse macrophage RAW264.7 cells. In this study, we assessed its effects on the production of tumor necrosis factor α (TNF‐α), prostaglandin E2 (PGE2), and the expression of cyclooxygenase‐2 (COX‐2) in LPS‐activated RAW 264.7 macrophages. Isovitexin inhibited the release of TNF‐α, a proinflammatory cytokine, upon LPS activation with a 50% inhibitory concentration (IC50) of 78.6 μM. Isovitexin markedly reduced LPS‐stimulated PGE2 production in a concentration‐dependent manner, with an IC50 of 80.0 μM. The expression of COX‐2 was also inhibited by isovitexin treatment. Our results suggest that suppression of ROS‐mediated COX‐2 expression by isovitexin is beneficial in reducing inflammation and carcinogenesis.

The renin-angiotensin system mediates hyperoxia-induced collagen production in human lung fibroblasts.

A high concentration of oxygen can cause lung injury and lead to pulmonary fibrosis. Angiotensin (Ang) II induces human lung fibroblast proliferation and stimulates collagen synthesis. However, the role of the renin-angiotensin system (RAS) in the pathogenesis of hyperoxia-induced collagen production is unclear. The aims of this study were to investigate the effects of hyperoxia on the components of the RAS and collagen expression in human lung fibroblasts (MRC-5). Hyperoxia increased total collagen, collagen type I, and alpha-smooth muscle actin (alpha-SMA) mRNA and protein expression. RAS components and Ang II production were also significantly increased after hyperoxic exposure. Hyperoxia induced Ang II type 1 receptor (AT1R) expression but did not alter AT2R expression, furthermore, silencing of AT1R signaling with small interfering RNA suppressed hyperoxia-induced phosphorylated-ERK (p-ERK) 1/2, alpha-SMA, and collagen type I expression. Ang II increased p-ERK 1/2 and collagen type I expression, and these increases were inhibited by the AT1R inhibitor, losartan, but not by the AT2R inhibitor, PD123319 under both normoxic and hyperoxic conditions. These data suggest Ang II-mediated signaling transduction via AT1R is involved in hyperoxia-induced collagen synthesis in human lung fibroblasts.

Chymase mediates paraquat-induced collagen production in human lung fibroblasts

Survivors of paraquat poisoning may be left with pulmonary fibrosis and a restrictive type of pulmonary dysfunction. Chymase converts angiotensin (Ang) I to Ang II, which is closely involved with lung fibrosis. The role played by chymase in paraquat-induced lung fibrosis is unclear. We examined the effects of paraquat on chymase, renin-angiotensin system components, and collagen expression in murine and human lung fibroblasts (MRC-5). Lung chymase and collagen type I mRNA and protein expression were significantly increased and angiotensin-converting enzyme (ACE) mRNA and protein expression were comparable between the control and paraquat-treated mice 1 and 3 weeks after administration. Paraquat significantly upregulated angiotensinogen mRNA expression in a dose-dependent manner while ACE activity and protein expression were similar in MRC-5 cells. Furthermore, paraquat enhanced Ang II and collagen type I mRNA and protein expression, alpha-smooth muscle actin, and chymase protein and chymase small interfering RNA inhibited these effects. The cDNA sequence of chymase in MRC-5 cells is identical to that in human mast cells. This study found increased chymase expression in paraquat-treated human lung fibroblasts and confirmed in vitro and in an in vivo paraquat model of lung fibrosis that chymase generates Ang II and enhances collagen expression. These data suggest a role for chymase in the pathogenesis of paraquat-induced lung fibrosis.

Methylprednisolone effects on oxygenation and histology in a rat model of acute lung injury

We examined the effects of methylprednisolone on gas exchange, pressure-volume curve, lavage fluid inflammatory cell counts, protein content, surfactant pool size, and lung histology in a rat model of paraquat-induced lung injury. Twenty-three adult male Sprague-Dawley rats received intraperitoneal paraquat injection (35 mg/kg) and were randomly divided into three groups: (1). control group received no further treatment; (2). 1-dose methylprednisolone group received a concomitant intraperitoneal methylprednisolone injection (30 mg/kg); (3). 3-dose methylprednisolone group received a concomitant and daily intraperitoneal methylprednisolone injection (30 mg/kg) for three doses. Three days after paraquat injection, the rat was ventilated for 90 min, a static pressure-volume curve and bronchoalveolar lavage was performed, and postmortem histology was examined. Surfactant pool size of the 3-dose methylprednisolone group was significantly increased when compared with the control and 1-dose methylprednisolone groups. Methylprednisolone treatment increased oxygenation and the value was statistically significant for 3-dose methylprednisolone group at 90 min of ventilation. Inflammatory cell counts in bronchoalveolar lavage fluid and lung injury score were decreased as the methylprednisolone dose increased. We conclude that high-dose methylprednisolone treatment increased surfactant pool size and improved lung histology of paraquat-injured lungs but this augmentation could not significantly improve oxygenation throughout the ventilation period.

Activation of the renin-angiotensin system in hyperoxia-induced lung fibrosis in neonatal rats

Background: Oxygen toxicity plays an important role in lung injury and may lead to bronchopulmonary dysplasia. We previously demonstrated that hyperoxia activated the renin-angiotensin system (RAS) in cultured human fetal lung fibroblasts. Objective: To examine whether the upregulation of RAS components is associated with hyperoxia-induced lung fibrosis in neonatal Sprague-Dawley rats. Methods: Experimental rat pups were exposed to 1 week of >95% O2 and a further 2 weeks of 60% O2. Control pups were exposed to room air over the same periods. Lung tissues were taken for biochemical and histochemical assays on postnatal days 7 and 21. Results: Hyperoxia significantly increased total collagen content and the expression of type I collagen and alpha smooth muscle actin when compared to control rats. RAS components including angiotensinogen, angiotensin-converting enzyme, angiotensin II, and angiotensin II type 1 receptor were significantly upregulated by hyperoxia. The results also demonstrated that only the extracellular signal-regulated kinase (ERK) signaling pathway was activated by hyperoxia exposure. p38 mitogen-activated protein kinase and c-Jun N-terminal kinase were not activated. Conclusions: Local RAS activation is involved in the pathogenesis of hyperoxia-induced lung fibrosis in neonatal rats. ERK phosphorylation might mediate angiotensin II type 1 receptor activation.

Angiotensin II type 1 receptor antagonist attenuates lung fibrosis in hyperoxia-exposed newborn rats.

Bronchopulmonary dysplasia (BPD) remains a major cause of morbidity and mortality during the first year of life, and many infants have significant respiratory problems throughout childhood. Currently no effective therapy is clinically available to prevent the long-term pulmonary sequelae of BPD. Previous research has demonstrated that the renin-angiotensin system is up-regulated in human lung fibroblasts. Angiotensin II type 1 receptor (AT1R) antagonists and AT1R short interfering RNA diminished hyperoxia-increased collagen expression, whereas AT2R antagonists did not have any effects on these hyperoxia-induced changes. The in vivo therapeutic effects of AT1R antagonists on hyperoxia-induced lung fibrosis remain unknown. The present study assessed the effects of an AT1R antagonist (losartan) on preventing hyperoxia-induced lung fibrosis in newborn rats. Rat pups were exposed to 7 days of >95% O2 and an additional 2 weeks of 60% O2. AT1R antagonist-treated pups were injected intraperitoneally with losartan at a dose of 10 mg/kg/day from postnatal days 1 to 7 and a dose of 5 mg/kg/day from postnatal days 8 to 21. Control group pups were injected with an equal volume of normal saline. AT1R antagonist treatment attenuated the hyperoxia-induced lung fibrosis on postnatal days 7 and 21 and also decreased the hyperoxia-induced expression of extracellular signal-regulated protein kinase and α-smooth muscle actin. AT1R antagonist treatment did not affect body weight or lung weight of the rats. These data suggest that AT1R antagonist may offer a novel therapeutic strategy for preventing hyperoxia-induced lung fibrosis.

Retinoic Acid Fails to Reverse Oligohydramnios-Induced Pulmonary Hypoplasia in Fetal Rats

All-trans retinoic acid (ATRA) stimulates platelet-derived growth factor (PDGF)-A expression and enhances alveolarization in rat lungs. On d 16 of gestation, pregnant Sprague-Dawley rats were randomly assigned to either a retinoic acid group (intragastric ATRA at 10 mg/kg body weight) or a vehicle group. We punctured each amniotic sac, and fetuses in the opposite uterine horn served as controls. On d 21 of gestation, the fetuses were delivered by cesarean section. Rats subjected to oligohydramnios exhibited significantly lower lung weights and lung/body weight ratios, and ATRA had no effects on the body or lung weights of oligohydramnios-exposed rats. Lung PDGF-A and -B mRNA expression was significantly lower in oligohydramnios-exposed rats compared with control littermates of maternal vehicle-treated dams. Maternal retinoic acid treatment significantly increased PDGF-A and -B mRNA expression in control and oligohydramnios-exposed rats compared with all rats and oligohydramnios-exposed rats of maternal vehicle-treated dams, respectively. Rats exposed to oligohydramnios exhibited a significantly lower generation of alveolar saccules than did control rats in the maternal retinoic acid– and vehicle-treated groups. In this model, maternal retinoic acid treatment showed no positive effects on oligohydramnios-induced pulmonary hypoplasia in the pseudoglandular stage.

Paraquat increases connective tissue growth factor and collagen expression via angiotensin signaling pathway in human lung fibroblasts

Survivors of paraquat poisoning are left with pulmonary fibrosis which results in a restrictive type of long-term pulmonary dysfunction. Connective tissue growth factor (CTGF) is a key growth factor that initiates tissue repair and underlies the development of lung fibrosis. Angiotensin (ANG) II may induce CTGF expression in the heart and kidney and plays an important role in the pathogenesis of lung fibrosis. The biological effects of ANG II are mediated by ANG II type 1 receptor (AT1R) and AT2R. The aims of this study were to investigate the effects of paraquat on ANG II, ANG II receptors, CTGF, and collagen expressions and to assess the role of ANG II receptors in paraquat-induced collagen synthesis in human lung fibroblasts (MRC-5). MRC-5 cells were incubated with various concentrations of paraquat with or without the ANG II receptor antagonist, saralasin. Paraquat increased ANG II production and AT1R mRNA and protein expression and decreased AT2R mRNA expression. Furthermore, paraquat treatment increased CTGF and collagen mRNA and protein expression in a dose-dependent manner and saralasin inhibited these effects. These results indicate that paraquat increases CTGF and collagen expression by activating angiotensin signaling pathway in human lung fibroblasts.

Oligohydramnios Decreases Platelet-Derived Growth Factor Expression in Fetal Rat Lungs

Objective: To evaluate the effects ofexperimental oligohydramnios on lung growth, expression of platelet-derived growth factor (PDGF) and its receptors, and lung morphology in fetal rats. Methods: On day 16 of gestation, we anesthetized timed pregnant Sprague-Dawley dams and punctured uterine wall and fetal membranes of each uterine sac which resulted in oligohydramnios. The fetuses in the opposite uterine horn served as controls. On days 19 and 21 of gestation, the fetuses were delivered by cesarean section and weighed, and the lungs were dissected free and weighed. Results: Rats exposed to oligohydramnios exhibited significantly lower lung/body weight ratios on days 19 and 21 of gestation and significantly lower radial saccular counts on day 21 of gestation than did the control rats. Lung PDGF-A and PDGF-B gene and protein expression and elastin level were significantly decreased in rats exposed to oligohydramnios on days 19 and 21 of gestation. The PDGF receptor alpha and beta gene expression levels were significantly decreased in rats exposed to oligohydramnios on day 19 of gestation. Conclusion: A decreased PDGF expression may be important in the pathogenesis of oligohydramnios-induced pulmonary hypoplasia and suggests that supplementation may provide useful therapeutic strategies.

Prevention of cellular oxidative damage by an aqueous extract of Anoectochilus formosanus.

Abstract: Anoectochilus formosanus (AF) is a popular folk medicine in Taiwan whose pharmacological effects have been characterized. In this work we investigated the antioxidant properties of an aqueous extract prepared from AF. The AF extract was capable of scavenging H2O2 in a dose‐dependent manner. We induced oxidative stress in HL‐60 cells, either by the addition of hydrogen peroxide (H2O2) or by the xanthine/xanthine oxidase reaction. Apoptosis caused by oxidative damage was displayed by DNA fragmentation on gel electrophoresis, and the apoptotic fraction was quantified with flow cytometry. The cell damage induced by oxidative stress was prevented by the plant extract in a concentration‐dependent manner. Furthermore, the proteolytic cleavage of poly(ADP‐ribose) polymerase during the apoptotic process was also inhibited by AF extract. Our results provide the basis for determining an AF extract to be an antioxidant.