Nanoparticles prepared from the water extract of Gusuibu (Drynaria fortunei J. Sm.) protects osteoblasts against insults and promotes cell maturation

Our previous study showed that Gusuibu (Drynaria fortunei J. Sm.) can stimulate osteoblast maturation. This study was further designed to evaluate the effects of nanoparticles prepared from the water extract of Gusuibu (WEG) on osteoblast survival and maturation. Primary osteoblasts were exposed to 1, 10, 100, and 1000 μg/mL nanoparticles of WEG (nWEG) for 24, 48, and 72 hours did not affect morphologies, viability, or apoptosis of osteoblasts. In comparison, treatment of osteoblasts with 1000 μg/mL WEG for 72 hours decreased cell viability and induced DNA fragmentation and cell apoptosis. nWEG had better antioxidant bioactivity in protecting osteoblasts from oxidative and nitrosative stress-induced apoptosis than WEG. In addition, nWEG stimulated greater osteoblast maturation than did WEG. Therefore, this study shows that WEG nanoparticles are safer to primary osteoblasts than are normal-sized products, and may promote better bone healing by protecting osteoblasts from apoptotic insults, and by promoting osteogenic maturation

Lipoteichoic acid induces surfactant protein-A biosynthesis in human alveolar type II epithelial cells through activating the MEK1/2-ERK1/2-NF-κB pathway

<p>Abstract</p> <p>Background</p> <p>Lipoteichoic acid (LTA), a gram-positive bacterial outer membrane component, can cause septic shock. Our previous studies showed that the gram-negative endotoxin, lipopolysaccharide (LPS), could induce surfactant protein-A (SP-A) production in human alveolar epithelial (A549) cells.</p> <p>Objectives</p> <p>In this study, we further evaluated the effect of LTA on SP-A biosynthesis and its possible signal-transducing mechanisms.</p> <p>Methods</p> <p>A549 cells were exposed to LTA. Levels of SP-A, nuclear factor (NF)-κB, extracellular signal-regulated kinase 1/2 (ERK1/2), and mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1 were determined.</p> <p>Results</p> <p>Exposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability. Meanwhile, when exposed to 30 μg/ml LTA for 1, 6, and 24 h, the biosynthesis of SP-A mRNA and protein in A549 cells significantly increased. As to the mechanism, LTA enhanced cytosolic and nuclear NF-κB levels in time-dependent manners. Pretreatment with BAY 11–7082, an inhibitor of NF-κB activation, significantly inhibited LTA-induced SP-A mRNA expression. Sequentially, LTA time-dependently augmented phosphorylation of ERK1/2. In addition, levels of phosphorylated MEK1 were augmented following treatment with LTA.</p> <p>Conclusions</p> <p>Therefore, this study showed that LTA can increase SP-A synthesis in human alveolar type II epithelial cells through sequentially activating the MEK1-ERK1/2-NF-κB-dependent pathway.</p

Effects of lipopolysaccharide (LPS) on the translocation and transactivation of GATA-2.

<p>RAW 264.7 cells were exposed to 100 ng/ml LPS for 1, 3, and 6 h. Amounts of nuclear GATA-2 were immunodetected (A, <i>top panel</i>). Levels of nuclear PCNA were measured as the internal control. These immunorelated proteins were quantified and statistically analyzed (<i>bottom panel</i>). RAW 264.7 cells were treated with 100 ng/ml LPS, the translocation of GATA-2 from the cytoplasm to nuclei were analyzed using confocal microscopy (B). The transactivation activity of GATA-2 was assayed using an EMSA analysis (C). These DNA-protein bands were quantified and statistically analyzed (D). The immunoblotting, confocal, and DNA-protein binding results shown are a representative of at least 3 experiments, and the other statistically analyzed results are a compilation of 6 replications. Each value represents the mean ± SD. An asterisk (*) indicates that the value significantly differed from the respective control, <i>p</i> < 0.05.</p

Roles of MyD88 in lipopolysaccharide (LPS)-induced activation of MEK1/2.

<p>RAW 264.7 cells were subjected to MyD88 small interfering (si) RNA for 24 and 48 h. Levels of MyD88 were immunodetected (A, <i>top panel</i>). β-Actin was measured as the internal control (<i>bottom panel</i>). These protein bands were quantified and statistically analyzed (B). RAW 264.7 cells were exposed to LPS, MyD88 siRNA, and a combination of MyD88 siRNA and LPS. Amounts of phosphorated MEK1/2 were immunodetected (C, <i>top panel</i>). MEK1 was measured as the internal control (<i>bottom panel</i>). These protein bands were quantified and statistically analyzed (D). The immunoblotting results shown are a representative of 6 experiments, and the other statistically analyzed results are a compilation of 6 replications. Each value represents the mean ± SD. An asterisk (*) and pound sign (#) respectively indicate that the value significantly (<i>p</i> < 0.05) differed from the respective control and LPS-treated group.</p

Roles of GATA-2 in lipopolysaccharide (LPS)-induced interleukin (IL)-1β mRNA expression.

<p>RAW 264.7 cells were subjected to GATA-2 small interference (si) RNA for 24 and 48 h. Levels of GATA-2 were immunodetected (A, <i>top panel</i>). β-Actin was measured as the internal control (<i>bottom panel</i>). These protein bands were quantified and statistically analyzed (B). RAW 264.7 cells were exposed to LPS, GATA-2 siRNA, and a combination of GATA-2 siRNA and LPS. Amounts of IL-1β mRNA were determined using an RT-PCR analysis (C, <i>top panel</i>). β-Actin mRNA was analyzed as the internal control (<i>bottom panel</i>). These DNA bands were quantified and statistically analyzed (D). A real-time PCR analysis was conducted to confirm the roles of GATA-2 (E). Effects of GATA-2 siRNA on LPS-induced IL-1β production were determined by ELISA (F). The immunoblotting results shown are a representative of 6 experiments, and the other statistically analyzed results are a compilation of 6 replications. Each value represents the mean ± SD. An asterisk (*) and pound sign (^#) indicate that a value significantly (<i>p</i> < 0.05) differed from the respective control and LPS-treated group, respectively.</p

Effects of MyD88 small interference (si) RNA and MAPK inhibitors on translocation of GATA-2.

<p>RAW 264.7 cells were exposed to lipopolysaccharide (LPS), MyD88 siRNA, and a combination of MyD88 siRNA and LPS. Amounts of GATA-2 were immunodetected (A, <i>top panel</i>). PCNA was measured as the internal control (<i>bottom panel</i>). These protein bands were quantified and statistically analyzed (B). RAW 264.7 cells were pretreated with 10 µM MAPK inhibitors, including SB203580 (SB), SP600125 (SP), and PD98059 (PD), for 1 h and then exposed to LPS. Nuclear GATA-2 was immunodetected (C, <i>top panel</i>). Amounts of PCNA were measured as the internal control (<i>bottom panel</i>). These protein bands were quantified and statistically analyzed (D). The immunoblotting results shown are a representative of 6 experiments, and the other statistically analyzed results are a compilation of 6 replications. Each value represents the mean ± SD. An asterisk (*) and pound sign (^#) respectively indicate that the value significantly (<i>p</i> < 0.05) differed from the respective control and LPS-treated group.</p

Roles of GATA-2 in lipopolysaccharide (LPS)-induced interleukin (IL)-1β mRNA expression in primary macrophages.

<p>Peritoneal macrophages prepared from mice were identified using an immunocytochemical confocal analysis of the F4/80 protein, a macrophage-specific marker (A, <i>top panel</i>). After exposure to 100 ng/ml LPS for 1, 3, and 6 h, the levels of IL-1β mRNA in primary macrophages were quantified using a real-time PCR analysis (A, <i>bottom panel</i>). The amounts of IL-1β protein were determined using ELISA (B). The transactivation activity of GATA-2 was assayed using an EMSA analysis (C). Primary macrophages were treated with LPS, GATA-2 siRNA, and a combination of GATA-2 siRNA and LPS, and IL-1β mRNA were quantified (D). The immunoblotting, confocal, and DNA-protein binding results shown are a representative of 6 experiments, and the other statistically analyzed results are a compilation of 6 replications. Each value represents the mean ± SD. An asterisk (*) and pound sign (^#) respectively indicate that the value significantly (<i>p</i> < 0.05) differed from the respective control and LPS-treated group.</p