Yen-Jen Sung

Magnolol inhibits Mac-1 (CD11b/CD18)-dependent neutrophil adhesion : Relationship with its antioxidant effect

Magnolol, a phenolic compound isolated from a Chinese herbal drug, Magnolia officinalis, has been shown to protect rat heart from ischemia/reperfusion injury. Neutrophil adhesion plays a crucial process during this inflammatory response. To evaluate whether magnolol prevents ischemia/reperfusion injury by inhibiting neutrophil adhesion, we determined whether magnolol can inhibit adhesion of phorbol-12-myristate-13-acetate (PMA)-activated human neutrophils to a fibrinogen-coated surface in a dose-dependent manner. Using flow cytometric analysis, we observed that magnolol pretreatment (10 min at 37 degrees C) diminished PMA (100 ng/ml)-induced Mac-1 upregulation. PMA also induced rapid intracellular accumulation of superoxide (O2-.) and hydrogen peroxide (H2O2) in neutrophils; magnolol pretreatment attenuated the accumulation of these two substances. Inhibition of reactive oxygen species by superoxide dismutase and/or catalase, which decompose O2-. and H2O2, respectively, also abolished Mac-1 upregulation and neutrophil adhesion. We conclude that magnolol inhibits neutrophil adhesion and that this can account for its anti-ischemia/reperfusion injury effect. We propose that the inhibitory effect of magnolol on neutrophil adhesion to the extracellular matrix is mediated, at least in part, by inhibition of the accumulation of reactive oxygen species, which in turn suppresses the upregulation of Mac-1 that is essential for neutrophil adhesion.

TGF-β1 blockade of microglial chemotaxis toward Aβ aggregates involves SMAD signaling and down-regulation of CCL5

BackgroundOveractivated microglia that cluster at neuritic plaques constantly release neurotoxins, which actively contribute to progressive neurodegeneration in Alzheimers disease (AD). Therefore, attenuating microglial clustering can reduce focal neuroinflammation at neuritic plaques. Previously, we identified CCL5 and CCL2 as prominent chemokines that mediate the chemotaxis of microglia toward beta-amyloid (Aβ)aggregates. Although transforming growth factor-β1 (TGF-β1) has been shown to down-regulate the expression of chemokines in activated microglia, whether TGF-β1 can reduce the chemotaxis of microglia toward neuritic plaques in AD remains unclear.MethodsIn the present study, we investigated the effects of TGF-β1 on Aβ-induced chemotactic migration of BV-2 microglia using time-lapse recording, transwell assay, real-time PCR, ELISA, and western blotting.ResultsThe cell tracing results suggest that the morphological characteristics and migratory patterns of BV-2 microglia resemble those of microglia in slice cultures. Using this model system, we discovered that TGF-β1 reduces Aβ-induced BV-2 microglial clustering in a dose-dependent manner. Chemotactic migration of these microglial cells toward Aβ aggregates was significantly attenuated by TGF-β1. However, these microglia remained actively moving without any reduction in migration speed. Pharmacological blockade of TGF-β1 receptor I (ALK5) by SB431542 treatment reduced the inhibitory effects of TGF-β1 on Aβ-induced BV-2 microglial clustering, while preventing TGF-β1-mediated cellular events, including SMAD2 phosphorylation and CCL5 down-regulation.ConclusionsOur results suggest that TGF-β1 reduces Aβ-induced microglial chemotaxis via the SMAD2 pathway. The down-regulation of CCL5 by TGF-β1 at least partially contributes to the clustering of microglia at Aβ aggregates. The attenuating effects of SB431542 upon TGF-β1-suppressed microglial clustering may be mediated by restoration of CCL5 to normal levels. TGF-β1 may ameliorate microglia-mediated neuroinflammation in AD by preventing activated microglial clustering at neuritic plaques.

Tetrandrine ameliorates ischaemia‐reperfusion injury of rat myocardium through inhibition of neutrophil priming and activation

We have previously shown that tetrandrine (TTD), a bisbenzyltetrahydroiosquinoline isolated from the Chinese herb Stephania tetrandra, inhibits neutrophil adhesion, Mac‐1 expression, and reactive oxygen species (ROS) production. To examine whether inhibition of neutrophil function may confer upon TTD the ability to prevent myocardial ischaemia‐reperfusion (MI/R) injury, experiments were performed on rats subjected to coronary ligation followed by reperfusion for induction of MI/R injury. Intravenous administration of TTD (0.1 and 1.0 mg kg−1) 15 min prior to coronary ligation completely prevented MI/R‐associated mortality. TTD pretreatment also significantly reduced MI/R‐induced ventricular tachyarrhythmia, myocardial infarct size, and neutrophil infiltration. However, TTD pretreatment did not influence mean arterial blood pressure, heart rate, or product of pressure‐rate, indicating that TTD extenuated MI/R through mechanisms independent of modulating haemodynamics or myocardial oxygen demand. Peripheral blood neutrophils were isolated for ex vivo examination of shape change and Mac‐1 upregulation of neutrophils, two sensitive indicators of proinflammatory priming, as well as N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP)‐induced adhesion and ROS production, parameters commonly used for the assessment of neutrophil activation. Neutrophils from MI/R animals showed significant shape change and Mac‐1 upregulation, both of which were prevented by TTD‐pretreatments. On the other hand, fMLP‐induced adhesion and ROS production of neutrophils were markedly enhanced by MI/R but diminished in TTD‐pretreated animals. These data suggest that the protective effect of TTD against MI/R injury can be accounted for by inhibition of neutrophil priming and activation, thereby abolishing subsequent infiltration and ROS production that cause MI/R injury.

Induction of p38 Mitogen-Activated Protein Kinase-Mediated Apoptosis Is Involved in Outgrowth of Trophoblast Cells on Endometrial Epithelial Cells in a Model of Human Trophoblast-Endometrial Interactions

Abstract During embryo implantation in species with hemochorial placentation, such as the mouse and human, trophoblast cells of the attached blastocyst penetrate the luminal epithelium of the endometrium before invasion into the endometrial stroma. Signs of apoptosis were demonstrated in luminal endometrial epithelial cells (EEC) adjacent to the trophoblast cells; however, the signaling mechanisms leading to apoptosis in EEC remain unclear. Because mitogen-activated protein kinases (MAPK) were shown to mediate apoptosis in several model systems and found to be activated in the uterus during decidualization, the possible involvement of MAPK during trophoblast-EEC interactions was studied. By coculturing BeWo human trophoblast spheroids with RL95-2 human EEC monolayers to mimic the blastocyst-endometrial interaction, we found that most spheroids rapidly attached to EEC monolayers and then progressively expanded, with marked dislodgment of EEC adjacent to the spreading trophoblast cells. Immunoblotting analysis showed that both p38 MAPK and extracellular signal-regulated kinase (ERK) were activated in EEC after coculture. However, only SB203580 (a p38 MAPK inhibitor), but not PD98059 (an ERK inhibitor), inhibited trophoblast outgrowth on EEC monolayers through the suppression of p38 MAPK activation in EEC. Furthermore, trophoblast expansion caused prominent EEC apoptosis at the spheroid-EEC interface, as detected by annexin V labeling and valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (which binds activated caspases) staining, and SB203580 significantly decreased the percentage of apoptotic cells. Our results, based on a model of human trophoblast-EEC interactions, establish that trophoblast cells cause activation of p38 MAPK in EEC and, consequently, induce apoptosis and displacement of EEC, a process that may facilitate implantation.

Intercellular calcium waves mediate preferential cell growth toward the wound edge in polarized hepatic cells

During liver regeneration, hepatocytes sense the damage and initiate proliferation of the quiescent cells through poorly understood mechanisms. Here, we have used cultured hepatic cells to study the roles played by intercellular calcium in mediating wound-healing processes. Well-differentiated and polarized Hep-G2 cells repaired an experimentally induced wound by induction of cell divisions. The resulting cellular growth did not occur evenly across the healing cell lawn; instead, proliferations were three times more active within 150-200 microm from the wound edge than further away; this periwound preferential cell growth was not observed in the poorly differentiated and/or nonpolarized cells. We have provided experimental evidence demonstrating that the wounding procedure itself could elicit a propagating calcium wave, and interestingly, blocking this injury-associated intercellular calcium communication could effectively inhibit the biased cell growth along the margin of the wound. A photolithography-based patterned cell culture system was employed to help delineate the mechanisms underlying this type of calcium signaling. In conclusion, our results suggested that intercellular communications via propagating calcium waves coordinate regenerative cell proliferations in response to hepatic tissue losses.

Collagen IV significantly enhances migration and transplantation of embryonic stem cells: involvement of α2β1 integrin-mediated actin remodeling.

Embryonic stem (ES) cell transplantation represents a potential means for the treatment of degenerative diseases and injuries. As appropriate distribution of transplanted ES cells in the host tissue is critical for successful transplantation, the exploration of efficient strategies to enhance ES cell migration is warranted. In this study we investigated ES cell migration under the influence of various extracellular matrix (ECM) proteins, which have been shown to stimulate cell migration in various cell models with unclear effects on ES cells. Using two mouse ES (mES) cell lines, ESC 26GJ9012-8-2 and ES-D3 GL, to generate embryoid bodies (EBs), we examined the migration of differentiating cells from EBs that were delivered onto culture surfaces coated with or without collagen I, collagen IV, Matrigel, fibronectin, and laminin. Among these ECM proteins, collagen IV exhibited maximal migration enhancing effect. mES cells expressed α2 and β1 integrin subunits and the migration enhancing effect of collagen IV was prevented by RGD peptides as well as antibodies against α2 and β1 integrins, indicating that the enhancing effect of collagen IV on cell migration was mediated by α2β1 integrin. Furthermore, staining of actin cytoskeleton that links to integrins revealed well-developed stress fibers and long filopodia in mES cells cultured on collagen IV, and the actin-disrupting cytochalasin D abolished the collagen IV-enhanced cell migration. In addition, pretreatment of undifferentiated or differentiated mES cells with collagen IV resulted in improved engraftment and growth after transplantation into the subcutaneous tissue of nude mice. Finally, collagen IV pretreatment of osteogenically differentiated mES cells increased osteogenic differentiation-like tissue and decreased undifferentiation-like tissue in the grafts grown after transplantation. Our results demonstrated that collagen IV significantly enhanced the migration of differentiating ES cells through α2β1 integrin-mediated actin remodeling and could promote ES cell transplantation efficiency, which may be imperative to stem cell therapy.

A longitudinal cohort study of incidence rates of inguinal hernia repair in 0- to 6-year-old children.

BACKGROUND/PURPOSE This study provides epidemiologic data on the incidence of inguinal hernia repair in preschool children using the Taiwan National Health Insurance Research Database. We believe that the data on hernia repair in said database provide a close approximation of the true incidence of inguinal hernia in young children. METHOD A cohort of 1,073,891 deidentified individuals was randomly selected from an insured population of 23 million. Subjects born during the period 1997-2004 were followed from birth to 6 years. The chi-square test and logistic regression modeling were used for statistical analyses. RESULT A total of 92,308 individuals were born during the study period. Of these individuals, 3881 underwent hernia repairs. The cumulative incidence of hernia repair in children aged 0 to 6 years was 4.20%/7 years. The boy/girl ratio was 4.27:1 and the unilateral/bilateral ratio was 3.77:1. The incidence of hernia repair among boys was highest during the first year of life, but then decreased with age. In contrast, the incidence among girls remained stable during the first 6 years of life. Boys younger than 1 year had more bilateral repairs than boys in other age groups (p<0.0001) and girls had significantly more bilateral repairs than boys (p<0.0001). Subjects with a history of preterm birth also had a higher incidence of hernia repair than subjects who were born at full term (odds ratio=2.34, p<0.0001). CONCLUSION Yearly incidence of hernia repair was obtained from a nationwide database. Some of the observations have not been reported elsewhere.

Apical Hypertrophic Cardiomyopathy: Correlations Between Echocardiographic Parameters, Angiographic Left Ventricular Morphology, and Clinical Outcomes

Echocardiographic parameters could be implicated in the development of apical asynergy (characterized by apical sequestration or apical aneurysm) and worse cardiovascular outcome in patients with apical hypertrophic cardiomyopathy (ApHCM).

Crystallization and preliminary X-ray diffraction analysis of recombinant hepatitis E virus-like particle

Hepatitis E virus (HEV) accounts for the majority of enterically transmitted hepatitis infections worldwide. Currently, there is no specific treatment for or vaccine against HEV. The major structural protein is derived from open reading frame (ORF) 2 of the viral genome. A potential oral vaccine is provided by the virus-like particles formed by a protein construct of partial ORF3 protein (residue 70-123) fused to the N-terminus of the ORF2 protein (residues 112-608). Single crystals obtained by the hanging-drop vapour-diffusion method at 293 K diffract X-rays to 8.3 A resolution. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 337, b = 343, c = 346 A, alpha = beta = gamma = 90 degrees , and contain one particle per asymmetric unit.

Non-classical estrogen receptors action on human dermal fibroblasts.

OBJECTIVE To study the possible non-genomic effect of selective estrogen receptor modulators on human dermal fibroblasts (HDF). MATERIALS AND METHODS WS1 cells were used to test the effect of raloxifene. The mRNA expressions of estrogen receptor (ER) α and β and G protein-coupled ER 1(GRP30) were examined by reverse transcription polymerase chain reaction. Apoptosis was identified by TUNEL assay and FACS analysis. MAPK and PI3 K/Akt pathways were determined by immunoblotting analysis. RESULTS Neither ERα nor ERβ, but GPR30 was detected in WS1 cells. Raloxifene increased apoptosis, which was blocked by pertussis toxin, an inhibitor of G protein, or by LY294002. Phosphorylated p38 MAPK and Akt were also increased after raloxifene treatment. CONCLUSION SERMs could induce apoptosis of HDF through G protein and PI3 K/Akt signaling, which may help understand the role of SERMs on the skin.