Protective effect of geranylgeranylacetone against hydrogen peroxide-induced oxidative stress in human neuroblastoma cells.

AIMS: Heat shock protein 70 (HSP70), one of the major HSPs, has been reported to suppress apoptosis and formation of pathogenic proteins in neurodegenerative disorders. Geranylgeranylacetone (GGA), an anti-ulcer drug, induces HSP70 and thereby protects against cellular damage in various diseases. We investigated the effect of GGA on hydrogen peroxide (H2O2)-induced neurotoxicity in human neuroblastoma SH-SY5Y cells. MAIN METHODS: H2O2-induced neuronal toxicity was measured by a CCK-8 assay and Hoechst 33342 staining. We also assessed oxidative stress and apoptosis by measuring reactive oxygen species (ROS) generation with 2',7'-dichlorofluorescein diacetate (DCFH-DA), caspase-3 activity, and mitogen-activated protein kinase (MAPK) pathway. KEY FINDINGS: GGA showed a concentration-dependent inhibition on H2O2-induced apoptotic cell death. H2O2-induced induction of HSP70 was enhanced by GGA pretreatment. GGA effectively suppressed the up-regulation of Bax and down-regulation of Bcl-2. GGA also blocked the H2O2-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). In addition, GGA attenuated H2O2-induced ROS generation and caspase-3 activity. SIGNIFICANCE: These results demonstrate that GGA protects SH-SY5Y cells from H2O2-induced apoptosis, at least in part by enhancing HSP70 production. Neuroprotective properties of GGA indicate that this compound may be a potential therapeutic agent for the treatment and prevention of neurodegenerative diseases.

A 635-nm light-emitting diode (LED) therapy inhibits bone resorptive osteoclast formation by regulating the actin cytoskeleton.

Bone diseases such as osteoporosis are mainly caused by upregulated activity of osteoclasts. The present study was designed to examine the effects of light-emitting diode (LED) irradiation on the formation and activity of multinucleated osteoclasts, specifically "round-shaped" osteoclast cells (ROC) in different cell types derived from mouse. After 635-nm LED irradiation, the cell viability was evaluated by MTT assay. The amount of total tartrate-resistant acid phosphatase (TRAP) + osteoclast and the number of ROC cells were also estimated by TRAP solution assay and TRAP staining, respectively. Actin rings were stained with rhodamine-conjugated phalloidin, and resorption assay was performed by dentin slices. In addition, gene expression levels between the control and irradiation groups were evaluated by RT-PCR. In a morphological analysis, the formation of ROC was significantly inhibited by 635-nm LED irradiation in the different cell types. Actin rings were seen at cell peripheries in most ROC cells of the control group, but patches containing disorganized actin were found in the irradiation group. Both the number of ROCs and bone resorption activity were much lower in the irradiation group than in the control group. Also, the gene expression levels involved in actin ring formation such as integrin ß3 and c-Src decreased in RT-PCR analysis. Overall, 635-nm LED therapy may play a pivotal role in regulating bone remodeling, and it may prove to be a valuable tool to prevent bone loss in osteoporosis and other resorptive bone diseases.

The potential of mouse skin-derived precursors to differentiate into mesenchymal and neural lineages and their application to osteogenic induction in vivo.

Although previous studies indicate that skin-derived precursors (SKPs) are multipotent dermal precursors that share similarities with neural crest stem cells (NCSCs), a shared ability for multilineage differentiation toward neural crest lineages between SKPs and NCSCs has not been fully demonstrated. Here, we report the derivation of SKPs from adult mouse skin and their directed multilineage differentiation toward neural crest lineages. Under controlled in vitro conditions, mouse SKPs were propagated and directed toward peripheral nervous system lineages such as peripheral neurons and Schwann cells, and mesenchymal lineages, such as osteogenic, chondrogenic, adipogenic, and smooth muscle cells. To ask if SKPs could generate these same lineages in vivo, a mixture of SKP-derived mesenchymal stem cells and hydroxyapatite/tricalcium phosphate was transplanted into the rat calvarial defects. Over the ensuing 4 weeks, we observed formation of osteogenic structure in the calvarial defect without any evidence of teratomas. These findings demonstrate the multipotency of adult mouse SKPs to differentiate into neural crest lineages. In addition, SKP-derived mesenchymal stem cells represent an accessible, potentially autologous source of precursor cells for tissue-engineered bone repair.

Intracellular acidification is associated with changes in free cytosolic calcium and inhibition of action potentials in rat trigeminal ganglion.

The effect of intracellular acidification and subsequent pH recovery in sensory neurons has not been well characterized. We have studied the mechanisms underlying Ca(2+)-induced acidification and subsequent recovery of intracellular pH (pH(i)) in rat trigeminal ganglion neurons and report their effects on neuronal excitability. Glutamate (500 µM) and capsaicin (1 µM) increased intracellular Ca(2+) concentration ([Ca(2+)](i)) with a following decrease in pH(i). The recovery of [Ca(2+)](i) to the prestimulus level was inhibited by LaCl(3) (1 mM) and o-vanadate (10 mM), a plasma membrane Ca(2+)/ATPase (PMCA) inhibitor. Removal of extracellular Ca(2+) also completely inhibited the acidification induced by capsaicin. TRPV1 was expressed only in small and medium sized trigeminal ganglion neurons. mRNAs for Na(+)/H(+) exchanger type 1 (NHE1), pancreatic Na(+)-HCO(3)(-) cotransporter type 1 (pNBC1), NBC3, NBC4, and PMCA types 1-3 were detected by RT-PCR. pH(i) recovery was significantly inhibited by pretreatment with NHE1 or pNBC1 siRNA. We found that the frequency of action potentials (APs) was dependent on pH(i). Application of the NHE1 inhibitor 5'-(N-ethyl-N-isopropyl) amiloride (5 µM) or the pNBC1 inhibitor 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid (500 µM) delayed pH(i) recovery and decreased AP frequency. Simultaneous application of 5'-(N-ethyl-N-isopropyl) amiloride and 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid almost completely inhibited APs. In summary, our results demonstrate that the rise in [Ca(2+)](i) in sensory neurons by glutamate and capsaicin causes intracellular acidification by activation of PMCA type 3, that the pH(i) recovery from acidification is mediated by membrane transporters NHE1 and pNBC1 specifically, and that the activity of these transporters has direct consequences for neuronal excitability.

P2X7 Receptor-mediated Membrane Blebbing in Salivary Epithelial Cells.

High concentrations of ATP induce membrane blebbing. However, the underlying mechanism involved in epithelial cells remains unclear. In this study, we investigated the role of the P2X7 receptor (P2X7R) in membrane blebbing using Par C5 cells. We stimulated the cells with 5 mM of ATP for 1~2 hrs and found the characteristics of membrane blebbing, a hallmark of apoptotic cell death. In addition, 500 microM Bz-ATP, a specific P2X7R agonist, induced membrane blebbing. However, 300 microM of Ox-ATP, a P2X7R antagonist, inhibited ATP-induced membrane blebbing, suggesting that ATP-induced membrane blebbing is mediated by P2X7R. We found that ATP-induced membrane blebbing was mediated by ROCK I activation and MLC phosphorylation, but not by caspase-3. Five mM of ATP evoked a biphasic [Ca(2+)](i) response; a transient [Ca(2+)](i) peak and sustained [Ca(2+)](i) increase secondary to ATP-stimulated Ca(2+) influx. These results suggest that P2X7R plays a role in membrane blebbing of the salivary gland epithelial cells.

Bioactivity and osteoblast responses of novel biomedical nanocomposites of bioactive glass nanofiber filled poly(lactic acid).

Biomedical nanocomposites constituted of bioactive ceramic and resorbable polymer have shown promise for the successful regeneration of bone tissues. We developed herein a novel nanocomposite made up of a bioactive glass in a nanofibrous form and a degradable synthetic polymer, poly(lactic acid) (PLA). The glass nanofiber with a bioactive composition was generated via an electrospinning process with an average diameter of approximately 320 nm. The nanofiber was homogenized with PLA solution at various concentrations (up to 35% nanofiber), followed by drying and thermal pressing to produce dense nanocomposites. The nanocomposites showed an internal morphology of uniformly dispersed nanofibers within the PLA matrix. The nanocomposites induced rapid formation of a hydroxycarbonate apatite layer on the surface under a simulated physiological medium. As the amount of bioactive nanofiber increased (from 5 to 25%), the in vitro bioactivity of the nanocomposite was improved. The osteoblast responses to the nanocomposites (compositions with 5 and 25% nanofiber) were assessed in terms of cell proliferation, differentiation, and mineralization. Osteoblasts attached and grew well on the nanocomposites and secreted collagen protein at initial culturing periods. The differentiation of cells, as assessed by the expression of alkaline phosphatase, was significantly improved on the nanocomposites as compared to those on pure PLA. Moreover, the mineralized product by the cells was observed to be significantly higher on the nanocomposites with respect to pure PLA. The newly developed nanocomposite constituted of bioactive nanofiber and degradable polymer is considered as a promising bone regeneration matrix with its excellent bioactivity and osteoblast responses.