Cadmium Activates Reactive Oxygen Species-dependent AKT/mTOR and Mitochondrial Apoptotic Pathways in Neuronal Cells / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To examine the role of Cd-induced reactive oxygen species (ROS) generation in the apoptosis of neuronal cells.</p><p><b>METHODS</b>Neuronal cells (primary rat cerebral cortical neurons and PC12 cells) were incubated with or without Cd post-pretreatment with rapamycin (Rap) or N-acetyl-L-cysteine (NAC). Cell viability was determined by MTT assay, apoptosis was examined using flow cytometry and fluorescence microscopy, and the activation of phosphoinositide 3'-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and mitochondrial apoptotic pathways were measured by western blotting or immunofluorescence assays.</p><p><b>RESULTS</b>Cd-induced activation of Akt/mTOR signaling, including Akt, mTOR, p70 S6 kinase (p70 S6K), and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). Rap, an mTOR inhibitor and NAC, a ROS scavenger, blocked Cd-induced activation of Akt/mTOR signaling and apoptosis of neuronal cells. Furthermore, NAC blocked the decrease of B-cell lymphoma 2/Bcl-2 associated X protein (Bcl-2/Bax) ratio, release of cytochrome c, cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (Endo G).</p><p><b>CONCLUSION</b>Cd-induced ROS generation activates Akt/mTOR and mitochondrial pathways, leading to apoptosis of neuronal cells. Our findings suggest that mTOR inhibitors or antioxidants have potential for preventing Cd-induced neurodegenerative diseases.</p>

Involvement of the Ca2+ signaling pathway in osteoprotegerin inhibition of osteoclast differentiation and maturation

The purpose of this study was to determine whether the Ca2+ signaling pathway is involved in the ability of osteoprotegerin (OPG) to inhibit osteoclast differentiation and maturation. RAW264.7 cells were incubated with macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-kappaB ligand (RANKL) to stimulate osteoclastogenesis and then treated with different concentrations of OPG, an inhibitor of osteoclast differentiation. The intracellular Ca2+ concentration [Ca2+]i and phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the different treatment groups were measured by flow cytometry and Western blotting, respectively. The results confirmed that M-CSF + RANKL significantly increased [Ca2+]i and CaMKII phosphorylation in osteoclasts (p < 0.01), and that these effects were subsequently decreased by OPG treatment. Exposure to specific inhibitors of the Ca2+ signaling pathway revealed that these changes varied between the different OPG treatment groups. Findings from the present study indicated that the Ca2+ signaling pathway is involved in both the regulation of osteoclastogenesis as well as inhibition of osteoclast differentiation and activation by OPG.

Cadmium induces apoptosis in primary rat osteoblasts through caspase and mitogen-activated protein kinase pathways

Exposure to cadmium (Cd) induces apoptosis in osteoblasts (OBs); however, little information is available regarding the specific mechanisms of Cd-induced primary rat OB apoptosis. In this study, Cd reduced cell viability, damaged cell membranes and induced apoptosis in OBs. We observed decreased mitochondrial transmembrane potentials, ultrastructure collapse, enhanced caspase-3 activity, and increased concentrations of cleaved PARP, cleaved caspase-9 and cleaved caspase-3 following Cd treatment. Cd also increased the phosphorylation of p38-mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases (ERK)1/2 and c-jun N-terminal kinase (JNK) in OBs. Pretreatment with the caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, ERK1/2 inhibitor (U0126), p38 inhibitor (SB203580) and JNK inhibitor (SP600125) abrogated Cd-induced cell apoptosis. Furthermore, Cd-treated OBs exhibited signs of oxidative stress protection, including increased antioxidant enzymes superoxide dismutase and glutathione reductase levels and decreased formation of reactive oxygen species. Taken together, the results of our study clarified that Cd has direct cytotoxic effects on OBs, which are mediated by caspase- and MAPK pathways in Cd-induced apoptosis of OBs.

Regulation of matrix metalloproteinase-9 protein expression by 1alpha,25-(OH)2D3 during osteoclast differentiation

To investigate 1alpha,25-(OH)2D3 regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were administered to induce the differentiation of RAW264.7 cells into osteoclasts. The cells were incubated with different concentrations of 1alpha,25-(OH)2D3 during culturing, and cell proliferation was measured using the methylthiazol tetrazolium method. Osteoclast formation was confirmed using tartrate-resistant acid phosphatase (TRAP) staining and assessing bone lacunar resorption. MMP-9 protein expression levels were measured with Western blotting. We showed that 1alpha,25-(OH)2D3 inhibited RAW264.7 cell proliferation induced by RANKL and M-CSF, increased the numbers of TRAP-positive osteoclasts and their nuclei, enhanced osteoclast bone resorption, and promoted MMP-9 protein expression in a concentration-dependent manner. These findings indicate that 1alpha,25-(OH)2D3 administered at a physiological relevant concentration promoted osteoclast formation and could regulate osteoclast bone metabolism by increasing MMP-9 protein expression during osteoclast differentiation.

Inhibitory effects of osteoprotegerin on osteoclast formation and function under serum-free conditions

The purpose of this study was to determine whether osteoprotegerin (OPG) could affect osteoclat differentiation and activation under serum-free conditions. Both duck embryo bone marrow cells and RAW264.7 cells were incubated with macrophage colony stimulatory factor (M-CSF) and receptor activator for nuclear factor kappaB ligand (RANKL) in serum-free medium to promote osteoclastogenesis. During cultivation, 0, 10, 20, 50, and 100 ng/mL OPG were added to various groups of cells. Osteoclast differentiation and activation were monitored via tartrate-resistant acid phosphatase (TRAP) staining, filamentous-actin rings analysis, and a bone resorption assay. Furthermore, the expression osteoclast-related genes, such as TRAP and receptor activator for nuclear factor kappaB (RANK), that was influenced by OPG in RAW264.7 cells was examined using real-time polymerase chain reaction. In summary, findings from the present study suggested that M-CSF with RANKL can promote osteoclast differentiation and activation, and enhance the expression of TRAP and RANK mRNA in osteoclasts. In contrast, OPG inhibited these activities under serum-free conditions.

Oxidative stress and apoptotic changes of rat cerebral cortical neurons exposed to cadmium in vitro / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the cytotoxic mechanism of cadmium (Cd) on cerebral cortical neurons.</p><p><b>METHODS</b>The primary cultures of rat cerebral cortical neurons were treated with different concentrations of cadmium acetate (0, 5, 10, and 20 micromol/L), and then the cell viability, apoptosis, ultrastructure, intracellular [Ca2+], and reactive oxygen species (ROS) levels, mitochondrial membrane potential (delta psi), activities of catalase (CAT) and superoxide dismutase (SOD) were measured.</p><p><b>RESULTS</b>A progressive loss in cell viability and an increased number of apoptotic cells were observed. In addition, Cd-induced apoptotic morphological changes in cerebral cortical neurons were also demonstrated by Hoechst 33258 staining. Meanwhile, ultrastructural changes were distortion of mitochondrial cristae and an unusual arrangement. Simultaneously, elevation of intracellular [Ca2+]i and ROS levels, depletion of Delta Psi were revealed in a dose-dependent manner during the exposure. Moreover, CAT and SOD activities in the living cells increased significantly.</p><p><b>CONCLUSION</b>Exposure of cortical neurons to different doses of Cd led to cellular death, mediated by an apoptotic mechanism, and the apoptotic death induced by oxidative stress may be a potential reason. And the disorder of intracellular homeostasis caused by oxidative stress and mitochondrial dysfunction may be a trigger for apoptosis in cortical neurons.</p>

Lipid peroxidation and ultrastructural modifications in brain after perinatal exposure to lead and/or cadmium in rat pups / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To assess lipid peroxidation and ultrastructural modifications in rat brains following perinatal exposure to lead (Pb) and/or cadmium (Cd).</p><p><b>METHODS</b>Female rats were divided into four groups: control group, Pb (300 mg/L) group, Cd group (10 mg/L) and Pb+Cd (300 mg/L, 10 mg/L) group. The compounds were delivered in the drinking water throughout pregnancy and lactation.</p><p><b>RESULTS</b>The levels of compounds in blood and brain of the Pb+Cd group were similar to those of other groups, but the effects of Pb+Cd on pups' body and brain weights were higher than on other compounds. Electron microscopy revealed that Pb and Cd had effects on mitochondrial swelling, disruption and cristae loss, Nissl body dissolution, degenerated organelles and vacuoles, cytomembrane disappearance, and nuclear chromoplasm concentration. The activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), acetylcholinesterase (AChE) was decreased, whereas the activity of maleic dialdehyde (MDA) was increased.</p><p><b>CONCLUSION</b>Perinatal exposure to low doses of Pb and Cd can produce alterations in lipid peroxidation and ultrastructural modifications in rat brains, and exposure to both metals can result in greater damages.</p>

Effects of chemical constituent in roots of Achyranthes Bidentata / 中国中药杂志

The difference of ingredients between the roots from the SP1 plants of Achyranthes Bidentata carried by satellite and the control's were evaluated in this study. The techniques of ultraviolet-visible spectroscopy (UVS), infrared spectroscopy (IR), Xray diffraction (XRD) and SDS-PAGE were used to analyse the chemical constituent in the root of A. bidentata. The results demonstrated that the UVS, IR, XRD and protein fingerprints of the roots from A. bidentata were distinct with special characters. The difference of the IR, XRD and protein fingerprints could be discriminated the satellite plants roots from those of the control, however, there were no difference of the UVS fingerprints between the satellite plants roots and the control. This indicated that the kinds of chemical ingredients were not different between the two groups, but the contents of some chemical ingredients deceased in SP1 plants of A. bidentata carried by satellite.