Alpha-Lipoic Acid Attenuates Cadmium- and Lead-Induced Neurotoxicity by Inhibiting Both Endoplasmic-Reticulum Stress and Activation of Fas/FasL and Mitochondrial Apoptotic Pathways in Rat Cerebral Cortex.

Although many studies have reported toxic effects of cadmium (Cd) and lead (Pb) in the central nervous system, few studies have investigated the combined toxicity of Cd and Pb. The mechanisms by which these combined heavy metals induce toxicity, as well as effective means to exert neuroprotection from these agents, remain poorly understood. To investigate the protective effects of alpha-lipoic acid (α-LA) on Cd- and/or Pb-induced cortical damage in rats, 48 Sprague-Dawley rats were exposed to drinking water containing 50 mg/L of Cd and/or 300 mg/L of Pb for 12 weeks, in the presence or absence of α-LA co-treatment (50 mg/kg) via gavage. We observed that exposure to Cd and/or Pb decreased the brain weight/body weight ratio and increased Cd and/or Pb contents as well as ultrastructural damage to the cerebral cortex. Cd and/or Pb also induced endoplasmic-reticulum (ER) stress and activated Fas (CD95/APO-1)/Fas ligand (FasL) and mitochondrial apoptotic pathways. Furthermore, co-treatment of Cd and Pb further exacerbated part of these phenotypes than treatment of Cd or Pb alone. However, simultaneous supplementation with α-LA attenuated Cd and/or Pb-induced neurotoxicity by increasing the brain weight/body weight ratio, reducing Cd and/or Pb contents, ameliorating both nuclear/mitochondrial damage and ER stress, and attenuating activation of Fas/FasL and mitochondrial apoptotic pathways. Collectively, our results indicate that the accumulation of Cd and/or Pb causes cortical damage and that α-LA exerts protection against Cd- and/or Pb-induced neurotoxicity. These findings highlight that α-LA may be exploited for the treatment and prevention of Cd- and/or Pb-induced neurotoxicity.

The Effects of Autophagy and PI3K/AKT/m-TOR Signaling Pathway on the Cell-Cycle Arrest of Rats Primary Sertoli Cells Induced by Zearalenone.

A high concentration of Zearalenone (ZEA) will perturb the differentiation of germ cells, and induce a death of germ cells, but the toxic mechanism and molecular mechanism remain unclear. The Sertoli cells (SCs) play an irreplaceable role in spermatogenesis. In order to explore the potential mechanism of ZEA male reproductive toxicity, we studied the effects of ZEA on cell proliferation, cell-cycle distribution, cell-cycle-related proteins and autophagy-related pathway the PI3K/Akt/mTOR signaling in primary cultured rats SCs, and the effects of autophagy and PI3K/AKT/m TOR signaling pathway on the SCs cell-cycle arrest induced by ZEA treated with the autophagy promoter RAPA, autophagy inhibitor CQ, and the PI3K inhibitor LY294002, respectively. The data revealed that ZEA could inhibit the proliferation of SCs by arresting the cell cycle in the G2/M phase and trigger the autophagy via inhibiting the PI3K/Akt/m TOR signaling pathway. Promoting or inhibiting the level of autophagy could either augment or reverse the arrest of cell cycle. And it was regulated by PI3K/Akt/m TOR signaling pathway. Taken together, this study provides evidence that autophagy and PI3K/Akt/m TOR signaling pathway are involved in regulating rats primary SCs cell-cycle arrest due to ZEA in vitro. To some extent, ZEA-induced autophagy plays a protective role in this process.

1-α,25-dihydroxyvitamin D3 potentiates avian osteoclast activation by increasing the formation of zipper-like structure via Src/Rac1 signaling.

Avian bone metabolism diseases affect the development and production of chickens, and many of these diseases can be prevented and controlled by balanced nutrition and hormone medicine. The steroid hormone 1α,25-dihydroxyvitamin D3 plays a key role in maintaining the balance of avian bone metabolism. Clinically, 1α,25-(OH)2D3 has been used to treat several bone diseases. Although several previous studies have investigated the effects of 1α,25-(OH)2D3 on osteoclastogenesis, the mechanisms underpinning osteoclast (OC) activity remain largely unknown. Herein, we used molecular and cell biology approaches to demonstrate that 1α,25-(OH)2D3 increases avian OC formation and activity, and upregulates bone resorption-related genes. Moreover, 1α,25-(OH)2D3 regulates the OC cytoskeleton by increasing the formation of zipper-like structure in OC precursor cells to potentiate OC activity via the Src/Rac1 signaling pathway. These findings provide new insight into the role of 1α,25-(OH)2D3 in OC activity.

ROS-Mediated Cell Cycle Arrest and Apoptosis Induced by Zearalenone in Mouse Sertoli Cells via ER Stress and the ATP/AMPK Pathway.

Zearalenone (ZEA) can perturb the differentiation of cells, reduce the generation of reproductive cells and induce a death of germ cells, but the molecular mechanism remains unclear. In order to investigate the potential mechanism of ZEA-induced cell cycle arrest and apoptosis, we studied the effects of ZEA on cell proliferation, cell-cycle distribution, cell-cycle-related proteins, cell death, cell apoptosis, ROS generation and the ATP/AMPK pathway in Sertoli cells. The role of ROS, ER stress and the ATP/AMPK pathway in ZEA-induced cell-cycle arrest and cell apoptosis was explored by using the antioxidant NAC, ER stress inhibitor 4-PBA and the AMPK inhibitor dorsomorphin, respectively. The results revealed that ZEA inhibited the cell proliferation, influenced the distribution of the cell cycle and induced cell apoptosis through the ATP/AMPK pathway. The ATP/AMPK pathway was regulated by ER stress that was induced by ROS generation after exposure to ZEA. Taking these together, this study provided evidence that ROS regulated the process of ZEA-induced cell cycle arrest and cell apoptosis through ER stress and the ATP/AMPK signal ways.

Is laparoscopic hepatectomy superior to open hepatectomy for hepatocellular carcinoma?

The low perioperative morbidity and shorter hospital stay associated with laparoscopic hepatectomy have made it an often-used option at many liver centers, despite the fact that many patients with hepatocellular carcinoma have cirrhosis, which makes the procedure more difficult and dangerous. Type of surgical procedure proves not to be a primary risk factor for poor outcomes after hepatic resection for hepatocellular carcinoma, the available evidence clearly shows that laparoscopic hepatectomy is an effective alternative to the open procedure for patients with early-stage hepatocellular carcinoma, even in the presence of cirrhosis. Whether the same is true for patients with intermediate or advanced disease is less clear, since laparoscopic major hepatectomy remains a technically demanding procedure.

Cadmium Activates Reactive Oxygen Species-dependent AKT/mTOR and Mitochondrial Apoptotic Pathways in Neuronal Cells.

OBJECTIVE: To examine the role of Cd-induced reactive oxygen species (ROS) generation in the apoptosis of neuronal cells. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

1α,25-Dihydroxyvitamin D3 inhibits the differentiation and bone resorption by osteoclasts generated from Wistar rat bone marrow-derived macrophages.

The steroid hormone 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3] plays an important role in maintaining a balance in calcium and bone metabolism. To study the effects of 1α,25-(OH)2D3 on osteoclast (OC) formation and bone resorption, OC differentiation was induced in bone marrow-derived mononuclear cells from Wistar rats with the addition of macrophage colony stimulating factor and receptor activator for nuclear factor-κB ligand in vitro. Cells were then treated with 1α,25-(OH)2D3 at 10-9, 10-8 or 10-7 mol/l. OCs were identified using tartrate-resistant acid phosphatase staining and activity was monitored in the absorption lacunae by scanning electron microscopy. Expression levels of functional proteins associated with bone absorption, namely carbonic anhydrase II, cathepsin K and matrix metalloproteinase-9 were evaluated by western blot analysis. The results showed that 1α,25-(OH)2D3 inhibited the formation and activation of OCs in a dose-dependent manner and downregulated the expression levels of bone absorption-associated proteins.

Cadmium-induced autophagy is mediated by oxidative signaling in PC-12 cells and is associated with cytoprotection.

Oxidative stress induced by cadmium (Cd) is a common phenomenon that has been observed in numerous studies. However, the underlying mechanism remains unknown. Recently, exposure of PC-12 cells to Cd has been shown to activate autophagy, which acts as a temporary survival pathway under stressful conditions by delaying the occurrence of apoptosis. The present study investigated the impact of oxidative stress on Cd­induced autophagy in PC-12 cells. The results demonstrated that Cd­induced autophagy (following treatment with Cd for 4 h), increased the levels of intracellular reactive oxygen species (ROS), decreased the mitochondrial membrane potential and resulted in apoptosis. A treatment with chloroquine (CQ; an autophagic inhibitor) sensitized the PC­12 cells to Cd, due to the increased production of ROS, which was associated with the incapacity to reduce mitochondrial and cell death. N-acetyl-L-cysteine, an antioxidant agent, decreased Cd-induced autophagy and reduced intracellular ROS levels, but enhanced CQ­induced apoptotic cell death. These findings indicate that moderate levels of ROS are essential in the regulation of Cd-induced autophagy, which subsequently enhances cell survival. Thus, the results of the present study provide an insight for future investigation of Cd-induced neurotoxicity.

Cadmium-induced autophagy promotes survival of rat cerebral cortical neurons by activating class III phosphoinositide 3-kinase/beclin-1/B-cell lymphoma 2 signaling pathways.

Autophagy is an evolutionarily conserved response that can be activated in response to heavy metal. Thus, the present study investigated the effect of autophagy on neurotoxic damage caused by cadmium (Cd) in rat cerebral cortical neurons. The results indicated that the viability of cortical neurons treated with Cd was markedly decreased in a dose-and time-dependent manner. The present study provided evidence that cortical neurons treated with Cd underwent autophagy: The conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3) to LC3-II, an increase in the punctate distribution of endogenous LC3-II and the presence of autophagosomes were identified. Combined treatment with Cd and chloroquine, an autophagy inhibitor, reduced the amount of autophagocytosis and cell activity, whereas rapamycin, an autophagy inducer, reduced Cd-mediated cytotoxicity. Furthermore, it was found that beclin-1 and class III phosphoinositide 3 kinase (PI3K) levels were increased, while levels of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) were decreased after Cd treatment. LY294002, a specific inhibitor of PI3K, prevented the decline in Bcl-2 production and the increase in levels of beclin-1, class III PI3K and autophagy following Cd treatment. In conclusion, the results of the present study suggested that Cd can induce cytoprotective autophagy by activating the class III PI3K/beclin-1/Bcl-2 signaling pathway, and that the autophagy pathway can serve as a sensitive biomarker for nervous system injury after exposure to Cd.

Immunosuppressive Effect of Hypodermin C on Complement Component 3 In Vitro.

Hypodermins A (HA), B (HB) and C (HC) are the major proteases secreted by first-instar larvae of Hypoderma lineatum (Diptera: Oestridae). These proteases are involved in the larval migration in the tissue, and prevent the activation of the host immune response. We previously showed that the recombinant HA functions as an immunosuppressive agent which could inhibit the rejection of xenotransplants. In the current study, we cloned the cDNA sequence of HC, which was transfected in Cos7 cells using the pEF1α-IRES-AcGFP expression vector. The Cos7 cells stably expressed HC, and were more resistant to lysis by guinea pig C3 than the control cells. The HC protease degraded the guinea pig C3, and inhibited the complement pathway in vitro. The DNA binding sites of HC were identified using an electrophoretic mobility shift assay. Our findings suggest that the recombinant HC might be useful as an immunosuppressive agent for the inhibition of the xenotransplant rejection.

Cadmium induces the differentiation of duck embryonic bone marrow cells into osteoclasts in vitro.

This study aimed to determine the in vitro effect of cadmium on the differentiation of duck embryonic bone marrow cells into osteoclasts. Bone marrow cells were harvested from 23-day old Gaoyou duck embryos and were cultured with either 50 nmol/L cadmium alone or different cadmium concentrations (0, 5, 10, 20 and 50 nmol/L) in combination with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). Tartrate-resistant acid phosphatase (TRAP) staining, pit formation assay with bovine cortical bone slices, and co-staining with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin and Hoechst 33258 were performed to determine the number of TRAP-positive cells and bone resorption activity. Cadmium at a concentration ⩾ 10 nmol/L in the presence of M-CSF and RANKL significantly increased in a concentration-dependent manner both the number of TRAP-positive cells (35-160%) and bone resorption activity (36-261%) (P<0.05). High cadmium concentrations in the presence of M-CSF and RANKL markedly promoted the formation of filamentous (F)-actin rings in differentiated osteoclasts. In conclusion, cadmium promotes in vitro the differentiation of duck embryonic osteoclasts in the presence of M-CSF and RANKL.

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

To investigate 1α,25-(OH)2D3 regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor kB 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 1α,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 1α,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 1α,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 kB 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 κB (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.

Cadmium-induced apoptosis in primary rat cerebral cortical neurons culture is mediated by a calcium signaling pathway.

Cadmium (Cd) is an extremely toxic metal, capable of severely damaging several organs, including the brain. Studies have shown that Cd disrupts intracellular free calcium ([Ca(2+)]i) homeostasis, leading to apoptosis in a variety of cells including primary murine neurons. Calcium is a ubiquitous intracellular ion which acts as a signaling mediator in numerous cellular processes including cell proliferation, differentiation, and survival/death. However, little is known about the role of calcium signaling in Cd-induced apoptosis in neuronal cells. Thus we investigated the role of calcium signaling in Cd-induced apoptosis in primary rat cerebral cortical neurons. Consistent with known toxic properties of Cd, exposure of cerebral cortical neurons to Cd caused morphological changes indicative of apoptosis and cell death. It also induced elevation of [Ca(2+)]i and inhibition of Na(+)/K(+)-ATPase and Ca(2+)/Mg(2+)-ATPase activities. This Cd-induced elevation of [Ca(2+)]i was suppressed by an IP3R inhibitor, 2-APB, suggesting that ER-regulated Ca(2+) is involved. In addition, we observed elevation of reactive oxygen species (ROS) levels, dysfunction of cytochrome oxidase subunits (COX-I/II/III), depletion of mitochondrial membrane potential (ΔΨm), and cleavage of caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) during Cd exposure. Z-VAD-fmk, a pan caspase inhibitor, partially prevented Cd-induced apoptosis and cell death. Interestingly, apoptosis, cell death and these cellular events induced by Cd were blocked by BAPTA-AM, a specific intracellular Ca(2+) chelator. Furthermore, western blot analysis revealed an up-regulated expression of Bcl-2 and down-regulated expression of Bax. However, these were not blocked by BAPTA-AM. Thus Cd toxicity is in part due to its disruption of intracellular Ca(2+) homeostasis, by compromising ATPases activities and ER-regulated Ca(2+), and this elevation in Ca(2+) triggers the activation of the Ca(2+)-mitochondria apoptotic signaling pathway. This study clarifies the signaling events underlying Cd neurotoxicity, and suggests that regulation of Cd-disrupted [Ca(2+)]i homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.

Influence of osteoprotegerin on differentiation, activation, and apoptosis of Gaoyou duck embryo osteoclasts in vitro.

ABSTRACT The aim of this study was to determine the influence of osteoprotegerin (OPG) on the differentiation, activation, and apoptosis of Gaoyou duck embryo osteoclasts cultured in vitro. Bone marrow cells were harvested from 23-d-old Gaoyou duck embryos and cultured in the presence of different concentrations of OPG (group A: no added factors, group B: 30 ng/mL of OPG, and group C: 100 ng/mL of OPG). Tartrate-resistant acid phosphatase (TRAP) staining, pit formation assay, and co-staining with tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin and Hoechst 33258 were all performed to determine the number of TRAP-positive cells, bone resorption activity, and the level of apoptosis, respectively. The number of TRAP-positive cells and the net expansion of pit formations area peaked on d 7 of culture in all 3 groups. The number of osteoclasts and the total volume of pit formations in OPG-treated groups were significantly lower compared with group A (P < 0.05). At each time point, the net expansion of pit formations area correlated with the number of TRAP-positive cells. The OPG inhibited the de novo formation of filamentous (F)-actin rings and promoted the disruption of existing F-actin rings in mature osteoclasts. In addition, OPG induced apoptosis in mature osteoclasts, as demonstrated by morphological changes in the nuclei. In osteoclast precursors, OPG inhibited differentiation and downregulated the formation of F-actin rings. In mature osteoclasts, OPG suppressed activation and enhanced the development of apoptosis, observed as a decrease in the number of TRAP-positive cells, the disruption of F-actin rings and morphological changes of the nuclei.

Osteoprotegerin influences the bone resorption activity of osteoclasts.

The aim of the present study was to determine whether osteoprotegerin (OPG) influences the bone resorption activity of osteoclasts. RAW264.7 cells were induced by macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-κB ligand (RANKL) and 0, 10, 20, 50 and 100 ng/ml OPG were added into various groups in the presence of the two cytokines. The OPG treatment was continued for 24 h. Osteoclast differentiation and activation were estimated via TRAP staining assay, TRITC-conjugated phalloidin staining, resorption activity analysis. Furthermore, the expression levels of the osteoclastic bone resorption-related genes MMP-9, cathepsin K and carbonic anhydrase II (CA II) were examined using real-time polymerase chain reaction (PCR). The data demonstrated that high concentrations of OPG could inhibit the differentiation and activation of osteoclasts. Furthermore, real-time PCR analysis illustrated that OPG decreased the expression of MMP-9 and cathepsin K in different concentrations of OPG and it decreased the expression of CA II genes at 10 and 20 ng/ml concentrations of OPG. For the time gradient study, OPG decreased the expression of MMP-9 and CA II genes but not that of the cathepsin K gene. In summary, the resorption activity of osteoclasts was suppressed by high concentrations of OPG and, at the molecular level, OPG decreased the expression of osteoclastic bone resorption-related genes.

Effects of RANKL, osteoprotegerin, calcium and phosphorus on survival and activation of Muscovy duck osteoclasts in vitro.

The aim of this study was to determine whether receptor activator of nuclear factor NF-kappaB ligand (RANKL), osteoprotegerin (OPG) and a calcium:phosphorus (Ca:P) ratio of 2:1 could affect survival and activation of Muscovy duck osteoclasts (OCs). Bone marrow cells were obtained from 5-day-old Muscovy ducks and cultured with (Group A) No added factors, (B) 30ng/mL soluble RANKL (sRANKL), (C) 30ng/mL sRANKL and 10ng/mL OPG, (D) 10ng/mL OPG, (E) 50ng/mL OPG, (F) 100ng/mL OPG and (G) 30ng/mL sRANKL, 6mmol/L Ca and 3mmol/L P. sRANKL promoted the survival of OCs on day 2, whereas the number of OCs decreased with addition of OPG in a dose-dependent manner. OPG and Ca:P (2:1) both inhibited OC survival induced by RANKL. RANKL stimulated bone resorption by OCs, whereas OPG, but not Ca:P (2:1), inhibited the activity of OCs induced by RANKL. RANKL promotes the survival and activation of OCs from Muscovy ducks, whereas OPG and, to a lesser extent, Ca:P (2:1) reduce the life span and inhibited the activation of OCs induced by RANKL.