Guangfei Xu

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces microglial nitric oxide production and subsequent rat primary cortical neuron apoptosis through p38/JNK MAPK pathway.

It has been widely accepted that microglia, which are the innate immune cells in the brain, upon activation can cause neuronal damage. In the present study, we investigated the role of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in regulating microglial nitric oxide production and its role in causing neuronal damage. The study revealed that TCDD stimulates the expression of inducible nitric oxide synthase (iNOS) as well as the production of nitric oxide (NO) in a dose- and time-dependent manner. Further, a rapid activation of p38 and JNK MAPKs was found in HAPI microglia following TCDD treatment. Blockage of p38 and JNK kinases with their specific inhibitors, SB202190 and SP600125, significantly reduced TCDD-induced iNOS expression and NO production. In addition, it was demonstrated through treating rat primary cortical neurons with media conditioned with TCDD treated microglia that microglial iNOS activation mediates neuronal apoptosis. Lastly, it was also found that p38 and JNK MAPK inhibitors could attenuate the apoptosis of rat cortical neurons upon exposure to medium conditioned by TCDD-treated HAPI microglial cells. Based on these observations, we highlight that the p38/JNK MAPK pathways play an important role in TCDD-induced iNOS activation in rat HAPI microglia and in the subsequent induction of apoptosis in primary cortical neurons.

2, 3, 7, 8-Tetrachlorodibenzo-P-Dioxin (TCDD) Induces Premature Senescence in Human and Rodent Neuronal Cells via ROS-Dependent Mechanisms

The widespread environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent toxicant that causes significant neurotoxicity. However, the biological events that participate in this process remain largely elusive. In the present study, we demonstrated that TCDD exposure triggered apparent premature senescence in rat pheochromocytoma (PC12) and human neuroblastoma SH-SY5Y cells. Senescence-associated β-galactosidase (SA-β-Gal) assay revealed that TCDD induced senescence in PC12 neuronal cells at doses as low as 10 nM. TCDD led to F-actin reorganization and the appearance of an alternative senescence marker, γ-H2AX foci, both of which are important features of cellular senescence. In addition, TCDD exposure altered the expression of senescence marker proteins, such as p16, p21 and p-Rb, in both dose- and time-dependent manners. Furthermore, we demonstrated that TCDD promotes mitochondrial dysfunction and the accumulation of cellular reactive oxygen species (ROS) in PC12 cells, leading to the activation of signaling pathways that are involved in ROS metabolism and senescence. TCDD-induced ROS generation promoted significant oxidative DNA damage and lipid peroxidation. Notably, treatment with the ROS scavenger N-acetylcysteine (NAC) markedly attenuated TCDD-induced ROS production, cellular oxidative damage and neuronal senescence. Moreover, we found that TCDD induced a similar ROS-mediated senescence response in human neuroblastoma SH-SY5Y cells. In sum, these results demonstrate for the first time that TCDD induces premature senescence in neuronal cells by promoting intracellular ROS production, supporting the idea that accelerating the onset of neuronal senescence may be an important mechanism underlying TCDD-induced neurotoxic effects.

2,3,7,8-TCDD induces neurotoxicity and neuronal apoptosis in the rat brain cortex and PC12 cell line through the down-regulation of the Wnt/β-catenin signaling pathway.

TCDD exposure has various toxic effects on in the human nervous system resulting in various developmental and behavioral deficits. However the underlying molecular mechanism of TCDD-induced adverse effects on the CNS and associated signaling pathways remains largely unknown. Herein we analyzed acute TCDD exposure in the rat brain cortex to investigate whether misregulation of the Wnt/β-catenin signaling pathway plays a role in neurotoxicity. Western blot and immunohistochemical experiments revealed a significant down-regulation of β-catenin and phospho-glycogen synthase kinase-3β (pSer9-GSK-3β) after TCDD exposure. TUNEL assay results showed apoptosis occurs mainly at day 7 after TCDD treatment. Immunofluorescent labeling indicated that β-catenin was localized mainly in the neurons; co-localization of β-catenin and active caspase-3 was found following TCDD exposure. Further, TCDD exposure decreased the level of pSer9-GSK-3β and β-catenin, and increased apoptosis in the PC12 neuronal cell line in a dose-dependent manner. Interestingly the application of lithium chloride, a GSK-3β inhibitor, reversed the suppressive effect of TCDD on β-catenin in PC12 cells and primary cortical neurons restoring cell viability and protecting cells from apoptosis as compared to untreated controls. Taken together, these results indicate that the canonical Wnt/β-catenin signaling pathway may play an important role in TCDD-induced neurotoxicity and neuronal apoptosis.

The PERK-eIF2α signaling pathway is involved in TCDD-induced ER stress in PC12 cells

Studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces apoptotic cell death in neuronal cells. However, whether this is the result of endoplasmic reticulum (ER) stress-mediated apoptosis remains unknown. In this study, we determined whether ER stress plays a role in the TCDD-induced apoptosis of pheochromocytoma (PC12) cells and primary neurons. PC12 cells were exposed to different TCDD concentrations (1, 10, 100, 200, or 500nM) for varying lengths of time (1, 3, 6, 12, or 24h). TCDD concentrations much higher than 10nM (100, 200, or 500nM) markedly increased glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP) levels, which are hallmarks of ER stress. We also evaluated the effects of TCDD on ER morphology in PC12 cells and primary neurons that were treated with different TCDD concentrations (1, 10, 50, or 200nM) for 24h. Ultrastructural ER alterations were observed with transmission electron microscopy in PC12 cells and primary neurons treated with high concentrations of TCDD. Furthermore, TCDD-induced ER stress significantly promoted the activation of the PKR-like ER kinase (PERK), a sensor for the unfolded protein response (UPR), and its downstream target eukaryotic translation initiation factor 2 α (eIF2α); in contrast, TCDD did not appear to affect inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6), two other UPR sensors. Importantly, TCDD significantly inhibited eIF2α phosphorylation and triggered apoptosis in PC12 cells after 6-24h of treatment. Salubrinal, which activates the PERK-eIF2α pathway, significantly enhanced eIF2α phosphorylation in PC12 cells and attenuated the TCDD-induced cell death. In contrast, knocking down eIF2α using small interfering RNA markedly enhanced TCDD-induced cell death. Together, these results indicate that the PERK-eIF2α pathway plays an important role in TCDD-induced ER stress and apoptosis in PC12 cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin stimulates proliferation of HAPI microglia by affecting the Akt/GSK-3β/cyclin D1 signaling pathway

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental toxin that induces apoptosis of neurons and a pro-inflammatory response in microglial cells. First, we found that TCDD induced proliferation of HAPI microglial cells in a dose- and time-dependent manner. Flow cytometry analysis showed that this proliferation by TCDD was due to mainly enhancing the G1 to S phase transition. Next, it was found that TCDD treatment led to up-regulation of cyclin D1, which induces cell cycle progression from G1 to S phase, in a time-dependent manner. As for molecular mechanism, we revealed that TCDD was capable of inducing Akt phosphorylation and activation, resulting in phosphorylation and inactivation of glycogen synthase kinase-3β (GSK-3β). Inactivated GSK-3β attenuated proteasomal degradation of cyclin D1 by reducing Thr(286)-phosphorylated cyclin D1 levels. Moreover, inactivated GSK-3β increased cyclin D1 gene transcription by increasing its transcription factor β-catenin in the nucleus. Further, blockage of phosphoinositide 3-kinase/Akt kinase with their specific inhibitors, LY294002 and Akt 1/2 kinase inhibitor, significantly reduced TCDD-enhanced proliferation of HAPI microglial cells. In conclusion, TCDD stimulates proliferation of HAPI microglial cells by affecting the Akt/GSK-3β/cyclin D1 signaling pathway.

TRAM1 protect HepG2 cells from palmitate induced insulin resistance through ER stress-JNK pathway.

Excess serum free fatty acids (FFAs) are fundamental to the pathogenesis of insulin resistance. Chronic endoplasmic reticulum (ER) stress is a major contributor to obesity-induced insulin resistance in the liver. With high-fat feeding (HFD), FFAs can activate chronic endoplasmic reticulum (ER) stress in target tissues, initiating negative crosstalk between FFAs and insulin signaling. However, the molecular link between insulin resistance and ER stress remains to be identified. We here reported that translocating chain-associated membrane protein 1 (TRAM1), an ER-resident membrane protein, was involved in the onset of insulin resistance in hepatocytes. TRAM1 was significantly up-regulated in insulin-resistant liver tissues and palmitate (PA)-treated HepG2 cells. In addition, we showed that depletion of TRAM1 led to hyperactivation of CHOP and GRP78, and the activation of downstream JNK pathway. Given the fact that the activation of ER stress played a facilitating role in insulin resistance, the phosphorylation of Akt and GSK-3β was also analyzed. We found that depletion of TRAM1 markedly attenuated the phosphorylation of Akt and GSK-3β in the cells. Moreover, application with JNK inhibitor SP600125 reversed the effect of TRAM1 interference on Akt phosphorylation. The accumulation of lipid droplets and expression of two key gluconeogenic enzymes, PEPCK and G6Pase, were also determined and found to display a similar tendency with the phosphorylation of Akt. Glucose uptake assay indicated that knocking down TRAM1 augmented PA-induced down-regulation of glucose uptake, and inhibition of JNK using SP600125 could block the effect of TRAM1 on glucose uptake. These data implicated that TRAM1 might protect HepG2 cells against PA-induced insulin resistance through alleviating ER stress.

Downregulation of the Wnt/β-catenin signaling pathway is involved in manganese-induced neurotoxicity in rat striatum and PC12 cells

Manganese (Mn) is an essential trace element. However, exposure to excessive Mn may cause neurodegenerative disorders called manganism. Accumulating evidence indicated that dysregulation of Wnt/β‐catenin signaling was tightly associated with the onset of neurodegenerative disorders. However, whether aberrant Wnt/β‐catenin signaling contributes to Mn‐induced neurotoxicity remains unknown. The present study investigates the involvement of Wnt/β‐catenin signaling in Mn‐induced neurotoxicity. Western blot and immunohistochemistry analyses showed a remarkable downregulation of p‐Ser9‐glycogen synthase kinase‐3β (GSK‐3β) and β‐catenin in rat striatum after Mn exposure. TUNEL assay revealed significant neuronal apoptosis following treatment with 25 mg/kg Mn. Immunofluorescent staining showed that β‐catenin was expressed predominantly in neurons, and colocalization of β‐catenin and active caspase‐3 was observed after Mn exposure. Furthermore, Mn exposure resulted in PC12 cells apoptosis, which was accompanied by reduced levels of cellular β‐catenin and p‐GSK‐3β. Accordingly, the mRNA level of the prosurvival factor survivin, a downstream target gene of β‐catenin, was synchronously decreased. More importantly, blockage of GSK‐3β activity with the GSK‐3β inhibitor lithium chloride could attenuate Mn‐induced downregulation of β‐catenin and survivin as well as neuronal apoptosis. Overall, the present study demonstrates that downregulation of Wnt/β‐catenin signaling pathway may be of vital importance in the neuropathological process of Mn‐induced neurotoxicity.

2,3,7,8-Tetrachlorodibenzo-p-dioxin promotes astrocyte activation and the secretion of tumor necrosis factor-α via PKC/SSeCKS-dependent mechanisms.

2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is a ubiquitous environmental pollutant that could induce significant toxic effects in the human nervous system. However, the underlying molecular mechanism has not been entirely elucidated. Reactive astrogliosis has implicated in various neurological diseases via the production of a variety of pro‐inflammatory mediators. Herein, we investigated the potential role of TCDD in facilitating astrocyte activation and the underlying molecular mechanisms. We showed that TCDD induced rapid astrocyte activation following TCDD exposure, which was accompanied by significantly elevated expression of Src‐Suppressed‐C Kinase Substrate (SSeCKS), a protein involved in protein kinase C (PKC)‐mediated Nuclear Factor kappa B signaling, suggesting a possible involvement of PKC‐induced SSeCKS activation in TCDD‐triggered reactive astroglia. In keeping with the finding, we found that the level of phosphorylated Nuclear Factor kappa B p65 was remarkably increased after TCDD treatment. Furthermore, interference of SSeCKS attenuated TCDD‐induced inducible nitric oxide synthase, glial fibrillary acidic protein, phospho‐p65 expression, and tumor necrosis factor‐α secretion in astrocytes. In addition, pre‐treatment with PKC inhibitor also attenuated TCDD‐induced astrocyte activation, as well as SSeCKS expression. Interestingly, we found that TCDD treatment could lead to SSeCKS perinuclear localization, which could be abolished after treatment with PKC inhibitor. Finally, we showed that inhibition of PKC activity or SSeCKS expression would impair TCDD‐triggered tumor necrosis factor‐α secretion. Our results suggested that TCDD exposure could lead to astrocyte activation through PKC/SSeCKS‐dependent mechanisms, highlighting that astrocytes might be important target of TCDD‐induced neurotoxicity.

2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced inflammatory activation is mediated by intracellular free calcium in microglial cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been known to induce inflammatory signaling in a number of cell types and tissues. However, the adverse effects of TCDD on the central nervous system (CNS) have not been entirely elucidated. In this study, using reverse transcriptase PCR (RT-PCR) and ELISA, we showed that TCDD up-regulated the expression and secretion of tumor necrosis factor-alpha (TNF-α) in a time-dependent manner in cultured HAPI microglial cells. TCDD also caused a fast (within 30min as judged by the increase in its mRNA level) activation of cytosolic phospholipase A2 (cPLA2). This initial action was accompanied by up-regulation of cyclooxygenase-2 (COX-2), an important inflammation marker within 1h after TCDD treatment. These pro-inflammatory responses were inhibited by two types of Ca(2+) blockers, bis-(o-aminophenoxy) ethane-N,N,N,N-tetra-acetic acid acetoxymethyl ester (BAPTA-AM) and nifedipine, thus, indicating that the effects are triggered by initial increase in the intracellular concentration of free Ca(2+) ([Ca(2+)]i). Further, TCDD exposure could induce phosphorylation- and ubiquitination-dependent degradation of IкBα, and the translocation of NF-κB p65 from the cytosol to the nucleus in this microglial cell line. Thus, the NF-κB signaling pathway can be activated after TCDD treatment. However, Ca(2+) blockers also obviously attenuated NF-κB activation and transnuclear transport induced by TCDD. In concert with these results, we highlighted that the secretion of pro-inflammatory cytokine and NF-κB activation induced by TCDD can be mediated by elevation of [Ca(2+)]i in HAPI microglial cells.

Effect of Intermittent versus Chronic Calorie Restriction on Tumor Incidence: A Systematic Review and Meta-Analysis of Animal Studies.

Both chronic calorie restriction (CCR) and intermittent calorie restriction (ICR) have shown anticancer effects. However, the direct evidence comparing ICR to CCR with respect to cancer prevention is controversial and inconclusive. PubMed and Web of Science were searched on November 25, 2015. The relative risk (RR) [95% confidence interval (CI)] was calculated for tumor incidence, and the standardised mean difference (95% CI) was computed for levels of serum insulin-like growth factor-1 (IGF-1), leptin, and adiponectin using a random-effects meta-analysis. Sixteen studies were identified, including 11 using genetically engineered mouse models (908 animals with 38–76 weeks of follow-up) and 5 using chemically induced rat models (379 animals with 7–18 weeks of follow-up). Compared to CCR, ICR decreased tumor incidence in genetically engineered models (RRu2009=u20090.57; 95% CI: 0.37, 0.88) but increased the risk in chemically induced models (RRu2009=u20091.53, 95% CI: 1.13, 2.06). It appears that ICR decreases IGF-1 and leptin and increases adiponectin in genetically engineered models. Thus, the evidence suggests that ICR exerts greater anticancer effect in genetically engineered mouse models but weaker cancer prevention benefit in chemically induced rat models as compared to CCR. Further studies are warranted to confirm our findings and elucidate the mechanisms responsible for these effects.