Meng Mao

Involvement of the PTEN-AKT-FOXO3a pathway in neuronal apoptosis in developing rat brain after hypoxia-ischemia.

The proapoptotic function of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) phosphatase has been linked to its capacity to antagonize the phosphatidylinositol-3-kinase–Akt signaling pathway. Previous studies have shown that the Forkhead transcriptional factor (FOXO3a) is a critical effector of the PTEN-mediated tumor suppressor. However, whether the PTEN–Akt–FOXO3a pathway is involved in neuronal apoptosis in developing rat brain after hypoxia–ischemia (HI) is unclear. In this study, we generated an HI model using postnatal day 10 rats. Immunohistochemistry and western blot were used to detect the expression of total and phosphorylated PTEN, Akt, and FOXO3a, as well as its target gene Bim. We found that dephosphorylation of PTEN was accompanied by dephosphorylation of Akt and FOXO3a, which induced FOXO3a translocation into the nucleus and upregulated the expression of Bim. Furthermore, we found that PTEN inhibition by bisperoxovanadium significantly increased the phosphorylation of Akt and FOXO3a, decreased the nuclear translocation of FOXO3a, and inhibited Bim expression after HI. Moreover, the downregulation of Bim caused by PTEN inhibition attenuated cellular apoptosis in developing rat brain. Our findings suggest that the PTEN–Akt–FOXO3a pathway is involved in neuronal apoptosis in neonatal rat brain after HI. Agents targeting PTEN may offer a promise to rescue neurons from HI brain damage.

Neuroprotective effect of brain-derived neurotrophic factor mediated by autophagy through the PI3K/Akt/mTOR pathway

Brain‑derived neurotrophic factor (BDNF) has been demonstrated to be a potent growth factor that is beneficial in neuronal functions following hypoxia‑ischemia (HI). Mature BDNF triggers three enzymes, mitogen‑activated protein kinase (MAPK), phosphatidylinositol 3‑kinase (PI3K) and phosphoinositide phospholipase C-γ (PLCγ), which are its predominant downstream regulators. The PI3K‑Akt signaling pathway is upstream of the mammalian target of rapamycin (mTOR), which is important in the induction of autophagy. However, whether the neuroprotective effect of BDNF is mediated by autophagy through the PI3K/Akt/mTOR pathway remains to be elucidated. Cortical neurons were cultured following isolation from pregnant rats (gestational days 16‑18). The induction of autophagy following BDNF treatment was analyzed by microtubule‑associated protein light chain 3 (LC3) conversion and autophagosome formation. The phosphorylation of Akt, mTOR and ribosomal protein S6 kinase (p70S6K) was analyzed in cultured cells with or without BDNF treatment. Cell viability was determined by a Cell Counting Kit‑8 for estimating the protective effect of BDNF. Results demonstrated that autophagy was induced in cells with oxygen deprivation. BDNF promoted cell viability via the upregulation of autophagy. Moreover, LC3 upregulation was related to Akt/mTOR/p70S6K inhibition by BDNF. In conclusion, the results suggested that the neuroprotective effect of BDNF was mediated by autophagy through the PI3K/Akt/mTOR pathway.

PI3K/Akt signaling pathway is required for neuroprotection of thalidomide on hypoxic–ischemic cortical neurons in vitro

Thalidomide, a derivative of glutamic acid, is used for immunomodulatory therapy in various diseases through inhibition of tumor necrotic factor-α (TNF-α) release. However, the effects of thalidomide in central nervous system (CNS) diseases such as stroke or hypoxic-ischemic encephalopathy (HIE) are unknown. In this study, we aimed to test whether thalidomide protects against hypoxic-ischemic neuronal damage and the possible signaling pathway involved in neuroprotection. Primary cultured cortical neurons of rats were treated with oxygen and glucose deprivation (OGD) for 3h to mimic hypoxic-ischemic injury in vivo. Neuronal apoptosis was measured with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. The expression of total caspase-3 (C3), cleaved caspase-3 (CC3), Akt, phosphorylated-Akt (p-Akt) and Bcl-2 protein were detected by Western blots. We found that OGD treatment increased the expression of CC3 and induced neuronal apoptosis. Both neuronal apoptosis and CC3 expression peaked at 24h after OGD. Furthermore, we found that thalidomide protected neurons against apoptosis by decreasing CC3 and increasing Bcl-2 expression in a dose-dependent manner. Meanwhile, we found that thalidomide induced p-Akt expression, which could be inhibited by PI3K specific inhibitor, LY294002. In addition, inhibition of PI3K increased CC3 but decreased Bcl-2 expression. In summary, thalidomide has anti-apoptotic effects on cortical neurons after OGD by modulating CC3 and Bcl-2 expression through activation of PI3K/Akt pathway.

Relationship between HIF-1α expression and neuronal apoptosis in neonatal rats with hypoxia–ischemia brain injury

Hypoxia inducible factor-1alpha (HIF-1alpha) plays an important role in maintaining oxygen equilibrium. Pathologic conditions such as hypoxia or ischemia have been reported to cause cellular apoptosis as well as to regulate HIF-1alpha. However, the relationship between HIF-1alpha and neuronal apoptosis in neonatal rats with hypoxia-ischemia brain injury is unclear. We hypothesized that HIF-1alpha will be differentially regulated depending upon the stimuli, such as hypoxia alone versus hypoxia-ischemia (HI), and thus play a role in neuronal apoptosis in developing rat brain. To test this hypothesis, we subjected postnatal day 10 (P10) rats to either hypoxia (8%O(2) and 92%N(2) for 2.5 h) or HI (ligating the right common carotid artery followed by hypoxia). Rat brains from hypoxia, HI, and sham controls were collected to detect HIF-1alpha expression and cellular apoptosis using immunohistochemistry, Western blot analysis, and TdT-mediated dUTP-biotin nick end labeling (TUNEL). We found that HIF-1alpha expression was upregulated at 4 h, peaked at 8 h, and declined at 24 h after hypoxia/HI compared with sham controls. Moreover, HIF-1alpha expression was significantly stronger in hypoxia-alone-treated rats than that in HI-treated rats. Meanwhile, we found that cellular apoptosis was more severe in HI-treated rats than that in hypoxia-treated rats. Furthermore, cellular apoptosis was prominent at 24 h in either hypoxia or HI but more severe in HI-treated rats. Our findings that cellular apoptosis increases with downregulation of HIF-1alpha suggest that HIF-1alpha may play a protective role in regulating cellular apoptosis in neonatal hypoxia-ischemia brain damage (HIBD).

The involvement of phosphoinositid 3-kinase/Akt pathway in the activation of hypoxia-inducible factor-1α in the developing rat brain after hypoxia–ischemia

Hypoxia inducible transcription factor (HIF)-1alpha plays an important role in maintaining oxygen homeostasis. However, the pathways involved in the regulation of HIF-1alpha are not clear. Since phosphoinositid 3-kinase/Akt (PI3K/Akt) pathway has been shown to be a common pathway involved in cell signaling, we therefore hypothesized that PI3K/Akt pathway is involved in the regulation of HIF-1alpha in developing rat brain after hypoxia-ischemia (HI). To test this hypothesis, we subjected postnatal day 10 rats to HI by ligating common carotid artery followed by hypoxia. Rat brains were collected to detect the expression of HIF-1alpha and its target gene, vascular endothelial growth factor (VEGF), as well as PI3K/Akt using immunohistochemistry and Western blot analysis. We found that the expression of HIF-1alpha and VEGF was significantly upregulated and peaked at 8 h after HI compared with sham controls. However, the expression of p-Akt peaked at 4 h, earlier than that seen in HIF-1alpha expression. Furthermore, we found that HIF-1alpha and VEGF protein were significantly inhibited after blocking the PI3K/Akt pathway using a specific inhibitor, wortmannin. Our findings suggest that the PI3K/Akt pathway is involved in the regulation of HIF-1alpha and its target gene VEGF in the developing rat brain after HI.

Signaling pathway involved in hypoxia-inducible factor-1α regulation in hypoxic-ischemic cortical neurons in vitro

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a key transciptional regulator of cellular and systemic oxygen homeostasis. Previous studies have shown that the regulation of HIF-1alpha is involved in the activation of PI3K/Akt pathway in some cells. However, whether this pathway plays a role in modulating HIF-1alpha in cultured cortical neurons during hypoxia-ischemia (HI) remains unclear. We therefore investigated the relationship between phosphoinositid 3-kinase/Akt (PI3K/Akt) pathway and HIF-1alpha expression in cultured neurons using an oxygen and glucose deprivation (OGD) model. In this study, cortical neurons cultured in vitro were subjected to OGD for 3h followed by reperfusion. The expressions of HIF-1alpha, VEGF, total Akt and phosphorelated-Akt (p-Akt) were detected by RT-PCR, Western blot and immunocytochemistry. We found that HIF-1alpha and VEGF were increased at 4h and peaked at 8h after OGD. Meanwhile, p-Akt increased and peaked at 4h after reperfusion, preceding HIF-1alpha expression. Pretreatment with wortmannin, a PI3K/Akt pathway inhibitor, significantly inhibited p-Akt expression and further attenuated both transcription and translation of HIF-1alpha and VEGF. Collectively, our findings suggested that PI3K/Akt signaling pathway might be involved in HIF-1alpha regulation after OGD in cultured cortical neurons.

Lack of association between ABCB1 gene polymorphisms and pharmacoresistant epilepsy: An analysis in a western Chinese pediatric population

OBJECTIVE The genetic polymorphisms of the ABCB1 (ATP-binding cassette B1) gene encoding P-glycoprotein have been proposed to be associated with pharmacoresistance phenotype in epilepsy patients. P-glycoprotein, a transmembrane transporter, works as an efflux pump by limiting antiepileptic drugs across the blood brain barrier, with correspondingly lowering drug concentrations in epileptogenic loci. In this study, we analyzed whether the three single nucleotide polymorphisms (C1236T, G2677T/A, and C3435T) in the ABCB1 gene were associated with pharmacoresistant epilepsy in a western Chinese pediatric population. METHODS A total of 350 children with epilepsy who had been prescribed antiepileptic drugs for at least 1year were included. Of this patient group 193 were drug responsive and 157 were drug resistant according to the presence of seizures. Genotypes of the three loci of ABCB1 gene were detected in 368 age- and sex-matched normal children and 350 epileptic children using the polymerase chain reaction (PCR)-restriction fragment length polymorphism technique. Normal population sample populace from the same ethnicity and territory was genotyped to check for population stratification. The allele, genotype, haplotype, and diplotype frequencies of ABCB1 polymorphisms were compared between drug-resistant and drug-responsive subjects. RESULTS No significant differences were observed in the frequencies of genotype, allele, haplotype, or diplotype of ABCB1 polymorphisms between patients with drug-resistant and drug-responsive epilepsy (p>0.05). CONCLUSION The above three polymorphisms in the ABCB1 gene were not found to be significantly associated with drug resistant epilepsy in a western Chinese pediatric population.

Enhanced migration and CXCR4 over-expression in fibroblasts with telomerase reconstitution

Telomerase reconstitution shows great potential for cell treatment and tissue engineering. Although the effects of telomerase on cell lifespan are well documented, the effects of telomerase on cellular biological characteristics, such as cellular migration, are relatively unknown. In this study, we tried to investigate if telomerase is involved in the regulation of fibroblast migration and the mechanism behind it. We found that when stimulated with a chemokine, CXCL12, the rate of migration was significantly higher in fibroblasts with telomerase reconstitution than that in fibroblasts without. Furthermore, the CXCL12 receptor, CXCR4, and multiple down-stream factors (Rho family members), were upregulated in the telomerase reconstituted fibroblasts. We concluded for the first time that telomerase reconstitution increased fibroblast migration through activation of CXCL12/CXCR4 axis and Rho family. The finding that fibroblasts with telomerase reconstitution have enhanced migration may have broad implications for cell therapy.

Proapoptotic Role of Human Growth and Transformation-Dependent Protein in the Developing Rat Brain After Hypoxia-Ischemia

Background and Purpose— Human growth and transformation-dependent protein (HGTD-P) is a new proapoptotic protein and an effector of cell death induced by hypoxia-ischemia (HI). The function of HGTD-P has been investigated in human prostate cancer cells and mouse neurons cultured in vitro. However, whether HGTD-P is involved in regulating the apoptosis of rat neurons is not clear, and the relevance of HGTD-P in HI animal models is still unknown. Therefore, in the present study, we tried to elucidate the role that HGTD-P plays in apoptosis of rat neurons subjected to HI, both in culture and in the developing rat brain in vivo. Methods— Samples from primary cultured neurons and postnatal day 10 rat brains with HI were collected. RT-PCR, Western blotting, and immunocytochemistry were used to detect the expression and distribution of rat HGTD-P, cleaved caspase 3, and apoptosis- inducing factor (AIF). MTT assay, DAPI, TUNEL, and flowcytometry were used to detect cell viability and apoptosis. Results— We found that HI upregulated the mRNA and protein levels of HGTD-P in rat neurons in vitro and in vivo. Antisense oligonucleotides (AS) targeted to HGTD-P inhibited the expression of HGTD-P, thus rescuing neuronal viability and attenuating neuronal apoptosis. In addition, we found that HGTD-P played its proapoptotic role by activating caspase 3 and inducing the translocation of AIF to nuclear. Conclusions— Our findings show that HGTD-P plays a proapoptotic role in the developing rat brain after HI and that it may be a potential target in treating HI-induced brain damage.

Effect of neuroserpin in a neonatal hypoxic-ischemic injury model ex vivo

Hypoxia-ischemia (HI) occurring in immature brains stimulates the expression of tissue-type plasminogen activator (tPA). Neuroserpin is a selected inhibitor of tPA in the central nerves system. However, the role that neuroserpin plays and the possible mechanisms involved during neonatal HI are poorly defined. In this study, an oxygen-glucose deprivation and reoxygenation (OGD/R) model was generated with cultured rat cortical neurons mimicking neonatal HI injury ex vivo, and an acute neuronal excitatory injury was induced by exposure to a high concentration of N-methyl-D-aspartic acid (NMDA). Cells received either neuroserpin or MK-801, an antagonist of the NMDA receptor, during OGD/R, and were incubated with or without neuroserpin after NMDA exposure. Cell viability and morphology were detected by a Cell Counting Kit-8 and immunohistochemical staining, respectively. TPA expression and activity were also assessed. We found that MK-801 alleviated injuries induced by OGD/R, suggesting an excitatory damage involvement. Neuroserpin provided a dose-dependent neuroprotective effect in both OGD/R and acute excitatory injuries by inhibiting the activity of tPA, without affecting neuronal tPA expression. Neuroserpin protected neurons against OGD/R even after a delayed administration of 3h. Collectively, our data indicate that neuroserpin protects neurons against OGD/R. mainly by inhibiting tPA-mediated acute neuronal excitotoxicity.