Role of TPEN in Amyloid-ß25-35-Induced Neuronal Damage Correlating with Recovery of Intracellular Zn2+ and Intracellular Ca2+ Overloading.

The overproduction of neurotoxic amyloid-ß (Aß) peptides in the brain is a hallmark of Alzheimer's disease (AD). To determine the role of intracellular zinc ion (iZn2+) dysregulation in mediating Aß-related neurotoxicity, this study aimed to investigate whether N, N, N', N'­tetrakis (2­pyridylmethyl) ethylenediamine (TPEN), a Zn2+­specific chelator, could attenuate Aß25-35­induced neurotoxicity and the underlying mechanism. We used the 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay to measure the viability of primary hippocampal neurons. We also determined intracellular Zn2+ and Ca2+ concentrations, mitochondrial and lysosomal functions, and intracellular reactive oxygen species (ROS) content in hippocampal neurons using live-cell confocal imaging. We detected L-type voltage-gated calcium channel currents (L-ICa) in hippocampal neurons using the whole­cell patch­clamp technique. Furthermore, we measured the mRNA expression levels of proteins related to the iZn2+ buffer system (ZnT-3, MT-3) and voltage-gated calcium channels (Cav1.2, Cav1.3) in hippocampal neurons using RT-PCR. The results showed that TPEN attenuated Aß25-35­induced neuronal death, relieved the Aß25-35­induced increase in intracellular Zn2+ and Ca2+ concentrations; reversed the Aß25-35­induced increase in ROS content, the Aß25-35­induced increase in the L-ICa peak amplitude at different membrane potentials, the Aß25-35­induced the dysfunction of the mitochondria and lysosomes, and the Aß25-35­induced decrease in ZnT-3 and MT-3 mRNA expressions; and increased the Cav1.2 mRNA expression in the hippocampal neurons. These results suggest that TPEN, the Zn2+-specific chelator, attenuated Aß25-35­induced neuronal damage, correlating with the recovery of intracellular Zn2+ and modulation of abnormal Ca2+-related signaling pathways.

TPEN attenuates amyloid-ß25-35-induced neuronal damage with changes in the electrophysiological properties of voltage-gated sodium and potassium channels.

To understand the role of intracellular zinc ion (Zn2+) dysregulation in mediating age-related neurodegenerative changes, particularly neurotoxicity resulting from the generation of excessive neurotoxic amyloid-ß (Aß) peptides, this study aimed to investigate whether N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a Zn2+-specific chelator, could attenuate Aß25-35-induced neurotoxicity and the underlying electrophysiological mechanism. We used the 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay to measure the viability of hippocampal neurons and performed single-cell confocal imaging to detect the concentration of Zn2+ in these neurons. Furthermore, we used the whole-cell patch-clamp technique to detect the evoked repetitive action potential (APs), the voltage-gated sodium and potassium (K+) channels of primary hippocampal neurons. The analysis showed that TPEN attenuated Aß25-35-induced neuronal death, reversed the Aß25-35-induced increase in intracellular Zn2+ concentration and the frequency of APs, inhibited the increase in the maximum current density of voltage-activated sodium channel currents induced by Aß25-35, relieved the Aß25-35-induced decrease in the peak amplitude of transient outward K+ currents (IA) and outward-delayed rectifier K+ currents (IDR) at different membrane potentials, and suppressed the steady-state activation and inactivation curves of IA shifted toward the hyperpolarization direction caused by Aß25-35. These results suggest that Aß25-35-induced neuronal damage correlated with Zn2+ dysregulation mediated the electrophysiological changes in the voltage-gated sodium and K+ channels. Moreover, Zn2+-specific chelator-TPEN attenuated Aß25-35-induced neuronal damage by recovering the intracellular Zn2+ concentration.

The synergistic anti-asthmatic effects of glycyrrhizin and salbutamol.

AIM: To investigate the efficacy of glycyrrhizin (GL) combined with salbutamol (SA) as an anti-asthma therapy. METHODS: Rat lung beta2-adrenergic receptor (beta(2)-AR) mRNA level was measured by real-time RT PCR. Intracellular cAMP accumulation was evaluated with a reporter gene assay. An in vitro acetylcholine-induced guinea pig tracheal strip contraction model was used to test the relaxing effects of GL and SA. The anti-inflammatory effects of GL and SA were tested using tumor necrosis factor-alpha-induced NF-kappaB transcriptional activation reporter assay, I-kappaB Western blotting and interleukin-8 ELISA. An in vivo guinea pig asthma model was used to prove further the synergistic effect of GL and SA. RESULTS: GL (0.3 micromol/L) increased mRNA levels of beta(2)-AR in vivo and the accumulation of cAMP in vitro. The combination of GL and SA also resulted in significant complementary anti-inflammatory effects via inhibition of NF-kappaB activation, degradation of I-kappaB and production of interleukin-8. A significant synergistic effect of the combination was detected both in vitro and in vivo in a guinea pig mode. CONCLUSION: The results demonstrate that GL and SA have synergistic anti-asthmatic effects and offer the possibility of a therapeutic application of GL in combination with beta(2)-AR agonists in the treatment of asthma.