Chemical profiling with HPLC-FTMS of exogenous and endogenous chemicals susceptible to the administration of chotosan in an animal model of type 2 diabetes-induced dementia.

In our previous study, the daily administration of chotosan (CTS), a Kampo formula consisting of Uncaria and other 10 different crude drugs, ameliorated cognitive deficits in several animal models of dementia including type 2 diabetic db/db mice in a similar manner to tacrine, an acetylcholinesterase inhibitor. The present study investigated the metabonomics of CTS in db/db mice, a type 2 diabetes model, and m/m mice, a non-diabetes control strain, to identify the exogenous and endogenous chemicals susceptible to the administration of CTS using high performance liquid chromatography equipped with an orbitrap hybrid Fourier transform mass spectrometer. The results obtained revealed that the systemic administration of CTS for 20 days led to the distribution of Uncalia plant-derived alkaloids such as rhynchophylline, hirsuteine, and corynoxeine in the plasma and brains of db/db and m/m mice and induced alterations in four major metabolic pathways; i.e., (1) purine, (2) tryptophan, (3) cysteine and methionine, (4) glycerophospholipids in db/db mice. Moreover, glycerophosphocholine (GPC) levels in the plasma and brain were significantly higher in CTS-treated db/db mice than in vehicle-treated control animals. The results of the in vitro experiment using organotypic hippocampal slice cultures demonstrated that GPC (10-30 µM), as well as tacrine, protected hippocampal cells from N-methyl-d-aspartate-induced excitotoxicity in a manner that was reversible with the muscarinic receptor antagonist scopolamine, whereas GPC had no effect on the activity of acetylcholinesterase in vitro. Our results demonstrated that some CTS constituents with neuropharmacological activity were distributed in the plasma and brain tissue following the systemic administration of CTS and may subsequently have affected some metabolic pathways including glycerophospholipid metabolism and cognitive function in db/db mice. Moreover, the present metabonomic analysis suggested that GPC is a putative endogenous chemical that may be involved in the tacrine-like actions of CTS in the present diabetic animal model.

Possible involvement of VEGF signaling system in rescuing effect of endogenous acetylcholine on NMDA-induced long-lasting hippocampal cell damage in organotypic hippocampal slice cultures.

In our previous study, elevation of endogenous acetylcholine (ACh) by tacrine (THA) rescued NMDA-induced long-lasting hippocampal cell damage via muscarinic M1 receptors. However, the detailed molecular mechanism underlying the effect of ACh is unclear. This study investigated possible involvement of the VEGF signaling system in the rescuing effect of ACh on N-methyl-d-aspartate (NMDA)-induced long-lasting hippocampal cell damage using organotypic hippocampal slice cultures (OHSCs). As previously reported, NMDA pretreatment caused long-lasting hippocampal cell damage in OHSCs in a manner reversible by treatment with THA. The protein kinase C (PKC) inhibitor Ro31-8220, but not the extracellular signal-regulated kinase (ERK) inhibitor U0126, dose-dependently and almost completely abolished the effect of THA. The rescuing effect of THA was also partially but significantly blocked by Ki8751, a selective inhibitor of type 2 vascular endothelial growth factor (VEGF) receptor (VEGFR-2) tyrosine kinase. NMDA pretreatment elevated the expression level of HIF1α, whereas it decreased the expression of VEGF-A. Moreover, NMDA pretreatment reduced the level of phosphorylated VEGFR-2 without apparently affecting the level of VEGFR-2 or ß-actin. These NMDA pretreatment-induced changes were significantly attenuated by THA treatment. Immunohistochemical analysis conducted 6days after NMDA pretreatment revealed that VEGF-A and VEGFR-2 were mainly expressed on astrocytes and neurons, respectively, in OHSCs. In OHSCs pretreated with NMDA, THA treatment induced a morphological and activation-related change in astrocytes expressing VEGF-A. The present results demonstrate that endogenous acetylcholine plays a rescuing role towards excitotoxicity-induced long-lasting hippocampal cell damage in part via paracrine VEGF signaling between astrocytes and hippocampal neurons or autocrine VEGF signaling in hippocampal neurons in OHSCs.

Endogenous acetylcholine rescues NMDA-induced long-lasting hippocampal cell damage via stimulation of muscarinic M(1) receptors: elucidation using organic hippocampal slice cultures.

This study aimed to investigate a recuing role of cholinergic systems in the excitotoxicity-induced hippocampal cell damage. Organotypic hippocampal slice cultures (OHSCs) were prepared from 7-day-old mice and exposed to N-methyl-d-aspartate (NMDA) for 24h. After washing out the NMDA, OHSCs were incubated in medium containing test drugs for 0-6 days. Hippocampal cell damage was evaluated by propidium iodide staining, immunofluorescence, and Western blotting. NMDA (1-10 µM) dose-dependently damaged hippocampal cells. The toxic effect of 3 µM NMDA was also observed at 3-6 days, even after washing out NMDA, and was blocked by MK-801 from day 3 to day 6. Post-treatments with tacrine, donepezil, and galantamine reduced the NMDA-induced long-lasting hippocampal cell damage. The effect of tacrine was induced in a manner dependent on the incubation period after NMDA treatment and was confirmed by Nissl staining and immunostaining with NeuN, a marker of mature neurons. The effect of tacrine was attenuated by scopolamine and a muscarinic M(1) receptor antagonist, pirenzepine, but not by a muscarinic M(3) receptor antagonist, darifenacin, or a nicotinic receptor antagonist, mecamylamine. The protein kinase C inhibitor Ro-31-8220 abolished the effect of tacrine. The pretreatment with 3 µM NMDA had no effect on the expression level of presynaptic cholinergic markers, choline acetyltransferase and vesicular acetylcholine transporter, in OHSCs. These results suggest that a low concentration of NMDA causes long-lasting hippocampal cell damage and that endogenous acetylcholine plays, via muscarinic M(1) receptor, a rescuing role in the excitotoxicity-induced long-lasting hippocampal cell damage.