We examined effect of iridoid glucosides, aucubin, catalpol, geniposide and gardenoside, and their enzymic hydrolysates on neurite outgrowth of PC12h cells. Except for aucubin, these glucosides induced neurite outgrowth at 0.1 microgram/ml and above in medium after 3 d of treatment. Hydrolysates of the four glucosides all caused neuritogenesis. Geniposide hydrolysate enhanced responses of cells to carbachol and KCl-induced depolarization in terms of cytoplasmic free-calcium concentration. The aglucone of geniposide, genipin, also promoted neurite outgrowth in a dose-dependent manner (ED50 = 0.7 microM). The neuritogenic effect of genipin was partially or considerably inhibited in the presence of H-89 and genistein. All the results presented suggest that certain iridoid compounds can induce neuronal differentiation in PC12h cells through activation of components of the intracellular signal transduction pathway.
Prominent neurite outgrowth induced by genipin, a plantderived iridoid, was substantially inhibited by addition of N G -nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase (NOS) inhibitor, and carboxy-PTIO, an NO scavenger, in PC12h cells. Increases of the NADPH-diaphorase activity and neuronal and inducible NOS proteins in cells preceded the neurite outgrowth after addition of genipin to medium. NO donors could induce the neurite outgrowth dose-dependently in the cells. On the other hand, an inhibitor of soluble guanylate cyclase (SGC), which is known to be a stimulatory target of NO, abolished greatly the genipin-induced neurite outgrowth. Addition of extracellular signal-regulated kinase (ERK) kinase inhibitors could almost completely abolish the neurite induction. L-NAME remarkably depressed genipin-stimulated phosphorylation of ERK-1 and -2. A neuritogenic effect of nerve growth factor (NGF) in PC12h cells was also remarkably inhibited by the NOS inhibitor, NO scavenger and SGC inhibitor. These ®ndings suggest that induced NO production followed by cyclic GMP-mediated stimulation of the mitogenactivated protein kinase (MAPK) cascade is implicated in the neuritogenesis by genipin and NGF in PC12h cells.
It is now generally documented that the cause of Alzheimer's disease is progressive deposition (histopathologically identified as senile plaque) of the amyloid b protein (Ab) (Ab1-42 and Ab1-40) with aging or due to genetic background in vulnerable brain regions, including the hippocampus and cortex. This deposition is strongly suspected to contribute to disruption of the neuronal network as a result of ubiquitous apoptotic cell death induced by Ab.1,2) The addition of Ab and its hydrophobic core fragments such as Ab25-35 (a monodecapeptide with an amino acid sequence identical to that from 25 to 35 of Ab) has been reported to induce significant cell death in approximately one week in primary cultures of rat hippocampal neurons. [3][4][5] Geniposide and genipin, iridoid compounds found in many plants such as Gardenia jasminoides, have been demonstrated in our laboratory to have neurotrophic, nerve growth factor (NGF)-like activity in their neuritogenesis and signaling promotion in PC12h cells, with genipin having the stronger effect.6,7) Administration of genipin counteracted the trauma-and scopolamine-induced impairment of memory in mice. 8) In some reported experiments, NGF and other neurotrophic factors reversed the deficit in spatial learning and memory in aged rats.9,10) We investigated whether these two iridoids can affect the neuronal toxicity of Ab in cultured hippocampal neurons.Hippocampal neurons from 18-d rat embryos were prepared as described previously.11) Hippocampal cells were maintained for 2 d in 48-well culture plates with Dulbecco's modified Eagle medium (DMEM) supplemented with 10% precolostrum new-born calf serum, selected for neuronal populations by incubation with cytosine-b-D-arabinofuranoside 10 mM for 24 h in Ca 2ϩ , Mg 2ϩ -free phosphate-buffered saline, pH 7.4 (CMF-PBS), and then treated with Ab25-35 (40 mM) in CMF-PBS for 2 d without serum. Ab25-35 was prepared using a peptide synthesizer according to the method described previously.11) It was dissolved in CMF-PBS at a concentration of 1.8 mM (most of the Ab was in fine suspension).Toxic effect on the cultures was evaluated by estimation of lactate dehydrogenase (LDH) release during 2 d of culture in the presence of Ab25-35 with and without test compounds as specified below and according to the method described previously.
12)Geniposide was kindly provided by Toyo F.C.C. Co. (Tokyo, Japan) and genipin was purchased from Wako Pure Chemical Industries (Osaka, Japan). They were dissolved in distilled water and refrigerated until use.The addition of Ab25-35 40 mM to the hippocampal neuron cultures markedly increased LDH release (Fig. 1, Ab25-35 only). Our colleagues previously found in cultured hippocampal cells that Ab25-35-induced plasma membrane impairment was blocked by the addition of the endonuclease inhibitor aurintricarboxylic acid, indicating that the cell degeneration was a result of apoptotic cell death.13) The neurotoxicity of Ab was effectively attenuated by the addition of genipin (20 mM or more) to the medium (Fig. 1A). ...
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