Western Blot Analysis == Western blot analysis was performed as described previously [18]. on PC12 cells using protein specific inhibitors and Western blot analysis. The mitogen-activated kinase/ERK kinase (MEK) inhibitor U0126 and the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002 significantly decreased neurite outgrowth. At the same time, SG increased phosphorylation of CREB in protein level. Thus, SG-induced neuritogenic activity depends on the activation of the extracellular-regulated protein kinase (ERK), cAMP responsive element-binding protein (CREB) and PI3K signalling pathways in PC12 cells. Keywords:1-O-(myristoyl) glycerol, neuritogenic activity,Ilisha elongate, PC12 cells, 1-O-(stearoyl) glycerol, ERK == 1. Introduction == With the development of aging society, Alzheimers disease (AD) has become a leading cause of dementia in the older population. The costs associated with treating dementia worldwide totaled over $600 billion in 2010 2010 [1]. The most popular selling drugs currently approved by the Food and Drug Administration (USA) for AD treatment include donepezil, rivastigmine, galantamine and memantine. Donepezil, rivastigmine and galantamine are cholinesterase inhibitors, whilst memantine is usually anN-methyl-d-aspartic acid (NMDA) receptor antagonist [2]. However, these drugs can only improve symptoms or slow down AD progression, not cure the disease [3]. Neurotrophic factors are considered potential therapeutic drugs for the treatment of neurodegenerative disorders. The targeted delivery of nerve growth factor (NGF) to basal forebrain cholinergic neurons has been reported to prevent cell death, stimulate synaptic cholinergic function and promote cognitive improvement in Rabbit polyclonal to AATK animals [4,5]. However, the application of NGF as a drug is limited because it cannot cross the blood brain barrier (BBB). Thus, finding small molecules that have neurotrophic properties much like those of NGF (NGF mimics) [68] and can cross the BBB is usually of great importance. Natural products are a great source of compounds for medical uses. Many potential lead compounds against AD have been found from natural products on land [912], however, marine organisms remain largely untapped for their potential for AD treatment [13]. Thus, isolation of active compounds from marine sources and determination of lead compounds for AD drug development are advantageous endeavors. In our laboratory, the PC12 cell bioassay system has long been established as a reliable bioassay system for the investigation of potential drugs with NGF mimicking activity. During our screening of active samples from marine natural products, the head of a fish,Ilisha elongata, was found to show neuritogenic activity against PC12 cells.I. elongate, belonging to family Pristigasteridae, is one of the main commercial fishes in coastal fisheries of the Indian Ocean, East China Sea and Russian waters [1416]. In this paper, we report the isolation, structural elucidation and biological activity of MG obtained from the head ofI. elongate. We further synthesised a series of compounds based on the structure of MG and obtained the lead Dimethylenastron compound SG by studying the structure-activity associations (SARs) of the monoglycerides. The mechanism of action of neurite outgrowth induced by SG on PC12 cells was finally investigated. == 2. Results and Conversation == == 2.1. Isolation and Structural Elucidation of 1-O-(Myristoyl) Glycerol == The head ofI. elongatewas freeze-dried, ground to a powder and extracted with methanol to obtain a crude extract. The extract was partitioned between EtOAc and H2O. The active EtOAc layer was subjected to silica open-column chromatography. The active fraction contained monoglyceride derivatives, as determined by high-resolution (HR)-ESI-TOF LC/MS analysis. The derivative with the highest content of active monoglycerides was obtained by HPLC purification. Only this compound was adequate for structural elucidation. The compound was identified as MG by spectroscopic analysis, and its spectroscopic data were comparable with Dimethylenastron those reported in the literature (Physique 1A) [17]. == Physique 1. == Chemical structure of MG, dose-dependent responses and photomicrographs of the NGF mimicking activity of MG 48 h after treatment. (A) Chemical structure of MG; (B) Percentage of neurite outgrowths of PC12 cells treated with MG at concentrations of 1 1, 3, 10 and 30 M. C: solvent Dimethylenastron control (0.5% DMSO); NGF (40 ng/mL): positive control; and (C) Photomicrographs of PC12 cells obtained under a phase-contrast microscope 48 h after treatment: (a) solvent control (0.5% DMSO); (b) NGF (40 ng/mL); (c) MG (10 M). Impartial experiments were repeated three times. Each value represents the imply SEM of three replicates.**and***indicate significant differences relative to the control atp< 0.01 andp< 0.001, respectively. == 2.2. NGF Mimic Activity of 1-O-(Myristoyl) Glycerol == The NGF mimicking activity of MG was tested on PC12 cells using NGF as a positive control and dimethyl sulfoxide (DMSO) as a negative control.Physique 1Bshows the percentage of neurite outgrowth of PC12 cells Dimethylenastron induced by MG at concentrations ranging from 1 to 30 M. MG.