Isorhynchophylline Protects PC12 Cells Against Beta-Amyloid-Induced Apoptosis via PI3K/Akt Signaling Pathway

Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 163057, 8 pages http://dx.doi.org/10.1155/2013/163057 Research Article Isorhynchophylline Protects PC12 Cells Against Beta-Amyloid-Induced Apoptosis via PI3K/Akt Signaling Pathway Yan-Fang Xian,1 Zhi-Xiu Lin,1 Qing-Qiu Mao,1 Jian-Nan Chen,2 Zi-Ren Su,2 Xiao-Ping Lai,2 and Paul Siu-Po Ip1 1 2 School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong College of Chinese Medicines, Guangzhou University of Chinese Medicine, 510006 Guangzhou, China Correspondence should be addressed to Zhi-Xiu Lin; and Paul Siu-Po Ip; Received 3 June 2013; Revised 12 September 2013; Accepted 23 September 2013 Academic Editor: Karl Wah-Keung Tsim Copyright © 2013 Yan-Fang Xian et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The neurotoxicity of amyloid-𝛽 (A𝛽) has been implicated as a critical cause of Alzheimer’s disease. Isorhynchophylline (IRN), an oxindole alkaloid isolated from Uncaria rhynchophylla, exerts neuroprotective effect against A𝛽25–35 -induced neurotoxicity in vitro. However, the exact mechanism for its neuroprotective effect is not well understood. The present study aimed to investigate the molecular mechanisms underlying the protective action of IRN against A𝛽25–35 -induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. Pretreatment with IRN significantly increased the cell viability, inhibited the release of lactate dehydrogenase and the extent of DNA fragmentation in A𝛽25–35 -treated cells. IRN treatment was able to enhance the protein levels of phosphorylated Akt (p-Akt) and glycogen synthase kinase-3𝛽 (p-GSK-3𝛽). Lithium chloride blocked A𝛽25–35 induced cellular apoptosis in a similar manner as IRN, suggesting that GSK-3𝛽 inhibition was involved in neuroprotective action of IRN. Pretreatment with LY294002 completely abolished the protective effects of IRN. Furthermore, IRN reversed A𝛽25–35 induced attenuation in the level of phosphorylated cyclic AMP response element binding protein (p-CREB) and the effect of IRN could be blocked by the PI3K inhibitor. These experimental findings unambiguously suggested that the protective effect of IRN against A𝛽25–35 -induced apoptosis in PC12 cells was associated with the enhancement of p-CREB expression via PI3K/Akt/GSK-3𝛽 signaling pathway. 1. Introduction Alzheimer’s disease (AD) is the most common form of neurodegenerative disorders of the brain and affects an estimated 26.6 million people across the globe in 2006 [1]. The neuropathological hallmarks of AD include massive accumulation of beta-amyloid (A𝛽) in senile plaques, abnormal tau filaments in neurofibrillary tangles, and extensive neuronal loss [2, 3]. A𝛽 is a 39- to 43-amino acid peptide fragment derived from sequential proteolysis of amyloid precursor protein (APP) through cleavage by 𝛽-secretase and 𝛾-secretase [4]. Recent studies have suggested that A𝛽 plays an important role in the pathogenesis of AD [5]. A𝛽 accumulation has been causatively implicated in the neuronal dysfunction and neuronal loss that underlie the clinical manifestations of AD [6]. A correlation among memory deficits, A𝛽 elevation, and amyloid plaques on transgenic has been reported in previous studies [7, 8]. Therefore, inhibition of A𝛽-induced neuronal degeneration may provide clinical benefits to AD patients. Isorhynchophylline (IRN, Figure 1), an oxindole alkaloid, has been identified as the main active ingredient responsible for the biological activities of Uncaria rhynchophylla [9, 10]. IRN has also been reported to protect against the ischemiaand glutamate-induced neuronal damage or death [9, 11], and inhibition of 5-HT receptor [12, 13]. Previous studies in our laboratory has demonstrated that IRN protected rat pheochromocytoma (PC12) cells against the A𝛽25–35 induced oxidative stress, mitochondrial dysfunction, apoptosis, calcium influx, and tau protein hyperphosphorylation 2 Evidence-Based Complementary and Alternative Medicine concentration of 20 𝜇M was then added to the culture for an additional 24 h. In experiments involving kinase inhibitors, the inhibitors LY294002 (50 𝜇M) or LiCl (10 mM) were added 1 h prior to IRN (50 𝜇M) and/or A𝛽25–35 (20 𝜇M) treatment. H N N H CH3 O OCH3 H3 CO O Figure 1: Chemical structure of isorhynchophylline (IRN). [14, 15]. However, the molecular mechanisms underlying the protective effect of IRN against the neurotoxicity induced by A𝛽25–35 have not been fully understood. In this study, we aimed to elucidate the molecular signaling pathway involved in the neuroprotective effect of IRN. 2. Materials and Methods 2.1. Chemicals and Reagents. Isorhynchophylline (IRN, purity ⩾ 98%) was purchased from Chengdu Mansite Pharmaceutical Co. Ltd. (Chengdu, Sichuan, China). Its identity was confirmed by comparing its H1 NMR spectra with the published data [16]. Nerve growth factor (NGF), LY294002 (LY), lithium chloride (LiCl), and 𝛽-amyloid peptide (A𝛽25–35 ) were purchased from SigmaAldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Gibco (Grand Island, NY, USA). Unless otherwise indicated, all other reagents were of analytical grade and were obtained from Sigma-Aldrich. 2.2. Peptide Preparation. A𝛽25–35 , which is the most toxic peptide fragment derived from amyloid precursor protein, was dissolved in deionized distilled water at the concentration of 1 mM. The stock solution was diluted to desired concentrations immediately before use and added to cell culture medium. 2.3. Cell Culture and Drug Treatment. The PC12 cells were obtained from the American Type Culture Collection (Rockville, MD, USA). They were maintained in DMEM medium supplemented with penicillin (100 U/mL), streptomycin (100 𝜇g/mL), 6% FBS, and 6% horse serum at 37∘ C in a humidified atmosphere of 95% air and 5% CO2 . Unless otherwise specified, the cells were seeded onto 24-well culture plate at a density of 8 × 104 cells/well. PC12 cells were differentiated with 50 ng/mL NGF in serum-free DMEM for 3 days [15]. IRN and all inhibitors were dissolved in DMSO and diluted with culture medium. The final concentration of DMSO in the test solutions was less than 0.1%. The cells were incubated with different concentrations of IRN (final concentrations: 1, 10, and 50 𝜇M) for 2 h. A𝛽25–35 at a final 2.4. Cell Viability Assay. Cell viability was measured using a CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). In brief, PC12 cells were seeded onto a 96-well culture plate at a density of 2 × 104 cells/well. Cells were washed with D-Hanks solution after drug treatment. Then, 100 𝜇L of serum-free medium and 20 𝜇L of CellTiter 96 AQ (...truncated)