The Protective Effect of Geniposide on Human Neuroblastoma Cells in The

Sun et al. BMC Complementary and Alternative Medicine 2013, 13:152 http://www.biomedcentral.com/1472-6882/13/152 RESEARCH ARTICLE Open Access The protective effect of geniposide on human neuroblastoma cells in the presence of formaldehyde Ping Sun1†, Jin-yan Chen3†, Jiao Li1, Meng-ru Sun3, Wei-chuan Mo2, Kai-li Liu2, Yan-yan Meng1, Ying Liu2, Feng Wang1, Rong-qiao He2* and Qian Hua1* Abstract Background: Formaldehyde can induce misfolding and aggregation of Tau protein and β amyloid protein, which are characteristic pathological features of Alzheimer’s disease (AD). An increase in endogenous formaldehyde concentration in the brain is closely related to dementia in aging people. Therefore, the discovery of effective drugs to counteract the adverse impact of formaldehyde on neuronal cells is beneficial for the development of appropriate treatments for age-associated cognitive decline. Methods: In this study, we assessed the neuroprotective properties of TongLuoJiuNao (TLJN), a traditional Chinese medicine preparation, against formaldehyde stress in human neuroblastoma cells (SH-SY5Y cell line). The effect of TLJN and its main ingredients (geniposide and ginsenoside Rg1) on cell viability, apoptosis, intracellular antioxidant activity and the expression of apoptotic-related genes in the presence of formaldehyde were monitored. Results: Cell counting studies showed that in the presence of TLJN, the viability of formaldehyde-treated SH-SY5Y cells significantly recovered. Laser scanning confocal microscopy revealed that the morphology of formaldehyde-injured cells was rescued by TLJN and geniposide, an effective ingredient of TLJN. Moreover, the inhibitory effect of geniposide on formaldehyde-induced apoptosis was dose-dependent. The activity of intracellular antioxidants (superoxide dismutase and glutathione peroxidase) increased, as did mRNA and protein levels of the antiapoptotic gene Bcl-2 after the addition of geniposide. In contrast, the expression of the apoptotic-related gene - P53, apoptotic executer - caspase 3 and apoptotic initiator - caspase 9 were downregulated after geniposide treatment. Conclusions: Our results indicate that geniposide can protect SH-SY5Y cells against formaldehyde stress through modulating the expression of Bcl-2, P53, caspase 3 and caspase 9, and by increasing the activity of intracellular superoxide dismutase and glutathione peroxidase. Keywords: Formaldehyde impairment, Geniposide, Neuroprotection Background apoptosis [1]. Reactive aldehydes are associated with the Reactive aldehydes are generated by several endogenous etiology of several neurological and psychiatric disorders and exogenous processes in biological systems. They target including chronic alcohol abuse, vascular dementia and cell membranes, induce lipid peroxidation, mitochondrial Alzheimer’s disease (AD) [2-4]. dysfunction and oxidative stress, and eventually lead to cell Formaldehyde is one of the reactive aldehydes with important clinical implications. He et al. summarized that formaldehyde can influence nerve function and that * Correspondence: [email protected]; [email protected] †Equal contributors it is linked to the development of psychiatric disorders 2State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, [5]. An epidemiological study has shown that histology Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing technicians and workers who are chronically exposed to 100101, China 1School of Preclinical Medicine, Beijing University of Chinese Medicine, 11 Bei formaldehyde exhibit a marked decline in cognitive ability San Huan Dong Road, Chaoyang District, Beijing 100029, China [6]. Formaldehyde concentrations in the hippocampus and Full list of author information is available at the end of the article © 2013 Sun et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sun et al. BMC Complementary and Alternative Medicine 2013, 13:152 Page 2 of 12 http://www.biomedcentral.com/1472-6882/13/152 urine of AD patients are significantly higher than those of performance liquid chromatography (HPLC; Figure 1) healthy aged individuals [7]. The activity of mitochondrial [14]. The purified geniposide and ginsenoside Rg1 aldehyde dehydrogenases, the main metabolic enzyme of were from the National Institutes for Food and Drug formaldehyde, is significantly increased in the putamen of Control (Beijing, China). The cell counting Kit-8 AD patients [1]. Furthermore, formaldehyde induces the (CCK-8) for measurement of cell viability was from hyperphosphorylation and aggregation of neuronal Tau pro- Dojindo Co. (Kumamoto, Japan). Hoechst33258 was from tein [8] and aggregation of Tau protein in vitro and in vivo Sigma and Alexa Fluor 488 Phalloidin was from Invitrogen [9-11]. These misfolded amorphous deposits are able to (Carlsbad, CA, USA). The primary antibody against Bcl-2 induce apoptosis of human neuroblastoma cells (SH-SY5Y was purchased from Cell Signaling Technology (Waltham, cell line) and primary rat hippocampal cells [9]. Formalde- MA, USA) and the β-actin antibody was from Beyotime hyde can also promote the misfolding, oligomerization, and Institute of Biotechnology (Jiangsu, China). The enhanced fibrillogenesis of β amyloid protein (Aβ) [12]. Therefore, chemiluminescence (ECL) substrate was from Pierce formaldehyde is closely related to age-associated dementia. (Boston, MA, USA) and polyvinylidene difluoride (PVDF) Recently, the discovery of effective drugs to counteract the membrane was purchased from Millipore (Billerica, adverse impacts of formaldehyde on the central nervous MA, USA). The annexin V apoptosis detection kit, system and promoting the survival of neuronal cells, has total superoxide dismutase (SOD) assay kit with become of great interest to many neuropharmacologists. WST-1, cellular glutathione peroxidase (GSHPx) assay kit TongLuoJiuNao (TLJN), a traditional Chinese medi- and catalase assay kit were from the Beyotime Institute of cine (TCM), is clinically efficacious in the treatment of Biotechnology (Jiangsu, China). The Taq DNA Polymerase ischemic cerebral stroke and dementia (Chinese SFDA: and TansStartTM Green qPCR SuperMix were purchased 2004 L01620). It has been reported that TLJN can protect from Transgen Inc. (Beijing, China). The primers for brain tissue against ischemia after the induction of middle Bcl-2, P53, caspase 3, caspase 9, β-actin and 18 s RNA cerebral artery occlusion in rats, by reducing not only were designed and synthesized by Shanghai Sangon infarct volume but also penumbra size [13], and alleviate Biotech Co. Ltd (Shanghai, China). the damage to ischemia/reperfusion-injured neurons [14]. Moreover, TLJN can improve learning and memory ability, Cell culture promote the degradation of Aβ, and clear amyloid plaques SH-SY5YcellsweregrowninDulbecco’s modified eagle from the AD rat brain [15]. The main ingredients of TLJN medium (DMEM) containing 10% (v/v) fetal bovine serum are geniposide and ginsenoside Rg1 [14]. Ginsenoside Rg1 (Invitrogen) and maintained in 5% (v/v) CO2/90% (v/v) has neuroprotective effects in mesencephalic dopaminergic humid air at 37°C. Cells were plated onto 96-well culture cells stressed with glutamate [16], while geniposide can plates at a density of 1 × 104 cells/well for cytotoxicity protect PC12 cells from hydrogen peroxide-induced cell assays or 60-mm culture dishes at a density of 1 × 106 cells/ death via its involvement in the PI3K signaling pathway well for western blotting, RT-PCR, flow cytometry, and and its ability to activate the glucagon-like peptide 1 total SOD detection. After 24 h, the culture medium was receptor (GLP-1R) [15]. Geniposide is also able to regulate replaced with serum-free DMEM supplemented with insulin secretion through activating GLP-1R [16]. Thus, 0.12 mM formaldehyde, and different concentrations of TLJN (geniposide and ginsenoside Rg1) is likely to be an drugs were used to treat cells as indicated in the results. appropriate drug for the treatment of dementia. However, whether it has any protective effects on reactive aldehyde- Cell viability assay treated neuronal cells, in particular formaldehyde-stressed The viability of cells was evaluated using the CCK-8 neurons, remains unclear. kit. Briefly, 10 μL of the CCK-8 solution was added Direct addition of formaldehyde to the cell culture to each well of the 96-well plate and left to incubate medium is a common cell culture model used in studies for4hintheincubatorat37°C.Accordingtothe on aldehyde toxicity [15]. Here, we examined the manufacturer, simultaneous absorbance readings were neuroprotective effects of TLJN and its main ingredients, measured at 450 and 630 nm using a microplate geniposide and ginsenoside Rg1, on formaldehyde-stressed reader. The difference between the two readings was SH-SY5Y cells. used in the final analysis. Methods Cell morphology analysis Materials SH-SY5Y cells (plated at 3 × 105) were grown in DMEM TLJN was provided by Kangyuan Pharmaceutical containing 10% (v/v) fetal bovine serum in 5% (v/v) Engineering Co. Ltd (Beijing, China). The concentra- CO2/90% (v/v) humid air at 37°C. After 12 h of plating, tions of geniposide (4.95 mg/mL) and ginsenoside the culture medium was replaced with serum-free Rg1 (1.02 mg/mL) in TLJN were determined by high DMEM in the presence

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