http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2014; 52(5): 628–636 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.860555
ORIGINAL ARTICLE
Actinidia callosa peel (kiwi fruit) ethanol extracts protected neural cells apoptosis induced by methylglyoxal through Nrf2 activation Chia-Chen Lee, Bao-Hong Lee, and She-Ching Wu
Abstract
Keywords
Context: Methylglyoxal (MG) is a reactive dicarbonyl compound generated as an intermediate of glycolysis during the physical glycation in the diabetic condition. MG itself has been commonly implicated in the development of diabetic neuropathy. Several active compounds in Actinidia callosa have been found to inhibit glycation and MG-protein reaction. Objective: This study investigated the protective effects of A. callosa (kiwi fruits) peel ethanol extracts (ACE) on MG-induced Neuro-2A cell apoptosis. Materials and methods: The Neuro-2A cells pre-treated by ACE (50–200 mg/mL) or allylisothiocyanate (AITC) (50 mM) for 6 h, in turn, the cells were treated with MG (250 mM) for 24 h. Results: ACE or AITC treatment markedly inhibited the generation of reactive oxygen species (ROS) and the elevation of caspase-3 and capase-9 levels induced by MG in Neuro-2A cells. ACE and AITC elevated Bcl2 and inhibited Bax expressions in MG-induced Neuro-2A cells. ACE elevated Nrf2 transcriptional activity and nuclear translocation in MG-induced Neuro-2A cells. Nrf2 down-stream molecules including HO-1 and GCL were elevated by ACE or AITC treatment in MG-induced Neuro-2A cells. The protective effects of ACE on MG-induced Neuro-2A apoptosis were attenuated while Nrf2 knockdown. Discussion and conclusion: We established the first evidence that ACE might contribute to the prevention of the development of diabetic neuropathy by blocking the MG-mediated intracellular glycation system.
Actinidia callosa peel ethnaolic extracts, apoptosis, diabetic neuropathy complication, methylglyoxal, Neuro-2A cell, nuclear factor-erythroid 2-related factor-2
Introduction Methylglyoxal (MG) is a reactive dicarbonyl compound physiologically generated as an intermediate of glycolysis during glycation. MG is a potent precursor of advanced glycation endproducts (AGEs) formation and is found at high levels in blood or tissue from experimental models of diabetes (Singh et al., 2001). MG induced AGEs formation has been commonly implicated in the development of diabetic microvascular complications including neuropathy, retinopathy, and atherosclerosis (Uchida et al., 1997). MG accumulation is often seen under conditions of hyperglycemia, impaired glucose metabolism, and oxidative stress (Abordo et al., 1999). Recently, several types of foods and drinks including coffee, cream, and cake have been shown to result in high MG levels in the plasma, thus causing both nutritional and health concerns. Coffee has been reported to contain high level of MG (230 mM) (17.7 mg/L) (Wang & Chang, 2010). In addition, approximate 5100 mg/g of MG was determined in honey (Adams et al., 2009). In Spain, dietary exposure of
Correspondence: She-Ching Wu, Department of Food Science, National Chiayi University, No. 300 Syue fu Rd, Chiayi City 60004, Taiwan, ROC. Tel: +886 5 2717622. Fax: +886 5 2717590. E-mail: [email protected].
History Received 22 May 2013 Accepted 21 October 2013 Published online 6 February 2014
the Spanish population to glyoxal and MG from cookies was estimated to be 213 and 216 mg/person/day, respectively (Arrbias-Lorenzo & Morales, 2010). Wine and beer were found to have 1556 and 1000 mg/L of MG, respectively (Barros et al., 1999; de Revel & Bertrand, 1993). Plasma of diabetic patients contain high levels of MG (200 mg/L) when comparing to normal human (5 mg/L) (Khuhawar et al., 2006). These data suggest that MG from dietary foods is accumulated in plasma in normal humans and the level may be equal to diabetic patients. Neuropathy is a common complication in the progression of diabetes mellitus, and neuronal apoptosis has been widely investigated in a recent study (Sharifi et al., 2007). High level of MG has been found in type 2 diabetic patients, leading to cell apoptosis (Wang et al., 2007). Evidence for a connection between neuronal apoptosis and diabetic neuropathy is gradually becoming established (Delaney et al., 2001; Schmeichel et al., 2003; Vincent et al., 2004). Consequently, understanding the effect of biological glycation on neuronal apoptosis has important implications for the prevention and treatment of diabetic neuropathy. Kiwi fruits (Actinidia callosa) contain a large amount of polyphenolic acid (Barberis et al., 2012) and show antioxidation activity (Fiorentino et al., 2009). Recently, A. callosa has been reported to down-regulate blood glucose and inhibits 3T3-L1 pre-adipocyte differentiation (Abe et al., 2010;
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Department of Food Science, National Chiayi University, Chiayi, Taiwan, ROC
DOI: 10.3109/13880209.2013.860555
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Shirosaki et al., 2008). Several active compounds in A. callosa have been found to inhibit glycation and MG-protein reaction, including 6-(2-pyrrolidinone-5-yl)()-epicatechin, 8-(2-pyrrolidinone-5-yl)-()-epicatechin, p-hydroxybenzoic acid, (þ)-pinoresinol, flavan-3-ols, (þ)-catechin, ()-epicatechin, and proanthocyanidin B (Jang et al., 2009). In addition, only a few studies have investigated the function of A. callosa peel. One study found that the ethanol extracts of A. callosa peel could improve sleep via a GABAergic mechanism (Yang et al., 2013). Currently, the effect of Actinidia callosa peel on protecting neural cells against MG-induced apoptosis in Neuro-2A cells was investigated. AITC has been reported to activate Nrf2, which was used as a positive control.
Materials and methods Materials Fetal bovine serum (FBS) was purchased from Life Technologies (Auckland, New Zealand). Dimethyl sulfoxide (DMSO) was purchased from Wako Pure Chemical Industries (Saitama, Japan). MG, 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), Triton X-100, trypsin, and 20 –70 -dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma Chemical Co. (St Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), penicillin, and streptomycin were purchased from HyClone Laboratories (Logan, UT). AntiNrf2 antibody for rat was purchased from Bioss Inc. (Woburn, MA). Anti-GAPDH antibody, anti-HO-1 antibody, anti-GCL antibody, anti-Bax antibody, anti-Bcl2 antibody, and Nrf2 siRNA were purchased from Santa Cruz (Burlingame, CA). Preparation of water extracts Actinidia callosa (kiwi fruit) was purchased from a local market. Approximately 25 g of peel of A. callosa was collected and mixed with 250 mL 95% ethanol for 24 h extraction at 4 C. The extracts were concentrated by vacuumdried and freeze-dried to powder. The A. callosa peel ethanol extracts (ACE) were stored at 20 C until used. Cell culture and treatment Neuro-2A neuroblastoma cell line was obtained from the Bioresource Collection and Research Center (BCRC, Food Industry Research and Development Institute, Hsin Chu, Taiwan). Cells were grown in DMEM medium, supplemented with 10% heat-inactivated FBS, glutamine (2 mM), and L-glutamine (2 mM) at 37 C, in a humidified atmosphere of 95% air and 5% CO2. Cells were treated by MG with ACE or AITC for different times, and the effects of ACE or AITC were investigated in the following experiments. Cell viability assay Neuro-2A cell viability was determined with MTT assays. MTT solution as added to each well for an additional incubation of 4 h at 37 C. After the addition of DMSO, the absorbance at 570 (formation of formazan) was measured (Hsu et al., 2010).
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Intracellular reactive oxygen species (ROS) production The intracellular ROS scavenging activity of sample was measured using the oxidant-sensitive fluorescent probe DCFHDA (Lee et al., 2011). Neuro-2A cells were harvested by the trypsin–EDTA solution (0.05% trypsin and 0.02% EDTA in PBS), washed with PBS twice. An aliquot of the suspension (195 mL) was mixed with DCFH-DA (20 mM final concentration). After washing, the cell pellet was mixed PBS and the ROS level was assayed by FACScan flowcytometry (BectonDickinson Lmmunocytometry Systems, San Jose, CA). Western immunoblotting Cells were lysed in ice-cold lysis buffer containing 20 mM TrisHCl (pH 7.4), 1% of Triton X-100, 0.1% of SDS, 2 mM EDTA, 10 mM NaF, 1 mM phenyl-methanesulfonyl fluoride, 500 mM sodium vanadate, and 10 mg/mL aprotinin overnight. Then the cell extract was centrifuged (12 000 g, 10 min) to recover the supernatant. The supernatant was taken as the cell extract. The cell protein was resolved on 10% SDS-PAGE and transferred to a polyvinyldiene fluoride membrane. Membranes were blocked with 5% nonfat milk for 1 h and incubated with primary antibodies for 4 h; subsequently, the membrane was washed three times each for 5 min in phosphate-buffered saline with Tween 20 (PBST), shaken in a solution of horseradish peroxidase (HRP)-linked secondary antibody for 1 h, and washed three more times each for 5 min in PBST. Proteins were detected by enhanced chemiluminescent reagent (Millipore, Billerica, MA). Colorimetric estimation of caspase-3, -8, and -9 activities Caspase-3, -8, and -9 activities were determined using kits from Biovision (Mountain View, CA). Cells were treated MG with or without ACE, and then cells were suspended in 50 mL of cell lysis buffer and incubated on ice for 10 min. Following centrifugation at 10 000 g for 1 min, the supernatant (cytosolic extract) was transferred to a fresh tube and put on ice for immediate assay. Protein concentration was assayed using 50200 mg of standard protein in 50 mL of cell lysis buffer for each assay. The cytosolic extract was mixed with 50 mL of reaction buffer (containing 10 mM dithiothreitol) and 5 mL of 4 mM substrate (200 mM final concentration), incubated at 37 C for 1 h and its absorbance at 405 nm was measured. Apoptosis analysis For apoptosis detection, Neuro-2A cells were harvested and washed in ice-cold PBS hence cells were re-suspended in 200 mL of binding buffer before being incubated in 5 mL of Annexin V-FITC (BD Biosciences, San Jose, CA) solution and 5 mL of PI (BD Biosciences, San Jose, CA) at room temperature for 15 min in the dark. Then 300 mL of binding buffer was added. Cells were analysed by flow cytometry. Untreated cells were used as the control for double staining (Hsu et al., 2010). Nrf2 luciferase assay A DNA fragment containing three copies of the antioxidant response element-4 (ARE4) from glutamatecysteine ligase
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(GCL) gene was subcloned into a pGL3-promoter vector to construct pGL3-ARE4-Luc. Cells were transiently transfected by a DNA mixture containing 2 mg of pGL3-ARE4-Luc and 0.5 mg of control plasmid pRL-TK (Promega, Madison, WI) with the lipofectamine-2000 transfection reagent in serumfree medium (Invitrogen, Carlsbad, CA). Luciferase activity was conducted utilizing the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). Luciferase activity of pRL-TK was used to normalize the transfection efficiency.
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Nrf2 knockdown by specific siRNA Neuro-2A cells were transfected with lipofectamine RNAiMAX transfection reagent to interfere Nrf2 gene (Invitrogen, Carlsbad, CA). The sequence of specific small interfering RNA (siRNA) for Nrf2 was transfected into Neuro-2A, and the control group was transfected with scramble siRNA. Cell lysates were subjected to Western blotting.
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Significant differences between means were determined by Duncan’s multiple range tests. Results were considered statistically significant at p50.05.
Results Cell viability of MG-treated Neuro-2A cells Recently, MG has been reported to result in oxidative stress and apoptosis in Neuro-2A cell (Huang et al., 2008a,b). We found that MG markedly induced a toxic effect in Neuro-2A cells in dose (0500 mM)- and time (024 h)-dependent manners (Figure 1A and B). As shown in Figure 1(C), ACE and AITC both did not exert cytotoxic protection in Neuro-2A cells co-treated with MG (250 mM) for 24 h. However, ACE (200 mg/mL) and AITC (50 mM) pre-treatment for 6 h showed protective effects in Neruo-2A cells before MG induction (24 h) (Figure 1D).
Statistical analysis
Anti-apoptotic effects of ACE in MG-induced Neruo-2A cells
Experimental results were the average of triplicate analyses. The data were recorded as mean SD and analyzed with a by statistical analysis system (SAS Inc., Cary, NC). A one-way analysis of variance was performed by ANOVA procedures.
To further elucidate the mechanism of action of ACE on Neruo-2A cells, we examined the effect of ACE on apoptosis by performing Annexin V-FITC/PI double staining. This staining method along with flow cytometry enables the
Figure 1. The cytotoxic effect of MG on Neuro-2A cells. (A) Neuro-2A cells were treated by various concentrations of MG for 24 h. (B) Neuro-2 A cells were treated with MG (250 mM) for various times (6, 12, 18, and 24 h). (C) Cell viability of Neuro-2 A cells co-treated with MG and ACE or AITC for 24 h. (D) Cell viability of Neuro-2 A cells pre-treated with ACE or AITC for 6 h before MG 24 h induction. MG: methylglyoxal. Data are shown as mean SD (n ¼ 3). Different letters indicate significant differences (p50.05).
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Figure 2. The effects of ACE (50–200 mg/mL) and AITC (50 mM) prevented MG-induced apoptosis in Neuro-2A cells. The Neuro-2A cells pre-treated by ACE or AITC for 6 h, in turn, the cells were treated with MG (250 mM) for 24 h. MG, methylglyoxal; AITC, allyl-isothiocyanate; ACE, Actinidia callosa peel ethanol extracts. Data are shown as mean SD (n ¼ 3). Different letters indicate significant differences (p50.05).
quantitative assessment of living (Annexin V-FITC negative/ PI negative), early apoptotic (Annexin V-FITC positive/PI negative), late apoptotic/necrotic (Annexin V-FITC positive/ PI positive), and dead (Annexin V-FITC negative/PI positive) cells (Lee et al., 2011). The protective effects of 6 h ACE or AITC pre-treatment on MG-induced Neruo-2A cell apoptosis are shown in Figure 2. The cells in the lower right quadrant represent early apoptosis, while those in the upper right quadrant represent late apoptosis. The results indicated that MG induction (24 h) clearly resulted in early apoptosis (17.3%), late apoptosis (36.2%), and necrosis (10.2%) in Neruo-2A cells. But pre-treatment (6 h) of ACE (200 mg/mL) or AITC (50 mM) effectively protected MG resulting in early apoptosis (6.5 and 3.3%, respectively) and late apoptosis (3.1 and 6.7%, respectively). Effects of ACE and AITC on ROS production and caspase-3, -8, and -9 activities In this study, we investigated whether ROS production was involved in the mechanism of MG (250 mM)-induced Neuro2A cell apoptosis and estimated the inhibitory effects of ACE or AITC on ROS production. As illustrated in Figure 3(A), the incubation of Neuro-2A cells with 250 mM MG for 24 h resulted in an increase in ROS generation by DCFH-DA stain, and we found that pre-treatment (6 h) of ACE (200 mg/mL) or AITC (50 mM) markedly lowered ROS production in MG-induced Neuro-2A cells.
In most of the apoptotic process, caspase-3 has been shown to play a pivotal role in the terminal, execution phase of apoptosis induced by diverse stimuli. Caspase-8 and caspase9 are activators/initiators of the death receptor pathway and the mitochondrial pathway, respectively (Fumarola & Guidotti, 2004; Thornberry & Lazebnik, 1998). The effects of ACE and AITC on caspase-3, -8, and -9 activities in MG-induced Neuro-2A cells were examined. As shown in Figure 3(B–D), ACE (200 mg/mL) and AITC (50 mM) pretreatment (6 h) significantly attenuated caspase-3 and -9 activities in MG-induced Neuro-2A cells, but caspase-8 activity was not affected by MG, ACE, or AITC. Improvement of ACE or AITC on Bcl2 and Bax ration We found that MG treatment (250 mM) for 24 h markedly attenuated Bcl2 and elevated Bax levels in Neuro-2A cells, but these effects were up- and down-regulated by ACE (200 mg/mL) and AITC (50 mM) pre-treatment (6 h) in MGinduced Neuro-2A cells (Figure 4). Regulations of ACE and AITC on Nrf2 activation Nrf2 is an essential component of antioxidant responsive element (ARE)-mediated induction, including the regulation of antioxidant enzymes. Nrf2 controls the expression and coordinates the induction of a number of genes, including those that express stress response proteins and detoxifying enzymes. Therefore, the nuclear abundance of Nrf2 is tightly
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Figure 3. Inhibitory effects of ACE (50–200 mg/mL) and AITC (50 mM) on (A) ROS production, (B) caspase-3, (C) caspase-9, and (D) caspase-8 activities in MG (250 mM)-induced Neuro-2A cells. The Neuro-2A cells pre-treated with ACE or AITC for 6 h. In turn, the cells were treated with MG for 24 h. MG, methylglyoxal; AITC, allyl-isothiocyanate; ACE, Actinidia callosa peel ethanol extracts. Data are shown as mean SD (n ¼ 3). Different letters indicate significant differences (p50.05).
regulated through the control of the mechanisms of nuclear export and degradation of Nrf2. It has been found that MG induction may down-regulate Nrf2 in vivo (Lee et al., 2012). Nrf2 is essential for heme oxygenase-1 (HO-1), glutamylcysteine ligase (GCL), and glyoxalase-1 expression (Keum et al., 2003; Kobayashi & Yamamoto, 2005; Vander-Jagt & Hunsaker, 2003). Glyoxalase-1 catalyzes the conversion of MG to D-lactic acid (Vander-Jagt & Hunsaker, 2003). As shown in Figure 5, ACE (200 mg/mL) and AITC (50 mM) pretreatment (6 h) effectively avoided decreases in GCL and HO-1 expression in MG-induced Neuro-2A cells. But the glyoxalase level was not affected by MG, ACE, or AITC treatment in MG-induced Neuro-2A cells. We found that ACE (200 mg/mL) and AITC (50 mM) treatment for 24 h significantly elevated Nrf2 reporter activity in Neuro-2A cells with or without MG induction (Figure 6). In addition, after treatment with ACE or AITC for 6–24 h, the translocation of Nrf2 from cytoplasm into the nucleus was increased in a time-dependent manner (Figure 7A). MG (250 mM) induction markedly lowered the nuclear Nrf2 level of Neuro-2A cells, but ACE (200 mg/mL) and AITC (50 mM) pre-treatment for 6 h significantly recovered the efficacy of Nrf2 translocation into nucleolus in MG-induced Neuro-2A cells (Figure 7B).
In order to confirm the mechanism of ACE (200 mg/mL) activated Nrf2 in MG (250 mM)-induced Neuro-2A cells, we utilized siRNA to silence Nrf2 expression in Neuro-2A cells. The results indicated that treatment with Nrf2 siRNA for 48 h inhibits Nrf2 expression in Neuro-2A cells (Figure 8A). As shown in Figure 8(B–E), we found that Nrf2 knockdown severely reduced cell viability, elevated ROS production, promoted caspase-3, and caspase-9 activities in MG (250 mM)-induced Neuro-2A cells compared to the MG alone group. In addition, the inhibition of ACE (200 mg/mL) and AITC (50 mM) resulted in cytotoxicity, ROS production, and attenuation of caspase-3, and caspase-9 activation in MGinduced Neuro-2A cells while Nrf2 was silenced. These results suggest that Nrf2 mediates an important response in ACE treatment for preventing Neuro-2A cells damage caused by MG.
Discussion MG is a well-known toxic compound leading to cell apoptosis. Studies have shown that MG-induced apoptosis in human leukemia 60 cells, Schwann cells, rat hippocampal neurons, and Nuuro-2A cells (Kang et al., 1996; Fukunaga et al., 2005; Di Loreto et al., 2008; Ota et al., 2007). This
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Figure 4. Effects of ACE (50–200 mg/mL) and AITC (50 mM) on Bcl2 and Bax levels in MG (250 mM)-induced Neuro-2A cells. The Neuro-2A cells pre-treated with ACE or AITC for 6 h. In turn, the cells were treated with MG for 24 h. MG, methylglyoxal; AITC, allyl-isothiocyanate; ACE, Actinidia callosa peel ethanol extracts. Data are shown as mean SD (n ¼ 3). Different letters indicate significant differences (p50.05).
evidence indicates that MG may result in diabetic neuropathy and memory impairment (Huang et al., 2008b). Phenolic acids and antioxidants have been found to show inhibitory effects on MG-induced Neuro-2A cell death, such as chlorogenic acid, syringic acid, and vanillic acid (Huang et al., 2008a). To clarify the effects of antioxidation on the molecular mechanisms of MG-induced Neuro-2A cell apoptosis, recent studies have evaluated the protein levels associated with apoptosis, including caspase-3 and caspase-9 expression (Huang et al., 2008a). Evidence suggests that production of ROS and activation of the caspase cascade triggers apoptotic processes in various cells (Hsu et al., 2010). Caspase-3, a member of the caspase family, has been shown to play a key role in apoptosis induced by a variety of stimuli (Lee et al., 2011; Hsu et al., 2010). Our results indicated that treatment with MG clearly elevated caspase-3 and capase-9 levels in Neuro-2A cells, but ACE or AITC treatment markedly inhibited expression of these proteins thereby preventing apoptosis (Figures 2 and 3). Some members of the Bcl2 family, such as Bcl2, is an apoptotic regulator to suppress cell death, while other
Figure 5. Effects of ACE (50–200 mg/mL) and AITC (50 mM) on glyoxalase, GCL, and HO-1 expression in MG (250 mM)-induced Neuro2A cells. The Neuro-2A cells were pre-treated with ACE or AITC for 6 h. In turn, the cells are treated with MG for 24 h. MG, methylglyoxal; AITC, allyl-isothiocyanate; ACE, Actinidia callosa peel ethanol extracts. Data are shown as mean SD (n ¼ 3). Different letters indicate significant differences (p50.05).
homologues including Bax and Bak exhibit powerful death promoting abilities. The Bcl2 family proteins play a central role in the process of cell apoptosis by interfering with caspases. It is well established that the ratio between Bcl2 and Bax is an important factor in the regulation of apoptosis rather than the level of each protein separately (Saikumar et al., 1998). Bax is a 21-kDa protein promoting mitochondrial membrane permeability, which has been demonstrated to accelerate apoptotic cell death. The ratio of Bcl2 to Bax, rather than the levels of the individual proteins, is considered
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Figure 6. Promotion of ACE (50–200 mg/mL) and AITC (50 mM) on Nrf2 transcriptional activity in MG (250 mM)-induced Neuro-2A cells. (A) The Neuro-2A cells were treated by ACE or AITC for various times. In turn, luciferase activity was determined. (B) Cells were pre-treated with ACE or AITC for 6 h, and then Neuro-2A cells were induced by MG for 24 h. MG, methylglyoxal; AITC, allyl-isothiocyanate; ACE, Actinidia callosa ethanol extracts. Data are shown as mean SD (n ¼ 5). Different letters indicate significant differences (p50.05).
Figure 7. Elevation of Nrf2-nuclear translocation in MG (250 mM)-induced Neuro-2A cells treated with ACE or AITC. (A) The Neuro-2A cells were treated with ACE for various times (6, 12, 18, and 24 h), subsequently, nuclear Nrf2 level was measured. (B) Neuro-2A cells were pre-treated with ACE (50 and 200 mg/mL) or AITC (50 mM) for 6 h, and then Neuro-2A cells were induced by MG for 24 h. MG, methylglyoxal; AITC, allyl-isothiocyanate; ACE, Actinidia callosa peel ethanol extracts. Data are shown as mean SD (n ¼ 5). Different letters indicate significant differences (p50.05).
critical in the determination of survival/death of cells (Scorrano et al., 2003). Our results indicated that ACE and AITC elevated Bcl2 and inhibited Bax expression in MGinduced Neuro-2A cells (Figure 4).
Nrf2 plays an important role in improving glucose tolerance and insulin resistance in Nrf2-knockout mice fed with high-fat diet for 180 days (Chartoumpekis et al., 2011). Nrf2 activation has been confirmed to improve insulin
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Figure 8. (A) The inhibitory effect of Nrf2 siRNA for Nrf2 expression in Neuro-2A cells. Effects of ACE (200 mg/mL) and AITC (50 mM) on (B) cell viability, (C) ROS production, (D) caspase-3, and (E) caspase-9 activities in MG (250 mM)-induced Neuro-2A cells treated by Nrf2 siRNA. MG, methylglyoxal; AITC, allyl-isothiocyanate; ACE, Actinidia callosa peel ethanol extracts. Data are shown as mean SD (n ¼ 5). Different letters indicate significant differences (p50.05).
sensitivity and metabolic syndrome (Yu et al., 2012). Hence, these results suggested that Nrf2 has potential for protecting diabetic neuropathy. We found that ACE elevated Nrf2 transcriptional activity and nuclear translocation in MG-induced Neuro-2A cells (Figures 6 and 7). The expression of Nrf2 down-stream was elevated by ACE or AITC treatment in MG-induced Neuro-2A cells, including HO-1 and GCL (Figure 5). In addition, the same results have been observed in Nrf2-silenced Neuro-2A cells. We found that the protective effects of ACE on MG-induced Neuro-2A apoptosis were attenuated with Nrf2 knockdown (Figure 8). Several lines of evidence indicate that Nrf2 phosphorylation (serine 40) can facilitate MG metabolism and its eventual degradation by upregulating glyoxalase-1 and antioxidant enzymes (Vander-Jagt et al., 2003). We considered that ACE may lead to Nrf2 phosphorylation thereby improving Neuro-
2A cell viability. The potentially protective mechanism of ACE for MG-induced Neuro-2A cell apoptosis is shown in Figure 9.
Conclusions In conclusion, the last-decade of research has demonstrated that neuropathy is a neurodegenerative disease. Increasing evidence suggests that neuronal cell-cycle regulatory failure followed by apoptosis is an important mechanism in the development of diabetic neuropathy complications. The data presented above show that MG was cytotoxic for neuronal cells in the progression of diabetic neuropathy. In the current study, we found that Nrf2 was activated by ACE and AITC treatment in MG-induced Neuro-2A cells, leading to increased HO-1 and GCL expression and suppressing
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Figure 9. The potential mechanism of ACE for protecting Neuro-2A cell apoptosis caused by MG induction.
MG-induced cell death. Taken together, ACE and AITC showed cytoprotective effects against MG-induced neuronal cell apoptosis, suggesting that the peel of kiwi fruits might be a potential strategy in the prevention of diabetic neuropathy complications.
Declaration of interest No competing financial interests exist.
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Huang SM, Chuang HC, Wu CH, Yen GC. (2008a). Cytoprotective effects of phenolic acid on methylglyoxal-induced apoptosis in neuro2A cells. Mol Nutr Food Res 52:940–9. Huang SM, Hsu CL, Chuang HC, et al. (2008b). Inhibitory effect of vanillic acid on methylglyoxal-mediated glycation in apoptotic neuro2A cells. NeuroToxicology 29:1016–22. Jang DS, Lee GY, Lee YM, et al. (2009). Flavan-3-ols having a gammalactam from the roots of Actinidia arguta inhibit the formation of advanced glycation endproducts in vitro. Chem Pharm Bull 57: 397–400. Kang Y, Edwards LG, Thornalley PJ. (1996). Effect of methylglyoxal on human leukaemia 60 cell growth: Modification of DNA G1 growth arrest and induction of apoptosis. Leuk Res 20:397–405. Keum YS, Owuor ED, Kim BR, et al. (2003). Involvement of Nrf2 and JNK1 in the activation of antioxidant responsive element (ARE) by chemopreventive agent phenethyl isothiocyanate (PEITC). Pharm Res 20:1351–6. Khuhawar MY, Kandhro AJ, Khand FD. (2006). Liquid chromatographic determination of glyoxal and methylglyoxal from serum of diabetic patients using meso-stilbenediamine as derivatizing agent. Anal Lett 39:2205–15. Kobayashi M, Yamamoto M. (2005). Molecular mechanisms activating the Nrf2-Keap1 pathway of antioxidant gene regulation. Antioxid Redox Signal 7:385–94. Lee BH, Hsu WH, Chang YY, et al. (2012). Ankaflavin: A natural novel PPARgamma agonist upregulates Nrf2 to attenuate methylglyoxalinduced diabetes in vivo. Free Radic Biol Med 53:2008–16. Lee BH, Huang YY, Wu SC. (2011). Hepatoprotective activity of fresh Polygonum multiflorum against Hep G2 hepatocarcinoma cells proliferation. J Food Drug Anal 19:26–32. Ota K, Nakamura J, Li W, et al. (2007). Metformin prevents methylglyoxal-induced apoptosis of mouse Scheann cells. Biochem Biophys Res Commun 25:270–5. Saikumar P, Dong Z, Patel Y. (1998). Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury. Oncogene 17:3401–5. Schmeichel AM, Schmelzer JD, Low PA. (2003). Oxidative injury and apoptosis of dorsal root ganglion neurons in chronic experimental diabetic neuropathy. Diabetes 52:165–71. Scorrano L, Korsmeyer SJ. (2003). Mechanisms of cytochrome c release by proapoptotic BCL-2 family members. Biochem Biophys Res Commun 304:437–44. Sharifi AM, Mousavi SH, Farhadi M, Larijani B. (2007). Study of high glucose-induced apoptosis in PC12 cells: Role of bax protein. J Pharmacol Sci 104:258–62. Shirosaki M, Koyama T, Yazawa K. (2008). Anti-hyperglycemic activity of kiwifruit leaf (Actinidia deliciosa) in mice. Biosci Biotechnol Biochem 72:1099–102. Singh R, Barden A, Mori T, Beilin L. (2001). Advanced glycation endproducts: A review. Diabetologia 44:129–46. Thornberry NA, Lazebnik Y. (1998). Caspase: Enemies within. Science 281:1312–16. Uchida K, Khor OT, Oya T, et al. (1997). Protein modification by a Maillard reaction intermediate methylglyoxal. Immunochemical detection of fluorescent 5-methylimidazolone derivatives in vivo. FEBS Lett 410:313–18. Vander-Jagt D, Hunsaker L. (2003). Methylglyoxal metabolism and diabetic complications: Roles of aldose reductase, glyxalase-I, betaine aldehyde dehydrogenase and oxoaldehyde dehydrogenase. Chem Biol Interact 143/144:341–51. Vincent AM, Russell JW, Low P, Feldman EL. (2004). Oxidative stress in the pathogenesis of diabetic neuropathy. Endocr Rev 25:612–28. Wang H, Meng QH, Gordon JR, et al. (2007). Proinflammatory and proapoptotic effects of methylglyoxal on neutrophils from patients with type 2 diabetes mellitus. Clin Biochem 40:123–29. Wang J, Chang T. (2010). Methylglyoxal content in drinking coffee as a cytotoxic factor. J Food Sci 75:H167–71. Yang H, Lee YC, Han KS, et al. (2013). Green and gold kiwifruit peel ethanol extracts potentiate pentobarbital-induced sleep in mice via a GABAergic mechanism. Food Chem 136:160–3. Yu ZW, Li D, Ling WH, Jin TR. (2012). Role of nuclear factor (erythroid-derived 2)-like 2 in metabolic homeostasis and insulin action: A novel opportunity for diabetes treatment? World J Diabetes 3:19–28.
ORIGINAL ARTICLE
Actinidia callosa peel (kiwi fruit) ethanol extracts protected neural cells apoptosis induced by methylglyoxal through Nrf2 activation Chia-Chen Lee, Bao-Hong Lee, and She-Ching Wu
Abstract
Keywords
Context: Methylglyoxal (MG) is a reactive dicarbonyl compound generated as an intermediate of glycolysis during the physical glycation in the diabetic condition. MG itself has been commonly implicated in the development of diabetic neuropathy. Several active compounds in Actinidia callosa have been found to inhibit glycation and MG-protein reaction. Objective: This study investigated the protective effects of A. callosa (kiwi fruits) peel ethanol extracts (ACE) on MG-induced Neuro-2A cell apoptosis. Materials and methods: The Neuro-2A cells pre-treated by ACE (50–200 mg/mL) or allylisothiocyanate (AITC) (50 mM) for 6 h, in turn, the cells were treated with MG (250 mM) for 24 h. Results: ACE or AITC treatment markedly inhibited the generation of reactive oxygen species (ROS) and the elevation of caspase-3 and capase-9 levels induced by MG in Neuro-2A cells. ACE and AITC elevated Bcl2 and inhibited Bax expressions in MG-induced Neuro-2A cells. ACE elevated Nrf2 transcriptional activity and nuclear translocation in MG-induced Neuro-2A cells. Nrf2 down-stream molecules including HO-1 and GCL were elevated by ACE or AITC treatment in MG-induced Neuro-2A cells. The protective effects of ACE on MG-induced Neuro-2A apoptosis were attenuated while Nrf2 knockdown. Discussion and conclusion: We established the first evidence that ACE might contribute to the prevention of the development of diabetic neuropathy by blocking the MG-mediated intracellular glycation system.
Actinidia callosa peel ethnaolic extracts, apoptosis, diabetic neuropathy complication, methylglyoxal, Neuro-2A cell, nuclear factor-erythroid 2-related factor-2
Introduction Methylglyoxal (MG) is a reactive dicarbonyl compound physiologically generated as an intermediate of glycolysis during glycation. MG is a potent precursor of advanced glycation endproducts (AGEs) formation and is found at high levels in blood or tissue from experimental models of diabetes (Singh et al., 2001). MG induced AGEs formation has been commonly implicated in the development of diabetic microvascular complications including neuropathy, retinopathy, and atherosclerosis (Uchida et al., 1997). MG accumulation is often seen under conditions of hyperglycemia, impaired glucose metabolism, and oxidative stress (Abordo et al., 1999). Recently, several types of foods and drinks including coffee, cream, and cake have been shown to result in high MG levels in the plasma, thus causing both nutritional and health concerns. Coffee has been reported to contain high level of MG (230 mM) (17.7 mg/L) (Wang & Chang, 2010). In addition, approximate 5100 mg/g of MG was determined in honey (Adams et al., 2009). In Spain, dietary exposure of
Correspondence: She-Ching Wu, Department of Food Science, National Chiayi University, No. 300 Syue fu Rd, Chiayi City 60004, Taiwan, ROC. Tel: +886 5 2717622. Fax: +886 5 2717590. E-mail: [email protected].
History Received 22 May 2013 Accepted 21 October 2013 Published online 6 February 2014
the Spanish population to glyoxal and MG from cookies was estimated to be 213 and 216 mg/person/day, respectively (Arrbias-Lorenzo & Morales, 2010). Wine and beer were found to have 1556 and 1000 mg/L of MG, respectively (Barros et al., 1999; de Revel & Bertrand, 1993). Plasma of diabetic patients contain high levels of MG (200 mg/L) when comparing to normal human (5 mg/L) (Khuhawar et al., 2006). These data suggest that MG from dietary foods is accumulated in plasma in normal humans and the level may be equal to diabetic patients. Neuropathy is a common complication in the progression of diabetes mellitus, and neuronal apoptosis has been widely investigated in a recent study (Sharifi et al., 2007). High level of MG has been found in type 2 diabetic patients, leading to cell apoptosis (Wang et al., 2007). Evidence for a connection between neuronal apoptosis and diabetic neuropathy is gradually becoming established (Delaney et al., 2001; Schmeichel et al., 2003; Vincent et al., 2004). Consequently, understanding the effect of biological glycation on neuronal apoptosis has important implications for the prevention and treatment of diabetic neuropathy. Kiwi fruits (Actinidia callosa) contain a large amount of polyphenolic acid (Barberis et al., 2012) and show antioxidation activity (Fiorentino et al., 2009). Recently, A. callosa has been reported to down-regulate blood glucose and inhibits 3T3-L1 pre-adipocyte differentiation (Abe et al., 2010;
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Department of Food Science, National Chiayi University, Chiayi, Taiwan, ROC
DOI: 10.3109/13880209.2013.860555
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Shirosaki et al., 2008). Several active compounds in A. callosa have been found to inhibit glycation and MG-protein reaction, including 6-(2-pyrrolidinone-5-yl)()-epicatechin, 8-(2-pyrrolidinone-5-yl)-()-epicatechin, p-hydroxybenzoic acid, (þ)-pinoresinol, flavan-3-ols, (þ)-catechin, ()-epicatechin, and proanthocyanidin B (Jang et al., 2009). In addition, only a few studies have investigated the function of A. callosa peel. One study found that the ethanol extracts of A. callosa peel could improve sleep via a GABAergic mechanism (Yang et al., 2013). Currently, the effect of Actinidia callosa peel on protecting neural cells against MG-induced apoptosis in Neuro-2A cells was investigated. AITC has been reported to activate Nrf2, which was used as a positive control.
Materials and methods Materials Fetal bovine serum (FBS) was purchased from Life Technologies (Auckland, New Zealand). Dimethyl sulfoxide (DMSO) was purchased from Wako Pure Chemical Industries (Saitama, Japan). MG, 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), Triton X-100, trypsin, and 20 –70 -dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma Chemical Co. (St Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), penicillin, and streptomycin were purchased from HyClone Laboratories (Logan, UT). AntiNrf2 antibody for rat was purchased from Bioss Inc. (Woburn, MA). Anti-GAPDH antibody, anti-HO-1 antibody, anti-GCL antibody, anti-Bax antibody, anti-Bcl2 antibody, and Nrf2 siRNA were purchased from Santa Cruz (Burlingame, CA). Preparation of water extracts Actinidia callosa (kiwi fruit) was purchased from a local market. Approximately 25 g of peel of A. callosa was collected and mixed with 250 mL 95% ethanol for 24 h extraction at 4 C. The extracts were concentrated by vacuumdried and freeze-dried to powder. The A. callosa peel ethanol extracts (ACE) were stored at 20 C until used. Cell culture and treatment Neuro-2A neuroblastoma cell line was obtained from the Bioresource Collection and Research Center (BCRC, Food Industry Research and Development Institute, Hsin Chu, Taiwan). Cells were grown in DMEM medium, supplemented with 10% heat-inactivated FBS, glutamine (2 mM), and L-glutamine (2 mM) at 37 C, in a humidified atmosphere of 95% air and 5% CO2. Cells were treated by MG with ACE or AITC for different times, and the effects of ACE or AITC were investigated in the following experiments. Cell viability assay Neuro-2A cell viability was determined with MTT assays. MTT solution as added to each well for an additional incubation of 4 h at 37 C. After the addition of DMSO, the absorbance at 570 (formation of formazan) was measured (Hsu et al., 2010).
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Intracellular reactive oxygen species (ROS) production The intracellular ROS scavenging activity of sample was measured using the oxidant-sensitive fluorescent probe DCFHDA (Lee et al., 2011). Neuro-2A cells were harvested by the trypsin–EDTA solution (0.05% trypsin and 0.02% EDTA in PBS), washed with PBS twice. An aliquot of the suspension (195 mL) was mixed with DCFH-DA (20 mM final concentration). After washing, the cell pellet was mixed PBS and the ROS level was assayed by FACScan flowcytometry (BectonDickinson Lmmunocytometry Systems, San Jose, CA). Western immunoblotting Cells were lysed in ice-cold lysis buffer containing 20 mM TrisHCl (pH 7.4), 1% of Triton X-100, 0.1% of SDS, 2 mM EDTA, 10 mM NaF, 1 mM phenyl-methanesulfonyl fluoride, 500 mM sodium vanadate, and 10 mg/mL aprotinin overnight. Then the cell extract was centrifuged (12 000 g, 10 min) to recover the supernatant. The supernatant was taken as the cell extract. The cell protein was resolved on 10% SDS-PAGE and transferred to a polyvinyldiene fluoride membrane. Membranes were blocked with 5% nonfat milk for 1 h and incubated with primary antibodies for 4 h; subsequently, the membrane was washed three times each for 5 min in phosphate-buffered saline with Tween 20 (PBST), shaken in a solution of horseradish peroxidase (HRP)-linked secondary antibody for 1 h, and washed three more times each for 5 min in PBST. Proteins were detected by enhanced chemiluminescent reagent (Millipore, Billerica, MA). Colorimetric estimation of caspase-3, -8, and -9 activities Caspase-3, -8, and -9 activities were determined using kits from Biovision (Mountain View, CA). Cells were treated MG with or without ACE, and then cells were suspended in 50 mL of cell lysis buffer and incubated on ice for 10 min. Following centrifugation at 10 000 g for 1 min, the supernatant (cytosolic extract) was transferred to a fresh tube and put on ice for immediate assay. Protein concentration was assayed using 50200 mg of standard protein in 50 mL of cell lysis buffer for each assay. The cytosolic extract was mixed with 50 mL of reaction buffer (containing 10 mM dithiothreitol) and 5 mL of 4 mM substrate (200 mM final concentration), incubated at 37 C for 1 h and its absorbance at 405 nm was measured. Apoptosis analysis For apoptosis detection, Neuro-2A cells were harvested and washed in ice-cold PBS hence cells were re-suspended in 200 mL of binding buffer before being incubated in 5 mL of Annexin V-FITC (BD Biosciences, San Jose, CA) solution and 5 mL of PI (BD Biosciences, San Jose, CA) at room temperature for 15 min in the dark. Then 300 mL of binding buffer was added. Cells were analysed by flow cytometry. Untreated cells were used as the control for double staining (Hsu et al., 2010). Nrf2 luciferase assay A DNA fragment containing three copies of the antioxidant response element-4 (ARE4) from glutamatecysteine ligase
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(GCL) gene was subcloned into a pGL3-promoter vector to construct pGL3-ARE4-Luc. Cells were transiently transfected by a DNA mixture containing 2 mg of pGL3-ARE4-Luc and 0.5 mg of control plasmid pRL-TK (Promega, Madison, WI) with the lipofectamine-2000 transfection reagent in serumfree medium (Invitrogen, Carlsbad, CA). Luciferase activity was conducted utilizing the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). Luciferase activity of pRL-TK was used to normalize the transfection efficiency.
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Nrf2 knockdown by specific siRNA Neuro-2A cells were transfected with lipofectamine RNAiMAX transfection reagent to interfere Nrf2 gene (Invitrogen, Carlsbad, CA). The sequence of specific small interfering RNA (siRNA) for Nrf2 was transfected into Neuro-2A, and the control group was transfected with scramble siRNA. Cell lysates were subjected to Western blotting.
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Significant differences between means were determined by Duncan’s multiple range tests. Results were considered statistically significant at p50.05.
Results Cell viability of MG-treated Neuro-2A cells Recently, MG has been reported to result in oxidative stress and apoptosis in Neuro-2A cell (Huang et al., 2008a,b). We found that MG markedly induced a toxic effect in Neuro-2A cells in dose (0500 mM)- and time (024 h)-dependent manners (Figure 1A and B). As shown in Figure 1(C), ACE and AITC both did not exert cytotoxic protection in Neuro-2A cells co-treated with MG (250 mM) for 24 h. However, ACE (200 mg/mL) and AITC (50 mM) pre-treatment for 6 h showed protective effects in Neruo-2A cells before MG induction (24 h) (Figure 1D).
Statistical analysis
Anti-apoptotic effects of ACE in MG-induced Neruo-2A cells
Experimental results were the average of triplicate analyses. The data were recorded as mean SD and analyzed with a by statistical analysis system (SAS Inc., Cary, NC). A one-way analysis of variance was performed by ANOVA procedures.
To further elucidate the mechanism of action of ACE on Neruo-2A cells, we examined the effect of ACE on apoptosis by performing Annexin V-FITC/PI double staining. This staining method along with flow cytometry enables the
Figure 1. The cytotoxic effect of MG on Neuro-2A cells. (A) Neuro-2A cells were treated by various concentrations of MG for 24 h. (B) Neuro-2 A cells were treated with MG (250 mM) for various times (6, 12, 18, and 24 h). (C) Cell viability of Neuro-2 A cells co-treated with MG and ACE or AITC for 24 h. (D) Cell viability of Neuro-2 A cells pre-treated with ACE or AITC for 6 h before MG 24 h induction. MG: methylglyoxal. Data are shown as mean SD (n ¼ 3). Different letters indicate significant differences (p50.05).
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Figure 2. The effects of ACE (50–200 mg/mL) and AITC (50 mM) prevented MG-induced apoptosis in Neuro-2A cells. The Neuro-2A cells pre-treated by ACE or AITC for 6 h, in turn, the cells were treated with MG (250 mM) for 24 h. MG, methylglyoxal; AITC, allyl-isothiocyanate; ACE, Actinidia callosa peel ethanol extracts. Data are shown as mean SD (n ¼ 3). Different letters indicate significant differences (p50.05).
quantitative assessment of living (Annexin V-FITC negative/ PI negative), early apoptotic (Annexin V-FITC positive/PI negative), late apoptotic/necrotic (Annexin V-FITC positive/ PI positive), and dead (Annexin V-FITC negative/PI positive) cells (Lee et al., 2011). The protective effects of 6 h ACE or AITC pre-treatment on MG-induced Neruo-2A cell apoptosis are shown in Figure 2. The cells in the lower right quadrant represent early apoptosis, while those in the upper right quadrant represent late apoptosis. The results indicated that MG induction (24 h) clearly resulted in early apoptosis (17.3%), late apoptosis (36.2%), and necrosis (10.2%) in Neruo-2A cells. But pre-treatment (6 h) of ACE (200 mg/mL) or AITC (50 mM) effectively protected MG resulting in early apoptosis (6.5 and 3.3%, respectively) and late apoptosis (3.1 and 6.7%, respectively). Effects of ACE and AITC on ROS production and caspase-3, -8, and -9 activities In this study, we investigated whether ROS production was involved in the mechanism of MG (250 mM)-induced Neuro2A cell apoptosis and estimated the inhibitory effects of ACE or AITC on ROS production. As illustrated in Figure 3(A), the incubation of Neuro-2A cells with 250 mM MG for 24 h resulted in an increase in ROS generation by DCFH-DA stain, and we found that pre-treatment (6 h) of ACE (200 mg/mL) or AITC (50 mM) markedly lowered ROS production in MG-induced Neuro-2A cells.
In most of the apoptotic process, caspase-3 has been shown to play a pivotal role in the terminal, execution phase of apoptosis induced by diverse stimuli. Caspase-8 and caspase9 are activators/initiators of the death receptor pathway and the mitochondrial pathway, respectively (Fumarola & Guidotti, 2004; Thornberry & Lazebnik, 1998). The effects of ACE and AITC on caspase-3, -8, and -9 activities in MG-induced Neuro-2A cells were examined. As shown in Figure 3(B–D), ACE (200 mg/mL) and AITC (50 mM) pretreatment (6 h) significantly attenuated caspase-3 and -9 activities in MG-induced Neuro-2A cells, but caspase-8 activity was not affected by MG, ACE, or AITC. Improvement of ACE or AITC on Bcl2 and Bax ration We found that MG treatment (250 mM) for 24 h markedly attenuated Bcl2 and elevated Bax levels in Neuro-2A cells, but these effects were up- and down-regulated by ACE (200 mg/mL) and AITC (50 mM) pre-treatment (6 h) in MGinduced Neuro-2A cells (Figure 4). Regulations of ACE and AITC on Nrf2 activation Nrf2 is an essential component of antioxidant responsive element (ARE)-mediated induction, including the regulation of antioxidant enzymes. Nrf2 controls the expression and coordinates the induction of a number of genes, including those that express stress response proteins and detoxifying enzymes. Therefore, the nuclear abundance of Nrf2 is tightly
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Figure 3. Inhibitory effects of ACE (50–200 mg/mL) and AITC (50 mM) on (A) ROS production, (B) caspase-3, (C) caspase-9, and (D) caspase-8 activities in MG (250 mM)-induced Neuro-2A cells. The Neuro-2A cells pre-treated with ACE or AITC for 6 h. In turn, the cells were treated with MG for 24 h. MG, methylglyoxal; AITC, allyl-isothiocyanate; ACE, Actinidia callosa peel ethanol extracts. Data are shown as mean SD (n ¼ 3). Different letters indicate significant differences (p50.05).
regulated through the control of the mechanisms of nuclear export and degradation of Nrf2. It has been found that MG induction may down-regulate Nrf2 in vivo (Lee et al., 2012). Nrf2 is essential for heme oxygenase-1 (HO-1), glutamylcysteine ligase (GCL), and glyoxalase-1 expression (Keum et al., 2003; Kobayashi & Yamamoto, 2005; Vander-Jagt & Hunsaker, 2003). Glyoxalase-1 catalyzes the conversion of MG to D-lactic acid (Vander-Jagt & Hunsaker, 2003). As shown in Figure 5, ACE (200 mg/mL) and AITC (50 mM) pretreatment (6 h) effectively avoided decreases in GCL and HO-1 expression in MG-induced Neuro-2A cells. But the glyoxalase level was not affected by MG, ACE, or AITC treatment in MG-induced Neuro-2A cells. We found that ACE (200 mg/mL) and AITC (50 mM) treatment for 24 h significantly elevated Nrf2 reporter activity in Neuro-2A cells with or without MG induction (Figure 6). In addition, after treatment with ACE or AITC for 6–24 h, the translocation of Nrf2 from cytoplasm into the nucleus was increased in a time-dependent manner (Figure 7A). MG (250 mM) induction markedly lowered the nuclear Nrf2 level of Neuro-2A cells, but ACE (200 mg/mL) and AITC (50 mM) pre-treatment for 6 h significantly recovered the efficacy of Nrf2 translocation into nucleolus in MG-induced Neuro-2A cells (Figure 7B).
In order to confirm the mechanism of ACE (200 mg/mL) activated Nrf2 in MG (250 mM)-induced Neuro-2A cells, we utilized siRNA to silence Nrf2 expression in Neuro-2A cells. The results indicated that treatment with Nrf2 siRNA for 48 h inhibits Nrf2 expression in Neuro-2A cells (Figure 8A). As shown in Figure 8(B–E), we found that Nrf2 knockdown severely reduced cell viability, elevated ROS production, promoted caspase-3, and caspase-9 activities in MG (250 mM)-induced Neuro-2A cells compared to the MG alone group. In addition, the inhibition of ACE (200 mg/mL) and AITC (50 mM) resulted in cytotoxicity, ROS production, and attenuation of caspase-3, and caspase-9 activation in MGinduced Neuro-2A cells while Nrf2 was silenced. These results suggest that Nrf2 mediates an important response in ACE treatment for preventing Neuro-2A cells damage caused by MG.
Discussion MG is a well-known toxic compound leading to cell apoptosis. Studies have shown that MG-induced apoptosis in human leukemia 60 cells, Schwann cells, rat hippocampal neurons, and Nuuro-2A cells (Kang et al., 1996; Fukunaga et al., 2005; Di Loreto et al., 2008; Ota et al., 2007). This
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Figure 4. Effects of ACE (50–200 mg/mL) and AITC (50 mM) on Bcl2 and Bax levels in MG (250 mM)-induced Neuro-2A cells. The Neuro-2A cells pre-treated with ACE or AITC for 6 h. In turn, the cells were treated with MG for 24 h. MG, methylglyoxal; AITC, allyl-isothiocyanate; ACE, Actinidia callosa peel ethanol extracts. Data are shown as mean SD (n ¼ 3). Different letters indicate significant differences (p50.05).
evidence indicates that MG may result in diabetic neuropathy and memory impairment (Huang et al., 2008b). Phenolic acids and antioxidants have been found to show inhibitory effects on MG-induced Neuro-2A cell death, such as chlorogenic acid, syringic acid, and vanillic acid (Huang et al., 2008a). To clarify the effects of antioxidation on the molecular mechanisms of MG-induced Neuro-2A cell apoptosis, recent studies have evaluated the protein levels associated with apoptosis, including caspase-3 and caspase-9 expression (Huang et al., 2008a). Evidence suggests that production of ROS and activation of the caspase cascade triggers apoptotic processes in various cells (Hsu et al., 2010). Caspase-3, a member of the caspase family, has been shown to play a key role in apoptosis induced by a variety of stimuli (Lee et al., 2011; Hsu et al., 2010). Our results indicated that treatment with MG clearly elevated caspase-3 and capase-9 levels in Neuro-2A cells, but ACE or AITC treatment markedly inhibited expression of these proteins thereby preventing apoptosis (Figures 2 and 3). Some members of the Bcl2 family, such as Bcl2, is an apoptotic regulator to suppress cell death, while other
Figure 5. Effects of ACE (50–200 mg/mL) and AITC (50 mM) on glyoxalase, GCL, and HO-1 expression in MG (250 mM)-induced Neuro2A cells. The Neuro-2A cells were pre-treated with ACE or AITC for 6 h. In turn, the cells are treated with MG for 24 h. MG, methylglyoxal; AITC, allyl-isothiocyanate; ACE, Actinidia callosa peel ethanol extracts. Data are shown as mean SD (n ¼ 3). Different letters indicate significant differences (p50.05).
homologues including Bax and Bak exhibit powerful death promoting abilities. The Bcl2 family proteins play a central role in the process of cell apoptosis by interfering with caspases. It is well established that the ratio between Bcl2 and Bax is an important factor in the regulation of apoptosis rather than the level of each protein separately (Saikumar et al., 1998). Bax is a 21-kDa protein promoting mitochondrial membrane permeability, which has been demonstrated to accelerate apoptotic cell death. The ratio of Bcl2 to Bax, rather than the levels of the individual proteins, is considered
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Figure 6. Promotion of ACE (50–200 mg/mL) and AITC (50 mM) on Nrf2 transcriptional activity in MG (250 mM)-induced Neuro-2A cells. (A) The Neuro-2A cells were treated by ACE or AITC for various times. In turn, luciferase activity was determined. (B) Cells were pre-treated with ACE or AITC for 6 h, and then Neuro-2A cells were induced by MG for 24 h. MG, methylglyoxal; AITC, allyl-isothiocyanate; ACE, Actinidia callosa ethanol extracts. Data are shown as mean SD (n ¼ 5). Different letters indicate significant differences (p50.05).
Figure 7. Elevation of Nrf2-nuclear translocation in MG (250 mM)-induced Neuro-2A cells treated with ACE or AITC. (A) The Neuro-2A cells were treated with ACE for various times (6, 12, 18, and 24 h), subsequently, nuclear Nrf2 level was measured. (B) Neuro-2A cells were pre-treated with ACE (50 and 200 mg/mL) or AITC (50 mM) for 6 h, and then Neuro-2A cells were induced by MG for 24 h. MG, methylglyoxal; AITC, allyl-isothiocyanate; ACE, Actinidia callosa peel ethanol extracts. Data are shown as mean SD (n ¼ 5). Different letters indicate significant differences (p50.05).
critical in the determination of survival/death of cells (Scorrano et al., 2003). Our results indicated that ACE and AITC elevated Bcl2 and inhibited Bax expression in MGinduced Neuro-2A cells (Figure 4).
Nrf2 plays an important role in improving glucose tolerance and insulin resistance in Nrf2-knockout mice fed with high-fat diet for 180 days (Chartoumpekis et al., 2011). Nrf2 activation has been confirmed to improve insulin
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DOI: 10.3109/13880209.2013.860555
Prevention of neuro-2A cells by peel of kiwi fruits
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Figure 8. (A) The inhibitory effect of Nrf2 siRNA for Nrf2 expression in Neuro-2A cells. Effects of ACE (200 mg/mL) and AITC (50 mM) on (B) cell viability, (C) ROS production, (D) caspase-3, and (E) caspase-9 activities in MG (250 mM)-induced Neuro-2A cells treated by Nrf2 siRNA. MG, methylglyoxal; AITC, allyl-isothiocyanate; ACE, Actinidia callosa peel ethanol extracts. Data are shown as mean SD (n ¼ 5). Different letters indicate significant differences (p50.05).
sensitivity and metabolic syndrome (Yu et al., 2012). Hence, these results suggested that Nrf2 has potential for protecting diabetic neuropathy. We found that ACE elevated Nrf2 transcriptional activity and nuclear translocation in MG-induced Neuro-2A cells (Figures 6 and 7). The expression of Nrf2 down-stream was elevated by ACE or AITC treatment in MG-induced Neuro-2A cells, including HO-1 and GCL (Figure 5). In addition, the same results have been observed in Nrf2-silenced Neuro-2A cells. We found that the protective effects of ACE on MG-induced Neuro-2A apoptosis were attenuated with Nrf2 knockdown (Figure 8). Several lines of evidence indicate that Nrf2 phosphorylation (serine 40) can facilitate MG metabolism and its eventual degradation by upregulating glyoxalase-1 and antioxidant enzymes (Vander-Jagt et al., 2003). We considered that ACE may lead to Nrf2 phosphorylation thereby improving Neuro-
2A cell viability. The potentially protective mechanism of ACE for MG-induced Neuro-2A cell apoptosis is shown in Figure 9.
Conclusions In conclusion, the last-decade of research has demonstrated that neuropathy is a neurodegenerative disease. Increasing evidence suggests that neuronal cell-cycle regulatory failure followed by apoptosis is an important mechanism in the development of diabetic neuropathy complications. The data presented above show that MG was cytotoxic for neuronal cells in the progression of diabetic neuropathy. In the current study, we found that Nrf2 was activated by ACE and AITC treatment in MG-induced Neuro-2A cells, leading to increased HO-1 and GCL expression and suppressing
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C.-C. Lee et al.
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Figure 9. The potential mechanism of ACE for protecting Neuro-2A cell apoptosis caused by MG induction.
MG-induced cell death. Taken together, ACE and AITC showed cytoprotective effects against MG-induced neuronal cell apoptosis, suggesting that the peel of kiwi fruits might be a potential strategy in the prevention of diabetic neuropathy complications.
Declaration of interest No competing financial interests exist.
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