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Protective effects of silk lutein extract from Bombyx mori cocoons on β-Amyloid peptide-induced apoptosis in PC12 cells
Biomedicine & Pharmacotherapy 103 (2018) 582–587
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Protective effects of silk lutein extract from Bombyx mori cocoons on βAmyloid peptide-induced apoptosis in PC12 cells
T
Nongnuch Singhranga, Chainarong Tocharusb,c, Sarinthorn Thummayotd, ⁎ Manote Sutheerawattananondae, Jiraporn Tocharusa, a
Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand c Center for Research and Development of Natural Products for Health, Chiang Mai University d Division of Anatomy, Faculty of Medical Science, University of Phayao, 56000, Thailand e School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand b
A R T I C LE I N FO
A B S T R A C T
Keywords: Alzheimer’s disease Silk lutein extract β-Amyloid PC12 cells Apoptosis Neurodegeneration Oxidative stress Bombyx mori cocoons
Beta-amyloid (Aβ) peptide, the hallmark of Alzheimer’s disease (AD), invokes a cascade of oxidative damage to neurons and eventually leads to neuronal death. This study evaluated the protective effects of lutein extract from yellow cocoons of Bombyx mori, and its underlying mechanisms against was investigated to assess its protective effects and the underlying mechanisms against Aβ25-35-induced neuronal cell death in cultured rat pheochromocytoma (PC12) cells. Aβ25-35-induced neuronal toxicity is characterized by decrease in cell viability, increase in intracellular reactive oxygen species (ROS) production, activation of mitochondrial death pathway, and activation the phospholyration of mitogen-activated protein kinase (MAPKs) pathway. Pretreatment with silk lutein extract significantly attenuated Aβ25-35-induced loss of cell viability, apoptosis, MAPKs pathway activation and ROS production. Taken together, our present study suggests that silk lutein extract protects PC12 cells from Aβ25-35-induced neurotoxicity via the reduction of the ROS production, and subsequent attenuation of the mitochondrial death pathway and reduces the activation of the MAPK kinase pathways. This compound might beneficial as potential therapeutic agent to prevent or retard the development and progression of AD.
1. Introduction
p38 MAPK. Excessive activation of MAPKs signaling pathways are the major signals in the neuronal cell death [13,14]. Thus, inhibition of these pathways could be beneficial for preventing or attenuating cell death in AD. Lutein is a group of carotenoids, found in the diets such as yellow corn, egg yolk and other fruits or green leafy vegetables [15]. Lutein is a lipid- soluble antioxidant, obtained from diet or supplements. Lutein exists mostly in the lens and macular. Recently, it has been suggested that lutein exerted its biological activity not only in the anti-oxidative effect but also in the anti-inflammatory effect [16–19]. It has been reported that lutein reduced oxidative damage in H2O2-induced human lens epithelial cells [20]. In addition, lutein administration reduced the level of ROS in retinal neural cells in lipopolysaccharide (LPS) –treated mice and in the diabetic retina [21,22]. Moreover, lutein protected the retinal ganglion cells and inner retinal cells from diabetes-induced cell death and maintains cell survival [22]. Recently, lutein also has been shown to exert a neuroprotective effect against ischemic stroke induced by cerebral ischemia/reperfusion in mice [23]. In addition, the antiamyloidogenic of activity of carotenoids such as cryptocapsin,
Alzheimer’s disease (AD) is an age-related neurodegenerative disease characterized by the progressive degeneration and loss of neurons in the brain. The pathological hallmarks of AD are a degeneration of cholinergic neurons, an accumulation of beta amyloid (Aβ) deposition in senile plaques and neurofibrillary tangle lesions in specific areas of the brain [1–3]. The pathogenesis of AD is not fully clear; Aβ has been recognized as one of the major causes of AD pathology [4–6]. The neurotoxic effects of Aβ have been reported in the in vivo and in vitro studies by triggering oxidative stress, impairing mitochondria function, thus increasing apoptotic cell death [7,8]. Therefore, numerous antioxidants have been demonstrated to be useful for the prevention or attenuation of AD development or progression [9–11]. In addition, oxidative stress is the initiating factor and upstream of the activation of the mitogen-activated protein kinase family members (MAPKs) in many kinds of cells [12]. The three major mammalian MAPKs are extracellular signal-regulated kinases 1 and 2 (ERK1/2), cJun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and ⁎
Corresponding author. E-mail address: [email protected] (J. Tocharus).
https://doi.org/10.1016/j.biopha.2018.04.045 Received 7 January 2018; Received in revised form 6 April 2018; Accepted 6 April 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
Biomedicine & Pharmacotherapy 103 (2018) 582–587
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plate and exposed to silk lutein extract for 2 h, which was followed by 10 μM Aβ25-35. Prior to the detection of ROS, the medium was removed, and PBS containing 20 μM 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) was added to each well, followed by incubation at 37 °C for 2 h in the dark. Fluorescence intensity was measured using a microplate reader (DTX 800, Beckman coulter, Austria) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.
cryptocapsin-5,6-epoxide and zeaxanthin was reported [24]. However, whether the administration of silk lutein reduces cell death in Aβ-induced neuronal cells and has a neuroprotective effect mechanism remains to be elucidated. 2. Materials and methods 2.1. Chemicals
2.6. Western blot analysis Dulbecco's Modified Eagle Medium: a nutrient Mixture F-12 (DMEM/F12), fetal bovine serum (FBS), fetal horse serum (FHS), trypsin, penicillin and streptomycin were purchased from GIBCO-BRL (Gaithersburg, MD, USA). The following antibodies were used for Western blotting: anti-ERK, anti-JNK, anti-p38, phospho-ERK, phosphoJNK, phospho-p38, β-actin, anti-mouse, anti-rabbit IgG peroxidaseconjugated secondary antibody (Millipore, Bedford, MA, USA). Specific inhibitors of ERK (PD98059), p38 (SB203580), JNK (SP60012) were purchased from Calbiochem (San Diego, CA, USA). Aβ25–35, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO, USA).
The cells were cultured at a density of 5 × 105 cells/ml in a 60 mm culture dish at 37 °C overnight. The cells were treated with silk lutein extract for 2 h, treated with or without 10 μM Aβ25-35 for 24 h, and then collected. The cells were lysed in RIPA buffer containing 1% NP40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 40 mM β-glycerophosphate, 50 mM sodium fluoride, 2 mM sodium orthovanadate and 1x protease inhibitor at 4 °C with rigorous shaking for 15 min. The cell lysate was centrifuged at 13,000 rpm at 4 °C for 20 min. The protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Equal amounts of proteins (50 μg) were electrophoresed in a 10–15% SDS polyacrylamide gel and then transferred to a PVDF membrane (Immobilon-P, Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk and probed with the indicated antibodies (anti-β-actin monoclonal antibody, anti-ERK1/2 monoclonal antibody, anti- pERK1/2 monoclonal antibody, anti-p JNK1/2 monoclonal antibody, anti- p38 MAPK monoclonal antibody, anti-JNK1/2 polyclonal antibody and anti-p-p38 MAPK polyclonal antibody) overnight at 4 °C followed by using probed with the indicated secondary antibodies. Detection was carried out using an enhanced chemiluminescence detection kit (Millipore, Bedford, MA, USA).
2.2. Cell culture PC12 cells, a rat pheochromocytoma cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in DMEM/F12, containing 10% heat-inactivated fetal horse serum, 5% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin. Cultures were incubated at 37 °C and 5% CO2 in a fully-humidified incubator. Aβ25–35 was dissolved in water at a concentration of 1 mM and stored at −20 °C. Before being used in any treatment, Aβ25–35 was diluted with phosphate buffer saline (PBS) and aggregated at 37 °C for 5 days.
2.7. Apoptosis detection assay by flow cytometry 2.3. Preparation of lutein extract The number of apoptotic cells was determined by flow cytometry. The cells at a density of 1 × 105 cells/ml at 37 °C were treated with silk lutein extract for 2 h prior to treatment in the presence or absence of 10 μM Aβ25-35 for 24 h. The percentage of cells undergoing apoptosis after the treatment was determined using the MuseTM Annexin-V and Dead cell assay kit and the Bcl2 levels (EMD Millipore, Bioscience). To determine the caspase-3 levels, the cells were incubated with caspase-3/7 working reaction assay (EMD Millipore Biosciences) for 10 min in the dark at room temperature. To determine the activation Bcl-2, the cells were incubated with Bcl-2 working reaction assay (EMD Millipore Biosciences) for 10 min in the dark at room temperature. Finally, the number of the apoptotic cells, the caspase-3 positive cells, and activated Bcl-2 positive cells were analyzed by using Muse Cell Analyzer (Merck Millipore, MA, USA), according to the manufacturer’s instruction.
Silk lutein extract was prepared from yellow silk cocoons of Thai silk worms (Bombyx mori) according to pending patent no. PCT/ TH2010/000048. Silk lutein extract was prepared from yellow silk cocoons as described previously [24]. Briefly, the silk cocoons were degummed at high pressure and temperature and then extracted with a solvent mixture of hexane, ethyl alcohol and ethyl acetate. The obtained solution was re-extracted with 10% NaCl to remove water-soluble impurities. The extract was concentrated at room temperature and then freeze-dried. The concentration of silk lutein was quantified by reverse-phase HPLC (Agilent HP 1100 series) using a LiChrospher®100 reverse-phase C18 column as stationary and acetonitrile/methanolethyl acetate as mobile phase and monitored at 445 nm. The HPLC chromatogram has been shown in previous study [24]. The silk lutein extract was kept in darkness at −20 °C until use.
2.8. Statistical analysis 2.4. Cell viability assay All data are expressed as mean ± SEM of three independent experiments. The statistical difference was analyzed using one-way analysis of variance (ANOVA) followed by Post Hoc Dunnett’s test for comparing the significance between the individual groups (p < 0.05).
Cell viability was measured by quantitative colorimetric assay with MTT. PC12 cells were cultured at a density of 1.5 × 104 cells/well in 96-well plate overnight. The various concentrations of silk lutein extract were added to PC12 cells. After 24 h, 10 mg/ml of MTT were added to each well and further incubated for 4 h at 37 °C in a humidified 5% CO2 and 95% air atmosphere. The formazan was solubilized with dimethyl sulfoxide (DMSO) and was determined by measuring the absorbance at 540 nm using a microplate reader (Bio-Tek Instruments, Winoaski, VT, USA).
3. Results 3.1. Effect of silk lutein extract on Aβ25-35-induced cytotoxicity in PC12 cells Aβ25-35-induced cytotoxicity was determined by measuring MTT reduction assay in PC12 cells cultured for 24 h with Aβ25-35 (1–50 μM) (Fig. 1A). Aβ25-35 reduced cell viability in a concentration-dependent manner. Exposure to Aβ25-35 at the concentration of 10 μM resulted in 63.38 ± 1.2% of relative cell viability which concentration was later
2.5. Measurement of reactive oxygen species (ROS) The production of ROS was determined using the DCF assay. The cells were plated for 24 h at a density of 2 × 104 cells/well in 96-well 583
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Fig. 2. Effect of silk lutein extract on Aβ25-35 –induced intracellular ROS. Cells were pretreated with various concentrations (0, 0.2, 2, 20, and 200 μM) of silk lutein extract for 2 h, followed by Aβ25-35 treated. Intracellular ROS levels were determined by measuring DCF fluorescence intensity, which measured fluorescent spectrophotometrically with the excitation at 485 nm and emission at 535 nm. The values present the mean ± SEM from 3 independent experiments. ***p < 0.001, in comparison with the control treatment; #p < 0.05, ## p < 0.01,###p < 0.001, in comparison with the Aβ25-35 treatment alone.
Fig. 1. Protective effect of silk lutein extract on Aβ25-35 –induced cytotoxicity in PC12 cells. Cell viability was determined by MTT assay. (A) PC12 cells were treated with various concentrations of Aβ25-35 (1, 5, 10 and 50 μM) for 24 h. (B) PC12 cells were pretreated with various concentrations (0, 0.2, 2, 20 and 200 μM) of silk lutein extract for 2 h, followed by Aβ25-35 treated for 24 h. The values present the mean ± SEM from 3 independent experiments. ***p < 0.001, in comparison with the control treatment; ###p < 0.001, in comparison with the Aβ25-35 treatment alone.
of apoptotic cells. Pretreatment with silk lutein extract significantly increased the levels of active caspase3/7 in a dose-dependent manner (Fig. 3E). These results suggest that silk lutein extract markedly reduced the apoptosis of Aβ25-35-induced PC cells.
used in all subsequent experiments. To investigate the neuroprotective effects of silk lutein extract on Aβ25-35-induced cytotoxicity in PC12 cell, cells were pretreated with or without those compounds at various concentrations (0.2, 2, 20 and 200 μM) for 2 h, followed by treatment with 10 μM of Aβ25-35 for 24 h. Silk lutein extract increased cell viability in the concentration-dependent manner. The neuroprotective effect of silk lutein at concentration of 200 μM was found to be similar to that of control as shown in Fig. 1B. However, silk lutein extract alone did not show any cytotoxicity (data not shown).
3.4. Effect of silk lutein extract on MAPKs signaling pathway To explore the mechanisms underlying the protective effect of silk lutein extract against Aβ25-35–induced cell death, MAP kinase are implicated in oxidative-stress-induced cell death. The phosphorylation of MAPK molecules, ERK1/2, P38 MAPK and JNK, in Aβ25-35–stimulated PC12 cells were examined. Treatment of PC12 cells with Aβ25-35 significantly increased the levels of phosphorylated ERK1/2 (pERK1/2), p38 MAPK (p-p38) and JNK (pJNK) (P < 0.05 as compared with the control). Pretreatment of PC12 cells with silk lutein extract at the concentration of 0.2, 2, 20 and 200 μM significantly attenuated Aβ25-35induced phosphorylation of pERK1/2 (Fig. 4A), p-p38 (Fig. 4B) and pJNK (Fig. 4C) while their non-phosphorylated forms remained unchanged. Similar results were observed in Aβ25-35–induced PC12 cells treated with PD98059 (ERK inhibitor), SB20358 (p38 inhibitor) and SP600125 (JNK inhibitor).
3.2. Effect of silk lutein extract on Aβ25-35-induced reactive oxygen species (ROS) Elevation of intracellular ROS has been implicated in cell death and ROS were determined by using H2DCFDA. To determine the effect of silk lutein extract on intracellular ROS production, we investigated the production of ROS following Aβ25-35 exposure. The level of ROS significantly increased 1.3 fold after exposure to Aβ25-35 (10 μM). Pretreatment with silk lutein extract at 0.2, 2, 20 and 200 μM for 2 h before exposure to Aβ25-35 markedly reduced the levels of ROS production to 1.10, 1.03, 1.00 and 0.99 fold of control, respectively. Silk lutein extract dose-dependently inhibited Aβ25-35-induced shift of fluorescent intensity, implicating its role in preventing the accumulation of ROS. These findings indicate that pretreatment with silk lutein extract can effectively prevent Aβ25-35 –induced ROS production (Fig. 2).
4. Discussion Aβ, the major component of senile plaques that form in the brains of patients with AD, may cause neuronal cell death via the generation of ROS and the elevation of calcium [24,25]. Several studies have shown that oxidative stress plays an important role in neuronal degeneration in AD [25]. Previous studies, both in vivo and in vitro, showed that Aβ25-35 treatment caused a significant increase in the level of ROS leading to oxidative damaged to DNA, lipids and protein, thereby impairing cell function [8,26–28]. Therefore, numerous antioxidants have been demonstrated to be useful for the prevention or attenuation of AD development or progression [9–11]. Our present study was the first to demonstrate the neuroprotective effects of silk lutein and its possible molecular mechanism in PC12 cells against Aβ25-35 –induced neuronal apoptosis. Treatment with 10 μM Aβ25-35 inhibited cell viability leading to an increase of cell death (60–70%) in cultured neuronal PC12 cells which was almost completely restored by treatment with 200 μM silk lutein extract. These results indicated that silk lutein extract did significantly protect PC12 cells from Aβ25-35 -induced cytotoxicity. Aβ associated with senile plaques formed in the brains of patients
3.3. Effect of silk lutein extract on the expression of apoptotic signaling pathway The induction of apoptosis after treatment of the cells with Aβ25-35 was determined by flow cytometry. The results showed that silk lutein extract significantly decreased the number of apoptotic cells in a dosedependent manner, compared to the only Aβ25-35 treatment (Fig. 3 A, B and C). The level of Bcl-2 proteins regulates the apoptotic cell death. We then investigated the expression of Bcl-2 protein in silk luteinmediated neuroprotection against Aβ25-35 –induced neurotoxicity. After PC12 cells were treated with Aβ25-35 for 24 h, the expression of Bcl-2 was significantly decreased. With the pretreatment with silk lutein extract at the concentration of 0.2, 2, 20 and 200 μM, the Bcl-2 expression was significantly increased (Fig.3D). Moreover, the active caspase 3/7 level was also significantly decreased, which correlates to the number 584
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Fig. 3. The protective effect of silk lutein extract on Aβ25-35- induced apoptosis in the PC12 cells. (A) PC12 cells were pretreated with or without silk lutein extract at the concentrations of 0.2, 2, 20 and 200 μM for 2 h before being treated with 10 μM Aβ25-35 for 24 h. The apoptotic cells were analyzed using an Annexin V/PI staining assay (A, B and C). The Bcl-2 level (D) and the caspase-3 level (E) by flow cytometry. The values present the mean ± SEM from 3 independent experiments. ***p < 0.001, in comparison with the control treatment; #p < 0.05, ##p < 0.01,###p < 0.001, in comparison with the Aβ25-35 treatment alone.
suggested that antioxidants may suppress Aβ25–35-induced ROS generation, thereby decreasing neurotoxicity [10,11,30,31]. Recent study demonstrated that lutein prevented Aβ25-35 -induced neurotoxicity mediated by modulating Nrf2 and NF-kB expression in cerebrovascular endothelial cells [33]. It is well-known that neuronal apoptosis is a leading pathway for Aβ-induced neurotoxicity; therefore the prevention of Aβ-triggered apoptosis is a strategy for the treatment of AD. The mitochondrial pathway of apoptosis is regulated by the Bcl-2 family proteins, consisting of several proteins including anti-apoptotic proteins such as BclXL and pro-apoptotic proteins Bcl-2 and pro-apoptotic proteins including Bax [32–34]. The balance between Bax and Bcl-2 plays a
with AD may lead to the generation of ROS, mitochondria dysfunction and ultimately to subsequent cause neuronal cell death [4,6,26,29]. Our findings indicate that, similar to other reports, Aβ25-35 was found to cause an elevation of oxidative stress as characterized by excessive production of intracellular ROS [7,27]. Interestingly, pretreatment with silk lutein extract significantly suppressed Aβ25–35-induced ROS accumulation dose-dependently, suggesting that the scavenging effect of silk lutein extract may be a significant contributor to cytoprotective effects. This was probably due to several double bonds in silk lutein which directly react with ROS to scavenge radicals. Consistent with a previous report, lutein suppresses the generation of ROS in diabetic retina and in H2O2-induced human lens epithelial cells [20,21]. Several studies 585
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Fig. 4. Effect of silk lutein extract on the phosphorylation of expression of MAPKs kinase pathway in Aβ25-35 induced PC12 cells. Cells were pretreated with 0, 0.2, 2, 20 and 200 μM of silk lutein extract for 2 h prior Aβ25-35 10 μM was added. The cell lysates were subjected to SDS-PAGE followed by Western blot with antibodies for ERK (A) The PC12 cells were prior treated with PD98059 (50 μM) for 1 h; pre-incubation was carried out with silk lutein extract (0.2, 2, 20 and 200 μM) for 2 h, and the cells were exposed to Aβ25-35 (10 μM) for another 24 h, p38 (B) The PC12 cells were prior treated with SB203580 (30 μM) for 1 h; pre-incubation was carried out with silk lutein extract (0.2, 2, 20 and 200 μM) for 2 h, and the cells were exposed to Aβ25-35 (10 μM) for another 24 h, JNK (C) The PC12 cells were prior treated with SP600125 (10 μM) for 1 h; pre-incubation was carried out with silk lutein extract (0.2, 2, 20 and 200 μM) for 2 h, and the cells were exposed to Aβ25-35 (10 μM) for another 24 h, and with antibodies for these phosphorylated forms, respectively. β-Actin was used as a loading control. The relative density of phosphorylated forms of ERK, p38 and JNK were normalized to total values of ERK, p38 and JNK. The values present the mean ± SEM from 3 independent experiments. ***p < 0.001, in comparison with the control treatment; #p < 0.05, ##p < 0.01, in comparison with the Aβ25-35 treatment alone.
SP98059 which are specific inhibitors of phosphorylated JNK, phosphorylated p38 and phosphorylated ERK1/2, respectively. Recent studies support a role of MAPK pathway in the mechanism of oxidative stress-induced neuronal cell death [37,38]. Together, these results suggest that silk lutein extract protects PC12 cells against neurotoxicity by regulating Aβ25-35-induced apoptotic signaling. In conclusion, our present study demonstrates that silk lutein extract effectively suppresses Aβ25-35-induced neuronal cell death from oxidative stress and attenuates pro-apoptosis signaling pathways, at least partly, via preventing ROS generation and attenuating a cell death mitochondria pathway and inhibiting phosphorylation of JNK/p38/ ERK signaling pathways. Therefore, silk lutein extract may protect neuronal cells against oxidative stress-induced neurodegeneration.
critical role in maintaining cell integrity and controlling cell survival [30,34], and the disruption of this balance causes loss of mitochondrial membrane potential and releases cytochrome c. The release of cytochrome c protein activates the caspase-9 and caspase-3 proteins, which leads to the induction of cell death. Our results found that Aβ25-35 increased the Bcl-2 expression in PC12 cells which is consistent with the previous studies [31,35,36]. Pre-treatment with silk lutein extract suppress the Bcl-2 expression. We then observed that pretreating cells with silk lutein extract significantly increased the level of caspase-3 in Aβ25-35-treated cells, suggesting the neuroprotective of silk lutein extract in vitro. Numerous studies have shown that Aβ induce apoptosis in cultured neurons, possibly via activation of the mitogen-activated protein kinase (MAPK) family of serine/threonine kinases pathway (such as c-Jun Nterminal kinase (JNK), p38 mitogen activated protein kinase (p38 MAPK) and (ERK1/2)) [35]. A previous study demonstrated that abnormal levels of phosphorylated JNK and p38 in the brain of AD patients are associated with oxidative stress. In our study, Aβ25-35 activated JNK, P38 and ERK1/2 compared to untreated cells. Pretreatment with silk lutein extract appeared to significantly block the Aβ25-35-induced activation of p-JNK, p-p38, and ERK1/2. Similar results were observed in the cells pretreated with PD600125, SB203580 and
Conflict of interest Authors have no conflict of interest. Acknowledgements This work was supported by the Agricultural Research Development Agency (Public Organization), Thailand. Chiang Mai University, 586
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Thailand is also gratefully acknowledged.
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Protective effects of silk lutein extract from Bombyx mori cocoons on βAmyloid peptide-induced apoptosis in PC12 cells
T
Nongnuch Singhranga, Chainarong Tocharusb,c, Sarinthorn Thummayotd, ⁎ Manote Sutheerawattananondae, Jiraporn Tocharusa, a
Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand c Center for Research and Development of Natural Products for Health, Chiang Mai University d Division of Anatomy, Faculty of Medical Science, University of Phayao, 56000, Thailand e School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand b
A R T I C LE I N FO
A B S T R A C T
Keywords: Alzheimer’s disease Silk lutein extract β-Amyloid PC12 cells Apoptosis Neurodegeneration Oxidative stress Bombyx mori cocoons
Beta-amyloid (Aβ) peptide, the hallmark of Alzheimer’s disease (AD), invokes a cascade of oxidative damage to neurons and eventually leads to neuronal death. This study evaluated the protective effects of lutein extract from yellow cocoons of Bombyx mori, and its underlying mechanisms against was investigated to assess its protective effects and the underlying mechanisms against Aβ25-35-induced neuronal cell death in cultured rat pheochromocytoma (PC12) cells. Aβ25-35-induced neuronal toxicity is characterized by decrease in cell viability, increase in intracellular reactive oxygen species (ROS) production, activation of mitochondrial death pathway, and activation the phospholyration of mitogen-activated protein kinase (MAPKs) pathway. Pretreatment with silk lutein extract significantly attenuated Aβ25-35-induced loss of cell viability, apoptosis, MAPKs pathway activation and ROS production. Taken together, our present study suggests that silk lutein extract protects PC12 cells from Aβ25-35-induced neurotoxicity via the reduction of the ROS production, and subsequent attenuation of the mitochondrial death pathway and reduces the activation of the MAPK kinase pathways. This compound might beneficial as potential therapeutic agent to prevent or retard the development and progression of AD.
1. Introduction
p38 MAPK. Excessive activation of MAPKs signaling pathways are the major signals in the neuronal cell death [13,14]. Thus, inhibition of these pathways could be beneficial for preventing or attenuating cell death in AD. Lutein is a group of carotenoids, found in the diets such as yellow corn, egg yolk and other fruits or green leafy vegetables [15]. Lutein is a lipid- soluble antioxidant, obtained from diet or supplements. Lutein exists mostly in the lens and macular. Recently, it has been suggested that lutein exerted its biological activity not only in the anti-oxidative effect but also in the anti-inflammatory effect [16–19]. It has been reported that lutein reduced oxidative damage in H2O2-induced human lens epithelial cells [20]. In addition, lutein administration reduced the level of ROS in retinal neural cells in lipopolysaccharide (LPS) –treated mice and in the diabetic retina [21,22]. Moreover, lutein protected the retinal ganglion cells and inner retinal cells from diabetes-induced cell death and maintains cell survival [22]. Recently, lutein also has been shown to exert a neuroprotective effect against ischemic stroke induced by cerebral ischemia/reperfusion in mice [23]. In addition, the antiamyloidogenic of activity of carotenoids such as cryptocapsin,
Alzheimer’s disease (AD) is an age-related neurodegenerative disease characterized by the progressive degeneration and loss of neurons in the brain. The pathological hallmarks of AD are a degeneration of cholinergic neurons, an accumulation of beta amyloid (Aβ) deposition in senile plaques and neurofibrillary tangle lesions in specific areas of the brain [1–3]. The pathogenesis of AD is not fully clear; Aβ has been recognized as one of the major causes of AD pathology [4–6]. The neurotoxic effects of Aβ have been reported in the in vivo and in vitro studies by triggering oxidative stress, impairing mitochondria function, thus increasing apoptotic cell death [7,8]. Therefore, numerous antioxidants have been demonstrated to be useful for the prevention or attenuation of AD development or progression [9–11]. In addition, oxidative stress is the initiating factor and upstream of the activation of the mitogen-activated protein kinase family members (MAPKs) in many kinds of cells [12]. The three major mammalian MAPKs are extracellular signal-regulated kinases 1 and 2 (ERK1/2), cJun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and ⁎
Corresponding author. E-mail address: [email protected] (J. Tocharus).
https://doi.org/10.1016/j.biopha.2018.04.045 Received 7 January 2018; Received in revised form 6 April 2018; Accepted 6 April 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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plate and exposed to silk lutein extract for 2 h, which was followed by 10 μM Aβ25-35. Prior to the detection of ROS, the medium was removed, and PBS containing 20 μM 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) was added to each well, followed by incubation at 37 °C for 2 h in the dark. Fluorescence intensity was measured using a microplate reader (DTX 800, Beckman coulter, Austria) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.
cryptocapsin-5,6-epoxide and zeaxanthin was reported [24]. However, whether the administration of silk lutein reduces cell death in Aβ-induced neuronal cells and has a neuroprotective effect mechanism remains to be elucidated. 2. Materials and methods 2.1. Chemicals
2.6. Western blot analysis Dulbecco's Modified Eagle Medium: a nutrient Mixture F-12 (DMEM/F12), fetal bovine serum (FBS), fetal horse serum (FHS), trypsin, penicillin and streptomycin were purchased from GIBCO-BRL (Gaithersburg, MD, USA). The following antibodies were used for Western blotting: anti-ERK, anti-JNK, anti-p38, phospho-ERK, phosphoJNK, phospho-p38, β-actin, anti-mouse, anti-rabbit IgG peroxidaseconjugated secondary antibody (Millipore, Bedford, MA, USA). Specific inhibitors of ERK (PD98059), p38 (SB203580), JNK (SP60012) were purchased from Calbiochem (San Diego, CA, USA). Aβ25–35, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO, USA).
The cells were cultured at a density of 5 × 105 cells/ml in a 60 mm culture dish at 37 °C overnight. The cells were treated with silk lutein extract for 2 h, treated with or without 10 μM Aβ25-35 for 24 h, and then collected. The cells were lysed in RIPA buffer containing 1% NP40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 40 mM β-glycerophosphate, 50 mM sodium fluoride, 2 mM sodium orthovanadate and 1x protease inhibitor at 4 °C with rigorous shaking for 15 min. The cell lysate was centrifuged at 13,000 rpm at 4 °C for 20 min. The protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Equal amounts of proteins (50 μg) were electrophoresed in a 10–15% SDS polyacrylamide gel and then transferred to a PVDF membrane (Immobilon-P, Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk and probed with the indicated antibodies (anti-β-actin monoclonal antibody, anti-ERK1/2 monoclonal antibody, anti- pERK1/2 monoclonal antibody, anti-p JNK1/2 monoclonal antibody, anti- p38 MAPK monoclonal antibody, anti-JNK1/2 polyclonal antibody and anti-p-p38 MAPK polyclonal antibody) overnight at 4 °C followed by using probed with the indicated secondary antibodies. Detection was carried out using an enhanced chemiluminescence detection kit (Millipore, Bedford, MA, USA).
2.2. Cell culture PC12 cells, a rat pheochromocytoma cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in DMEM/F12, containing 10% heat-inactivated fetal horse serum, 5% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin. Cultures were incubated at 37 °C and 5% CO2 in a fully-humidified incubator. Aβ25–35 was dissolved in water at a concentration of 1 mM and stored at −20 °C. Before being used in any treatment, Aβ25–35 was diluted with phosphate buffer saline (PBS) and aggregated at 37 °C for 5 days.
2.7. Apoptosis detection assay by flow cytometry 2.3. Preparation of lutein extract The number of apoptotic cells was determined by flow cytometry. The cells at a density of 1 × 105 cells/ml at 37 °C were treated with silk lutein extract for 2 h prior to treatment in the presence or absence of 10 μM Aβ25-35 for 24 h. The percentage of cells undergoing apoptosis after the treatment was determined using the MuseTM Annexin-V and Dead cell assay kit and the Bcl2 levels (EMD Millipore, Bioscience). To determine the caspase-3 levels, the cells were incubated with caspase-3/7 working reaction assay (EMD Millipore Biosciences) for 10 min in the dark at room temperature. To determine the activation Bcl-2, the cells were incubated with Bcl-2 working reaction assay (EMD Millipore Biosciences) for 10 min in the dark at room temperature. Finally, the number of the apoptotic cells, the caspase-3 positive cells, and activated Bcl-2 positive cells were analyzed by using Muse Cell Analyzer (Merck Millipore, MA, USA), according to the manufacturer’s instruction.
Silk lutein extract was prepared from yellow silk cocoons of Thai silk worms (Bombyx mori) according to pending patent no. PCT/ TH2010/000048. Silk lutein extract was prepared from yellow silk cocoons as described previously [24]. Briefly, the silk cocoons were degummed at high pressure and temperature and then extracted with a solvent mixture of hexane, ethyl alcohol and ethyl acetate. The obtained solution was re-extracted with 10% NaCl to remove water-soluble impurities. The extract was concentrated at room temperature and then freeze-dried. The concentration of silk lutein was quantified by reverse-phase HPLC (Agilent HP 1100 series) using a LiChrospher®100 reverse-phase C18 column as stationary and acetonitrile/methanolethyl acetate as mobile phase and monitored at 445 nm. The HPLC chromatogram has been shown in previous study [24]. The silk lutein extract was kept in darkness at −20 °C until use.
2.8. Statistical analysis 2.4. Cell viability assay All data are expressed as mean ± SEM of three independent experiments. The statistical difference was analyzed using one-way analysis of variance (ANOVA) followed by Post Hoc Dunnett’s test for comparing the significance between the individual groups (p < 0.05).
Cell viability was measured by quantitative colorimetric assay with MTT. PC12 cells were cultured at a density of 1.5 × 104 cells/well in 96-well plate overnight. The various concentrations of silk lutein extract were added to PC12 cells. After 24 h, 10 mg/ml of MTT were added to each well and further incubated for 4 h at 37 °C in a humidified 5% CO2 and 95% air atmosphere. The formazan was solubilized with dimethyl sulfoxide (DMSO) and was determined by measuring the absorbance at 540 nm using a microplate reader (Bio-Tek Instruments, Winoaski, VT, USA).
3. Results 3.1. Effect of silk lutein extract on Aβ25-35-induced cytotoxicity in PC12 cells Aβ25-35-induced cytotoxicity was determined by measuring MTT reduction assay in PC12 cells cultured for 24 h with Aβ25-35 (1–50 μM) (Fig. 1A). Aβ25-35 reduced cell viability in a concentration-dependent manner. Exposure to Aβ25-35 at the concentration of 10 μM resulted in 63.38 ± 1.2% of relative cell viability which concentration was later
2.5. Measurement of reactive oxygen species (ROS) The production of ROS was determined using the DCF assay. The cells were plated for 24 h at a density of 2 × 104 cells/well in 96-well 583
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Fig. 2. Effect of silk lutein extract on Aβ25-35 –induced intracellular ROS. Cells were pretreated with various concentrations (0, 0.2, 2, 20, and 200 μM) of silk lutein extract for 2 h, followed by Aβ25-35 treated. Intracellular ROS levels were determined by measuring DCF fluorescence intensity, which measured fluorescent spectrophotometrically with the excitation at 485 nm and emission at 535 nm. The values present the mean ± SEM from 3 independent experiments. ***p < 0.001, in comparison with the control treatment; #p < 0.05, ## p < 0.01,###p < 0.001, in comparison with the Aβ25-35 treatment alone.
Fig. 1. Protective effect of silk lutein extract on Aβ25-35 –induced cytotoxicity in PC12 cells. Cell viability was determined by MTT assay. (A) PC12 cells were treated with various concentrations of Aβ25-35 (1, 5, 10 and 50 μM) for 24 h. (B) PC12 cells were pretreated with various concentrations (0, 0.2, 2, 20 and 200 μM) of silk lutein extract for 2 h, followed by Aβ25-35 treated for 24 h. The values present the mean ± SEM from 3 independent experiments. ***p < 0.001, in comparison with the control treatment; ###p < 0.001, in comparison with the Aβ25-35 treatment alone.
of apoptotic cells. Pretreatment with silk lutein extract significantly increased the levels of active caspase3/7 in a dose-dependent manner (Fig. 3E). These results suggest that silk lutein extract markedly reduced the apoptosis of Aβ25-35-induced PC cells.
used in all subsequent experiments. To investigate the neuroprotective effects of silk lutein extract on Aβ25-35-induced cytotoxicity in PC12 cell, cells were pretreated with or without those compounds at various concentrations (0.2, 2, 20 and 200 μM) for 2 h, followed by treatment with 10 μM of Aβ25-35 for 24 h. Silk lutein extract increased cell viability in the concentration-dependent manner. The neuroprotective effect of silk lutein at concentration of 200 μM was found to be similar to that of control as shown in Fig. 1B. However, silk lutein extract alone did not show any cytotoxicity (data not shown).
3.4. Effect of silk lutein extract on MAPKs signaling pathway To explore the mechanisms underlying the protective effect of silk lutein extract against Aβ25-35–induced cell death, MAP kinase are implicated in oxidative-stress-induced cell death. The phosphorylation of MAPK molecules, ERK1/2, P38 MAPK and JNK, in Aβ25-35–stimulated PC12 cells were examined. Treatment of PC12 cells with Aβ25-35 significantly increased the levels of phosphorylated ERK1/2 (pERK1/2), p38 MAPK (p-p38) and JNK (pJNK) (P < 0.05 as compared with the control). Pretreatment of PC12 cells with silk lutein extract at the concentration of 0.2, 2, 20 and 200 μM significantly attenuated Aβ25-35induced phosphorylation of pERK1/2 (Fig. 4A), p-p38 (Fig. 4B) and pJNK (Fig. 4C) while their non-phosphorylated forms remained unchanged. Similar results were observed in Aβ25-35–induced PC12 cells treated with PD98059 (ERK inhibitor), SB20358 (p38 inhibitor) and SP600125 (JNK inhibitor).
3.2. Effect of silk lutein extract on Aβ25-35-induced reactive oxygen species (ROS) Elevation of intracellular ROS has been implicated in cell death and ROS were determined by using H2DCFDA. To determine the effect of silk lutein extract on intracellular ROS production, we investigated the production of ROS following Aβ25-35 exposure. The level of ROS significantly increased 1.3 fold after exposure to Aβ25-35 (10 μM). Pretreatment with silk lutein extract at 0.2, 2, 20 and 200 μM for 2 h before exposure to Aβ25-35 markedly reduced the levels of ROS production to 1.10, 1.03, 1.00 and 0.99 fold of control, respectively. Silk lutein extract dose-dependently inhibited Aβ25-35-induced shift of fluorescent intensity, implicating its role in preventing the accumulation of ROS. These findings indicate that pretreatment with silk lutein extract can effectively prevent Aβ25-35 –induced ROS production (Fig. 2).
4. Discussion Aβ, the major component of senile plaques that form in the brains of patients with AD, may cause neuronal cell death via the generation of ROS and the elevation of calcium [24,25]. Several studies have shown that oxidative stress plays an important role in neuronal degeneration in AD [25]. Previous studies, both in vivo and in vitro, showed that Aβ25-35 treatment caused a significant increase in the level of ROS leading to oxidative damaged to DNA, lipids and protein, thereby impairing cell function [8,26–28]. Therefore, numerous antioxidants have been demonstrated to be useful for the prevention or attenuation of AD development or progression [9–11]. Our present study was the first to demonstrate the neuroprotective effects of silk lutein and its possible molecular mechanism in PC12 cells against Aβ25-35 –induced neuronal apoptosis. Treatment with 10 μM Aβ25-35 inhibited cell viability leading to an increase of cell death (60–70%) in cultured neuronal PC12 cells which was almost completely restored by treatment with 200 μM silk lutein extract. These results indicated that silk lutein extract did significantly protect PC12 cells from Aβ25-35 -induced cytotoxicity. Aβ associated with senile plaques formed in the brains of patients
3.3. Effect of silk lutein extract on the expression of apoptotic signaling pathway The induction of apoptosis after treatment of the cells with Aβ25-35 was determined by flow cytometry. The results showed that silk lutein extract significantly decreased the number of apoptotic cells in a dosedependent manner, compared to the only Aβ25-35 treatment (Fig. 3 A, B and C). The level of Bcl-2 proteins regulates the apoptotic cell death. We then investigated the expression of Bcl-2 protein in silk luteinmediated neuroprotection against Aβ25-35 –induced neurotoxicity. After PC12 cells were treated with Aβ25-35 for 24 h, the expression of Bcl-2 was significantly decreased. With the pretreatment with silk lutein extract at the concentration of 0.2, 2, 20 and 200 μM, the Bcl-2 expression was significantly increased (Fig.3D). Moreover, the active caspase 3/7 level was also significantly decreased, which correlates to the number 584
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Fig. 3. The protective effect of silk lutein extract on Aβ25-35- induced apoptosis in the PC12 cells. (A) PC12 cells were pretreated with or without silk lutein extract at the concentrations of 0.2, 2, 20 and 200 μM for 2 h before being treated with 10 μM Aβ25-35 for 24 h. The apoptotic cells were analyzed using an Annexin V/PI staining assay (A, B and C). The Bcl-2 level (D) and the caspase-3 level (E) by flow cytometry. The values present the mean ± SEM from 3 independent experiments. ***p < 0.001, in comparison with the control treatment; #p < 0.05, ##p < 0.01,###p < 0.001, in comparison with the Aβ25-35 treatment alone.
suggested that antioxidants may suppress Aβ25–35-induced ROS generation, thereby decreasing neurotoxicity [10,11,30,31]. Recent study demonstrated that lutein prevented Aβ25-35 -induced neurotoxicity mediated by modulating Nrf2 and NF-kB expression in cerebrovascular endothelial cells [33]. It is well-known that neuronal apoptosis is a leading pathway for Aβ-induced neurotoxicity; therefore the prevention of Aβ-triggered apoptosis is a strategy for the treatment of AD. The mitochondrial pathway of apoptosis is regulated by the Bcl-2 family proteins, consisting of several proteins including anti-apoptotic proteins such as BclXL and pro-apoptotic proteins Bcl-2 and pro-apoptotic proteins including Bax [32–34]. The balance between Bax and Bcl-2 plays a
with AD may lead to the generation of ROS, mitochondria dysfunction and ultimately to subsequent cause neuronal cell death [4,6,26,29]. Our findings indicate that, similar to other reports, Aβ25-35 was found to cause an elevation of oxidative stress as characterized by excessive production of intracellular ROS [7,27]. Interestingly, pretreatment with silk lutein extract significantly suppressed Aβ25–35-induced ROS accumulation dose-dependently, suggesting that the scavenging effect of silk lutein extract may be a significant contributor to cytoprotective effects. This was probably due to several double bonds in silk lutein which directly react with ROS to scavenge radicals. Consistent with a previous report, lutein suppresses the generation of ROS in diabetic retina and in H2O2-induced human lens epithelial cells [20,21]. Several studies 585
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Fig. 4. Effect of silk lutein extract on the phosphorylation of expression of MAPKs kinase pathway in Aβ25-35 induced PC12 cells. Cells were pretreated with 0, 0.2, 2, 20 and 200 μM of silk lutein extract for 2 h prior Aβ25-35 10 μM was added. The cell lysates were subjected to SDS-PAGE followed by Western blot with antibodies for ERK (A) The PC12 cells were prior treated with PD98059 (50 μM) for 1 h; pre-incubation was carried out with silk lutein extract (0.2, 2, 20 and 200 μM) for 2 h, and the cells were exposed to Aβ25-35 (10 μM) for another 24 h, p38 (B) The PC12 cells were prior treated with SB203580 (30 μM) for 1 h; pre-incubation was carried out with silk lutein extract (0.2, 2, 20 and 200 μM) for 2 h, and the cells were exposed to Aβ25-35 (10 μM) for another 24 h, JNK (C) The PC12 cells were prior treated with SP600125 (10 μM) for 1 h; pre-incubation was carried out with silk lutein extract (0.2, 2, 20 and 200 μM) for 2 h, and the cells were exposed to Aβ25-35 (10 μM) for another 24 h, and with antibodies for these phosphorylated forms, respectively. β-Actin was used as a loading control. The relative density of phosphorylated forms of ERK, p38 and JNK were normalized to total values of ERK, p38 and JNK. The values present the mean ± SEM from 3 independent experiments. ***p < 0.001, in comparison with the control treatment; #p < 0.05, ##p < 0.01, in comparison with the Aβ25-35 treatment alone.
SP98059 which are specific inhibitors of phosphorylated JNK, phosphorylated p38 and phosphorylated ERK1/2, respectively. Recent studies support a role of MAPK pathway in the mechanism of oxidative stress-induced neuronal cell death [37,38]. Together, these results suggest that silk lutein extract protects PC12 cells against neurotoxicity by regulating Aβ25-35-induced apoptotic signaling. In conclusion, our present study demonstrates that silk lutein extract effectively suppresses Aβ25-35-induced neuronal cell death from oxidative stress and attenuates pro-apoptosis signaling pathways, at least partly, via preventing ROS generation and attenuating a cell death mitochondria pathway and inhibiting phosphorylation of JNK/p38/ ERK signaling pathways. Therefore, silk lutein extract may protect neuronal cells against oxidative stress-induced neurodegeneration.
critical role in maintaining cell integrity and controlling cell survival [30,34], and the disruption of this balance causes loss of mitochondrial membrane potential and releases cytochrome c. The release of cytochrome c protein activates the caspase-9 and caspase-3 proteins, which leads to the induction of cell death. Our results found that Aβ25-35 increased the Bcl-2 expression in PC12 cells which is consistent with the previous studies [31,35,36]. Pre-treatment with silk lutein extract suppress the Bcl-2 expression. We then observed that pretreating cells with silk lutein extract significantly increased the level of caspase-3 in Aβ25-35-treated cells, suggesting the neuroprotective of silk lutein extract in vitro. Numerous studies have shown that Aβ induce apoptosis in cultured neurons, possibly via activation of the mitogen-activated protein kinase (MAPK) family of serine/threonine kinases pathway (such as c-Jun Nterminal kinase (JNK), p38 mitogen activated protein kinase (p38 MAPK) and (ERK1/2)) [35]. A previous study demonstrated that abnormal levels of phosphorylated JNK and p38 in the brain of AD patients are associated with oxidative stress. In our study, Aβ25-35 activated JNK, P38 and ERK1/2 compared to untreated cells. Pretreatment with silk lutein extract appeared to significantly block the Aβ25-35-induced activation of p-JNK, p-p38, and ERK1/2. Similar results were observed in the cells pretreated with PD600125, SB203580 and
Conflict of interest Authors have no conflict of interest. Acknowledgements This work was supported by the Agricultural Research Development Agency (Public Organization), Thailand. Chiang Mai University, 586
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Thailand is also gratefully acknowledged.
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