- Email: [email protected]
Krill oil protects PC12 cells against methamphetamine-induced neurotoxicity by inhibiting apoptotic response and oxidative stress
Accepted Manuscript Krill oil protects PC12 cells against methamphetamine induced neurotoxicity by inhibiting apoptotic response and oxidative stress
Qi Xiong, Qin Ru, Xiang Tian, Mei Zhou, Lin Chen, Yi Li, Chaoying Li PII: DOI: Reference:
S0271-5317(17)31175-2 doi:10.1016/j.nutres.2018.07.006 NTR 7920
To appear in:
Nutrition Research
Received date: Revised date: Accepted date:
22 December 2017 4 July 2018 9 July 2018
Please cite this article as: Qi Xiong, Qin Ru, Xiang Tian, Mei Zhou, Lin Chen, Yi Li, Chaoying Li , Krill oil protects PC12 cells against methamphetamine induced neurotoxicity by inhibiting apoptotic response and oxidative stress. Ntr (2018), doi:10.1016/j.nutres.2018.07.006
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Krill oil protects PC12 cells against methamphetamine induced neurotoxicity by inhibiting apoptotic response and oxidative stress
1
RI
PT
Qi Xiong1 , Qin Ru1, Xiang Tian1, Mei Zhou1, Lin Chen1, Yi Li2, Chaoying Li1
2
Wuhan Mental Health Center, Wuhan, Hubei Province, China 430022
NU
SC
Wuhan Institutes of Biomedical Sciences, Jianghan University, Wuhan City, Hubei Province, China 430000
MA
Corresponding author: Chaoying Li Tel: +86-13628678552, Email: [email protected]; [email protected] Qi Xiong’s email: [email protected]
D
Qin Ru’s email: [email protected]
PT E
Xiang Tian’s email: [email protected] Mei Zhou’s email: [email protected]
CE
Lin Chen’s email: [email protected]
AC
Yi Li’s email: [email protected]
ACCEPTED MANUSCRIPT
List of abbreviations
METH; methamphetamine
PT
KO; krill oil EPA; eicosapentaenoic acid
RI
DHA; docosahexaenoic acid
SC
FBS; fetal bovine serum
NU
MTT; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide GSH; Glutathione
MA
SOD; superoxide dismutase
PT E
DMSO; dimethyl sulfoxide
D
MDA; Malomdialdehvde
ROS; reactive oxygen species
AC
CE
ANOVA; analysis of variance
ACCEPTED MANUSCRIPT Abstract Methamphetamine (METH) exposure can cause severe effects to the nervous system; however, the underlying molecular mechanism of neurotoxicity caused by METH is still unclear. Oxidative stress and apoptosis are linked in the pathophysiology of many
PT
neurodegenerative diseases. Krill oil (KO) benefits human health via its strong antioxidant
RI
ability. Therefore, we hypothesized that KO supplementation might effectively prevent
SC
METH-induced neurotoxicity via the inhibition of apoptotic responses and oxidative damages. In this study, PC12 cells were exposed to both METH (3 mM) and KO (0.1, 0.2,
NU
0.4, 0.8 μg/ml) in vitro for 24 h, and the following parameters were measured to detect
MA
apoptosis and oxidative stress responses that were triggered by METH: cell viability, the oxidative enzyme system, NO production, ROS production, apoptosis, mitochondrial
D
membrane potential and protein expression of cleaved caspase-3. The results indicate that
PT E
KO mitigates the apoptotic response post-METH exposure in PC 12 cells by increasing cell viability, decreasing protein expression of cleaved caspase-3, reducing apoptotic rates,
CE
and decreasing dissipation of mitochondrial membrane potential. In addition, the study
AC
revealed increases in SOD and GSH activity, and decreases in MDA content, NO and ROS production, suggesting that KO is beneficial in reducing oxidative stress, which may also play a role in the regulation of METH-triggered apoptotic response. Consequently, these data indicate that KO could potentially alleviate METH-induced neurotoxicity via the reduction of apoptotic responses and oxidative damages. Keywords: Methamphetamine; PC12 cells; Krill Oil; Neurotoxicity; Apoptosis; Oxidative damage
ACCEPTED MANUSCRIPT Introduction Methamphetamine (METH) is a highly addictive psychostimulant drug that is abused by many people. METH abuse has caused severe societal problems in recent decades and has drawn intense worldwide attention [1, 2]. METH mainly targets the dopaminergic
PT
system and abnormal use of METH induces neurotoxicity in different regions of the brain
RI
[3, 4]. Previous studies found that METH-triggered neurotoxic effects may be caused by
SC
the pro-inflammatory cytokines [5], oxidative stress [6] as well as apoptosis [7] in dopaminergic neurons. In addition, METH exposure is a significant risk factor that
NU
promotes susceptibility to neurodegenerative diseases, such as Parkinson’s disease [8, 9].
MA
However, the precise molecular mechanism of METH-induced neurotoxicity in the
D
nervous system remains to be elucidated.
PT E
Krill are small crustaceans that possess a large amount of phospholipids and are particularly abundant in the Arctic and Antarctic polar seas [10]. Antarctic Krill,
CE
Euphausia superba, is thought to be the most important and prolific species [11]. Krill is a sustainable source of omega-3 polyunsaturated fatty acids (n-3 PUFAs) and is especially
AC
1
rich in long-chain PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) which naturally occur in phospholipid form [12, 13]. The oil is extracted from Antarctic krill; the major component of the n-3 PUFAs in Krill Oil (KO) is associated with phospholipids, while the natural antioxidant astaxanthin is another important component [14]. The antioxidant effect of KO is substantial due to the protective nature of the phospholipid bilayer, which helps prevent unsaturated bonds from oxidizing. During the
ACCEPTED MANUSCRIPT past decades, studies have demonstrated that KO provides health benefits, including treatment of chronic inflammation [15, 16], arthritis [16] and hyper-lipidemia [17].
In
addition, KO has also been proven to have an impact on memory improvement in the rat model [18], as well as decreasing levels of lipid peroxide and reactive oxygen species in
PT
rats [19, 20]. Moreover, Jayathilake demonstrated that KO extract could inhibit cell
RI
proliferation and induce apoptosis of human colorectal cancer cells [21]. Genotoxicity
SC
studies have also confirmed the safety of KO [22]; however, the effect of KO on
NU
METH-induced neurotoxicity had not been investigated prior to this study.
MA
In the current study, we hypothesized that KO could mitigate METH-induced neurotoxicity via the reduction of apoptosis and oxidative stress in dopaminergic cells. To
D
address this, rat adrenal pheochromocytoma (PC12) cells were used as a cell culture
PT E
model because they possess much of the biochemical machinery associated with dopaminergic neurons [23]. The PC12 cells were exposed to METH (3 mM) and different
CE
concentrations of KO (0.1, 0.2, 0.4, 0.8 μg/ml) for 24 h, while the following apoptotic and
AC
oxidative stress-related parameters were measured: cell viability, mitochondrial membrane potential, apoptosis, the oxidative enzyme system, NO production, reactive oxygen species (ROS) production, and protein expression of cleaved caspase-3. The results show that KO has potential as a therapeutic substance to alleviate METH induced neurotoxicity in future clinical practice.
2
Methods and materials
ACCEPTED MANUSCRIPT 2.1 Reagents METH was obtained from the Hubei Public Security Bureau. RPMI-1640 medium, fetal bovine serum (FBS), trypsin, penicillin, and streptomycin were purchased from Life Technologies
(Carlsbad,
CA,
USA).
PT
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DCFH-DA and
RI
dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Corp (St Louis, MO,
SC
USA). The Muse Annexin V & Dead Cell Assay Kit (MCH100105) and the Muse MitoPotential Kit (MCH100110) were obtained from the Millipore Corporation
NU
(Darmstadt, Germany). Glutathione (GSH), Superoxide Dismutase (SOD) and
MA
Malomdialdehvde (MDA) Assay Kits were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). An NO Assay Kit was obtained from the
D
Beyotime Institute of Biotechnology (Haimen, China). All other chemicals were standard
PT E
analytical grade. SuperbaTM Krill Oil (KO) was kindly provided by the Aker Biomarine
AC
2.2 Cell lines
CE
Antarctic Company (Norway); its major components are described in Table 1.
PC12 cells, which are derived from a pheochromocytoma of the rat adrenal medulla, were obtained from the Shanghai Institute of Cell Library (Shanghai, China) and were plated and maintained in RPMI-1640 medium supplemented with 20% (v/v) fetal bovine serum (Gibco, USA) and the antibiotics penicillin (100 U/ml) and streptomycin (100 μg/ml). The cells were maintained at 37 ℃ and 5% (v/v) CO2 in a humid environment. The cells were then harvested and plated in 96-, 24-, or 6-well culture plates for different
ACCEPTED MANUSCRIPT experimental requirements.
2.3
Exposure of cells to METH and KO A stock solution of Krill oil was prepared in ethanol at a concentration of 10 mg/ml.
PT
Different concentrations of Krill Oil (0.1, 0.2, 0.4, 0.8 μg/ml) were chosen based on the
RI
results from our previous research (data not shown). The PC12 cells were then treated
SC
either with 3 mM METH [9] or with the combinations of 3 mM METH and KO (0.1, 0.2, 0.4, 0.8 μg/ml) for 24 h in a 37 ˚C and 5% (v/v) CO2 incubator. The cells were then
MA
NU
harvested and utilized in the following experiments.
2.4 Measurement of cell viability viability
was
measured
by
D
Cell
PT E
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in the current study [24]. Briefly, the cells were harvested and counted, then seeded on a 96-well
CE
plate at a density of 5×103 (Corning, USA) overnight in a 37 ˚C and 5% (v/v) CO2
AC
incubator. Different concentrations of KO were prepared with dilutions ranging from 0.1 to 0.8 μg/ml. The METH (3 mM) was diluted in RPMI-1640 and KO was further diluted in RPMI-1640 with METH to obtain the final concentrations (0.1, 0.2, 0.4, 0.8 μg/ml). After 24 h incubation, 20 μl of MTT (0.5 mg/ml) reagent was added to each well. The cells were further incubated for 4 h in the 37 ˚C incubator in the dark, the supernatants were carefully removed, 150 μl of DMSO was added to each well and mixed thoroughly. Absorbance was measured at 490 (A490) nm using a microplate reader (Thermal Scientific,
ACCEPTED MANUSCRIPT USA). The relative cell viability (%) was calculated as (A490 of treated samples/ A490 of untreated samples) × 100%.
2.5 Measurements of the oxidative enzyme system
PT
The oxidative enzyme system (MDA, SOD and GSH) was used to examine the oxygen
RI
reactivity of PC12 cells treated with METH. PC12 cells were seeded in 6 well plates at a
SC
density of 5×104 cells/well overnight in a 37 ˚C and 5% (v/v) CO2 incubator. After 24 h incubation with either METH (3 mM) alone or the combination of METH (3 mM) and
NU
different concentrations of KO (0.1, 0.2, 0.4, 0.8 μg/ml), cells were collected and the
MA
levels of MDA, SOD and GSH were measured. The cell samples were prepared for the experiments as follows: MDA - cells were harvested and centrifuged at 1000 rpm for 5
D
min at room temperature (RT), supernatants were removed and the cells were
PT E
resuspended in 300 μL phosphate buffer saline (PBS). The cells were then sonicated and centrifuged again to obtain cell samples; SOD - cells were first harvested and centrifuged
CE
at 1000 rpm for 5 min at room temperature, supernatants were removed and the cells were
AC
resuspended in 1 mL PBS. The cells were then mixed with 100 μL lysis buffer on ice for 40 min. Finally the cells were centrifuged at 5000 rpm for 15 min to obtain cell samples; GSH - cells were first harvested and centrifuged at 1000 rpm for 5 min at RT, supernatants were removed and the cells were resuspended in 1 mL PBS. The cells were then mixed with 100 μL lysis buffer on ice for 40 min. Finally, the cells were centrifuged at 5000 rpm for 15 min to obtain cell samples. The levels of MDA, GSH and SOD were further measured as described in a previous study [25], using corresponding commercial
ACCEPTED MANUSCRIPT detection kits (Nanjing Jiancheng Bioengineering Institute, China). The absorbances for MDA, GSH and SOD were measured at different wavelengths (532 nm, 420 nm and 405 nm). The results were then corrected for their protein levels based on the reference
PT
manual for a BCA protein assay kit (Beyotime Institute of Biotechnology, China).
RI
2.6 Measurement of NO production
SC
Quantification of sodium nitrite was used as an indirect way to determine the production of NO generated by 3 mM METH. Briefly, cultured PC12 cells were seeded in
NU
24-well plates at a density of 3 ×105 cells/well overnight in a 37 ˚C and 5% (v/v) CO2
MA
incubator. The cells were then exposed to METH/KO, and unexposed cells were used as negative controls. Twenty four hours later, the supernatants were collected to determine
D
the production of NO (Beyotime Institute of Biotechnology, China). Procedures were
PT E
followed according to our previous study [26]. Generally, the supernatants were transferred to 96-well plates, standard samples were prepared at different concentrations
CE
(0, 1, 2, 5, 10, 20, 40, 60 and 100 μM) and added at a volume of 50 μL/well. All other test
AC
samples were also prepared at volumes of 50 μL/well. Then, 50 μL of Griess Reagent I and 50 μL of Griess Reagent II were added to each standard sample and test sample, accordingly. The results were read by a microplate reader at 540 nm. The production of NO for each group was calculated according to the standard curve.
2.7 Measurement of ROS production Intracellular ROS level was detected using a fluorescent probe DCFH-DA
ACCEPTED MANUSCRIPT (Sigma-Aldrich, MO, USA). The protocol from a previously study was followed [27]. Generally, PC12 cells were seeded in 6-well plates at a density of 5×104 cells/well overnight in a 37 ˚C and 5% (v/v) CO2 incubator. After 24 h incubation with either METH (3 mM) alone or the combination of METH (3 mM) and different concentrations of KO
PT
(0.1, 0.2, 0.4, 0.8 μg/mL), each supernatant was completely removed, and the cells were
RI
washed with PBS three times. Then, DCFH-DA was dissolved in 10 nmol/L DMSO, and
SC
100 μmol/L DCFH-DA, which was further dissolved in RPMI-1640 complete medium, was added into each group (500 μL/well) and the cells were incubated in a 37 ˚C and 5% (v/v)
NU
CO2 incubator for 1 h. After the DCFH-DA was removed from each well, and each well
MA
was washed with PBS three times to completely remove the DCFH-DA. The cells were then dissolved and harvested with 1 mL PBS. Finally, the fluorescent signal was read at
PT E
Ortenberg, Germany).
D
Ex485nm/Em538nm, using a FLUOstar® Omega Microplate Reader (BMG LABTECH,
CE
2.8 Measurement of cell apoptosis
AC
The Muse Annexin V & Dead Cell Kit (EMD Millipore Corporation, Germany) was used to detect apoptotic cell death, as described by a previous study [28]. Briefly, PC12 cells were seeded in 6-well culture plates at a density of 5×104 cells/well and incubated overnight at 37 ˚C and 5% (v/v) CO2. Then, cells were treated with either METH (3 mM) alone or the combination of METH (3 mM) and different concentrations of KO (0.1, 0.2, 0.4, 0.8 μg/ml) for another 24 h. After 24 h incubation, the cells were harvested and centrifuged at 1000 rpm for 5 min at room temperature, then the supernatants were
ACCEPTED MANUSCRIPT removed and 100 μL RPMI-1640 complete medium was added. They were then thoroughly mixed with another 100 μL of MuseTM Annexin-V & Dead Cell reagent for 20 min at room temperature. Finally, after incubation, the cells were analyzed immediately
RI
2.9 Measurement of mitochondrial membrane potential
PT
by the Muse™ Cell Analyzer (EMD Millipore Corporation, Germany).
SC
Changes in the mitochondrial membrane potential （ΔΨm）during the early stages of apoptosis were analyzed by the Muse MitoPotential assay (EMD Millipore Corporation,
NU
Germany). The detailed protocol was followed as previously described [28]. PC12 cells
MA
were seeded in 6-well culture plates at a density of 5×104 cells/well overnight in a 37 ˚C and 5% (v/v) CO2 incubator. The cells were then treated with different concentrations of
D
Krill Oil (0.1, 0.2, 0.4, 0.8 μg/ml)/METH (3 mM) in 6-well culture plates for 24 h. First,
PT E
the cells were harvested and centrifuged at 1000 rpm for 5 min at room temperature, then the supernatants were removed and replaced by 100 μL PBS, then prepared
CE
MuseTM MitoPotential working solution, by diluting the dye 1:1000 with 1X Assay
AC
Buffer. An amount of 95 μL of MuseTM MitoPotential working solution was added to 100 μL of cells, and the cell samples were incubated at 37˚C for 20 min. Finally, 5 μl of MuseTM MitoPotential 7-AAD dye was also added to each sample and the cells were incubated for another 5 min at RT. Changes in the mitochondrial membrane potential were determined by flow cytometry (EMD Millipore Corporation, Germany).
ACCEPTED MANUSCRIPT 2.10 Western blot analysis of cleaved caspase-3 PC12 cells were treated with either METH (3 mM) or the combination of METH (3 mM) and different concentrations of KO (0.1, 0.2, 0.4, 0.8 μg/ml) for 24 h in a 37 ˚C and 5% (v/v) CO2 incubator. Protein extracts were prepared in 50 mM tris pH 8, 150
PT
μM NaCl, 0.5% sodium deoxycholate, 20% (v/v) protease inhibitor Cocktail and 10%
RI
(v/v) PMSF (Sigma, USA). Protein concentrations from different groups were
SC
determined by BCA assay kit (Boster Company, China). Western blot analysis of protein expression was performed as previously described [29]. SDS-PAGE loading
NU
buffer was added to each group, and boiled for 5 min. Then, 50 μg of the protein
MA
samples were separated on a 12% SDS-PAGE gel and transferred to PVDF membrane (Merck Millipore, USA). For immunoblotting, membranes were blocked with 5% (w/v)
D
milk in tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST) for 1 h at RT.
PT E
Primary antibodies, including rabbit anti-cleaved caspase-3 (Cell Signaling, USA , 1:500) and rabbit anti-β-actin (Cell Signaling, USA, 1:1000) were also incubated with
CE
PVDF membranes in 5% (w/v) milk in TBST at 4 ˚C overnight. Then, the membranes
AC
were washed and incubated with a 1:3000 dilution of HRP-conjugated anti-rabbit secondary antibodies (Boster Company, China) in 5% (w/v) milk in TBST for 1 h at RT. After washing in TBST an additional three times, the membranes were incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher, USA), and the bands were visualized by using an imaging analysis system (Gene Company, Hong Kong). The intensity of each band was quantitatively determined by GenTools Image Analyzer Software (Gene Company, Hong Kong). β-actin was used as a loading control.
ACCEPTED MANUSCRIPT Therefore, the density ratio indicated the relative intensity of each band against β-actin. 2.11 Statistical analyses Data are expressed as the means ± standard error of the mean ( x ±SEM). A one way analysis of variance (ANOVA), followed by a post hoc Tukey test, was applied to all the
PT
experiments to examine the significance of the data. Statistical analysis was performed
RI
using SPSS 16.0 (IBM Corporation, New York, USA) and the criterion for significance
SC
was set at P < .05. For the primary result measure, cell viability, a sample size of 9 cell samples provided statistical power of at least 82%, detecting a 10% difference, with a
NU
probability of < .05. This sample size provided at least this level of statistical power for
Results
D
3
MA
the other results as well.
PT E
3.1 The effect of KO on the viability of PC12 cells post METH treatment The MTT assay was used to evaluate cell viability after treatment with METH and
CE
different concentrations of KO (Fig. 1). Compared with the control group, the cell survival
AC
rate of METH-only exposed PC12 cells decreased significantly (57.71 ± 7.72 vs 100.12 ± 11.21, P < .01), while the survival rates of PC12 cells all increased significantly after KO treatment (0.1, 0.2, 0.4, 0.8 μg/mL), in contrast with the METH-only treated group. Specifically, 0.8 μg/mL KO raised the cell survival rate the highest degree, up to 91.21 ± 6.83, compared with the METH-only treated group (57.71 ± 7.72, P < .01). In addition, the cell survival rates of 0.2 μg/mL KO and 0.4 μg/mL KO (85.12 ± 6.24 vs 85.34 ± 4.61) were similar post-METH treatment, yet they were significantly increased in comparison
ACCEPTED MANUSCRIPT with METH-only treated group (P < .01) 3.2 The effect of KO on the oxidative enzyme system in PC 12 cells after METH exposure Table 2 represents the results from the MDA, SOD and GSH oxidative stress
PT
experiments. It shows that the levels of MDA increased significantly post METH treatment
RI
in comparison with the control group (3.03 ± 0.03 vs 1.27 ± 0.02, P < .01). In contrast, with
SC
the METH-only treated group, all concentrations of KO post METH treatment decreased MDA content significantly (1.52 ± 0.06, 1.28 ± 0.10, 1.32 ± 0.04, 1.37 ± 0.06 vs 3.03 ±
NU
0.03, P < .01). In addition, METH alone reduced SOD activities significantly in PC12 cells
MA
compared with the control group (54.50 ± 1.01 vs 73.20 ± 1.46, P < .01), and all concentrations of KO significantly increased SOD activities (P < .05, P < .01), while 0.2
D
μg/mL KO raised the SOD activities the highest, up to 79.10 ± 3.52, compared with the
PT E
METH-only treated group (54.50 ± 1.01, P < .01). Finally, the METH-only treated group experienced significantly decreased GSH activities in contrast with the control group
CE
(40.98 ± 0.50 vs 45.98 ± 1.07, P < .05), and all concentrations of KO significantly
AC
increased GSH activities (P < .05, P < .01), while 0.4 μg/mL KO increased the GSH activities the highest, up to 51.57 ± 2.06, compared with the METH-only treated group (40.98 ± 0.50, P < .01).
3.3 The effect of KO on NO Production in PC 12 cells after METH exposure Figure 2 represents the NO responses to different concentrations of KO with/without METH. Compared with the control group, the METH-treated group showed a significant
ACCEPTED MANUSCRIPT increase in NO production (2.41 ± 0.10 vs 1.91 ± 0.04, P < .01). When the concentrations of KO were between 0.2 - 0.8 μg/mL, NO levels decreased, in contrast with the METH-only treated group (1.18 ± 0.24, 0.85 ± 0.08, 1.21 ± 0.12 vs 2.41 ± 0.10 P < .01). Finally, no significant difference was detected when the concentration of KO was 0.1
RI
PT
μg/mL compared to the METH-only treated group.
SC
3.4 The effect of KO on ROS Production in PC 12 cells after METH exposure To attribute whether apoptotic cell death induction by METH in PC12 cells is caused by
NU
oxidative stress, ROS was examined. Figure 3 shows that METH induced greater levels of
MA
ROS generation compared with that of the control group (1.83 ± 0.07 vs 1.01 ± 0.04, P < .01). In addition, incremental increases in the concentration of KO, decreased the levels
D
of ROS production significantly in comparison with the METH group (1.37 ± 0.10, 1.24 ±
PT E
0.09, 1.18 ± 0.09, 1.06 ± 0.04 vs 1.83 ± 0.07, all P < .01). Therefore, the above results indicate that all of the KO concentrations decrease the levels of ROS production in PC12
AC
CE
cells after METH treatment.
3.5 The effect of KO on apoptosis response in PC 12 cells after METH exposure Flow cytometry, which detects apoptotic cells by Annexin V/7-AAD, was also applied to this study to detect the METH-induced apoptotic effect in PC12 cells. Data in Figure 4 shows that a significant rise in apoptotic rates was induced by METH, compared with the control (24.83 ± 2.60 vs 3.67 ± 1.38, P < .01). However, when the concentrations of KO were between 0.2 - 0.8 μg/mL, they significantly decreased the apoptotic rates in
ACCEPTED MANUSCRIPT comparison with METH-only treated group (16.12 ± 1.63, 14.65 ± 1.58, 12.10 ± 2.10 vs 24.83 ± 2.60, P < .05). No significant difference in the apoptotic rate was detected when the concentration of KO was 0.1 μg/mL compared to the METH-only treated group.
PT
3.6 The effect of KO on mitochondrial membrane potential in PC 12 cells after METH
RI
exposure
SC
ΔΨm is an important indicator of mitochondrial dysfunction, and the loss of ΔΨ is an indicator and a hallmark for apoptosis. Figure 5 shows that METH induced a significant
NU
increase in total depolarization of the mitochondria compared with the control group
MA
(31.27 ± 3.82 vs 14.62 ± 3.42, P < .05 ), which indicates that a significant dissipation of ΔΨ is triggered by METH. In addition, the concentrations of KO that are between 0.2-0.8
D
μg/mL significantly decreased the total depolarization of mitochondria in contrast with the
PT E
METH treated group (20.92 ± 0.43, 20.27 ± 0.22, 15.35 ± 2.9 vs 31.27 ± 3.82, P < .05). However, a concentration of 0.1 μg/mL KO showed a lack of significant statistical
AC
CE
difference.
3.7 The effect of KO on the protein expression of cleaved caspase-3 in PC 12 cells after METH exposure
To evaluate the apoptotic response in PC12 cells exposed to METH and the protective effect of KO, the protein expression of cleaved caspase-3 was investigated by Western Blot. As shown in Figure 6A and 6B, the protein expressions of cleaved caspase-3 in PC12 cells treated by METH were significantly increased compared with that of the control group
ACCEPTED MANUSCRIPT (121.45 ± 4.60 vs 100.02 ± 0.02, P < .05). In contrast, all concentrations of KO significantly decreased protein expression of cleaved caspase-3 (86.17 ± 3.62, 71.27 ± 5.65, 62.86 ± 5.95), while 0.8 μg/mL KO decreased the protein expression level of cleaved caspase-3 the most,
Discussion
RI
Cell death and neurological damage have been linked to oxidative stress and the
SC
imbalance between the generation of free radicals and antioxidant defenses [30]. The
NU
current study investigated, for the first time, the protective effect of KO in preventing PC12 cells from experiencing a METH-triggered apoptotic response and oxidative damage.
MA
Previous studies have demonstrated that over exposure to METH may result in decreased cell viability. Pitaksalee demonstrated that cell viability of SH-SY5Y cells decreased
PT E
D
significantly post 24 h METH exposure in a dose dependent manner [31]. Similar results were also detected in another study [32]. In the current study, MTT results showed that
CE
METH decreased cell viability significantly compared with the control group, while cell death could be blocked when the concentrations of KO were between 0.1 and 0.8 μg/mL. Therefore, our data demonstrates that a METH-induced decrease in cell viability can be
AC
4
PT
to 54.93 ± 4.18, compared with the METH-only treated group (121.45 ± 4.60, P < .01).
rescued by KO.
METH-induced neuronal injury is also related to cell apoptosis, as demonstrated by an increased percentage of apoptotic rates in SH-SY5Y cells and significantly elevated protein expressions of cleaved caspase-3, cleaved PARP and Bax/Bcl-2 in PC12 cells [32-34]. In agreement with these results, our results indicate that cell apoptotic rates and protein levels
ACCEPTED MANUSCRIPT of cleaved caspase-3 increase significantly post METH 24 h treatment in PC12 cells when compared with the control group. However, compared with the METH-only treated group, KO abolishes these apoptotic effects, when the concentration range is 0.2-0.8 μg/mL, which indicates that KO might have a potential protective effect on PC12 cells against
RI
PT
METH-induced apoptosis.
SC
Mitochondrial impairment plays an important role in METH-induced dopaminergic neurotoxicity, which is also correlated with apoptosis [35]. The stability of ∆Ψm is
NU
important in maintaining normal cell function and the reduction of ∆Ψm has a significant
MA
impact on the apoptotic process [36]. Related studies [32, 37] have found that a significant loss of ∆Ψm is detected after METH treatment in PC12 cells, while METH induces
D
mitochondrial dysfunction in the form of a marked decrease in human T cell ∆Ψm. ∆Ψm
PT E
has also been measured by flow cytometry in groups treated with both METH and KO/METH in current research. Our results show that METH significantly increases
CE
depolarization of the mitochondria compared with the control group, which indicates that a
AC
significant dissipation of ∆Ψm is triggered post METH treatment, while KO effectively decreases this effect when the concentrations utilized are between 0.2 and 0.8 μg/mL. This indicates that KO might reduce METH induced PC12 cell apoptosis via a mitochondria-mediated pathway.
Former studies have demonstrated that exposure to METH may lead to oxidative stress [38, 39]. The balance between oxidants and antioxidants most likely plays a vital role in
ACCEPTED MANUSCRIPT both apoptosis and inflammation [40, 41]. Cellular antioxidants include both low-weight molecular and macromolecules, such as GSH and SOD, which are responsible for decreasing concentrations of ROS/RNS, and increasing resistance to neurodegenerative diseases in the central nervous system [42]. Meanwhile, the production of MDA is used to
PT
measure the level of oxidative stress in an organism [43]. The free radical NO plays an
RI
important role in oxidative stress and the mechanism underlying oxidative stress-involved
SC
cell apoptosis [44, 45]. Additionally, KO contains large amount of astaxanthin, which is a powerful antioxidant [46]. Venkatraman found that KO exposed mice have higher liver
NU
SOD and GPX mRNA expression than the lipid-only control group [47]. In addition,
MA
disturbances in the levels of ∆Ψm are also correlated with an excess of ROS production [48]. The current study shows similar results, in that METH exposure decreases SOD and
D
GSH significantly, while increasing MDA content and production of NO and ROS
PT E
significantly, compared with the control group. Generally, KO (0.2, 0.4, and 0.8 μg/mL) reverses the above results in contrast with the METH-only treated group, while the NO
CE
production was decreased significantly in 0.4 μg/mL KO treated group compared with
AC
other KO treated groups after METH exposure, yet the protective effect of KO against oxidative stress after METH treatment in PC12 cells was not in a dose dependent manner.
One limitation of the present study is that the current results illustrate the protective effect of KO in alleviating the apoptotic response and oxidative damage in PC12 cells only in vitro. In the future, animal models will also be applied to our study to comprehensively evaluate the protective effect of KO in the nervous system. In addition, the current results
ACCEPTED MANUSCRIPT did not demonstrate that KO mitigates METH-induced neurotoxicity in PC12 cells in a dose dependent manner, yet we still found that KO (0.1, 0.2, 0.4, 0.8 μg/mL) demonstrates protective effects, by inhibiting apoptosis and oxidative damage, as seen in most of our experimental results. In the future, by modifying the experimental concentrations of KO,
PT
we plan to detect the relationship between KO concentration and its protective effect on
SC
RI
PC12 cells with METH-induced neurotoxicity.
In conclusion, oxidative stress and apoptosis are correlated in the pathophysiology of
NU
many neurodegenerative diseases [49]. Our study found that both apoptosis and oxidative
MA
damage are triggered by 24 h METH treatment in PC12 cells, as evidenced by decreases in cell viability, increases in protein expression of cleaved caspase-3 and apoptotic rates, loss
D
of mitochondrial membrane potential, as well as decreases in SOD and GSH activity,
PT E
elevated MDA content, and decreases in NO and ROS production. Consequently, we demonstrate the hypothesis that KO supplementation might effectively prevent
CE
METH-induced neurotoxicity via the inhibition of apoptotic responses and oxidative
AC
damage. Meanwhile, we try to highlight the potential and specific health benefits of KO. Although the exact mechanism of the anti-apoptotic and antioxidant properties of KO is unclear, the mitochondria-mediated pathway may be involved in these two correlated pathological processes. Therefore, it necessitates further investigation in the future.
ACCEPTED MANUSCRIPT Acknowledgment The authors thank Weiling Li for her helpful manuscript editing assistance and Aker BioMarine Company (Norway) for the kind gift of SuperbaTM Krill Oil in this study. This work was supported by Hubei Provincial Department of Education guidance project
AC
CE
PT E
D
MA
NU
SC
RI
PT
(B2017273). The authors declare no conflicts of interest.
ACCEPTED MANUSCRIPT REFERENCES [1] Panenka WJ, Procyshyn RM, Lecomte T, MacEwan CW, Flynn SW, Honer WC, et al. Methamphetamine use: a comprehensive review of molecular, preclinical and clinical findings. Drug Alcohol Depen 2013;120:167-79.
PT
[2] Wang Q, Wei LW, Xiao HQ, Xue Y, Du SH, Liu YG, et al. Methamphetamine induces
RI
hepatotoxicity via inhibiting cell division, arresting cell cycle and activating apoptosis: In
SC
vivo and in vitro studies. Food Chem Toxicol 2017;105:61-72.
[3] Kita T, Takeshima M, Wagner GC, Hozumi H, Miyazaki I, Asanuma M. New
NU
perspectives on the mechanism of methamphetamine-induced neurotoxicity. Nihon
MA
Shinkei Seishin Yakurigaku Zasshi 2008;28:49–61.
[4] Nara A, Aki T, Funakoshi T, Uemura K. Methamphetamine induces macropinocytosis
D
in differentiated SH-SY5Y human neuroblastoma cells. Brain Res 2010;1352:1-10.
receptor
5,
PT E
[5] Shah A, Silverstein PS, Singh DP, Kumar A. Involvement of metabotropic glutamate AKT/P13K
signaling
and
NF-kappa
B
pathway
in
CE
methamphetamine-mediated increase in IL-6 and IL-8 expression in astrocytes. J
AC
neuroinflamm 2012;9:1-10. [6] Shah A, Kumar S, Simon SD, Singh DP, Kumar A. HIV gp120- and methamphetamine-mediated oxidative stress induces astrocyte apoptosis via cytochrome P450 2E1. Cell death Dis 2013;4:1-15. [7] Roos WP, Thomas AD, Kaina B. DNA damage and the balance between survival and death in cancer biology. Nat Rev Cancer 2016;16:20-33. [8] Callaghan RC, Cunningham JK, Sykes J, Kish SJ. Increased risk of Parkinson’s
ACCEPTED MANUSCRIPT disease in individuals hospitalized with conditions related to the use of methamphetamine or other amphetamine-type drugs. Drug Alcohol Depen 2012;120:35–40. [9] Xu X, Huang E, Tai YC, Zhao X, Chen XB, Chen CX, et al. Nupr1 modulates methamphetamine-induced dopaminergic neuronal apoptosis and autophagy through
PT
CHOP-Trib3-Mediated endoplasmic reticulum stress signaling pathway. Front Mol
RI
Neurosci 2017;10:203-18.
SC
[10] Burri L, Johnsen L. Krill products: An overview of animal studies. Nutrients 2015;7: 3300-21.
NU
[11] Nicol S, Foster J, Kawaguchi S. The fishery for Antarctic krill—recent developments.
MA
Fish Fish 2012;13:30-40.
[12] Ulven SM, Kirkhus B, Lamglait A, Basu S, Elind E, Haider T, et al. Metabolic
D
effects of krill oil are essentially similar to those of fish oil but at lower dose of EPA and
PT E
DHA in healthy volunteers. Lipids. 2011;46:37-46. [13] Berge K, Musa-Veloso K, Harwood M, Hoem N, Burri L. Krill oil supplement
CE
lowers serum triglycerides without increasing low-density lipoprotein cholesterol in
AC
adults with borderline high or high triglyceride levels. Nutr Res 2014;34:126-33. [14] Grimstad T, Bjorndal B, Cacabelos D, Aasprong OG, Janssen EA, Omdal R, et al. Dietary supplementation of krill oil attenuates inflammation and oxidative stress in experimental ulcerative colitis in rats. Scand J Gastroenterol 2012;47:49-58. [15] Banni S, Carta G, Murru E, Cordeddu L, Giordano E, Sirigu AR, et al. Krill oil significantly decreases 2-arachidonoylglycerol plasma levels in obese subjects. Nutr Metab 2011;8:1-6.
ACCEPTED MANUSCRIPT [16] Deutsch L. Evaluation of the effect of Neptune krill oil on chronic inflammation and arthritic symptons. J Am Coll Nutr 2007;26:39-48. [17] Bunea R, EI FK, Deutsch L. Evaluation of the effects of neptune krill oil on the clinical course of hyperlipidemia. Altern Med Rev. 2004;9:420-28.
PT
[18] Wibrand K, Berge K, Messaoudi M, Duffaud A, Panja D, Bramham CR, et al.
RI
Enhanced cognitive function and antidepressant-like effects after krill oil supplementation
SC
in rats. Lipids Health Dis 2013;12:6-18.
[19] Gamoh S. Krill-derived phospholipids rich in n-3 fatty acid improve spatial memory
NU
in adult rats. J Agric Sci-Cambridge 2011;3:3–12.
MA
[20] Polotow TG, Poppe SC, Vardaris CV, Ganini D, Guariroba M, Mattei R, et al. Redox status and neuro inflammation indexes in cerebellum and motor cortex of wistar rats
D
supplemented with natural sources of omega-3 fatty acids and astaxanthin: fish oil, krill
PT E
oil, and Algal biomass. Mar Drugs 2015;13:6117-37. [21] Jayathilake AG, Senior PV, Su XQ. Krill oil extract suppresses cell growth and
AC
328-38.
CE
induces apoptosis of human colorectal cancer cells. BMC complement Alternm 2016;16:
[22] Robertson B, Burri L, Berge K. Genotoxicity test and subchronic toxicity study with superba krill oil in rats. Toxicol Rep 2014;1:764–76. [23] Roth JA, Horbinski C, Higgins D, Lein P, Garrick MD. Mechanisms of manganese-induced rat pheochromocytoma (PC12) cell death and cell differentiation. Neurotoxicology 2002;23:147-57. [24] Meng HZ, Ni XF, Yu HN, Wang SS, Shen SR. Effects of astaxanthin on oxidative
ACCEPTED MANUSCRIPT stress induced by Cu2+ in prostate cells. J Zhejiang Univ Sci b 2002;18:161-71. [25] Reglero MM, Taggart MA, Gonzalez M, Mateo R. Heavy metal exposure in large game from a lead mining area: effects on oxidative stress and fatty acid composition in liver. Environ Pollut 2009;157:1388-95.
PT
[26] Xiong Q, Ru Q, Chen L, Yue K, Tian X, Ma BM, et al. Combined effects of fine
RI
particulate matter and lipopolysaccharide on apoptotic responses in NR8383 macrophages.
SC
J Toxicol Env Heal A 2015;78:43-52.
[27] Ng YW, Say YH. Palmitic acid induces neurotoxicity and gliatoxicity in SH-SY5Y
NU
human neuroblastoma and T98G human glioblastoma cells. Peer J 2018;26:1-21.
MA
[28] Kim D, Song J, Ahn C, Kang Yh, Chun CH, Jin EJ. Peroxisomal dysfunction is associated with up-regulation of apoptotic cell death via miR-223 induction in knee
D
osteoarthritis patients with type 2 diabetes mellitus. Bone 2014;64:124-31.
PT E
[29] Price RJ, Lillycrop KA, Burdge GC. Folic acid supplementation in vitro induces cell type-specific changes in BRCA1 and BRCA2 mRNA expression, but does not alter DNA
CE
methylation of their promoters or DNA repair. Nutr Res 2015;35:532-44.
AC
[30] Barros MP, Poppe SC, Bondan EF. Neuroprotective properties of the marine carotenoid astaxanthin and omega-3 fatty acids, and perspectives for the natural combination of both in krill oil. Nutrients 2014;6:1293-1317. [31] Pitaksalee R, Sanvarinda Y, Sinchai T, Sanvarinda P, Thampithak A, Jantaratnotai N, et al. Autophagy inhibition by caffeine increases toxicity of methamphetamine in SH-SY5Y neuroblastoma cell line. Neurotox Res 2015;27:421-29. [32] Tian X, Ru Q, Xiong Q, Yue K, Chen L, Ma BM, et al. Neurotoxicity induced by
ACCEPTED MANUSCRIPT methamphetamine-heroin combination in PC12 cells. Neurosci Lett 2017;647:1-7. [33] Huang WY, Xie WB, Qiao DF, Qiu PM, Huang EP, Li B, et al. Caspase-11 plays an essential role in methamphetamine-induced dopaminergic neuron apoptosis. Toxicol Sci 2015;145:68-79.
PT
[34] Wongprayoon P, Govitrapong P. Melatonin protects SH-SY5Y neuronal cells against
RI
methamphetamine-induced endoplasmic reticulum stress and apoptotic cell death.
SC
Neurotox Res 2017;31:1-10.
[35] Thrash-Williams B, Ahuja M, Karuppagounder SS, Uthayathas S, Suppiramaniam V,
NU
Dhanasekaran M. Assessment of therapeutic potential of amantadine in methamphetamine
MA
induced neurotoxicity. Neurochem Res 2013;38:2084-94. [36] Green DR, Reed JC. Mitochondria and apoptosis. Science 1998;281:1309-12.
D
[37] Potula R, Hawkins BJ, Cenna JM, Fan SS, Dykstra H, Ramirez SH, et al.
PT E
Methamphetamine causes mitochondrial oxidative damage in human T lymphocytes leading to functional impairment. J Immunol 2010;185:2867-76.
CE
[38] Buonocore G, Perrone S, Tataranno ML. Oxygen toxicity: Chemistry and biology of
AC
reactive oxygen species. Semin Fetal Neonatal Med 2010;15:186–90. [39] Wells PG, McCallum GP, Lam KC, Henderson JT, Ondovcik SL. Oxidative DNA damage and repair in teratogenesis and neurodevelopmental deficits. Birth Defects Res C 2010;90:103–9. [40] Conim A, Alves-Pereira, I, Ferreira R. Important role of antioxidant enzymes in the saccharomyces cerevisiae survival to the toxicity of vanadium pentoxide. Toxicol Lett 2010;196:304-10.
ACCEPTED MANUSCRIPT [41] Ni S, Wang D, Qiu X, Pang L, Song Z, Guo K. Bone marrow mesenchymal stem cells protect against bleomycin-induced pulmonary fibrosis in rat by activating Nrf2 signaling. Int J Clin Exp Pathol 2015;8:7752-61. [42] Koutsilieri E, Scheller C, Tribl F, Riederer P. Degeneration of neuronal cells due to oxidative
PT
stress-Microglial contribution. Parkinsonism Relat D 2002;8:401-6.
RI
[43] Rio DD, Stewart AJ, Pellegrini N. A review of recent studies on malondialdehyde as toxic
SC
molecule and biological marker of oxidative stress. Nutr Metab Cardiov AS 2005; 15:316-28. [44] Albina JE, Cui S, Mateo RB, Reichner JS. Nitric oxide-mediated apoptosis in murine
NU
peritoneal macrophages. J Immunol 1993;150:5080–85.
MA
[45] Jothiramajayam M, Sinha S, Ghosh M, Nag A, Jana A, Makherjee A. Sodium fluoride promotes apoptosis by generation of reactive oxygen species in human lymphocytes. J Toxicol
D
Environ Health A 2014;77:1269–80.
PT E
[46] Grynbaum MD, Hentschel P, Putzbach K, Rehbein J, Krucker M, Nicholson G, et al. Unambiguous detection of astaxanthin and astaxanthin fatty acid esters in krill (Euphausia superb
CE
Dana). J Sep Sci 2005;28:1685-93.
AC
[47] Venkatraman JT, Chandrasekar B, Kim JD, Fernandes G. Effects of n-3 and n-6 fatty acids on the activities and expression of hepatic antioxidant enzymes in autoimmune-prone NZB × NZW F1 mice. Lipids 1994;29:561–68. [48] Radi E, Formichi P, Battisti C, Federico A. Apoptosis and oxidative stress in neurodegenerative disease. J Alzheimers Dis 2014;42:125-52. [49] Sinha K, Das J, Pal PB, Sil PC. Oxidativestress: The mitochondria-dependent and mitochondriain dependent pathways of apoptosis. Arch Toxicol 2013;87:1157-80.
ACCEPTED MANUSCRIPT Figure Legends
Figure 1. Effect of krill oil on cell viability of PC12 cells post METH treatment.
Cells were treated with MA (3 mM) /MA + krill oil (0.1, 0.2, 0.4, 0.8 μg/mL) for 24 h, and then cells were
PT
further incubated with MTT (0.5 mg/ml) for 4 h in the 37 ˚C incubator at dark. The cell viability was evaluated by MTT reduction assay. Values are means ± SEM (n = 9). Data were analyzed by one way
RI
ANOVA followed using a post hoc test of Tukey. Statistical significance: ##P < .01 compared to the
SC
control group, **P < .01 compared to the METH group.
NU
Figure 2. Effect of krill oil on NO Production in PC 12 cells after METH exposure.
MA
Cells were treated with MA (3 mM) /MA + krill oil (0.1, 0.2, 0.4, 0.8 μg/mL) for 24 h, the supernatant from each group was collected, and then all the samples were further incubated with Griess Reagent I and Griess Reagent II reagents (50 μL for each reagent) to determine the production of NO. Values are means ±
D
SEM (n = 9). Data were analyzed by one way ANOVA followed using a post hoc test of Tukey. Statistical
PT E
significance: ##P < .01 compared to the control group, **P < .01 compared to the METH group.
CE
Figure 3. Effect of krill oil on ROS Production in PC 12 cells after METH exposure
AC
Cells were treated with MA (3 mM) /MA + krill oil (0.1, 0.2, 0.4, 0.8 μg/mL) for 24 h, the probe DCFH-DA (100 μmol/L) was incubated with cells for 1 h in a 37 ˚C and 5% (v/v) CO2 incubator, and then the fluorescent signal was obtained to evaluate the intracellular ROS level in different groups. Values are means ± SEM (n = 9). Data were analyzed by one way ANOVA followed using a post hoc test of Tukey. Statistical significance: ##P < .01 compared to the control group, **P < .01 compared to the METH group.
ACCEPTED MANUSCRIPT Figure 4. Effect of krill oil on apoptosis response in PC 12 cells after METH exposure. Cells were treated with MA (3 mM) /MA + krill oil (0.1, 0.2, 0.4, 0.8 μg/mL) for 24 h and analyzed by Muse Annexin V & Dead Cell assay. A representative dot plots in live, dead, late apoptotic/dead and early apoptotic phase is shown in the upper panel, and the mean percentage of the cell death is expressed by a histogram in the lower panel. Values are means ± SEM (n = 9). Data were analyzed by one way ANOVA followed using a post hoc test of Tukey. Statistical significance: ##P < .01 compared to the control group,
PT
*P < .05 compared to the METH group.
RI
Figure 5. Effect of krill oil on mitochondrial membrane potential in PC 12 cells after
SC
METH exposure.
NU
Cells were treated with MA (3 mM) /MA + krill oil (0.1, 0.2, 0.4, 0.8 μg/mL) for 24 h and analyzed by Muse MitoPotential assay. A representative dot plots in live, depolarized/live, depolarized/dead and dead phase is shown in the upper panel, and the mean percentage of the total depolarization is expressed by a
MA
histogram in the lower panel. Values are means ± SEM (n = 9). Data were analyzed by one way ANOVA followed using a post hoc test of Tukey. Statistical significance: #P < .05 compared to the control group, *P
PT E
D
< .05 compared to the METH group.
Figure 6. Effect of krill oil on the protein expression of cleaved caspase 3 in PC 12 cells
AC
CE
after METH exposure.
Cell extracts from PC12 cells treated with MA (3 mM) /MA + krill oil (0.1, 0.2, 0.4, 0.8 μg/mL) for 24 h and analyzed by Western Blot with anti-cleaved caspase 3 (1:500) and anti-β-actin (1:1000) antibodies (A). Protein data values were normalized to actin level and reported in relative protein levels of cleaved caspase 3 with a histogram (B). Values are means ± SEM (n = 5). Data were analyzed by one way ANOVA followed using a post hoc test of Tukey. Statistical significance: #P < .05 compared to the control group, **P < .01 compared to the METH group.
ACCEPTED MANUSCRIPT Table 1 The main compositions and analytical specifications of krill oil LIMITS ≥40 ≥5 ≥22
UNIT g/100g g/100g g/100g
EPA
≥12
g/100g
DHA
≥5.5
g/100g
Astaxanthin esters
≥100
μg astx-diol/g oil3
Krill Oil’s specification is provided by Aker BioMarine Company (Norway)
SC NU MA D PT E CE AC
The superba
RI
TM
PT
COMPOSITION Total phospholipids Choline Total omega-3 fatty acids
ACCEPTED MANUSCRIPT Table 2 Effect of krill oil (KO) on the oxidative enzyme system (MDA content, SOD activity and GSH activity) in PC 12 cells after METH exposure SOD (U/mg protein) 73.20±1.46 54.50±1.01##
GSH (μmol/g protein)
1.52±0.06**
78.30±4.92**
49.30±2.18*
1.28±0.10**
79.10±3.52**
1.32±0.04**
70.03±5.88*
51.57±2.06**
1.37±0.06**
76.87±4.65**
49.10±0.92**
RI
PT
45.98±1.07 40.98±0.50#
49.23±0.54**
SC
Control METH METH+KO (0.1μg/ml) METH+KO (0.2μg/ml) METH+KO (0.4μg/ml) METH+KO (0.8μg/ml)
MDA (nmol/mg protein) 1.27±0.02 3.03±0.03##
NU
Group
AC
CE
PT E
D
MA
Cells were treated with MA (3 mM) /MA + KO (0.1, 0.2, 0.4, 0.8 μg/mL) for 24 h, then cell samples were collected for measurements of the levels of MDA, GSH and SOD using corresponding commercial detection kits. For each individual measurement, values are means ± SEM (n = 9). Data were analyzed by one way ANOVA followed using a post hoc test of Tukey. Statistical significance: ## P < .01, compared to the control group, # P < .05, compared to the control group, ** P < .01, compared to the METH group, * P < .05, compared to the METH group.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Qi Xiong, Qin Ru, Xiang Tian, Mei Zhou, Lin Chen, Yi Li, Chaoying Li PII: DOI: Reference:
S0271-5317(17)31175-2 doi:10.1016/j.nutres.2018.07.006 NTR 7920
To appear in:
Nutrition Research
Received date: Revised date: Accepted date:
22 December 2017 4 July 2018 9 July 2018
Please cite this article as: Qi Xiong, Qin Ru, Xiang Tian, Mei Zhou, Lin Chen, Yi Li, Chaoying Li , Krill oil protects PC12 cells against methamphetamine induced neurotoxicity by inhibiting apoptotic response and oxidative stress. Ntr (2018), doi:10.1016/j.nutres.2018.07.006
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Krill oil protects PC12 cells against methamphetamine induced neurotoxicity by inhibiting apoptotic response and oxidative stress
1
RI
PT
Qi Xiong1 , Qin Ru1, Xiang Tian1, Mei Zhou1, Lin Chen1, Yi Li2, Chaoying Li1
2
Wuhan Mental Health Center, Wuhan, Hubei Province, China 430022
NU
SC
Wuhan Institutes of Biomedical Sciences, Jianghan University, Wuhan City, Hubei Province, China 430000
MA
Corresponding author: Chaoying Li Tel: +86-13628678552, Email: [email protected]; [email protected] Qi Xiong’s email: [email protected]
D
Qin Ru’s email: [email protected]
PT E
Xiang Tian’s email: [email protected] Mei Zhou’s email: [email protected]
CE
Lin Chen’s email: [email protected]
AC
Yi Li’s email: [email protected]
ACCEPTED MANUSCRIPT
List of abbreviations
METH; methamphetamine
PT
KO; krill oil EPA; eicosapentaenoic acid
RI
DHA; docosahexaenoic acid
SC
FBS; fetal bovine serum
NU
MTT; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide GSH; Glutathione
MA
SOD; superoxide dismutase
PT E
DMSO; dimethyl sulfoxide
D
MDA; Malomdialdehvde
ROS; reactive oxygen species
AC
CE
ANOVA; analysis of variance
ACCEPTED MANUSCRIPT Abstract Methamphetamine (METH) exposure can cause severe effects to the nervous system; however, the underlying molecular mechanism of neurotoxicity caused by METH is still unclear. Oxidative stress and apoptosis are linked in the pathophysiology of many
PT
neurodegenerative diseases. Krill oil (KO) benefits human health via its strong antioxidant
RI
ability. Therefore, we hypothesized that KO supplementation might effectively prevent
SC
METH-induced neurotoxicity via the inhibition of apoptotic responses and oxidative damages. In this study, PC12 cells were exposed to both METH (3 mM) and KO (0.1, 0.2,
NU
0.4, 0.8 μg/ml) in vitro for 24 h, and the following parameters were measured to detect
MA
apoptosis and oxidative stress responses that were triggered by METH: cell viability, the oxidative enzyme system, NO production, ROS production, apoptosis, mitochondrial
D
membrane potential and protein expression of cleaved caspase-3. The results indicate that
PT E
KO mitigates the apoptotic response post-METH exposure in PC 12 cells by increasing cell viability, decreasing protein expression of cleaved caspase-3, reducing apoptotic rates,
CE
and decreasing dissipation of mitochondrial membrane potential. In addition, the study
AC
revealed increases in SOD and GSH activity, and decreases in MDA content, NO and ROS production, suggesting that KO is beneficial in reducing oxidative stress, which may also play a role in the regulation of METH-triggered apoptotic response. Consequently, these data indicate that KO could potentially alleviate METH-induced neurotoxicity via the reduction of apoptotic responses and oxidative damages. Keywords: Methamphetamine; PC12 cells; Krill Oil; Neurotoxicity; Apoptosis; Oxidative damage
ACCEPTED MANUSCRIPT Introduction Methamphetamine (METH) is a highly addictive psychostimulant drug that is abused by many people. METH abuse has caused severe societal problems in recent decades and has drawn intense worldwide attention [1, 2]. METH mainly targets the dopaminergic
PT
system and abnormal use of METH induces neurotoxicity in different regions of the brain
RI
[3, 4]. Previous studies found that METH-triggered neurotoxic effects may be caused by
SC
the pro-inflammatory cytokines [5], oxidative stress [6] as well as apoptosis [7] in dopaminergic neurons. In addition, METH exposure is a significant risk factor that
NU
promotes susceptibility to neurodegenerative diseases, such as Parkinson’s disease [8, 9].
MA
However, the precise molecular mechanism of METH-induced neurotoxicity in the
D
nervous system remains to be elucidated.
PT E
Krill are small crustaceans that possess a large amount of phospholipids and are particularly abundant in the Arctic and Antarctic polar seas [10]. Antarctic Krill,
CE
Euphausia superba, is thought to be the most important and prolific species [11]. Krill is a sustainable source of omega-3 polyunsaturated fatty acids (n-3 PUFAs) and is especially
AC
1
rich in long-chain PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) which naturally occur in phospholipid form [12, 13]. The oil is extracted from Antarctic krill; the major component of the n-3 PUFAs in Krill Oil (KO) is associated with phospholipids, while the natural antioxidant astaxanthin is another important component [14]. The antioxidant effect of KO is substantial due to the protective nature of the phospholipid bilayer, which helps prevent unsaturated bonds from oxidizing. During the
ACCEPTED MANUSCRIPT past decades, studies have demonstrated that KO provides health benefits, including treatment of chronic inflammation [15, 16], arthritis [16] and hyper-lipidemia [17].
In
addition, KO has also been proven to have an impact on memory improvement in the rat model [18], as well as decreasing levels of lipid peroxide and reactive oxygen species in
PT
rats [19, 20]. Moreover, Jayathilake demonstrated that KO extract could inhibit cell
RI
proliferation and induce apoptosis of human colorectal cancer cells [21]. Genotoxicity
SC
studies have also confirmed the safety of KO [22]; however, the effect of KO on
NU
METH-induced neurotoxicity had not been investigated prior to this study.
MA
In the current study, we hypothesized that KO could mitigate METH-induced neurotoxicity via the reduction of apoptosis and oxidative stress in dopaminergic cells. To
D
address this, rat adrenal pheochromocytoma (PC12) cells were used as a cell culture
PT E
model because they possess much of the biochemical machinery associated with dopaminergic neurons [23]. The PC12 cells were exposed to METH (3 mM) and different
CE
concentrations of KO (0.1, 0.2, 0.4, 0.8 μg/ml) for 24 h, while the following apoptotic and
AC
oxidative stress-related parameters were measured: cell viability, mitochondrial membrane potential, apoptosis, the oxidative enzyme system, NO production, reactive oxygen species (ROS) production, and protein expression of cleaved caspase-3. The results show that KO has potential as a therapeutic substance to alleviate METH induced neurotoxicity in future clinical practice.
2
Methods and materials
ACCEPTED MANUSCRIPT 2.1 Reagents METH was obtained from the Hubei Public Security Bureau. RPMI-1640 medium, fetal bovine serum (FBS), trypsin, penicillin, and streptomycin were purchased from Life Technologies
(Carlsbad,
CA,
USA).
PT
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DCFH-DA and
RI
dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Corp (St Louis, MO,
SC
USA). The Muse Annexin V & Dead Cell Assay Kit (MCH100105) and the Muse MitoPotential Kit (MCH100110) were obtained from the Millipore Corporation
NU
(Darmstadt, Germany). Glutathione (GSH), Superoxide Dismutase (SOD) and
MA
Malomdialdehvde (MDA) Assay Kits were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). An NO Assay Kit was obtained from the
D
Beyotime Institute of Biotechnology (Haimen, China). All other chemicals were standard
PT E
analytical grade. SuperbaTM Krill Oil (KO) was kindly provided by the Aker Biomarine
AC
2.2 Cell lines
CE
Antarctic Company (Norway); its major components are described in Table 1.
PC12 cells, which are derived from a pheochromocytoma of the rat adrenal medulla, were obtained from the Shanghai Institute of Cell Library (Shanghai, China) and were plated and maintained in RPMI-1640 medium supplemented with 20% (v/v) fetal bovine serum (Gibco, USA) and the antibiotics penicillin (100 U/ml) and streptomycin (100 μg/ml). The cells were maintained at 37 ℃ and 5% (v/v) CO2 in a humid environment. The cells were then harvested and plated in 96-, 24-, or 6-well culture plates for different
ACCEPTED MANUSCRIPT experimental requirements.
2.3
Exposure of cells to METH and KO A stock solution of Krill oil was prepared in ethanol at a concentration of 10 mg/ml.
PT
Different concentrations of Krill Oil (0.1, 0.2, 0.4, 0.8 μg/ml) were chosen based on the
RI
results from our previous research (data not shown). The PC12 cells were then treated
SC
either with 3 mM METH [9] or with the combinations of 3 mM METH and KO (0.1, 0.2, 0.4, 0.8 μg/ml) for 24 h in a 37 ˚C and 5% (v/v) CO2 incubator. The cells were then
MA
NU
harvested and utilized in the following experiments.
2.4 Measurement of cell viability viability
was
measured
by
D
Cell
PT E
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in the current study [24]. Briefly, the cells were harvested and counted, then seeded on a 96-well
CE
plate at a density of 5×103 (Corning, USA) overnight in a 37 ˚C and 5% (v/v) CO2
AC
incubator. Different concentrations of KO were prepared with dilutions ranging from 0.1 to 0.8 μg/ml. The METH (3 mM) was diluted in RPMI-1640 and KO was further diluted in RPMI-1640 with METH to obtain the final concentrations (0.1, 0.2, 0.4, 0.8 μg/ml). After 24 h incubation, 20 μl of MTT (0.5 mg/ml) reagent was added to each well. The cells were further incubated for 4 h in the 37 ˚C incubator in the dark, the supernatants were carefully removed, 150 μl of DMSO was added to each well and mixed thoroughly. Absorbance was measured at 490 (A490) nm using a microplate reader (Thermal Scientific,
ACCEPTED MANUSCRIPT USA). The relative cell viability (%) was calculated as (A490 of treated samples/ A490 of untreated samples) × 100%.
2.5 Measurements of the oxidative enzyme system
PT
The oxidative enzyme system (MDA, SOD and GSH) was used to examine the oxygen
RI
reactivity of PC12 cells treated with METH. PC12 cells were seeded in 6 well plates at a
SC
density of 5×104 cells/well overnight in a 37 ˚C and 5% (v/v) CO2 incubator. After 24 h incubation with either METH (3 mM) alone or the combination of METH (3 mM) and
NU
different concentrations of KO (0.1, 0.2, 0.4, 0.8 μg/ml), cells were collected and the
MA
levels of MDA, SOD and GSH were measured. The cell samples were prepared for the experiments as follows: MDA - cells were harvested and centrifuged at 1000 rpm for 5
D
min at room temperature (RT), supernatants were removed and the cells were
PT E
resuspended in 300 μL phosphate buffer saline (PBS). The cells were then sonicated and centrifuged again to obtain cell samples; SOD - cells were first harvested and centrifuged
CE
at 1000 rpm for 5 min at room temperature, supernatants were removed and the cells were
AC
resuspended in 1 mL PBS. The cells were then mixed with 100 μL lysis buffer on ice for 40 min. Finally the cells were centrifuged at 5000 rpm for 15 min to obtain cell samples; GSH - cells were first harvested and centrifuged at 1000 rpm for 5 min at RT, supernatants were removed and the cells were resuspended in 1 mL PBS. The cells were then mixed with 100 μL lysis buffer on ice for 40 min. Finally, the cells were centrifuged at 5000 rpm for 15 min to obtain cell samples. The levels of MDA, GSH and SOD were further measured as described in a previous study [25], using corresponding commercial
ACCEPTED MANUSCRIPT detection kits (Nanjing Jiancheng Bioengineering Institute, China). The absorbances for MDA, GSH and SOD were measured at different wavelengths (532 nm, 420 nm and 405 nm). The results were then corrected for their protein levels based on the reference
PT
manual for a BCA protein assay kit (Beyotime Institute of Biotechnology, China).
RI
2.6 Measurement of NO production
SC
Quantification of sodium nitrite was used as an indirect way to determine the production of NO generated by 3 mM METH. Briefly, cultured PC12 cells were seeded in
NU
24-well plates at a density of 3 ×105 cells/well overnight in a 37 ˚C and 5% (v/v) CO2
MA
incubator. The cells were then exposed to METH/KO, and unexposed cells were used as negative controls. Twenty four hours later, the supernatants were collected to determine
D
the production of NO (Beyotime Institute of Biotechnology, China). Procedures were
PT E
followed according to our previous study [26]. Generally, the supernatants were transferred to 96-well plates, standard samples were prepared at different concentrations
CE
(0, 1, 2, 5, 10, 20, 40, 60 and 100 μM) and added at a volume of 50 μL/well. All other test
AC
samples were also prepared at volumes of 50 μL/well. Then, 50 μL of Griess Reagent I and 50 μL of Griess Reagent II were added to each standard sample and test sample, accordingly. The results were read by a microplate reader at 540 nm. The production of NO for each group was calculated according to the standard curve.
2.7 Measurement of ROS production Intracellular ROS level was detected using a fluorescent probe DCFH-DA
ACCEPTED MANUSCRIPT (Sigma-Aldrich, MO, USA). The protocol from a previously study was followed [27]. Generally, PC12 cells were seeded in 6-well plates at a density of 5×104 cells/well overnight in a 37 ˚C and 5% (v/v) CO2 incubator. After 24 h incubation with either METH (3 mM) alone or the combination of METH (3 mM) and different concentrations of KO
PT
(0.1, 0.2, 0.4, 0.8 μg/mL), each supernatant was completely removed, and the cells were
RI
washed with PBS three times. Then, DCFH-DA was dissolved in 10 nmol/L DMSO, and
SC
100 μmol/L DCFH-DA, which was further dissolved in RPMI-1640 complete medium, was added into each group (500 μL/well) and the cells were incubated in a 37 ˚C and 5% (v/v)
NU
CO2 incubator for 1 h. After the DCFH-DA was removed from each well, and each well
MA
was washed with PBS three times to completely remove the DCFH-DA. The cells were then dissolved and harvested with 1 mL PBS. Finally, the fluorescent signal was read at
PT E
Ortenberg, Germany).
D
Ex485nm/Em538nm, using a FLUOstar® Omega Microplate Reader (BMG LABTECH,
CE
2.8 Measurement of cell apoptosis
AC
The Muse Annexin V & Dead Cell Kit (EMD Millipore Corporation, Germany) was used to detect apoptotic cell death, as described by a previous study [28]. Briefly, PC12 cells were seeded in 6-well culture plates at a density of 5×104 cells/well and incubated overnight at 37 ˚C and 5% (v/v) CO2. Then, cells were treated with either METH (3 mM) alone or the combination of METH (3 mM) and different concentrations of KO (0.1, 0.2, 0.4, 0.8 μg/ml) for another 24 h. After 24 h incubation, the cells were harvested and centrifuged at 1000 rpm for 5 min at room temperature, then the supernatants were
ACCEPTED MANUSCRIPT removed and 100 μL RPMI-1640 complete medium was added. They were then thoroughly mixed with another 100 μL of MuseTM Annexin-V & Dead Cell reagent for 20 min at room temperature. Finally, after incubation, the cells were analyzed immediately
RI
2.9 Measurement of mitochondrial membrane potential
PT
by the Muse™ Cell Analyzer (EMD Millipore Corporation, Germany).
SC
Changes in the mitochondrial membrane potential （ΔΨm）during the early stages of apoptosis were analyzed by the Muse MitoPotential assay (EMD Millipore Corporation,
NU
Germany). The detailed protocol was followed as previously described [28]. PC12 cells
MA
were seeded in 6-well culture plates at a density of 5×104 cells/well overnight in a 37 ˚C and 5% (v/v) CO2 incubator. The cells were then treated with different concentrations of
D
Krill Oil (0.1, 0.2, 0.4, 0.8 μg/ml)/METH (3 mM) in 6-well culture plates for 24 h. First,
PT E
the cells were harvested and centrifuged at 1000 rpm for 5 min at room temperature, then the supernatants were removed and replaced by 100 μL PBS, then prepared
CE
MuseTM MitoPotential working solution, by diluting the dye 1:1000 with 1X Assay
AC
Buffer. An amount of 95 μL of MuseTM MitoPotential working solution was added to 100 μL of cells, and the cell samples were incubated at 37˚C for 20 min. Finally, 5 μl of MuseTM MitoPotential 7-AAD dye was also added to each sample and the cells were incubated for another 5 min at RT. Changes in the mitochondrial membrane potential were determined by flow cytometry (EMD Millipore Corporation, Germany).
ACCEPTED MANUSCRIPT 2.10 Western blot analysis of cleaved caspase-3 PC12 cells were treated with either METH (3 mM) or the combination of METH (3 mM) and different concentrations of KO (0.1, 0.2, 0.4, 0.8 μg/ml) for 24 h in a 37 ˚C and 5% (v/v) CO2 incubator. Protein extracts were prepared in 50 mM tris pH 8, 150
PT
μM NaCl, 0.5% sodium deoxycholate, 20% (v/v) protease inhibitor Cocktail and 10%
RI
(v/v) PMSF (Sigma, USA). Protein concentrations from different groups were
SC
determined by BCA assay kit (Boster Company, China). Western blot analysis of protein expression was performed as previously described [29]. SDS-PAGE loading
NU
buffer was added to each group, and boiled for 5 min. Then, 50 μg of the protein
MA
samples were separated on a 12% SDS-PAGE gel and transferred to PVDF membrane (Merck Millipore, USA). For immunoblotting, membranes were blocked with 5% (w/v)
D
milk in tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST) for 1 h at RT.
PT E
Primary antibodies, including rabbit anti-cleaved caspase-3 (Cell Signaling, USA , 1:500) and rabbit anti-β-actin (Cell Signaling, USA, 1:1000) were also incubated with
CE
PVDF membranes in 5% (w/v) milk in TBST at 4 ˚C overnight. Then, the membranes
AC
were washed and incubated with a 1:3000 dilution of HRP-conjugated anti-rabbit secondary antibodies (Boster Company, China) in 5% (w/v) milk in TBST for 1 h at RT. After washing in TBST an additional three times, the membranes were incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher, USA), and the bands were visualized by using an imaging analysis system (Gene Company, Hong Kong). The intensity of each band was quantitatively determined by GenTools Image Analyzer Software (Gene Company, Hong Kong). β-actin was used as a loading control.
ACCEPTED MANUSCRIPT Therefore, the density ratio indicated the relative intensity of each band against β-actin. 2.11 Statistical analyses Data are expressed as the means ± standard error of the mean ( x ±SEM). A one way analysis of variance (ANOVA), followed by a post hoc Tukey test, was applied to all the
PT
experiments to examine the significance of the data. Statistical analysis was performed
RI
using SPSS 16.0 (IBM Corporation, New York, USA) and the criterion for significance
SC
was set at P < .05. For the primary result measure, cell viability, a sample size of 9 cell samples provided statistical power of at least 82%, detecting a 10% difference, with a
NU
probability of < .05. This sample size provided at least this level of statistical power for
Results
D
3
MA
the other results as well.
PT E
3.1 The effect of KO on the viability of PC12 cells post METH treatment The MTT assay was used to evaluate cell viability after treatment with METH and
CE
different concentrations of KO (Fig. 1). Compared with the control group, the cell survival
AC
rate of METH-only exposed PC12 cells decreased significantly (57.71 ± 7.72 vs 100.12 ± 11.21, P < .01), while the survival rates of PC12 cells all increased significantly after KO treatment (0.1, 0.2, 0.4, 0.8 μg/mL), in contrast with the METH-only treated group. Specifically, 0.8 μg/mL KO raised the cell survival rate the highest degree, up to 91.21 ± 6.83, compared with the METH-only treated group (57.71 ± 7.72, P < .01). In addition, the cell survival rates of 0.2 μg/mL KO and 0.4 μg/mL KO (85.12 ± 6.24 vs 85.34 ± 4.61) were similar post-METH treatment, yet they were significantly increased in comparison
ACCEPTED MANUSCRIPT with METH-only treated group (P < .01) 3.2 The effect of KO on the oxidative enzyme system in PC 12 cells after METH exposure Table 2 represents the results from the MDA, SOD and GSH oxidative stress
PT
experiments. It shows that the levels of MDA increased significantly post METH treatment
RI
in comparison with the control group (3.03 ± 0.03 vs 1.27 ± 0.02, P < .01). In contrast, with
SC
the METH-only treated group, all concentrations of KO post METH treatment decreased MDA content significantly (1.52 ± 0.06, 1.28 ± 0.10, 1.32 ± 0.04, 1.37 ± 0.06 vs 3.03 ±
NU
0.03, P < .01). In addition, METH alone reduced SOD activities significantly in PC12 cells
MA
compared with the control group (54.50 ± 1.01 vs 73.20 ± 1.46, P < .01), and all concentrations of KO significantly increased SOD activities (P < .05, P < .01), while 0.2
D
μg/mL KO raised the SOD activities the highest, up to 79.10 ± 3.52, compared with the
PT E
METH-only treated group (54.50 ± 1.01, P < .01). Finally, the METH-only treated group experienced significantly decreased GSH activities in contrast with the control group
CE
(40.98 ± 0.50 vs 45.98 ± 1.07, P < .05), and all concentrations of KO significantly
AC
increased GSH activities (P < .05, P < .01), while 0.4 μg/mL KO increased the GSH activities the highest, up to 51.57 ± 2.06, compared with the METH-only treated group (40.98 ± 0.50, P < .01).
3.3 The effect of KO on NO Production in PC 12 cells after METH exposure Figure 2 represents the NO responses to different concentrations of KO with/without METH. Compared with the control group, the METH-treated group showed a significant
ACCEPTED MANUSCRIPT increase in NO production (2.41 ± 0.10 vs 1.91 ± 0.04, P < .01). When the concentrations of KO were between 0.2 - 0.8 μg/mL, NO levels decreased, in contrast with the METH-only treated group (1.18 ± 0.24, 0.85 ± 0.08, 1.21 ± 0.12 vs 2.41 ± 0.10 P < .01). Finally, no significant difference was detected when the concentration of KO was 0.1
RI
PT
μg/mL compared to the METH-only treated group.
SC
3.4 The effect of KO on ROS Production in PC 12 cells after METH exposure To attribute whether apoptotic cell death induction by METH in PC12 cells is caused by
NU
oxidative stress, ROS was examined. Figure 3 shows that METH induced greater levels of
MA
ROS generation compared with that of the control group (1.83 ± 0.07 vs 1.01 ± 0.04, P < .01). In addition, incremental increases in the concentration of KO, decreased the levels
D
of ROS production significantly in comparison with the METH group (1.37 ± 0.10, 1.24 ±
PT E
0.09, 1.18 ± 0.09, 1.06 ± 0.04 vs 1.83 ± 0.07, all P < .01). Therefore, the above results indicate that all of the KO concentrations decrease the levels of ROS production in PC12
AC
CE
cells after METH treatment.
3.5 The effect of KO on apoptosis response in PC 12 cells after METH exposure Flow cytometry, which detects apoptotic cells by Annexin V/7-AAD, was also applied to this study to detect the METH-induced apoptotic effect in PC12 cells. Data in Figure 4 shows that a significant rise in apoptotic rates was induced by METH, compared with the control (24.83 ± 2.60 vs 3.67 ± 1.38, P < .01). However, when the concentrations of KO were between 0.2 - 0.8 μg/mL, they significantly decreased the apoptotic rates in
ACCEPTED MANUSCRIPT comparison with METH-only treated group (16.12 ± 1.63, 14.65 ± 1.58, 12.10 ± 2.10 vs 24.83 ± 2.60, P < .05). No significant difference in the apoptotic rate was detected when the concentration of KO was 0.1 μg/mL compared to the METH-only treated group.
PT
3.6 The effect of KO on mitochondrial membrane potential in PC 12 cells after METH
RI
exposure
SC
ΔΨm is an important indicator of mitochondrial dysfunction, and the loss of ΔΨ is an indicator and a hallmark for apoptosis. Figure 5 shows that METH induced a significant
NU
increase in total depolarization of the mitochondria compared with the control group
MA
(31.27 ± 3.82 vs 14.62 ± 3.42, P < .05 ), which indicates that a significant dissipation of ΔΨ is triggered by METH. In addition, the concentrations of KO that are between 0.2-0.8
D
μg/mL significantly decreased the total depolarization of mitochondria in contrast with the
PT E
METH treated group (20.92 ± 0.43, 20.27 ± 0.22, 15.35 ± 2.9 vs 31.27 ± 3.82, P < .05). However, a concentration of 0.1 μg/mL KO showed a lack of significant statistical
AC
CE
difference.
3.7 The effect of KO on the protein expression of cleaved caspase-3 in PC 12 cells after METH exposure
To evaluate the apoptotic response in PC12 cells exposed to METH and the protective effect of KO, the protein expression of cleaved caspase-3 was investigated by Western Blot. As shown in Figure 6A and 6B, the protein expressions of cleaved caspase-3 in PC12 cells treated by METH were significantly increased compared with that of the control group
ACCEPTED MANUSCRIPT (121.45 ± 4.60 vs 100.02 ± 0.02, P < .05). In contrast, all concentrations of KO significantly decreased protein expression of cleaved caspase-3 (86.17 ± 3.62, 71.27 ± 5.65, 62.86 ± 5.95), while 0.8 μg/mL KO decreased the protein expression level of cleaved caspase-3 the most,
Discussion
RI
Cell death and neurological damage have been linked to oxidative stress and the
SC
imbalance between the generation of free radicals and antioxidant defenses [30]. The
NU
current study investigated, for the first time, the protective effect of KO in preventing PC12 cells from experiencing a METH-triggered apoptotic response and oxidative damage.
MA
Previous studies have demonstrated that over exposure to METH may result in decreased cell viability. Pitaksalee demonstrated that cell viability of SH-SY5Y cells decreased
PT E
D
significantly post 24 h METH exposure in a dose dependent manner [31]. Similar results were also detected in another study [32]. In the current study, MTT results showed that
CE
METH decreased cell viability significantly compared with the control group, while cell death could be blocked when the concentrations of KO were between 0.1 and 0.8 μg/mL. Therefore, our data demonstrates that a METH-induced decrease in cell viability can be
AC
4
PT
to 54.93 ± 4.18, compared with the METH-only treated group (121.45 ± 4.60, P < .01).
rescued by KO.
METH-induced neuronal injury is also related to cell apoptosis, as demonstrated by an increased percentage of apoptotic rates in SH-SY5Y cells and significantly elevated protein expressions of cleaved caspase-3, cleaved PARP and Bax/Bcl-2 in PC12 cells [32-34]. In agreement with these results, our results indicate that cell apoptotic rates and protein levels
ACCEPTED MANUSCRIPT of cleaved caspase-3 increase significantly post METH 24 h treatment in PC12 cells when compared with the control group. However, compared with the METH-only treated group, KO abolishes these apoptotic effects, when the concentration range is 0.2-0.8 μg/mL, which indicates that KO might have a potential protective effect on PC12 cells against
RI
PT
METH-induced apoptosis.
SC
Mitochondrial impairment plays an important role in METH-induced dopaminergic neurotoxicity, which is also correlated with apoptosis [35]. The stability of ∆Ψm is
NU
important in maintaining normal cell function and the reduction of ∆Ψm has a significant
MA
impact on the apoptotic process [36]. Related studies [32, 37] have found that a significant loss of ∆Ψm is detected after METH treatment in PC12 cells, while METH induces
D
mitochondrial dysfunction in the form of a marked decrease in human T cell ∆Ψm. ∆Ψm
PT E
has also been measured by flow cytometry in groups treated with both METH and KO/METH in current research. Our results show that METH significantly increases
CE
depolarization of the mitochondria compared with the control group, which indicates that a
AC
significant dissipation of ∆Ψm is triggered post METH treatment, while KO effectively decreases this effect when the concentrations utilized are between 0.2 and 0.8 μg/mL. This indicates that KO might reduce METH induced PC12 cell apoptosis via a mitochondria-mediated pathway.
Former studies have demonstrated that exposure to METH may lead to oxidative stress [38, 39]. The balance between oxidants and antioxidants most likely plays a vital role in
ACCEPTED MANUSCRIPT both apoptosis and inflammation [40, 41]. Cellular antioxidants include both low-weight molecular and macromolecules, such as GSH and SOD, which are responsible for decreasing concentrations of ROS/RNS, and increasing resistance to neurodegenerative diseases in the central nervous system [42]. Meanwhile, the production of MDA is used to
PT
measure the level of oxidative stress in an organism [43]. The free radical NO plays an
RI
important role in oxidative stress and the mechanism underlying oxidative stress-involved
SC
cell apoptosis [44, 45]. Additionally, KO contains large amount of astaxanthin, which is a powerful antioxidant [46]. Venkatraman found that KO exposed mice have higher liver
NU
SOD and GPX mRNA expression than the lipid-only control group [47]. In addition,
MA
disturbances in the levels of ∆Ψm are also correlated with an excess of ROS production [48]. The current study shows similar results, in that METH exposure decreases SOD and
D
GSH significantly, while increasing MDA content and production of NO and ROS
PT E
significantly, compared with the control group. Generally, KO (0.2, 0.4, and 0.8 μg/mL) reverses the above results in contrast with the METH-only treated group, while the NO
CE
production was decreased significantly in 0.4 μg/mL KO treated group compared with
AC
other KO treated groups after METH exposure, yet the protective effect of KO against oxidative stress after METH treatment in PC12 cells was not in a dose dependent manner.
One limitation of the present study is that the current results illustrate the protective effect of KO in alleviating the apoptotic response and oxidative damage in PC12 cells only in vitro. In the future, animal models will also be applied to our study to comprehensively evaluate the protective effect of KO in the nervous system. In addition, the current results
ACCEPTED MANUSCRIPT did not demonstrate that KO mitigates METH-induced neurotoxicity in PC12 cells in a dose dependent manner, yet we still found that KO (0.1, 0.2, 0.4, 0.8 μg/mL) demonstrates protective effects, by inhibiting apoptosis and oxidative damage, as seen in most of our experimental results. In the future, by modifying the experimental concentrations of KO,
PT
we plan to detect the relationship between KO concentration and its protective effect on
SC
RI
PC12 cells with METH-induced neurotoxicity.
In conclusion, oxidative stress and apoptosis are correlated in the pathophysiology of
NU
many neurodegenerative diseases [49]. Our study found that both apoptosis and oxidative
MA
damage are triggered by 24 h METH treatment in PC12 cells, as evidenced by decreases in cell viability, increases in protein expression of cleaved caspase-3 and apoptotic rates, loss
D
of mitochondrial membrane potential, as well as decreases in SOD and GSH activity,
PT E
elevated MDA content, and decreases in NO and ROS production. Consequently, we demonstrate the hypothesis that KO supplementation might effectively prevent
CE
METH-induced neurotoxicity via the inhibition of apoptotic responses and oxidative
AC
damage. Meanwhile, we try to highlight the potential and specific health benefits of KO. Although the exact mechanism of the anti-apoptotic and antioxidant properties of KO is unclear, the mitochondria-mediated pathway may be involved in these two correlated pathological processes. Therefore, it necessitates further investigation in the future.
ACCEPTED MANUSCRIPT Acknowledgment The authors thank Weiling Li for her helpful manuscript editing assistance and Aker BioMarine Company (Norway) for the kind gift of SuperbaTM Krill Oil in this study. This work was supported by Hubei Provincial Department of Education guidance project
AC
CE
PT E
D
MA
NU
SC
RI
PT
(B2017273). The authors declare no conflicts of interest.
ACCEPTED MANUSCRIPT REFERENCES [1] Panenka WJ, Procyshyn RM, Lecomte T, MacEwan CW, Flynn SW, Honer WC, et al. Methamphetamine use: a comprehensive review of molecular, preclinical and clinical findings. Drug Alcohol Depen 2013;120:167-79.
PT
[2] Wang Q, Wei LW, Xiao HQ, Xue Y, Du SH, Liu YG, et al. Methamphetamine induces
RI
hepatotoxicity via inhibiting cell division, arresting cell cycle and activating apoptosis: In
SC
vivo and in vitro studies. Food Chem Toxicol 2017;105:61-72.
[3] Kita T, Takeshima M, Wagner GC, Hozumi H, Miyazaki I, Asanuma M. New
NU
perspectives on the mechanism of methamphetamine-induced neurotoxicity. Nihon
MA
Shinkei Seishin Yakurigaku Zasshi 2008;28:49–61.
[4] Nara A, Aki T, Funakoshi T, Uemura K. Methamphetamine induces macropinocytosis
D
in differentiated SH-SY5Y human neuroblastoma cells. Brain Res 2010;1352:1-10.
receptor
5,
PT E
[5] Shah A, Silverstein PS, Singh DP, Kumar A. Involvement of metabotropic glutamate AKT/P13K
signaling
and
NF-kappa
B
pathway
in
CE
methamphetamine-mediated increase in IL-6 and IL-8 expression in astrocytes. J
AC
neuroinflamm 2012;9:1-10. [6] Shah A, Kumar S, Simon SD, Singh DP, Kumar A. HIV gp120- and methamphetamine-mediated oxidative stress induces astrocyte apoptosis via cytochrome P450 2E1. Cell death Dis 2013;4:1-15. [7] Roos WP, Thomas AD, Kaina B. DNA damage and the balance between survival and death in cancer biology. Nat Rev Cancer 2016;16:20-33. [8] Callaghan RC, Cunningham JK, Sykes J, Kish SJ. Increased risk of Parkinson’s
ACCEPTED MANUSCRIPT disease in individuals hospitalized with conditions related to the use of methamphetamine or other amphetamine-type drugs. Drug Alcohol Depen 2012;120:35–40. [9] Xu X, Huang E, Tai YC, Zhao X, Chen XB, Chen CX, et al. Nupr1 modulates methamphetamine-induced dopaminergic neuronal apoptosis and autophagy through
PT
CHOP-Trib3-Mediated endoplasmic reticulum stress signaling pathway. Front Mol
RI
Neurosci 2017;10:203-18.
SC
[10] Burri L, Johnsen L. Krill products: An overview of animal studies. Nutrients 2015;7: 3300-21.
NU
[11] Nicol S, Foster J, Kawaguchi S. The fishery for Antarctic krill—recent developments.
MA
Fish Fish 2012;13:30-40.
[12] Ulven SM, Kirkhus B, Lamglait A, Basu S, Elind E, Haider T, et al. Metabolic
D
effects of krill oil are essentially similar to those of fish oil but at lower dose of EPA and
PT E
DHA in healthy volunteers. Lipids. 2011;46:37-46. [13] Berge K, Musa-Veloso K, Harwood M, Hoem N, Burri L. Krill oil supplement
CE
lowers serum triglycerides without increasing low-density lipoprotein cholesterol in
AC
adults with borderline high or high triglyceride levels. Nutr Res 2014;34:126-33. [14] Grimstad T, Bjorndal B, Cacabelos D, Aasprong OG, Janssen EA, Omdal R, et al. Dietary supplementation of krill oil attenuates inflammation and oxidative stress in experimental ulcerative colitis in rats. Scand J Gastroenterol 2012;47:49-58. [15] Banni S, Carta G, Murru E, Cordeddu L, Giordano E, Sirigu AR, et al. Krill oil significantly decreases 2-arachidonoylglycerol plasma levels in obese subjects. Nutr Metab 2011;8:1-6.
ACCEPTED MANUSCRIPT [16] Deutsch L. Evaluation of the effect of Neptune krill oil on chronic inflammation and arthritic symptons. J Am Coll Nutr 2007;26:39-48. [17] Bunea R, EI FK, Deutsch L. Evaluation of the effects of neptune krill oil on the clinical course of hyperlipidemia. Altern Med Rev. 2004;9:420-28.
PT
[18] Wibrand K, Berge K, Messaoudi M, Duffaud A, Panja D, Bramham CR, et al.
RI
Enhanced cognitive function and antidepressant-like effects after krill oil supplementation
SC
in rats. Lipids Health Dis 2013;12:6-18.
[19] Gamoh S. Krill-derived phospholipids rich in n-3 fatty acid improve spatial memory
NU
in adult rats. J Agric Sci-Cambridge 2011;3:3–12.
MA
[20] Polotow TG, Poppe SC, Vardaris CV, Ganini D, Guariroba M, Mattei R, et al. Redox status and neuro inflammation indexes in cerebellum and motor cortex of wistar rats
D
supplemented with natural sources of omega-3 fatty acids and astaxanthin: fish oil, krill
PT E
oil, and Algal biomass. Mar Drugs 2015;13:6117-37. [21] Jayathilake AG, Senior PV, Su XQ. Krill oil extract suppresses cell growth and
AC
328-38.
CE
induces apoptosis of human colorectal cancer cells. BMC complement Alternm 2016;16:
[22] Robertson B, Burri L, Berge K. Genotoxicity test and subchronic toxicity study with superba krill oil in rats. Toxicol Rep 2014;1:764–76. [23] Roth JA, Horbinski C, Higgins D, Lein P, Garrick MD. Mechanisms of manganese-induced rat pheochromocytoma (PC12) cell death and cell differentiation. Neurotoxicology 2002;23:147-57. [24] Meng HZ, Ni XF, Yu HN, Wang SS, Shen SR. Effects of astaxanthin on oxidative
ACCEPTED MANUSCRIPT stress induced by Cu2+ in prostate cells. J Zhejiang Univ Sci b 2002;18:161-71. [25] Reglero MM, Taggart MA, Gonzalez M, Mateo R. Heavy metal exposure in large game from a lead mining area: effects on oxidative stress and fatty acid composition in liver. Environ Pollut 2009;157:1388-95.
PT
[26] Xiong Q, Ru Q, Chen L, Yue K, Tian X, Ma BM, et al. Combined effects of fine
RI
particulate matter and lipopolysaccharide on apoptotic responses in NR8383 macrophages.
SC
J Toxicol Env Heal A 2015;78:43-52.
[27] Ng YW, Say YH. Palmitic acid induces neurotoxicity and gliatoxicity in SH-SY5Y
NU
human neuroblastoma and T98G human glioblastoma cells. Peer J 2018;26:1-21.
MA
[28] Kim D, Song J, Ahn C, Kang Yh, Chun CH, Jin EJ. Peroxisomal dysfunction is associated with up-regulation of apoptotic cell death via miR-223 induction in knee
D
osteoarthritis patients with type 2 diabetes mellitus. Bone 2014;64:124-31.
PT E
[29] Price RJ, Lillycrop KA, Burdge GC. Folic acid supplementation in vitro induces cell type-specific changes in BRCA1 and BRCA2 mRNA expression, but does not alter DNA
CE
methylation of their promoters or DNA repair. Nutr Res 2015;35:532-44.
AC
[30] Barros MP, Poppe SC, Bondan EF. Neuroprotective properties of the marine carotenoid astaxanthin and omega-3 fatty acids, and perspectives for the natural combination of both in krill oil. Nutrients 2014;6:1293-1317. [31] Pitaksalee R, Sanvarinda Y, Sinchai T, Sanvarinda P, Thampithak A, Jantaratnotai N, et al. Autophagy inhibition by caffeine increases toxicity of methamphetamine in SH-SY5Y neuroblastoma cell line. Neurotox Res 2015;27:421-29. [32] Tian X, Ru Q, Xiong Q, Yue K, Chen L, Ma BM, et al. Neurotoxicity induced by
ACCEPTED MANUSCRIPT methamphetamine-heroin combination in PC12 cells. Neurosci Lett 2017;647:1-7. [33] Huang WY, Xie WB, Qiao DF, Qiu PM, Huang EP, Li B, et al. Caspase-11 plays an essential role in methamphetamine-induced dopaminergic neuron apoptosis. Toxicol Sci 2015;145:68-79.
PT
[34] Wongprayoon P, Govitrapong P. Melatonin protects SH-SY5Y neuronal cells against
RI
methamphetamine-induced endoplasmic reticulum stress and apoptotic cell death.
SC
Neurotox Res 2017;31:1-10.
[35] Thrash-Williams B, Ahuja M, Karuppagounder SS, Uthayathas S, Suppiramaniam V,
NU
Dhanasekaran M. Assessment of therapeutic potential of amantadine in methamphetamine
MA
induced neurotoxicity. Neurochem Res 2013;38:2084-94. [36] Green DR, Reed JC. Mitochondria and apoptosis. Science 1998;281:1309-12.
D
[37] Potula R, Hawkins BJ, Cenna JM, Fan SS, Dykstra H, Ramirez SH, et al.
PT E
Methamphetamine causes mitochondrial oxidative damage in human T lymphocytes leading to functional impairment. J Immunol 2010;185:2867-76.
CE
[38] Buonocore G, Perrone S, Tataranno ML. Oxygen toxicity: Chemistry and biology of
AC
reactive oxygen species. Semin Fetal Neonatal Med 2010;15:186–90. [39] Wells PG, McCallum GP, Lam KC, Henderson JT, Ondovcik SL. Oxidative DNA damage and repair in teratogenesis and neurodevelopmental deficits. Birth Defects Res C 2010;90:103–9. [40] Conim A, Alves-Pereira, I, Ferreira R. Important role of antioxidant enzymes in the saccharomyces cerevisiae survival to the toxicity of vanadium pentoxide. Toxicol Lett 2010;196:304-10.
ACCEPTED MANUSCRIPT [41] Ni S, Wang D, Qiu X, Pang L, Song Z, Guo K. Bone marrow mesenchymal stem cells protect against bleomycin-induced pulmonary fibrosis in rat by activating Nrf2 signaling. Int J Clin Exp Pathol 2015;8:7752-61. [42] Koutsilieri E, Scheller C, Tribl F, Riederer P. Degeneration of neuronal cells due to oxidative
PT
stress-Microglial contribution. Parkinsonism Relat D 2002;8:401-6.
RI
[43] Rio DD, Stewart AJ, Pellegrini N. A review of recent studies on malondialdehyde as toxic
SC
molecule and biological marker of oxidative stress. Nutr Metab Cardiov AS 2005; 15:316-28. [44] Albina JE, Cui S, Mateo RB, Reichner JS. Nitric oxide-mediated apoptosis in murine
NU
peritoneal macrophages. J Immunol 1993;150:5080–85.
MA
[45] Jothiramajayam M, Sinha S, Ghosh M, Nag A, Jana A, Makherjee A. Sodium fluoride promotes apoptosis by generation of reactive oxygen species in human lymphocytes. J Toxicol
D
Environ Health A 2014;77:1269–80.
PT E
[46] Grynbaum MD, Hentschel P, Putzbach K, Rehbein J, Krucker M, Nicholson G, et al. Unambiguous detection of astaxanthin and astaxanthin fatty acid esters in krill (Euphausia superb
CE
Dana). J Sep Sci 2005;28:1685-93.
AC
[47] Venkatraman JT, Chandrasekar B, Kim JD, Fernandes G. Effects of n-3 and n-6 fatty acids on the activities and expression of hepatic antioxidant enzymes in autoimmune-prone NZB × NZW F1 mice. Lipids 1994;29:561–68. [48] Radi E, Formichi P, Battisti C, Federico A. Apoptosis and oxidative stress in neurodegenerative disease. J Alzheimers Dis 2014;42:125-52. [49] Sinha K, Das J, Pal PB, Sil PC. Oxidativestress: The mitochondria-dependent and mitochondriain dependent pathways of apoptosis. Arch Toxicol 2013;87:1157-80.
ACCEPTED MANUSCRIPT Figure Legends
Figure 1. Effect of krill oil on cell viability of PC12 cells post METH treatment.
Cells were treated with MA (3 mM) /MA + krill oil (0.1, 0.2, 0.4, 0.8 μg/mL) for 24 h, and then cells were
PT
further incubated with MTT (0.5 mg/ml) for 4 h in the 37 ˚C incubator at dark. The cell viability was evaluated by MTT reduction assay. Values are means ± SEM (n = 9). Data were analyzed by one way
RI
ANOVA followed using a post hoc test of Tukey. Statistical significance: ##P < .01 compared to the
SC
control group, **P < .01 compared to the METH group.
NU
Figure 2. Effect of krill oil on NO Production in PC 12 cells after METH exposure.
MA
Cells were treated with MA (3 mM) /MA + krill oil (0.1, 0.2, 0.4, 0.8 μg/mL) for 24 h, the supernatant from each group was collected, and then all the samples were further incubated with Griess Reagent I and Griess Reagent II reagents (50 μL for each reagent) to determine the production of NO. Values are means ±
D
SEM (n = 9). Data were analyzed by one way ANOVA followed using a post hoc test of Tukey. Statistical
PT E
significance: ##P < .01 compared to the control group, **P < .01 compared to the METH group.
CE
Figure 3. Effect of krill oil on ROS Production in PC 12 cells after METH exposure
AC
Cells were treated with MA (3 mM) /MA + krill oil (0.1, 0.2, 0.4, 0.8 μg/mL) for 24 h, the probe DCFH-DA (100 μmol/L) was incubated with cells for 1 h in a 37 ˚C and 5% (v/v) CO2 incubator, and then the fluorescent signal was obtained to evaluate the intracellular ROS level in different groups. Values are means ± SEM (n = 9). Data were analyzed by one way ANOVA followed using a post hoc test of Tukey. Statistical significance: ##P < .01 compared to the control group, **P < .01 compared to the METH group.
ACCEPTED MANUSCRIPT Figure 4. Effect of krill oil on apoptosis response in PC 12 cells after METH exposure. Cells were treated with MA (3 mM) /MA + krill oil (0.1, 0.2, 0.4, 0.8 μg/mL) for 24 h and analyzed by Muse Annexin V & Dead Cell assay. A representative dot plots in live, dead, late apoptotic/dead and early apoptotic phase is shown in the upper panel, and the mean percentage of the cell death is expressed by a histogram in the lower panel. Values are means ± SEM (n = 9). Data were analyzed by one way ANOVA followed using a post hoc test of Tukey. Statistical significance: ##P < .01 compared to the control group,
PT
*P < .05 compared to the METH group.
RI
Figure 5. Effect of krill oil on mitochondrial membrane potential in PC 12 cells after
SC
METH exposure.
NU
Cells were treated with MA (3 mM) /MA + krill oil (0.1, 0.2, 0.4, 0.8 μg/mL) for 24 h and analyzed by Muse MitoPotential assay. A representative dot plots in live, depolarized/live, depolarized/dead and dead phase is shown in the upper panel, and the mean percentage of the total depolarization is expressed by a
MA
histogram in the lower panel. Values are means ± SEM (n = 9). Data were analyzed by one way ANOVA followed using a post hoc test of Tukey. Statistical significance: #P < .05 compared to the control group, *P
PT E
D
< .05 compared to the METH group.
Figure 6. Effect of krill oil on the protein expression of cleaved caspase 3 in PC 12 cells
AC
CE
after METH exposure.
Cell extracts from PC12 cells treated with MA (3 mM) /MA + krill oil (0.1, 0.2, 0.4, 0.8 μg/mL) for 24 h and analyzed by Western Blot with anti-cleaved caspase 3 (1:500) and anti-β-actin (1:1000) antibodies (A). Protein data values were normalized to actin level and reported in relative protein levels of cleaved caspase 3 with a histogram (B). Values are means ± SEM (n = 5). Data were analyzed by one way ANOVA followed using a post hoc test of Tukey. Statistical significance: #P < .05 compared to the control group, **P < .01 compared to the METH group.
ACCEPTED MANUSCRIPT Table 1 The main compositions and analytical specifications of krill oil LIMITS ≥40 ≥5 ≥22
UNIT g/100g g/100g g/100g
EPA
≥12
g/100g
DHA
≥5.5
g/100g
Astaxanthin esters
≥100
μg astx-diol/g oil3
Krill Oil’s specification is provided by Aker BioMarine Company (Norway)
SC NU MA D PT E CE AC
The superba
RI
TM
PT
COMPOSITION Total phospholipids Choline Total omega-3 fatty acids
ACCEPTED MANUSCRIPT Table 2 Effect of krill oil (KO) on the oxidative enzyme system (MDA content, SOD activity and GSH activity) in PC 12 cells after METH exposure SOD (U/mg protein) 73.20±1.46 54.50±1.01##
GSH (μmol/g protein)
1.52±0.06**
78.30±4.92**
49.30±2.18*
1.28±0.10**
79.10±3.52**
1.32±0.04**
70.03±5.88*
51.57±2.06**
1.37±0.06**
76.87±4.65**
49.10±0.92**
RI
PT
45.98±1.07 40.98±0.50#
49.23±0.54**
SC
Control METH METH+KO (0.1μg/ml) METH+KO (0.2μg/ml) METH+KO (0.4μg/ml) METH+KO (0.8μg/ml)
MDA (nmol/mg protein) 1.27±0.02 3.03±0.03##
NU
Group
AC
CE
PT E
D
MA
Cells were treated with MA (3 mM) /MA + KO (0.1, 0.2, 0.4, 0.8 μg/mL) for 24 h, then cell samples were collected for measurements of the levels of MDA, GSH and SOD using corresponding commercial detection kits. For each individual measurement, values are means ± SEM (n = 9). Data were analyzed by one way ANOVA followed using a post hoc test of Tukey. Statistical significance: ## P < .01, compared to the control group, # P < .05, compared to the control group, ** P < .01, compared to the METH group, * P < .05, compared to the METH group.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6