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Food Safety and Toxicology
Retardation of axonal and dendritic outgrowth is associated with MAPK signaling pathway in offspring mice following maternal exposure to nano titanium dioxide Yingjun Zhou, Jianhui Ji, Chunmei Chen, and Fashui Hong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06992 • Publication Date (Web): 31 Jan 2019 Downloaded from http://pubs.acs.org on February 1, 2019
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Journal of Agricultural and Food Chemistry
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Retardation of axonal and dendritic outgrowth is associated with
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MAPK signaling pathway in offspring mice following maternal
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exposure to nano titanium dioxide
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Yingjun Zhou1, 2, 3, 4, #, Jianhui J1, 2, 3, 4, #, Chunmei Chen1, 2, 3, 4, Fashui Hong1, 2, 3, 4*
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1
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Environmental Protection, Huaiyin Normal University, Huaian 223300, China
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2 Jiangsu
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Normal University, Huaian 223300, China
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3 Jiangsu
Jiangsu Collaborative Innovation Center of Regional Modern Agriculture &
Key Laboratory for Food Safety and Nutrition Function Evaluation, Huaiyin
Key Laboratory for Eco-Agricultural Biotechnology around Hongze Lake,
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Huaiyin Normal University, Huaian 223300, China
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4 School
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#Y.J,
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* Corresponding author, E-mail:
[email protected] of Life Sciences, Huaiyin Normal University, Huaian 223300, China
and J.H contributed equally to this work.
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Abstract
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Exposure to nano titanium oxide (TiO2) has been proven to suppress brain growth in
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mouse offspring; however, whether retardation of axonal or dendritic outgrowth is
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associated with activation of the mitogen-activated protein kinase (MAPK) pathway
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remains unclear. In the present study, pregnant mice were exposed to nano-TiO2 at
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1.25, 2.5 and 5 mg/kg body weight, respectively, and the molecular mechanism of
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axonal or dendritic outgrowth retardation was investigated. The results suggested that
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nano-TiO2 crossed the blood-fetal barrier and blood-brain barrier and deposited in the
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brain of offspring, which retarded axonal and dendritic outgrowth, including the
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absence of axonal outgrowth, and decreased dendritic filament length, dendritic
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branching number and dendritic spine density. Importantly, maternal exposure to
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nano-TiO2 increased phosphorylated (p)-extracellular signal-regulated kinase1/2
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(ERK1/2, +24.35% to +59.4%), p-p38 (+60.82% to 181.85%) and p-c-jun N-terminal
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kinase (JNK, +28.28% to 97.28%) expression in offspring hippocampus. These
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findings suggested that retardation of axonal and dendritic outgrowth in mouse
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offspring caused by maternal exposure to nano-TiO2 may be related to excessive
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activation of the ERK1/2/MAPK signaling pathway. Therefore, the potential toxicity
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of nano-TiO2 is a concern, especially in pregnant woman or children who are exposed
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to nano-TiO2.
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Key words: Nano titanium dioxide; Offspring mice, Axonal outgrowth; Dendritic
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outgrowth; MAPK signaling pathway
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INTRODUCTION
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Nano-TiO2 particles have been widely used in the food industry as they can improve
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food freshness and quality, and can trace and detect pathogens or contaminants.
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However, the human health problems related to nano-TiO2 particles cannot be ignored.
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3, 4
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1, 2
Many studies have shown that nano-TiO2 can reach the brain and induce 4-7
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neurotoxicity.
For example, nano-TiO2 can penetrate the blood-brain barrier and
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enter the central nervous system (CNS), resulting in oxidative stress in nerve cells,
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secretion of toxic factors, and nerve function damage. 4, 8 Nano-TiO2 also disrupts the
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pathway of glutamate metabolism, leading to decreases in animal memory and spatial
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cognitive ability.
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long-term exposure to such substances during pregnancy or lactation will inevitably
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affect fetal and infant CNS development.
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involves a period of cell division and differentiation, and fetal immune capacity is
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very low. In addition, the fetus is more vulnerable to external toxic substances than
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adults, and CNS development can be retarded and damaged.
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to nano-TiO2 has been shown to lead to neurotoxicity and impairments in learning,
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memory and behavior in offspring. 14-18 Recently, our research team demonstrated that
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maternal exposure to nano-TiO2 resulted in nano-TiO2 crossing the blood-brain
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barrier and blood-placental barrier, and depositing in the brain of offspring leading to
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apoptosis and excessive autophagy of neurons, upregulation of the expression of
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related factors, downregulation of dendritic development-related protein expression
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Nano-TiO2 is widely used in maternal and infant products, and
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It is well-known that fetal growth
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Maternal exposure
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and inhibition of brain development. 19-21 However, it has not been confirmed whether
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inhibition of axonal and dendritic outgrowth induced by maternal exposure to
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nano-TiO2 involves regulation by the ERK1/2/MAPK signaling pathway.
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It is well-known that the neuron is the basic unit in the structure and function of
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the hippocampus. The axon usually needs to grow and extend a long distance to reach
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its dominant position. Dendrites on the neuron also produce complex dendritic fields
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to receive and integrate multiple signal inputs. In order to function properly, the
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hippocampus needs to establish correct synaptic connections between neurons. The
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branching pattern of the dendrite determines the number of axons and synapses it
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receives from neurons in different brain regions. Therefore, the complex morphology
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of dendrites is the structural basis of dendritic function, and the mechanism of axonal
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and dendritic outgrowth is an important topic in the study of dendritic function. The
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ERK1/2/MAPK signaling pathway is an important signaling pathway during CNS
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development. As suggested, extracellular signal-regulated kinase (ERK) plays a
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regulatory role in the formation and changes in dendritic structure of hippocampal
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neurons.
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N-terminal kinase (JNK) and p38 MAPK. In the CNS, ERK1/2 is the center of many
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signaling pathways in the MAPK cascade, which is mainly activated by growth
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factors and is associated with cell proliferation, differentiation and development,
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whereas JNK and p38MAPK are preferentially activated by various environmental
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stresses and inflammatory cytokines, which in turn induce neuronal cell death.
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Therefore, we hypothesized that the retardation of axonal and dendritic outgrowth of
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Mitogen-activated protein kinases (MAPKs) include ERK1/2, c-jun
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neurons in offspring hippocampus induced by maternal exposure to nano-TiO2 may be
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related to activation of the ERK1/2/MAPK signaling pathway.
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Accordingly, in the present study, different doses (1.25, 2.5, 5 mg/kg body
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weight [bw]) of nano-TiO2 were used to continuously expose maternal mice from
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prenatal day 7 to postnatal day (PND) 21, who stopped lactation 21 days after delivery,
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and the changes in hippocampal morphology and dendritic development were
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observed under an optical microscope. The expression of ERK1/2/MAPK signaling
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pathway-related factors in the hippocampus was examined to determine the
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mechanism of axonal and dendritic outgrowth retardation induced by maternal
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exposure to nano-TiO2.
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MATERIALS AND METHODS
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Chemicals
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Nano-TiO2 (TiO2 content >99.5%) was donated by Professor Yang Ping (College of
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Chemistry, Soochow University, China) and was characterized in our previous reports.
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36-38
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hydrodynamic diameter, 174.8 m2/g surface area, and 43.7 mV Zeta potential.
Nano-TiO2 featured an anatase phase, 6.5 nm particle size, 45.04 nm
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Animals and treatment
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CD-1ICR pregnant mice (Specific Pathogen-Free, SPF) were purchased from the
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Animal Center of Suzhou University (Suzhou, China), and were confined in the same
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room in separate cages, where they were allowed free access to water and sterilized
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food. The pregnant mice were randomly divided into four subgroups (5 mice in each
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group),
with
0.5%
w/v
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hydroxypropylmethylcellulose (HPMC) (Sigma-Aldrich, St. Louis, MO, USA) and
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the three experimental groups were treated with 1.25, 2.5 and 5 mg/kg bw nano-TiO2
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suspensions, and the dose selection was made according to our previous studies. 39 All
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mice were weighed, and the 0, 1.25, 2.5, and 5 mg/kg bw nano-TiO2 suspensions were
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administered to mice via intragastric feeding every day from prenatal day 7 to PND
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21. Offspring animals were separated from their mothers at PND 21 and maintained in
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cages in an isolated animal room at 60±10% relative humidity, a 12 h light/dark cycle,
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and the room temperature was set at 24±2°C. All animal experiments were approved
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by the Animal Experimental Committee of Soochow University (Ethical approval
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number 2111270).
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Preparation of brain and hippocampus
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After feeding, all offspring animals were weighed and cervical dislocation was
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performed following anesthesia by intravenous injection of pentobarbital sodium 80
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mg/kg bw. The brain was removed, weighed, and the hippocampus then removed. The
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brain coefficient was calculated as the ratio of brain (wet weight, mg) versus body
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weight (g).
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Assay of titanium content
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Approximately 25 mg of offspring brain tissues were digested and their titanium
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contents were measured using inductively coupled plasma-mass spectrometry
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(ICP-MS; Thermo Elemental X7; Thermo Electron Co., Waltham, MA, USA). 34
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Observation of hippocampal and dendritic morphology
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To determine pathologic morphology and axonal outgrowth, hippocampi (5 from each
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group) were embedded in paraffin blocks, which were then sliced (5 μm), placed onto
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glass slides and stained using hematoxylin-eosin. The stained sections were examined
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by a histopathologist, using an optical microscope (Nikon U-III Multi-point Sensor
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System, Japan).
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To determine dendritic outgrowth, Golgi-Cox staining was used to evaluate 40
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dendritic morphology, according to Gibb’s method.
Offspring hippocampi (5 from
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each group) were suspended in Golgi-Cox solution. After 14 days, all hippocampi
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were removed at room temperature and in darkness, and then immersed in 30%
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sucrose solution for 5 days with vibration. The hippocampal tissues were sliced (60
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μm) and then developed and mapped using the floating method, and rinsed with
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distilled water for 10 min and then mounted, dried, dehydrated and sealed. Finally,
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according to the method described in a previous study, 41 images were observed and
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collected, and dendritic morphology and outgrowth were assessed using an Olympus
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BX-51 microscope equipped with a computer-controlled motorized stage and
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Neurolucida software (MBF Biosciences, Williston, VT, USA).
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Western blot assay
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Proteins in offspring hippocampi were extracted using a Cell Lysis Kit (Thermo
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Fisher Scientific, USA) and their concentrations were measured with the BCA protein
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assay kit (Thermo Fisher Scientific Inc., IL, USA). Western blot assays were
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performed as described previously.19-21 Primary antibodies including anti–ERK1/2
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(1:2000), anti-p38 (1:1000), anti-JNK (1:1000), anti-p-ERK1/2 (1:2000), and
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anti-p-JNKp54/p46 (1:2000) were obtained from Thermo Fisher Scientific Inc. (IL,
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USA), and anti-p-p38 (1:1000) was obtained from Abcam Trading (Shanghai)
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Company Ltd., Shanghai, China). The expression of β-actin, as the loading control,
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was detected using an anti--actin antibody (1:5000, Thermo Fisher Scientific Inc., IL,
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USA).
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Statistical analysis
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All the results were based on at least triplicate experiments (five or more repetitions
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per experimental group) and expressed as mean ± standard deviation (SD). SPSS 19.0
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(SPSS, Chicago, IL, USA) was used to analyze multiple sets of data. One way
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ANOVA was used to determine statistical significance. The data were considered
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statistically significant at p
