Reactive oxygen species mediate nitric oxide production through ERK/JNK MAPK signaling in HAPI microglia after PFOS exposure Cheng Wang, Xiaoke Nie, Yan Zhang, Ting Li, Jiamin Mao, Xinhang Liu, Yiyang Gu, Jiyun Shi, Jing Xiao, Chunhua Wan, Qiyun Wu PII: DOI: Reference:
S0041-008X(15)30019-3 doi: 10.1016/j.taap.2015.06.012 YTAAP 13402
To appear in:
Toxicology and Applied Pharmacology
Received date: Revised date: Accepted date:
9 January 2015 6 June 2015 12 June 2015
Please cite this article as: Wang, Cheng, Nie, Xiaoke, Zhang, Yan, Li, Ting, Mao, Jiamin, Liu, Xinhang, Gu, Yiyang, Shi, Jiyun, Xiao, Jing, Wan, Chunhua, Wu, Qiyun, Reactive oxygen species mediate nitric oxide production through ERK/JNK MAPK signaling in HAPI microglia after PFOS exposure, Toxicology and Applied Pharmacology (2015), doi: 10.1016/j.taap.2015.06.012
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 Reactive oxygen species mediate nitric oxide production through ERK/JNK MAPK signaling in HAPI
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microglia after PFOS exposure
Cheng Wang , Xiaoke Nie1, Yan Zhang1, Ting Li2, Jiamin Mao2, Xinhang Liu3, Yiyang Gu2, Jiyun Shi2, Jing Xiao3, Chunhua Wan1, Qiyun Wu1*
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*Correspondence to: Qiyun Wu, Department of Nutrition and Food Hygiene, School of Public Health, Nantong University, Nantong, Jiangsu 226001, People’s Republic of China. Email: [email protected] Phone: +86-0513-85012177 Fax: +86-0513-85012180
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1 Department of Nutrition and Food Hygiene, School of Public Health, Nantong University, Nantong, Jiangsu 226001, People’s Republic of China
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2 Department of Labor and Environmental Hygiene, School of Public Health, Nantong University, Nantong, Jiangsu 226001, People’s Republic of China
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3 Department of Occupational Medicine and Environmental Toxicology, School of Public Health, Nantong University, Nantong, Jiangsu 226001, People’s Republic of China
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ACCEPTED MANUSCRIPT Abstract
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Perfluorooctane sulfonate (PFOS), an emerging persistent contaminant that is commonly encountered during daily life, has been shown to exert toxic effects on the central nervous system (CNS). However, the molecular mechanisms underlying the neurotoxicity of PFOS remains largely unknown. It has been widely acknowledged that the inflammatory mediators released by hyper-activated microglia play vital roles in the pathogenesis of various neurological diseases. In the present study, we examined the impact of PFOS exposure on microglial activation and the release of proinflammatory mediators, including nitric oxide (NO) and reactive oxidative species (ROS). We found that PFOS exposure led to concentration-dependent NO and ROS production by rat HAPI microglia. We also discovered that there was rapid activation of the ERK/JNK MAPK signaling pathway in the HAPI microglia following PFOS treatment. Moreover, the PFOS-induced iNOS expression and NO production were attenuated after the inhibition of ERK or JNK MAPK by their corresponding inhibitors, PD98059 and SP600125. Interestingly, NAC, a ROS inhibitor, blocked iNOS expression, NO production, and activation of ERK and JNK MAPKs, which suggested that PFOS-mediated microglial NO production occurs via a ROS/ERK/JNK MAPK signaling pathway. Finally, by exposing SH-SY5Y cells to PFOS-treated microglia-conditioned medium, we demonstrated that NO was responsible for PFOS-mediated neuronal apoptosis.
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PFOS; microglia; ROS; iNOS; NO; ERK/JNK MAPK
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ACCEPTED MANUSCRIPT Introduction
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Perfluorooctane sulfonate (PFOS), an emerging pollutant, has been found to accumulate in both wildlife and humans due to its extensive use and extraordinary stability (J. P. Giesy and K. Kannan, 2001; F. Xiao et al., 2014). PFOS has been shown to have multiple toxic effects, including immunotoxicity, hepatotoxicity, developmental toxicity, and carcinogenicity (S. Fuentes et al., 2006; J. L. Butenhoff et al., 2012; E. Corsini et al., 2012; M. R. Qazi et al., 2013). In addition, accumulating evidence suggests that PFOS exposure can have adverse effects on the central nervous system (CNS), such as neuroendocrine dysfunction and neurobehavioral abnormalities (M. E. Austin et al., 2003; N. Onishchenko et al., 2011). To date, the majority of the studies have focused primarily on neurons. However, the effects of PFOS on microglia and the inflammatory response following PFOS exposure should also be addressed. In this study, we examined the underlying molecular mechanisms by which PFOS induced inflammatory responses in HAPI microglial cells. Microglial cells are resident phagocytic cells of the CNS that are primarily involved in immune surveillance. Once activated by injury, ischemia or inflammatory stimuli, microglial cells display macrophage-like characteristics, including the production of pro-inflammatory cytokines, phagocytosis and antigen presentation (G. A. Garden and T. Moller, 2006). However, not only do the uncontrolled and sustained production of inflammatory cytokines by microglia trigger neuronal cell death (A. M. Kaindl et al., 2012), but they also signal to astrocytes, thereby amplifying their inflammatory response and leading to more injurious accumulation of neurotoxins in the CNS tissues (D. van Rossum and U. K. Hanisch, 2004). Increasing evidence has shown that activated microglia and the attendant neurotoxic substances, including nitric oxide (NO), cause neuronal death (V. L. Dawson et al., 1994; Y. Li et al., 2013). NO, which is synthesized via nitric oxide synthases (NOSs), is involved in various pathological and physiological processes in the CNS (V. Calabrese et al., 2007). The NOSs have three family members, termed inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS) (O. W. Griffith and D. J. Stuehr, 1995). It was reported that in response to a variety of stimuli, inflammatory and immune cells mainly express iNOS (C. Nathan and Q. W. Xie, 1994). A recent study suggested that activated microglia kill neurons through iNOS as a result of the release of NO (A. Bal-Price and G. C. Brown, 2001). It has also been shown that PFOS activates the inflammatory response in astrocytes (H. C. Zeng et al., 2011) and mediates the expression of iNOS in human–hamster hybrid cells (X. Wang et al., 2013). Therefore, we considered that it was worth exploring whether PFOS exposure could activate microglia and induce iNOS expression. The mitogen-activated protein kinases (MAPKs) are classified into three components: P38 kinase, extracellular signal-regulated kinases ERK1/2 (P44/P42) and c-Jun amino-terminal kinase JNK (P46/P54), which have been reported to be involved in the release of immune-related cytotoxic factors, such as NO, COX-2, IL-1βand TNF-αwhen activated by various pro-inflammatory signals (Y. Choi et al., 2009; L. Yan et al., 2013). Some studies have reported that PFOS can induce the phosphorylation of all members of the MAPK family in zebrafish embryos and cerebellar granule cells (X. Shi and B. Zhou, 2010; Y. J. Lee et al., 2013). However, very few studies have addressed the role of MAPK signaling in activated microglia following PFOS exposure. Reactive oxygen species (ROS), including superoxide anion (.O2-), hydroxyl free radical (.OH) and hydrogen peroxide (H2O2), can act as second messengers and play a vital role in the maintenance 3
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of many physiological functions. However, the overproduction of ROS is detrimental to organisms. For example, an excess of ROS production was recently reported to contribute directly to neuronal damage and neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases (K. W. Zeng et al., 2010). Furthermore, substantial ROS production is involved in controlling the expression of several inflammatory mediators. ROS have been shown to act as a signal initiation molecule to enhance NO production (H. Eguchi et al., 2011). Mounting evidence suggests that MAPKs play a role as a bridge in the ROS-mediated production of NO (S. U. Kim et al., 2013). Oxidative stress has been shown to contribute to the neurotoxicity following PFOS exposure (C. Liu et al., 2007). Thus, we thought that it would be intriguing to explore whether PFOS exposure could induce ROS production, as well as ROS-dependent MAPK activation. In the present study, we explored the molecular mechanisms underlying the PFOS-mediated inflammatory response in HAPI microglia. We demonstrated that PFOS-mediated NO production via iNOS was associated with ERK/JNK activation, which was dependent on ROS production. In addition, we also detected the effects of the conditioned medium from PFOS-treated HAPI microglial cells treated with or without a NOS inhibitor (L-NMMA) on SH-SY5Y cells. We found that L-NMMA enhanced the cell viability and attenuated PFOS-induced apoptosis. In summary, our results demonstrated that ROS-mediated NO production was dependent on ERK/JNK activation following PFOS treatment, and that NO was directly responsible for PFOS-mediated neurotoxicity.
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Materials and Methods
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Chemicals The following chemicals were purchased from their suppliers: PFOS (potassium salt, purity 98%) (Sigma–Aldrich, Switzerland); PD98059 (Sigma–Aldrich, St. Louis, MO, USA); SP600125 (Sigma–Aldrich, St. Louis, MO, USA); TUNEL, In Situ Cell Death Detection Kit (Roche Applied Science, Mannheim, Germany); NG-monomethyl-L-arginine (L-NMMA; Dojindo Chemical, Kumamoto, Japan) and N-acetyl-L-cysteine (NAC; Sigma, St. Louis, MO, USA). Cell culture and treatment Rat HAPI microglial cells, a MG-like rat cell line which was obtained from Professor Qin Shen at Nantong University, Jiangsu Province, China (P. Cheepsunthorn et al., 2001; Z. Cui et al., 2010), were propagated in Dulbecco's modified Eagle's medium(DMEM; Invitrogen, Grand Island, NY, USA) containing 10% fetal bovine serum (Sigma–Aldrich, St. Louis, MO, USA), 100μg/ml streptomycin and 100U/ml penicillin at 37 °C in a humidified incubator with 5% CO2/95% air. The HAPI microglial cells were treated with different concentrations of PFOS (0.1, 1, 5, 10, 20, 50, 100, 200 nM) dissolved in DMSO for 6 h or with 20 nM PFOS for 0, 1, 3, 6, 8 or 12 h. Cultures containing only 0.05% DMSO were used as a control. For assays of ERK/JNK MAPK inhibition, the cells were pre-treated with an ERK inhibitor (PD98059) or JNK inhibitor (SP600125) for 60 min, followed by PFOS treatment for 6 h. Before being treated with PFOS or DMSO, the cells were pre-incubated with serum-free medium. After their indicated treatments, the cells and remaining medium were harvested and stored at –20 °C until use. The SH-SY5Y cell line was derived from human brain neuroblastoma (R. A. Ross et al., 1983) and was maintained at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4
ACCEPTED MANUSCRIPT 100U/ml penicillin, 100μg/ml streptomycin and 10% fetal bovine serum (Sigma–Aldrich, St. Louis, MO, USA) in a humidified incubator with 5% CO2/95% air.
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Measurement of NO The production of NO was determined by measuring a stable oxidative end-product of NO, nitrite, in culture supernatant fluid with the Griess reagents (1% sulphanilamide, 5% phosphoric acid,
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and 0.1% naphthylethylenediamine). Briefly, 100μl of supernatant fluid from stimulated HAPI cells was mixed with the same volume of Griess reagents in a 96-well plate. Ten minutes later, the absorbance of every well was measured using an automated microplate reader at 540 nm. The concentration of nitrite was calculated in accordance with a nitrite standard curve based on sodium nitrite. Culture medium only was used as the blank control, and each experiment was performed in triplicate.
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Measurement of cellular reactive oxygen species (ROS) The intracellular ROS generation was measured using a fluorescent marker, DCFH-DA (Genmed, Arlington, MA, USA). Briefly, after the indicated treatments, cells seeded on glass coverslips were washed three times with serum-free DMEM and incubated with DCFH-DA at 37 °C for 30 min. Subsequently, the nuclei were counterstained with Hoechst-33258 (Invitrogen, 1:1000). After being washed, the cells were immediately examined using a fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany). The level of ROS fluorescence in each group was determined using a flow cytometric analysis. The experiments were repeated in triplicate.
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Cell viability assay Cell viability was determined by the Cell Counter Kit-8 (CCK-8) assay (Dojindo Laboratories, Japan). Briefly, SH-SY5Y cells were seeded at a density of 5 × 103 cells/well in 96-well plates, and then were incubated at 37 ◦C with conditioned medium from PFOS-treated HAPI microglial cells for 24 h. Thereafter, 10μl of CCK-8 solution was added to every well, then the cells were incubated for 2 h in an incubator, and the absorbance was determined using a quantified microplate reader. All results were confirmed by replication in at least three independent experiments. Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling (TUNEL) assay The TUNEL assay was applied to determine the level of apoptosis by demonstrating apoptotic bodies in the SH-SY5Y cells using the In Situ Cell Death Detection Kit (Roche Applied Science, Mannheim, Germany). Briefly, before being fixed with 4% formaldehyde and incubated at room temperature for 40 min, SH-SY5Y cells were exposed to PFOS-induced HAPI microglial cells’ conditioned medium for 24 h. To guarantee that there were constant concentrations of NO, the PFOS-treated HAPI microglial cells’ conditioned medium was refreshed every 8 h. After several rinses in phosphate-buffered saline (PBS) and permeabilization in 0.1% Triton X-100 solution on ice for 5 min, 50 μl of TUNEL reaction mixture was added to the cells on coverslips, which were incubated for 60 min at 37 ◦C in a dark humidified chamber. Thereafter, the cells were rinsed three times in PBS, followed by incubation with Hoechst for 20 min at room temperature. Apoptotic cells were detected as localized bright green cells (positive cells) by a fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany). 5
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Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated from HAPI microglial cells treated with DMSO or PFOS using TRIzol reagent (Invitrogen, Shanghai, China) in accordance with the manufacturer’s instructions. Extracted RNA was reverse transcribed for cDNA synthesis using an Omniscript RT kit (Qiagen, Germany) in accordance with the manufacturer’s protocol. PCR amplifications were performed with specific primers according to standard methods by Taq DNA polymerase. The primers sequences used were: iNOS: forward primer, 5’-GCTTTGTGCGGAGTGTCAGT-3’; reverse primer, 5’-CCTCCTTTGAGCCCTCTGTG-3’; GAPDH: forward primer, 5’-TGATGACATCAAGAAGGTGGTGAAG-3’; reverse primer, 5’-TCCTTGGAGGCCATGTGGGCCAT-3’. The amplification steps for iNOS were 30 cycles of 30 s at 94 ◦C, 1 min at 60 ◦C, and 72 ◦C for 1 min. The amplification steps for GAPDH were 30 cycles of 30 s at 94 ◦C, 30 s at 55 °C and 30 s at 72 ◦C. The PCR products for each sample were analyzed by electrophoresis in a 1.5% agarose gel. Gels were stained with ethidium bromide and analyzed under ultraviolet light. The intensity of the mRNA expression was measured by densitometry and then corrected with the corresponding GAPDH mRNA measurement.
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Western blot analysis After the indicated treatments, cells were washed with ice-cold PBS, lysed with lysis buffer for 1 min on ice, and subsequently thoroughly scraped with a cell scraper to remove them from the surface. Then, the protein samples were centrifuged at 13,000 rpm at 4 °C for 15 min. The resulting supernatants were transferred to new tubes, and the protein concentration was determined using a BCA protein assay kit (Thermo Scientific). Thereafter, equal amounts of protein were separated by 10% or 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, USA). To block non-specific binding, the membranes were immersed in 5% non-fat dry milk or bovine serum albumin (BSA) in PBS containing 0.5% Tween 20 (PBS-T), followed by incubation with one of the following primary antibodies at 4°C overnight or at room temperature for 8 h. The rabbit anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:1000) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); the rabbit monoclonal anti-rat iNOS (1:800), rabbit monoclonal anti-rat p38 (1:1000), rabbit anti-rat phospho-p38 (1:1000), rabbit anti-rat JNK (1:1000), rabbit anti-rat phospho-JNK (1:1000), rabbit anti-rat phosphor-ERK (1:1000), and rabbit anti-rat ERK (1:1000) were obtained from Cell signaling (MA, USA). The membranes were then washed with PBS-T and incubated with horseradish peroxidase conjugated anti-rabbit or anti-mouse antibodies at room temperature for 2 h. The blots were visualized using an enhanced chemiluminescence (ECL) kit (Pierce, Rockford, USA). The relative intensity of protein bands was quantified by densitometry (ImageJ, NIH, Bethesda, MD) and then standardized using the corresponding GAPDH intensity as a control. Statistical analysis The results are expressed as the mean ± SEM for three independent experiments. Statistical significance was assessed by a one-way analysis of variance (ANOVA) followed by Duncan’s post-hoc test. A value of P
S0041-008X(15)30019-3 doi: 10.1016/j.taap.2015.06.012 YTAAP 13402
To appear in:
Toxicology and Applied Pharmacology
Received date: Revised date: Accepted date:
9 January 2015 6 June 2015 12 June 2015
Please cite this article as: Wang, Cheng, Nie, Xiaoke, Zhang, Yan, Li, Ting, Mao, Jiamin, Liu, Xinhang, Gu, Yiyang, Shi, Jiyun, Xiao, Jing, Wan, Chunhua, Wu, Qiyun, Reactive oxygen species mediate nitric oxide production through ERK/JNK MAPK signaling in HAPI microglia after PFOS exposure, Toxicology and Applied Pharmacology (2015), doi: 10.1016/j.taap.2015.06.012
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 Reactive oxygen species mediate nitric oxide production through ERK/JNK MAPK signaling in HAPI
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microglia after PFOS exposure
Cheng Wang , Xiaoke Nie1, Yan Zhang1, Ting Li2, Jiamin Mao2, Xinhang Liu3, Yiyang Gu2, Jiyun Shi2, Jing Xiao3, Chunhua Wan1, Qiyun Wu1*
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*Correspondence to: Qiyun Wu, Department of Nutrition and Food Hygiene, School of Public Health, Nantong University, Nantong, Jiangsu 226001, People’s Republic of China. Email: [email protected] Phone: +86-0513-85012177 Fax: +86-0513-85012180
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1 Department of Nutrition and Food Hygiene, School of Public Health, Nantong University, Nantong, Jiangsu 226001, People’s Republic of China
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2 Department of Labor and Environmental Hygiene, School of Public Health, Nantong University, Nantong, Jiangsu 226001, People’s Republic of China
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3 Department of Occupational Medicine and Environmental Toxicology, School of Public Health, Nantong University, Nantong, Jiangsu 226001, People’s Republic of China
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ACCEPTED MANUSCRIPT Abstract
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Perfluorooctane sulfonate (PFOS), an emerging persistent contaminant that is commonly encountered during daily life, has been shown to exert toxic effects on the central nervous system (CNS). However, the molecular mechanisms underlying the neurotoxicity of PFOS remains largely unknown. It has been widely acknowledged that the inflammatory mediators released by hyper-activated microglia play vital roles in the pathogenesis of various neurological diseases. In the present study, we examined the impact of PFOS exposure on microglial activation and the release of proinflammatory mediators, including nitric oxide (NO) and reactive oxidative species (ROS). We found that PFOS exposure led to concentration-dependent NO and ROS production by rat HAPI microglia. We also discovered that there was rapid activation of the ERK/JNK MAPK signaling pathway in the HAPI microglia following PFOS treatment. Moreover, the PFOS-induced iNOS expression and NO production were attenuated after the inhibition of ERK or JNK MAPK by their corresponding inhibitors, PD98059 and SP600125. Interestingly, NAC, a ROS inhibitor, blocked iNOS expression, NO production, and activation of ERK and JNK MAPKs, which suggested that PFOS-mediated microglial NO production occurs via a ROS/ERK/JNK MAPK signaling pathway. Finally, by exposing SH-SY5Y cells to PFOS-treated microglia-conditioned medium, we demonstrated that NO was responsible for PFOS-mediated neuronal apoptosis.
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Keywords:
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PFOS; microglia; ROS; iNOS; NO; ERK/JNK MAPK
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ACCEPTED MANUSCRIPT Introduction
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Perfluorooctane sulfonate (PFOS), an emerging pollutant, has been found to accumulate in both wildlife and humans due to its extensive use and extraordinary stability (J. P. Giesy and K. Kannan, 2001; F. Xiao et al., 2014). PFOS has been shown to have multiple toxic effects, including immunotoxicity, hepatotoxicity, developmental toxicity, and carcinogenicity (S. Fuentes et al., 2006; J. L. Butenhoff et al., 2012; E. Corsini et al., 2012; M. R. Qazi et al., 2013). In addition, accumulating evidence suggests that PFOS exposure can have adverse effects on the central nervous system (CNS), such as neuroendocrine dysfunction and neurobehavioral abnormalities (M. E. Austin et al., 2003; N. Onishchenko et al., 2011). To date, the majority of the studies have focused primarily on neurons. However, the effects of PFOS on microglia and the inflammatory response following PFOS exposure should also be addressed. In this study, we examined the underlying molecular mechanisms by which PFOS induced inflammatory responses in HAPI microglial cells. Microglial cells are resident phagocytic cells of the CNS that are primarily involved in immune surveillance. Once activated by injury, ischemia or inflammatory stimuli, microglial cells display macrophage-like characteristics, including the production of pro-inflammatory cytokines, phagocytosis and antigen presentation (G. A. Garden and T. Moller, 2006). However, not only do the uncontrolled and sustained production of inflammatory cytokines by microglia trigger neuronal cell death (A. M. Kaindl et al., 2012), but they also signal to astrocytes, thereby amplifying their inflammatory response and leading to more injurious accumulation of neurotoxins in the CNS tissues (D. van Rossum and U. K. Hanisch, 2004). Increasing evidence has shown that activated microglia and the attendant neurotoxic substances, including nitric oxide (NO), cause neuronal death (V. L. Dawson et al., 1994; Y. Li et al., 2013). NO, which is synthesized via nitric oxide synthases (NOSs), is involved in various pathological and physiological processes in the CNS (V. Calabrese et al., 2007). The NOSs have three family members, termed inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS) (O. W. Griffith and D. J. Stuehr, 1995). It was reported that in response to a variety of stimuli, inflammatory and immune cells mainly express iNOS (C. Nathan and Q. W. Xie, 1994). A recent study suggested that activated microglia kill neurons through iNOS as a result of the release of NO (A. Bal-Price and G. C. Brown, 2001). It has also been shown that PFOS activates the inflammatory response in astrocytes (H. C. Zeng et al., 2011) and mediates the expression of iNOS in human–hamster hybrid cells (X. Wang et al., 2013). Therefore, we considered that it was worth exploring whether PFOS exposure could activate microglia and induce iNOS expression. The mitogen-activated protein kinases (MAPKs) are classified into three components: P38 kinase, extracellular signal-regulated kinases ERK1/2 (P44/P42) and c-Jun amino-terminal kinase JNK (P46/P54), which have been reported to be involved in the release of immune-related cytotoxic factors, such as NO, COX-2, IL-1βand TNF-αwhen activated by various pro-inflammatory signals (Y. Choi et al., 2009; L. Yan et al., 2013). Some studies have reported that PFOS can induce the phosphorylation of all members of the MAPK family in zebrafish embryos and cerebellar granule cells (X. Shi and B. Zhou, 2010; Y. J. Lee et al., 2013). However, very few studies have addressed the role of MAPK signaling in activated microglia following PFOS exposure. Reactive oxygen species (ROS), including superoxide anion (.O2-), hydroxyl free radical (.OH) and hydrogen peroxide (H2O2), can act as second messengers and play a vital role in the maintenance 3
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of many physiological functions. However, the overproduction of ROS is detrimental to organisms. For example, an excess of ROS production was recently reported to contribute directly to neuronal damage and neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases (K. W. Zeng et al., 2010). Furthermore, substantial ROS production is involved in controlling the expression of several inflammatory mediators. ROS have been shown to act as a signal initiation molecule to enhance NO production (H. Eguchi et al., 2011). Mounting evidence suggests that MAPKs play a role as a bridge in the ROS-mediated production of NO (S. U. Kim et al., 2013). Oxidative stress has been shown to contribute to the neurotoxicity following PFOS exposure (C. Liu et al., 2007). Thus, we thought that it would be intriguing to explore whether PFOS exposure could induce ROS production, as well as ROS-dependent MAPK activation. In the present study, we explored the molecular mechanisms underlying the PFOS-mediated inflammatory response in HAPI microglia. We demonstrated that PFOS-mediated NO production via iNOS was associated with ERK/JNK activation, which was dependent on ROS production. In addition, we also detected the effects of the conditioned medium from PFOS-treated HAPI microglial cells treated with or without a NOS inhibitor (L-NMMA) on SH-SY5Y cells. We found that L-NMMA enhanced the cell viability and attenuated PFOS-induced apoptosis. In summary, our results demonstrated that ROS-mediated NO production was dependent on ERK/JNK activation following PFOS treatment, and that NO was directly responsible for PFOS-mediated neurotoxicity.
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Materials and Methods
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Chemicals The following chemicals were purchased from their suppliers: PFOS (potassium salt, purity 98%) (Sigma–Aldrich, Switzerland); PD98059 (Sigma–Aldrich, St. Louis, MO, USA); SP600125 (Sigma–Aldrich, St. Louis, MO, USA); TUNEL, In Situ Cell Death Detection Kit (Roche Applied Science, Mannheim, Germany); NG-monomethyl-L-arginine (L-NMMA; Dojindo Chemical, Kumamoto, Japan) and N-acetyl-L-cysteine (NAC; Sigma, St. Louis, MO, USA). Cell culture and treatment Rat HAPI microglial cells, a MG-like rat cell line which was obtained from Professor Qin Shen at Nantong University, Jiangsu Province, China (P. Cheepsunthorn et al., 2001; Z. Cui et al., 2010), were propagated in Dulbecco's modified Eagle's medium(DMEM; Invitrogen, Grand Island, NY, USA) containing 10% fetal bovine serum (Sigma–Aldrich, St. Louis, MO, USA), 100μg/ml streptomycin and 100U/ml penicillin at 37 °C in a humidified incubator with 5% CO2/95% air. The HAPI microglial cells were treated with different concentrations of PFOS (0.1, 1, 5, 10, 20, 50, 100, 200 nM) dissolved in DMSO for 6 h or with 20 nM PFOS for 0, 1, 3, 6, 8 or 12 h. Cultures containing only 0.05% DMSO were used as a control. For assays of ERK/JNK MAPK inhibition, the cells were pre-treated with an ERK inhibitor (PD98059) or JNK inhibitor (SP600125) for 60 min, followed by PFOS treatment for 6 h. Before being treated with PFOS or DMSO, the cells were pre-incubated with serum-free medium. After their indicated treatments, the cells and remaining medium were harvested and stored at –20 °C until use. The SH-SY5Y cell line was derived from human brain neuroblastoma (R. A. Ross et al., 1983) and was maintained at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4
ACCEPTED MANUSCRIPT 100U/ml penicillin, 100μg/ml streptomycin and 10% fetal bovine serum (Sigma–Aldrich, St. Louis, MO, USA) in a humidified incubator with 5% CO2/95% air.
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Measurement of NO The production of NO was determined by measuring a stable oxidative end-product of NO, nitrite, in culture supernatant fluid with the Griess reagents (1% sulphanilamide, 5% phosphoric acid,
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and 0.1% naphthylethylenediamine). Briefly, 100μl of supernatant fluid from stimulated HAPI cells was mixed with the same volume of Griess reagents in a 96-well plate. Ten minutes later, the absorbance of every well was measured using an automated microplate reader at 540 nm. The concentration of nitrite was calculated in accordance with a nitrite standard curve based on sodium nitrite. Culture medium only was used as the blank control, and each experiment was performed in triplicate.
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Measurement of cellular reactive oxygen species (ROS) The intracellular ROS generation was measured using a fluorescent marker, DCFH-DA (Genmed, Arlington, MA, USA). Briefly, after the indicated treatments, cells seeded on glass coverslips were washed three times with serum-free DMEM and incubated with DCFH-DA at 37 °C for 30 min. Subsequently, the nuclei were counterstained with Hoechst-33258 (Invitrogen, 1:1000). After being washed, the cells were immediately examined using a fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany). The level of ROS fluorescence in each group was determined using a flow cytometric analysis. The experiments were repeated in triplicate.
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Cell viability assay Cell viability was determined by the Cell Counter Kit-8 (CCK-8) assay (Dojindo Laboratories, Japan). Briefly, SH-SY5Y cells were seeded at a density of 5 × 103 cells/well in 96-well plates, and then were incubated at 37 ◦C with conditioned medium from PFOS-treated HAPI microglial cells for 24 h. Thereafter, 10μl of CCK-8 solution was added to every well, then the cells were incubated for 2 h in an incubator, and the absorbance was determined using a quantified microplate reader. All results were confirmed by replication in at least three independent experiments. Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling (TUNEL) assay The TUNEL assay was applied to determine the level of apoptosis by demonstrating apoptotic bodies in the SH-SY5Y cells using the In Situ Cell Death Detection Kit (Roche Applied Science, Mannheim, Germany). Briefly, before being fixed with 4% formaldehyde and incubated at room temperature for 40 min, SH-SY5Y cells were exposed to PFOS-induced HAPI microglial cells’ conditioned medium for 24 h. To guarantee that there were constant concentrations of NO, the PFOS-treated HAPI microglial cells’ conditioned medium was refreshed every 8 h. After several rinses in phosphate-buffered saline (PBS) and permeabilization in 0.1% Triton X-100 solution on ice for 5 min, 50 μl of TUNEL reaction mixture was added to the cells on coverslips, which were incubated for 60 min at 37 ◦C in a dark humidified chamber. Thereafter, the cells were rinsed three times in PBS, followed by incubation with Hoechst for 20 min at room temperature. Apoptotic cells were detected as localized bright green cells (positive cells) by a fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany). 5
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Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated from HAPI microglial cells treated with DMSO or PFOS using TRIzol reagent (Invitrogen, Shanghai, China) in accordance with the manufacturer’s instructions. Extracted RNA was reverse transcribed for cDNA synthesis using an Omniscript RT kit (Qiagen, Germany) in accordance with the manufacturer’s protocol. PCR amplifications were performed with specific primers according to standard methods by Taq DNA polymerase. The primers sequences used were: iNOS: forward primer, 5’-GCTTTGTGCGGAGTGTCAGT-3’; reverse primer, 5’-CCTCCTTTGAGCCCTCTGTG-3’; GAPDH: forward primer, 5’-TGATGACATCAAGAAGGTGGTGAAG-3’; reverse primer, 5’-TCCTTGGAGGCCATGTGGGCCAT-3’. The amplification steps for iNOS were 30 cycles of 30 s at 94 ◦C, 1 min at 60 ◦C, and 72 ◦C for 1 min. The amplification steps for GAPDH were 30 cycles of 30 s at 94 ◦C, 30 s at 55 °C and 30 s at 72 ◦C. The PCR products for each sample were analyzed by electrophoresis in a 1.5% agarose gel. Gels were stained with ethidium bromide and analyzed under ultraviolet light. The intensity of the mRNA expression was measured by densitometry and then corrected with the corresponding GAPDH mRNA measurement.
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Western blot analysis After the indicated treatments, cells were washed with ice-cold PBS, lysed with lysis buffer for 1 min on ice, and subsequently thoroughly scraped with a cell scraper to remove them from the surface. Then, the protein samples were centrifuged at 13,000 rpm at 4 °C for 15 min. The resulting supernatants were transferred to new tubes, and the protein concentration was determined using a BCA protein assay kit (Thermo Scientific). Thereafter, equal amounts of protein were separated by 10% or 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, USA). To block non-specific binding, the membranes were immersed in 5% non-fat dry milk or bovine serum albumin (BSA) in PBS containing 0.5% Tween 20 (PBS-T), followed by incubation with one of the following primary antibodies at 4°C overnight or at room temperature for 8 h. The rabbit anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:1000) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); the rabbit monoclonal anti-rat iNOS (1:800), rabbit monoclonal anti-rat p38 (1:1000), rabbit anti-rat phospho-p38 (1:1000), rabbit anti-rat JNK (1:1000), rabbit anti-rat phospho-JNK (1:1000), rabbit anti-rat phosphor-ERK (1:1000), and rabbit anti-rat ERK (1:1000) were obtained from Cell signaling (MA, USA). The membranes were then washed with PBS-T and incubated with horseradish peroxidase conjugated anti-rabbit or anti-mouse antibodies at room temperature for 2 h. The blots were visualized using an enhanced chemiluminescence (ECL) kit (Pierce, Rockford, USA). The relative intensity of protein bands was quantified by densitometry (ImageJ, NIH, Bethesda, MD) and then standardized using the corresponding GAPDH intensity as a control. Statistical analysis The results are expressed as the mean ± SEM for three independent experiments. Statistical significance was assessed by a one-way analysis of variance (ANOVA) followed by Duncan’s post-hoc test. A value of P
