Neurochem Res DOI 10.1007/s11064-017-2419-1
ORIGINAL PAPER
Nrf2 Signaling Pathway Mediates the Antioxidative Effects of Taurine Against Corticosterone-Induced Cell Death in HUMAN SK-N-SH Cells Qinru Sun1 · Ning Jia2 · Jie Yang2 · Guomin Chen2
Received: 8 June 2017 / Revised: 6 October 2017 / Accepted: 9 October 2017 © Springer Science+Business Media, LLC 2017
Abstract Substantial evidence has shown that elevated circulating corticosteroids or chronic stress contributes to neuronal cell death, cognitive and mental disorders. However, the underlying mechanism is still unclear. Taurine is considered to protect neuronal cells from apoptotic cell death in neurodegenerative diseases and neuropsychiatric disorders. In the present study, the protective effects of taurine against corticosterone (CORT)-induced oxidative damage in SK-NSH neuronal cells were investigated. The results showed that CORT significantly induced cell death, which was blocked by pretreatment with taurine. Similarly, pretreatment with taurine suppressed CORT-induced apoptotic cell death decreasing the levels of intracellular reactive oxygen species and improving mitochondrial function. Pretreatment with taurine increased the expression of phosphorylated extracellular regulated protein kinases (ERK) as well as the nuclear translocation of nuclear factor (erythroid 2-derived)-like 2 (Nrf2) in the CORT rich environment. Furthermore, administration of the ERK inhibitor U0126 or transient (siRNA) silencing of Nrf2 blocked the protective effects of taurine on cell viability and expression levels of Nrf2 and heme oxygenase-1 (HO-1) in the CORT model of neuronal damage. These results suggest that the Nrf2 signaling pathway may play a role in the protection mechanism of taurine against CORT-induced neuronal oxidative damage. * Ning Jia
[email protected] 1
Institute of Forensic Medicine, Xi’an Jiaotong University Health Science Center, Xi’an 710061, Shaanxi, People’s Republic of China
Department of Human Anatomy, Histology and Embryology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an 710061, Shaanxi, People’s Republic of China
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Keywords Nrf2 · Taurine · Reactive oxygen species · Corticosterone · Mitochondria
Introduction Exposure of an organism to stressful conditions causes the adrenal glands to release high amounts of corticosteroids (cortisol in primates, corticosterone (CORT) in rats and mice). This hormone easily enters the brain and exerts powerful feedback effects on the hypothalamic–pituitary–adrenal (HPA) axis [1, 2]. Recent findings support that the dysfunction of HPA axis is one of the major causes in chronic neurodegenerative and neuropsychiatric diseases, such as Alzheimer’s disease, anxiety and major depression [3–6]. Besides, mice administrated with higher concentration of glucocorticoid (a class of corticosteroids) have displayed impaired long-term memory retrieval mediated by hippocampus. In addition, application of glucocorticoids impaired working memory mediated by the prefrontal cortex in human [7]. These data indicates that chronic high levels of plasma corticosteroids might be involved in neuronal damage in neurological diseases. Studies on both in vivo and in vitro have shown that excessive secretion of CORT weakens cellular antioxidant defense system and induces neuronal oxidative damage and cell death in certain brain regions, particularly in the hippocampus and prefrontal cortex [3, 8, 9]. The nuclear factor (erythroid 2-derived)-like 2 (Nrf2) is one of the master regulators of the antioxidant defense response by binding the antioxidant response element (ARE), which initiates transcription of a series of genes encoding factors associated with neuronal survival, such as heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidase (NQO-1) among others [10]. Several kinases including phosphatidylinositol 3-kinase (JNK), protein
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kinase C (PKC) and extracellular signal-regulated kinase (ERK) are able to induce Nrf2 dissociation from Kelchlike ECH-associated protein 1 (Keap1) and subsequent translocation to the nucleus by phosphorylating Nrf2 at serine and threonine residues [11, 12]. Our previous study has demonstrated that prenatally-stressed offspring rats showed more depression-like behaviors and lower level of ERK2 mRNA in the hippocampus and prefrontal cortex compared with the control rats [13, 14], accompanied with high levels of CORT in hippocampus, excessive intracellular reactive oxygen species (ROS) in hippocampal CA3 area and hippocampal neurons loss [15, 16]. These data strongly suggest that the decline of ERK is involved in oxidative damage in the hippocampus of prenatally-stressed rats accompany with high levels of plasma CORT. Taurine, a major free amino acid in the mammalian central nervous system, is increasingly being recognized as a crucial neuromodulator and antioxidant employed in experimental therapies against neuronal damage [17–19]. Studies have shown that taurine can protect neurons from glutamate-induced cytotoxicity via reducing the intracellular ROS and maintaining the mitochondrial membrane potential (ΔΨm) [20]. Our recent study indicates that administration of taurine prevents hippocampal neurons in offspring rats against oxidative damage induced by prenatal stress [21]. More intriguingly, other studies have suggested that taurine stimulates neural stem/progenitor cell proliferation and synapse development in the developing brain probably due to taurine-induced ERK1/2 phosphorylation [22]. However, whether activation of ERK and Nrf2 signaling pathway participate in the protection of taurine against CORT-induced neuronal death is still unclear. In the present study, we treated SK-N-SH cells with CORT to set up a CORT-induced neuronal cell death model. Meanwhile, SK-N-SH cells were pretreated with or without taurine. In addition, the cells were subjected to the ERK inhibitor U0126 treatment or transient (siRNA) silencing of Nrf2 prior to the incubation with taurine followed by CORT. The viability of cells was observed and the neuronal apoptotic cell death was detected. The levels of mitochondrial ROS, intracellular ROS, mitochondrial membrane potential (ΔΨm) and ATP generation were detected as well. Furthermore, the expression levels of pERK, ERK, Nrf2 and HO-1 were determined. The results showed that CORT increased cell death by reducing mitochondrial function, increasing ROS production and followed by neuronal apoptotic cell death. Taurine could block the effects of CORT on neuronal cells by enhancing the level of phosphorylation of ERK and activating the Nrf2 signaling pathway.
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Materials and Methods Neuronal Cell Culture and Experimental Grouping HUMAN SK-N-SH neuroblastoma cells were cultured in DMEM supplemented with 10% (v/v) heat-inactivated fetal calf serum (Invitrogen) and 100 U/mL penicillin/ streptomycin (Invitrogen). Cells were maintained at 37 °C in humidified 5% CO2 and 95% air. CORT (Sigma) was dissolved in DMSO and prepared a stock solution for different final concentrations (1, 2, 10, 20, 100, 200 and 1000 μM), with the concentration of DMSO less than 0.1%. Taurine (Sigma) was dissolved in fresh medium and prepared a stock solution for different final concentrations (0.1, 1, 10, 25, 50 mM) in culture medium. The ERK inhibitor, U0126 was added into the culture medium with the final concentration of 5 μM. The 100 U/mL catalasepolyethylene glycol (PEG-catalase) (Sigma) or 10 mM N-acetylcysteine (NAC) (Sigma) was pretreated into the culture medium [23, 24]. SK-N-SH cells were plated for 48 h before treatments. The SK-N-SH cells were treated with one or more of the above reagents and divided into 4 groups according to the various treatments: 1. control group (non-treatment); 2. CORT group (CORT treatment for 24 h); 3. taurine + CORT group (pretreatment with taurine for 2 h followed by CORT for 24 h); 4. (U0126 + taurine) + CORT group (pretreatment with U0126 and taurine for 2 h followed by CORT for 24 h). Measurement of Cell Viability SK-N-SH cells were cultured in a 96-well plate. Briefly, in each well, the MTT solution (final concentration of 0.5 mg/ mL) was incubated for 4 h at 37 °C. Subsequently, DMSO was used to terminate the reaction. The absorbance at 490 nm was recorded by using a multifunctional microplate reader (Bio-Rad). The results were presented as a percentage of control. Detection of Apoptotic Cell Death Apoptotic cell death was employed by using the In Situ Cell Death Detection Kit (TUNEL) according to the introduction (Roche). In brief, the cultured cells in coverslips were fixed in fresh prepared 4% paraformaldehyde in PBS for 20 min at room temperature. Next, cells were permeabilized in PBS contained 0.1% Triton X-100 and 0.1% sodium citrate for 2 min on ice. The coverslips were washed with PBS for 5 min and then observed under a fluorescence microscope.
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Determination of Mitochondrial ROS For determination of the mitochondrial redox state, after treatments, SK-N-SH cells were incubated with MitoSOX Red (5 μM) for 15 min at 37 °C. The fluorescence was read at 510 nm for excitation and 580 nm for emission with the spectrofluorimeter (BioTek/FLx800, USA). The intracellular fluorescence intensity was expressed as the fold increase compared to the control. Detection of Mitochondrial Membrane Potential (ΔΨm) ΔΨm was determined by using tetramethylrhodamine methyl ester (TMRM) (Invitrogen) staining. SK-N-SH cells were cultured in 8-well chamber slices. After treatments, 200 nM TMRM in phenol red-free medium was added for 30 min at 37 °C. Fluorescent signals were captured using a fluorescence microscope (Olympus). The intensity of TMRM was analyzed with the Image J software (NIH). Measurement of ATP Level The ATP level was measured by the ATP Determination Kit (Roche) according to the manufacturer’s instruction. After treatments, SK-N-SH cells were homogenized by using cell lysis buffer, incubated on ice for 15 min, and centrifuged at 14000xg for 15 min at 4 °C. Subsequent supernatants were collected and the chemiluminescence was measured by using a Beckman Coulter DTX880 (Beckman) with an integration time of 10 s. Measurement of Intracellular ROS To further explored which types of ROS were involved in CORT induced neuronal loss, the assay were employed with slight modified based on the method provided by Bartolini et al. [23, 24]. Briefly, to determine the H 2O2 and the effect of thiol depletion under cellular stress, PEG-catalase (PEGCAT; 50 U/mL) or N-acetylcysteine (NAC; 10 mM) was subjected to SK-N-SH cells 2 h prior to CORT, respectively. SK-N-SH cells were incubated with the fluorescent probe 2,7-dichlorofluoresceindiacetate (DCFH-DA) for 15 min at 37 °C. The fluorescence was read at 495 nm for excitation and 520 nm for emission by using the spectrofluorimeter (BioTek/FLx800, USA). Transient Transfection with siRNA Cells were plated in 6-well plates and grown to a confluence of 70–80%, and then were transfected with Nrf2-specific siRNA and negative control siRNA by using Hiperfect transfection reagent (Qiagen, Valencia, CA). 24 h later, the transfected cells were treated with either vehicle or taurine
(25 mM) for 2 h in the presence or absence of 200 μM CORT for 24 h. The SK-N-SH cells were treated with control siRNA or Nrf2 siRNA followed by CORT for 24 h and divided into 5 groups: 1. control group (non-treatment); 2. control siRNA + CORT group (transfection with control siRNA for 24 h, followed by CORT for 24 h); 3. Nrf2 siRNA + CORT group (transfection with Nrf2 siRNA for 24 h, followed by CORT for 24 h); 4. control siRNA + taurine + CORT group (transfection with control siRNA for 24 h, treatment with taurine for 2 h followed by CORT for 24 h); 5. Nrf2 siRNA + taurine + CORT group (transfection with Nrf2 siRNA for 24 h, treatment with taurine for 2 h followed by CORT for 24 h). Protein Extraction and Western‑Blotting Analysis Protein of whole cell was extracted by using a cell protein extraction kit (Beyotime) according to the manufacturer’s instructions. SK-N-SH cells from each group were washed twice by using ice-cold PBS, and then were homogenized at 1:5 (wt/vol) in an ice-cold lysis buffer. For isolation the nuclear fractions of SK-N-SH cells, a nuclear and cytoplasmic extraction kit (Beyotime) was used according to the manufacturer’s instructions. Briefly, the harvested neuronal cultures were incubated with the isolation buffer contained phenylmethylsulfonylfluoride for 20 min. The solution was centrifuged at 20,000×g and the pallet was resuspended with isolation buffer. The suspension was centrifuged at 20,000×g for 10 min and the supernatant was collected. Samples were subjected to SDS–PAGE and transferred to polyvinylidene fluoride membranes. The blots were probed with the following primary antibodies: rabbit anti-cleaved caspase-3 (Santa Cruz), mouse anti-ERK (CST), rabbit antipERK (CST), rabbit anti-Nrf2 (Abcam), mouse anti-HO-1 (Abcam), mouse anti β-actin (Sigma) rabbit anti-Lamin B (Santa Cruz) followed by incubation with species-matched horseradish peroxidase-conjugated secondary antibodies. The blots were developed with a chemiluminescence substrate solution (Thermo Scientific). The optical density of immunoreactive bands was quantified using the Quantity One software (Bio-Rad). Statistics Analysis All data were represented as mean ± SEM and analyzed by using the software of SPSS 11.0. One-way ANOVA was used to examine differences among the groups. A difference was considered significant at P < 0.05 level.
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Results Taurine Attenuated CORT‑Induced Neuronal Cell Death in SK‑N‑SH Cells We treated the SK-N-SH cells with different concentrations of CORT (1, 2, 10, 20, 100, 200 and 1000 μM) and determined the protective effect of taurine by employing MTT assay. As shown in Fig. 1a, cells treated with CORT 10 or 20 μM did not show obviously decline of cell viability compared with the control cells. Treatment with 200 μM CORT displayed nearly 40% reduction which was adopted in the subsequent experiments. Next, we tested whether application of taurine affect the viability of SKN-SH cells. There was no significant difference in cell viability among the cells treated with taurine (0, 1, 5, 10, 25, 50 mM) (Fig. 1b). Then, we examined whether taurine attenuates CORT-induced SK-N-SH cell death. As shown in Fig. 1c, pretreatment with taurine (10, 25 mM) significantly increased the cell viability in a dose-dependent manner.
Fig. 1 Taurine attenuated CORT-induced neuronal death in SKN-SH cells. SK-N-SH cells were plated for 48 h before treatments. SK-N-SH cells were treated with different concentrations of CORT (1, 2, 10, 20, 100, 200 and 1000 μM) a or taurine (0.1, 1, 10, 25 and 50 mM), b for 24 h; (c) SK-N-SH cells were pretreated with differ-
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Taurine Attenuated Neuronal Apoptotic Cell Death Induced by CORT in SK‑N‑SH Cells In order to explore whether CORT induced apoptotic cell death in SK-N-SH cells, we measured apoptotic cell death by TUNEL assay and the level of the cleaved caspase-3 by employing Western-blotting. As shown in Fig. 2a, the CORT group (treatment with 200 μM CORT) significantly increased the number of apoptotic cells compared with the control group, and pretreatment with taurine (10, 25 mM) reversed the increase of apoptotic cell death induced by CORT (P < 0.01). Consistent with this, the level of cleaved caspase-3 was increased in CORT group (P < 0.01), and pretreatment with taurine (10, 25 mM) significantly reduced the level of cleaved caspase-3 (P < 0.01) (Fig. 2b, c). Taurine Inhibited CORT‑Induced Intracellular ROS Accumulation To examine whether intracellular ROS participated in CORT induced cell death, we measured intracellular ROS levels. As shown in Fig. 3a, CORT significantly increased intracellular ROS accumulation which was inhibited by pretreatment of 10 or 25 mM taurine (P < 0.01). To further identify which
ent concentrations of taurine (0.1, 1, 10, 25 and 50 mM) for 2 h and then incubated with CORT (200 μM) for 24 h. The viability of the cells was identified further analyzed by MTT assay and is represented as the percentage of the control (non-treated) cells. #P < 0.01 versus control; *P < 0.01 versus CORT (n = 4)
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reduced the mitochondrial ROS levels (P < 0.01). As shown in Fig. 4b, c, cells treated with CORT showed significantly decreased ΔΨm and ATP level compared with the untreated cells (P < 0.01), which were reversed by pretreatment with 10, 25 mM taurine (P < 0.01). Taurine Activated the ERK and Nrf2 in CORT Milieu
Fig. 2 Taurine attenuated apoptotic cell death in the presence of CORT in SK-N-SH cells. a The comparison of the neuronal apoptosis percentage among different groups. b The representative immunoblot bands and the quantification analysis of cleaved caspase-3. #P < 0.01 versus control; *P < 0.01 versus CORT (n = 4)
types of ROS were formed, SK-N-SH cells were pretreated with 10 mM NAC or 100 U/mL PEG-CAT for 2 h and then incubated with CORT (200 μM) for 24 h. Pretreatment of 10 mM NAC or 100 U/mL PEG-CAT attenuated CORTinduced intracellular ROS accumulation (P < 0.01). Similarly, pretreatment of 10 mM NAC or 100 U/mL PEG-CAT attenuated CORT-induced cell death (shown in Fig. 3b) (P < 0.01). Taurine Reversed CORT‑Induced Mitochondrial Dysfunction in SK‑N‑SH Cells We further examined the function of mitochondria including mitochondrial ROS levels, ATP level and mitochondrial membrane potential (ΔΨm) in SK-N-SH cells. As shown in Fig. 4a, we found that the mitochondrial ROS levels were greatly increased in CORT group compared with that in the control group (P < 0.01), and 10, 25 mM taurine obviously
To further investigate the possible mechanism of taurine against CORT induced neurotoxicity, we co-treated the cells with taurine and the ERK inhibitor U0126 followed by CORT. We examined the ratio of pERK/ERK and the nuclear translocation of Nrf2 by using Western-blotting. As shown in Fig. 5a, the ratio of pERK/ERK was significantly decreased in the CORT group compared with the control group (P < 0.01). Pretreatment with 25 mM taurine recovered the ratio of pERK/ERK (P < 0.01) suppressed by CORT or U0126 (P < 0.01). In the presence of CORT, U0126 significantly blocked taurine-increased ratio of pERK/ERK in SK-N-SH cells (P < 0.01). As shown in Fig. 5b, compared with the control group, the expression of nuclear Nrf2 was markedly decreased in the presence of CORT (P < 0.01). Similarly, Co-treatment with U0126 blocked the increase of nuclear Nrf2 induced by taurine (P < 0.01). On the contrary, as shown in Fig. 5c, the expression level of cytosol Nrf2 was higher in the CORT group than that in the control group (P < 0.01). And co-treatment with U0126 blocked the decrease of cytosol Nrf2 induced by taurine (P < 0.01). Consistent with these results, 25 mM taurine did not take effect on attenuating apoptotic cell death, reducing accumulation of intracellular ROS and enhancing cell viability in SK-N-SH cells co-treatment with U0126 in CORT milieu (P < 0.01) (shown in Fig. 6a–c). Transient (siRNA) Silencing of Nrf2 Partially Blocked Taurine‑Rescued Cell Survival in the Presence of CORT To confirm the role of Nrf2 signaling pathway in the neuroprotective effects of taurine on neuronal cells in the presence of CORT, SK-N-SH cells were transfected with Nrf2 siRNA. As shown in Fig. 7a, b, transfection with Nrf2 siRNA significantly down-regulated the expression levels of nuclear Nrf2 and HO-1 compared to transfection with control siRNA in the presence of CORT (P < 0.01). Pretreatment with taurine partially recovered the expression levels of nuclear Nrf2 and HO-1 in SK-N-SH cells transfected with control siRNA (P < 0.01), which was blocked by transfected with Nrf2 SiRNA. As one of the Nrf2 targeted genes, HO-1 has been considered to play a pivotal role in repressing oxidative stress. To further investigate whether taurine effects on cell survival and apoptosis (upon CORT-induced neuronal toxicity), is promoted by Nrf2 signaling pathway. As shown
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Fig. 3 Taurine inhibited CORT-induced intracellular ROS accumulation. SK-N-SH cells were pretreated with 10 mM NAC or 100 U/mL PEG-CAT for 2 h and then incubated with CORT (200 μM) for 24 h. a The intracellular ROS levels were determined by H2DCFDA staining. Taurine inhibited CORT-induced intracellular ROS levels. Simi-
larly, pretreatment of 10 mM NAC or 100 U/mL PEG-CAT attenuated CORT-induced intracellular ROS accumulation. b The viability of the cells was identified further analyzed by MTT assay and is represented as the percentage of the control (non-treated) cells. #P < 0.01 versus control; *P < 0.01 versus CORT (n = 4)
Fig. 4 Taurine inhibited CORT-induced mitochondrial dysfunction. a The mitochondrial ROS was detected by using the MitoSOX Red staining method. b The ATP level was detected by the ATP Deter-
mination Kit. c The mitochondrial membrane potential (ΔΨm) was measured by TMRM staining. #P < 0.01 versus control; *P < 0.01 versus CORT (n = 4)
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Fig. 5 Taurine promoted ERK activation and Nrf2 translocation to nucleus which was inhibited by the ERK inhibitor U0126. a The representative immunoblot bands for pERK1/2, ERK1/2 and the comparison of the ratio of the pERK/ERK among different groups. b, c The representative immunoblot bands for nuclear Nrf2 and cyto-
sol Nrf2 and the quantification analysis. Protein expression levels were normalized to β-actin or Lamin B. $P < 0.01 versus taurine; #P < 0.01 versus control; *P < 0.01 versus CORT; &P < 0.01 versus taurine + CORT (n = 4)
in Fig. 7c, the reduced cell viability in SK-N-SH cells treated with CORT was partially recovered by pretreatment with taurine (P < 0.01), while such protection of taurine did not occur in cells transfected with Nrf2 siRNA. Similarly, the increased number of apoptotic cells in SK-N-SH cells treated with CORT was significantly reduced by pretreatment with taurine (P < 0.01), while transfecting with Nrf2 siRNA weakened the protection of taurine against CORT-induced apoptotic cell death (P < 0.01, Fig. 7d).
[25, 26]. However, chronic repeated challenges or stimuli can lead to excessive plasma CORT followed by neuronal damage in several brain areas and subsequent metabolic syndrome (MetS), anxiety or depression disorder and cognitive dysfunctions [27]. Hence, to seek an effective and feasible compound to block the neurotoxicity of CORT, we designed a series of cell experiments in the present study. Furthermore, we explored the protective mechanism of taurine against CORT. In this study, although less than 100 μM CORT did not change the cell viability, the SK-N-SH cells treated with 100, 200 μM CORT for 24 h showed lower cell viability than that in the control cells, respectively. Pretreatment with taurine (10, 25 mM) enhanced cell viability in the presence of CORT. These results illustrated that taurine inhibited CORT-induced cell death in the SK-N-SH cells. Furthermore, we found that the SK-N-SH cells treated with 100, 200 μM CORT for 24 h displayed more apoptotic cells and higher level of cleaved caspase-3 than that in the control
Discussion Corticosterone, the major endogenous ligand in rodents (cortisol in human) released by the adrenal cortex, is the target of the hypothalamic–pituitary–adrenal axis, which helps individuals to deal with the challenges of their environment by modulating glucose metabolism, cardiovascular function, immune function, and psychological processes
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Fig. 6 U0126 blocked the protection of taurine against CORT. a The comparison of the percentage of cell viability among different groups. b The comparison of the neuronal apoptosis percentage among differ-
ent groups. c The comparison of the intracellular ROS levels among different groups. #P < 0.01 versus control; *P < 0.01 versus CORT; &P < 0.01 versus taurine + CORT (n = 4)
cells. As expected, pretreatment with taurine (10, 25 mM) reduced apoptotic cell death and the level of cleaved caspase-3 in the CORT milieu. These results demonstrated that taurine could protect neuronal cells against CORT-induced apoptotic cell death. Although the protection of taurine against glutamate, A-beta or NMDA has been mentioned in studies from other research groups [19, 20, 28], the protective mechanism of taurine in neuronal cells against CORT remains to be explored. To explore the neuroprotective mechanism of taurine against CORT-induced SK-N-SH neuronal apoptotic cell death, we evaluated the intracellular ROS levels and the mitochondrial function. The results showed that administration of 200 μM CORT significantly increased the intracellular ROS levels, and pretreatment with taurine significantly reduced CORT-induced intracellular ROS accumulation. Notably, the thiol-antioxidants NAC or H2O2 scavenger PEG-CAT could attenuated CORT induced intracellular ROS accumulation, which implied thiol and H 2O2 were involved in CORT-induced ROS accumulation. Furthermore, the mitochondrial ROS was significantly increased by CORT, and taurine equally inhibited mitochondrial ROS accumulation in the presence of CORT. The ΔΨm and the
ATP level were significantly decreased in the CORT group compared with that in the control group, while pretreatment with taurine obviously reversed the decline of the ΔΨm and the ATP level in the SK-N-SH cells. These results indicated that taurine blocked neuronal apoptosis by improving mitochondrial function and reduced the level of intracellular ROS in the CORT rich environment. Mitochondria play important roles in buffering the intracellular ROS levels [29]. Once mitochondrial dysfunction occurs, the clearance of intracellular ROS is impaired which conversely results in higher level of ROS. Subsequently, excessive ROS worsen mitochondrial dysfunction that leads to mitochondria-dependent apoptotic cell death [30]. Taurine could break this vicious circle to protect SK-N-SH cells against CORT-induced apoptotic cell death. For further exploration, it is worth investigating whether the Nrf2 signaling pathway, a pivotal antioxidant defense pathway, is involved in the protection mechanism of taurine in the presence of CORT. The results showed that the ratio of pERK/ERK was obviously reduced in SK-N-SH cells treated with 200 μM CORT, and pretreatment with taurine increased the ratio of pERK/ERK in the presence of CORT. Likewise, treatment with 200 μM CORT decreased the expression of
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Fig. 7 Transient (siRNA) silencing of Nrf2 eliminated the protective effects of taurine by suppressing levels of nuclear Nrf2 and HO-1 in SK-N-SH cells in CORT rich environment. a The representative immunoblot bands for nuclear Nrf2 and the quantification analysis. b The representative immunoblot bands for HO-1 and the quantification
analysis. c Cell viability and d apoptotic cell death were examined by MTT assay and TUNEL assay respectively. #P < 0.01 versus control; *P < 0.01 versus control siRNA + CORT; $P < 0.01 versus Nrf2 siRNA + CORT; &P < 0.01 versus control siRNA + taurine + CORT (n = 4)
Nrf2 in SK-N-SH cells, which was reversed by pretreatment with taurine. Remarkably, the ERK inhibitor U0126 did not only block the role of taurine in raising the ratio of pERK/ERK, but also interrupted taurine-enhanced expression of Nrf2 in the CORT rich environment. These results demonstrated that the protection of taurine against CORT was mediated by the activation of ERK and Nrf2 signaling pathway. Nrf2-antioxidant response element (ARE) is an important adaptive pathway in response to stress. Under physiological conditions, Kelch-like ECH-associated protein 1 (Keap1) keeps the Nrf2 in the cytoplasm. However, when cells are exposed to oxidative stress in neurodegenerative
disease and neuropsychiatric diseases, Nrf2 can freely translocate into the nucleus to bind ARE, which is resulting in induction of many cytoprotective genes [31] and antioxidant genes [32], such as HO-1, NQO-1, glutamyl cysteine ligase catalytic subunit and glutamyl cysteine ligase modulatory subunit. In this study, the levels of Nrf2 and HO-1 were decreased in SK-N-SH cells treated with 200 μM CORT, which showed that CORT destroyed resistance of SK-N-SH cells to oxidative stress by down-regulating the Nrf2-ARE pathway. The Nrf2-ARE pathway can be activated by Akt, p38MAPK, and ERK to protect RAW 264.7 cells, HT22 cells and HepG2 cells from oxidative damage
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[33–35]. Encouragingly, in this present study, we found that co-treatment with U0126 blocked the protective roles of taurine in attenuating apoptotic cell death, reducing the accumulation of intracellular ROS and enhancing cell viability in SK-N-SH cells in the CORT rich environment. Furthermore, we found that down-regulating the expression of the Nrf2 by administrating Nrf2 siRNA blocked the increase of the expression of HO-1induced by taurine, although the expression of HO-1 was increased by taurine in the presence of CORT. This result demonstrated that taurine repressed oxidative stress and rescued mitochondrial function via upregulating the expression levels of Nrf2 and HO-1. This is consistent with studies from other groups [36, 37]. Taken together, the ERK and Nrf2 signaling pathway were involved in the protection of taurine in SK-N-SH cells against CORTinduced oxidative damage and apoptotic cell death. In conclusion, the above results suggest that CORT induces neuronal cell death by augmenting the levels of mitochondrial ROS and intracellular ROS, decreasing the ΔΨm and the ATP level in SK-N-SH cells. Taurine protects SK-N-SH cells against CORT by activation of ERK and Nrf2 signaling pathway to resist oxidative damage and apoptotic cell death. This study provides new evidence to support application of taurine to treat neurodegenerative and neuropsychiatric diseases. In future, the dosage form and strength of taurine-related additives or drugs need to be rigorously tested in animals and clinical trials.
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Acknowledgements This work was supported by the National Natural Science Foundation of China (Program Nos. 81500927 & 31200843), the Project Supported by Natural Science Basic Research Plan in Shaanxi Province of China (Program Nos. 2017JM8017 & 2016JM8041) and the Fundamental Research Funds for the Central Universities (No. xjj2015077).
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Compliance with Ethical Standards
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Conflict of interest The authors declare that they have no conflict of interest. 15.
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