Biomedicine & Pharmacotherapy 108 (2018) 1216–1224
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Brahma-related gene 1 ameliorates the neuronal apoptosis and oxidative stress induced by oxygen-glucose deprivation/reoxygenation through activation of Nrf2/HO-1 signaling
T
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Feng Lia, , Jing Liangb, Dongfang Tangc a
Anesthesiology Department, First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 277 West Yanta Road, Shaanxi Province, 710061, China Radiotherapy Department, Shaanxi Provincial Tumor Hospital, Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an, Shaanxi Province, 710061, China c Neurosurgery Department, Fuwai Central China Cardiovascular Hospital, Zhengzhou, Henan Province, 450000, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Brg1 Cerebral ischemia and reperfusion HO-1 Nrf2 OGD/R
Accumulating evidence suggests that brahma-related gene 1 (Brg1) is a critical regulator of cell apoptosis and oxidative stress in response to various insults; however, whether Brg1 regulates neuronal apoptosis and oxidative stress during cerebral ischemia/reperfusion injury remains unclear. This study aimed to investigate the expression, biological function, and regulatory mechanism of Brg1 in regulating neuronal apoptosis and oxidative stress induced by oxygen-glucose deprivation/reoxygenation (OGD/R), an in vitro cellular model of cerebral ischemia and reperfusion injury. The results showed that Brg1 expression was altered in response to OGD/R treatment. Overexpression of Brg-1 increased cell viability, attenuated apoptosis, and reduced reactive oxygen species (ROS) production in neurons, thus showing a protective effect against OGD/R-induced injury. In contrast, knockdown of Brg1 significantly inhibited cell viability, increased apoptosis, and promoted ROS production in neurons following OGD/R treatment. Moreover, these results showed that Brg1 promoted the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and increased the expression of heme oxygenase-1 (HO-1). However, knockdown of Nrf2 or HO-1 significantly abrogated Brg1-mediated neuroprotective effects. Taken together, these results demonstrate that Brg1 ameliorates OGD/R-induced neuronal injury in vitro by promoting the activation of Nrf2/HO-1 signaling. The study highlights a potential role of Brg1 in regulating cerebral ischemia/reperfusion injury in vivo.
1. Introduction Cerebral ischemia or reperfusion injury usually arises after a stroke, cardiac arrest, or surgical anesthesia and can cause severe damage to the brain including neuronal apoptosis, oxidative stress, inflammation, and neurotoxicity [1]. Cerebral ischemia or reperfusion injury remains a serious global socioeconomic problem due to its high morbidity, mortality, and disability rates [2]. A combination of environmental factors and genes contribute to the development of cerebral ischemia or reperfusion injury; however, the mechanisms underlying cerebral ischemia or reperfusion injury-induced neuronal damage are not completely understood. Therefore more effective and promising candidate targets involved in cerebral ischemia or reperfusion injury need to be identified to help develop novel therapeutic strategies for this disease.
Brahma-related gene 1 (Brg1), a catalytic subunit of SWI2/SNF2like chromatin-remodeling complexes, is an important regulator of gene expression as it interferes with the chromatin architecture of target promoters [3]. Brg1 is found within various multiprotein complexes that regulate DNA replication, repair, recombination, and transcriptional regulation [3]. It has been reported to interact with a diverse group of nuclear proteins including nuclear receptors, transcription factors, and chromatin-modifying enzymes [4]. Therefore Brg1 is involved in multiple cellular processes including cell proliferation, apoptosis, and differentiation [5–7]. Brg1 is implicated in numerous diseases including cancer, liver fibrosis, and cardiac disease [8–12]. Accumulating evidence shows that Brg1 regulates reactive oxygen species (ROS) production and oxidative stress by interacting with nuclear factor erythroid 2-related factor 2 (Nrf2) [13,14]. Importantly,
Abbreviations: Brg1, Brahma-related gene 1; HO-1, heme oxygenase-1; Nrf2, nuclear factor erythroid 2-related factor 2; OGD/R, oxygen-glucose deprivation/ reoxygenation; ROS, reactive oxygen species ⁎ Corresponding author. E-mail address:
[email protected] (F. Li). https://doi.org/10.1016/j.biopha.2018.09.144 Received 24 May 2018; Received in revised form 19 September 2018; Accepted 26 September 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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Fig. 1. Brg1 expression is altered in neurons in response to OGD/R treatment. Relative mRNA (A) and protein (B) expression levels of Brg1 in HT22 neurons were determined by RT-qPCR and western blotting, respectively. HT22 neurons were subjected to OGD for 8 h and then reoxygenation for 6, 12, and 24 h. Cells cultured under normoxic conditions were used as the control group. Effect of OGD/R treatment on mRNA (C) and protein (D) expression level of Brg1 in primary mouse hippocampal neurons was detected by RT-qPCR and western blotting, respectively. *P < 0.05 versus control.
and reduced ROS production in neurons, while Brg1 knockdown significantly inhibited cell viability, increased apoptosis, and promoted ROS production in neurons following OGD/R treatment. Moreover, Brg1 promoted the nuclear translocation of Nrf2 and increased the expression of HO-1; however, knockdown of Nrf2 or HO-1 significantly abrogated the Brg1-mediated neuroprotective effects. Taken together, these results demonstrate that Brg1 ameliorated OGD/R-induced neuronal injury in vitro by promoting the activation of Nrf2/HO-1 signaling.
several studies have shown that Brg1 exerts protective roles in organ (i.e. liver and heart) ischemia/reperfusion injury [10,15]. Nrf2 is a master transcription factor that regulates the cellular defense against oxidative stress [16]. Under oxidative stress, Nrf2 is stabilized and translocated into the nucleus where it subsequently activates the transcription of antioxidant enzymes including heme oxygenase-1 (HO-1), S-transferase, superoxide dismutase, and glutathione peroxidase [17]. Nrf2 was suggested as a critical regulator of cerebral ischemia/reperfusion injury, and its expression is altered during the induction of cerebral ischemia/reperfusion injury in vivo [18]. Loss of Nrf2 accelerates cerebral infarction and neurological deficits, while activation of Nrf2 protects against cerebral ischemia/ reperfusion injury [19–21]. Therefore Nrf2 has emerged as a promising therapeutic target for cerebral ischemia/reperfusion injury [22]. Excessive ROS production is considered as one of the leading causes of brain damage during cerebral ischemia or reperfusion injury [23]. Considering the regulatory effect of Brg1 on ROS production, it was hypothesized that Brg1 may play an important role in cerebral ischemia/reperfusion injury. Herein, we used oxygen-glucose deprivation/reoxygenation (OGD/R)-treated neurons, an in vitro model of cerebral ischemia and reperfusion injury, to investigate the biological function of Brg1 in regulating neuronal apoptosis and oxidative stress. The results showed that OGD/R treatment induced a significant decrease in Brg1 expression in neurons. Functional studies showed that overexpression of Brg-1 increased cell viability, attenuated apoptosis,
2. Materials and methods 2.1. Cell culture HT22, a murine hippocampal neuron line, was purchased from BeNa Culture Collection (Kunshan, China) and cultured according to the manufacturer’s recommended method. In brief, cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/ streptomycin, 4.5 g/L glucose plus glutamine, and sodium pyruvate. Primary mouse hippocampal neurons were isolated from postnatal 1–2 day old C57BL/6 mice according to the protocols previously described [24]. Briefly, hippocampal tissues were dissected into ice-cold Hank’s balanced salt solution (HBSS) and digested in 0.125% trypsin-EDTA solution at 37 °C for 15 min followed by incubation with DMEM/F12 1217
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Fig. 2. Brg1 protects neurons from OGD/R-induced injury in vitro. HT22 neurons were transfected with a Brg1 expression vector (Brg1) or empty vector (vector) for 24 h and then subjected to 24-h OGD/R treatment. (A) Brg1 protein expression was detected by western blotting. (B) The effect of Brg1 overexpression on cell viability was detected using the MTT assay. (C) The effect of Brg1 overexpression on cell apoptosis was assessed using the TUNEL assay. (D) The effect of Brg1 overexpression on ROS levels was determined using DCFH-DA. *P < 0.05.
95% air) at 37 °C for 6–24 h.
containing 10% FBS. Then, the tissues were triturated by pipetting and filtered through a 70-mm nylon cell strainer. Afterwards, the dissociated cells were re-suspended in fresh DMEM/F12 medium containing 10% FBS and 1% penicillin/streptomycin. After culture for 4 h, cells were maintained in Neurobasal medium with 2% B27 supplement, 4.5 g/L glucose, 0.5 mM L-glutamine and 1% penicillin/streptomycin. Cells were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37 °C.
2.3. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis Total RNA was isolated from samples using Trizol reagent (Invitrogen) following the manufacturer’s instructions. Synthesis of cDNA from total RNA was performed using MultiScribe Reverse Transcriptase (Applied Biosystems, Foster City, CA, USA). PCR amplification of cDNA was performed using Power SYBR Green PCR Master Mix (Applied Biosystems) on a 7900 H T Real-Time PCR System (Applied Biosystems). The thermal cycling protocols used were 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The primer sequences used in this study were: Brg1 forward 5′-AGATGGA GTAGCCCTTAGCA-3′ and reverse 5′-GAGGTCCCCTCTCTAGACA GTT-3′; Nrf2 forward 5′-ACAGTGCTCCTATGCGTGAA-3′ and reverse 5′-GAGCCTCTAAGCGGCTTGAA-3′; HO-1 forward 5′-TGCTAGCCTGGT GCAAGATA-3′ and reverse 5′-GCCAACAGGAAGCTGAGAGT-3′; and GAPDH forward 5′-GGCCTCCAAGGAGTAAGAAA-3′ and reverse 5′-GCCCCTCCTGTTATTATGG-3′. GAPDH was used as the internal control gene for normalization of gene expression. Relative gene expression was analyzed using the 2−ΔΔCt method.
2.2. Cell transfection and OGD/R induction The siRNAs targeting Brg1, Nrf2, and HO-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and transfected into cells as per the manufacturer’s instructions. The ORF of Brg1 cDNA was inserted into a pcDNA3.1 vector to obtain the Brg1 expression vector. The vector was transfected into cells using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, USA) as per the manufacturer’s instructions. The OGD/R protocol was performed to mimic in vitro ischemia/reperfusion-like conditions. In brief, cells were washed twice with PBS before being incubated in glucose-free DMEM in a hypoxic chamber (1% O2, 5% CO2, and 94% N2) at 37 °C for 8 h. The cells were then incubated in normal media under normoxic conditions (5% CO2 and 1218
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Fig. 3. Silencing of Brg1 enhances apoptosis and oxidative stress in OGD/R-treated neurons. HT22 neurons were transfected with Brg1-targeting siRNA or a negative control (NC) siRNA for 24 h and then subjected to 24-h OGD/R treatment. (A) Brg1 protein expression was examined by western blotting. (B) The effect of Brg1 silencing on cell viability was determined using the MTT assay. (C) The effect of Brg1 silencing on cell apoptosis was detected using the TUNEL assay. (D) The effect of Brg1 silencing on ROS production was detected using DCFH-DA. *P < 0.05.
and cultured overnight. After the indicated procedures, 10 μl of MTT solution (5 mg/mL) was added to each well and the cells were incubated for 4 h at 37 °C. The formazan crystals were then solubilized by DMSO (150 μl/well). The absorbance at 570 nm was read using a microplate reader (Bio-Rad, Sunnyvale, CA, USA), and the absorbance values of control cells were set at 100%.
2.4. Western blot analysis Total protein was extracted from cells using cell lysis buffer (Beyotime Biotechnology, Haimen, China), and nuclear fractions were extracted using a protein extraction kit (Beyotime Biotechnology) according to the manufacturer’s protocols. Protein concentration was quantified using a bicinchoninic acid method. Equal amounts of proteins were loaded and electrophoresed on a 10% SDS polyacrylamide gel. The separated proteins were subsequently transferred to a PVDF membrane, which was blocked with 5% nonfat milk in TBST for 45 min at 37 °C. Afterward, the membrane was incubated overnight at 4 °C with primary antibodies against Brg1 (Abcam, Cambridge, MA, USA), Nrf2 (Abcam), Lamin B2 (Abcam), HO-1 (Santa Cruz Biotechnology), and GAPDH (Santa Cruz Biotechnology). After washing with TBST, the membrane was incubated with secondary antibodies (Abcam) at room temperature for 1 h. Protein bands were detected using enhanced chemiluminescence reagents (GE Healthcare BioSciences, Pittsburgh, PA, USA). Images were analyzed using a densitometer and Image-Pro Plus 6.0 software.
2.6. Detection of cell apoptosis Cell apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. Cells on glass slides were fixed with 4% paraformaldehyde for 30 min and permeabilized by 0.3% Triton X-100 for 5 min at room temperature. Afterward, each sample was incubated with 50 μl of TUNEL detection solution for 1 h at 37 °C in the dark. TUNEL-positive cells were counted under a fluorescence microscope. 2.7. Measurement of ROS production ROS production was measured using 6-carboxy-2070-dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma, St. Louis, MO, USA). After the indicated treatments, 100 μl of cell samples were washed with PBS and stained with DCFH-DA (10 μM) for 30 min at 37 °C in the dark. The fluorescence intensity was measured using a fluorescence spectrophotometer.
2.5. Measurement of cell viability Cell viability was detected using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. In brief, cells were seeded into 96-well plates at a density of 5 × 103 cells/well 1219
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Fig. 4. Brg1 mediates Nrf2/HO-1 signaling in OGD/R-treated neurons. (A) The effect of Brg1 overexpression on nuclear Nrf2 protein expression was detected by western blotting. Lamin B2 was used as the loading control. The effect of Brg1 overexpression on HO-1 mRNA (B) and protein (C) expression was detected by RTqPCR and western blotting, respectively. (D) The effect of Brg1 silencing on nuclear Nrf2 protein expression was examined by western blotting. The effect of Brg1 silencing on HO-1 mRNA (E) and protein (F) expression was detected by RT-qPCR and western blotting, respectively. *P < 0.05.
overexpression of Brg1. The results showed that transfection of the Brg1 expression vector significantly upregulated Brg1 expression in HT22 neurons (Fig. 2A). Interestingly, Brg1 overexpression significantly recovered the cell viability impaired by OGD/R treatment (Fig. 2B). Moreover, the TUNEL assay showed that Brg1 overexpression markedly suppressed OGD/R-induced apoptosis (Fig. 2C). In addition, OGD/Rinduced ROS production in neurons was also significantly reduced by Brg1 overexpression (Fig. 2D). To confirm that Brg1 confers neuroprotection effect under OGD/R conditions, we detected the effect of Brg1 overexpression on OGD/R-induced injury in primary mouse hippocampal neurons. The results showed that Brg1 overexpression significantly attenuated OGD/R-induced apoptosis and ROS production in primary mouse hippocampal neurons (Supplemental Fig. 2). These results suggest that Brg1 overexpression can improve cell viability and inhibit cell apoptosis and ROS production in OGD/R-treated neurons.
2.8. Statistical analysis Results are expressed as the mean ± standard deviation. Statistical analyses were performed using SPSS software (version 19.0; SPSS Inc., Chicago, IL, USA). Differences were determined using a one-way analysis of variance or Student’s t-test. Differences were regarded statistically significant when P < 0.05. 3. Results 3.1. Effect of OGD/R treatment on Brg1 expression in neurons To investigate the potential relevance of Brg1 in OGD/R-treated neurons, we examined the expression of Brg1 in response to OGD/R treatment. Results showed that OGD/R treatment significantly decreased the viability of HT22 neurons in a time-dependent manner (Supplemental Fig. 1). We found that Brg1 expression was significantly increased in HT22 neurons in response to 6-h OGD/R treatment but gradually decreased after 12- and 24-h OGD/R treatment (Fig. 1A and B). Consistently, similar results were obtained using primary mouse hippocampal neurons (Fig. 1C and D). These data indicate that Brg1 may play an important role in OGD/R-treated neurons.
3.3. Loss of Brg1 accelerates OGD/R-induced neuronal injury To validate the protective role of Brg1 in OGD/R-treated neurons, loss-of-function experiments were performed using Brg1-targeting siRNAs. The results showed that transfection of Brg1-targeting siRNAs significantly decreased the expression of Brg1 (Fig. 3A). Silencing of Brg1 aggravated the decreased cell viability (Fig. 3B) and enhanced apoptosis (Fig. 3C) and ROS production (Fig. 3D) in OGD/R-treated neurons. Consistently, similar effects mediated by Brg1 knockdown were confirmed in primary mouse hippocampal neurons (Supplemental Fig. 3). These results suggest that loss of Brg1 accelerates OGD/R-
3.2. Brg1 overexpression protects neurons from OGD/R-induced injury To investigate the exact biological function of Brg1 in OGD/Rtreated neurons, we performed gain-of-function experiments by 1220
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Fig. 5. Knockdown of Nrf2 blocks Brg1-mediated neuroprotective effects. HT22 neurons were co-transfected with a Brg1 expression vector and Nrf2 siRNA for 24 h before they were subjected to 24-h OGD/R treatment. (A) Nrf2 protein expression was detected using western blotting. (B) Cell viability was detected using the MTT assay. (C) ROS production was assessed by DCFH-DA. (D) HO-1 protein expression was detected using western blotting. *P < 0.05.
activation of Nrf2, we detected the effect of Nrf2 silencing on Brg1mediated neuroprotective effects. Silencing of Nrf2 significantly blocked the regulatory effect of Brg1 overexpression on cell viability, ROS production, and HO-1 expression (Fig. 5A–D). These results indicate that Brg1 protects neurons against OGD/R in a Nrf2-dependent manner.
induced neuronal injury. 3.4. Brg1 regulates Nrf2 nuclear translocation and HO-1 expression Brg1 was reported to antagonize apoptosis and oxidative stress through the activation of Nrf2/HO-1 signaling [13]. Therefore it was hypothesized that Brg1 attenuated OGD/R-induced injury by activating Nrf2/HO-1 signaling. The results showed that Brg1 overexpression significantly promoted nuclear Nrf2 protein expression (Fig. 4A). We found that HO-1 expression was increased in HT22 neurons following OGD/R treatment (Supplemental. Fig. 4). Furthermore, Brg1 overexpression further increased the mRNA and protein expression of HO-1 in OGD/R-treated neurons (Fig. 4B and C). In contrast, Brg1 silencing showed the opposite effect (Fig. 4D–F). These results suggest that Brg1 regulates Nrf2/HO-1 signaling in OGD/R-treated neurons.
3.6. HO-1 knockdown abolishes Brg1-mediated neuroprotective effects To further confirm whether Nrf2/HO-1 signaling was involved in Brg1-mediated neuroprotective effects, the effect of HO-1 silencing on Brg1-mediated neuroprotective effects was assessed. The results showed that HO-1 knockdown significantly reversed the regulatory effect of Brg1 overexpression on cell viability and ROS production (Fig. 6A–C). These results suggest that HO-1 is essential for Brg1mediated neuroprotective effects.
3.5. Nrf2 knockdown blocks Brg1-mediated neuroprotective effects To investigate whether Brg1 inhibits OGD/R-induced injury by 1221
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Fig. 6. HO-1 knockdown abolishes Brg1-mediated neuroprotective effects. HT22 neurons were co-transfected with a Brg1 expression vector and HO-1 siRNA for 24 h before they were subjected to 24-h OGD/R treatment. (A) Ho-1 protein expression was detected using western blotting. (B) Cell viability was detected using the MTT assay. (C) ROS production was assessed by DCFH-DA. *P < 0.05. (D) Schematic presentation of the role of Brg1 in regulating Nrf2/HO-1 signaling in response to OGD/R treatment in neurons. Brg1 increases the nuclear translocation of Nrf2 and interacts with Nrf2 at the promoter region of HO-1, which facilitates the transcription of HO-1 and leads to protective effects against OGD/R-induced apoptosis and oxidative stress in neurons.
4. Discussion
cells [33,34]. In the present study, Brg1 overexpression improved cell viability and inhibited the OGD/R treatment-induced apoptosis of neurons. These results support an antiapoptotic role of Brg1; however, the precise role of Brg1 may be disparate in different cell types and contexts. Brg1 is reported to regulate gene expression by mediating critical interactions with nuclear receptors and transcription factors [35,36]. Intriguingly, recent studies demonstrated that Brg1 is capable of interacting with Nrf2 to activate antioxidant signaling [13,37]. Brg1 is reported to interact with Nrf2 to promote the recruitment of RNA polymerase II to the HO-1 promoter, thus activating the expression of HO-1 [13,38]. Nrf2/HO-1 activation accelerates the clearance of excessive ROS and relieves oxidative stress in pathological processes. Notably, Brg1-mediated Nrf2/HO-1 signaling was reported in various pathological processes. Brg1 overexpression alleviates hepatic ischemia/reperfusion injury by activating Nrf2/HO-1 signaling [39], while Brg1 knockdown inhibits HO-1 expression in hepatocytes with hypoxia or reoxygenation treatment [10]. Moreover, Brg1 overexpression alleviates acute lung injury in vivo by upregulating the expression and nuclear translocation of Nrf2 [40]. Adiponectin was shown to inhibit hyperglycemia-induced oxidative stress, myocardial apoptosis, cardiac hypertrophy, and cardiac dysfunction by promoting Brg1 and Nrf2/HO-1 signaling [14]. Diabetes and hyperglycemia-induced dysregulation of Brg1 significantly blocked the cardioprotective effects of sevoflurane postconditioning or emulsified isoflurane postconditioning-mediated activation of Nrf2/HO-1 signaling [15,41]. Consistent with these findings, the results of this study demonstrate that Brg1 overexpression inhibits OGD/R-mediated ROS production in association with the activation of Nrf2/HO-1 signaling. Overexpression of
This study provides compelling evidence that Brg1 plays an important role in regulating OGD/R-induced neuronal injury. The results demonstrate that Brg1 overexpression prevented OGD/R-induced apoptosis and ROS production in neurons in vitro. Furthermore, the underlying mechanism was associated with its regulatory effect on Nrf2/HO-1 signaling (Fig. 6D). Therefore Brg1-mediated Nrf2/HO-1 signaling may be critically involved in regulating OGD/R-induced neuronal injury. Accumulating evidence has suggested that Brg1 functions as a transcriptional coregulator that is implicated in the activation and suppression of gene expression via the modulation of chromatin in various cellular processes [3]. Singh et al. reported that Brg1 enables early embryo growth by inhibiting genes that induce apoptosis and cell growth arrest [6]. Brg1 promotes the proliferation and survival of various cancer cells by regulating the expression of HIF-1α/VEGF-A, SMAD6, AKT, BCL-2, and cyclin proteins [12,25–27]. Brg1 overexpression increases the viability and proliferation of primary myoblasts by upregulating the expression of PAX7 [28]. Brg1 also promotes myocardial proliferation and regeneration by inhibiting cyclin-dependent kinase inhibitors [29] and inhibits apoptosis and senescence in cancer cells by suppressing p53 and PTEN [30,31]. These findings suggest that Brg1 functions as a positive regulator of cell survival. However, there are also studies that demonstrate a proapoptotic role of Brg1; for example, one study reported that Brg1 promoted the apoptosis of human rheumatoid fibroblast-like synoviocytes by interacting with and promoting p53 [32]. Brg1 has also been suggested to be a tumor suppressor that induces growth arrest and apoptosis in various cancer 1222
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Appendix A. Supplementary data
Brg1 increased Nrf2 nuclear translocation and HO-1 expression. Notably, Nrf2 knockdown significantly blocked the inhibitory effect of Brg1 on apoptosis and ROS production, thus indicating that Brg1mediated neuroprotective effects are Nrf2 dependent. Our study suggests that Brg1 may protect against ischemia/reperfusion injury in the brain by promoting Nrf2/HO-1 signaling in cerebral, which is same as its molecular mechanism in heart. Nrf2/HO-1 signaling has becoming as a promising therapeutic target for treatment of cerebral ischemia/reperfusion injury. Activation of Nrf2/HO-1 signaling is involved in neuroprotective drugs-mediated protection effect against cerebral ischemia/reperfusion injury [42,43]. The Nrf2 activator, tert-Butylhydroquinone, has been shown to protects against oxidative stress and reduces infarct size and neurological deficits during cerebral ischemia/reperfusion injury [19,20]. HO-1 is a cytoprotective protein that prevents apoptosis and oxidative stress caused by various pathological stimuli [44]. Both mRNA and protein expression of HO-1 are induced in brain following ischemia and reperfusion injury [45–47]. Overexpression of HO-1 has been shown to attenuate neuron apoptosis and brain damage after cerebral ischemia/ reperfusion injury [48–50]. Importantly, the results from this study showed that HO-1 knockdown partially reversed the Brg1-mediated neuroprotective effects, thus indicating that HO-1 contributes to Brg1mediated antioxidant reactions. Collectively, our study provides evidence that Brg1 may serve as a promising target to activate Nrf2/HO-1 signaling for treatment of cerebral ischemia/reperfusion injury. Brg1 plays an important role in the nervous system. Central nervous system-specific knockout of Brg1 induced growth arrest and neuronal degeneration in mice [51]. Moreover, Brg1 also regulates the differentiation of neural stem cells and neurogenesis [52]. However, whether Brg1 is involved in regulating neuronal injury during cerebral ischemia or reperfusion injury remains poorly understood. This study found that Brg1 was essential for neuronal survival during OGD/R treatment, which provided in vitro ischemia or reperfusion-like conditions. Consistently, a recent study reported that Brg1 expression is controlled by miR-144-3p, which is involved in attenuating OGD/R-induced neuronal injury [53]. Therefore Brg1 may play a role in cerebral ischemia or reperfusion injury in vivo. Certain limitations of our study should be noted. Although the direct regulatory mechanism of Brg1 on HO-1 expression has been well documented, our study doesn’t provide direct evidence showing Brg1 directly interacts with HO-1 to promote HO-1 expression. Therefore, the direct regulatory mechanism of Brg1 on HO-1 expression in neurons remains to be determined. Many questions remain concerning the function of Brg1. For instance, how Brg1 is regulated under the OGD/R conditions? Interestingly, recent studies have shown that Brg1 expression is controlled by miRNAs under various conditions [53–55]. Considering that miRNAs are dysregulated under OGD/R conditions, we can speculate that the altered expression of Brg1 under OGD/R conditions may be caused by dysregulated miRNAs in neurons. Therefore, the regulation of Brg1 under OGD/R conditions in neurons requires further investigation. In conclusion, these results demonstrate that OGD/R induced neuronal injury and was accompanied by decreased Brg1 expression. Brg1 overexpression protected neurons from OGD/R injury by activating Nrf2 and its downstream gene, HO-1. These in vitro data indicate that Brg1 may be involved in regulating cerebral ischemia/reperfusion injury in vivo. Brg1 overexpression to activate Nrf2/HO-1-mediated antioxidant signaling may be a potential therapy to alleviate neuronal injury during cerebral ischemia/reperfusion injury; however, the precise role and regulatory mechanism of Brg1/Nrf2/HO-1 in cerebral ischemia/reperfusion injury require further in vivo investigation using animal models.
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