Biochemical and Biophysical Research Communications
SENP1 inhibits the IH-induced apoptosis and nitric oxide production in BV2 microglial cells
Introduction
Intermittent hypoxia (IH), as a hallmark of obstructive sleep apnea, has been reported that could induce oxidative stress [1] and inflammation [2]. The long-term IH will result in neuronal cell death [3] and eventually central nervous system (CNS) degeneration. CNS impairments arising from neuronal apoptosis in the learning and memory regions of the brain bring to significant cognitive and behavioral deficits [1], [4], [5]. Several studies have focused on the roles of IH in multiple pathophysiological processes. However, the molecular mechanisms underlying the IH-induced neuronal apoptosis remain largely unknown.
It has been reported that IH induces the activation of microglial cells [5]. Microglial cells in CNS are macrophage-like resident immune cells, which can be activated upon trauma, stroke and infection through responding to various cellular factors, including cytokines, chemokines, nitric oxide (NO), and reactive oxygen intermediates [6], [7], [8]. Microglial cells show a ‘resting’ phenotype characterized by ramified morphology in the healthy adult CNS [9]. It plays important role in the maintenance and resolution of brain tissue homeostasis [10], as well as clearance of dead cells and secretion of neurotrophins in the normal conditions [11]. While in the pathological conditions, microglial cells are activated for increasing the production of inflammatory cytokines [12]. Further, the hyper-activated microglial cells will activate the surrounding normal microglia and propagate the production of pro-inflammatory factors via a feed-forward manner [13]. Besides, IH induces the expression of nitric oxide synthase (iNOS) to produce reactive nitrogen species and abnormal high levels of glutamate, which ultimately result in excitotoxicity in CNS especially in hippocampal neurons [5]. As reported, microglial cells are major sources of iNOS in the CNS neuroinflammation [14]. Additionally, iNOS has been shown that was expressed in microglia upon ischemia-induced hypoxia [12]. Taken together, identification of compounds that modulate microglial reaction under pathological conditions is highly desirable for the development of therapeutic agents [10].
SUMOylation has been shown as a modulator of hypoxic response [15]. Sentrin-specific proteases (SENP)s are proteases that participate in the regulation of SUMOylation by generating mature small ubiquitin-related modifiers (SUMO) for protein conjugation (endopeptidase activity) and removing conjugated SUMO from targets (isopeptidase activity) [16]. So it's essential to reveal SUMOylation and the roles of SENPs in microglial cells under IH condition. Hence, uncovering the potential roles of SENPs in IH conditions would provide more intensive view of understanding the mechanisms of IH-induced CNS damage. In this study, we firstly detected the expression levels of SENPs in microglial cells under IH and normoxia conditions. We found that SENP1 was significantly down-regulated in cells exposure to IH. Subsequently, we detected the effect of IH for the activation of microglia and investigated the potential roles of SENP1 in the SENP1-overexpressing cell lines. We found the apoptosis-inducing and activating role of IH on microglia. Additionally, the effect of IH on BV-2 including apoptosis, iNOS expression and NO induction can be attenuated by SENP1 overexpression. The results of the present study are of both theoretical and therapeutic significance to explore the potential roles of SENP1 under IH condition and elucidated the mechanisms underlying microglial survival and activation.
Section snippets
Cell culture
BV2 microglia cells were gifts from Professor Zeng-qiang Yuan (State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences). The cells were plated onto 6-well cell culture plates in Dulbecco's Modified Eagle Medium (Gibco, Grand Island, NY, USA) containing 1% penicillin and streptomycin (Invitrogen, Carlsbad, CA, USA) and 10% fetal bovine serum (Gibco, Grand Island, NY, USA) in an incubator with 5% CO2 at 37 °C. Cells were fed with fresh medium for
SENP1 is downregulated in BV2 cells exposure to IH
We detected the expression levels of SENPs in BV2 cells treated in different conditions: IH condition and normoxia condition for 8 h via RT-PCR assay. As shown in the Fig. 1A, the expression of SENP1, rather than other SENPs, was significantly decreased by IH (the relative expression levels were decreased from 1 to 0.22 ± 0.08) compared to the normoxia condition. The following Western blotting assay also showed that the levels of SENP1 were decreased in BV2 under IH condition (the normalized
Discussion
In the present study, we revealed that IH triggers the expression of iNOS, as well as the production of NO in microglial cells. Since iNOS was expressed only in microglia cells under inflammatory conditions, the release of iNOS was considered as a significant marker of microglial activation [12]. High expression levels of iNOS in microglia causes NO inhibition of neuronal respiration and results in neuronal depolarization and glutamate release, which induce neuronal death and CNS dysfunction [5]
Acknowledgments
This work was supported by the Science Foundation of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. We thanks to Prof. Dr. Jin-ke Cheng (Department of Biochemistry and Molecular Cell Biology, Shanghai Jiao Tong University School of Medicine) for his assistance in this study.
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