Sulfiredoxin-1 protects primary cultured astrocytes from ischemia-induced damage
Introduction
Stroke is one of the leading causes of death and results in severe long-term disability in many countries. Both in ischemic and hemorrhagic stroke, the primary insult elicits an inflammatory response that contributes to secondary brain damage. Although many anti-inflammatory approaches have been investigated in animal models, none of these has demonstrated protective efficacy in stroke patients. The focus of these approaches on neuronal-specific mechanisms might be one of the reasons for failure (Barreto et al., 2011). There is now compelling evidence that astrocytes not only influence the immune and inflammatory events that occur in stroke, but also regulate neuronal and microglial inflammatory responses (Kim et al, 2010, Sofroniew, 2014). Thus precisely controlling the duration and extent of the inflammatory responses by astrocytes could be a good strategy in reducing brain damage and improving recovery.
Sulfiredoxin-1 (Srxn1), conserved in eukaryotes, is an endogenous antioxidant protein. It has been suggested to be a potential therapeutic agent for several diseases, including brain disorder (Findlay et al., 2005). Induction of Srxn1 expression contributes to neuroprotective ischemic preconditioning in response to oxygen-glucose deprivation in vitro and following a brief ischemic episode in vivo (Bell et al., 2011). Our previous study showed that Srxn1 can protect PC12 cells from H2O2-induced oxidative stress and is involved in Prdxs activity (Li et al., 2013). Srxn1 catalyzes the adenosine triphosphate (ATP)-dependent reduction of the hyperoxidized sulfonic acid inactive form of 2-Cys peroxiredoxin PrxSO2/3 into sulfonic PrxSOH (Lowther, Haynes, 2011, Soriano et al, 2008, Soriano et al, 2009). It also has a role in the reduction of glutathionylation, a post-translational oxidative modification that occurs on numerous proteins in Parkinson's disease (Findlay et al., 2005). Baek et al. (2012) reported that Srxn1-depleted cells show an activation of mitochondria-mediated apoptotic pathways, including mitochondria membrane potential collapse, cytochrome c release, and caspase activation. Each of these characteristics of Srxn1 theoretically represents protective mechanisms. However, which of these mechanisms accounts for protection by Srxn1 remains unclear. Recently, Srxn1 has been shown to participate in the maintenance of redox homeostasis in macrophages and other inflammatory mediators (Abbas et al., 2011). There is genetic evidence showing that Srxn1 affects the expression of cytokines, chemokines, interleukins, and various inflammatory genes in Srxn1 null mice (Planson et al., 2011). Thus Srxn1 may influence host response to inflammation.
Astrocytes are more resistant to focal cerebral ischemia than neurons. Interestingly, although the baseline level of Srxn1 showed no difference between astrocytes and neurons, the level of Srxn1 when induced is much higher in astrocyte cultures than in neuron cultures (Bell et al, 2011, Gürer et al, 2009, Soriano et al, 2008). These biochemical findings suggest an important role for Srxn1 in reactive astrocytes, which we examined in this study using astrocyte-rich primary cultures. We propose that loss of Srxn1 expression in astrocytes may cause excessive inflammatory responses and thereby contribute to oxygen-glucose deprivation followed by recovery (OGD/R)- and hydrogen peroxide (H2O2)-induced cell death.
Section snippets
Astrocyte-rich primary cultures
Primary cultures were prepared in compliance with animal welfare regulations of the current international laws. The animal protocol was approved by the Institutional Animal Care and Use Committee of Chongqing Medical University, China. All efforts were made to minimize suffering. Astrocyte-rich primary cultures were prepared from Sprague-Dawley rats born 1 to 2 days as described elsewhere (Park et al., 2009). Briefly, cortices were dissociated into a cell suspension using trypsin digestion and
Knockdown of Srxn1 in astrocyte-rich primary cultures
To ascertain whether the Srxn1 mRNA and protein level is reduced in astrocytes upon exposure of cells to siRNA to Srxn1, q-PCR and western blotting were used. Fig. 1A shows q-PCR data for Srxn1 mRNA level in the following experimental groups: Control, Scramble shRNA, Lenti-Srxn1-sh1, Lenti-Srxn1-sh2, Lenti-Srxn1-sh3, and Lenti-Srxn1-sh4. Compared with the Control shRNA group, considerable reduction in Srxn1 is observed in both Lenti-Srxn1-sh3 group and Lenti-Srxn1-sh4 groups (P < 0.05).
Discussion
Inflammation occurs in this necrotic brain tissue, following the breakdown of the blood–brain barrier (BBB) and infiltration of blood immune cells. Post-ischemic inflammation is an essential step in the delayed phase of ischemic injury. Recently, studies have shown that some small molecules may play an important role in brain inflammation (Shichita et al, 2012a, Shichita et al, 2012b). Our present data suggest that Srxn1, an endogenous small protein, may cause excessive astrocyte inflammatory
Acknowledgments
This work was supported by grants from National Nature Science Foundation of China (No. 81271460 and 81301125).
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