Full length articleRole of interleukin-10 in the neuroprotective effect of the Angiotensin Type 2 Receptor agonist, compound 21, after ischemia/reperfusion injury
Introduction
Angiotensin type 2 (AT2R) receptor stimulation with the non-peptide agonist, compound 21 (C21), has been shown to provide neuroprotection and functional recovery after experimental ischemic stroke in rodents (Alhusban et al., 2015, Joseph et al., 2014, McCarthy et al., 2014, Min et al., 2014). C21 reduced infarct size after both permanent and temporary middle cerebral artery occlusion (MCAO). In addition, C21 reduced neuronal apoptosis and decreased mortality after stroke (Schwengel et al., 2016). Nevertheless, the mechanism underlying C21-mediated neuroprotection is still not known. In our hands, we reported an upregulation of the neuroprotective cytokine, interleukin (IL)ā10 with C21 treatment at 24Ā h in the ischemic hemisphere after 3Ā h MCAO. Moreover, we reported an increase in the number of IL-10 positive cells at 7 days with a single dose of C21 after 90-min MCAO (Alhusban et al., 2015). Whether the increased IL-10 was causally related to the improved outcome remained unknown.
IL-10 is an anti-inflammatory cytokine that mediates its actions through activation of the JAK1-STAT3 (Janus Kinase 1 - Signal Transducer and Activator of Transcription 3) signaling pathway (Sabat et al., 2010). IL-10 has been shown to provide direct neuroprotection in vivo and in vitro. Administration of exogenous IL-10 centrally and systemically decreases the infarct size in rats after permanent focal ischemia (Spera et al., 1998), while IL-10 knockout mice show larger infarct volume following middle cerebral artery occlusion (Grilli et al., 2000). Moreover, post-ischemic IL-10 gene transfer attenuates brain infarction in rats subjected to focal and global ischemia (Ooboshi et al., 2005). Interestingly, neuroprotection by systemic immune cells such as regulatory T and B cells have also been shown to be mediated through IL-10 production (Bodhankar et al., 2013, 2014; Liesz et al., 2009; Liesz et al., 2013). In vitro, IL-10 protects murine cortical and cerebellar neurons from excitotoxic damage and oxygen glucose deprivation (OGD) by activating phosphatidylinositide 3-kinases (PI-3K) and signal transducer and activator of transcription 3 (STAT-3) pathways (Bachis et al., 2001, Grilli et al., 2000, Sharma et al., 2011).
Studies examining the protective role of AT2R stimulation using in vitro neuronal injury models are inconclusive. The peptide AT2R agonist, CGP-42112, but not C21, protected primary cortical neurons against glucose deprivation (Lee et al., 2012). In addition, Wu et al. showed neuroprotection with angiotensin type 1 (AT1R) receptor blockers (ARBs) but not CGP-42112 pretreatment against OGD/reoxygenation injury (Wu et al., 2010); these effects involved AT1R blockade and not indirect AT2R stimulation (Wang et al., 2014, Wu et al., 2010).
In this study, we aimed to determine the contribution of IL-10 to the neuroprotective effect of C21 using temporary MCAO in vivo. In addition, we tested the direct neuroprotective effect of C21 in vitro in an ischemia/reoxygenation injury model.
Section snippets
Materials and methods
Experiments were approved by the Care of Experimental Animal Committee of Augusta University/Institutional Animal Care and Use Committee (IACUC) of the Charlie Norwood Veterans Affairs Medical Center, Augusta, GA.
IL-10 is involved in C21-mediated neuroprotection in vivo
We have previously shown that C21 provides neuroprotection and IL-10 upregulation after stroke (Alhusban et al., 2015). To examine the involvement of IL-10 in C21-mediated neuroprotection, Wistar rats were subjected to experimental conditions outlined in (Fig. 1.A) as described in the methods section. As we have previously reported, C21 treated rats showed a reduction in infarct size compared to saline treatment. IL-10 neutralization abrogated the neuroprotective effect of C21 supporting the
Discussion
Our data prove a causal role of IL-10 in the neuroprotective and anti-inflammatory effects of C21 after temporary MCAO in vivo. In addition, the current results show direct neuroprotection with C21 treatment after OGD/reoxygenation injury in vitro.
Recent reports have pointed out the involvement of IL-10 in C21-mediated beneficial effects in renal inflammation, and myocardial ischemia models through AT2R stimulation (Curato et al., 2010, Dhande et al., 2013, Namsolleck et al., 2013). C21 has
Conflict of interest
The authors declare no conflict of interest. The contents do not represent the views of the Department of Veterans Affairs or the United States Government.
Acknowledgements
This study was supported by Veterans Affairs (VA) Merit Review (BX000891), RO1-NS063965 and R21-NS088016 to SCF. AE is a Research Career Scientist at the Charlie Norwood Veterans Affairs Medical Center in Augusta, Georgia. This work was supported in part by VA Merit Award (BX000347), VA Research Career Scientist Award, and NIH award (R01NS083559) to AE.
The authors thank Vicore Pharma (Gƶteborg, Sweden) for the supply of C21 and AstraZeneca for the supply of candesartan.
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