AG-490 Western blot results showed that the expressions

Western blot results showed that the expressions of SOCS1 and SOCS3 significantly increased after administration of rGas6 24h after MCAO (Fig. 7A–C p<0.05 vs. MCAO+Vehicle). The expressions of TRAF3, TRAF6 and proinflammatory cytokines IL-1β, IL-6 and TNF-α were significantly decreased after rGas6 treatment (Fig. 7A, D–H p<0.05 vs. MCAO+Vehicle). SOCS1-siRNA and SOCS3-siRNA were injected ICV 48h before MCAO, and rats were treated with rGas6, SOCS1-siRNA and SOCS3-siRNA abolished the effects of rGas6, leading to increased expression of TRAF3 and TRAF6 (Fig. 7A, D, E p<0.05 vs. Gas6). R428 also reduced the expression of STAT1, SOCS1 and SOCS3, and increased the levels of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) by lowering the inhibition of TRAF3 and TRAF6 (Fig. 7A–H p<0.05 vs. Gas6).
Discussion After ischemic stroke, inflammation has been shown to be an important secondary injury mediator in patients, as well as in animals (Herz et al., 2014, Jin et al., 2010). Following AG-490 ischemia, microglia become activated, producing a significant production of neurotoxic molecules and proinflammatory cytokines, causing additional brain damage (Herz et al., 2014). Targeting and preventing inflammation would seem to be a logical therapeutic goal for limiting cerebral infarct volume and neurological dysfunction after ischemic stroke. In this study, we investigated the role of rGas6 in preventing neuroinflammation after MCAO. Herein, we showed that rGas6 treatment of MCAO reduces brain infarction, improves short-term neurological scores, as well as long-term functional behavior (spatial learning, memory abilities and movement coordination). We also found that rGas6 provided its anti-inflammatory effects by suppressing inflammation via inhibition of the TLR/TRAF/NF-κB signaling pathway at the level of TRAF. Our results suggest that Gas6 may be a potential treatment for ischemic stroke patients. Growth arrest specific protein 6 (Gas6) is a known activator of TAM receptors (Tyro3, Axl and Mer) (Stitt et al., 1995), which decrease inflammatory response (Rothlin et al., 2007), and also stimulate cell growth, inhibit apoptosis, mediate efferocytosis, and stimulate hemostasis (Goruppi et al., 1996, Anderson et al., 2003). Gas6 can activate all TAM receptors, but it has the greatest affinity for Axl (Axl>Tyro3>>>Mer). Our double immunofluorescent staining of Gas6 and Axl with NeuN, GFAP, and Iba-1 demonstrated that Gas6 and Axl were positively expressed by neurons, astrocytes and microglia/macrophages. Interestingly, after MCAO, the expression of Gas6 and Axl was increased in microglia/macrophages but not neurons or astrocytes. Microglia, which are mediators of neuroinflammation, are mobilized in response to many CNS injuries, and play a dual role in inflammation; microglia can resolve and also exacerbate CNS disease (Ginhoux et al., 2010). Previous studies have also shown Gas6 expression on several cell types within the rat central nervous system and also Axl expression by microglia (Prieto et al., 1999, Ji et al., 2013). During inflammation after brain ischemia-reperfusion injury, Gas6 and Axl expression was briefly elevated before reducing to levels lower than baseline values after MCAO; as a result, the expression of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) were higher after MCAO than sham surgery. Following injury, the inflammatory cascade includes release of inflammatory cytokines and neutrophil infiltration into the ischemic brain, which can exacerbate the pathogenesis of cerebral ischemic injury (Terao et al., 2008). The high levels of IL-1β, IL-6 and TNF-α have been well recognized as a crucial factor in contributing to organ injury after ischemia-reperfusion (Kinsey et al., 2008). Known as an innate immune regulator, Axl is not active during until inflammation occurs, after which Axl becomes a mediator of inflammation by damping the inflammatory response and maintaining immune system homeostasis (Lemke and Rothlin, 2008). In this study, we observed that rGas6 treatment can lead to neurobehavioral improvement, as well as amelioration of brain infarction after MCAO in short and long-term experiments; these findings are consistent with other experimental inflammatory models (Giangola et al., 2013, Llacuna et al., 2010). The study by Yan Shen et al. demonstrated an inhibitory effect by Gas6 on acute inflammatory cytokine secretion by macrophages (Shen et al., 2016). Another study, in a sepsis model, confirmed that Gas6 is an anti-inflammatory agent and can prevent organ dysfunction (Giangola et al., 2013).