Then we try to provide evidence to show that the

Then, we try to provide evidence to show that the mechanism of GLT-1 down-regulation after lethal OGD may be related to the activation of p38 MAPK in astrocyte-neuron co-cultures. Firstly, it was observed that p-p38 MAPK expression in astrocytes was significantly increased after lethal OGD by western blot in the present study. There is increasing evidence showed that p38 MAPK activation induced by lethal OGD played a critical role in triggering cell death (Lu et al., 2011; Nito et al., 2012; Yang et al., 2016). For example, neonatal rat hippocampal slice cultures exposed to OGD led to p38 MAPK activation that preceded increases in superoxide generation and neuronal death (Lu et al., 2011). The p38 MAPK activation in rat primary-cultured hippocampal neurons induced by OGD significantly reduced neuron viability and effectively caused LDH release (Zhao et al., 2018). Secondly and notably, our present study showed that decreasing p38 MAPK function and expression in astrocytes, by pretreatment with SB203580 or p38 MAPK siRNA, dose dependently suppressed the neuronal injury induced by lethal OGD. Feng et al found that depressing p38 MAPK function in neurons, rather than astrocytes, by SB203580 also prevented necroptosis induced by OGD in primary cortical neurons (Feng et al., 2017). Inhibition of p38α MAPK with SB203580 or its siRNA attenuated the OGD-induced motor neuron cell death (Guo and Bhat, 2007). However, much more other studies reported that p38 MAPK activation protected neurons that underwent ischemic preconditioning both in organs being preconditioned (Guan et al., 2016, 2014; Maslov and Lishmanov, 2012; Zhang et al., 2017) and in organs far from the preconditioned region (Sun et al., 2010). Furthermore, inhibition of p38 MAPK activation via siRNA or its antagonist SB203580 significantly attenuated the protective effect on retinal function induced by ischemic preconditioning in rats (Dreixler et al., 2009), blocked the glutamate receptor ischemic tolerance induced by cerebral ischemic preconditioning (Zhang et al., 2017), and decreased the neuronal protective effect induced by sulbactam or Buyang Huanwu Decoction against lethal OGD (Qi et al., 2018). The discrepancy showed two-edged effects of p38 MAPK on lethal brain ischemia and sublethal brain ischemia. We hypothesized that lethal ischemia may cause excessive activation of p38 MAPK which was involved in cerebral ischemic injury, while sublethal brain ischemia may cause moderate activation of p38 MAPK which usually lead to neuroprotection. To verify this assumption, we studied the characteristics of p-p38 MAPK expression in lethal OGD and sublethal OGD. Our results (see supplemental information) showed that the p38 MAPK activation lasted to 6 h after sublethal OGD (45-min OGD), and the peak value was 5.58 times of control level. However, p-p38 MAPK up-regulation after lethal OGD (4-h OGD) lasted to at least 48 h that was the end point observed in this study, and the peak value was 12.45 times of control level (Fig. 4 in the present study). This excessive activation of p38 MAPK should be responsible for the neuronal injury induced by lethal OGD. Thirdly, we studied the relationship between excessive activation of p38 MAPK and GLT-1 down-regulation after lethal OGD. Double immunofluorescence clearly showed that p-p38 MAPK and GLT-1 were co-expressed in astrocyte. This provided a possibility that p38 MAPK may regulate GLT-1 after lethal OGD. And our results support this possibility: with pre-administration of SB203580 or p38 MAPK siRNA, the down-regulation of GLT-1 induced by lethal OGD was inverted, and meanwhile the neuronal survival was significantly improved. As a support to this result, a study reported that formaldehyde decreased GLT-1 expression or inhibited glutamate uptake through p38 MAPK activation in cultured astrocytes (Song et al., 2010). Taken together, these findings revealed that the mechanism of GLT-1 down-regulation mediating OGD injury depended on the excessive activation of p38 MAPK.