3.1. The expression of SPRED1 protein in cerebral cortex is down-regulated after cerebral ischemia
To investigate the role of SPRED1 in cerebral ischemia, we established MCAO model in rats to simulate the ischemia-reperfusion of brain. A filament with silicone tip was inserted into the middle cerebral artery of rats for 60 minutes, and then the filament was removed for reperfusion to form the model[1]. Representative images of brain slices stained with TTC and ischemic infarct area of white part showed the success of MCAO model (Fig. 1A). Neuro-inflammation and oxidative damage were two important reasons for the loss of nerve cells after ischemia-reperfusion of brain [13, 14]. In the cerebral cortex tissue, the activation of iNOS and the down-regulated SOD1、SOD2 indicated the increased levels of inflammation and stress in brain tissue (Fig. 1B). Associated with this, malondialdehyde (MDA), one of the most important products of intracellular lipid peroxidation after oxidative damage [15], was expanded significantly (Fig. 1C). By contrary, the antioxidant enzyme activity of superoxide dismutase (SOD) and catalase [16] were substantially impaired (Fig. 1D, E). These data suggested that ischemia-reperfusion successfully triggered the oxidative stress to the brain.
Interestingly, the ischemic brain tissues and the sham brain tissues were sectioned respectively and then stained them through IHC experiment by SPRED1 antibody. As shown in Fig. 1F, SPRED1 is high expressed in sham group but low expressed in the cerebral cortex area of MACO rats. Meanwhile, we split the tissues of cerebral cortex and extract the protein to perform the immunoblotting analysis, results demonstrated again that SPRED1 protein level decreased obviously after cerebral ischemia (Fig. 1G, H). This indicates that cerebral ischemia represses the expression of SPRED1 in brain.
3.2. Oxidative stress induced the down-regulation of SPRED1 in BV2 cells
At the SPR domain of C-terminus, SPRED1 proteins contain a conserved cysteine-rich domain (Fig. 2A), indicating that SPRED1 is very likely to be regulated by oxidative stress that is one of the main secondary damages to nerve cell after ischemia-reperfusion. Thus, we explored the effect of H2O2 induction on SPRED1 in BV2 cells. As shown in Fig. 2B, C, the expression of SPRED1 emerged a trend of continuous decrease first and then recovery by the treatment of 100µM H2O2 for 24h. Interestingly, with the treatment of H2O2, corresponding changes in both SOD1 and SOD2 was similar to SPRED1 protein, especially the behavior of SOD2 is almost identical to SPRED1 (Fig. 2D, E). This reveals that the expression of SPRED1 is strictly regulated by the oxidative stress.
Additionally, as the negative regulator of MAPK, the down-regulation of SPRED1 failed to support the expression of p38MAPK, one of the key kinases responding to inflammation signal and stress signal after cerebral ischemia [17]. However, with the treatment to 12h and 24h, the p65 subunit of NF-κB gets a consistent recovery along with the SPRED1 (Fig. 2D, F). NF-κB similarly plays an important role in transducing the signal of neuro-inflammatory, oxidative stress and cell survival in the CNS[18]. This result prompts a potential correlation between oxidative stress, SPRED1 and NF-κB.
3.3. SPRED1 over-expressed can enhance the induction of p65 upon H2O2 treatment
To investigate the relevance between SPRED1 and p65, BV2 cells were transfected by SPRED1 over-expressed plasmid vector to raise its expression. However, the up-regulated SPRED1 seemingly showed a limited impact on the expression of p38MAPK and p65, but dramatically affected them upon treating by H2O2 simultaneously (Fig. 3A, B). Atsushi Hirao et al. found that the presence of SPRED1 in hematopoietic stem cells is more inclined to maintain intracellular homeostasis, while the deletion of SPRED1 can enhance the self-renewal of cells [19]. Thus, when H2O2 re-suppressed the overexpressed SPRED1, these cells in homeostasis were probably re-activated and thus become more active, triggering a significant increase in p38 and p65 proteins (Fig. 3A, B).
As a major immune cell in the CNS, BV2 cell can secrete pro-inflammatory cytokines such as TNF-α and IL-1β, etc [5]. We found H2O2-treatment weakened their abilities to secrete TNF-α and IL-1β, to some extent, which may be due to the existence of oxidative damage (Fig. 3C, D). Curiously, overexpression of SPRED1 did not alter the secretion of these two cytokines in BV2 cells, but in parallel H2O2 treatment after SPRED1 over-expressed, the secretion of TNF-α and IL-1β fall into a decline instead (Fig. 3C, D). That suggests up-regulated NF-κB after H2O2 induced the degradation of the overexpressed SPRED1 is more inclined to reduce the secretion of pro-inflammatory cytokines.
3.4 H2O2-mediated SRPED1 inactivation contributes to resisting oxidative stress injury
In order to further determine the role of SPRED1 in response to oxidative stress injury in BV2 cells, we examined cell viability and apoptosis after overexpressed SPRED1 as well as in parallel treatment of H2O2. Compared to H2O2 treatment alone, the viability of BV2 cells was enhanced in parallel H2O2 treatment after SPRED1 overexpression (Fig. 4A). Detection of cell necrosis, apoptosis and survival also support a similar conclusion, although overexpression of SPRED1 alone did not improve cell condition, including cellular activity and generative status (Fig. 4B, C). NF-κB mediated Bax/Bcl2 signal is critical for neuronal survival after cerebral ischemia[20]. However, H2O2 induction promoted the level of Bax but repressed Bcl2. SPRED1 overexpression with parallel H2O2 treatment played an opposite role, promoting Bcl2 but repressing Bax (Fig. 4D, E),. This finding seems to indicate that SPRED1 is not so important in normal BV2 cells, but when conditionality causes its degradation, it can play a unique regulatory role, re-activating the cell instinct to resist the damage.
Discussion
Cerebral ischemia is a common disease in the elderly, and its pathological manifestations are the impairment of motor, sensory and autonomic functions caused by the loss of central ischemic areas and peripheral neurons [21]. However, cerebral ischemia and the restoration of cerebral blood flow will lead to the damage of neuronal structure and function in the ischemic area again, where includes many pathological mechanisms like mitochondrial dysfunction, oxidative stress, excitatory amino acid toxicity, inflammatory response, etc [22]. Oxidative stress and its related molecular events play an important role in the pathological process of ischemic stroke, which not only triggers many direct damages such as lipid peroxidation, protein and DNA oxidation, but also leads to the initiation of inflammatory and cell death pathways through multiple cell signaling effects[23–25]. Here, we found that H2O2-mediated SRPED1 inactivation contributes to resisting oxidative stress injury, indicating the potential role of SPRED1 in preventing cerebral ischemia-induced oxidative stress injury to nerve cells.
Sprouty-related protein with EVH1 domain (SPRED1) is an identified negative Ras-MAPK-ERK regulator associated with neurofibromatosis 1 (NF-1)-like human syndrome and has been found highly enriched in CNS germinal zones during neurogenesis, while SPRED1 knockdown will increases the self-renewal of neural stem cells and promote the proliferation of progenitor cell autonomously [11, 12]. Additionally, SPRED1 has inhibitory effects on the Ras-ERK pathway induced by multiple growth factors, Ras-MAPK pathway is one of the receptor tyrosine protein kinase pathways, which plays an important role in the signal transmission of cell growth, survival, proliferation and differentiation[26]. However, sprouty-related structural domain of SPRED1, the SPR domain, is rich in cysteine residues (Cys) that are easily regulated by H2O2 as well as other ROS[12].
In fact, our investigation revealed that H2O2-simulated oxidative stress readily reduced SPRED1 protein levels in BV2 cells, and SPRED1 in cortical tissues was also shown to be largely inhibited in an MCAO-simulated model of cerebral ischemia-reperfusion injury. However, overexpressed SPRED1 in BV2 cells exhibited a limited effect on p38 and p65 proteins level as well as on the cell viability and apoptosis, seeming to be effective in maintaining the cellular homeostasis. Interestingly, once H2O2 induces the degradation of overexpressed SPRED1 in BV2 cells, the original cytotoxic effect of H2O2 is greatly inhibited, and the cell's instinct to resist oxidative toxicity was instead enhanced. Furthermore, the regulation of intracellular homeostasis by SPRED1 may involve multiple mechanisms, besides the widely known Ras-MAPK pathway, NF-κB and the dependent pathways are also the important objects to be regulated.