Discussion
Wip1 is an important oncogenic protein that has already been found to be highly expressed in multiple types of tumour cells in original research.24 Wip1 has been recently reported to negatively regulate peripheral inflammation.25 In addition, in recent years, an increasing number of studies have begun to focus on the role of Wip1 in the CNS. In our previous study, we found that Wip1 played a critical role in protecting against brain injury under high-altitude hypoxic conditions by regulating the inflammatory reaction.21 Wip1-KO enhanced the levels of inflammatory cytokines in brain tissue after high-altitude hypoxic injury. In addition, Qui et al have shown that Wip1-KO downregulates the expression of some proteins in the Wnt/β-Catenin signalling pathway 7 days after permanent middle cerebral artery occlusion (pMCAO).26 However, the effect of Wip1 on brain ischaemia/reperfusion injury, especially on the brain inflammatory response, remains to be elucidated. In the present study, we found that Wip1 protein levels were obviously increased on the third day after MCAO but had dropped to basal levels by the seventh day after brain ischaemia/reperfusion in the cortex. We examined the infarct volumes on the third and seventh days. Wip1-KO mice exhibited greater infarct volumes than WT mice at these two time points, possibly as a result of neurogenesis induced by Wip1-KO in the late stages.26 In addition, Wip1-KO exacerbated brain ischaemia/reperfusion injury by activating microglia and the NF-κB signalling pathway.
Wip1-KO also increased apoptosis of nerve cells in the acute phase of brain ischaemia/reperfusion and increased mortality in the late stage. These results indicate that Wip1-KO is proapoptotic under ischaemia/reperfusion conditions. However, Wip1-KO was not found to aggravate neuronal damage in mice in another study.26 The reason for the different findings may be that Wip1 was observed in a pMCAO model in the other study. pMCAO always results in more severe injury than temporary MCAO, and the injury is irreversible. Furthermore, Wip1 might be involved in different signalling pathways under different pathological conditions. Many papers have shown that increased levels of inflammation are harmful under ischaemic conditions.27–29 Our results suggested that Wip1-KO led to slight increases in TNF-α levels and significant increases in IL-6 levels in brain tissue, but no significant changes in these inflammatory factors were found in the peripheral circulation. These results suggested that the exacerbation of inflammation in the brain was due to increased release of inflammatory factors from brain cells rather than to peripheral inflammation.
Microglia are immune cells that reside in brain tissue and play a key role in regulating brain inflammation after ischaemia/reperfusion. The effect of Wip1 on microglial/macrophage polarisation status after ischaemia/reperfusion has not been reported previously. Our results strongly indicate that compared with WT conditions, Wip1-KO excessively activated microglia/macrophages in vivo and in vitro and caused more microglia/macrophages to express the M1 phenotype, as indicated by the substantially greater release of proinflammatory factors. On the one hand, excessive activation of microglia can exacerbate inflammation; on the other hand, it can result in excessive release of glutamate, further leading to excitatory neural toxicity and resulting in the death of neurons. However, neuroinflammation is a double-edged sword that is both detrimental and beneficial to neurons. The balance of the physiological role and neurotoxic role of glutamate requires further investigation.
The NF-κB signalling pathway plays a crucial role in regulating inflammation. Wip1 has been reported to suppress NF-κB signalling by preventing IκB dissociation from NF-κB or dephosphorylating the p65 subunit of NF-κB.30–32 Classically, activated microglia are also named M1 microglia, which contribute to the generation of various proinflammatory cytokines, such as TNF-α and IL-6, by stimulating the NF-κB signalling pathway. In the present study, we found that Wip1-KO could activate NF-κB signalling after brain ischaemia/reperfusion, which might be the reason for the increased brain inflammation in Wip1-KO mice after brain ischaemic injury. Interestingly, our results showed that Wip1 depletion had no effect on NF-kB in the Wip1-KO sham group. Normally, NF-κB is also regulated by p38, IKK (inhibitor of nuclear factor kappa-B kinase), and other kinases, which might be the reason that Wip1 deletion had no significant influence on NF-κB in the Wip1-KO sham group. However, Wip1 can be overexpressed under stress conditions and is thus involved in the DNA repair process and repression of gene expression. However, Wip1 is a phosphatase. Alteration of the phosphorylation status of Wip1 may also play a role in regulating gene expression. More studies are needed to clarify these possibilities.
In summary, the present study is the first to show that Wip1-KO is harmful to the brain after cerebral ischaemia, possibly because it increases the numbers of activated microglia/macrophages and increases brain inflammation after brain ischaemia/reperfusion. Therefore, these results provide new insights regarding the mechanism of brain ischaemia/reperfusion and suggest that the prognosis of stroke may be affected by the regulation of Wip1 expression. Further study is needed to explore whether Wip1 overexpression can exert a neuroprotective effect after ischaemic brain injury and to determine the balanced expression level of Wip1.