In past majority of studies in cerebral ischemic injury, the attention had been mainly focused on the fate of neurons. The role of astrocytes had been neglected for a long time, despite of the fact that the number of astrocytes in CNS is over fivefold of neurons. In the recent 25 years, a revolutionary understanding has been developed with more and more focus in the physiology and pathology of astrocytes. Nowadays astrocytes are considered as “the principal housekeeping cells” in CNS 
. Structurally connecting the entire CNS, astrocytes also perform supportive functions such as blood-brain barrier formation, synaptic transmission 
, regulation of blood flow 
, maintenance of the homeostasis in synaptic interstitial fluid 
, and energy metabolism 
. Therefore, the intact functional status of astrocytes is crucial for neuronal survival after various injuries.
In the previous works, it has been confirmed by separate teams that NDRG2 is localized in astrocytes in healthy cerebrum 
. Our previous study also reported that the NDRG2 signals were further enhanced in reactive astrocytes of penumbra after transient middle cerebral artery occlusion (MCAO) in rats 
. In the present study, we employed IL-6-differentiated C6 glioma cells as mature astrocytes and injured them in an OGD model, as referred previously 
. After OGD 4h followed by re-oxygenation, we observed a significant increase of both TUNEL-positive staining and NDRG2 expression in a time-dependent manner, parallel to the finding in previous in vivo
, indicating that this OGD model in IL-6-differentiated C6 glioma cells could mimic the I/R injury induced by transient focal cerebral ischemia in rats.
The phenomenon of NDRG2 signals co-localized with TUNEL-positive cells in ischemic penumbra suggested that NDRG2 might be involved in cellular apoptosis induced by ischemia 
. In the present study, we constructed NDRG2 over-expression and silencing systems to verify the role of NDRG2 in cellular apoptosis and proliferation. The results showed that NDRG2 over-expression could inhibit the proliferation of astrocytes after OGD, while its silencing made an opposite effect. Moreover, over-expression of NDRG2 enhanced the increase of Bax/Bcl-2 ratio after OGD while NDRG2 silencing attenuated such an increase. Bax and Bcl-2 are two important members of Bcl-2 family that is closely associated with cellular fate 
. Bcl-2 acts as an anti-apoptotic signal and Bax is pro-apoptotic. Therefore, the Bax/Bcl-2 ratio is considered as the switch to determine the cell death 
. Our present data showed that the decreased survival rate in up-regulated NDRG2 astrocytes after OGD exposure was due to the pro-apoptotic effect of Bax. On the other hand, it was interesting that the expression of Bcl-2 was uninfluenced, no matter when NDRG2 was in a level of up- or down-regulation. The interaction between NDRG2 and Bcl-2 still needs to be clarified.
So far we have demonstrated that NDRG2 promoted the cellular apoptosis after OGD. Our findings are in line with that reported by Wang et al
in A549 cells 
, however, inconsistent with that reported by Liu et al
in cervical cancer Hela cells 
. Liu and colleagues found that the over-expression of NDRG2 resulted in increased surviving rate, decreased percentage of apoptotic cells and lowered Bax/Bcl-2 ratio after irradiation exposure. On the contrary, NDRG2 silencing contributed to decreased cells survival, increased apoptosis and higher Bax/Bcl-2 ratio. These might be the consequences of different inherent characteristics among different types of tumor cell and different mechanisms associated with various injury-causing factors, such as irradiation, OGD, and ischemia, etc. It still needs to be further investigated.
In resting status, NDRG2 expression was observed mainly in the plasma membrane and cytoplasm 
. Upon cell stress like hypoxia and ischemia, the translocation of NDRG2 from the cytoplasm to the nucleus will occur 
. Using the technique of cell fraction assay, we confirmed this phenomenon quantitatively. Such a stress-accompanying change generally indicates the activation of intracellular signaling pathways. The mechanisms and effects of NDRG2 nuclear translocation are still unknown. Present data has not shown that there exists any nuclear localization signal in NDRG2 protein, which is the most common form of nuclear import elements. Therefore, it is presumed that NDRG2 might have its own motif to guide its nuclear translocation under particular conditions. Wang et al
found the segment of residue 101–178 in NDRG2 pivotal to its translocation 
. Recently, Hwang et al
demonstrated that helix α6 of hNDRG2 might contribute to the translocation, based on the knowledge of the three-dimensional crystal structure 
p53 is the master regulator of cell death by inducing apoptosis and its biological effects are mainly explained by its activity as a transcription factor 
. Bax is one of its downstream target genes associated with pro-apoptotic effect. It was documented that p53 could regulate the Bax transcription in focal ischemia and experimental Parkinson’s disease 
. To verify whether OGD-induced NDRG2 up-regulation is associated with p53, we constructed p53 over-expression and silencing systems. It appeared that p53 silencing obviously suppressed the up-regulation of NDRG2 after OGD. Interestingly, over-expression of p53 did not further strengthen the uprising of NDRG2 after OGD. From these data, we can demonstrate that OGD-induced NDRG2 uprising was regulated by p53 expression, which was consistent with Liu’s report 
. It is noteworthy that over-expression of p53 could not lead to a higher NDRG2 expression than that observed in simply OGD-treated cells. Several upstream regulators acting on NDRG2 promoter were reported, such as HIF-1α and p53 transactional activated NDRG2 while Myc transactional suppressed NDRG2 
. On the other hand, although p53 is commonly labeled as a pro-apoptotic gene, it could transcriptionally activate some anti-apoptotic genes, such as HB-EGF, DcR1 and DcR2 
. Taken all these into consideration, the p53 pathway is a complex network.
In conclusion, NDRG2 is a novel regulator of apoptosis. It plays an important role in the p53-related pro-apoptotic effect when the astrocytes originated from C6 glioma cells are exposed to OGD. And the pro-apoptotic effect of NDRG2 is independent of Bcl-2. Our findings bring insight to the roles of NDRG2 in ischemic-hypoxic injury and provide potential targets for future clinical therapies on stroke.