Abstract
Neuroserpin (NSP) reportedly exerts neuroprotective effects in cerebral ischemic animal models and patients; however, the mechanism of protection is poorly understood. We thus attempted to confirm neuroprotective effects of NSP on astrocytes in the ischemic state and then explored the relative mechanisms. Astrocytes from neonatal rats were treated with oxygen-glucose deprivation (OGD) followed by reoxygenation (OGD/R). To confirm the neuroprotective effects of NSP, we measured the cell survival rate, relative lactate dehydrogenase (LDH) release; we also performed morphological methods, namely Hoechst 33342 staining and Annexin V assay. To explore the potential mechanisms of NSP, the release of nitric oxide (NO) and TNF-α related to NSP administration were measured by enzyme-linked immunosorbent assay. The proteins related to the NF-κB, ERK1/2, and PI3K/Akt pathways were investigated by Western blotting. To verify the cause-and-effect relationship between neuroprotection and the NF-κB pathway, a NF-κB pathway inhibitor sc3060 was employed to observe the effects of NSP-induced neuroprotection. We found that NSP significantly increased the cell survival rate and reduced LDH release in OGD/R-treated astrocytes. It also reduced NO/TNF-α release. Western blotting showed that the protein levels of p-IKKBα/β and P65 were upregulated by the OGD/R treatment and such effects were significantly inhibited by NSP administration. The NSP-induced inhibition could be significantly reversed by administration of the NF-κB pathway inhibitor sc3060, whereas, expressions of p-ERK1, p-ERK2, and p-AKT were upregulated by the OGD/R treatment; however, their levels were unchanged by NSP administration. Our results thus verified the neuroprotective effects of NSP in ischemic astrocytes. The potential mechanisms include inhibition of the release of NO/TNF-α and repression of the NF-κB signaling pathways. Our data also indicated that NSP has little influence on the MAPK and PI3K/Akt pathways.
Highlights
Thrombolysis induced by tissue plasminogen activator is an important strategy for treating cerebral ischemia (CI)
Gelderblom et al recently reported that an unbalanced expression of NSP and tissue plasminogen activator (tPA) caused a negative outcome in experimental CI, which is believed to be associated with increased microglial activation [5]
The maximum effect was observed with 5 ng/mL of NSP, but no further improvements were observed at a dose over 5 ng/mL (Fig 1A)
Summary
Thrombolysis induced by tissue plasminogen activator (tPA) is an important strategy for treating cerebral ischemia (CI). Rodriguez-Gonzalez et al (2011) demonstrated that NSP had neuroprotective effects in CI patients [4] and OGD-treated mixtures of neurons and astrocytes in rats by attenuating tPA-mediated mechanisms of inflammation and disrupting the blood–brain barrier in an ischemic model in vitro [1]. They found that a high serum NSP level indicates a better outcome in CI patients [7]. These previous clinical and bench studies verified the neuroprotective properties of NSP from different angles, and are helpful in approaching the mechanisms of NSP; there have been no studies to date on the effects of NSP on astrocytes in vitro
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