Expression of MANF and NF-κB p65 in cerebral cortex cerebral ischemia-reperfusion injury in rats

ObjectiveTo investigate the role of expression and changes of mesencephalic astrocyte-derived neurotrophic factor (MANF) and nuclear factor (NF)-κB p65 in cerebral cortex of rats with cerebral ischemia-reperfusion injury (CIRI).

MethodsThirty adult male SD rats were divided into Sham group and cerebral ischemia-reperfusion group (I/R group) according to random number table, 15 rats in each group.The model of focal CIRI in rats was established by modified thread embolization methods.In the Sham group, the thread embolization was not inserted into the internal carotid artery, and the other treatments were the same as the I/R group.Behavioral changes were observed by neurological deficit score 24 h reperfusion after 2 h ischemia, cerebral infarction area was observed by cranial magnetic resonance (MRI) and TTC staining, and cerebral cortex histopathological changes and cell damage in ischemic area were observed by HE staining.The relative protein levels of MANF and NF-κB p65 were detected by Western blotting, and the protein expression levels of NF-κB p65, CHOP and MANF were detected by immunohistochemistry.The subcellular localization of MANF and NF-κB p65 were observed by immunofluorescence staining.The apoptosis of cerebral cortex cells in the ischemic area was observed by TUNEL method, and the levels of TNF-α, IL-1β and IL-6 in serum of rats were detected by ELISA.

ResultsCompared with the Sham group, the rats in the I/R group showed increased neurological deficit scores (P < 0.05), the T2 phase of cranial MRI presented high signal cerebral infarction areas, the cerebral infarction foci areas appeared white in TTC staining, the HE staining of the cerebral cortex of the ischemic side showed a decrease in the number of neuronal cells, some of which showed vacuolated alterations, and the cerebral cortex was obviously edematous, which indicated that the model was established successfully.Moreover, compared with the Sham group, the contents of MANF and NF-κB P65 protein in ischemic cerebral cortex of rats in the I/R group were significantly increased (P < 0.01 and P < 0.05) and colocalized in the nucleus, the apoptosis rate of cerebral cortex cells on the ischemic side was significantly increased (P < 0.01), and the expression levels of TNF-α, IL-1β and IL-6 in serum were significantly increased (P < 0.01).

ConclusionsMANF and NF-κB p65 expression are upregulated in CIRI and undergo nuclear translocation.MANF may interact with NF-κB p65 in the nucleus to inhibit inflammation and alleviate CIRI.In addition, MANF may also attenuate neuronal apoptosis through the inhibition of ERS, thus alleviating CIRI.