Research On The Role Of MANF And NF-κB P65 In Cerebral Ischemia-reperfusion Injury In Rats

Objective: To observe the role of Mesencephalic astrocyte-derived neurotrophic factor(MANF)and Nuclear transcription factor-κB p65(NF-κB p65)expression and changes in the cerebral cortex in the rat Cerebral ischemia-reperfusion injury(CIRI)model,with the aim of providing new perspectives for preventive measures and therapeutic use in ischemic stroke.Methods: Thirty mature male SD rats,aged 6-8 weeks,weighing 200-230 g,were categorized into two groups based on randomization: the sham-operated group(Sham group)and the cerebral ischemia-reperfusion group(I/R group),with 15 rats per group.After isoflurane inhalation anesthesia,the rats were fixed on the operating table,and the right common carotid artery(CCA),external carotid artery(ECA)and internal carotid artery(ICA)were dissected through a ventral median incision in the neck layer by layer.The proximal end of the CCA and the root of the ECA were ligated with surgical sutures,and a bolt(nylon thread with a diameter of 0.235 mm),which had been blunted at the head end in advance,was slowly inserted from the CCA into the ICA and stopped when a slight resistance was felt(insertion depth of about 18-20 mm),followed by ligation of the ICA with sutures placed in advance below the ICA,and blockage of the middle cerebral artery.Stitched incision was made and after 2 h of embolization,the wire peg was pulled out a little so that the insertion depth was about 15 mm and middle cerebral artery reperfusion was started.The Sham group did not insert the wire bolus into the ICA,and other operations were performed in the same way as the I/R group.The rats in both groups were selected to observe the neurological deficits by Longa method,and the area of cerebral infarcts by cranial magnetic resonance(MRI)and triphenyl-2H-tetrazolium chloride(TTC)staining after 24 h of reperfusion.Histopathological alterations and cell damage were detected in the ischemic brain cortex of rats under htoxylin eosin(HE)staining,and the relative contents of MANF and NF-κB p65 proteins in the ischemic brain cortex were examined by Western blotting.In the ischemic areas of the brain cortex,immunohistochemistry was used to measure the expression of MANF,NF-κB p65 and C/EBP-homologous protein(CHOP)and immunofluorescence staining was used to observe the subcellular localization of MANF and NF-κB p65.TUNEL method has been applied to monitor the apoptosis of cells in the cerebral cortex in the ischemic area,and enzyme-linked immunosorbent assay(ELISA)method has been performed to test the contents of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in the serum samples of both groups of rats.Results: 1.Compared with the Sham group,the rats in the I/R group showed severe neurological impairment with a dramatic increment in Longa score(P < 0.05),and the area of cerebral infarction showed high signal in the T2 phase of cranial MRI,as well as a white color in the area of brain infarction in TTC staining.In the I/R group,HE staining of the ischemia brain cortex showed a decline in the number of neuronal cells,some cells appeared vacuole-like changes,and the brain cortex was obviously edematous,which indicated that the model was successfully established.2.Compared with the Sham group,the content of MANF and NF-κB p65 protein was significantly upregulated(P < 0.05)and co-localized in the nucleus of the ischemia lateral cerebral cortex,and the apoptosis rate of the ischemia lateral cerebral cortex cells was obviously elevated(P < 0.05),as well as the serum contents of TNF-α,IL-1β and IL-6 were dramatically raised(P < 0.05)in the I/R group.Conclusion: 1.MANF and NF-κB p65 are upregulated in CIRI and undergo nuclear translocation,and MANF may interact with NF-κB p65 in the nucleus to suppress the inflammatory response and mitigate CIRI.2.MANF may also alleviate CIRI by inhibiting endoplasmic reticulum stress(ERS)and attenuating neuronal apoptosis.