Study On The Activation Of Small Ubiquitin-like Modifier And Its Neuroprotection Effects On Cerebral Ischemia

Objective:The small ubiquitin-like protein(SUMOs) family belongs to a type of small molecular proteins possessing the similar structure but different functions with the ubiquitin protein family. In the occurrence and development processes of diseases related to the oxidative stress, SUMOs play a healing role for the damaged cells through the covalent binding with their target proteins. The oxidative stress reaction is often induced obviously in the early stage after cerebral ischemia, implying that the activation of a large number of the SUMOs family members might occur and play a specific protective role of neurons after the ischemic cerebrovascular disease, especially in the acute stress period. In this study, we detected the activation and neuroprotective functions of the SUMOs protein family members after the ischemic cerebrovascular disease using the in vitro and in vivo experimental models, providing references for the future research of the neuroprotective drugs.Methods: The hippocampal neuronal cells from the SD neonatal rat within the first 24 hours of birth were separated and cultured, and then the purity of the neurons was detected using the immunofluorescence method. The oxygen-glucose deprivation(OGD) neuron model was established, and the number of apoptotic cells was monitored by the in situ apoptosis method. The protein expression levels of SUMO-1 and SUMO-2/3 were tested by the Western Blot analysis, and their localization changes were analyzed with the cell immunofluorescence double staining assay. The SUMOs gene was specifically knocked out in the neurons of the middle cerebral artery occlusion(MCAO) model, and the animal nerve function scores of the SUMOs gene knockout mice and wild type mice were compared. The mouse brain tissues were dissected and the volume of infarction was compared. Then the apoptosis of hippocampal neurons was detected by TUNEL method.Results: The rat hippocampal neuronal cells were successfully obtained with the primary adherent cell cultivation method, and identified in the Immunofluorescence assay with a high expression of the neuronal marker proteins, the neuron-specific enolase(NSE) and microtubule-associated protein 2(MAP-2), but no expression of the glial cell marker protein, the glial fibrillary acidic protein(GFAP). The purity of neurons was greater than 96%. The in situ apoptosis data showed that the apoptosis cells in OGD neurons were obvious more than those in the normal control cells(P<0.01). Western Blot assay showed that SUMO-2/3 in the OGD neurons was significantly increased(P<0.01), but SUMO-1 presented no significant change(P>0.05). The immunofluorescence experiments displayed the shift of SUMO-2/3 from cytoplasm to nucleus in OGD neurons. And the experiment results of the MCAO animal model indicated that the neuron-specific SUMO(SUMO-1 or SUMO-2/3) gene knockout mice treated with MCAO had a significantly lower animal neurological function score than the wild type MCAO mice(P<0.01). The cerebral infarction volume in neuron-specifical gene knockout of SUMO-1-3 mice sufferring the same MCAO operation was significantly greater than wild-type mice(P<0.01); And the apoptosis rate of hippocampal neurons was higher than that of wild mice(P<0.01).Conclusion:In the early stage after the ischemic cerebrovascular disease, SUMO-2/3, one of the small ubiquitin-like protein family members, is rapidly activated and plays a potential neuroprotective role, while SUMO-1 is almost not involved in the pathophysiological process of this illness. The exogenous SUMO-2/3 protein or drugs that can induce the expression of SUMO-2/3 may be developed and applied into clinic practice as a new promising nerve protection medicine.