UPS/SUMO System Mediates The Neuroprotection Induced By Isoflurane Preconditioning

In recent years, perioperative stroke has become one of the most serious complications of cardio-cerebral sugery, since it causes servere disability and high mortality. There is an urgent need for developing feasible and effective approach to prevent ischemic/ reperfusion insults during the perioperative period. In previous study, our research group has found the volatile anethetics including isoflurane and sevoflurane could induce the ischemic tolerance against ischemic and reperfusion injury. However, the mechanism of isoflurane preconditioning has not been fully understood yet.Ubiquiting proteasome system(UPS) and small ubiquitin- related modifier(SUMO) system were found to play a crucial role in protein ubiquitination and sumoylation. Previous studies demonstrated that accumulation of ubiquited protein promoted intracellular protein degradation, but conjugated-sumo proteins were essential for maintaining protein stability and integrity. A previous study reported that cellular SUMO protein modification was massive increased in the ground squirrel during hibernation torpor, which demonstrated that sumoylation process might be involved in the neuroprotective effect against ischemic injury. We speculate that UPS and SUMO pathway mediates the neuroprotection induced by isoflurane preconditioning.Therefore, the current study is to further investigate the role of UPS/SUMO protein regulation in the isoflurane preconditioning neuroprotection, hopefully to provide a new theoretical basis and research direction for the development of new drug and approaches.Experiment 1 Isoflurane precondioning induces neuprotection against oxygen-glucose deprivation injury in SH-SY5 Y cellsObjectives: To study the neuroprotective effect of single isoflurane preconditioning.Methods: All- trans-retinoic acid(RA) stimulated the human neuroblastoma cells(SH-SY5Y) to differenciate into neuron-like cells. The expression of neuron marker Neu N and βⅢ-Tublin were evaluated by Western blot and immunofluorescence staining. And then the differenciated neuron- like cells were used in the subsquent experiments. The cells were divided into four groups: Sham group, Iso group(single isoflurane inhale), OGD group(oxygen-glucose deprivation model), Iso PC group(isoflurane precondioning protection). The cells in Iso group and Iso PC group were exposed to 2% isoflurane for 2 hrs, and the cells in OGD group and Iso PC group were challenged by oxygen-glucose deprivation for 4 hrs at 24 hrs after isoflurane preconditioning. The cell in the sham group was cultured without any treatment. Cell viabilities were evaluated by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide(MTT) assay and cell apoptosis was measured by flow cytometry and caspase-3 expression at 24 hrs after reoxygenation.Results: Expression of neuronal protein marker were measured in RA-stimulated SH-SY5 Y cells at different times, and the expression of Neu N and βⅢ- Tublin was highest at 7 days after stimulation. Therefore, RA-stimulated cells for 7 days were used in the following experiments. O xygen-glucose model reduced the cell viabilities and increased the cell apoptosis of neuron- like cells. Precoditioning of isoflurane could elevate the cell survival rate.Conclusion: RA stimulated the human neuroblastoma cells to differencaite into neuron- like cells. Isoflurane preconditioning attenuated the injury induced by oxygenglucose deprivation.Experiment 2 Isoflurane preoconditioning induces neuroprotection against cerebral ischemic-reperfusion injury on ratsObjectives: To study the neuroprotection by isoflurane preconditioning against ischemicreperfusion injury.Methods: The SD rats were randomly divided into 4 groups: Sham group, Iso group(isoflurane inhale without MCAO model), MC AO group(ischemic-reperfusion damage group), Iso PC group(isoflurane preconditioning plus MCAO model). The rats in Iso group and Iso PC group were inhaled with 2% isoflurane 1 h for 5 days; the rats in MCAO group and Iso PC group were received middle cerebral artery occlusion model(MCAO) for 120 min at 24 hrs after isoflurane preoconditioning. The neurological behavirol scores and infarct volume were evaluated at 24, 48, 72 hrs and 7 days after reperfusion injury.Results: The neurological deficits and infarct volume were worse after ischemic-reperfusion damage, and isoflurane preconditioning significantly improved the neurological behavioral scores and reduced the infarct volume at 24, 48, 72 hrs and 7 days after MCAO.Conclusions: Isoflurane induced short-term neuroprotection against ischemic-reperfusion injury.Experiment 3 Isoflurane preconditioning induces ischemic tolerance via attenuating conjugated-ubiquitin protein aggregationObjectives: To study the role of UPS system in the process of isoflurane preconditioning and ischemic-reperfusion damage.Methods: The SD rats were randomly divided into 5 groups: Sham group, MCAO 24 h group, Iso PC 24 h group, MCAO 72 h group, Iso PC 72 h group(n=8). All animals were subjected to ischemic injury except Sham group. Preconditioned animals were exposed to 2% 1h- isoflurane preconditioning for 5 days at 24 hrs before MCAO model. The expression of conjugated- ubiquitin and free-ubiquitin protein were measured at 24 and 72 hrs after reperfusion by Western blot and immunohistochemistry staining respectively.Results: Ischemic injury increased the accumulation of conjugated-ubiquitin protein and isoflurane preconditioning inhibited the ubiquitination protein. The activities of conjugated ubiquitin immunohistochemistry staining in the Iso PC group was significantly lower than MCAO group.Conclusions: Atteunation of conjugated-ubiquitin protein aggregation may be involved in the neuroprotective induced by isoflurane preconditioning in cerebral ischemic-reperfusion injury model.Experiment 4 SUMO system is involved in the neuroprotection induced by isoflurane preconditioningObjectives: To study the role of SUMO system in the ischemic tolerance induced by isoflurane preconditioning.Methods: In vitro, the neuron- like cells were divided into 4 groups: S ham group, Iso group, O GD group and Iso PC group. The levels of SUMO-1, SUMO-2/3 and Ubc9 protein expression were assessed by Western blot and immunofluorescence staining at 24hrs after reoxygenation. In vivo, the SD rats were randomly divided into 6 groups: Sham group, Iso group, MC AO 4h group, Iso PC 4h group, MCAO 24 h group, Iso PC 24 h group(n=8). The expression of SUMO system and Ubc9 protein were measured by Western blot and immunofluorescence staining at 4 hrs and 24 hrs after reperfusion injury.Results: In vitro, the level conjugated-SUMO1 protein was decreased and the expression of free-SUMO protein was decreased after oxygen-glucose deprivation, and isoflurane preconditioning could elevate conjugated-SUMO1 expression and inhibit free-SUMO1 protein. Isoflurane preconditioning could attenuate the increase of conjugated-SUMO2/3 protein induced by hypoxia injury. The expression of Ubc9 was decrea sed at 4 hrs and 24 hrs after reoxygenation, isoflurane preconditioning could reverse this decrease at 4 hrs after OGD. In vivo, the expression of conjugated-SUMO1 was decrease and the conjugated-SUMO2/3 was increased after ischemic-reperfusion damage. Iso flurane preconditioning could elevate the conjugated-SUMO1 protein expression but had no effect on SUMO2/3 protein level. The expression of Ubc9 protein was higher in Iso PC group than MCAO group.Conclusions: Isoflurane preconditioning induced ischemic tolerance by regulation of SUMO system and Ubc9 protein.Experiment 5 Down-regulation of Ubc9 attenuated the neuroprotective effect of isoflurane preconditioning against ischemic-reperfusion injuryObjectives: To study the regulative mechanism of Ubc9 in the process of ischemic damage and isoflurane preconditioning.Methods: In vitro, the differenciated SH-SY5 Y cells were divided into 8 groups: Sham group, Iso group, OGD group, Iso PC group, Lenti+OGD group(Lentivirus-Ubc9 + oxygen-glucose deprivation model), Lenti+Iso PC group(Lentivirus-Ubc9 + isoflurane preconditioning group), si RN A+OGD group(si RNA-Ubc9 + oxygen-glucose deprivation model), si RNA+Iso PC group(si RNA-Ubc9+isoflurane preconditioning group). The viabilities and apoptosis of cells were measured by MTT assay and caspase-3 expression at 24 hrs after reoxygenation. In vivo, si RNA-Ubc9 was intracerebroventricular microinjected at 24 hrs before MC AO model. The SD rats were divided into 6 groups: Sham group, Iso group, MCAO group, Iso PC group, si RNA+MCAO group(si RN A-Ubc9 + MCAO model group), si RNA+Iso PC group(si RNA-Ubc9+isoflurane preconditioning group)(n=8). The neurological deficits and infarct volume were assessed at 24 hrs after reperfusion injury.Results: In vitro, Ubc9 lentivirus-transfected cells showed the increase of SUMO-1 and Ubc9 protein expression. The expressions of SUMO-1 and Ubc9 protein were decreased in si RNA-Ubc9 transfected cells. Up-regulation of Ubc9 has the similar neuroprotective effect, inhibition of Ubc9 protein could attenuate the neuroprotection of isoflurane preconditioning. In vivo, intracerebroventricular microinjection of si RNA-Ubc9 could inhibit the expression of Ubc9 protein level. The neurological behavioral score and infarct volume were worse in si RNA+Iso PC group than Iso PC group.Conclusions: Over-expression of Ubc9 allieviated the ischemic-reperfusion injury, but knockdown of Ubc9 protein markedly attenuated the neuroprotection of isoflurane preconditioning.