The Role Of SUMO-specific Protease 6 (SENP6) In Cerebral Ischemia-reperfusion Injury

Background: Ischemic stroke is an important cause of death and disability in human beings.Most of the causes are cerebral vascular blood flow obstruction and reperfusion injury,resulting in neuronal apoptosis and loss,which leads to a series of signs of nerve defects.Our early studies found that in the animal model of cerebral ischemia reperfusion and the oxygen-glucose deprived cell reoxygenation cell model,the ANXA1 molecule in neurons was abnormally modified or demodified,such as increased phosphorylation and decreased SUMOylation,and these abnormal modifications or demodifications could mediate the occurrence of neuronal apoptosis.This study will investigate the mechanism of reduced SUMOylation and cerebral ischemia-reperfusion injury.The SUMOylation modification cycle mainly includes activation,binding,connection and sumoylation.This study takes the SUMOylation as the entry point to study the expression changes of the SUMOylation enzyme SENP6 in cerebral ischemia reperfusion injury.Senp6-C1030 s was used to inhibit SENP6 activity in vivo,and the changes of neural signs and learning and memory ability in mice were observed.The effect of SENP6 on cerebral ischemia-reperfusion injury was proposed.Objective: To explore the influence of SENP6 on cerebral ischemia reperfusion injury,to study the influence of SENP6 expression on cerebral ischemia infarction area,to explore the influence of SENP6 expression on learning and memory ability after cerebral ischemia injury and the influence of SENP6 expression on motor function of animals.Methods:(1)Western blot and PCR were used to detect the expression of SENP6 at protein and RNA levels;(2)The animal model of transient focal cerebral ischemia-reperfusion injury was established by injecting lentivirus stereotactic with virus and middle cerebral artery embolization.(3)Infarcted volume was measured by 2,3,5-triphenyltetrazole(TTC)staining,and statistical analysis was performed.(4)To confirm the effect of focal cerebral ischemia injury on learning and memory function through water maze and new object recognition experiment;(5)The motor function of the animals was further studied through the wheel test,and the motor function of the animals was investigated by using the neural function score MNSS.Results: 1.Oxygen and glucose deprivation/reoxygenation induced increased SENP6 expression in neurons.Senp6 m RNA and protein expression were detected by q PCR and Western Blotting,respectively,after 1 hour of cerebral artery embolization and 0,3,6,12,24,48 and 72 hours of oxygenation.The results showed that:(1)OGD induced high expression of SENP6 m RNA and protein levels in neurons.Compared with the control group,the expression levels of SENP6 m RNA and protein in OGD group were significantly increased.After oxygen and glucose deprivation,the expression of SENP6 m RNA and protein increased with the increase of reoxygenation time.(2)The correlation analysis between the expression of SENP6 protein and reoxygenation time at 0,3,6,12 and 24 hours showed that the expression of SENP6 protein increased with the increase of reoxygenation time.There was a significant correlation between the time of reoxygenation and SENP6 expression(R2=0.9028).The results suggested that OGD/reoxygenation could induce the increase of SENP6 m RNA and protein expression.2.Inhibition of SENP6 activity reduces cerebral infarction volume after cerebral ischemia.LV-Cam KⅡ-SENP6-WT(overexpressing SENP6)and LV-Cam KⅡ-SNEP6-C1030s(expressing SNEP6 enzyme activity mutant)were injected into CA1,cerebral cortex and striatum of mice,respectively.TTC staining was used to measure the cerebral infarct volume 24 h after cerebral ischemia reperfusion.The results showed that the volume of cerebral infarction was significantly increased in rats with cerebral ischemia/reperfusion injury,and the volume of cerebral infarction was further increased in MCAO induced by overexpression of SENP6.Compared with MCAO+Vector group,the volume of cerebral infarction in mice with overexpression of MCAO+SENP6-WT was significantly increased.Overexpression of SENP6-C1030 s significantly inhibited cerebral infarction in mice.Compared with MCAO+Vector group,cerebral infarction volume in mice with MCAO+ SENP6-C1030 s significantly decreased.These results suggest that SENP6 is involved in the occurrence of cerebral infarction after cerebral ischemia.3.Inhibition of SENP6 activity has a protective effect on learning and memory function after cerebral ischemia reperfusion injury.LV-Cam KⅡ-SENP6-WT(overexpressing SENP6)and LV-Cam KⅡ-SENP6-C1030s(expressing SENP6 enzyme activity mutant)were injected into the hippocampus CA1 region,cerebral cortex and striatum of mice,respectively.After 24 hours of cerebral ischemia and reperfusion,the spatial memory ability of mice was measured by water maze test.New object recognition detection to explore the cognitive ability of mice.The results showed that in the water maze experiment,the spatial memory ability of mice was significantly reduced by cerebral ischemia/reperfusion injury,such as: Overexpression of SENP6 further impaired MCAO spatial memory ability.Compared with mice in MCAO+Vector group,MCAO can achieve a latency of escape latency and a latency of crossing platform.The escape latency of MCAO+SENP6-WT mice in water maze was significantly increased,and the number of crossing platform was significantly decreased.However,overexpression of SENP6-C1030 s could significantly reverse MCAO and cause spatial memory loss.Compared with MCAO+Vector group,MCAO+SENP6-C1030 s group significantly reduced escape latency in water maze and increased the number of crossing platform.The results of the new object identification experiment were consistent with the results of the water maze: Overexpression of SENP6 can aggravate the decrease of new object recognition ability caused by MCAO.Overexpression of SENP6-C1030 s can invert the phenomenon of MCAO causing the decrease of new object recognition ability in mice,which is manifested as a significant increase of new object recognition index.These results suggest that SENP6 is involved in the process of impaired learning and memory ability after cerebral ischemia/reperfusion injury.4.Inhibition of SENP6 activity has a protective effect on motor function after cerebral ischemia reperfusion injury.LV-Cam KⅡ-SENP6-WT(overexpressing SENP6)and LVCAMK SAB-SENP6-C1030s(expressing SNEP6 enzyme activity mutant)were injected into the hippocampus CA1 region,cerebral cortex and striatum of mice,respectively.After 24 h of cerebral ischemia reperfusion,the signs of neurological impairment in mice were firstly evaluated by MNSS.Neurological function score showed that cerebral ischemia/reperfusion injury caused obvious signs of nerve deficit in mice,and overexpression of SENP6 aggravated the signs of nerve deficit caused by MCAO.Compared with MCAO+Vector group,MCAO+SENP6-WT group significantly increased the MNSS neurological function score.Overexpression of SENP6-C1030 s could significantly reverse the signs of neurological impairment caused by MCAO.Compared with MCAO+Vector group,MCAO+ SENP6-C1030 s group significantly reduced the MNSS neurological function score.Then the motor coordination ability of mice was detected by Rotarod,and the time of falling off the Rotarod was observed.The results showed: MCAO treatment weakened the motor coordination ability of mice,which was manifested as a decrease in the residence time at the rotator,and the overexpression of SENP6 aggravated the decrease in the motor coordination ability caused by MCAO.Compared with the MCAO+Vector group,the residence time at the rotator was significantly reduced in MCAO+SENP6-WT group.Overexpression of SENP6-C1030 s significantly reduced motor coordination caused by the reversal of MCAO.Compared with MCAO+Vector group,the residence time in the rotograph was significantly increased in MCAO+SENP6-C1030 s group.These results suggest that SENP6 is involved in the process of nerve defect and motor coordination decline after cerebral ischemia/reperfusion injury.Conclusion: Based on the high expression of SENP6 in cerebral ischemia reperfusion injury,this study overexpressed wild-type SENP6 in vivo and the enzyme activity mutant SENP6-C1030S,to observe the role of SENP6 in cerebral ischemia reperfusion injury.The results showed that the overexpression of SENP6 aggravated the signs of MCAO nerve defect,increased the volume of cerebral infarction,and impaired the learning and memory ability of mice.Inhibition of SENP6 activity could reverse the nerve damage induced by MCAO.These results suggest that the SENP6 desumoylation modification may be involved in the process of cerebral ischemic nerve injury,and the inhibition of SENP6 functional activity may be a potential target for the treatment of cerebral ischemic diseases.