Researching Of The Role Of UCP2-SIRT3 Pathway In Cerebral Ischemia-Reperfusion Damage

Research BackgroundStroke is the second leading cause of death in the world.Its specific mechanisms and treatment measures have always been the focus of medical research.The main mechanism of brain injury caused by cerebral ischemia in ischemia-reperfusion injury involves complex mechanisms such as energy deficiency,oxidative stress injury,cell excitotoxicity,and inflammatory effects.As the energy supply barrier and oxidative stress injury are considered as the key mechanisms of cerebral ischemia-reperfusion injury,it has attracted people’s attention.UCP2(mitochondrial uncoupling protein 2)is one of the members of the uncoupling protein family.Its main function is to promote proton entry into mitochondria,decrease membrane potential,and regulate ATP production as proton channel proteins.UCP2 is highly expressed in brain and other tissues,and is activated under a variety of stress conditions.The enhanced activity of UCP2 can increase the level of NAD+(nicotinamide adenine dinucleotide)in mitochondria.NAD+ is one of the important coenzymes in the cell productivity reaction and participates in intracellular glycolysis,oxidative phosphorylation and other processes.Therefore,NAD+ levels are closely related to ATP productionIt is currently believed that the level of NAD+ regulates the activity of the Sirtuins family of proteins.The Srituins family is the third class of deacetylases whose activation depends on intracellular NAD+ levels.The Srituins family is involved in the regulation of cellular oxidative stress,energy production,and cell survival/death.SIRT3(NAD-dependent deacetylase sirtuin-3,mitochondrial deacetylase 3)is one of Sirtuins family proteins located in mitochondria and participates in the regulation of mitochondrial energy metabolism and redox status.Therefore,the energy metabolism and oxidative stress regulated by UCP2-SIRT3 pathway may be one of the mechanisms of nerve cell injury.It is currently believed that in the process of cell damage,the initial energy metabolism,oxidative stress do not show a linear relationship with the extent of cell damage.In the early and mid-term of the injury process,these changes will make a compensatory response,and this enhanced compensatory response will often promote cell dysfunction in the long term and damage the cells.These changes appear to increase first and then decrease throughout the cell.Therefore,we need to continue to explore the mechanism of energy metabolism,oxidative stress leading to nerve cell damage.Research purposes The thread occlusion method was used to replicate the middle cerebral artery occlusion(MCAO)model in male c57 mice.TTC staining and HE staining were performed,and neurological behavior scores were performed to analyze the brain injury after ischemia-reperfusion in mice.Experimental method 1.6-week-old c57BL/6 male mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co.,Ltd.and randomly divided into 5 groups:(1)sham-operation;(2)ischemia 1h reperfusion 24 h group(ischemia-1h);(3)ischemia 2h-reperfusion 24 h group(ischemia-2h);(4)genipin treated –sham-operation(g-sham-operation);(5)genipin treated with ischemia for 1h-perfusion for 24h(g-ischemia-1h).The MCAO model of c57 mice was established by a occlusion method.After 24 hours of reperfusion,the neurobehavioral scores were scored for each group of mice.TTC and HE staining were used to observe the cerebral infarct size in each group of mice and the brain histological changes in the infarcted area.2.Changes of NAD+/NADH,ATP,GSH,SOD activity and SIRT3 activity in brain tissue were detected by chemiluminescence.3.The changes of UCP2,SIRT3 and cleaved-caspase3 levels in brain tissue were detected by Western Bolt assay.Experimental results and discussion: 1.The neurobehavioral score,TTC staining and HE staining showed that after ischemia-reperfusion,the brain damage of mice was aggravated with the prolongation of ischemic time,and the cerebral infarct area was expanded with the prolongation of ischemic time;After treatment,brain damage was significantly improved in the g-ischemia-1h group,and the area of cerebral infarction was reduced.2.The results of ATP in brain tissue showed that after ischemia-reperfusion,the ATP content in brain tissue of mice decreased with the prolongation of ischemic time;after genipin treatment,the ATP level in brain tissue of g-ischemia-1h group has no statistical difference with sham-operation,indicating that genipin can effectively maintain the ATP content in brain tissue of mice with ischemia-reperfusion injury.3.GSH level and SOD activity test results showed that after ischemia-reperfusion,GSH content and SOD activity in the brain tissue of mice decreased significantly with prolongation of ischemic time;after given genipin,g-ischemia-1h group showed no significant difference or increase in GSH content and SOD activity with g-sham-operation group,indicating that genipin can effectively reduce the level of oxidative stress in the brain of ischemia-reperfusion mice.4.NAD+/NADH and SIRT3 activity assay results showed that compared with the sham-operation group,the ratio of NAD+/NADH and the activity of SIRT3 in the brain of ischiemia-1h mice was increased;after the treatment with genipin,the g-ischemia-1h group Brain tissue NAD+/NADH ratio and SIRT3 activity decreased;Western Bolt results showed that compared with the sham-operation group,the expression of UCP2 and cleaved-caspase3 protein in the brain of ischiemia-1h group was significantly increased,but SIRT3 protein expression was not significantly changed;after given to genipin,The expression of UCP2 protein in the brain of the g-ischemia-1h group was decreased,while the expression of cleaved-caspase3 and SIRT3 protein had no significant changes.Conclusion: 1.After 1/2 hour cerebral ischemia-reperfusion,ATP concentration decreased,GSH level and SOD activity decreased.After 1h of cerebral ischemia-reperfusion,UCP2 expression and SIRT3 activity increased,and NAD+/NADH levels increased.After 2h of cerebral ischemia-reperfusion,UCP2 expression and SIRT3 activity decreased,and NAD+/NADH levels decreased.It is suggested that UCP2 and SIRT3 play compensatory early and play an injury role in late stage,suggesting that UCP2,SIRT3,mediating oxidative stress/energy metabolism disorder are related to cerebral ischemia-reperfusion tissue injury;2.Genipin can inhibit the expression of UCP2 activity after 1h of ischemia-reperfusion,it can maintain ATP levels,reduce NAD + / NADH levels and SIRT3 activity,enhance energy metabolism,inhibit oxidative stress,thereby reducing the degree of brain tissue damage,and further support The above hypothesis.In summary,we speculate that UCP2-SIRT3 signaling pathway regulates cerebral ischemia-reperfusion cell injury through energy metabolism/oxidative stress.