Mitochondrial Autophagy Through The PINK1/Parkin Pathway Reduce Cerebral Ischemia Reperfusion Injury By Genipin In HtrA2/Omi Mutant Mice

Background:Ischemic stroke is the third leading cause of death in the world,and its incidence is positively correlated with age.The lack of blood in the brain in a short period of time causes local brain tissue damage.Although reperfusion therapy can save the cells that are about to die,it will cause a second damage to the nerve cells.Reactive oxygen species（reactive oxygen species,ROS）increase rapidly during Cerebral Ischemia Reperfusion Injury（Ischemia Reperfusion Injury,IRI）,and the antioxidant defense capacity is overwhelmed,causing protein oxidation and the formation of protein carbonyl groups.Since mitochondria are the target of ROS,increased ROS will cause damaged mitochondrial accumulation and mitochondrial dysfunction,which is a key event when IRI induces nerve cell damage.It is worth noting that mitochondrial autophagy can selectively degrade the accumulated or damaged mitochondria,and is a necessary way to remove the damaged mitochondria.The PINK1/Parkin pathway is the main pathway of mitochondrial autophagy.Voltage-dependent anion channel 1（Voltage-dependent anion-selective channel protein 1,VDAC1）is a pore-like protein located in the outer membrane of mitochondria,and is the only way for metabolic molecules in cells to enter and exit mitochondria.VDAC1 is important for PINK1/Parkin-mediated autophagy in damaged mitochondria.After IRI,PINK1 cannot cross the inner membrane of mitochondria due to the damage of mitochondrial membrane potential,so it accumulates in the outer membrane of mitochondria and recruits Parkin into mitochondria,which further mediates the ubiquitination of VDAC1 protein,thereby converting p62/SQSTM1 and LC3-II recruits to mitochondria,initiates mitochondrial autophagy and clears damaged mitochondria.Therefore,mitochondrial autophagy may play a protective role against ischemia-reperfusion injury.Uncoupling protein 2（UCP2）is an anionic carrier protein,which is a member of the uncoupling protein family.In cerebral ischemia-reperfusion injury,the increase of UCP2 reduces the proton gradient,increases the ratio of NAD⁺/NADH,accelerates the oxidation of substrates,reduces the generation of ROS and reduces the damage.However,it has been reported in the literature that GNP as a UCP2 inhibitor enhances the expression of antioxidant enzyme proteins,reduces oxidative stress and enhances the expression of LC3-II,reduces the accumulation of p62 protein and enhances autophagy flux to alleviate damage.This shows that the mechanism of UCP2’s action when IRI occurs may not be clear.There is a serine protease Omi（also known as Htr A2）,which is located in the mitochondrial membrane space.In the Parkinson’s disease model mnd2 mice(Htr A2^-/-),Omi’s protease domain Ser276 has a non-functional missense mutation at Cys,the protease activity is reduced,causing the imbalance of mitochondrial homeostasis,and a large amount of ROS is produced to activate the stress response of mitochondrial origin.Omi can cleave apoptotic family proteins or interact with them to induce autophagy.Therefore,in IRI,the increase of Omi expression may also reduce IRI by inducing autophagy.In this study,Omi gene mutant mice Wild Type and Heterozygote were pretreated with GNP to replicate the IRI model.Therefore,it is explored that Htr A2/Omi cooperates with UCP2 to play a protective role in IRI by regulating mitochondrial autophagy.Method:The Htr A2/Omi gene mutant mice were randomly divided into eight grou ps:Wild Type sham operation(Htr A2^+/+_sham);Wild Type Ischemia 1.5h reperfu sion 24h group(Htr A2^+/+_MCAO-1.5h);Heterozygous sham operation(Htr A2^+/-_sham);Heterozygous Ischemia 1.5h reperfusion 24h group(Htr A2^+/-_MCAO-1.5h);GNP p reconditioning Wild Type sham operation(Htr A2^+/+_sham G+);GNP preconditioni ng Wild Type Ischemia 1.5h reperfusion 24h group(Htr A2^+/+_MCAO-1.5hG+);GNP preconditioning Heterozygous sham operation(Htr A2^+/-_sham G+);GNP precondit ioning Heterozygous Ischemia 1.5h reperfusion 24h group(Htr A2^+/-_MCAO-1.5hG+);each group has 6 mices.GNP pretreatment:GNP was injected into mice intr aperitoneally at a dose of 40 mg/kg,Once every two days,for a total of two weeks,followed by sham operation and an MCAO model of Ischemia for 1.5 hours and reperfusion for 24 hours.1.Detect the area of cerebral infarction and the level of oxidized protein damage in eight groups of mice through the protein hydroxylation kit2.Through immunofluorescence co-localization,eight groups of mice Omi and UCP2 co-localization changes were detected.3.Through protein immune blotting,RT-q PCR and immunofluorescence co-localization,the changes in the levels of autophagy flux-related proteins p62,LC3-II and m RNA in eight groups of mice were detected to evaluate the changes in autophagy flux.4.The changes of PINK1 and Parkin levels in mitochondrial autophagy in eight groups of mice were detected by Western blot,RT-q PCR and immunofluorescence co-localization methods,and then the changes of mitochondrial autophagy levels were evaluated.Results:1.The results of the protein hydroxylation kit showed that the protein oxidative damage of the Htr A2^+/-_MCAO-1.5hG+group and the Htr A2^+/+_MCAO-1.5hG+group was reduced compared with the GNP model group without GNP pretreatment,and the Htr A2^+/-_MCAO-1.5h G+group was significantly reduced.2.Immunoblotting was used to detect LC3-II and p62 cerebral cortex proteins.The protein expressions of LC3-II and p62 in Wild-type MCAO-1.5h group were all decreased,while those in Heterozygous MCAO-1.5h group were increased.GNP can up-regulate the expression of LC3-II protein and inhibit the accumulation of p62protein in MCAO-1.5h Heterozygous group.The RT-q PCR results showed that the trend of LC3-II and p62 m RNA in Heterozygous MCAO-1.5h group was the same as that of protein results.After GNP was given,the LC3-II m RNA expression and p62m RNA expression in the Heterozygous MCAO-1.5h group increased.Through tissue immunofluorescence,it was found that the number of LC3-II positive cells in the Wild-Type MCAO-1.5h group increased,and the co-localization with VDAC1increased significantly,while the trend in the Heterozygous MCAO-1.5h group was exactly the opposite.However,GNP administration significantly increased LC3-II positive cells and colocalization levels in the Heterozygous MCAO-1.5h group.3.Western blotting and RT-q PCR detection of PINK1 protein and m RNA in the cerebral cortex showed that the expression of PINK1 was increased after Wild-Type cerebral ischemia-reperfusion injury.while the expression of PINK1 protein did not increase significantly after cerebral ischemia-reperfusion injury in Heterozygotes,but after administration of GNP,the expression of PINK1 protein increased significantly.The expression of PINK1 protein and m RNA increased in the Heterozygous MCAO-1.5h group.Tissue fluorescence colocalization found that the colocalization level of PINK1 and VDAC1 in the Wild-Type MCAO-1.5h group was significantly up-regulated,while the Heterozygous MCAO-1.5h group had an opposite trend with the Wild-Type.However,GNP can reverse the decrease in the colocalization level of PINK1 and VDAC1 in the Heterozygous MCAO-1.5h group.4.Western blotting and RT-q PCR detection of Parkin protein and m RNA in the cerebral cortex showed that the expression of Parkin was increased after Wild-Type cerebral ischemia-reperfusion injury.while the expression of Parkin protein did not increase significantly after cerebral ischemia-reperfusion injury in Heterozygotes,but after administration of GNP,the expression of Parkin protein increased.The expression of Parkin protein and m RNA increased in the heterozygotic MCAO-1.5h group.Through tissue fluorescence colocalization,it was found that the colocalization level of Parkin and VDAC1 in the wild-type MCAO-1.5h group was significantly up-regulated,while the Heterozygous MCAO-1.5h group had an opposite trend with the Wild-Type.However,GNP can reverse the decrease in the colocalization level of Parkin and VDAC1 in the Heterozygous MCAO-1.5h group.5.Immunofluorescence co-localization found that the co-localization level of the UCP2 and Omi in Heterozygotes after ischemia-reperfusion injury was less than that of the Wild Type,and the co-localization level of the two in MCAO was significantly enhanced after administration of GNP.Conclusions:1.Genipin can reduce Cerebral Ischemia Reperfusion.2.Genipin increase the LC3-II expression,reduces the accumulation of p62,restores autophagic flux,and reduces Cerebral ischemia-reperfusion injury in Htr A2/Omi mutant mice.3.The brain protection of Genipin may be achieved by inhibiting UCP2 to affect the expression of Htr A2/Omi and enhancing the PINK1/Parkin signaling pathway of mitochondrial autophagy.