Research On The Mechanism Of Htra2/Omi Cooperating With UCP2 To Regulate Mitochondrial Dynamics For Resisting Cerebral Ischemia Reperfusion Injury

When the brain tissue recovers its blood flow after ischemia,it is often accompanied by ischemia/reperfusion injury（I/R）which leads to neuron apoptosis.The mechanism of ischemia-reperfusion injury is also very complex,involving the production of oxygen free radicals,calcium overload,mitochondrial permeability opening,the release of proapoptosis factors and obvious inflammatory response.More and more studies have shown that mitochondrial are involved in the pathophysiological process of ischemia-reperfusion injury.After ischemia,reoxygenation can increase the level of oxidative stress,decrease the ability of antioxidation,and lead to mitochondrial injury,thus aggravating cell apoptosis.Therefore,targeting mitochondrial may be an effective neuroprotective strategy in the treatment of ischemia-reperfusion injury.As the balance between mitochondrial fusion and fission,mitochondrial dynamics plays an important role in the normal physiological activities of cells.Mitochondrial fusion can make the damaged mitochondrial DNA and other components complement with the normal mitochondrial,thus maintaining the normal mitochondrial activity.On the other hand,mitochondrial fission can purposefully separate and remove damaged mitochondrial to maintain homeostasis in the process of controlling mitochondrial quality.When apoptosis signal stimulation such as DNA damage,endoplasmic reticulum stress,and a large number of oxygen free radicals occur,it will activate the general mitochondrial fission,accompanied by the destruction of mitochondrial ridge and the enhancement of outer membrane permeability（MOMP）,thus releasing mitochondrial apoptotic factors and starting the apoptosis process.Therefore,the dynamic change of mitochondrial dynamics may play a role in the apoptosis of ischemia-reperfusion injury cells.There is an important protease HtrA2/Omi in the mitochondrial membrane space.HtrA2/Omi is a member of HtrA family and has serine protease hydrolysis activity.HtrA2/Omi can modify or degrade the misfolded proteins damaged by ROS in the membrane gap.In our previous studies,we found that H₂O₂ induced oxidative stress of PC12 can cause high expression of HtrA2/Omi,and through the combination with OPA1,destroy the dynamic balance of mitochondrial,promote the point aggregation of mitochondrial,and induce the increase of apoptosis.But at the same time,we detected the cerebral cortex of HtrA2/Omi gene mutant mice,and found that the loss of HtrA2/Omi function would cause the imbalance of the proportion of the long and short body of OPA1,and lead to the disappearance of mitochondrial crista structure and the increase of apoptosis.Therefore,it is not clear how the protease HtrA2/Omi regulates mitochondrial dynamics and participates in neuronal apoptosis.We noticed that uncoupling protein 2（UCP2）,located in the inner membrane of mitochondrial.When cerebral ischemia-reperfusion injury occurs,it will activate the expression of UCP2,thus increasing the ratio of NAD⁺/NADH,and then accelerating the oxidation of substrate,and reduced the production of ROS in redox reaction.It has been found that cells deficiency in UCP2 can induce mitochondrial fission and increased apoptosis in cultured proximal tubular epithelial cells.This suggests that UCP2 may also be involved in the regulation of mitochondrial dynamics and apoptosis.However,it has also been reported that genipin,an inhibitor of UCP2,can effectively improve the apoptosis induced by cerebral ischemia-reperfusion injury in C57 mice.It can be seen that the specific mechanism of UCP2 in ischemia-reperfusion injury is still unclear.Therefore,we speculate that UCP2 may be involved in the regulation of apoptosis through mitochondrial dynamics in oxidative stress injury.In this study,we used HtrA2/Omi gene mutation mice and pretreatment with genipin,then replicate the model of cerebral ischemia-reperfusion injury,to observe the changes of mitochondrial dynamics and explore the mechanism of HtrA2/Omi cooperating with UCP2 in regulating mitochondrial dynamics in cerebral ischemia-reperfusion injury.Methods:1.Mice were randomly divided into 8 groups:wild-type sham operation group（WT Sham）,wild-type ischemia reperfusion group（WT I/R-1.5h）,heterozygote sham operation group（HtrA2^Heteroetero sham）,heterozygote ischemia reperfusion group（HtrA2^Heteroetero I/R-1.5h）,genipin pretreatment wild-type sham operation group（WT sham G+）,genipin pretreatment wild-type ischemia reperfusion group（WT I/R-1.5h G+）,genipin pretreatment heterozygote sham operation group（HtrA2^Heteroetero sham G+）,genipin pretreatment heterozygote ischemia reperfusion group（HtrA2^Heteroetero I/R-1.5h G+）,each group has 6 mice.Genipin pretreatment means that mice were injected with genipin at a weight of 40 mg/kg every other day for two weeks.Then mice were treated with sham surgery or ischemia for 1.5h and then reperfusion for 24h.2.TTC staining was used to detect the cerebral infarction area,and TUNEL staining were used to observe the cell apoptosis,Western blot used to detect the expression of apoptosis related proteins.3.SOD activity and MDA content of each group were detected to evaluate the level of oxidative stress.The expression changes of HSP90 were detected to evaluate the degree of mitochondrial damage.The expression changes in mRNA and protein of mitochondrial fission（Fis1,Drp1）and mitochondrial fusion（Mfn1,Mfn2）protein were detected to evaluate the changes of mitochondrial dynamics.Results:1.TTC staining showed that the infarct area of HtrA2^Heteroetero I/R group was significantly larger than that of WT I/R group,but after pretreatment with genipin,the infarct area of WT and HtrA2^Heteroetero decreased.TUNEL staining showed that the number of apoptosis cells in HtrA2^Heteroetero I/R G+group and WT I/R G+group was significantly reduced respectively compared with the control group.2.Compared with WT I/R-1.5h group,the level of Bax/Bcl-2 increased significantly in HtrA2^Heteroetero I/R-1.5h group.However,after pretreatment with genipin,the level of Bax/Bcl-2 was still increased after I/R,but the degree of increase was obviously weakened,especially in HtrA2^Heteroetero I/R-1.5h G+group.At the same time,there was no significant difference between HtrA2^Heteroetero I/R-1.5h G+group and WT I/R-1.5h G+group in the level of Bax/Bcl-2.3.Compared with WT I/R group,the SOD activity of HtrA2^Heteroetero I/R-1.5h group decreased more significantly,and the MDA level increased more significantly.After pretreatment with genipin,Compared with the WT sham G+group,the activity of SOD increased significantly while level of MDA without significant changes in WT I/R G+group.4.After I/R,HSP90 protein expression decreased,especially in HtrA2^Heteroetero I/R-1.5h group.After pretreatment with genipin,there was no significant decrease in WT I/R G+group while HSP90 protein expression increased significantly in HtrA2^Heteroetero I/R-1.5h G+group.5.After I/R,the mRNA and protein expression levels of Fis1 and Drp1 in WT increased,while the mRNA and protein expression levels of Mfn1 and Mfn2decreased.After pretreatment with genipin,the mRNA level of Drp1 increased,the mRNA and protein expression level of Fis1 decreased,the mRNA expression level of Mfn1 decreased while the protein expression slightly increased,and the mRNA and protein expression level of Mfn2 decreased.6.After I/R,the mRNA and protein expression levels of Fis1 and Drp1 of HtrA2^heteroetero increased,while the mRNA and protein expression levels of Mfn1 and Mfn2decreased.After pretreatment with genipin,the protein expression of Fis1 and Drp1decreased significantly in HtrA2^heteroetero I/R group,and the mRNA and protein expression of Mfn1 and Mfn2 also decreased.Conclusions:1.TTC staining showed that the decrease of infarct area and apoptotic level after pretreatment with genipin.It suggests that genipin can reduce cortical damage due to the dysfunction of HtrA2/Omi by inhibiting apoptosis after I/R.2.Genipin may increase the expression of HSP90,inhibit the expression of mitochondrial fission and fusion protein,so as to alleviate the mitochondrial damage caused by HtrA2/Omi dysfunction in the process of cerebral I/R.3.When cerebral I/R occurs,genipin may inhibit mitochondrial fusion in HtrA2/Omi mutant mice.4.Genipin can inhibit UCP2 while it may also reduce mitochondrial fission after I/R.Therefore,protease HtrA2/Omi may cooperate with UCP2 to regulate mitochondrial dynamics,reduce apoptosis caused by mitochondrial injury,and resist cerebral ischemia-reperfusion injury.