The Role Of HtrA2/Omi In Regulating Mitochondrial Unfolded Protein Response And Its Role In Cerebral Ischemia Reperfusion Injury

Research Background:Mitochondria are important organelles of oxidation reduction for eukaryotic cells,it is the source of the body to produce energy.The balance of mitochondrial homeostasis is essential for the body to maintain normal operation of the cells,correspond to the stimulus of the trauma and mitigate cell death.When mitochondria cannot maintain homeostasis balance,such as the lack of mitochondrial quality control proteins,the damage of antioxidant defense system,the accumulation of mitochondrial unfolded proteins,or the mitochondrial biogenesis cannot function well,will lead to serious mitochondrial dysfunction,results in many different kinds of diseases,like neural degeneration disease,cancer,aging,vascular diseases,diabetes,heart failure,which is the serious threat to human health.Htr A2/Omi（High temperature requirement factor A2,Htr A2）is an evolutionary conservative mitochondrial serine protease,synthesized from the endoplasmic reticulum,located in the mitochondrial intermembrane space（IMS）.As a mitochondrial quality control protein,Htr A2/Omi can recognize hydrophobic surfaces of misfolded proteins,function as the role of molecular chaperone,resist stress,have the function of the cell protection.But when the mitochondria is damaged,and mitochondrial membrane permeability increased,Htr A2/Omi released from mitochondria and into the cytoplasm and（or）the cell nucleus,via the AVPS structure domain,Htr A2/Omi can combine and suppress XIAP（X-linked inhibitor apoptosis protein）,cause the activation of Caspase9,action the function of pro-apoptotic,finally led to cell death.In addition,Htr A2/Omi can also mediate apoptosis of Caspase-independent by the direct effect of serine protease,which is its unique way,can obviously different from other mitochondria pro-apoptotic protein,like cytochrome C（Cytochrome-C）,AIF（Apoptosis inducing factor,AIF）and Smac/DIABLO（Second mitochondria-derived activator of caspases,Smac/DIABLO）.Therefore,to explore the mechanism of brain Htr A2/Omi in mitochondria helps us to clarify the pathogenesis of nervous system diseases and cerebrovascular diseases,which would provide certain theoretical basis for clinical prevention and treatment.Research Objective:1.By using suture-occluded method to copy MCAO（Middle cerebral artery occlusion,MCAO）model in mice of Wild type,and through TTC staining,Western blot experiment method to discuss the mechanism of Htr A2/Omi in Neuronopathies of cerebral ischemia reperfusion injury on mitochondrial pathway.2.By using HtrA2^mnd2 mice to detect the mitochondrial morphology changes,mitochondrial pathway related gene and protein expression changes of homozygote mice in the case of Htr A2/Omi gene mutations.To further explore the nerve protective effect of Htr A2/Omi and its effect on mitochondrial homeostasis regulation mechanism.Experimental Methods:1.Heterozygote of HtrA2^mnd2 mice were imported from The Jackson Laboratory in2014.Breeding and authenticating their offspring mice,then divided into 3 groups: Wild type,Heterozygote and Homozygote.Record the growth state and behavior characteristics respectively,and using electron microscope technology to observe mitochondrial morphological differences,and using PCR Array screening for gene technology,Western blot methods to detect the difference of mitochondrial stress and apoptosis related gene and protein among different genotypes in mice,to discuss the affect of Htr A2/Omi gene mutations on central nervous system.2.Wild type mice,3 groups at random:control group（Sham-operation）,1.5 h ischemia reperfusion for 24 h group（Ischemia-1.5 h）,Ucf-101 treatment group（Ucf-101）.By using suture-occluded method to copy MCAO（Middle cerebral artery occlusion,MCAO）model in mice,the use of nerve function score to detect the state behavior in mice,TTC staining method to observe the mice brain cerebral infarction area change and Western blot method to detect the expression changes of related proteins on mitochondrial pathway,to explore the role of Htr A2/Omi in cerebral ischemia-reperfusion injury.3.Wild type and Heterozygote,according to the genotype can be divided into 4 groups at random:the control group（Sham-operation）,1 h ischemia reperfusion for 24 h group（Ischemia-1 h）,1.5 h ischemia reperfusion for 24 h group（Ischemia-1.5 h）,3 h ischemia reperfusion for 24 h group（Ischemia-3 h）,By using suture method to copy middle cerebral artery occlusion（MCAO）model in mice,the use of nerve function score to detect the state behavior in mice and TTC staining method to observe the brain cerebral infarctionarea change,to explore the affect of Htr A2/Omi in cerebral ischemia-reperfusion injury.Experimental Result :1.The growth of mice status display: Homozygous of HtrA2^mnd2 mice has obvious neurodegenerative phenotype,the major performance is muscle atrophy,hunched over,ataxia and repetitive movements.And the weight of Htr A2/Omi Homozygous mice began to decrease at about 20 days,about 30 days,it was reduced to 1/3 below of other newborn mouse,organ-body ratios of thymus and spleen was decreased obviously.2.The use of Htr A2/Omi specific inhibitors Ucf-101,by using suture method to copy Wild type MCAO model in mice,the mice nerve function score,TTC staining and Western blot results show that compared with the Ischemia-1.5 h model group,the Longa score and cerebral infarction area is significantly reduced in Ucf-101 treatment group,the expression of Mature-Htr A2/Omi,XIAP cracking belt XIAP-45 k Da,Cleaved-Caspase9 and Cleaved-Caspase3 is significantly decreased in Ucf-101 treatment group,It suggests that Htr A2/Omi may play a crucial role in cerebral ischemia-reperfusion injury and the use of Htr A2/Omi specific inhibitors Ucf-101 can significantly reduce the brain damage of cerebral ischemia-reperfusion.3.By using suture method to copy Wild type MCAO model in mice,along with the extension of brain ischemia time,the protein expression in lesion side of cortex shows that the expression of Mature-Htr A2/Omi,Cleaved-Caspase3 and XIAP-45 kda is increased gradually;the expression of S-Opa1 / L-Opa1 is increased gradually and became obvious rise in Ischemia-1.5 h;while the expression of Hsp10 and Hsp60 in Ischemia-1 h increased obviously,then decreased,the expression of Clpp is increased more obviously in Ischemia-1.5 h.It suggests that in cerebral ischemia-reperfusion injury,the body is likely by means of Htr A2/Omi to start the protective reaction of heat shock protein and the mitochondrial stress reaction,and then cracking mitochondria fusion protein,eventually lead to apoptosis,cause brain damage.4.Comparative observation of nerve function score and TTC staining in MCAO model between wild type and heterozygous mice results showed that with the extension of brain ischemia time,the Longa score and cerebral infarction area of two genotypes（Wild type and Heterozygote）are gradually increased.And under the same conditions,the Longa score and cerebral infarction area of Heterozygote is larger than that of Wild type,and thedifference in Ischemia-1.5 h is obvious.It states that Htr A2/Omi gene mutation may increase the sensitivity of cerebral ischemia reperfusion injury in Heterozygote,which aggravated the brain damage,therefore,Htr A2/Omi may have neuroprotective effect.5.30 days HtrA2^mnd2 mice cortical electron microscopy（sem）results display :Compared with Wild type,For Homozygote mice,the distribution of mitochondria is uneven and few in number,membrane structure is disappeared,ridge structures is disappeared,nuclear membrane is incomplete and shrinking obvious,that declares the mitochondria of Homozygous of HtrA2^mnd2 mice were severely damaged,which may be the significant cause of death for Homozygous of HtrA2^mnd2 mice.6.30 days HtrA2^mnd2 mice cortical PCR Array detection results showed that:After Htr A2/Omi gene mutations,the significantly lower of Tomm40（Iconic gene of mitochondria）and Mfn1（mitochondrial split gene）,the obviously up-regulated of Pmaip1（Noxa）（apoptosis）,the low expression of Ucp2,Ucp3 and other molecular changes in gene transcription,further illustrate Htr A2/Omi plays an important role in maintaining mitochondrial structure and function.7.30 days HtrA2^mnd2 mice cortical Western blot results showed that:Compared with Wild type,The expression of heat shock protein Hsp10,Hsp60 and Hsp90,mitochondrial complex I and II,uncoupling protein Ucp2 and mitochondrial stress protein Clpp is significantly reduced in homozygous of HtrA2^mnd2 mice,the expression of Cleaved-Caspase9 and Cleaved-Caspase3 is increased,It suggests that in the case of Htr A2/Omi gene mutations,the mitochondria might not be able to effectively activate their protection mechanism,or these protection unable to counteract those damage effects.Thus,the neuroprotective effect of Htr A2/Omi may through the reaction of mitochondrial heat shock protein,mitochondrial stress reaction and mitochondrial respiratory chain pathways involved in the regulation of mitochondrial homeostasis balance.Conclusion:1.The successful breeding and identification of HtrA2^mnd2 mice.2.In cerebral ischemia-reperfusion injury,Htr A2/Omi through the mitochondrial unfolded protein response plays an important role in protecting the mitochondria,and with the injury aggravated,amount of Htr A2/Omi have been released into the cytoplasm,by the combination of XIAP and cracking the mitochondria fusion protein,play a role ofpromoting apoptosis.3.The neuroprotective effect of Htr A2/Omi may through the reaction of mitochondrial unfolded protein response and mitochondrial respiratory chain pathways involved in the regulation of mitochondrial homeostasis balance.