The Role Of Omi/HtrA2 On Cerebral Cortex Based On Oxidative Stress

Mitochondria are both the site of energy metabolism and the main source of reactive oxygen species.In mitochondrial dysfunction,oxidative phosphorylation dysfunction,decreased ATP synthesis,and increased production of reactive oxygen species further aggravate mitochondrial dysfunction.The presence of various mitochondrial proteins and enzymes in mitochondria is the basis for safeguarding the normal function of mitochondria.Current research shows that the mitochondrial serine protease Omi/HtrA2 is an important protease that exists in the mitochondrial membrane space of mammals and has the function of clearing misfolded proteins or helping them to fold correctly.However,when stimulated by apoptotic signals,Omi/HtrA2 is released from the mitochondria to the cytoplasm and directly cleaves apoptosis inhibitory protein?XIAP?,promoting programmed cell apoptosis.Motor neuron degeneration 2?mnd2?homozygous mice have severe striatum neurodegeneration,motor neuron damage,and short survival time,which is related to the significant loss of Omi protease activity caused by the missense mutation S276 C in Omi.,but the specific mechanism is not clear.Therefore,this experiment mainly from the perspective of oxidative stress to explore the mechanism of mnd2 homozygous mouse central nervous system damage caused by Omi/HtrA2 mutationMethod:1.mnd2 mice were divided into three groups according to genotypes: wild type?+/+?,heterozygous?+/-?,homozygous?-/-?.HE staining was used to observe changes in mouse brain cortical cells between different groups.Transmission electron microscopy was used to observe changes in mitochondria in mouse brain cortex between different groups.The mitochondrial energy metabolism related genes and Omi/HtrA2 gene expression were examined by PCR Array and RT-PCR.The kit was used to detect SOD activity and MDA content,reflecting the oxidative stress in the cerebral cortex.Western blot was used to detect the expression of mitochondrial stress protein,mitochondrial fission fusion protein,mitochondrial respiratory chain complex,and apoptosis-related protein in mouse brain cortex.Tunel staining was used to examine the apoptosis of cerebral cortical cells in each group.To investigate the mechanism of central nerve damage in mnd2 homozygous mice.2.H₂O₂ replication PC12 cells oxidative stress injury model,and give Ucf-101 inhibition of Omi / Htr A2 expression,to explore the impact of oxidative stress on Omi.The experiment was divided into three groups: control group,hydrogen peroxide group,hydrogen peroxide plus ucf-101 group.The morphology of the cells between the different groups was observed by an inverted microscope.Mitochondrial stress and expression of apoptosis-related proteins were examined in each group by immuno-imprinting.Result:1.Compared with wild-type mice,mnd2 homozygous mice exhibited no increase in body weight,growth arrest,and died within 30-40 days,with brain atrophy and other symptoms.The results of HE staining showed that the number of cells in the cerebral cortex of homozygous mice was reduced,disordered,and cells were shrinking.Further electron microscopy showed that the homozygous mouse mitochondria did not have a complete sacral structure,as indicated by the arrow,the sacral structure was destroyed,and the folded intimal structure was completely lost.This shows that mitochondrial damage may be an important cause of brain injury.2.Western Blot results showed that compared with the wild type,mnd2 homozygous mouse fusion protein Mfn1,Mfn2,Opa1 protein expression decreased,mitotic protein Drp1 protein expression levels increased,indicating that mnd2 homozygous mice mitochondrial fusion disruption This may be an important cause of mitochondrial structural damage.3.Compared with the wild type,the SOD activity of mnd2 homozygous mice decreased,but the content of MDA increased,indicating that homozygous mice increased free radical production,while the scavenging ability decreased and ROS levels increased.Western Blot was used to detect the expression of oxidative stress-related genes.The results showed that homozygous mice had reduced levels of Sirt3,SOD1,SOD2 protein compared to wild-type mice,indicating that the homozygous mice had reduced antioxidant capacity.4.Western Blot results showed that Hsp60,Hsp90,Hsp10,Clp P and other mitochondrial stress-related proteins were reduced in homozygous mice compared to wild-type mice,indicating that homozygous mice have reduced ability to repair mitochondria and eliminate misfolded proteins.5.The results of PCR Array and Western blot showed that the expression of mitochondrial respiratory chain complex-related genes in the cortex of mnd2 homozygous mice was reduced,which is an important cause of energy metabolism disorders.6..Western Blot results showed that homozygous mice increased the expression of Cleaved-casepase3,Cleaved-casepase8,Cleaved-casepase9,and increased XIAP short body.Tunel staining showed an increase in apoptotic cells in homozygous mice,indicating that homozygous mice have more severe cortical damage.7.PC12 cells were stimulated by H₂O₂.Observed under an inverted microscope,the cell status was deteriorated.After inhibiting Omi/HtrA2,the status was improved.Western Blot results showed that compared with the control group,the expression of Sirt3 protein in the H₂O₂ group was decreased,indicating that the antioxidant capacity was decreased;the expression of the mitochondrial stress-related proteins HSP60,Clp P protein was decreased;and the pro-apoptotic proteins Cleaved-Casepase3,Omi/HtrA2,and XIAP cleavage fragments.Increased expression indicates increased damage to PC12 cells.Compared with H₂O₂ group,the expression of Sirt3 protein in H₂O₂+ucf-101 group increased,the expression of mitochondrial stress protein Hsp60 and Clp P increased,and the expression of Cleaved-Casepase3,Omi/HtrA2,and XIAP cleavage fragments decreased.This indicates that Omi/HtrA2 is involved in oxidative stress-induced apoptosis.In conclusion:1.Omi/HtrA2 mutations increase the production of free radicals,resulting in decreased scavenging capacity,resulting in increased ROS levels,resulting in mitochondrial structure and dysfunction,leading to apoptosis,and impaired cerebral cortical damage in homozygous mice.2.Omi/HtrA2 is involved in oxidative stress-induced apoptosis.