Pumilio 2 Protein Participates In The Oxidative Stress Of Neuronaldamage After Intracerebral Hemorrhage

Objective:The incidence rate of intracerebral hemorrhage is high,and the mortality and disability rates are high.Oxidative stress is an important mechanism leading to brain damage after intracerebral hemorrhage.Oxidative stress leads to neuronal damage,ultimately leading to neurological dysfunction.Pumilio 2（PUM2）plays an important role in the central nervous system,regulating mitochondrial function.In this study,the data of intracerebral hemorrhage tissue chip were used to predict that PUM2 gene was involved in the process of intracerebral hemorrhage injury at the transcriptome level.In the model of collagenase induced intracerebral hemorrhage in mice,oxidative stress occurred in the tissues around the hematoma,and the expression of PUM2 protein was reduced;Further utilizing H₂O₂to treat N2a cells,a cell model of oxidative stress damage was established,demonstrating that oxidative stress leads to a decrease in PUM2 protein expression and mitochondrial dynamic imbalance.Methods:1.Bioinformatics analysis:Download the GSE24265 chip data from the GEO database,use R language to retrieve bioinformatics analysis packages such as lima,ggplot2,igraph,ggraph and obtain differentially expressed genes（DEGs）from intracerebral hemorrhage and Control tissues.By conducting GO and KEGG enrichment analysis on these DEGs,the pathways of the key are obtained.In addition,differential genes related to PUM2 gene are screened out and GO analysis is conducted to enrich into biological processes related to PUM2 gene.2.In vivo experiment:（1）ICH animal models were prepared by injecting type IV collagenase,and the sham group was injected with an equal amount of physiological saline.After 12 hours of modeling,the construction of the model was evaluated using the Bederson grading method.Evaluate neurological function at 1 day,3 days,4 days,and 7 days after intracerebral hemorrhage;Western Blot was used to detect the expression of proapoptotic protein BAX,and a tissue injury apoptosis model was successfully constructed;（2）One day after operation,SOD activity was detected using a reagent kit to evaluate changes in oxidative stress;Western Blot detection of PUM2 protein expression changes at various time points;immunofluorescence method was applied to detect PUM2 protein and SOD,and then perform statistical analysis on the fluorescence intensity of PUM2 protein and SOD,respectively.3.In vitro experiment:（1）H₂O₂with a concentration of 0-1000 umol/L was selected to act on N2a cells for 4 hours,and the cell viability was detected by CCK8 method.Finally,H₂O₂with a concentration of 400 umol/L was selected to act on N2a cells for 4 hours,and the cell morphology was observed by inverted phase contrast microscopy;The expression of apoptosis-related proteins BAX and Bcl-2 was detected by Western Blot assay to construct a cell injury and apoptosis model;（2）Using a kit to detect SOD activity and fluorescent probe method to detect the expression of total ROS in cells,evaluate the changes in oxidative stress;The expression of PUM2 protein was detected by Western Blot method;Immunofluorescence method was used to stain PUM2 protein and SOD,and the fluorescence intensity of both was statistically analyzed;（3）Western Blot method was used to detect the expression of mitochondrial membrane fusion protein OPA1,MFN1,and mitochondrial fission protein MFF,and evaluate the mitochondrial dynamic imbalance dynamics.Results:1.Bioinformatics analysis:A total of 436 DEGs were obtained from intracerebral hemorrhage tissue and Control tissue,including 275 upregulated genes and 161downregulated genes.The level of PUM2 gene transcriptome was low in the tissues of patients with intracerebral hemorrhage.GO analysis of differentially expressed genes related to PUM2 gene revealed that the biological process of enriching the PUM2 gene is often related to regulating RNA stability.2.In vivo experiments:（1）Compared with the sham group,the ICH group mice at each time point showed varying degrees of neurological deficits（P<0.05）.Western Blot detection showed an increase in the expression of proapoptotic protein BAX in the brain tissue of the ICH group at each time point,and a brain tissue injury apoptosis model was successfully constructed;（2）One day after ICH,the total SOD activity in the brain tissue decreased（P<0.05）,and oxidative stress damage occurred in the brain tissue after ICH;Western Blot detection showed a significant decrease in PUM2 protein expression in the brain tissue of the ICH group at various time points compared to the sham group（P<0.01）;Immunofluorescence staining showed a decrease in fluorescence intensity of PUM2 protein,P<0.01,and a decrease in fluorescence intensity of SOD,P<0.05.3.In vitro experiments:（1）As the concentration of H₂O₂increases,CCK8 gradually decreases,P<0.01,and cell viability decreases;Compared with the Control group,N2a cells were treated with H₂O₂at a concentration of 400umol/L for 4 hours.The H₂O₂group showed morphological changes,manifested as cell shrinkage and enlarged intercellular gaps;The Western Blot method detected an increase in the expression of pro apoptotic protein BAX（P<0.05）and a decrease in the expression of anti apoptotic protein Bcl-2（P<0.01）,successfully establishing a neuronal injury and apoptosis model;（2）On the basis of this model,it was found that compared to the Control group,the total SOD activity in the H₂O₂injured group decreased（P<0.05）;The fluorescence intensity of ROS increased by P<0.01,and the cells underwent oxidative stress damage after being stimulated with H₂O₂;Western Blot assay showed a decrease in PUM2 protein expression,P<0.05;Immunofluorescence staining showed a decrease in the fluorescence intensity of PUM2 protein,P<0.05,and a decrease in the fluorescence intensity of SOD,P<0.05;The oxidative stress injury of cells leads to a decrease in PUM2 protein expression;（3）Western Blot method detected that the expression of mitochondrial outer membrane fusion protein OPA1 increased,P<0.01,the expression of inner mitochondrial membrane fusion protein MFN1 increased,P<0.05,and the expression of mitochondrial fission protein MFF decreased,P<0.05.The mitochondrial dynamics imbalance occurred when cells were damaged by oxidative stress.Conclusion:After intracerebral hemorrhage,oxidative stress of neuronal damage leads to a decrease in PUM2 protein expression,which may be involved in the process of neuronal mitochondrial damage.