Analysis Of Transcriptome Characteristics Of Cerebral Injury After CA/ROSC Based On RNA-seq Technique And Effect Of UTI Intervention

Background and Objective: CA is common and fatal.Brain damage after CA is one of the main pathophysiological changes of post-CA/ROSC syndrome.Currently,there are no effective drugs or technologies for brain function recovery,which not only afflicts medical workers,but also increases the burden of society and families.It has always been the focus and difficulty in the field of basic and clinical research on acute and critical diseases.Thus it is important to find new interventions and further study their mechanisms.The mechanism and pathophysiology of brain functional injury after CA/ROSC have not been fully clarified,many previous studies only briefly detected a few brain function indicators or several factors of a mechanism pathway,which is difficult to comprehensively demonstrate the specific mechanism.Thus it’s difficult to provide clinical guidance because of the obvious heterogeneity of research results.UTI has an inhibitory effect on a variety of proteases,sugars and lipid hydrolases,as well as anti-inflammatory,anti-apoptotic and free radical scavenging effects.UTI is widely used in the therapy for sepsis,severe acute pancreatitis and multiple organ dysfunction.Studies have confirmed that UTI can protect various kinds of brain injuries through antioxidant and anti-inflammatory responses,but the mechanisms are still unclear.RNA-seq is one of necessary techniques to study basic biology and disease mechanism based on second generation high-throughput sequencing technology,which can be used to study a particular species or organize.And transcriptome analysis can be used to study the structure and function of genes on a holistic level,and further reveal the molecular mechanism of disease occurrence and specific biological process.In this research,we used RNA-seq to screen significant DEGs and further explore the mechanism of brain injury after CA/ROSC and the intervention effect of UTI.No relevant reports have been found through literature search,so this study has innovative significance and certain clinical practical value.Methods: In this study,111 SD rats were randomly divided into control group（n=9）,sham group（n=28）,model group（n=46）and UTI group（n=28）.CA/ROSC models were successfully established in model group and UTI group.UTI group was intravenously injected with UTI（100000U/kg）within 2min after successful ROSC while model group was injected with the same amount of saline.The sham group performed other operations except CA/CPR,and the control group received no treatment.Observe and compare survival rates between different groups for two weeks.Survival condition of rats in each group were compared.The pathological characteristics of rats at 1d,3d and 7d after surgery were studied by wet and dry weight method and HE staining method.Serum levels of inflammatory cytokines such as IL-6 and TNF-α in each group were determined using Aim Plex multi-immunoassay.Left cerebral RNA in each group was extracted and sequenced on 1d and 3d after surgery.Then GO and KEGG enrichment analysis were performed according to DEGs in RNA-seq results.And further analyze the mechanisms of brain injury after CA/ROSC and the intervention effect of UTI.DEGs such as CCL2 and Plg were selected and verified by WB and IHC.Results: There was no significant difference in basic parameter values among all groups.The overall survival rate of CA/ROSC rats was significantly higher in UTI group than that in model group（50% vs 30%）.The survival rate of rats in the sham group was 100% within 2 weeks.Survival rates of rats in the model group on 1d,3d,6d,10 d and 14 d after modeling were respectively 70%（7/10）,50%（5/10）,50%（5/10）,30%（3/10）,30%（3/10）.Survival rates in UTI group on 1d,3d,6d,10 d and 14 d were80%（8/10）,60%（6/10）,60%（6/10）,50%（5/10）,50%（5/10）.Compared with the sham group,the brain water content in the model group showed a trend of increasing and then slightly decreasing on 1d,3d and 7d.In terms of HE staining results of cerebral cortex,the most serious one was on the third day after CA/ROSC.The intercellular space of nerve cells increased,and a large number of cell bodies became vacuolar degeneration.The results showed that serum levels of IL-6 and TNF-α in the model group increased on 3d.UTI can significantly reduce the content of IL-6（5.9267±1.21138 vs 1204.8567±372.76861,P<0.01）and TNF-α（2.3267±0.64933 vs 8.5533±1.98953,P<0.05）in serum.GO and KEGG pathway enrichment analysis revealed that DEGs mainly belonged to IL-17 inflammatory signaling pathway and neuroactive ligand-receptor interaction signaling pathway.During the period of 1～3d after CA/ROSC,the expression of IL-17 signaling pathway was continuously up-regulated,while the expression of neuroactive ligand receptor interaction pathway was continuously down-regulated.After UTI intervention,immune pathways（IL-17 signaling and Toll-like receptor signaling）and infectious disease（influenza A and measles）were significantly down-regulated,and the neuroactive ligand-receptor interaction pathway was significantly up-regulated.WB results showed that the expression of CCL2 increased in model group（3d）and decreased after UTI intervention（P<0.05）;Plg decreased after 3 days of modeling and increased significantly after UTI intervention（P<0.05）.The expression of Plg showed a consistent trend in IHC results,and IHC also suggested that the protein was expressed in the cytoplasm/membrane of neuronal cells.Conclusions:（1）The pathological damage of rats in the model group showed a trend of gradual aggravation on 3d and then slightly relief on 7d after CA/ROSC.The injury degree of rats in the first three days after CA/ROSC was more serious,and the third day was the most one.Therefore,this time is selected as the observation phase point of our follow-up study.（2）DEGs between the model group and the control group were mainly enriched in IL-17 signaling pathway and neuroactive ligand-receptor interaction pathway,which means that the damage was mainly caused by the abnormal regulation of immune-inflammatory response and signaling molecules and interaction.（3）UTI can improve the pathological damage of cerebral cortex and hippocampus after CA/ROSC as well as improve the survival condition of CA/ROSC rats by modulating immune-inflammatory response and signaling molecules and interaction pathways.