Effect Of Celastrol On LncRNAs And MRNAs Profiles In Transient Middle Cerebral Artery Occlusion Mice Model

Background and aims:Ischemic stroke,the most common type of stroke,is a serious cerebrovascular disease with high morbidity,morbidity,and mortality.Celastrol exhibits potent antiinflammatory and antioxidant effects and plays an active role in many diseases,but its therapeutic effect on ischemic stroke and the underlying mechanisms are not yet fully understood.Long non-coding RNAs(lncRNAs)are a class of RNA molecules more than 200 nucleotides in length and without protein-coding ability,which are closely related to the development of many diseases by regulation of gene transcription.In this study,we performed bioinformatics analysis of lncRNA and mRNA expression profiles in mice with transient cerebral ischemia and post-treatment of celastrol,in order to elucidate celastrol plays an important role in ischemic stroke and provide a theoretical basis for the potential regulatory mechanisms.Methods:Transient middle cerebral artery occlusion(tMCAO)model was established to simulate transient cerebral ischemia in mice.The cerebral infarction area was measured by TTC staining.The motor function of mice was assessed by neurological deficit score and rotarod test.We performed lncRNA and mRNA analysis by RNA sequencing(RNA-Seq)in sham,tMCAO,and tMCAO+celastrol groups.Gene ontology(GO)analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment,and network analysis were used for bioinformatic analysis of the identified deregulated genes.Nine differentially expressed genes were selected from the ceRNA network and lncRNA-mRNA interaction network,and they were further verified by qRT-PCR in brain tissue,primary neurons,primary astrocytes,and BV2 cell lines,respectively.Results:1.Celastrol significantly reduced the cerebral infarction volume and improved the neurological score and motor function of tMCAO model mice.2.A total of 50 differentially expressed lncRNAs and 696 differentially expressed mRNAs were identified between the tMCAO and sham group.A total of 544 differentially expressed lncRNAs and 324 differentially expressed mRNAs were identified between the tMCAO and tMCAO+ celastrol group.3.The results of GO analysis suggested that inflammatory response and immune response played important roles in the biological process of ischemic stroke.The results of KEGG pathway analysis indicated that signaling pathways associated with inflammation were the main pathways in which celastrol exerted neuroprotective effects,including "IL-17 signaling pathway","TNF signaling pathway" and "NODlike receptor signaling pathway".4.Four differentially expressed lncRNAs and five differentially expressed mRNAs were selected from the ceRNA network or lncRNA-mRNA co-expression network for further validation in brain tissue,primary neurons,primary astrocytes,and BV2 cell lines,and qRT-PCR results suggested that some genes showed the same folding change pattern as in RNA-Seq results.Conclusions:These findings demonstrated that celastrol can effectively reduce cerebral ischemia-reperfusion injury.Bioinformatics analysis of lncRNA and mRNA in transient cerebral ischemia in mice provided new perspectives in the neuroprotective effects of celastrol and the underlying mechanisms.