Transcriptomic-based Mechanism Of β-caryophylene Against Cerebral Ischemia-reperfusion Injury

Objective: Experimental studies have shown that β-caryophyllene(BCP)improved neurological deficits of cerebral ischemia-reperfusion injury(CIRI)rats resulting from Middle Cerebral Artery Occlusion(MCAO).However,research on targets of BCP on CIRI has not been completed.In this study,the mRNA sequencing was used to distinguish various therapeutic multiple targets of BCP on CIRI,in order to provide more different potential therapeutic targets for the treatment of ischemic stroke.Methods: 250-300 g Sprague-Dawley rats(male)were randomly divided into 3 groups(n=3): sham operation group(Sham),cerebral ischemia reperfusion injury(CIRI),β-caryophyllene 306 mg.kg-1 group(BCP).The MCAO/R model of rats was constructed by suture method,ischemia for 1.5 h,and reperfusion for 24 h.The SD rats in the sham operation group were only separated without embolization treatment,the rest of the operations were as same as those in the other groups.The administration group was given intragastric administration 5 days before the model was established,and the drug was given again half an hour before the model.After 24 hours of cerebral ischemia and reperfusion,the dead rats and those without typical ischemia were removed.The surviving rats were scored and statistically calculated(double-blind method).The rats were perfused with paraformaldehyde.After that,the sections were sliced and stained with HE.The rats were anesthetized with4% sodium pentobarbital and sacrificed with CO2 asphyxiation.Then,the brain was taken out and stained with TTC to detect the cerebral infarct volume.The neurobehavioral score,cerebral infarction volume measurement and HE staining were used to confirm the protective effect of BCP on cerebral ischemic injury in CIRI rats.Then,the injured brain tissues of each group of rats were taken to identify the differentially expressed genes(DEGs)of BCP against CIRI through mRNA sequencing analysis.CIRI induced up-regulated genes(CIRI vs.Sham)and BCP-induced down-regulated genes(BCP vs CIRI)were identified.Significant DEGs were identified only that expressed in each of all samples.Gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)pathway analysis of significant DEGs were determined by cluster Profiler.Protein interactive network(PPI)was analyzed using the String tool and Hub genes was identified by cyto Hubba.Transcription factor(TF)regulatory network for the potential Hub genes was constructed.The hippocampal and cortical tissues were exfoliated as far as possible,and the cortical tissues of rats were retained,then the expression levels of Hub gene and related inflammatory cytokines were detected by Western-blot method and ELISA kit.Result: After mRNA sequencing,a total of 411 DEGs were filtered based on the two intersection series(CIRI vs.Sham and CIRI vs.BCP),with Pax1,Cxcl3 and Ccl20 are the most remarkable ones reversed by BCP.GO analysis was represented by DEGs involved in multiple biological process such as extra-cellular matrix organization,leukocyte migration,regulation of angiogenesis,reactive oxygen species metabolic process,etc.KEGG analysis showed that DEGs participated several signaling pathways including MAPK signaling pathway(rno04010),Cytokine-cytokine receptor interaction(rno04060),JAK-STAT signaling pathway(rno04630),and others.The protein-protein interaction(PPI)network consisted of 339 nodes and 1,945 connections,and top ten Hub genes were identified by cyto Hubba such as STAT3,MMP-9 and ICAM1.Subsequently,a TFs-mi RNAs-targets regulatory network was established,involving 6 TFs,5 mi RNAs,and 10 hub genes,consisting of several regulated models such as Brd4-rno-let-7e-Mmp9,Brd4-rno-let-7i-Stat3,and Hnf4a-rno-let-7e-Icam1.Finally,western blot demonstrated that BCP could inhibit the increased STAT3,MMP-9 and ICAM1 expression in rat brains after CIRI.And ELISA also showed that BCP inhibited the expression of CIRI-induced inflammatory cytokines IL1-βand TNF-α.Conclusion: In conclusion,this study shows that the intervention of BCP can significantly reduce neurologic deficit,improve the cerebral ischemia,and a total of ten hub genes were found closely related to the treatment of BCP on CIRI.Prudent experimental validation suggests that the BCP might have the neuro-protective effects in CIRI by decreasing the expression of STAT3,MMP-9,ICAM1.In a sense,this study reveals the possible targets of BCP anti-CIRI after cerebral ischemia reperfusion injury,which may provide new treatment strategies and research methods for ischemic stroke.