Differentially expressed genes induced by β-caryophyllene in a rat model of cerebral ischemia-reperfusion injury.

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. Differentially expressed genes (DEGs) were identified from RNA-seq 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 cytoHubba. Transcription factor (TF) regulatory network for the potential Hub genes was constructed. Western blot and ELISA were used to verified hub genes and relative inflammatory cytokines. After mRNA sequencing, a total of 411 DEGs were filtered based on the 2 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 1945 connections, and top ten Hub genes were identified by cytoHubba such as TIMP1, MMP-9, and STAT3. Subsequently, a TFs-miRNAs-targets regulatory network was established, involving 6 TFs, 5 miRNAs, 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-7b -Timp1. Finally, western blot demonstrated that BCP could inhibit the increased TIMP1, MMP-9 and STAT3 expression in rat brains after I/R. ELISA represented that BCP could suppress inflammatory cytokines caused by CIRI and present anti-oxidative property. 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 MMP-9 and TIMP-1, STAT3. In a sense, this study reveals that the MMP-9/TIMP-1 signaling pathway may be involved in the injury after CIRI and thus provides a new treatment strategy as well as a researching method for stroke.