The Mechanism Of Rapamycin Nano-micelle Ophthalmic Solution In Alleviating Corneal Allograft Rejection:an RNA-seq Analysis

Objective:Using RNA sequencing (RNA-seq) technology to reveal the mechanism of rapamycin nano-micelle ophthalmic solution in alleviating corneal allograft rejection from the transcription level,providing an experimental basis for the clinical application of drugs.Methods:We performed penetrating keratoplasty on mice randomly divided into three groups:a syngeneic penetrating keratoplasty group (Syn),an allogeneic penetrating keratoplasty group (Allo),and an allograft group treated with rapamycin nano-micelle eye drops (Rapa).Three corneal samples in each group (each sample contains three corneas)were subjected to high-throughput RNA-seq.Under the conditions of |Log2FC|> 0.585 and P<0.05,we screened differentially expressed genes (DEGs).We then undertook Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.The quantitative real-time PCR (qPCR) was employed to validate the expression of the selected target genes at the transcript level.Results:Compared to the Syn group,the Allo group had 1,375 significantly up-regulated genes and 627 significantly down-regulated genes.Following rapamycin intervention,the Rapa group had 737 significantly up-regulated genes and 1,058 significantly down-regulated genes.GO enrichment analysis showed that DEGs up-regulated in the Allo group were significantly enriched in immune system processes,immune responses,and regulation of immune system processes.Following rapamycin intervention,DEGs down-regulated in the Rapa group were also found to be enriched in terms of the aforementioned biological processes.KEGG enrichment analysis showed that up-regulated DEGs in the Allo group were mainly enriched in Cytokine-cytokine receptor interactions,Herpes simplex infection,Cell adhesion molecules,Chemokine signaling pathways and NOD-like receptor signaling pathway,etc;After rapamycin intervention,the down-regulated DEGs were mainly enriched in Cytokine-cytokine receptor interaction,Chemokine signaling pathways,Natural killer cell mediated cytotoxicity,T cell receptor signaling pathway and NF-kappa B signaling pathway,etc.The down-regulated DEGs in Allo group were mainly enriched in Metabolic pathways,axon guidance,Hippo signaling pathway,Rap1 signaling pathway and AMPK signaling pathway,etc;After rapamycin intervention,the up-regulated DEGs were mainly enriched in PI3K-Akt signaling pathway,Signaling pathways regulating pluripotency of stem cells,Wnt signaling pathway,Rapl signaling pathway and Hippo signaling pathway,etc.We undertook RT-PCR on CD52,KLRD1,KLHL6,Fcerlg,CCR1,and CCL3 to confirm the expression of these genes at the transcript levels,and we found the results to be consistent with those in RNA sequencing These findings suggest that the results of transcriptome sequencing are reliable.Conclusion:In this study,we found that differentially expressed genes such as CD52,KLRD1,KLHL6,Fcerlg,CCR1,and CCL3 are up-regulated during rejection,and down-regulated following rapamycin intervention.KEGG analysis showed that Cytokine-cytokine receptor interactions and Hippo signaling pathways are involved in the occurrence of corneal allograft rejection and in the process by which rapamycin alleviates rejection.Our results provide a transcriptomic basis for further clarifying the mechanism of rapamycin and searching for potential therapeutic targets.