Page 68 - ARNM-3-1

Advances in Radiotherapy
& Nuclear Medicine Aspirin’s protective effect on RISI

To understand the functional implications of these 2.9. ASP treatment and evaluation of protective
shifts, the Reactome pathway enrichment analysis was effects
conducted. GSEA was used to evaluate pathway activations To assess the protective effects of ASP on RISI, mice in the
across different major cell types post-irradiation. Core ASP treatment group were gavaged with ASP (20 mg/kg,
pathways such as extracellular matrix organization, once daily) for seven consecutive days before radiation
chemokine signaling, fatty acyl-CoA metabolism, Rho exposure. The corresponding control group received an
GTPase activity, and cornified envelope formation were equal volume of saline. One day after the final gavage,
identified. The average expression levels of core genes both groups were irradiated with a single dose of 20 Gy
involved in these pathways were calculated for epithelial X-ray to establish the RISI model. The severity of RISI was
cells, fibroblasts, T cells, monocytes, and endothelial
cells. The differential activation of these pathways was monitored daily post-irradiation, and the modified RISI
visualized to highlight the primary mechanisms impacted scoring system was used to evaluate the effects of ASP
by radiation and to illustrate potential targets for ASP’s treatment on the development and progression of skin
injury.
protective effects.
2.7. Cell subtype annotation and analysis 3. Results
For finer subclassification, specific marker genes were 3.1. Overall differential analysis
used to define each cluster . For instance, IFE cells were The differential analysis of irradiated versus control
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identified based on Krt10, Lor, and Pgsl1 expression, groups revealed significant changes in cell clustering and
and further subdivided into IFE basal (IFE B), and IFE gene expression patterns. The scRNA-seq data visualized
keratinized (IFE K) based on differential expression through t-SNE plots showed a distinct separation between
profiles. Similarly, the upper hair follicle (uHF) was the control and irradiated groups, indicating substantial
distinguished using Krt17, Krt79, and Cyb5r3, and transcriptional reprogramming post-irradiation.
sebaceous gland (SG) markers included Mgst1 and Scd1. Specifically, the control group included 5479 cells,
For each of these clusters, additional gene markers were whereas the irradiated group showed a reduction to
used to refine subclassification, incorporating well- 3543 cells, highlighting a significant decrease in cellular
established gene markers such as Krt14, Krt15, and Lrig1 population after radiation exposure (Figure 1A). Heatmap
for infundibulum and bulge regions. visualization further illustrated marked differences in
Further subclassification was performed by analyzing gene expression profiles, with notable clusters of genes
comprehensive gene expression profiles, which are visualized either upregulated or downregulated between the two
in the subsequent tSNE plots and heatmaps. Cluster- conditions. The contrasting colors of the heatmap
specific gene expression was validated with previously emphasize the clear distinction in expression levels,
reported datasets, which include marker genes for dermal suggesting major transcriptional changes following
and epidermal skin cell types . For clusters displaying irradiation (Figure 1B). The differential gene expression
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ambiguous classifications, marker gene expression was analysis identified a significant number of differentially
further analyzed to provide a more precise categorization . expressed genes (DEGs) between the irradiated and control
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groups. This set of DEGs includes both upregulated genes
2.8. Pseudotime analysis (n = 257) and downregulated genes (n = 500) (Table S1),
To further investigate the progression and differentiation which were further analyzed for pathway enrichment.
of IFE cells during radiation-induced injury, pseudotime Enrichment analysis showed that upregulated genes were
trajectory analysis was conducted using Monocle . The predominantly involved in pathways such as “extracellular
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scRNAseq data used for this analysis was composed matrix organization” and “chemokine receptors bind
of IFE cells, IFE B1, and IFE B2 cells. Gene expression chemokines” pathways, reflecting increased tissue
data were extracted and organized into a cell data set remodeling and immune signaling activity. On the other
object in Monocle, with further dimension reduction hand, downregulated genes were enriched in pathways
performed using DDRTree. Cells were then ordered along related to “Rho GTPases activate NADPH oxidases” and
a pseudotemporal trajectory to model the progression of “fatty acid metabolism,” indicating suppression of cellular
cellular states during injury and recovery. Differential gene processes linked to oxidative stress responses and lipid
expression analysis identified genes involved in radiation metabolism (Figure 1C). GSEA provided further validation
response pathways, with Krt14, Krt10, and Mki67 serving for the differential pathway activation. The normalized
as key markers for identifying cellular progression along enrichment scores (NES) indicate a shift in pathway
the trajectory. activity between the two groups, with some pathways such

Volume 3 Issue 1 (2025) 60 doi: 10.36922/arnm.5829