[Analysis of the number, type, and functional heterogeneity of senescent cells in the radiation-induced skin wounds in mice].

Abstract

Objective: To investigate the number, type, and functional heterogeneity of senescent cells in the radiation-induced skin wounds in mice. Methods: The study was an experimental study. Forty male p16-diphtheria toxin receptor-tdTomato (p16^DTR/Tom) transgenic mice aged 6-8 weeks, which could be used to trace senescent cells, were divided into 35 Gy group and 50 Gy group (with 20 mice in each group) according to the random number table method, and 35 or 50 Gy X-ray irradiation was applied to the skin of the right hind limb of the mice to establish 3 or 4 degree of radiation-induced skin injury model, respectively. The positive area percentage of senescent cells in the wound tissue of mice in two groups was detected before irradiation and at 10, 20, and 30 d after irradiation; at 10 d after irradiation, the co-localization of endothelial cells (ECs), mononuclear macrophages (MMs), keratinocytes (KCs), fibroblasts (Fbs) and senescent cells in the wound tissue of mice in 50 Gy group was observed by immunofluorescence method. Nine male p16^DTR/Tom transgenic mice aged 6-8 weeks were divided into unirradiated group without any treatment and 35 Gy group and 50 Gy group with the same treatment as above (with 3 mice in each group) according to the random number table method. The wound tissue of mice in 35 Gy group and 50 Gy group at 10 d after irradiation and the normal skin tissue of mice in unirradiated group at the corresponding time point was taken, and the senescence percentages of KCs, Fbs, ECs, and MMs were detected by flow cytometry. Bioinformatics analysis was performed on publicly available single-cell transcriptome sequencing data from normal skin tissue of healthy rats (setting as control group) and mixed wound tissue of rats for 7 and 14 d after irradiation with 30 Gy electron beams (setting as irradiated group), and the two groups of cells were subjected to senescence assessment to screen for senescent cells, the correlation between the expression profiles of senescence-associated secretory phenotypes (SASPs) of various types of senescent cells in irradiated group was analyzed, and the differentially expressed genes (DEGs) with significantly differential expression between the senescent cells in irradiated group and the corresponding normal cells in control group were screened for gene ontology (GO) enrichment analysis. Results: The positive area percentage of senescent cells in the wound tissue of mice in 50 Gy group was significantly higher than that in 35 Gy group at 20 and 30 d after irradiation (with t values of -5.56 and -5.48, respectively, P<0.05). ECs, MMs, KCs, and Fbs co-localized with senescent cells in the wound tissue of mice in 50 Gy group at 10 d after irradiation. The senescence percentages of KCs, Fbs, ECs, and MMs in the wound tissue of mice in 50 Gy group at 10 d after irradiation were (21.07±9.49)%, (16.10±3.27)%, (16.90±5.29)%, and (34.13±8.76)%, respectively, which were significantly higher than (3.58±1.13)%, (4.13±0.19)%, (3.86±1.28)%, and (10.14±4.95)% in the normal skin tissue of mice in unirradiated group at the corresponding time point, with P values all <0.05. Bioinformatics analysis showed that the senescence scores of ECs, Fbs, KCs, macrophages, monocytes, and Schwann cells in the wound tissue of rats in irradiated group were significantly higher than those in the normal skin tissue of rats in control group (with Z values of -8.71, -9.58, -7.19, -8.82, -6.66, and -2.70, respectively, P<0.05), i.e., 6 types of senescent cells were screened. The SASPs expression profiles of monocytes and macrophages in the wound tissue of rats in irradiated group were significantly correlated (r=0.83, P<0.05), but there was no statistically significant correlation between the SASPs expression profiles of the remaining types of senescent cells (P>0.05). GO enrichment analysis showed that, compared with the corresponding normal cells in control group, the significantly up-regulated DEGs of the 6 types of senescent cells in the wound tissue of rats in irradiated group were significantly enriched in the regulation of apoptosis signaling pathway, the significantly up-regulated DEGs of multiple senescent cells were significantly enriched in the myeloid cell differentiation pathway, and the significantly down-regulated DEGs of multiple senescent cells were significantly enriched in the cell division-related pathway, with P values all <0.05. Conclusions: The number of senescent cells in the wounds of mice with radiation-induced skin injury is up-regulated, and the accumulation of senescent cells is radiation dose- and time-dependent; multiple types of cells including ECs, Fbs, KCs, and MMs can undergo senescence, and there are obvious differences in the function and SASP expression profiles among various types of senescent cells.