Senolytics (DQ) Mitigates Radiation Ulcers By Removing Senescent Cells

Objective: To explore the therapeutic effects of dasatinib and quercetin on radiation ulcers by removing senescent cells.Methods: 1.Our study first analyzed the correlation between radiation and the expression of senescence-related genes in human oral mucosa gene database GSE103412.2.Establish mouse radiation-induced oral mucositis and rat radiation-induced skin ulcer models respectively.For radiation-induced oral mucositis model,the head and neck of mouse were exposed to fractionated radiation of a 6-Gy dose/day for 5 days continuously.Mice were sacrificed at day 3,6,8,10,and the tongues were removed and analyzed.For radiation-induced skin ulcers model,right posterior limb of each rat was exposed to a single 40 Gy radiation under anesthesia.Rats were sacrificed at day 5,8,11,15 after irradiation and the right posterior limbs of the rat were removed.Subsequently,tongue tissues and rat skin tissues were used to analyze the structural changes by HE staining and detect the expression of cyclin-protein P16 by immunohistochemistry;Furthermore,the expression of senescence and senescence-related secreted phenotype(SASP)genes were detected by qRT-PCR.3.To evaluate the effect of senolytics on radiation ulcer,15 mice / rats were randomly divided into three groups(5/each group): unirradiated group,irradiated group and DQ group.Mice were sacrificed at day 10 and the tongues were removed and analyzed,tongues were stained with toluidine blue and photographed.The right posterior limb of each rat was acquired and photographed at day 15 after radiation or treatment.In addition,tongue tissues and skin tissues were used to analyze the structural changes by HE staining and detect the expression of γ-H2 AX and Ki67 protein by immunofluorescence and cyclin-protein P16 by immunohistochemistry;Finally,we analyzed the expression of senescence and SASP genes by qRT-PCR.4.Cell senescence model was established,HOK and human skin fibroblasts were exposed to a single dose of 8Gy irradiation.Cells and the cell supernatant were collected at 7 days after irradiation for further analysis.Cell senescence were detected by SA-β-gal staining,the expression of senescence and SASP genes were detected by qRT-PCR.Cell supernatant were collected to detect inflammation-related cytokines by ELISA.Subsequently,young HOK and human fibroblasts were co-cultured with young cell supernatant(Con-CM)and senescent cell supernatant(SASP-CM)respectively,SA-β-gal and qRT-PCR were operated to detect whether young HOK and human fibroblasts were senescent after co-cultured.Further,the expression of p-JAK1 and p-JAK2 protein of young and senescent cells were detected by Western Blot(WB).Senescent HOK and skin fibroblasts were treated with vehicle and JAKi,then cells and cell supernatant from senescent cells treated with vehicle(SASP-CM)and senescent cells treated with JAKi((SASP+JAKi)-CM)were collected,the expression of inflammation-related genes were detected by qRT-PCR.5.HOK and human fibroblasts were divided into unirradiated control group,unirradiated DQ group,irradiated control group and irradiated DQ group.After treatment with DQ for 24 hours,cell apoptosis was detected by flow cytometry,meanwhile,apoptosis was observed using calcein staining,and the expression of apoptosis-related proteins were detected by WB.6.CAL27 and MCF7 cells were divided into unirradiated control group,unirradiated DQ group,irradiated control group and irradiated DQ group.After treatment with DQ for 24 hours,cell apoptosis and cell cycle were detected by flow cytometry.Single cell cloning experiment was used to test its proliferation ability and colony forming ability.Results: 1.The heat map analysis of database GSE103412 indicated that the expression levels of CDKN2 A,TP53 and SASP-related genes were significantly upregulated after radiation therapy.2.To evaluate mouse radiation-induced oral mucositis and rat radiation-induced skin ulcer models,the tongue tissues and skin tissues were collected at different time points.HE staining showed that the irradiated tongue tissues exhibited severe destruction of the epithelial layer compared with normal epithelial morphology;and compared with normal skin tissues,irradiated skin tissues showed structure changes,inflammatory cells infiltrate and ulcers;These results indicate that models of radiation oral mucositis in mice and radiation skin ulcers in rats have been successfully established;Furthermore,tongue tissues and skin tissues immunohistochemical staining showed that the expression of senescence marker p16 was increased at different time points after radiation.qRT-PCR showed that the expression of p16,p21,and plasminogen activator inhibitor-1(PAI-1),as well as the SASP factors,were increased in irradiated tongue tissues and skin tissues compared with non-irradiated controls.3.Toluidine blue staining observed that DQ almost completely prevented the appearance of oral mucositis in irradiated mice.HE staining showed that the tongue epithelial layers of DQ treated mice were complete and continuous,and DQ also significantly reduced radiation-induced skin ulcers.Immunofluorescence staining revealed that compared with the irradiated group,the levels of DNA damage response marker protein γ-H2 AX were significantly reduced both in the tongue tissue and skin tissue of irradiated DQ-treated mice/rats,while the expression of Ki67 protein was significantly increased.In addition,qRT-PCR found that DQ significantly reduced the genes expression of senescence and SASP in mouse tongue tissues and rat skin tissues compared with the irradiation group.4.SA-β-gal staining showed lots of senescent cells became blue after radiation compared with young HOK and human fibroblasts.qRT-PCR showed that the expression of senescence genes was significantly increased after irradiation.Notably,ELISA results revealed that the expression of inflammation-related cytokines were increased in the cell supernatant after radiation.All the above indicated that the radiation-induced cell senescence model was successfully established.To find out whether senescent cells induce senescence and inflammation in adjacent healthy cells,co-culture experimental was performed.The result showed that exposure of non-senescent HOK and skin fibroblasts to SASP-CM induced SA-β-gal expression and senescent morphology compared with Con-CM.Cells were also collected for qRT-PCR analysis,which showed that CM derived from senescent cells caused upregulation of senescence genes and SASP genes relative to CM from non-senescent cells.In addition,the increased expression of p-JAK1 and p-JAK2 protein after radiation was detected by WB.Furthermore,qRT-PCR results showed that the expression of inflammation-related genes was reduced in(SASP+JAKi)-CM treated young HOK and human fibroblasts relative to SASP-CM treated young cells 5.Flow cytometry revealed that DQ eliminated 40-60% of senescent HOK and 10-20% of senescent skin fibroblasts,while DQ has no effect on the young HOK or skin fibroblasts.Similarly,calcein AM/PI staining observed that senescent HOK and fibroblast cell death caused by DQ was significantly increased compare with young cells.Moreover,WB result showed that the expression of apoptosis-related proteins PARP and cleaved caspase 3 were increased,while the expression of caspase 3 protein was decreased in senescent HOK and fibroblast cells after DQ treatment.6.Flow cytometry showed that DQ treatment increased the apoptosis rate in CAL27 and MCF-7 cells,whether they were exposed to radiation or not.Similarly,cell cycle detection showed that G1 phase arrest of CAL27 and MCF-7 cells were significantly increased after DQ treatment.Proliferation was measured by colony formation assays,which showed that DQ reduced the colony formation ability of both CAL27 and MCF-7 cells.Conclusion: 1.Biomarkers of senescence accumulate in human and animal radiation-induced ulcers.2.DQ treatment can mitigate radiation-induced ulcers via clearance of senescent cells.3.Senescent cells induce senescence and the SASP in adjacent cells.4.DQ treatment eliminates senescent cells by inducing apoptosis.5.DQ treatment can enhance cancer cell radiosensitivity.