The Role Of Cellular Senescence In The Progression Of Skin Radiation Ulcers And The Therapeutic Effect Of UMSC-CM

Backgrounds:Radiation skin ulcer is a kind of skin tissue damage caused by tumor radiotherapy,occupational exposure,nuclear accident and other high-dose radiation,which is characterized by continuous ulcer progression.At present,the pathological mechanism of radiation ulcer has not been fully clarified,and there is a lack of effective means of prevention and treatment.In the previous study,our group reported for the first time that senescent cells may be involved in the progression of skin radiation ulcer,but its pathological characteristics and mechanism need to be further studied.Cell senescence is divided into primary cell senescence and secondary cell senescence,and there are significant differences in their i nducing mechanism and secretory function,which leads to their different functions in different stages of the disease.Senescent cells can directly or indirectly release particles or vesicles containing cytokines,chemokines,growth factors and proteases,resulting in inhibition of the immune system,enhancement of inflammatory response and weakening of tissue regeneration,which seriously affect wound repair and regeneration.At present,the treatment of senescent cells mainly includes Senomorphic compound s targeting SASP and Senolytic compounds targeting senescent cells,but the clinical efficacy is not satisfactory due to the frequency of drug administration,bioavailability,drug metabolism and side effects.Mesenchymal stem cells and their secretory factors are widely used in tissue repair and regeneration.These secretory products contain biological components(such as mi RNA and proteins),which not only have the effects of antioxidation,anti-apoptosis and promoting proliferation,but also regulate some signal pathways(such as Wnt pathway),regulate stem cell activity and promote cell reprogramming.More importantly,they also have the advantages of low immunogenicity,easy storage and good safety.Therefore,this study suggests that cell senescence ma y be a potential therapeutic target for radiation ulcer,and the conditioned medium of mesenchymal stem cells is expected to become a potential acellular therapy.Methods:1.Establishment of a rat model of skin radiation ulcerThe left posterior limbs of male SD rats at 6 weeks received a single irradiation of40 Gy X-rays to establish a skin radiation ulcer model,and the observation was continued for 260 days.The ulcers were evaluated by skin injury scoring criteria of animal models.Samples were taken at different time points(0,8,16,20,30 and 60 days)after irradiation,and the pathological characteristics of skin tissue were observed by H&E staining.The fibrosis of skin was observed by Masson staining.The expression levels of γ-H2 AX,TUNEL and Ki67 were detected by immunofluorescence to evaluate DNA damage,cell apoptosis and cell proliferation,respectively.2.Study on the role and mechanism of cell senescence in radiation ulcerThe senescence of skin cells was detected by SA-β-gal staining and immunofluorescence staining of p16.The types of senescent cells at different time points after irradiation were detected by immunofluorescence co-staining of α-SMA,CD31 and F4/80 with p16 respectively.Senescent dermal fibroblasts were established by 8 Gy irradiation in vitro and subcutaneously injected into the back irradiated by 40 Gy and observed for 60 days.The effect of senescent cells on skin radiation ulcer was evaluated by gross morphology,immunofluorescence staining and Western blot detection.Dermal fibroblasts(Control)were isolated and irradiated by 8 Gy to establish an in vitro senescent cell model(Primary).The culture supernatant(Conditioned medium,CM)was used to induce normal dermal fibroblasts.The senescence of induced dermal fibroblasts(Secondary)was detected,and the culture supernatants were used to induce normal dermal fibroblasts(Tertiary).Normal dermal fibroblasts,radiation-induced senescent dermal fibroblasts and conditioned medium-induced senescent dermal fibroblasts were collected and used for RNA-seq analysis.3.Study on the effect of uMSC-CM on mitigating radiation ulcer of skinThe left posterior limbs of SD rats were irradiated with a single 40 Gy X-ray to establish a radiation ulcer model,and a control group,an irradiation group,and a uMSCCM administration group were established.The samples were taken at 0,13,30 and 60 days after irradiation,and the protective effect of uMSC-CM on skin ulcers was evaluated by H&E staining.The levels of angiopoietin-1(ANG-1)and IL-1 α in serum of rats were detected by ELISA.The senescence level of skin cells was detected by immunofluorescence staining and Western blotting of p16 and p21 protein expression.Fibroblasts were irradiated by 8 Gy and then treated with uMSC-CM(500 ng/m L)for 7 days.DNA damage was evaluated by γ-H2 AX immunofluorescence staining,and the senescence level of skin cells was detected by SA-β-gal staining,immunofluorescence staining and Western blot.Results:1.The model of radiation skin ulcer was successfully established by 40 Gy X-ray irradiationAfter a single dose of 40 Gy irradiation on the left posterior limbs of rats,the irradiated area showed redness,swelling,depilation,exudation,and ulceration,indicating that the radiation ulcer model was established successfully.After the symptoms of limbs’ ulcer gradually improved in 23-30 days,pigmentation,scab,desquamation and necrotic contracture appeared again,suggesting that the course of the disease showed two-stage characteristics,and the score of skin toxic damage was consistent with the symptoms of ulcer.The results of H&E staining showed that the inflammatory cells infiltrated and the thickness of epidermis increased in the early stage after irradiation,while the skin accessory organs such as hair follicles,blood vessels and sebaceous glands decreased significantly in the late stage.Masson staining showed that collagen deposition in skin tissue increased.The immunofluorescence staining results of DNA damage marker protein(γ-H2AX)and cell proliferation marker protein(Ki67)showed that DNA damage persisted and cell proliferation was significantly inhibited in radiation skin ulcers.2.Senescent cells promote the progression of radiation skin ulcersStaining for a recognized marker of cellular senescenc e(SA-β-gal),Western-blot and immunofluorescence staining of cell cycle arrest-related proteins(p16,p21)showed that the number of senescent cells continued to increase after irradiation.The results of immunofluorescence co-staining showed that different types of cells could senescence after irradiation,and the senescence of macrophages mainly appears in the early stage of irradiation injury,while the senescence of endothelial cells and fibroblasts continues to increase after irradiation.Senescent fibroblasts and normal fibroblasts were intradermically transplanted into irradiated back skin.The results showed that exogenous senescent cells could accelerate the progression of radiation skin ulcers.However,normal fibroblasts can effectively delay the progression of radiation skin ulcers.Immunofluorescence staining showed that there were more senescent cells in the back skin transplanted with senescent cells compared with the group irradiated alone.Consistently,the results of Western blot detection showed that the expression levels of senescence-related proteins in skin tissues exogenously injected with senescent cells were significantly increased.The results of cell SA-β-gal staining and p16 immunofluorescence staining showed that the conditioned medium derived from radiation-induced senescence cells and CMinduced senescence cells could induce normal fibroblasts to senescence.Cellular RNA sequencing analysis showed that both radiation-induced senescent cells and conditioned medium-induced senescent cells could up-regulate senescence and senescence-related pro-inflammatory genes,and KEGG pathway analysis showed that both radiation and CM induced senescent cells could activate p53,cell senescence,cancer and cell cycle signaling pathways.The above results show that senescent cells can promote the progression of radiation ulcer and conditioned medium from senescent cells can induce senescence of normal cells.However,the analysis of differential genes and pathways related to senescence showed that the senescence induced by conditioned medium was not the same as that induced by radiation.3.uMSC-CM mitigates radiation ulcers by inhibiting cellular senescenceIn vivo experiments found that uMSC-CM can effectively relieve skin radiation ulcers.Elisa assay found that uMSC-CM could mitigate the level of IL-1α in peripheral serum and rescue the level of ANG-1.H&E staining results showed that uMSC-CM could reduce epidermal edema,reduce inflammation,and promote the repair of skin tissue and appendages.The results of immunofluorescence stain ing and western blot experiments showed that uMSC-CM could inhibit skin cell senescence.In vitro,uMSCCM was found to reduce radiation-induced DNA damage.The results of SA-β-gal staining,q RT-PCR and Western blotting indicated that uMSC-CM treatment decreased the expression of senescence-related proteins and the transcription level of senescencerelated secretory factors induced by radiation,and improve the expression of nuclear protein Lamin B1.Conclusion:1.The animal model of radiation skin ulcer was successfully established by local irradiation of 40 Gy X-ray.Its pathological features are similar to those of clinical radiation skin ulcers,which can be used to study the mechanism and explore effective intervention strategies.2.The characteristics of cell senescence in radiation skin ulcers and its regulatory effect on the progression of radiation ulcer were revealed.3.It is proved that the conditioned medium derived from human umbilical cord mesenchymal stem cells can mitigate radiation skin ulcers by inhibiting cell senescence,and senescent cells may be a potential therapeutic target for radiation skin ulcers.