Involvement Of Cell Senescence In Radiation Ulcer And The Therapeutic Effect Of 3’-deoxyadenosine

BackgroundRadiation ulcer is a common radiation injury of skin or mucosa,which is mainly occurred in occupational or accident exposure,radiotherapy for bone marrow transplantation or cancer patients and nuclear radiation in wartime,etc.In recent years,despite the great improvement of radiotherapy technology,the continuous improvement of radiation equipment,the control of radiotherapy for benign diseases and the strengthening of the protection of normal tissues in the radiation area which make the probability of serious radiation damage gradually decreased,however,the non-cancer tissues of patients still suffer from various degrees of non-specific radiation damage because the application of radiotherapy in the treatment of malignant tumors is more and more ex tensive.Radiation ulcer accounted for 8.4%of the wounds that were difficult to heal,which was not uncommon in clinic.When ulcer occurs,local infection plus radiation effect of sustainability and side effects may happen,if not treated in time,can easily cause ulcer surface to increase and deepen,resulting in huge ulcer,and even cause systemic infection,acute bleeding and other life-threatening events.The physiological process of wound healing includes four stages:hemostasis,inflammation,prolife ration and remodeling.However,radiation ulcer cannot be carried out orderly and timely when compared with normal wound healing.Generally speaking,radiation ulcer can be blocked in the inflammatory stage,resulting in continuous inflammatory response which delays repair and healing.Radiation ulcer brings great pain to patients,damages their quality of life,and requires huge medical resources.These chronic wounds can last for years,and some can even lead to amputations.Tissue reconstruction of radiation ulcer poses a great challenge to plastic surgeons.Anti-inflammatory drugs,growth factors and local anesthesia are usually used to alleviate radiation ulcer in clinic,but the effect is not good.Palifermin,a recombinant human keratinocyte growth factor,is the only FDA approved targeted drug for the prevention of oral mucositis in bone marrow transplant patients,however,its receptor（FGFR2β）is often overexpressed in cancer cells,which may increase the risk of tumor growth.At the same time,hyperbaric oxygen therapy is also considered to be an effective method to reduce skin ulcer after radiotherapy,but the treatment time is very long.Therefore,there is an urgent need of a safe and effective drug to reduce radiation ulcer.At present,the mechanism of radiation ulcer is unclear.Ionizing radiation can directly induce DNA damage or increase the production of free radicals and reactive oxygen species（ROS）which promote gene expression modification and DNA damage indirectly.Different types and levels of DNA damage cause various cell responses,one is to promote cell survival by temporarily activating cell cycle checkpoint and DNA repair,the other is to activate cell death program or cell senescence process to inhibit the proliferation of irreparable cells.Cell senescence is an irreversible state of cell cycle arrest,radiation can induce sustained DNA damage response（DDR）which promote cell cycle arrest and cell senescence by activating p16^INK4a-RB and/or p53-p21^CIP1 signaling pathway.Cell senescence plays an important role in embryo development,organ formation,wound healing and tumor suppression.In tissues,the effects of cell senescence may be beneficial or harmful,depending on trigger factors,tissue and cell type.At the cellular level,the long-term existence of senescent cells in the tissue results in long-lasting inflammatory response,which contributes to tissue damage and delayed repair and regeneration.At the same time,senescent cells secrete a large number of pro-inflammatory factors,including cytokines,chemokines,matrix remodeling protease and growth factor,which are collectively referred to senescence-related secretory phenotype（SASP）.SASP can cause tissue dysfunction and wound deterioration by paracrine.However,the relationship between cell senescence and radiation ulcer is not clear.3’-deoxyadenosine（cordycepin）is a nucleoside analogue found in Cordyceps militaris.3’-deoxyadenosine has attracted people’s attention because of its potential therapeutic and biological value.3’-deoxyadenosine can interact with cancer,tumor,inflammation,oxidant,polyadenylation of m RNA and other drug targets,and has many pharmacological activities,such as anti-oxidation,anti-tumor,anti-inflammatory,neuroprotection,bone loss prevention and so on.However,whether 3’-deoxyadenosine can inhibit the cell damage induced by ionizing radiation and reduce radiation ulcer remain to be further explored.In this study,we first established three kinds of radiation ulcer models:radiation skin ulcer,intestinal ulcer and oral mucositis.We first found that DNA damage and cell senescence are persistent in radiation ulcer,and senescent cells may be involved in the development of radiation ulcer.Next,we established a radiation-induced cell senescence model of fibroblasts in vitro,and identified 3’-deoxyadenosine can effectively prevent radiation-induced DNA damage and cell senescence.At the same time,3’-deoxyadenosine can mitigate radiation ulcer and radiation-induced long-term damage.Finally,we probed into the mechanism of3’-deoxyadenosine in preventing cell senescence and mitigating radiation ulcer,and identified the possible target of 3’-deoxyadenosine,which provided a new experimental basis for the development of more effective radiation ulcer treatment strategy.Methods1.The distribution of senescent cells in radiation ulcerTo establish a large dose radiation ulcer model,the right posterior limb of Sprague Dawley（SD）rat was exposed to 40 Gy radiation.The general damage of skin at different time points was observed by HE staining;To assess the presence of DNA damage and senescent cells in the development of radiation ulcer,the expression of DNA damage related protein（γ-H2AX）was detected by immunofluorescence,β-SA-gal staining was assed,Western-blot was used to analyze the expression ofγ-H2AX and senescence related proteins（p16 and p21）,q RT-PCR were used to detect the mRNA levels of SASP such as IL-1β,IL-6 and TNF-α;To evaluate the role of senescent cells in the development of radiation ulcer,senescent cells were subcutaneously injected into the irradiated legs and the changes of the legs were observed.2.The effect of 3’-deoxyadenosine on preventing cell senescence in vitroThe in vitro cell senescence model was established by receiving different radiation doses（0,5,8Gy）;Western blot was used to detect the expression of antioxidan t-related protease such as SOD 1,SOD 2,GPX-1 and Catalase in cells treated with3’-deoxyadenosine for different time;The ability of 3’-deoxyadenosine to reduce radiation-induced total ROS and mitochondrial ROS were detected by flow cytometry;To detect the ability of 3’-deoxyadenosine to inhibit DNA damage induced by ionizing radiation,Western blot and immunofluorescence staining were used to identify the expression ofγ-H2AX;To evaluate the inhibitory effect of 3’-deoxyadenosine on cell senescence and SASP induced by ionizing radiation,Western blot was used to detect the expression of p16 and p21,SA-β-gal staining was assessed,qRT-PCR was used to detect the mRNA level of IL-1α,IL-1β,IL-6,IL-8,MMP-3,MMP-12 and PAI-1,and CCK-8was used to detect cell proliferation;The ability of 3’-deoxyadenosine to promote cell colony formation and reduce oxidative stress-induced apoptosis was evaluated by colony formation experiment and flow cytometry respectively.3.The effect of 3’-deoxyadenosine on mitigating radiation ulcer in vivoThe right posterior limb of SD rat was exposed to 40 Gy radiation,abdominal or head and neck area of C57BL/6J mouse was exposed to 12 or 15 Gy irradiation to establish skin ulcer,intestine ulcer and mucositis models,respectively;HE staining was used to observe the skin,intestines and tongue tissues,and then to evaluate the protective effect of3’-deoxyadenosine on skin,villi,crypt and tongue epithelium;Toluidine blue staining to detect the protective effect of 3’-deoxyadenosine on tongue epithelium;To identify the ability of 3’-deoxyadenosine to reduce DNA damage induced by ionizing radiation and maintain cell proliferation,immunofluorescence was used to detectγ-H2AX,Ki67,Brd U and p63;To evaluate the ability of 3’-deoxyadenosine to inhibit cell senescence and SASP induced by ionizing radiation in vivo,SA-β-gal staining was assessed,Western blot was used to detect the expression of p16 and p21,q RT-PCR was used to detect the expression p16,p21 and IL-1β,IL-6 and TNFα;To evaluate the ability of 3’-deoxyadenosine to reduce apoptosis and fibrosis induced by ionizing radiation in vivo,TUNEL and Masson trichrome staining was used,qRT-PCR was used to detect the m RNA expression of Collagen-1A,Collagen-3A and Fibronectin;To evaluate the effect of 3’-deoxyadenosine on local radiation-induced systemic inflammatory response,ELISA was used to detect the expression of IL-1β,IL-6 and TNF-αin serum.4.The mechanism of 3’-deoxyadenosine on inhibiting cell senescence and mitigating radiation ulcerTo assess whether 3’-deoxyadenosine inhibits cell senescence by activating FOXO and SIRT,Western-blot was used to detect Fox Os and Sirts related proteins,immunofluorescence staining and q RT-PCR were used to detect Fox Os;To evaluate the effect of 3’-deoxyadenosine on activating NRF2 and promoting the entry of NRF2 into the nucleus to promote the expression of downstream related genes,immunofluorescence was used to detect NRF2 in cells and tissues,Western blot was used to detect the expression of NRF2 and p-NRF2 in whole cell lysate,cytoplasm and nucleus respectively,Western blot analysis of TXN and NRF2 downstream related genes（GCLC,GCLM,HMOX1 and NQO1）were done,detection of m RNA levels of NRF2 downstream related genes（GSTA1,NQO1and Srx）by qRT-PCR was used,NRF2 transcriptional activity was measured by using a luciferase-based ARE controlled gene expression system;To identify 3’-deoxyadenosine can inhibit ionizing radiation induced cell senescence by activating NRF2,the ROS was detected,Western blot was used to detect p16 and p21 expression,β-SA-gal staining was assessed,q RT-PCR was used to detect SASP when NRF2 was inhibited by si NRF2;To identify 3’-deoxyadenosine can mitigate radiation ulcer by activating NRF2,ML-385 was used to inhibit the expression of NRF2 in vivo,the damage of skin,intestine and tongue was observed,qRT-PCR was used to detect p16,p21 and SASP.To clarify the effect of 3’-deoxyadenosine on NRF2 protein stability,qRT-PCR was used to detect NRF2,Western blot was used to detect NRF2 after synthesis of protein was inhibited by CHX;To clarify the effect of 3’-deoxyadenosine on activating NRF2 by promoting autophagic degradation of Keap1,q RT-PCR was used to detect Keap1,immunofluorescence was used to detect Keap1 in tissues,Western-blot was used to detect Keap1 and NRF2 after ubiquitin-proteasome and autophagy-lysosome pathways were inhibited by a proteasome inhibitor（MG-132）and distinct autophagy inhibitors（CQ and3MA）respectively,Western-blot was used to detect autophagy markers,autophagic vacuole formation was observed by TEM,Western-blot was used to detect cell senescence after p62and Atg 7 were inhibited by si-p62 and si-Atg7 respectively,ROS was detected by flow cytometry,β-SA-gal staining and colony forming ability were assed.To clarify 3’-deoxyadenosine can prevent cell senescence by activating AMPK,Western-blot was used to detect Akt、S6、ACC,AMPK and senescence associated protein,immunofluorescence was used to detect S6 in tissues,ROS was detected by flow cytometry,β-SA-gal staining and colony forming ability were assessed after AMPK was inhibited by Compound C;To identify 3’-deoxyadenosine can mitigate radiation ulcer by activating AMPK,Compound C was used to inhibit the expression of AMPK in vivo,then to observe the damage of skin,intestine and tongue,and qRT-PCR was used to detect p16,p21;To identify 3’-deoxyadenosine can bind toα1 andγ1 subunit of AMPK near the autoinhibitory domain（AID）,the binding ability of 3’-deoxyadenosine to each subunit of AMPK was evaluated by molecular docking using POCASA.To indirectly indicate that 3’-deoxyadenosine plays a protective role by binding with AMPKα1/γ1,AMPKα1 and AMPKγ1 were inhibited by si-RNA respectively,and then Western-blot was used to detect S6 and NRF2.Results1.High dose irradiation（40Gy）can result in skin ulcer which is difficult to heal,which provides us a stable model for screening drugs to alleviate radiation ulcer;Immunofluorescence staining and Western blot showed that DNA damage markers and proteins（γ-H2AX）were persistent in radiation ulcer;We found persistent senescent cell markers（SA-β-gal）and cell cycle arrest induced senescence related proteins（p16 and p21）in radiation ulcer;At the same time,qRT-PCR showed that the pro-inflammatory factors（IL-1β,IL-6 and TNFα）which related to cell senescence were highly expressed in the ulcerated skin;After senescent cells were subcutaneously injected into the irradiated legs,the development of radiation ulcer was accelerated obviously,and the degree of redness and ulcer was more serious.These results showed that senescent cells were involved in the development of radiation ulcer.Therefore,preventing cell senescence may represent a prospective strategy to mitigate radiation ulcer.2.We established an in vitro fibroblast cell senescence model induced by radiation,Western blot showed that 3’-deoxyadenosine significantly increased the expression of SOD1,SOD2,GPX-1 and Catalase in cells,suggesting that 3’-deoxyadenosine may reduce the oxidative stress induced by ionizing radiation.The total ROS and mitochondrial ROS were detected by DCFDA and Mito-sox red respectively and 3’-deoxyadenosine can significantly reduce the production of ROS in cells;Western blot and imm unofluorescence were used to detect the expression ofγ-H2AX,and 3’-deoxyadenosine can inhibit the level ofγ-H2AX induced by ionizing radiation and accelerate the repair of DNA damage;3’-deoxyadenosine decreased the expression of p16 and p21,and decreased the product ion ofβ-galactosidase;At the same time,the levels of IL-1α,IL-1β,IL-6,IL-8,MMP-3,MMP-12 and PAI-1 was decreased when cells were treated with 3’-deoxyadenosine;3’-deoxyadenosine can promote the colony forming ability in both normal cells and irradiated cells,and reduce the apoptosis induced by ionizing radiation and hydrogen peroxide,suggesting that 3’-deoxyadenosine can inhibit ionizing radiation-induced cell damage and maintain the function of stem cells.3.We have established three kinds of radiation ulcer models（rat skin ulcer,mouse oral mucositis and mouse intestinal ulcer）.We found that 3’-deoxyadenosine can effectively reduce the occurrence and development of three kinds of radiation ulcer and without toxic effect on other organs,3’-deoxyadenosine is a safe radiation protective agent.At the histological level,immunofluorescence staining was used to detectγ-H2AX,Ki67,BrdU or p63,and 3’-deoxyadenosine reduced DNA damage induced by ionizing radiation and maintained cell proliferation;SA-β-gal staining showed that 3’-deoxyadenosine reduced the expression of SA-β-gal in vivo,qRT-PCR and Western blot showed that 3’-deoxyadenosine inhibited the expression of p16,p21 and SASP;3’-deoxyadenosine can reduce the acute injury（apoptosis）of tissue caused by irradiation,and also inhibit the chronic injury（fibrosis）;ELISA was used to detect the expression of IL-1β,IL-6 and TNF-αin peripheral blood,and we found that 3’-deoxyadenosine can effectively reduce the systemic inflammatory response and chronic injury induced by local irradiation.4.At the cellular and histological level,we found that 3’-deoxyadenosine can promote the expression of NRF2,and reverse the protective effect of 3’-deoxyadenosine by interfering with the intracellular NRF2 and inhibiting the level of NRF2 in animals,indicating that 3’-deoxyadenosine can inhibit the radiation induced cell senescence and ulcer by activating NRF2;3’-deoxyadenosine promotes p62 dependent autophagic degradation of Keap1,which dissociates NRF2 and Keap1,and increases the entry of NRF2into the nucleus,then to bind with the oxidative response element（ARE）and activate the transcription of some antioxidant genes,so as to prevent radiation induced cell damage and senescence;3’-deoxyadenosine binds withα1 subunit andγ1 subunit near the auto inhibitory domain（AID）of AMPK,thus promoting the self-inhibitory function of AMPK,then to activate AMPK indirectly,and finally inducing autophagic degradation of Keap1.AMPK-NRF2 is a potential target to inhibit radiation induced cell senescence and mitigate radiation ulcer.Conclusion1.We found that DNA damage and senescent cells persist in animal radiation ulcer;We proved that senescent cells could promote the occurrence and development of radiatio n ulcer when senescent cells were subcutaneously injected into the irradiated legs.Therefore,preventing cell senescence may represent a prospective strategy to mitigate radiation ulcer.2.We identified 3’-deoxyadenosine can significantly inhibit the oxidative stress injury and cell senescence induced by ionizing radiation in an in vivo fibroblast cell senescence model.3.Three kinds of radiation ulcer models（skin ulcer,oral mucositis and intestinal ulcer）were established to further clarify that 3’-deoxyadenosine can mitigate radiation ulcer,inhibit cell senescence and DNA damage.3’-deoxyadenosine can be potential agent to prevent cell senescence and mitigate radiation.4.We identified 3’-deoxyadenosine can bind withα1 subunit andγ1 subunit near the auto inhibitory domain（AID）of AMPK,then to induce dissociation of NRF2 from Keap1and promote NRF2 transport to nucleus,thus inhibiting cell senescence and mitigating radiation ulcer by activating AMPK-NRF2 signaling pathway.This study revealed a new mechanism by which 3’-deoxyadenosine activates AMPK,and this mechanism is demonstrated to play an important role in the prevention of cell senescence and mitigation of radiation ulcer.Meanwhile,the mechanisms by which AMPK regulates antioxidant responses remain largely unknown,our study demonstrates that activating NRF2 represent an approach for AMPK to regulate antioxidant responses.AMPK-NRF2 is a potential target to inhibit radiation induced cell senescence and mitigate radiation ulcer.