Study On The Effect And Mechanism Of DHFR On Radiation Induced Skin Injury

The incidence rate of malignant tumor is increasing year by year in recent years,which is seriously harmful to human life and health.Radiotherapy is one of the important means of tumor treatment.While halting tumor growth,radiation therapy can also cause damage to the surrounding tissue.As the largest organ in the body,skin is vulnerable to attack from radiation.Actually,most patients have varying degrees of skin injury caused by radiation.Currently available methods for the treatment of radiation induced skin injury include local application of non-steroidal ointment,steroid ointment,antioxidant,hydrogel dressing and stem cell therapy.Patients suffering from radiation dermatitis have poor response to these treatments.Therefore,it is very important to explore the mechanism of radiation induced skin injury and find more effective treatment methods.Objective:The aim of this study is to clarify the effects of ionizing radiation on the expression of tetrahydrofolate(THF)and dihydrofolate reductase(DHFR),to explore the effect of DHFR on radiation-induced skin injury in vivo and in vitro,and to reveal the mechanism of inhibition of THF synthesis pathway by ionizing radiation,so as to provide a new target for the treatment of radiation-induced skin injury.Methods:(1)To explore the effects of ionizing radiation on THF and DHFR:1.ELISA method was used to detect the changes of THF in human skin tissues and skin cells after ionizing radiation.2.The expression of DHFR was verified by western blot analysis in the skin cells at different time points after exposure to ionizing radiation;(2)From the cell level to explore the effect of DHFR on the progression of radiation induced skin injury:1.Adenovirus overexpressing DHFR was synthesized and its expression was verified by immunofluorescence and western blot analysis;2.CCK-8 assays were used to determine the effect of DHFR on the proliferative capacity of cells after exposure to ionizing radiation;3.Flow cytometry was used to determine the effect of DHFR on cell apoptosis;4.The effect of DHFR on DNA damage repair in cells exposed to ionizing radiation was assessed by immunofluorescence;5.The effect of DHFR on mitochondrial structure and stability of membrane potential in cells exposed to ionizing radiation was examined using a mitochondria specific fluorescent probe.(3)To explore the effect of DHFR on the progression of radiation skin injury in vivo:1.Establish the radiation skin injury model of rats:choose 28 normal 6-week-old SD male rats and the rats received 35 Gy electron wire irradiation after shaving on the back;2.After irradiation,rats were randomly assigned to four groups:PBS injected group(PBS group),control adenovirus injected group(Ad-NC group),adenovirus overexpressing DHFR injected group(Ad-DHFR group),folic acid injected group(FA group).Each group was injected with the corresponding drugs in the irradiated area at the following time points:immediately,3,and 7 days after exposure;3.Within two months after receiving ionizing radiation,photographs of wounds were taken every 3 days and skin damage scoring was performed,and the curves of skin damage score changes were plotted;4.The rats were sacrificed 8 weeks after irradiation,and the wound tissue was excised.The levels of reactive oxygen species(ROS)and malondialdehyde(MDA)in the irradiated skin tissue were detected by using the ROS fluorescent probe and MDA detection kit.HE staining was used to detect the histomorphological changes of the irradiated skin.(4)High throughput sequencing was performed on both irradiated and non-irradiated WS1 cells to detect molecular changes at the intracellular transcript level.Bioinformatics methods were used to explore the mechanism of DHFR down-regulation induced by ionizing radiation.Results:(1)1.ELISA results showed that the THF content in human skin tissues and cells decreased after ionizing radiation.Western blot results showed that the expression of DHFR protein in skin cells was significantly decreased at 48 h after irradiation.(2)Western blot analysis and immunofluorescence confirmed that adenovirus-mediated DHRF overexpression was successful.DHFR overexpression could improve the viability of skin cells after irradiation,reduce the apoptotic rate of skin cells exposed to radiation,help to maintain the structural integrity of mitochondrial structure and the stability of the membrane potential in irradiated skin cells,and reduce DNA double strand break damage induced by ionizing radiation in vitro.(3)The skin exposed to radiation showed obvious congestive erythema two weeks later,and formed more obvious ulcers at about 4 weeks.Compared with the PBS group and the Ad-NC group,the Ad-DHFR group and the FA group had slower skin erythema and ulcer formation,lower skin damage scores,and lower levels of ROS and MDA in the skin tissue.HE staining showed that the thickness of the skin cuticle was smaller and the number of skin appendages was more in the Ad-DHFR group and the FA group.(4)Ionizing radiation modulated the expression of 2158 genes(1588 of which were up-regulated and 570 were down regulated)in WS1 cells.DHFR and methyltransferase METTL16 were significantly down-regulated genes,and there was a significant positive correlation between the expression levels of METTL16 and DHFR.Bioinformatics predicted that the DHFR mRNA region contained m6A methylation modification sites.Conclusions:It was found that ionizing radiation could decrease THF content and DHFR expression in skin.Overexpression of DHFR can alleviate radiation-induced skin injury.In addition,bioinformatics analysis suggested that m6A modification may play an important role in the regulation of DHFR expression by ionizing radiation.Therefore,this study is expected to have a deeper understanding of the mechanism of radiation-induced skin injury and provide a new treatment plan for reducing radiation-induced skin injury.