Establishment Of Animal Model Of Radiation Injury Of Skin And Study On The Therapeutic Effect Of Vitamin B₁₂ Ointment In The Treatment Of Radiation Injury Of Skin

Background:With the growth of the world population and the extension of the average life span of human beings,the number of patients with malignant tumors is increasing year by year.Malignant tumor has become an important disease that seriously threatens human health and life.Radiotherapy is one of the most important treatment methods for malignant tumors,which is suitable for most malignant tumors.According to research statistics,more than 50%of patients with malignant tumors need to receive radiotherapy during the course of the disease.However,while radiation destroys and inhibits tumors,it also inevitably destroys normal tissues and organs,leading to side effects.Radiation injury of skin is one of the common side effects of patients receiving radiotherapy,and 95%of patients will have different degrees of radiation injury of skin during radiotherapy.Acute radiation injury of skin usually occurs within 2-3 weeks after the start of radiotherapy.The clinical manifestations include mild skin erythema,dry desquamation,moist desquamation,and ulcer necrosis.Chronic radiation injury of skin can occur months or even years after radiotherapy.The clinical manifestations include telangiectasia,skin atrophy,fibrosis,edema,necrosis,fistula formation,and prolonged wound healing.Different from simple trauma and general burns,the damage caused by radiation on the skin and subcutaneous tissues usually causes infections and ulcers,which are difficult to cure,and lead to fibrosis and necrosis,and even physiological dysfunction,which seriously affects the quality of life of patients and restricts the smooth completion of treatment plan.Therefore,the prevention and treatment of radiation injury of skin has become a major problem in the field of radiotherapy.The treatment of radiation injury of skin includes general care,local treatment,dressing treatment,operation and so on.However,in the current clinical research on the treatment of radiation injury of skin,the effects of many treatment methods and drugs seem to be contradictory,and there is still no standard method to reach a consensus.Therefore,based on the current situation,the development of an efficient and safe specific drug for the treatment of radiation injury of skin has immeasurable economic and social benefits.More than 70 years ago,when looking for an"anti-pernicious anemia factor" for the treatment of pernicious anemia,Vitamin B12 was first discovered in liver extracts.Vitamin B12,also known as cobalamin,is a water-soluble vitamin.It is an important coenzyme in the process of cell synthesis of nucleic acid.It can catalyze molecular rearrangement,deoxynucleotides formation from nucleotides,demethylation and other reactions.In addition,Vitamin B12 can accelerate the proliferation of granulation tissue and the regeneration of epithelial cells,has a significant repair and regeneration function on damaged skin mucosal epithelial cells and vascular endothelial cells,and has nutrition and promote repair effects on damaged nerve sheaths.However,at present,there are few basic researches on the treatment of radiation injury of skin with Vitamin B12,and the specific mechanism of its treatment is still unclear,which is a key issue to be solved urgently.Objective:This study explored the therapeutic effect of Vitamin B12 ointment on radiation injury of skin by establishing an animal model of radiation injury of skin,in order to provide a theoretical basis for the future clinical application of Vitamin B12 ointment.Methods:(1)Establish an animal model of Ⅳ grade radiation injury of skin with mini fragrant pigs.Set up a drug-free group,a triethanolamine group,and a Vitamin B12 group,and apply medicine and observe and take pictures according to different groups.Radiation Therapy Oncology Group(RTOG)grading standard was used to evaluate the changes in the grade of skin damage in each group over time.(2)After applying the medicine and observing for 12 weeks,the skin tissues of the test areas of each group were cut out,and HE and Masson staining were performed to detect the histological changes of the skin in different groups.(3)After applying the medicine and observing for 12 weeks,the skin tissues of the tested areas of each group were cut,and immunohistochemistry and immunofluorescence staining were performed to detect the expression levels of NF-κB,COX-2,IL-6,and TGF-β in the skin tissues of different groups.(4)RT-qPCR was used to detect the expression levels of NF-κB,COX-2,IL-6 and TGF-β mRNA in the skin tissues of the tested areas in each group.(5)Western-blot was used to detect the expression levels of NF-κB,COX-2,IL-6,and TGF-β protein in the skin tissues of the tested areas in each group.Results:(1)Vitamin B12 ointment shows an excellent therapeutic effect in animal models of radiation injury of skin.After 12 weeks of medicine application and observation,there were significant differences in the appearance of the skin in each group.The skin of the drug-free group showed grade Ⅳ radiation injury,the triethanolamine group showed grade Ⅱ radiation injury,the Vitamin B12 group showed grade Ⅰradiation injury,which almost recovered to normal skin condition.(2)Vitamin B12 ointment treatment slowed down the tendency of epidermal thickening and collagen deposition after skin radiation injury.The results of HE staining showed that compared with normal skin,the epidermis of each group was thickened to different degrees.The epidermal thickness from high to low was drug-free group,the triethanolamine group,the Vitamin B12 group,and the normal skin.Masson staining showed a large amount of collagen deposition in the drug-free group.The Vitamin B12 group and the normal skin had the same collagen content,which was much lower than that of the drug-free group.(3)The expression level of molecules related to radiation injury of skin was significantly reduced after Vitamin B12 ointment treatment.In immunohistochemistry,Western-blot,and RT-qPCR detection,it was found that the expression levels of NF-κB and COX-2 in the Vitamin B12 group were significantly lower than those in the drug-free group.(4)After Vitamin B12 ointment treatment,the expression level of inflammation-related molecules was significantly reduced.In immunohistochemistry,Western-blot,and RT-qPCR detection,it was found that the expression of IL-6 in the Vitamin B12 group was significantly lower than that in the drug-free group.(5)The expression level of radiofibrosis-related molecules was significantly reduced after Vitamin B12 ointment treatment.In immunofluorescence,Western-blot,and RT-qPCR detection,it was found that the expression level of TGF-β in the Vitamin B12 group was significantly lower than that in the drug-free group.Conclusion:In conclusion,Vitamin B12 ointment can significantly reduce the expression levels of NF-κB,COX-2,IL-6,and TGF-β after skin radiation injury,and has strong anti-radiation,anti-inflammatory and anti-fibrosis effects,which has an excellent effect on the treatment of severe radiation injury of skin.The results of this study lay the foundation for future clinical research.The application of Vitamin B12 ointment as a specific drug for radiation injury of skin in clinical practice is especially worth looking forward to.