The Study On Mechanism Of NLRP3 Inflammasome In Radiation-induced Skin Injury

With the increasing use of radiotherapy,more and more attention has been paid to complications caused by radiotherapy.Among them,how to prevent and treat radiation skin injury is still an important problem to be solved,and its treatment was still lack of effective treatment means.At present,a large number of clinical studies and experimental studies have confirmed that the formation and healing process of wound after radiation have been delayed,but the specific molecular mechanisms have not been well understood yet.The establishment of appropriate animal models is essential to elucidate the pathogenesis and development of radioactive skin injury.As an inflammatory regulatory platform discovered in recent years,NLRP3 inflammasome plays a crucial role in inflammatory response,and activation of inflammasome can cause cell pyroptosis and promote the occurrence of inflammatory response.It has been reported that NLRP3 inflammasome were involved in the pathological process of various inflammatory skin diseases,but little has been known about the role of NLRP3 inflammasome in skin wound healing.In this study,high-energy X-ray with single high-dose irradiation on the right hind limb of rats was used to establish the radiation-induced skin injury model.We use this model to explore the activation and change pattern of NLRP3 inflammatory corpuscle after radiation,and to speculate its role in the inflammatory response of radioactive skin injury and its influence on the whole healing process.The detail information is as follows:? To establish a rat model of radiation-induced skin injuryThe electron beam and X-ray with the radiation dose of 32 Gy,38Gy and 45 Gy were used to explore the optimal dose and radiation type for the establishment of the animal model of radiation-induced skin injury,which irradiate the circular area with the diameter of 3cm on the right hind limb of Wistar male rats at a single large dose.The skin lesions of rats were detected by clinical symptoms and HE pathological examination.? To investigate the expression and change pattern of NLRP3 inflammasomeThe animal model of radiation-induced skin injury was established to observe the expression and change pattern of NLRP3 inflammasome in skin tissue and serum at 6h,12 h,24h,48 h,72h,14 d,15d and 18 d after irradition.? To observe the distribution of NLRP3 inflammasome protein in skin tissues and the apoptosis rate after irradiationImmunohistochemical staining and immunofluorescence double-labeling staining were used to investigate the distribution of NLRP3 inflammasome related proteins in skin tissues.The apoptosis rate in tissue after irradiation was measured by TUNEL.The main results are as follows:? No death in two groups while only 45 Gy group of animals in the electronic radiation group had radiation-induced skin injury.Different degrees of radiation-induced skin injury were observed in X-ray irradiation groups.H&E staining showed necrosis in epidermis,dermal edema,inflammatory immune cells infiltration and loss of skin adnexal,especially in38 Gy and 45 Gy groups.However,the skin wound in the 45 Gy group was too severe,which posed a risk of infection,and fewer patients have reached such a degree of injury clinically.Therefore,X-ray and 38 Gy radiation dose were selected subsequently.? After radiation(X-ray,38 Gy),the contents of NLRP3 and IL-1? in serum of irradiated animals were positively correlated with time;In combination with the symptoms of radiation-induced skin injury in rats,it was found that the severity of the wound was positively correlated with the content of IL-1?.? The apoptosis rate were increased significantly at 6h and 72 h after irradiation.? After irradiation,the activition of NLRP3 inflammasome in skin wound tissues were significantly enhanced,and the NLRP3 and ASC protein were mainly expressed in the epidermis and dermal appendages,caspase-1 protein was also expressed in the dermis layer;The NLRP3 inflammasome were mainly expressed in macrophages and epithelial cells in the epidermal layer and dermal skin appendages within 3d after radiation;NLRP3 inflammasome were mainly expressed in epidermal cells 14 days after irradiation while there was no expression of inflammation in macrophages in the dermis layer.? Caspase-1 was consistently highly expressed at 3 d after radiation,and the apoptosis rate reached the maximum at 3 d,suggesting that pyroptosis might occur at this time.A safe,stable and effective radiation-induced skin injury model can be established by single high-dose irradiation on the skin of the right hind limb of rats;Radiation can activate inflammasome in skin tissue,and promote the production and release of inflammatory factors,thus causing an inflammatory response;Radiation can induce not only apoptosis of cells in skin tissue,but also the caspase-1-dependent pyroptosis of cells;In the later stage of skin injury by radiation,the high expression of NLRP3 in the epidermal cells which were non-macrophages may be activated by factors such as IL-1?.As mentioned above,NLRP3 inflammasome may be one of the important links of radiation skin injury and inflammatory cascade reaction.