Molecular Mechanism Of HMGB1 In The Development Of Radiation Induced Lung Injury

Objective:To explore the role of HMGB1 in the development of radiation-induced lung injury model in mice,whether HMGB1 is involved in the latency and acute inflammation stages of radiation-induced lung injury,the main reasons caused variations of HMGB1 level at different stages,the receptors that HMGB1 binding to in the process of radiation-induced lung injury,and whether the inhibitors of HMGB1 have protective effects for radiation-induced lung injury.Method1)C57BL/6 mice were randomly divided into four groups: control group(Contorl,C): no radiotherapy or glycyrrhizin;Glycyrrhizin + Control group(CG): just administrate glycyrrhizin but no radiation;Radiation group(R): 20 Gy whole thorax radiation without glycyrrhizin;Glycyrrhizin + Radiation(RG): whole thorax radiation and administrated with glycyrrhizin.Observe the changes of HMGB1 in different stages of radiation induced lung injury and whether glycyrrhizin,an inhibitor of HMGB1,can alleviate radiation induced lung injury.2)Immunofluorescence was used to detect the translocation of HMGB1 in mice lung tissue and MLE-12 cells in the early stage after radiotherapy in vivo and vitro.Apoptosis was detected by TUENL assay in the early stage after radiation,analyze the correlation between the apoptosis and the translocation of HMGB1.Whether block the translocation of HMGB1 can alleviate the subsequent inflammation.3)Check the expression HMGB1 and its receptors RAGE and TLR4 in lung tissues during the latent and acute inflammation stage after radiation.Conditional culture method was used in vitro to observe which receptor was the HMGB1 binding for in the development of radiation induced lung injury.Results:1)Acute inflammation was observed at 12 weeks after radiation.The levels of HMGB1 in peripheral blood of C57 mice increased significantly at1 W,2W and 12 W after radiotherapy,but did not increase at 4W,which demonstrate HMGB1 played an important role in the latency and acute inflammation stages of radiation induced lung injury.Glycyrrhizin can decrease plasma HMGB1 levels in latency and acute inflammation stages after radiotherapy,reduce exudation of inflammatory cells in tracheal and alleviate pulmonary inflammation after radiation.2)Immunofluorescence staining showed that there was obvious translocation of HMGB1 in lung epithelial cells at 1W and 2W after radiotherapy,but it was subsided at 4W.Obvious translocation of HMGB1 was observed at 24 hours after radiotherapy in MLE-12 cells.Western-blot analysis also confirmed translocation of HMGB1 in MLE-12 cells after radiation.TUNEL assay indicated that apoptosis was not the reason for translocation and release of HMGB1.Glycyrrhizin can block translocation and release of HMGB1 and alleviate its chemotaxis.3)The expression of RAGE and TLR4 in lung tissue did not improve significantly at 4W after radiation.At 12 W after radiotherapy,the expression of RAGE receptors in lung tissue decreased significantly,while the expression of TLR4 receptors increased significantly,suggesting that HMGB1/TLR4 is an important pathway mediating radiation-induced lung injury.The level of s RAGE in peripheral blood increased significantly at 4W and 12 W after radiotherapy,suggesting that plasma s RAGE level may be a biomarker for predicting and determining the degree of radiation-induced lung injury.Conclusion:HMGB1 plays an important role in the latent and acute inflammation stages of radiation-induced lung injury.During the latent stage,radiation first caused nucleoplasm translocation of HMGB1 in epithelial cells,then the high concentration of HMGB1 in cytoplasm were released into extracellular environment.HMGB1 in outside cell chemoattracts immune inflammatory cells to the damaged lung tissues and were activated by binding the TLR4 receptor on the surface of inflammatory cells.HMGB1 and secrete cytokines,leading to acute radiation lung injury.Radiotherapy can damage lung epithelial cells,decrease the expression of RAGE receptor in lung tissue,and increase the expression of s RAGE in plasma,so the level of s RAGE in serum can be used as a biomarker to predict and determine the degree of radiation-induced lung injury.Target HMGB1 is a potential therapy to alleviate the radiation induced lung injury.