Molecular mechanisms of lung injury from ultra high and conventional dose rate pulsed radiation based on 4D DIA proteomics study

To investigate the mechanism of ultra-high dose rate pulsed radiation in radiation-induced lung injury (RILI), providing an experimental and theoretical basis for the application of FLASH-RT (FLASH-radiotherapy) in radiotherapy. C57BL/6J mice were randomly divided into three groups: a sham group, a FLASH-irradiated group, and a CONV-irradiated group. A whole-body irradiation with a single dose of 3 Gy (200 Gy/s for FLASH group and 0.3 Gy/s for CONV group) using electron rays was used to establish models of lung injury. After 3 months, lung tissues were stained with HE and Masson stains to observe pathological changes in lung tissue and subjected to 4D-Fast DIA quantitative proteomics, with the sequencing data validated by Western blot and immunofluorescence. The mice in FLASH group had less lung tissue damage and lower levels of fibrosis compared to the CONV group. Proteomic sequencing showed significant differences in CCT6b protein expression between the two irradiation groups. As verified by the WB and immunofluorescence assays, the expression level of CCT6b was significantly reduced in the CONV group of mice compared with the SHAM and FLASH groups. With the down-regulation of CCT6b, there was a notable decrease in the expression of E-cadherin, accompanied by an increase in the expression of alpha-smooth muscle actin and Vimentin. The differential response in the level of lung fibrosis caused by the two types of radiation may be related to the level of CCT6b expression, but the specific mechanism of action needs to be further investigated.