The Role And Mechanism Study Of Ubiquitin Specific Peptidase 11 In Radiation Induced Pneumonitis

Purpose:Radiation induced pneumonitis(RIP)is an important factor limiting radiotherapy for thoracic cancer.Ubiquitin specific peptidase 11(USP11)is reported to be involved in a variety of biological processes such as cell cycle regulation,DNA damage and repair,inflammatory response,but its role in RIP remains unclear.This study intends to explore the biological role and mechanism of USP11 in the development of RIP and find drugs targeting USP11 to treat RIP.Methods:1.The RIP models of USP11 knockout mice(Usp11-/-)and wild-type mice(Usp11+/+)were established by whole thorax irradiation with 20 Gy X-ray and total body irradiation with 9 Gy X-ray,respectively.RT-PCR,ELISA,immunohistochemical staining,electron microscope,fluorescence tracing and ABPAS staining were used to detect the changes of cytokines,tight junction and mucus layer in lung tissue after ionizing radiation,so as to clarify the effect of knockout USP11 expression on the occurrence of RIP and barrier destruction in vivo.2.HE staining,RT-PCR and ELISA experiments were used to investigate the effect of mitoxantrone(an inhibitor of USP11)on RIP in mice.3.Polychromatic immunofluorescence was used to analyze the expression of USP11 among various types of pulmonary parenchymal cells including alveolar type Ⅱ epithelial cells(ACEⅡ),vascular endothelial cells and fibroblasts,to speculate the probable effector cells of USP11.In the identified effector cells,the effects of up and down regulating USP 11 expression on the production and release of inflammatory factors and the changes of tight junction were verified by RT-PCR and Western Blot.Co-culture system was used to clarify whether USP11 participated in the regulation of neutrophil chemotaxis and apoptosis by paracrine of the effector cells after ionizing radiation,and the transition between radiation-induced pneumonitis fibrosis.4.Proteomics and protein ubiquitination modification were used to analyze the response of USP11 effector cells after ionizing radiation,and the results were verified by Western blot and Co-IP experiments.5.Furthermore,the potential target of USP11 was detected by TCGA database analysis,and the relationship between USP11 and C/EBPβ was detected by immunofluorescence and Co-IP experiment.Western Blot and RT-PCR were performed to explore the effect of USP11 on C/EBPβ stability and its role in the regulation of inflammatory response.Results:1.After whole thorax irradiation with 20 Gy X-ray for 2 and 3 weeks,the cytokines related to inflammatory reaction in mouse lung tissue increased significantly,indicating that the modeling of RIP in mice was established successful.The expression levels of IL-1β、IL-6 and IL-8 in the serum,lung tissue and bronchoalveolar lavage fluid of Usp11-/-were lower than those of Usp11+/+,and knockout of USP11 increased the thickness of mucus layer after ionizing radiation and reduced the permeability of mouse pulmonary vessels to fluorescent tracer FITC-dextran.2.The expression of IL-8,et al.in lung tissues of Usp11-/-mice was lower than that of Usp11+/+ under 9 Gy total body X-ray irradiation,the secretion of protective mucin was increased,and the destruction of tight junctions was reduced.3.As an inhibitor of USP11,mitoxantrone can reduce the expression of IL-1β,IL-6,and IL-8 in the serum of mice after radiation,thereby alleviating RIP.4.Immunofluorescence staining showed that USP11 was highly expressed in the mouse pulmonary vascular endothelial cells,and Western Blot experiment results showed that USP11 existed obvious radiation response effect in the microvascular endothelial HMEC-1 cell.5.The results in vitro showed that down regulating the expression of USP11 could reduce the release of inflammatory cytokines,the level of ROS,the damage of tight junction and permeability in endothelial cells caused by ionizing radiation.6.The results of co-cultured system showed that down regulating the expression of USP11 in endothelial cells reduced its recruitment to neutrophils,promoted the apoptosis of neutrophils and alleviated the fibrosis process of fibroblasts.7.The results of ubiquitination modification combined with proteomics suggested that overexpression of USP11 can stabilize a variety of de ubiquitination enzymes such as USP22,USP33 and OTUD5,and preliminarily clarified that USP11 participated in the occurrence of inflammatory response by stabilizing OTUD5.8.USP11 stablized C/EBPβ by deubiquitination to play a pro-inflammatory role.Co-IP experiment result showed that USP11 and C/EBPβ did not work through direct combination.We preliminarily confirmed that OTUD5 was involved in USP11 deubiquitination C/EBPβ.Conclusion:1.USP11 can increase the occurrence and progression of early RIPI both in vivo and in vitro.2.The mechanism involved may be related to USP11 stabilizing C/EBPβ protein by regulating OTUD5 and promoting the occurrence of RIP.3.We preliminarily revealed that mitoxantrone,an inhibitor of USP11,could alleviate RIP in mice.Based on the above results,this study will provide reliable theoretical support for the development of novel prevention and treatment strategies by targeting USP11-OTUD5-C/EBPβ for RIP.