Protective Role And Mechanism Of Nuclear Factor-erythroid 2-related Factor 2 Against Radiation-induced Lung Injury

Part I: Effect and mechanism of Nrf2-knockout on radiation-induced lung injury in miceObjective To establish a mouse model of radiation-induced lung injury(RILI)using wild-type mice and Nrf2-knockout mice,and to explore the effect and its mechanism of Nrf2-knockout on RILI in mice.Materials and methods Twenty-four wild-type mice(WT)and 24 Nrf2-knockout mice(KO)were divided into 4 groups respectively(6 per group): normal control group(CON),1 day after irradiation group(IR1d),7 days after irradiation group(IR7d)and 14 days after irradiation group(IR14d).Mice in CON received no treatment.Mice in IR1 d,IR7d,IR14 d group were subjected to 15 Gy chest X-ray irradiation to establish a mouse model of RILI,and then the mice were sacrificed on Day 1,Day 7 and Day 14 after X-ray irradiation.Lung tissues and serum of mice in each group were collected.The severity of RILI in mice was detected by HE staining.The collagen deposition in the lung tissues was detected by Masson staining.The content of collagen in the lung tissues was indirectly determined by hydroxyproline content measurement.Immunohistochemistry staining of F4/80 and MPO was performed to explore the infiltration of macrophages and neutrophils in the lung tissues.The levels of inflammatory cytokines in serum were detected by CBA method.The oxidative damage of lung tissues was detected by 8-OHd G and 4-HNE immunohistochemical staining and serum MDA level detection.Western blotting was used to detect the protein levels of Bax and Bcl-2 in lung tissue.Protein levels of Nrf2,SOD2,GPx1 and CAT antioxidant activity in lung tissues were detected by Western blotting and CAT activity kit.Protein levels of TGF-β1,Smad2/3,p-Smad2,p-smad3,Collagen I,Collagen III,and α-SMA in lung tissues were detected by Western blotting to investigate the activation of fibrosis signaling pathway and fibrotic phenotype after irradiation.Results After X-ray irradiation,the lung injury was obvious with damaged alveolar structure,thickened alveolar wall and hemorrhage.The injury score based on HE staining was increased(P<0.05).The model of RILI was well-established.The lung injury in Nrf2-knockout mice was aggravated compared with wild-type mice at the same time-point after radiation and the injury score based on HE staining was significantly increased(P<0.05).Collagen deposition and hydroxyproline content in lung tissues of mice were significantly increased after X-ray irradiation(P<0.05).Collagen deposition and hydroxyproline content in lung tissues of Nrf2-knockout mice increased significantly compared with wild-type mice(P<0.05).The infiltration of macrophages and neutrophils in lung tissues of mice increased after X-ray irradiation(P<0.05).Nrf2-knockout mice had significantly increased infiltration of macrophages and neutrophils in lung tissues at the same time-point after irradiation compared with wild-type mice(P<0.05).The serum levels of IL-12,IFN-γ,MCP-1,TNF,IL-6 and IL-10 in the mice were significantly increased after X-ray irradiation(P<0.05).The levels of inflammatory cytokines IL-12,IFN-γ,MCP-1,TNF and IL-6 were significantly higher in Nrf2-knockout mice at the same time-point after radiation than in wild-type mice(P<0.05),while level of anti-inflammatory cytokine,IL-10,was significantly lower than that of wild-type mice(P<0.05).Levels of 8-OHd G,4-HNE and MDA in lung tissues of mice were significantly increased after X-ray irradiation(P<0.05).8-OHd G,4-HNE and MDA levels of lung tissues at the same time-point after radiation were significantly higher in Nrf2-knockout mice than those in wild-type mice(P<0.05).The levels of anti-apoptotic protein Bcl-2 in lung tissues of mice decreased after X-Ray radiation and the levels of pro-apoptotic protein Bax increased(P<0.05).Compared with wild-type mice,the levels of Bcl-2 in lung tissues of mice decreased in Nrf2-knockout group,and the levels of Bax increased(P<0.05).After X-ray irradiation,the anti-oxidative proteins Nrf2,GPx1,SOD2 were significantly up-regulated and the CAT activity was significantly increased in the lung tissues of mice(P<0.05).The protein levels of GPx1,SOD2 and activity of CAT in Nrf2-knockout mice were significantly lower than those of wild-type mice at the same time-point after irradiation(P<0.05).The protein levels of TGF-β1,p-Smad2,p-Smad3,Collagen I,Collagen I and α-SMA were significantly up-regulated in lung tissues of mice after X-Ray radiation(P<0.05).The protein levels of TGF-β1,p-Smad2,p-Smad3,Collagen I,Collagen I and α-SMA were significantly higher in Nrf2-knockout mice than those in wild-type mice(P<0.05).Conclusion Nrf2 knockout aggravated RILI in mice caused by X-ray radiation.Loss of Nrf2 may aggravate RILI by impairing the antioxidative capacity,resulting in increased oxidative damage and immune imbalance.In addition,Nrf2 deficiency may promote the formation of myofibroblasts and the deposition of collagen by activating TGF-β1-Smad2/3 pathway,thereby promoting the progression of pulmonary fibrosis.Part II: Protective effect and mechanism of Nrf2 overexpression on radiation-induced lung injury in miceObjective To establish a mouse model of Nrf2 overexpression in lung tissue by intratracheal injection of Nfe2l2 overexpression lentivirus.Nrf2 overexpressing mice and wild-type mice were used to establish a model of radiation-induced lung injury(RILI)to explore the protective effect and underlying mechanism of Nrf2 overexpression on RILI.Materials and methods A mouse model of Nrf2 overexpression in lung tissue was established by intratracheal injection of Nfe2l2 overexpression lentivirus.Ten Nrf2 overexpressing mice(Lv-Nfe2l2)and 10 vector control mice(Lv-vector)were divided into two groups respectively: normal control group(CON),14 days after X-ray irradiation group(IR14d),5 mice in each group.The mice in CON group received no radiation.The mice in IR14 d group received 15 Gy chest radiation and the lung tissues and serum samples were collected on Day 14 after irradiation.The severity of RILI of mice was detected by HE staining.The collagen deposition in lung tissues was detected by Masson staining.The collagen content in lung tissues was indirectly determined by hydroxyproline content measurement.The infiltration of macrophages and neutrophils in lung tissues was detected by immunohistochemical staining of F4/80 and MPO.The serum levels of cytokines were detected by CBA method.The oxidative damage in lung tissues was detected by immunohistochemical staining of 8-OHd G and 4-HNE,and detection of MDA levels.The protein levels of Nrf2,SOD2,GPx1 and activity of CAT in lung tissues were detected by Western blotting and CAT kit to explore antioxidive capacity.Results Compared with CON group,lung injury of the mice was severe and HE staining score was increased on Day 14 after X-ray irradiation(P<0.05).Compared with vector control group,lung injury and HE staining score were decreased on Day 14 after radiation in Nrf2 overexpressing mice(P<0.05).On Day 14 after radiation,the collagen deposition and hydroxyproline content in lung tissues of Nrf2 overexpressing mice were significantly lower than those in vector control mice(P<0.05).Compared with CON group,pulmonary infiltration of macrophages and neutrophils increased significantly on Day 14 after X-Ray radiation(P<0.05).The infiltration of neutrophils and macrophages in lung tissues of Nrf2 overexpressing mice was significantly lower than those of vector control mice on Day 14 after radiation(P<0.05).The serum levels of IL-12,IFN-γ,MCP-1,TNF,IL-6 and IL-10 increased significantly on Day 14 after X-Ray radiation in vector control and Nrf2 overexpressing mice(P<0.05).The levels of IL-12,IFN-γ,MCP-1,TNF and IL-6 in mice were significantly lower in Nrf2 overexpressing group than those in vector control group on Day 14 after radiation,while the level of IL-10 was significantly higher than that of vector control group(P<0.05).The levels of 8-OHd G,4-HNE and MDA in lung tissues of vector control and Nrf2 overexpressing mice were significantly increased on Day 14 after X-ray irradiation(P<0.05).Compared with vector control group,the levels of 8-OHd G,4-HNE and MDA were significantly lower in Nrf2 overexpressing group on Day 14 after radiation(P<0.05).The protein levels of Nrf2,GPx1,SOD2 and activity of CAT in lung tissues of vector control and Nrf2 overexpressing mice were significantly increased on Day 14 after irradiation(P<0.05).The proteins levels of Nrf2,GPx1 and SOD2,and activity of CAT in Nrf2 overexpressing mice were significantly higher than those of vector control group on Day 14 after irradiation(P<0.05).Conclusion Nrf2 Overexpression alleviate RILI in mice caused by chest radiation.Nrf2 overexpression may reduce RILI by enhancing antioxidant capacity,mitigating oxidative damage caused by radiation,and regulating the pro-inflammatory and antiinflammatory rebalance.The antioxidant signalling pathway may be a promising target for the prevention and treatment of RILI.