The Role Of Bach1 In Regulating Macrophage Mitochondrial Homeostasis In Radiation-induced Lung Injury

Radiation-induced lung injury is an unavoidable complication of radiotherapy for thoracic malignancies,which severely limits the dose of radiation therapy for tumors and endangers the life of patients.The unclear mechanism of RILI,lacking of effective predictors and treatments that are of great significance to explore the molecular pathological mechanism of RILI and discover and identify effective drug targets.As the first line of defense in lung,macrophages maintain lung immune homeostasis.Mitochondria play a crucial role in the immune response of macrophages as the center of cellular energy metabolism.Mitochondria are extremely sensitive to ionizing radiation,which are significant targets of ionizing radiation.The energy deposition of ionizing radiation resulting large amounts of ROS in cells which imbalanced mitochondrial homeostasis.Mitochondrial homeostasis is a vital cause of lung tissue impairment.Therefore,effective protection of mitochondria and timely repair of damaged mitochondria,maintenance of mitochondrial homeostasis are key means to mitigate RILI.Bach1 regulates oxidative stress,regulating mitochondrial biogenesis,mitochondrial fusion division and other processes by mediating the expression of various mitochondrial functional genes,which is a key factor in maintaining the homeostasis of structure and function.The deletion of Bach1 gene has been shown to protect mitochondrial function to attenuate acute liver injury caused by sepsis and hyperoxia-induced acute lung injury.However,the effect and role of Bach1 gene in RILI are not yet clear.Therefore,in this study,the changes and role of Bach1 in RILI were clarified through in vivo and in vitro experiments,and the role of Bach1 in regulating mitochondrial homeostasis in macrophages in RILI was elucidated.In vivo experiments were conducted to construct a mouse RILI model under the conditions of a single dose of 20Gy of X-rays with a time point of two weeks of post-irradiation rearing.Wild type（WT）mice were randomly divided into control（Control）and irradiation（IR）groups to explore the changes of Bach1 in RILI.The results indicated structural destruction of lung tissue and increased macrophage infiltration with increased Bach1 protein content in the IR group compared to the Control group.Bach1 gene knockout mice(Bach1^-/-)were further used to verify the role of Bach1 in RILI and were randomly divided into wild type control（WT）,knockout control(Bach1^-/-),wild-radiation（WT-IR）,and knockout-irradiation(Bach1^-/--IR)groups.The results revealed that Bach1 knockdown attenuated RILI in the Bach1^-/--IR group compared to the WT-IR group with structural disruption of lung tissue,decreased antioxidant capacity,and suppressed inflammatory upregulation and mitochondrial homeostatic imbalance.In vitro experiments were performed by isolating mouse bone marrow-derived macrophages（BMDMs）and constructing radioactive cell models under the conditions of a single dose of 8Gy of X-rays with 24 hours post-irradiation as the time point.BMDMs from WT mice were randomly divided into control（Control）and irradiation（IR）groups,and the results displayed that macrophages were stimulated by ionizing radiation and activated leading to decreased antioxidant capacity,inflammatory damage and impairment of mitochondrial function.On this basis,BMDMs from WT mice and Bach1^-/-mice were randomly divided into wild type control（WT）,knockout control(Bach1^-/-),wild-irradiation（WT-IR）,and knockout-irradiation(Bach1^-/--IR)groups.The results showed that Bach1 knockdown inhibited the accumulation of ROS,decreased antioxidant capacity and reduced inflammatory factor release caused by ionizing radiation.To further clarify the effect of Bach1 knockdown on mitochondrial function,the results of detecting mitochondrial membrane potential,ATP content and mitochondrial Ca²⁺concentration showed that the decrease in mitochondrial membrane potential,ATP content and increase in mitochondrial Ca²⁺concentration were suppressed in Bach1^-/--IR compared to WT-IR group.In addition,BMDMs transfected with overexpressed Bach1 gene plasmid into WT mice were randomly divided into negative control（NC）,overexpression（OE）,negative-irradiation（NC-IR）,and overexpression-irradiation（OE-IR）groups.The results suggested that overexpression of the Bach1 gene resulted in a more pronounced decrease in antioxidant capacity,inflammatory factor release and mitochondrial damage.In summary,in vivo and in vitro experiments confirmed that the deletion of Bach1 gene could maintain mitochondrial homeostasis in macrophages caused by ionizing radiation and alleviated RILI.