The Role Of Complement In Radiation-induced Lung Injury

This research is divided into two parts:The first part systematically observed the changes of various complements in lung tissue within 6 months after ⁶⁰Coγ-ray irradiation（20Gy）in the chest of mice,explored the role of complement in radiation-induced lung injury,and identified important complements closely related to radiation-induced lung injury.In the second part,the important complement C3a,which was found to be closely related to radiation lung injury,was intervened to further verify and reveal its possible role and mechanism in regulating radiation lung injury.Methods:In the first batch of animal experiments,140 C57BL/6 mice were randomly divided into 14 groups（7 control groups,7 irradiation groups）,10 mice in each group.The irradiated group was given a single chest irradiation of 20 Gy with⁶⁰Coγray,and the content changes of complement C2,C3a,C4,C5a and complement complex C5B-9 in lung tissue homogenate were detected by enzyma-linked immunosorbent assay（ELISA）at 1 d、3 d、7 d、15 d、30 d、3 m and 6 m after irradiation respectively;Real-time quantitative PCR（RT-PCR）was used to detect intracellular complement m RNA levels in lung tissues after early irradiation.Western Blot was used to detect the expression of C3a,C5a and C3a receptor（C3a R）.In the second batch of animal experiments,120 mice were randomly divided into12 groups（3 blank control groups,3 C3a RA control groups,3 irradiation groups,and 3irradiation+C3a RA groups）,10 mice in each group.⁶⁰Coγ-rays were irradiated with 20Gy on the chest for a single time.The C3a RA control group and the irradiation+C3a RA group were given C3a R receptor inhibitor once a day.The respiratory function changes of the mice were detected at 1 m,3 m,and 6 m after irradiation,and the changes of body weight,lung weight and lung coefficient were measured.The lung tissue was digested with collagenase to prepare cell suspension,and the changes of CD4+and CD8+T cell subsets in lung tissue were detected by flow cytometry,and the lung tissue inflammation and fibrosis were analyzed by HE and Masson staining of lung tissue sections.Beas-2B lung epithelial cells were used for cell experiments.RT-PCR was used to detect the expression level of complement in lung epithelial cells after irradiation.Flow cytometry was used to detect the apoptosis and cell cycle changes of lung epithelial cells after complement C3a acted on them.Results:（1）The detection results of major complements（C2,C3,C4,C5）in mouse lung tissue at different times after a single chest irradiation with 20 Gy ⁶⁰Coγrays showed that:（1）Complement C2 increased significantly at 3 d after irradiation（p<0.05）;There was no significant change at other times,suggesting that complement C2 may be related to the transient emergency response caused by irradiation.（2）Complement C3a continued to increase from 3d to 6m,and at 7d and 6m,the level of C3a receptor（C3a R）was significantly lower than that of the control group,indicating that complement C3a may play an important role in radiation-induced lung injury.It is suggested that by regulating complement C3a and its receptor,it is possible to find a new way to effectively prevent and treat radiation-induced lung injury.（3）Complement C4 content increased significantly at 3 and 7 days after irradiation（p<0.01）;it returned to normal afterwards,suggesting that complement C4 may be related to the inflammatory reaction caused by chest irradiation in the early stage.（4）Complement C5a in mice was significantly higher than that in the control group at 6 months after irradiation（p<0.01）,while there was no difference between the other time points and the control group,which may be related to the degree of fibrosis,and further research is needed to confirm.（5）Complement complex C5b-9 increased on the 3rd day after irradiation,which was consistent with the response of complement C2 and C4 after irradiation.The relationship between them needs to be further studied.（2）γ-ray irradiation leads to increased apoptosis of lung epithelial cells and block of cell cycle G2/M phase;complement C3a aggravates radiation-induced lung injury.It is suggested that radiation damage may be protected by intervening complement C3a.（3）The complement C3a receptor inhibitor（C3a RA）was used to block C3a in vivo,and its protective effect on lung injury in mice induced by 20 Gy ⁶⁰Coγ-ray chest irradiation was observed.Abnormal tissue immune function,manifested as a decrease in CD4+T cells,an increase in CD8+T cells,and a decrease in the ratio of CD4+/CD+8;some respiratory function indexes were abnormal in 3 months.Pathological changes of lung injury such as alveolar wall thickening,alveolar cavity fusion,reduction in alveolar number,vasodilation,erythrocyte exudation,leukocyte infiltration,and interstitial collagen fiber hyperplasia were observed.（2）After C3a RA was administered to irradiated mice,compared with the irradiated group,the inflammatory response of lung tissue in the irradiated+C3a RA group was significantly improved at 1 month;some abnormal respiratory function indexes recovered at 3 months;and obvious pathological changes were observed.lighten.It indicated that by antagonizing the complement C3a receptor and interfering with the C3a pathway and its level,it may be an effective way to prevent and treat radiation-induced lung injury.Conclusion:（1）C2,C3a,C4,C5a,C5b-9 and other complements in mouse lung tissue increased in different phases afterγ-ray irradiation,among which C3a increased most obviously,suggesting that it may play an important role in radiation-induced lung injury effect.（2）By blocking the complement receptor C3a R,inhibiting the C3a-C3a R pathway can effectively inhibit the process of radiation-induced pulmonary fibrosis.