The Role Of Tankyrase2 In The Regulation Of AEC And LFB Apoptosis In Radiation-induced Lung Injury

Radiation pulmonary injury(RPI)is one of the most common complications of radiotherapy for chest cancers and pre-treatment for bone marrow transplantation,but also commonly results from nuclear radiation during the war and the usual nuclear accident.RPI often involves two stages,the reversible radioactive interstitial pneumonia(RIP)at early stage and irreversible pulmonary pulmonary fibrosis(RPF)at late stage.The incidence of conventional radiotherapy RPI is about 30%.In recent years,with the continuous improvement and optimization of radiotherapy,the incidence of RPI has decreased.However,once the RPI have occurred,it will seriously affect the quality of life of patients after radiotherapy,and even endanger their lives.In recent years,some progress has been made on RPI mechanism which is still far from elucidated.Moreover,safe and effective prevention and treatment drugs for RPI is also scarce.Thus,in-depth study on the mechanism of RPI to find a new target for RPI prevention and treatment is of important theoretical and practical significance to improve the clinical radiotherapy of chest tumors and the quality of life of patients.The occurrence of RPI is multicontogenic and has obvious species and individual differences.Apoptosis plays an important role in the process of pulmonary injury.Alveolar epithelial cell(AEC)is the main target cell of radiation injury in lung tissue.Lung fibroblasts(LFB)are important effector cells for the development of RPI.Previous study showed distinct expression of Tankyrase2 between fibrosis-prone and fibrosis-resistant mice during RPI process.As a telomere-binding protein,Tankyrase 2 play a role in a variety of fields for its structural features and distribution diversity in cells.By adjusting the length of telomere,Tankyrase 2 can affect chromosomal stability,which is closely related with cell senescence,death and cancer.Its PARP domain is the transition point for apoptosis and necrosis.Whether Tankyrase 2 has an effect in the process of RPI has not been reported in the literature.Here,we probe the role of Tankyrase2 in AEC and LFB apoptosis regulation during RPI process.Material and methods 1.Correlation studies on Tankyrase2 and apoptosis with RPF species difference.Sixty C57 BL / 6J and Sixty C3 H / HeN mice were randomly divided into two groups(control group and irradiation group),respectively.The 60 Co ? ray irradiation method was used to establish RPI model with 20 Gy per irradiation.Lung tissue is obtained after 15 min,1d,3d,7d irradiation,and then the apoptosis,DNA damage and expression of ?H2AX,TNKS2 and PARP are were detected by in situ terminal labeling,?H2AX immunoblotting and quantitative real-time PCR respectively to determine the role of DNA damage and apoptosis in RPI and their relation with RPF and to explore the correlation of Tankyrase2 with RPF tendency in mice.2.Study on the role of Tankyrase2 in the changes of AEC and LFB biological behavior.A549 cells and MRC-5 cells were cultured in vitro and irradiated with 60 Co ?-rays at doses of 5Gy,10 Gy and 20 Gy,respectively.In addition,another group of the cells were treated with 3-AB inhibitors,and the above irradiation method was utilized.The cell proliferation,DNA damage,apoptosis and expression of AIF,TNKS2,?H2AX and PARP were detected by MTT,?H2AX immunofluorescence,AO/EB staining,quantitative real-time PCR and immunoblotting at 30 min,1h,3h,6h,12 h,24h,48 h and 72 h after irradiation to illustrate the role of Tankyrase2 in radiation induced of AEC and LFB biological behavior changes.Results: 1.Difference of cellular apoptosis and Tankyrase2 expression level in lung tissue between C3H/HeN and C57BL/6J mice after irradiation1.1 Cellular apoptosis level in lung tissue of C3H/HeN and C57BL/6J mice was different after irradiation.After irradiation with 60 Co ?-ray,many apoptotic cells,mainly apoptotic epithelial cells,can be seen at the early stage(1-3d)and the number of apoptosis cells in the lung tissue of C57BL/6J mice was more than that of C3H/HeN mice.The number of apoptotic cells significantly increased at one month after irradiation(inflammatory stage),which was not significantly different between the two groups with epithelial cell and mononuclear macrophage as the main type.Three months after irradiation,that is early stage of fibrosis,the number of apoptotic cells in lung tissue of C57BL/6J mice was lower than that of C3H/HeN mice.Majority of apoptosis cells in C3H/HeN mice were macrophages.1.2 Differences of DNA damage level in lung tissue between C3H/HeN and C57BL/6J mice after irradiation.C57BL/6J mice had higher ?H2AX expression level than C3H/HeN mice at 1-7 days after irradiation.One months after irradiation,there was no difference in ?H2AX expression level between two groups.However,the expression of ?H2AX in lung tissue of C3H/HeN mice was higher than that of C57BL/6J mice at 3th month.1.3 Differences in expression of AIF in lung tissue between C3H/HeN and C57BL/6J mice after irradiation.The expression of AIF in lung tissue of C3 H / HeN mice was significantly higher than that of control group at 7d,1m and 3m after irradiation.The expression of AIF was not significantly different from that of control group at 1d and 3d after irradiation.The expression of AIF in lung tissue of C57BL/6J mice was significantly higher than that of control at all time point.The expression of AIF in lung tisuse of C3H/HeN mice was lower than that of C57BL/6J mice at 1d,3d and 1m.However,no AIF expression difference was detected between two groups.On the contrary,the expression of AIF in lung tissue of C3H/HeN mice was higher than that of C57BL/6J mice at 3m after irradiation.1.4 Differences in expression of TNKS2 in lung tissue between C3H/HeN and C57BL/6J mice after irradiation.At RNA level,TNKS2 expression in lung tissue of C3H/HeN mice after irradiation was higher than that of C57BL/6J mice(except 3th day).At protein level,TNKS2 expression in lung tissue of C3H/HeN mice after irradiation was also higher than that of C57BL/6J mice(except 1 month).1.5 Differences in expression of PARP in lung tissue between C3H/HeN and C57BL/6J mice after irradiation.The expression of PARP in lung tissue of C57BL/6J mice was higher than that of C3 H / HeN mice before irradiation.The expression of PARP in lung tissue of C3H/HeN mice was lower than that of C57BL/6J mice after irradiation(except 1st day).2.Differences of DNA damage and cellular apoptosis and expression level of Tankyrase2 and PARP in AEC and LFB after irradiation2.1 Effects of different doses of ?-ray irradiation on proliferation in A549 cells and MRC-5 cells.A549 cells and MRC-5 cells were irradiated by ? ray at dose of 5Gy,10 Gy and 20 Gy,the proliferation of A549 cells were all inhibited by the three doses.10 Gy and 20 Gy ? ray also inhibited MRC-5 cell proliferation 47 h and 72 h after irradiation.On the contrary,? ray irradiation at 5Gy promoted MRC-5 cells proliferation after 72 h.2.2 Effects of different doses of ?-ray irradiation on DNA damage in A549 cells and MRC-5 cells.DNA damage occurred in A549 cell 30 min,1h and 3h after irradiation at any dose,which was dose-dependent.The higher irradiation dose,the more DNA damage.Positive cells decreased 6h and 12 h after irradiation,which was still more than control group.DNA damage and expression?H2AX was only present in MRC-5 cells at few time piont.2.3 Effects of different doses of ?-ray irradiation on cellular apoptosis in A549 cells and MRC-5 cells :(1)The number of apoptotic cells in A549 cells at 20 Gy ?-ray irradiation was higher than that in 5Gy and 10 Gy ?-rays(except 12h).The number of apoptotic cells at 10 Gy ?-ray irradiation was higher than that of 5Gy ?-ray irradiation(except 3h).(2)The expression of AIF in A549 cells was higher than that in the control group both at 5Gy ?-ray and 10 Gy ?-ray irradiation,and the expression of AIF in A549 cells irradiated at 10 Gy ?-ray was higher than that at 5Gy ?-ray.(3)After 5Gy and 10 Gy ?-ray irradiation,the expression of AIF in A549 cells treated with inhibitor 3-AB was significantly lower than that of untreated cells.For inhibitor treated cells,the number of apoptotic cells at 10 Gy ?-ray irradiation was higher than that of 5Gy ?-ray irradiation.(4)No cellular apoptosis and expression of AIF were detected after different doses of ?-ray irradiation.2.4 Effects of different doses of ?-ray irradiation on TNKS2 and PARP expression in A549 cells and MRC-5 cells:(1)At RNA level,the expression of TNKS2 in A549 cells was lower than that in the control group at 1 to 12 hours after irradiation with 5 and 10 Gy ?-rays.However,the expression of TNKS2 in A549 cells was significantly higher than that in the control group at 30 min after irradiation.The protein expression level of Tankyrase2 in A549 cells was lower than that in the control group at 30 min and 12 h after irradiation at dose of 5Gy,which on the contrary was higher than that in the control group at 1h and 3 h.The protein expression level of TNKS2 in A549 cells irradiated by 10 Gy ?-rays was higher than that in the control group at any time point(except 12h).The expression of Tankyrase2 in 10 Gy ?-ray irradiation group was higher than that in 5Gy irradiation group at 30 min,1h,6h and 24 h.(2)The RNA expression level of TNKS2 in MRC-5 cells was higher than that in control group at any time point after irradiation with 5Gy ?-ray,so as with 10 Gy ?-ray irradiation(except 30 min).The expression of TNKS2 in MRC-5 cells irradiated with 5Gy ?-ray was higher than that with 10 Gy ?-ray at 30 min,1h and 12 h,but at 3h after irradiation,TNKS2 expression was much higher in 10 Gy ?-ray group than that in 5Gy ?-ray.The protein expression level of TNKS2 in MRC-5 cells was lower than that in control group at 30 min and 24 h,but higher at 1-12 h.The protein expression level of TNKS2 in MRC-5 cells irradiated by 10 Gy ?-rays was higher than that in the control group at any time point(except 12h)and was higher than that in 5Gy irradiation group at any time point.(3)PARP expression in A549 cells was lower than that in the control group at 30 min,3h and 12 h after irradiation with 5Gy?-ray,but higher than that in the control group at 1h and 6h after irradiation.The expression level of PARP in A549 cells irradiated by 10 Gy ?-rays was higher than that in the control group at any time point and was higher than that in 5Gy irradiation group excepting at 1h.PARP expression in MRC-5 cells was higher than that in the control group at 30 min,3h and 12 h after irradiation with 5Gy?-ray,but lower than that in the control group at 24 h after irradiation.The expression level of PARP in MRC-5 cells irradiated by 10 Gy ?-rays was higher than that in the control group at any time point and was higher than that in 5Gy irradiation group excepting at 6h,12 h and 24 h.Conclusion 1.The difference of cellular apoptosis level in lung tissue between C57BL/6J and C3H/HeN mice after ?-ray irradiation was one of the mechanisms for prone/anti-fibrosis in these two kinds of mice.C57BL/6J was susceptible to DNA damage and alveolar epithelial cells apoptosis,and however,macrophages and other pro-fibrosis cells were resistant to apoptosis.Thus,C57BL/6J was susceptible to RPF.On the contrary,less alveolar epithelial cells apoptosis occurred in the lung tissue of C3H/HeN mice,and macrophages and other pro-fibrosis cells induced by irradiation were eliminated through apoptosis,which may associated with fibrosis-resistance of C3H/HeN mice.2.Tankyrase2 is negatively correlated with RPF.3.AEC is more sensitive to ?-ray than LFB and susceptible to DNA damage and apoptosis.Tankyrase2 is positively correlated with AEC apoptosis regulation after irradiation,and is negatively correlated with LFB apoptosis regulation after irradiation.