TGFβ3 Protects Radiation-induced Lung Epithelial Cell Injury By Intervening TGFβ1 And Its Mechanism

Objective Radiotherapy is one of the main treatments for malignant cancer.Since ionizing radiation has an untargeted effect on cancer tissues,it not only kills cancer cells but also affects the cycle,apoptosis and DNA damage repair of normal cells around cancer tissues,leading to multi-system damage and even fibrosis.Studies have shown that transforming growth factor-β（TGFβ）plays an important role in radiation damage,and TGFβ1 in particular is considered to be a profibrotic factor.TGFβin the lab early in radioactive pulmonary fibrosis effect and the mechanism research,found that TGFβ3radioactive pulmonary fibrosis in mice has obvious protective effect,at the same time,also focus on the domestic and foreign related research reports the TGFβ3 inhibition of fibrosis,but its effect on radiation damage cells and its specific mechanism is not yet clear.In this study,lung epithelial cells were used as the target cells,and the possible mechanism of TGFβ3 protecting lung epithelial cells from radiation was explored by observing the combined effects of TGFβ3 and/or TGFβ1 and radiation,so as to provide experimental basis for the prevention and treatment of lung injury caused by radiation with TGFβ3.Methods Beas-2B cells were irradiated with ⁶⁰Co-γrays at the dose rate of 61.35c Gy/min,irradiation time of 9 min 2 s and dose of 6 Gy.Beas-2B cells were treated with TGFβ3 and/or TGFβ1 at a final concentration of 5 ng/ml,and the cell morphology was observed regularly.Real-time fluorescence quantitative PCR（RT-q PCR）was used to detect the expression levels of EMT-related genes.Molecular markers of epithelial features,such as cell Surface Protein C（SPC）,E-cadherin（E-cadherin）,tight conjunctin（ZO-1）,and molecular markers of interstitial features,such as N-cadherin,αsmooth muscle actin（α-SMA）,and Vimentin（Vimentin）.Collagen I expression level;Expression level of endogenous TGFβ1 in Beas-2B cells;MMP2/MMP9 expression level;And TGFBR2 expression level.CCK8 detected the proliferation of Beas-2B cells.Flow cytometry analysis on cell apoptosis and cell cycle.Three interfering si RNA of TGFBR2 were designed and synthesized,and verified and optimized by real-time fluorescence quantitative PCR（RT-q PCR）and Western blot.Results 1.Radiation-induced lung epithelial cell injury:Compared with the control group,⁶⁰Co-γ-ray（6 Gy）irradiation group significantly inhibited cell proliferation,significantly increased the proportion of apoptosis and G2/M phase of cell cycle,and significantly decreased the expression of molecular markers such as SPC,E-cadherin and ZO-1 that induced cell EMT showing epithelial characteristics.The expressions of interstitial molecular markers such as N-cadherin,α-SMA and Vimentin were significantly increased,and the expressions of pulmonary fibrosis related indexes were manifested as Collagen I,endogenous TGFβ1,TGFBR2 and MMP2/MMP9.2.TGFβ1 promotes radiation-induced lung epithelial cell injury:Compared with the ⁶⁰Co-γ-ray（6 Gy）group,In the ⁶⁰Co-γ-ray（6 Gy）+TGFβ1（5 ng/m L）group,the proportion of apoptosis and the proportion of G2/M phase of cell cycle were significantly increased,and the expression of E-cadherin and ZO-1,the molecular markers that induced EMT to show epithelial characteristics of cells,were significantly decreased.The expression of interstitial molecular markers such as N-cadherin was significantly increased.3.TGFβ3 inhibits radiation-induced lung epithelial cell injury:Compared with the⁶⁰Co-γ-ray（6 Gy）irradiation group,⁶⁰Co-γ-ray（6 Gy）irradiation+TGFβ3（5 ng/ml）irradiation group significantly reduced the proportion of apoptosis and the proportion of G2/M phase cells in the cell cycle,and significantly inhibited the expression of molecular markers such as SPC,which showed epithelial characteristics of EMT,significantly increased.The expression of interstitial molecular markers such asα-SMA and Vimentin was significantly decreased.Collagen I,MMP2/MMP9 and TGFBR2expressions were significantly reduced.4.TGFβ3 antagonizes the promoting effect of TGFβ1 on radiation-induced lung epithelial cell injury and its mechanism:Compared with the ⁶⁰Co-γ-ray（6 Gy）+TGFβ1（5 ng/ml）group,The proportion of apoptosis,the proportion of G2/M phase in cell cycle and the expression of endogenous TGFβ1 were significantly decreased in⁶⁰Co-γray（6 Gy）+TGFβ1（5 ng/ml）+TGFβ3（5 ng/ml）group.After TGFBR2-si RNA-1 was used to silence TGFBR2 of Beas-2B cells,radiation-induced apoptosis,G2/M cell cycle arrest and epithelial-mesenchymal transformation were still evident.However,TGFβ3（5 ng/ml）lost its protective effect on radiation-induced apoptosis,G2/M cell cycle arrest and epithelial-mesenchymal transformation.Conclusion（1）TGFβ1 promoted radiation-induced lung epithelial cell injury,including EMT induction,increased apoptosis and G2/M phase arrest.（2）TGFβ3inhibited radiation-induced lung epithelial cell injury,including inhibiting EMT,decreasing apoptosis and G2/M cell cycle arrest,and decreasing collagen fiber and MMP2/MMP9 expression levels.（3）TGFβ3 inhibits TGFβ1 in promoting radiation injury,including reducing apoptosis,G2/M cell cycle arrest,and reducing endogenous TGFβ1 expression level,suggesting that TGFβ3 can inhibit radiation injury by regulating endogenous TGFβ1 expression level.（4）Abnormally high expression of TGFBR2 was induced by radiation,and TGFβ3 significantly reduced the elevated expression level of TGFBR2 after radiation.After TGFBR2-si RNA-1 was used to silence TGFBR2 of Beas-2B cells,TGFβ3 lost its protective effect on radiation-induced apoptosis,G2/M cell cycle arrest and epithelial-mesenchymal transformation.These results showed that TGFβ3 inhibited the injury of lung epithelial cells induced by radiation through TGFBR2.