The Molecular Mechanism Of Epithelial Mesenchymal Transformation Induced By Radon Exposure In Bronchial Epithelial Cells And Protection Of Radon Exposure Injury

ObjectiveIn this study,the cell proliferation,migration ability,lung injury and the change of the EMT markers was observed by constructing radon-exposed cell and animal models as well as polydatin protection experimental models.The EMT process of human bronchial epithelial cells induced by radon exposure,lung tissue injury in radon inhalation mice and its mechanism was explored.It was to clarify the process and molecular mechanism of epithelial cell EMT induced by different doses of radon and its daughters irradiation at the molecular,cellular and overall levels to determine the role of EMT in radon-induced lung injury.At the same time,the preventive effect of polydatin in radon exposure injury was discussed,to provide theoretical basis and new ideas for preventing of radon exposure injury.Methods(1)The mouse model of radon exposure was constructed by using ecological radon chamber.Male BALB/c mice(six of each group)inhaledradon gas at 105 Bq/m3 for 10 hours/day and 6 days/week.When the cumulative exposure dose of radon reached 60 and 120 WLM,respectively,SOD activity and MDA content in mice serum were detected.HE and Masson staining were used to observe the pathological changes and the fibrin deposition of lung tissue caused by radon exposure.The expression of EMT markers in lung tissues was detected by immunohistochemistry and Western blot.(2)The cell model of radon exposure is constructed.The human bronchial epithelial cells(16HBE and BEAS-2B)are directly exposed to radon and its progeny at 20,000 Bq/m3 for 20min each time and once every three days.The repeated twice exposure is called first generation of radon exposure(Rn1).The sixth generation ofradon exposure(Rn6)and the twelfth generation of radon exposure(Rn12)were selected for the follow-up experiments.The ROS level of cell and SOD activity were detected by the related kits,ATP level was detected by chemiluminescence method,mitochondrial membrane potential(MMP)was detected by probe method,cell proliferation was detected by CCK8 method,cell adhesion was detected by the adhesion kit,and the cell migration ability after radon exposure was determined by Transwell migration and scratch tests.The mRNA changes of EMT marker genes were determined by RT-PCR.The protein changes of EMT markers were detected by immunofluorescence and Western blot assay.(3)The molecular mechanism of EMT in epithelial cells induced by radon exposure was explored by using the constructed radon exposed cells and animal models.Western blotting was used to analyze the protein expression of related pathways in radon exposed cells and lung tissues of radon infected mice.After the intervention with the pathway inhibitor,the proliferation ability of radon-exposed cells was detected by CCK8 method,the cell adhesion was determined by the adhesion kit,and EMT protein expression level s was detected by western blot method.(4)The radon-exposed polydatin intervention cell model was constructed.Polydatin with final concentrations of 100 μmol and 200 μmol was directly added to the constructed radon-exposed cell model for 24h.The cell was to detected cell proliferation,cell adhesion and cell migration after addition of polydatin,and to determine the expression of EMT-related genes and pathway proteins after the intervention of polydatin.(5)The polydatin radon exposure protective cell model was constructed.Before and after each radon exposure,cells were treated with 200μmol polydatin as a protective agent,and the other conditions were the same as those in the radon exposure group alone.Then,the cell proliferation capacity,adhesion property and migration ability in polydatin radon exposure protective cells were measured,and the expression level of EMT markers and related pathway proteins were detected.(6)The mouse model of polydatin radon exposure was constructed.Male BALB/c mice were intraperitoneally injected with 200 μL of polydatin solution of 50 mg/kg and 100 mg/kg half an hour before each radon inhalation.Each group has six mice.The other conditions were the same as that of radon gas inhalation group alone.When the cumulative dose of radon reached 120 WLM,the serum SOD activity and the MDA content of mice in each group were measured.HE and Masson staining were used to observe the pathological changes and fiber deposition in lung tissue.The expression of EMT important markers in lung tissue was detected by immunohistochemistry.(7)SPSS 20.0 software was used for statistical analysis.T test was used for significance test between two independent samples.Results among multiple groups were analyzed using one-way ANOVA.P<0.05 was considered statistically significant.Results(1)Compared with the control group,the serum MDA content of mice in radon exposure group was increased,and the SOD activity was decreased.The alveolar wall of the lung tissue became thinner and the pulmonary septum became thicker,and pulmonary bullae appeared.Inflammatory infiltration can be seen in lung tissue of radon exposure mice.The deposition of collagen fibers in the lung tissue and the expression of fibrosis index α-SMA were increased.The lung tissue showed that epithelial cell markers was decreased and interstitial cell markers was increased.The expression of p-PI3K,p-AKT,and p-mTOR was increased.The weight of mice in the 60 WLM group did not change significantly,while the weight of mice in the 120 WLM group was significantly reduced.(2)In radon exposed cells,the level of ROS was increased,but the activity of SOD was decreased.There was no significant difference in the levels of MMP and ATP between control group and radon exposed group.The ability of cell proliferation was improved,the cell adhesion decreased,and the cell migration ability was enhanced after radon exposure.In radon-exposed cells,E-cad expression was decreased,and FN1,Vimentin,N-cad,α-SMA,and Snail expression was increased.The balance of MMP2/TIMP2 in radon-exposed cells was broken.(3)The expression of p-PI3K,p-AKT,p-mTOR,and β-catenin was increased in radon-exposed cells.The expression of p-PI3K,p-AKT,and p-mTOR proteins also increased in the lung tissue of radon-exposed mice.After adding AKT or mTOR inhibitor,the radon exposed cells showed the weakened cell proliferation and the increased cell adhesion,the increased expression of E-cad,and the decreased expression of FN1,Vimentin,N-cad,α-SMA and Snail.(4)After the intervention with polydatin,the ROS level of radon exposed cells was decreased,and SOD activity was increased.The changes in cell adhesion were not significant in each group.Polydatin weakened the ability of cell migration.The expression of E-cad protein was increased in polydatin cells,while the expression of FN1,Vimentin,N-cad,α-SMA and Snail decreased.P-mTOR and p-AKT expression in the intervention group was decreased.(5)Compared with the radon exposure group,the levels of ROS,migration,and FN1,Vimentin,N-cad,α-SMA and Snail1 expression decreased significantly in the polydatin-protected cells.The SOD activity,cell adhesion,and E-cad protein expression was increased in the protected group.The expression of p-AKT and p-mTOR was decreased significantly in cells with polydatin.(6)Compared with mice in 120WLM group,the weight of mice with polydatin was increased,serum MDA content decreased,and SOD activity increased.In the radiation protection group of polydatin,the thickening degree of pulmonary bulla and pulmonary septum was reduced,and the deposition of collagen fibers in lung tissue was decreased.The expression of EMT interstitial cell markers(FN1,Vimentin,Snap)and fibrosis index α-SMA in lung tissue were decreased.p-PI3K,p-AKT,and p-mTOR protein expressions were significantly reduced.Conclusion(1)Early radon exposure can induce EMT in human bronchial epithelial cells and mouse lung tissue,and the degree of EMT gradually increases with the increase of cumulative dose of radon exposure.(2)The PI3K/AKT/mTOR pathway is involved in the occurrence of radon exposure-induced EMT in bronchial epithelial cells.(3)Polydatin reduce oxidative stress,attenuate EMT in bronchial epithelial cells and pulmonary fibrosis in mouse caused by radon exposure.It has a good radiation protection effect on radon exposure injury,and the radiation protection effect before and after exposure is better.The protective effect of polydatin may be related to its inhibition of PI3K/Akt/mTOR pathway activation.