The Mechanisms Of TGF-?3 Ameliorating Radiation-Induced Pulmonary Fibrosis By Epithelial-Mesenchymal Transition

OBJECTIVE:In recent years,due to the aggravation of environmental pollution,the incidence of thoracic tumors is increasing year by year,radiotherapy is the conventional means of treating thoracic tumors at present,however,radiation-induced pulmonary fibrosis is one of the most serious complications of chest radiotherapy.Because its morbidity is high,there is still lack of specific effective prevention and treatment measures and the prognosis of patients is poor,radiation-induced pulmonary fibrosis seriously threatens human health.Therefore,how to prevent and reduce the occurrence of radiation-induced pulmonary fibrosis is the goal that medical workers are striving for.Some studies have shown that transforming growth factor-?3?TGF-?3?has an inhibitory effect on radiation-induced pulmonary fibrosis now,However,it is unclear that what the specific mechanism is up to now.Numerous studies have proved that epithelial-mesenchymal transition?EMT?of alveolar epithelial cells?AECs?is one of the important causes of pulmonary fibrosis.In the present study,mice and alveolar epithelial cells?AECs?were induced by ⁶⁰Co?radiation to establish a radioactive pulmonary fibrosis animal model and alveolar epithelial cells EMT system,and we will explore whether TGF-?3 is inhibitory to radiation-induced pulmonary fibrosis through EMT pathway and possible specific signal transduction mechanism.METHOD:90 C57BL/6 female mice were randomly divided into control group,irradiation plus saline group?irradiation group?and irradiation plus TGF-?3 group?TGF-?3 group?.A single dose at 20Gy of ⁶⁰Co?radiation was administered to the entire thorax of each mouse in the irradiation group and TGF-?3 group.Thereafter,the irradiation group received intraperitoneal injections of 0.5ml saline every week and the TGF-?3 group received intraperitoneal injections of 1?g/kgbw human recombinant TGF-?3 every week.The mice in each group were sacrificed at 1,3 and 6 months after irradiation respectively.Pathological changes in the lungs were evaluated by HE stain and Masson's trichrome stain.The expression of EMT epithelium marked protein ZO-1 and interstitial marked protein N-cadherin were detected by immunohistochemistry in lung tissue of the mice.⁶⁰Co?radiation was administered to alveolar epithelial cells to establish a radiation-induced EMT cell system,and the system was treated by TGF-?3 and TGF-?1,The expression of EMT epithelium marked protein E-cadherin,interstitial marked protein N-cadherin,TGF-?/smad pathway related protein P-smad3 and smad3were detected by a flow cytometer and Western blot.The mRNA expression of TGF-?3 and TGF-?1 were measured by reverse transcription polymerase chain reaction?RT-PCR?.RESULTS:HE and Masson staining showed that alveolar septum obviously widen,alveolar walls thickening,alveolar structure severely damage and large amounts of collagen fibers deposited and other typical fibrosis pathological changes after irradiation.Compared with the irradiation group,there were the less severe pathological changes of the lung tissue in mice and the less collagen fiber deposition in the TGF-?3 group,which showed that we had successfully established radiation-induced pulmonary fibrosis model in mice,TGF-?3 had obviously inhibitory effect on radiation-induced pulmonary fibrosis in mice. The immunohistochemistry results showed that the expression of ZO-1was decreased significantly?P<0.05?and the expression of N-cadherin significantly increased?p<0.05?in the month 1,month 3 and month 6 after irradiation in lung tissues of mice.It indicated that EMT was closely related to the development of radiation-induced pulmonary fibrosis.And the expression of ZO-1 and N-cadherin in the TGF-?3 group was between the control group and the irradiated group and had statistically significant difference compared with the control group and the irradiated group?P<0.05?.It was indicated that TGF-?3 had obviously inhibitory effect on EMT induced by irradiation in lung tissue of mice.The human alveolar type II epithelial cell line A549 and Beas-2B were irradiated with ⁶⁰Co?radiation,and cellular morphology were observed by inverted phase contrast microscope to turn from Pebble shape to Fusiform shape,a myofibroblast-like morphology after irradiation.Flow cytometry results showed that the A549 cells became big and their contents increased,epithelia cells markers E-cadherin expression decreased and interstitial markers N-cadherin expression increased after irradiated with 8 Gy⁶⁰Co?radiation.Western blot results showed that epithelia cells markers E-cadherin expression decreased and interstitial markers N-cadherin expression increased in the A549 cells and Beas-2B cells after irradiated.The flow cytometry and the western blot results indicated that we had successfully established a radiation-induced alveolar epithelial cell EMT system.TGF-?3and TGF-?1 may induce A549 cells and Beas-2B cells to occur EMT.Howere,in the radiation-induced Beas-2B cell EMT system,the result showed that TGF-?1 promote the progress of EMT,but compared with TGF-?1,TGF-?3attenuated the progress of EMT.Although there was no significant difference in the expression of smad3 among groups,the expression of p-smad3 in TGF-?3 treatment group was significantly lower than that of TGF-?1treatment group in the radiation-induced Beas-2B cell EMT system,and these results suggested that TGF-?3 mediated EMT could be related to TGF-?/smad pathway in the radiation-induced Beas-2B cell EMT system.The combined effect of TGF-?3 and TGF-?1 showed a certain antagonism in the radiation-induced Beas-2B cell EMT system,which was the most obvious when combined with 1:1,and the expression of P-smad3 was lower than that of the two alone.The results of RT-PCR showed that exogenous TGF-?3reduced TGF-?1mRNA expression and increase TGF-?3mRNA expression.CONCLUSION:TGF-?3 can inhibit the progress of radiation-induced pulmonary fibrosis in mice via inhibiting EMT;TGF-?3 could inhibit EMT through inhibiting TGF-?/smad pathway in the radiation-induced Beas-2B cell EMT system;Exogenous TGF-?3 could decreased endogenous expression of TGF-?1 and increased the endogenous expression of TGF-?3,which could enhance the effect of TGF-?3 inhibiting radiation-induced EMT in the radiation-induced Beas-2B cell EMT system.