| Objective:Radiation-induced pulmonary fibrosis(RPF)is a frequently occurred complication from radiotherapy of thoracic tumors which may also occur in nuclear accident and the pre-treatment of bone marrow transplantation. Pulmonary fibrosis is not a unique pathologic process but rather than a common outcome resulting from the interplay between a variety of effector cells and inflammatory mediators which is normally involved in normal tissue repair. However, persistent and exaggerated wound healing cause an excess of fibroblast replication and extracellular matrix(ECM) deposition and ultimately lead to pulmonary fibrosis which threatening the health and life of patients. Therefore to define and clarify the regularity and molecular mechanism of RPF has a very important significance for predicting the clinical outcome and finding new therapeutic strategies. Transforming growth factor-β1(TGF-β1) has been proved to be one of the most important cytokine to cause fibrosis. However, unlike the profibrotic function of TGF-β1, TGF-β3 showed the potential anti-fibrotic properties especially in scar-less wound healing. Due to the lack of evidence for the role of transforming growth factors β3(TGF-β3) in RPF,we observed the effect of TGF-β3 on the experimental mice model of RPF and this will provide experimental basis and new clues for the prevention and clinical treatment on RPF. Methods & Materials: For the thoracic irradiation, the C57BL/6 mice were anesthetized by the intraperitoneal injections of 50 mg/kgbw 0.5% pentobarbital sodium. A single dose at 20 Gy of cobalt-60 γ radiation was administered to the entire thorax(215 c Gy/min; source surface distance: 3 meters) of each mouse. Organs above and below the thorax were shielded. The mice were randomly divided into three group, the control group; the irradiation plus saline group(irradiation) receiving intraperitoneal injections of 0.5ml saline every week after irradiation and the irradiation plus TGF-β3 Group(TGF-β3) receiving intraperitoneal injections of 1μg/kgbw human recombinant TGF-β3 every week after irradiation.Ten mice in each group were sacrificed at 1,3 and 6 months after irradiation. Pathological changes in the lungs were evaluated by HE stain and Masson’s trichrome stain. Alveolar macrophages, neutrophils and lymphocytes in bronchi alveolar lavage fluid(BALF) were counted. The cytokines and chemokines in BALF were examined by Bio-Plex Multiplex System. The secretions of MMP-9 and TIMP-1 were examined by immunohistochemistry. The treg in peripheral blood; the circulating fibrocytes, the Th17 and Treg in lung were examined by a flow cytometer.Human peripheral blood mononuclear cells(PBMC) were isolated from human buffy coats by Ficoll-Paque Plus in vitro. Circulating fibrocytes were analyzed by a flow cytometer to evaluate the effects on the differentiation of fibrocytes from PBMC of TGF-β1,TGF-β3 and co-cultured Treg cells.Results:(1) After irradiation,the weight gain of mice decreased; the lung weight and the lung coefficient increased slightly.(2) Routine blood test results showed that the number of WBC in the irradiation group and TGF-β3-treated group fluctuated; the HGB in the TGF-β3-treated group decreased significantly at 6m after irradiation(P<0.05); the number of PLT showed an increasing tendency.(3) The total number of cells and neutrophils in BALF tended to increase in the irradiation group and TGF-β3-treated group, but there were no statistically significant difference. The concentration of chemokines in BALF increased first and then decreased both in the irradiation group and TGF-β3-treated group. The concentration of IL-12 increased and IL-4 decreased significantly in the TGF-β3-treated group(P<0.05). The concentration of IL-17 in the irradiation group increased according to the time after irradiation, whereas in the TGF-β3-treated group it significantly increased(P<0.05) at 1m and then decreased. The concentration of VEGF in the radiation group tended to increase after irradiation which increased significantly(P<0.05) compared with control group at 1m and 6m.(4) The pathological changes of lungs showed that a large number of inflammatory infiltrated cells and little alveolar septal thickening were observed in the irradiated lungs at 1m after irradiation. At 3m and 6m, severer alveolar septal thickening, alveolar epithelial hyperplasia and irregular alveolar collapse were observed in the irradiation group with large amounts of collagen depositions. However, in the TGF-β3-treated group, only the infiltration of inflammatory cells rather than alveolar septal thickening were observed at 1m after irradiation, and alveolar septal thickening was delayed and appeared at 3m, while alveolar epithelial hyperplasia and irregular alveolar collapse were observed at 6m with less collagen depositions by Masson’s trichrome staining. The fibrillation between irradiation group and TGF-β3-treated group seemed to be similar, but the indications of fibrosis appeared later and were less severe in the TGF-β3-treated group than that in the irradiation group.(5) The immunohistochemistry results showed that the MMP-9 and TIMP-1 secretion were significantly increased both in the irradiation group and TGF-β3-treated group(P<0.05). The secretion of MMP-9 in the TGF-β3-treated group increased with the development of RPF and it was clearly higher than that in the irradiation group(P<0.05). There was no statistically significant difference on the secretion of TIMP-1 between irradiation group and TGF-β3-treated group.(6) From the observation period of 1m to 6 m, the number of fibrocytes increased significantly in the irradiation group than that in control group(P<0.05). However, TGF-β3 treatment clearly decreased the lung recruitment of fibrocytes in response to irradiation, and the inhibition is more prominent at the time points of 1 month compare with irradiation group(P<0.05).(7) The Treg ratio in peripheral blood tended to decrease both in the irradiation group and TGF-β3-treated group, but there was no significant difference compared with control group. The percentage of Th17 cells in lung CD4+ T cells decreased increased significantly in the irradiation group compared with control group from 1m to 6m(P<0.05). In the TGF-β3-treated group, the percentage of Th17 cells in lung CD4+ T cells showed a different pattern with peaking at 1 m, and then reduced at 3m and 6m. The percentage of Th17 cells was almost at the control level at 6 m, and it was significantly lower than that in the irradiation group(P<0.05). The percentage of Treg cells in lung CD4+ T cells increased significantly from 1m to 6 m in both irradiation group and TGF-β3-treated group(P<0.05).While in the TGF-β3-treated group it reached the peak at 3 month, and was clearly higher than that in the irradiation group(P<0.05). The ratio of Th17 cells/Treg cells gradually increased during the process of fibrosis and reached the peak at 6m in the irradiation group(P<0.05). In the TGF-β3-treated group, the ratio of Th17 cells/Treg cells was significantly higher than that in the irradiation group and control group at 1 m, but decreased significantly at 6m compared with irradiation group(P<0.05).(8) In vitro experimental results show that both TGF-β1 and TGF-β3 can promote the differentiation of human PBMC into fibrocyte, but the ability of TGF-β3 in inducting the differentiation of PBMC into fibrocyte was weaker than TGF-β1. In addition, TGF-β3 can partially antagonize TGF-β1-induced fibrocyte differentiation. Co-cultured Treg cells can promote the differentiation of fibrocyte at low ratio of Treg:PBMC, but when the ratio rose, Treg cells depressed the differentiation of fibrocyte without contact inhibition.Conclusions:(1) TGF-β3 attenuated the development of radiation-induced pulmonary fibrosis in mice.(2) TGF-β3 regulated the ratio of MMP-9/TIMP-1, Th1/Th2 type cytokines, Th17/Treg cell balance and decreased the recruitment of lung fibrocytes thus decelerated the progress of RPF.(3) TGF-β3 can partially antagonize TGF-β1-induced fibrocyte differentiation in vitro.(4) Co-cultured Treg regulated the differentiation of fibrocytes without contact inhibition in vitro. TGF-β3 may affect the differentiation of fibrocytes by regulating the ratio of Th17/Treg. |
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