| Objective Radiation-induced lung injury (RILI) is a major complication of thoracic radiotherapy, and the way to prevent or ameliorate radiation-induced lung injury needs further study. The purpose of this study was to construct a model of RILI using the C57BL/6 murine strain, and to explore whether a7-nAchR agonist GTS-21 is a radioprotective agent for RILI.Methods To construct RILI model in mice, C57BL/6 mice received 12 Gy thoracic irradiation, and were sacrificed at 1,3,7,14,21d and 3m,6m post-irradiation. The lung tissues were removed and processed for definitive analysis, including HE staining and Masson staining. To confirm the effect of a7-nAchR agonist GTS-21on RILI, the mice were received 12Gy irradiation and randomly divided into two groups. One was given 4mg/kg GTS-21 at 1 and 7day after radiation, another was given 0.9% normal saline at the same time as GTS-21 group for a control. Each of group were sacrificed at 1,3,7,14,21d and 3m,6m post-irradiation, and the sections were respectively detected the hydroxyproline content, and stained with hematoxylin and eosin (HE) and Masson's trichrome to assess the degree of inflammation and fibrosis. Serum concentrations of TNF-a, IL-1? and IL-6 were quantitatively measured using a Cytometric Bead Array (CBA) kit.Results Normal lung morphology was observed in non-irradiated mice, while lungs of the radiated mice with or without 0.9% normal saline showed little structure change at 1d after radiation. But at 3-7d after radiation, we could found the initial injury included edema of the alveolar walls, intra-alveolar hemorrhage, alveolar exudation and inflammatory cells accumulation in the alveolar space, and the inflammation response was continuing increase until 21d. At 3 months post-irradiation, the alveolar septa were thicker and alveolar spaces were smaller. Large deposits of collagen in the alveolar septa and bronchiolar area, with obliteration of the alveoli were observed at 6 months after irradiation. However, in GTS-21 group, we found the macrophage accumulation, interstitial edema, alveolar septal thickness, perivascular fibrosis, and collapse in radiation-treated lungs were inhibited compare with control group. Moreover, at 6m after radiation, the content of hydroxyproline in GTS-21 group was significantly decreased, comparing to control group (P<0.05). Finally, we found radiation-induced TNF-a, IL-1? and IL-6 in serum were also inhibited by GTS-21.Conclusion A mice model of radiation-induced lung injury was established successfully, and GTS-21 may reduce lung inflammation and fibrosis caused by radiation treatment.Objective Radiation-induced lung injury (RILI) is a continuous progression of events, and the induction and progression of inflammatory and fibrotic tissue reactions were largely due to the release of inflammatory factors which leading to cytokine cascade. So the inflammatory pathway plays an important role in RILI. Pulmonary macrophages are one of the most active cells in lung which can produce a large number of inflammation factors. Thus, the purpose of this part study was to explore the role of inflammation related proteins in protective effects of radiation-induced lung injury with GTS-21.Methods Real-time PCR was used to detect the mRNA levels of HMGB1, TLR-4, NF-?B, MyD88 and TGF-? in lung tissue from GTS-21 group and control group at different time after radiation. Western blot was used to detect the protein levels of HMGB1, TLR-4, TGF-?, MMP-2/9 and TIMP-1 in lung tissue from GTS-21 group and control group at different time after radiation. Immunohistochemistry was used to analyze NF-?B expression in each group at different time after radiation. Moreover, different concentrations of GTS-21 were given to RAW264.7 macrophages after 6Gy irradiation. Then, we used MTT, Flow cytometry to explore the effect of GTS-21 on proliferation and apoptosis of macrophages. And we used real-time PCR and western blot to detect the mRNA and protein levels of HMGB1, TLR-4, NF-?B and MyD88 in those irradiated RAW264.7cells.Results Comparing to the control group, the mRNA levels of HMGB1,TLR-4 and NF-?B were decreased at the early time of radiation pneumonitis, and the most significant difference was observed at 21d post-irradiation(P< 0.05). TGF-? was also decrease in GTS-21group at 3m and 6m post-irradiation when compared to control(P< 0.05). However, there did not have any different on MyD88 between GTS-21 and control groups. Immunohistochemical analysis showed that the NF-?B p65 was obviously expressed at 21 d after radiation in both two groups, but the expression of NF-?B p65 was significantly decreased in GTS-21 group at time points of 7,14,21d post-irradiation when compared to control group. The result from western blot showed that the protein levels of HMGB1, TLR-4 and MMP-2 in GTS-21 group were significantly decreased at 21d after radiation, while the TIMP-1 expression was increased in GTS-21 group when compared with control group. At 6m after radiation, the protein levels of TGF-P and TIMP-1 were decreased and MMP-2 levels were increased in GTS-21 group. However, we do not found any different between two groups on MMP-9 expression. At last, the RAW264.7 macrophages which treated with GTS-21 after radiation showed a decline in protein and mRNA expressions of HMGB1, NF-?B and TLR-4. But we also did not found any difference in MyD88 expression in those RAW264.7 macrophages. And the proliferation and apoptosis of macrophages were both inhibited by GTS-21.Conclusion GTS-21 reduced radiation pneumonitis and fibrosis by inhibiting HMGB1/TLR-4/NF-?B pathway which subsequently reduced TGF-? expression and balanced MMPs/TIMPs expression.Objective An immediate effect of tissue irradiation is the generation of reactive oxygen (ROS) that can produce oxidative damage to DNA, lipids, and proteins resulting in cell injury or death. Macrophages play an important role in producing ROS in RILI. The purpose of this part study was to identify the role of ROS in protective effects of radiation-induced lung injury with GTS-21.Methods Dihydroethidium (DHE) was used to detect the ROS level in lung tissue GTS-21 and control groups at 21 d after radiation. Real-time PCR was used to detect the mRNA levels of a7-AchR and NOX1/2/4 in two groups. Western blot was used to detect the protein levels of NOX1/2/4 and Hif-l?. Moreover, DHE was used to detect the ROS level in RAW264.7 macrophages which treated with or without GTS-21 after 6Gy irradiation.Results The DHE staining revealed that the ROS level in lung tissue of GTS-21 group was reduced at 21d after radiation when compare to control group. And the ROS level in RAW264.7 macrophages which treated with GTS-21 after 6Gy irradiation was also decreased when compared to the cells that radiated alone. The result from real-time PCR showed that the mRNA expression of a7-AchR was increased with inflammation degree, but the expression of ?7-AchR did not have any difference at any time point in these two groups. Moreover, the mRNA level of NOX-lwas totally reduced in GTS-21group (P<0.05), while the NOX-2 mRNA was also inhibited in GTS-21 group, especially at 21 d post-irradiation. There was no difference in NOX-4 expression between the two groups. The result from western blot showed that the protein levels of NOX-1 and NOX-2 were consistent with their mRNA expression. The protein level of NOX-4 still showed no difference between the two groups. The expression of Hif-la was significantly reduced at 3,7,21d after radiation in GTS-21 group when compared to control (P<0.05)Conclusion GTS-21 reduced radiation pneumonitis and fibrosis may also by inhibiting NOX-1 and NOX-2 expression which subsequently reduced ROS level and Hif-la expression. |
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