| Radiation-induced lung injury is inevitable in the process of thoracic-tumor patients who receive radiotherapy, and affect the increase of radiation dose, even cause the interruption of treatment; however, its pathogenesis is still not fully elucidated. This study used mammalian miniature pigs whose the dose-effect curve of single dose radiation are more’similar to human as well as the anatomical structure and physiology of lung as experimental subjects, applying double immunofluorescence staining, western blot, gelatin zymography, masson’s trichrome and enzyme-linked immunosorbent assay (ELISA) to observe alveolar epithelial type Ⅱ (AE2) cells transition into mesenchymal cells and matrix metalloproteinases (MMPs), to investigate the possible mechanism of radiation-induced lung injury in miniature pigs, and further to explore the radiology-protective mechanism of Sarcandra Glabra. on radiation-induced lung injury in miniature pigs from molecular level.Part 1:The inhibition of Sarcandra Glabra. on epithelial to mesenchymal cells in radiation-induced lung injury of miniature pigsObjective:Through observing the alveolar type Ⅱ epithelial cells into mesenchymal cells, to explore its possible role in radiation-induced lung injury, and further to probe radiology-protective mechanism of Sarcandra Glabra.Methods:According to the stochastic grouping’s method,60 miniature pigs were randomly divided into control group (control group), medicine radiotherapy group (drug group) and simple radiotherapy group (RT group),20 each group. After intraperitoneal anesthesia with 3% sodium pentobarbital (30mg/kg), miniature pigs in RT group and drug group received a single 15Gy dose of right-thoracic irradiation using 60Co γ-rays, the blank group without irradiation. Miniature pigs in drug group were treated with radiotherapy for a week followed oral given by formula granule solution in accordance with the 0.3g/kg body weight dose, meanwhile equal physiological saline to the other two groups, until the end of observation. At 4,8,12 and 24 week postirradiation, 5 minipigs respective and random from three groups were taken to harvest the venous blood from the porcine anterior vena cava, and then sacrificed to remove the lungs; the part was fixed in 10% neutral formalin for histological and immunohistochemical analysis; the other was snap frozen in liquid nitrogen and kept at -80℃ to use, left and right lung tissue were preserved and fixed separately. The pathologic changes of the lung tissue were observed by H&E staining, the protein expression of α-SMA was evaluated by immunohistochemistry, the protein expression of SP-A, TGFβ1, vimentin and E-cadherin was measured by western blot, the co-localization of the SP- A and α-SMA proteins in the alveoli of irradiated lung was analyzed using a laser scanning confocal microscope.Results:1.The general shape of lung:with the extension of post-irradiation time, the right lung of miniature pigs in RT group appeared edema and hyperemia, and from scattered punctate hemorrhage to large area hemorrhagic focus, finally developed into the pulmonary consolidation. The degree of right pulmonary lesions in drug group was relatively alleviated throughout the experiments, right lung significantly became smaller for atrophy, but obvious pulmonary consolidation was observed at 24W after radiation.2. H&E staining:At 4th week after radiation, main changes of lung tissue in RT group were exudation, characterized by pulmonary interstitial and pulmonary capillary hyperemia, edema and diffuse accumulation of inflammatory cells, with the extension of post-irradiation time, exudation lesions became worse, and fiber lesions were observed gradually. At 12th week after radiation, lung lesions developed fiber lesions, characterized by thickened alveolar wall; narrow or collapsed alveolar space; widened or fused alveolar septa and more fibroblasts. To 24th week after radiation, more reddish, funicular and swirled fibrinoid lesion appeared in the lung parenchyma, and obvious pulmonary consolidation was observed. At each time point, the pulmonary lesions in drug group was similar to that in RT group, but the degree of it was less.3. a-SMA protein expression:In RT group, a-SMA protein expression from the 4th week after radiation was significantly increased, and showed a rising trend gradually (vs control group P<0.05). After Sarcandra Glabra. intervention, the change trend of a-SMA protein expression at each point in time was not as good as it in RT group (vs control group and RT group respectively, all P<0.05).4. SP-A, TGβ1, vimentin and E-cadherin protein expression:After radiation, the protein expression of TGFβ1 and vimentin showed the trend of increasing with post-irradiated time, on the contrary, the protein expression of SP-A and E-cadherin reduced in a time-dependent manner, compared with control group, the difference of four proteins was statistically significant at each time point (all P<0.01). In drug group, the changed trends of four proteins were resemblance to it in RT group, and the extent of increase or reduce was less, compared with control group (P<0.01) and RT group (P<0.05) respectively at each time point, the difference was statistically significant.5. Co-localization of the SP-A and a-SMA proteins:Under confocal laser scanning microscope, SP-A positive cells with green fluorescent markers were observed in alveolar walls, in control group, and a small a-SMA positive cells or area with red fluorescent markers tags in alveolar-septum. In RT group, at 8W post-irradiation, more a-SMA positive cells with red fluorescent tags were observed in the pulmonary interstitial, interspersed with a certain number of SP-A positive cells with green fluorescent markers; at 12W post-irradiation, a-SMA positive cells with red fluorescent tags became more and more, and SP-A positive cells with green fluorescent markers kept reducing. After two kinds of fluorescence fused, at 8W after irradiation, some cells with orange tags can be seen in the pulmonary interstitial, and at the corresponding time point in drug group, SP-A positive cells with green fluorescent markers were more than it in RT group, a-SMA positive cells with red fluorescent tags and cells with orange tags were less than it in RT group. In control group, no cell with orange tags was observed.Conclusion:1. A single 15Gy dose of right-thoracic irradiation using 60Co y-rays can be successfully established radiation-induced lung injury miniature pigs model.2. During the progress of radiation-induced lung injury, alveolar type II epithelial cells achieved a mesenchymal phenotype, namely, epithelial to mesenchymal cells transition occurred, which promote the development of radiation-induced lung injury, at a certain extent, and accelerate the formation of radiation pulmonary fibrosis.3. Sarcandra Glabra.can reduce alveolar type Ⅱ epithelial cells into mesenchymal cells, that is, epithelial-mesenchymal transition may play the part of protection of radiation-induced lung injury by down-regulating TGFβ1 and vimentin protein expression, up-regulating SP-A and E-cadherin protein expression.Part 2:The inhibition of Sarcandra Glabra. on matrix metalloproteinase in radiation-induced lung injury of miniature pigsObjective:To study changes of protein expression and enzyme activity of MMP2, MMP9 in lung tissue during radiation-induced lung injury, and explore that Sarcandra Glabra. may play a role of radiological protection by influencing MMP2 and MMP9.Methods:The collagen deposition was measured by Masson’s trichrome and hydroxyproline detection using acidic hydrolysis. The protein expression of MMP2 and MMP9 were detected by western blot and ELISA, enzyme activity of MMP2 and MMP9 were tested by gelatin zymography.Results:1. Masson staining:Collagen fibers in the pulmonary interstitium of miniature pigs in RT group increased slightly from the 4th weeks after radiation, the concentrated area of collagen fibers increased gradually with the extension of post-irradiation time; to the 12th weeks after radiation, it was obviously more, and the scattered fibrosis lesions appeared. At the 24th weeks after radiation, widely distributed layered or spiral collagen accumulation was detected in the pulmonary interstitium, and fibrosis lesions were becoming more and more. Compared with RT group, the degree of pulmonary fibrosis in drug group were lighter in miniature pigs at each time point.2. Hydroxyproline content:In RT group and drug group, the hydroxyproline content in lung tissue increased with a time-dependent manner, but the hydroxyproline content in drug group was lower than it in RT group, and the differences of pairwise comparisons in the RT group, control group and drug group were statistically significant (all P<0.01).3. MMP2 and MMP9 concentration:MMP2 concentration in lung tissue of miniature pigs in RT group began to increase at 4th week after irradiation, and fell back at 12th week, but was still higher than that of control group and drug group, and compared with the other group respectively at four time points, the difference is statistically significant (P<0.01); MMP2 concentration in drug group was between RT group and control group. MMP9 concentration in lung tissue of miniature pigs showed an increased trend after irradiation, and it increased evidently at 24th week because of its basic value is low; at each time point after irradiation, the differences of pairwise comparisons in three groups were statistically significant (drug group vs control group, RT group respectively, P<0.05; others, P<0.01).4. MMP2 and MMP9 protein expression:In RT group, protein expression of MMP2 and MMP9 in lung tissue of miniature pigs showed a trend of increase after irradiation, and it fell back at 12th week and was higher than that of control group and drug group; protein expression of MMP9 obviously increased at 24W post-irradiation on account of its basic value is low, which coincided to the results of ELISA. At each time point after irradiation, the differences of pairwise comparisons in control groups, RT group and drug roup were statistically significant (MMP2:P<0.01; MMP9:at 12W post-irradiarion, P<0.05; others, P<0.01).5. MMP2 and MMP9 enzyme activity:In RT and drug group, enzyme activity of MMP2 and MMP9 in lung tissue of miniature pigs kept increasing after irradiation, and the extent of increase in drug group was relatively less. Enzyme activity of MMP2 in RT group compared with drug group, and the difference was statistically significant from the 8th week after radiation (P<0.05); the control group compared with RT group and drug group respectively, at each time point after irradiation, the differences were statistically significant (P<0.01). For enzyme activity of MMP9, the differences of pairwise comparisons in three groups were statistically significant (P<0.05).Conclusion:1. Irradiation can obviously cause the increase of collagen content in lung tissue of miniature pigs.2. Evident increase of concentration, protein expression and enzyme activity of MMP2 and MMP9 in lung tissue, during radiation-induced lung injury, led to the imbalance between synthesis and degradation of extracellular matrix, and further to promote the development of lung injury.3. Sarcandra Glabra. can slow down the progress of radiation pulmonary fibrosis to play the part of radiological protection by reducing hydroxyproline content and down-regulating protein expression and enzyme activity of MMP2 and MMP9.Part 3:Serological markers of radiation-induced lung injury and radiological protection of Sarcandra Glabra.Objective:To seek to better predict serum marker and Sarcandra Glabra.’s radiological protection of radiation-induced lung injury by detecting serum levels of TGFβ1, SP-A, SP-D, MMP2 and MMP9.Methods:The serum levels of TGFβ1, SP-A, SP-D, MMP2 and MMP9 were detected by ELISA, gelatinase activity of MMP2 and MMP9 in serum were tested by gelatin zymography.Results:1. the serum levels of TGFβ1, SP-A, SP-D, MMP2 and MMP9: After radiation, the serum levels of TGFβ1, SP-A and SP-D in drug group and RT group were gradually increased with the extension of post-irradiation time, however, the serum levels in drug group were lower than that in RT group; at each time point, the differences of pairwise comparisons in control group, drug group and RT group were statistically significant (all P<0.01). In RT group, the serum levels of MMP2 started to increase from the 4th week post-irradiation, but fell back at 12th week and began to rise at 24th week post-irradiation. In drug group, the trend of serum MMP2 was not as good as that in RT group, at each time point, the differences of pairwise comparisons in three groups were statistically significant (all P<0.01). After radiation, the serum levels of MMP9 increased in a time-dependent manner in RT group and drug group, namely, RT group>drug group>control group, and the differences of pairwise comparisons in three groups were statistically significant at each time point (at 12th week, P<0.05; others, P<0.01).2. Serum enzyme activity of MMP2 and MMP9:In RT group and drug group, serum enzyme activity of MMP2 and MMP9 was higher than control group, and increased with post-irradiated time. At 24W after radiation, serum enzyme activity of MMP2 and MMP9 in RT group and drug group, compared with control group, respectively increased by 7.9 times and 17.0 times,6.4 times and 14.3 times. At each time point, the differences of pairwise comparisons in three groups were statistically significant (MMP2: P<0.01; MMP9:at 4W post-irradiation, RT group vs control group, P<0.05; others, P<0.01).Conclusion:1. After irradiation, serum levels of TGFβ1, SP-A, SP-D, MMP2 and MMP9 alone as the serological markers of radiation-mduced lung injury has certain limitation, and joint detection can make up for each other’s weaknesses, which may contribute to the early diagnosis, prevention and treatment of radiation-induced lung injury. 2. Sarcandra Glabra. can play radiological protection on radiation-induced lung injury in miniature pigs by reducing serum levels of profibrogenic cytokine, protecting of alveolar type II epithelial cells and down-regulating serum levels and enzyme activity of MMP2 and MMP9. |
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