| RILI is a common complication of radiotherapy in thoracic tumors.Although a large number of studies have been done at present,the pathogenesis of RILI and the details of the treatment intervention have not been elucidated.It is difficult to start early diagnosis and targeted treatment.Current evidence indicates that inflammation plays a central role in the development and progression of RILI,especially in acute RILI.Local recruitment of inflammatory cells and production of inflammatory cytokines play an important role in mediating,amplifying and maintaining RILI.At the level of molecular biology,the process of RILI is that multiple effector cells in the lung mediate the transmission of intercellular damage information,initiate and maintain a variety of cytokine cascade effects,thereby causing damage that leads to pulmonary Fibrosis.Extracellular histone,a newly discovered molecule of DAMP,has distinctly different biological roles in mediating and noninfectious inflammatory reactions.It is a new field of research and it is speculated that it may participate as an important proinflammatory mediator in RILI development took place.In this study,we investigated the dynamic changes of plasma extracellular histone protein during RILI in mice and its relationship with inflammatory cytokines and lung injury,and explored the important role of extracellular histone proteins involved in and played a role in RILI.To investigate the changes of extracellular histones and their effects on systemic inflammation after radiotherapy for lung cancer patients.RILI treatment targeting extracellular histones may be a new and effective clinical pathway.This study is divided into four parts.First partObjective:To establish a mouse RILI animal model and evaluate it effectively to provide a reliable platform for studying radiation lung injury.Methods:Fifty male inbred C57BL/6 mice were randomly divided into two groups:control group and irradiation group,with 5 rats in each group at each time point.Mice were sacrificed after 1,3,5,8 week post-irradiation by 12Gy dose of linear accelerator,the lung tissues of mice in the control and irradiation groups were taken respectively.The histopathological changes in the lung were observed under a microscope using HE and VG staining methods,and the severity of alveolitis was performed according to the Szapiel method.Results:After irradiation of the entire chest of the mouse,the lung tissue undergoes a series of pathological changes.HE staining:At 1 week after irradiation,the alveolar space of the lung was slightly widened and the capillaries were dilated and congested.At the 3rd week after irradiation,the alveolar septum of the lung was widened,and the inflammatory cells infiltrated and the blood vessels dilated and congested.At 5 and 8 weeks after irradiation,the alveolar septum widened more clearly,part of the alveolar cavity collapsed,visible fibroblasts and lymphocytes and other inflammatory cell infiltration.Collagen fibers were pink after VG staining.At 1week after irradiation,there was a slight increase in collagen fibers in the alveolar septum of the lung;at 3 weeks after irradiation,the alveolar septum widened and the number of collagen fibers increased;at 5 and 8 weeks after irradiation,the alveolar septum widening was more pronounced.The number of collagen fibers increased significantly and some alveolar spaces collapsed.The degree of pulmonary alveolitis in mice was quantified by Szapiel grading.Statistical analysis showed that there was no significant difference in the degree of alveolitis between the irradiation group and the control group at the first week after irradiation(?~2=0.625,P=0.444),but with the increase of time,the difference was significant,and the degree of alveolitis was aggravated.The difference between the 3rd,5th,and 8th weeks was statistically significant(?~2=13.868,P=0.001).Summary:The RILI model can be constructed successfully by 12Gy dose of linear accelerator in the whole chest of C57BL/6 mice,which provides the foundation for the theoretical study of RILI and the screening of control targets.The second partObjective:This section investigates the dynamic changes of plasma extracellular histones during the process of mouse RILI and its relationship with related inflammatory factors and lung injury related indicators,and discusses the role of extracellular histones in RILI process.Methods:Fifty male inbred C57BL/6 mice were randomly divided into two groups:control group and irradiation group.Each group had 5 rats at each observation time point and were sacrificed at 1,3,5,and 8 weeks after irradiation with 12Gy dose.The MPO activity of the lung homogenates and plasma extracellular histones were detected by ELISA.The inflammatory factors(IL-1?,IL-6,TNF-?,and TGF-?1)were detected by Luminex.The dynamic changes,the use of immunohistochemical techniques to detect the lung tissue TNF-?,TGF-?1 and?-SMA protein expression levels.Results:The MPO activity of the lung homogenate began to increase after the first week of irradiation in mice,reaching the highest level in the third week,and began to decline slightly in the fifth week,but was still higher than the baseline level in the eighth week.The overall difference was Statistical significance(F=69.006,P=0.000),further analysis between groups,there was no significant difference between the 3rd,5th and 8th week time points(P>0.05).The change rule of extracellular histone at each time point in the irradiation group was:the expression began to increase in the first week after irradiation,reached the highest level in the third week,began to decrease slightly in the fifth week,decreased obviously in the eighth week and were still higher than the baseline level.The differences between the time points were statistically significant(F=563.096,P=0.000)The overall differences of IL-1?,IL-6,and TGF-?1 in the irradiated group were statistically significant(F=56.993,213.424,58.014,P=0.000),and further comparison among the groups was made.There was no statistically significant difference between week time points(P>0.05).The difference of TNF-?between the irradiated groups was statistically significant(F=505.622,P=0.000).Immunohistochemistry results showed that TNF-?,TGF-?1 and?-SMA all expressed brownish yellow in cytoplasm.TNF-?,TGF-?1 and?-SMA were very little expressed in lung tissue of control mice.The expression of TNF-?and TGF-?1protein was highest at the 3rd week after irradiation,while the peak of?-SMA protein expression was at 5th week after irradiation.After the initiation of fibrosis,the infiltration of inflammatory cells began to decrease after 8 weeks of irradiation.Extracellular histones and related inflammatory factors began to decline,and TNF-?,TGF-?1 and?-SMA protein expression levels began to decline,but were still higher than the basal level.The overall difference in staining index of TNF-?,TGF-?1 and?-SMA protein at different time points after irradiation was statistically significant(F=23.118,19.185,13.925,P=0.000).Summary:After whole-chest irradiation in mice,the lung homogenate MPO,plasma extracellular histones and inflammatory factors(IL-1?,IL-6,TNF-?and TGF-?1)all increased in varying degrees,and the changes were similar;The expression levels of TNF-?,TGF-?1 and?-SMA protein increased in different degrees,which was basically consistent with the formation of RILI.Extracellular histones were considered to play a role in RILI process.The third partObjective:This part of the study aims to investigate the protective effect of heparin on RILI mice.Methods:Seventy-five C57BL/6 mice were randomly divided into three groups:control group,single irradiation group and heparin-treated group.In the latter two groups,the whole chest was irradiated with 12Gy dose of linear accelerator and the heparin-treated mice were subcutaneously injected with 300U/kg of heparin per week on the 2nd day after irradiation.The mice in the control group and the irradiation group were at the same time.A one-time intraperitoneal injection of 0.9%sodium chloride solution or 300U/kg body weight of heparin was given intraperitoneally.At the 1st,3rd,5th,and 8th week after irradiation,lung tissues from each group were taken,and the histopathological changes and collagen deposition in the lungs were observed under HE and VG staining microscopy.The severity of alveolitis was measured according to the Szapiel method.The expression of TNF-?,TGF-?1 and?-SMA protein in lung tissues were detected by immunohistochemistry.Plasma extracellular histones were detected by ELISA and the dynamic changes of inflammatory cytokines(IL-1?,IL-6,TNF-?and TGF-?1)in each group were detected by Luminex.Results:According to the Szapiel method,the degree of alveolitis in three groups of mice was quantified.Statistical analysis showed that:at 1 week after irradiation,there was no significant difference in the degree of alveolitis between the 3 groups(?~2=3.278,P=0.194),but it increased with time(3rd,5th,and 8th weeks).Compared with the simple irradiation group,the degree of alveolitis in the heparin-treated mice was reduced,but it was still significantly higher than that in the control group.The differences were statistically significant(?~2=22.373,19.579,19.498,P=0.000,0.000,0.001).Compared with the simple irradiation group,heparin treatment reduced the alveolar wall thickness and interstitial edema in mice,and weakened collagen deposition.Except for the first week,at the remaining time points(3rd,5th,and 8th weeks),the staining indexes of TNF-?,TGF-?1 and?-SMA protein in the heparin-treated group were significantly lower than those in the control group(P<0.05)In the heparin treatment group,the levels of extracellular histones and inflammatory factors showed a decreasing trend at each observation time point,but they were still higher than the baseline level,and the differences were statistically significant(P<0.05).Summary:The application of heparin has certain preventive effects on early RILI,and its prevention and control may be achieved through the following ways:neutralization of extracellular histones,down-regulation of plasma proinflammatory cytokines in mice,and reduction of TNF-?,TGF-?1 and?-SMA in lung tissue.The expression,to varying degrees inhibit or reduce the alveolar inflammatory response,provides an effective target for RILI prevention.Fourth partObjective:To investigate the changes in extracellular histones and their effects on systemic inflammation after radiation therapy in patients with lung cancer.Methods:Blood samples were obtained from 45 non-surgical lung cancer patients at 5 time points(1 day before radiotherapy,1 day after radiotherapy,1,3,and5 weeks)and extracellular histones,MPO,and a variety of inflammatory responses were measured.Cytokines.In addition,serum from patients after radiotherapy was incubated with human lung epithelial cell line(BEAS-2B)and human monocyte cell line(U937)overnight,and specific anti-histone neutralizing antibodies were given to intervene to assess the degree of cell damage and cells.Factor generation and other indicators.Results:Compared with pre-radiotherapy,extracellular histone levels increased significantly at the first week after radiotherapy and peaked at the third week,accompanied by increased MPO and inflammatory cytokines,and extracellular histones at the 5th week.At the beginning of decline,inflammatory cytokines also began to decrease,but were still higher than pre-radiotherapy levels,and the differences were statistically significant(P<0.05).Dynamic changes in extracellular histones are closely related to these inflammatory markers.Compared with serum obtained before radiotherapy,serum after radiotherapy of patients can significantly cause damage to BEAS-2B cells and stimulate the production of cytokines in U937cell supernatant.Specific anti-histone neutralizing antibodies can significantly inhibit this damage effect.Summary:This study revealed a causal relationship between circulating histones and systemic inflammation.Extracellular histones may serve as markers of systemic inflammation associated with radiotherapy and represent therapeutic targets for the resolution of inflammation.conclusions:1.The RILI model can be successfully constructed by irradiating the full-thickness C57BL/6 mouse with 12Gy dose.2.After whole-chest irradiation in mice,the lung homogenate MPO,extracellular plasma histones and inflammatory cytokines IL-1?,IL-6,TNF-?,TGF-?1 all increased.The levels ofTNF-?,TGF-?1 and?-SMA protein expression increased,which were basically consistent with the formation of RILI.It suggests that extracellular histones play a pro-inflammatory role in the pathogenesis of RILI.3.The use of heparin can down-regulate proinflammatory cytokines in mice,reduce the expression of TNF-?,TGF-?1 and?-SMA in lung tissue,and inhibit or reduce the inflammatory reaction of alveoli to different extents.The early RILI has a certain control effect.4.Extracellular histones may serve as markers of systemic inflammation associated with radiotherapy and may be an important therapeutic target. |
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