| Radiation-induced lung injury (radiation-induced lung injury, RILI) is a common complication after thoracic radiotherapy, bone marrow transplantation pretreatment and wartime nuclear radiation. Clinically significant radiation lung injury occurs in up to30%of patients irradiated for lung cancer and in about10-15%of other thoracic tumor patients. Even the most advanced application of Conformal IMRT, it is still difficult to prevent its occurrence. Due to the limitation of understanding the mechanism, there have been no strategic advances in RILI treatment over the past decades. Currently, the only way available to decrease the risk of developing severe radiation pneumonitis is to minimize the dose of radiation or the areas of lungs that are exposed to the radiation.The disadvantage is that tumor target dose must be limited definitely, which will inevitably reduce the tumor control rate. In recent years, it is increasingly recognized that radiation-induced lung injury is associated with a variety of multi-cytokine mediated interactions between cells. Several studies showed that IL-6, TGF-β, IL-1, and TNF-α are the key cytokines involved in the pathogenesis of radiation-induced lung injury, and simultaneous inhibition of IL-6, TGF-β, as well as TNF-α attenuates the degree of radiation-induced lung injury. However, targeting a single cytokine has limited effect on lung injury prevention. It seems that there might be other important related cytokines playing important roles in radiation-induced lung injury. IL-17A is a newly discovered pro-inflammatory cytokines, which is expressed in fibrosis-related diseases. IL-17A antagonist reduced the degree of pneumonitis and pulmonary fibrosis in bleomycin-or silica-induced lung injury in mice.Based on these studies, we hypothesized that IL-17A may also play an important role in the process of radiation-induced lung injury. Literature search was rarely found research about the roles of IL-17A in radiation-induced lung injury.The purpose of the research is to confirm that IL-17A plays an important role in the pathogenesis of radiation-induced lung injury, thereby providing a reliable target for prevention and treatment of radiation-induced lung injury. Targeting at IL-17A may be a new and effective clinical approach for radiation-induced lung injury. The study is divided into three parts to elaborate. Part1Objective:The purpose of the study is to establish mice model of radiation-induced lung injury quickly and efficiently, evaluate for the method and provide a reliable platform for the field of radiation-induced lung injury.Methods:A total of90mice (C57BL/6, Inbred male) were randomly divided into three groups:(1) Sham irradiation group (n=20), abbreviated as Sham group;(2) Radiation group A (n=35), non-anesthesia, one-time high-dose chest irradiation15Gy, abbreviated as RT-A group.(3) Radiation group B (n=35), non-anesthesia, irradiation20Gy, abbreviated as RT-B group. Mice were sacrificed after1,4,8,16week post-irradiation by linear accelerator. Lung tissues were taken out from mice of RT-A, B and sham group. Pathological changes and collagen deposition in lung tissue were observed under the microscope by the methods of H&E and Masson. The severity of alveolitis and pulmonary fibrosis was quantified according to Szapiel scales. Hydroxyproline content was measured by alkaline hydrolysis to evaluate collagen deposition in the lung tissue. In terms of survival,50mice were randomly divided into three groups:(1) Sham group (n=10);(2) RT-A group (n=20).(3) RT-B group (n=20). Survival rates were observed in16weeks, and Kaplan-Meier method and log-rank test were used to perform survival analysis.Results:After the mice were irradiated, a series of pathological changes occurred in the lung tissue. General speaking, there are three stages in the process:exudative, proliferative and fibrosis stage. Within8weeks, lung tissue H&E stained showed mainly alveolitis performance:alveolar wall thickening, alveolar congestion, interstitial pulmonary edema and telangiectasia, inflammatory cell infiltration. And alveolitis gradually worsened with the time passed. In the8th week, lung sections Masson stained appeared spotty fibrosis and fibrotic lesions is more obvious at16weeks. According to Szapiel grading criterion and statistical analysis, alveolitis of group RT-A and B were more serious than group Sham, and group RT-B more severe than group RT-A. In terms of fibrosis, group RT-A and B were more severe than Sham group, and group RT-B more serious than group RT-A. The study found that hydroxyproline levels of lung tissues from irradiated mice were gradually increased with time, and hydroxyproline increasing trend was particularly evident in the8th and16th week which was consistent with the occurrence of spotty fibers in pathology. On both the time points, hydroxyproline content of group RT-B was significantly higher than that of group RT-A, and the difference was statistically significant (P<0.01). Terms of survival time, no sham irradiated mice died within the observation time.16weeks survival rate of mice of group RT-A was80%, and that of mice of group RT-B was40%. And mice of group RT-B died significantly earlier than group RT-A. There is a statistically significant difference between the two groups in survival (P=0.0241<0.05).Conclusion:The whole chest of C57BL/6mice were irradiated by15Gy dose of linear accelerator, which can successfully build radiation-induced lung injury model. The model provides the basis for research on the mechanisms and targeting drug to radiation-induced lung injury.Part2Objective:IL-17A, as a pro-inflammatory factor, play an important role in many diseases related to inflammation or proliferation. The objective is to investigate the expressions and roles of IL-17A in the process of radiation induced lung injury (puhnonitis or lung fibrosis).Method:A total of61C57BL/6mice were included in this study. They were radomly divided into two groups:1) Sham group, n=25;2) RT group, n=36. Mice were sacrificed for histological examination or molecular biology at the observed time (1st week,4th week,8th week, and16th week). Mice of RT group underwent15Gy total thoracic radiation, and Sham goup not irradiated. The expression of IL-17A mRNA was quantitatively detected by real-time RT-PCR, and2-△△Ct was used for Relative quantitative analysis. In addition, IL-17A protein levels in lung tissue were detected by Western blot and immunohistochemistry staining.Results:After receiving15Gy irradiation, IL-17A in lung tissue of mice shows varying degrees of over-expression at different stages by immunohistochemistry analysis. IL-17A positive particles located in the cytoplasm of alveolar macrophages, alveolar epithelial cells, lymphocytes and bronchial epithelial cells of lung tissue. Stained histological sections showed alveolitis gradually aggravated within8weeks, while the expression of IL-17A peak at4weeks, which suggested that IL-17A may participate in the process of the early radiation-induced lung injury. According to real-time PCR results, IL-17A mRNA expression were increased in varying degrees within the observation time, started at1week, peaked at4weeks, and began to decline at8weeks. IL-17A mRNA continued to decline at16weeks, but still higher than the baseline level. Semi-quantitative Western blot analysis showed that IL-17A protein expression in lung began to rise post-irradiation after one week, four weeks peaked, then the expression level began to decline. These results show that the expression of IL-17A protein is consistent with the mRNA results.Conclusion:IL-17A expression significantly elevated after irradiation to the whole chest of mice, which was consistent with the occurrence time of alveolitis and fibrosis. Thus, IL-17Amay participate in radiation induced lung injury.Part3Objective:Blocking IL-17A signaling pathway improved outcomes variety of inflammatory diseases significantly. The study is to investigate the effect of interleukin-17A (IL-17A) antibodies on radiation-induced lung injuries in mice.Method:The thoraces of C57BL/6mice were irradiated with15Gy dose only once, which established mice model of RILL The total of135mice were divided into Sham (n=30), radiation control (n=35), treatment (n=35, IL-17A-neutralizing antibody,4μg/mouse, IV,4days per month for4months) and placebo group (n=35) before a single dose irradiation (15Gy) to the thorax. Inflammation and collagen contents in the lung tissues were examined by H&E and Massion staining method, and the concentration of IL-17A, TGF-β1, and IL-6in broncho alveolar lavage fluid (BALF) were measured by ELISA method. The degree of alveolitis and fibrosis was judged according to an established grading scale. Hydroxyproline content was measured using the alkaline solution. In another50animals,180-day survival rate following the irradiation and treatment was calculated by Kaplan-Meier method.Results:Sixteen weeks after the irradiation and treatment, there was significant inflammatory cell infiltration and interstitial collagen depositions in the radiation control and placebo groups, whereas these changes were relatively mild in the treatment group. The percentage of grade II and III alveolitis in the treatment group (16%, P<0.05) was lower than in the radiation control (72%) or placebo group (64%). The mean Aschcroft fibrosis scores were2.8(treatment group),5.2(radiation control) and4.8(placebo group) respectively. The scores of treatment group was lower than that of radiation control (P＜0.001) or placebo group (P＜0.001). The IL-17A, TGF-β and IL-6concentrations in the treatment group were lower than in the radiation control and placebo group (P＜0.01) following the irradiation. The180-day mortality rate in the treatment group was lower than in the radiation control group16.7%vs75.0%(P<0.05). Conclusion:IL-17A antibody treatment alleviates radiation-induced pneumonitis and subsequent fibrosis, and improvise post-irradiation survival. IL-17A neutraulizing antibody play an protective effect on mice undergoing thoracic radiation. Targeting at IL-17A maybe is an effective way for the treatment of radiation-induced lung injury. |
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