Mechanism Study Of Integrin ?v?3 Inhibitor Cilengitide Attunates Radiation-induced Pulmonary Fibrosis

PART ? Integrin ?v?3 inhibitor Cilengitide attunates radiation-induced pulmonary fibrosis in a murine modelObjective: Radiation-induced pulmonary fibrosis(RIPF)is a common complication of radiation therapy and there is no effective treatment.Transforming growth factor-?1(TGF?1)is recognized as the most closely related cytokine to radiation-induced pulmonary fibrosis.It is reported that integrin ?v?3 inhibitor Cilengitide can inhibit the activation of TGF?1 and interfere with the occurrence of intestinal fibrosis.However,the effect of Cilengitide on radiation pulmonary fibrosis has not been reported at present.This study will use a mouse model of radiation pulmonary fibrosis to evaluate the effect of integrin ?v?3 inhibitor Cilengitide on radiation pulmonary fibrosis.Methods: We divided 150 C57BL/6 mice into 5 groups randomly: control group,normal administration group,irradiation group,irradiation plus low dose Cilengitide group,and irradiation plus high dose Cilengitide group The mice in the irradiation group were given 16 Gy whole thoracic irradiation,and mice in the administration group were intraperitoneally injected with 15 mg/kg or 75 mg/kg Cilengitide daily for 8 weeks.Each group of mice was recorded monthly for survival and weight status.Pulmonary fibrosis was assessed at 6th,16 th,and 20 th week after irradiation by HE and Masson staining.Pulmonary fibrosis in each group was evaluated at 20 weeks after irradiation using hydroxyproline content assay and ?-SMA immunohistochemistry.The levels of total TGF?1 and activated TGF?1 in serum of 20 th week irradiation mice in each group were measured at by enzyme-linked immunosorbent assay(ELISA).The expression of Tgfb1,Itgav,Itgb3,and Col1a1 m RNA in the right lung tissue of each group of 20 th week irradiation mice was measured by Real-Time PCR method.Results: Cilengitide administration benefit the survival of the irradation mice.Among them,irradiation plus low dose Cilengitide group(P < 0.001)was more effective than irradiation plus high dose Cilengitide group(P = 0.04).The results of HE and Masson staining of lung tissue at 6 weeks after radiotherapy indicated that Cilengitide had no significant intervention effect on the acute inflammatory phase of radiation-induced lung injury.At the 20 th week after radiotherapy,HE staining,Masson staining,?-SMA immunohistochemistry and lung tissue hydroxyproline result showed that daily intraperitoneal injection of Cilengitide could significantly reduce the degree of radiation pulmonary fibrosis in mice.Real-Time PCR showed that the Tgfb1,Itgav,and Col1a1 m RNA expression levels of irradiation group mouse were significantly higher than those in the control group,but after the administration of Cilengitide,the Tgfb1,Itgav,and Col1a1 m RNA expression levels were significantly decreased.Itgav is the most obvious(P < 0.001).Conclusion: In this part of the study,we use radiation pulmonary fibrosis mouse model and first found that integrin ?v?3 inhibitor Cilengitide can significantly attenuate radiation-induced pulmonary fibrosis which provides a theoretical basis for clinical use of Cilengitide treating RIPF.PART ? Cilengitide attunates radiation-induced pulmonary fibrosis via TGF?1/?v?3-PAI-1 pathwayObjective: In the previous part of the study,we found that integrin ?v?3 inhibitor Cilengitide can reduce the activation of serum TGF?1 and significantly reduce the degree of radiation pulmonary fibrosis in mice,but its specific mechanism is unclear.The purpose of this study was to identify the key target genes of TGF?1 to induce RIPF and to explore the regulatory role of Cilengitide in key genes.Methods: CCK-8 cell proliferation assay was used to determine the semi-inhibitory concentration(IC50)of Cilengitide against MRC-5 human embryonic lung fibroblasts.ELISA was performed to detect the content of total TGF?1 and activated TGF?1 in the supernatant of MRC-5 cells after 8Gy irradiation and Cilengitide treatment.Microarray and protein interaction analysis methods were used to find the key target genes of TGF?1 to induce RIPF.Western Blot and Real-Time PCR were used to detect the regulation of TGF?1 and Cilengitide on key target genes.Western Blot and si RNA transfection were used to detect the type I collagen after inhibition of key target gene expression.Results: CCK-8 cell proliferation assay showed that Cilengitide could significantly inhibit the proliferation of MRC-5 cells,and its IC50 was about 0.5u M.The results of ELISA showed that the total TGF?1 and activated TGF?1 content in the cell supernatant increased gradually after single dose of MRC-5 cells(0 Gy-10 Gy),and the total TGF?1 secreted by cells after 8 Gy irradiation Compared with the control group significant increased(P < 0.05)and activated TGF?1(P < 0.01)was the most obvious.However,after treated with Cilengitide,the total TGF?1 secreted by the cells did not change significantly compared with that of the 8Gy irradation,but the activated TGF?1 was significantly decreased(P < 0.001).Through microarray and protein interaction assays,we found that plasminogen activator inhibitor-1(PAI-1)is a key target gene of TGF?1 to induce RIPF.Western Blot and Real-Time PCR results showed that TGF?1 can regulate the expression of PAI-1 in MRC-5 cells,Cilengitide can inhibit the expression of PAI-1,and PAI-1 si RNA can significantly inhibit the expression of type I collagen.Conclusion: This study suggests that radiation can induce radiation-induced lung fibrosis by activating the TGF?1/?v?3-PAI-1 pathway in human embryonic lung fibroblasts.The integrin ?v?3 inhibitor Cilengitide can attenuate the occurrence of radiation pulmonary fibrosis by interfering with the expression of key proteins in the TGF?1/?v?3-PAI-1 pathway.PART ? Genetic variants in TGF?1/?v?3-PAI-1 pathway genes are associated with an increased risk of radiation pneumonitis in lung cancer patientsObjective: In prevous 2 parts we found in TGF?1/?v?3-PAI-1 pathway plays a crucial role in the process of lung injury,although its association with radiation pneumonitis(RP)is unclear.We hypothesized that genetic variants in TGF?1/?v?3-PAI-1 pathway genes may influence the risk of RP.Methods: In this study,169 lung cancer patients were genotyped for six single nucleotide polymorphisms in PAI-1 using the Sequenom Mass ARRAY system.The risk of RP was evaluated by Cox proportional hazards analyses.The cumulative RP probabilities by genotype were assessed using Kaplan-Meier analyses.Results: Univariate and multivariate analyses revealed that PAI-1:rs7242 GT/GG was correlated with an increased occurrence of grade ? 3 RP(crude hazard ratio = 3.331;95% confidence interval,1.168-9.497;P = 0.024).Conclusion: Our results indicated that PAI-1:rs7242 in the 3?-untranslated region of PAI-1 can be a predictor of grade ?3 RP before radiotherapy.