Objective: To investigate the effects of gene therapy with replication-defective adenovirus enclosing Egr-1 promoter and Smad7 cDNA on irradiation-induced pulmonary fibrosis.
Methods: The recombinant replication-defective adenovirus AD. Egr-Smad7 enclosing Egr-1 promoter and Smad7 cDNA was constructed. 288 C57BL mice were randomly divided into 6 groups: AD. Egr-Smad7 group (Group RA, receiving intratracheal instillation of AD. Egr-Smad7 of the dose of 10(9) pfu/0.1 ml), AD. Egr-Smad7 + radiation group (Group RAR, receiving intratracheal instillation of AD. Egr-Smad7 of the dose of 10(9) pfu/0.1 ml and then radiation to the chest 14 h later), replication-defective adenovirus group (Group AV, receiving intratracheal instillation of replication-defective adenovirus of the dose of 10(9) pfu/0.1 ml), replication-defective adenovirus + irradiation group (Group AVR, receiving intratracheal instillation of replication-defective adenovirus of the dose of 10(9) pfu/0.1 ml and then radiation to the chest 14 h later), blank control group (Group C), and pure irradiation group (Group R), each group was re-divided into 6 subgroups of 8 mice to be observed 0, 1, 2, 4, 8, and 12 weeks after the treatment. The mice were killed at different time points and their lungs were taken out. The levels of type I collagen, type III collagen, connective tissue growth factor (CTGF), and transforming growth factor-beta1 (TGF-beta1) were detected by ELISA. The level of hydroxyproline was examined by alkaline hydrolysis method. The lung tissues were stained with HE to undergo pathological examination.
Results: The TGF-beta1 levels of the irradiation groups all increased, peaking in the second week (all P < 0.05), all significantly higher than those of Group C. However, the TGF-beta1 levels at different time points of Group RAR were all significantly lower then those of the other irradiation groups. The CTGF levels of different groups at different time points were all significantly higher than those of Group C (P < 0.05 or P < 0.01), and the CTGF levels of Groups RA and AV were decreased to almost normal 12 weeks after the irradiation. The levels of type I collagen and type III collagen of the 1 and 2-week subgroups of Group RAR were significantly lower than those of Group C (all P < 0.01), then gradually increased, and were slightly higher than those Group C 12 weeks later. The levels of type I collagen and type III collagen at different time points of the other groups were all significantly higher than those of Group C (P < 0.05 or P < 0.01). However, The levels of type I collagen at different time points of Group RAR were all lower than those of the other groups except Group C, and the levels of type III collagen in the first to eighth weeks after irradiation of Group RAR were all lower than those of the other groups except Group C. The hydroxyproline level of the 1 and 2 week subgroups of Group RAR were significantly lower than those of Group C (all P < 0.01), and then gradually increased. The hydroxyproline levels of the other irradiation groups all gradually increased significantly, peaking at the 12 th week (all P < 0.01). 1 approximately 2 weeks after irradiation Groups RAR, RA, and AV showed remarkable pulmonary congestion changes, even more remarkable then those in Group R, 8 approximately 12 week later, fibrosis changes were found in Group R and AVR, and 12 weeks later the histological structure of lung of Group AV, RAR, and RA returned almost normal.
Conclusion: Radioactive rays induce Egr-1 promoter to regulated the expression of exogenous Smad7 gene that blocks the signal transduction of TGF-beta. Thus use of AD. Egr-Smad7 may become a novel strategy of gene therapy in prevention and treatment of pulmonary fibrosis.