Role Of Matrix Metalloproteinase 12 In Pulmonary Abnormal Remodeling After Early Radiation-induced Lung Injury And The Initiation Of Pulmonary Fibrosis And Exploration Of Therapeutic Targets

Objective To date, the initial mechanism of radiation-induced pulmonary fibrosis is unknown,which is the terminal phase of void remodeling after lung injury caused by irradiation.There seems to be an unknown link between increased expression of MMP-12 and the initial process of pulmonary fibrosis in early radiation- induced lung injury.The aim of this study was to explore the effect of MMP-12 in early remodeling, and the internal linkage between remodeling and initiation of fibrosis in early phase, in order to clarify the initial mechanism of radiation-induced pulmonary fibrosis; Furthermore, to investigate the intervention of Pirfenidone on early remodeling after radiation-induced lung injury.Materials and Methods Broad-spectrum gene chip was used to screen early lung injury-related genes after irradiation; On this basis, pulmonary tissue of rats was irradiated by 60Coγ-ray. Respectively, MMP-12 activity was examined by gelatin zymography, and protein expression of MMP-12 was measured by Western Blot and immunohistochemistry; the degradation and collapse of elastin were observed by tissue elastin specific staining; the"crosstalking"phenomenon was observed between alveolar type II cells and mesenchymal cells by transmission electron microscope; the content of fibrosis-related cytokines TGF-βl, IL-1β, TNF-αwas determined by ELISA; the expression ofα-SMA was examined by immunohistochemistry. Treated with 60Coγ-ray and Pirfenidone, alveolar type II cells (AT- II) and interstitial cells (AM and Fb) were separated from rats; MMP-12 activity was detected in supernatants from AT- II, interstitial cells by zymography. Treated with 60Coγ-ray and different doses of Pirfenidone, abnormal proliferation of human lung fibroblasts (HLF) was detected by MTT; TGF-βl andα-SMA expression were evaluated using immunocytochemical stain. Synthesis of TNF-α, IL-lβ, IFN-γwas measured by Western Blot; MMP-12 activity was detected in supernatants from HLF by zymography.Results (1)MMP-12 activity began to increase 1 week after radiation; Elastin, a part of the basement membrane of alveolar wall, began to degrade and collapse 1 week after radiation, which became worst at the 4th week; The"cross talking"phenomenon was found by electron microscope between alveolar typeⅡcells and mesenchymal cells; The expression of TGF-β1 andα-SMA were both elevated gradually as time elapsed within 4 weeks after radiation. (2) In vitro, Pirfenidone can significantly ease consolidation and exudative inflammation in pulmonary tissue of irradiated rats, suppress radiation-induced elevation of protein expression and activity of MMP-12, inhibit degradation and rupture of elastins on the basement membrane of alveolar wall, the"cross talking"biological response between parenchymal and mesenchymal cells as well as resulting release of fibrosis-related cytokines, and antagonize transfomation of Fb towards MFb(Myofibroblasts). (3)In vivo, Pirfenidone can suppress radiation-enhanced HLF proliferation and TGF-β1,α-SMA, TNF-α, IL-1β, IFN-γexpression as well as MMP-12 activity in supernatants from HLF in a dose-dependent manner. Pirfenidone can also downregulate MMP-12 activity in supernatants from pulmonary parenchymal (AT-Ⅱ) and interstitial cells(AM和Fb).Conclusion Early in radiation lung injury, activated macrophages express MMP-12, which increased activity of the latter makes elastic fibers on the basement membrane of alveolar wall degrade and break. Lung tissue begins to reconstruction. The gap of ECM forms, which causes alveolar epithelial and interstitial cells in alveolar septum to contact with each other, triggers the"cross talking"biological responses among these cells and results in the release of some fibrosis-related factors. They stimulate fibroblasts in alveolar septum activate, proliferate, wander, move to peripheral gap of ECM and transform towards MFb. These cells synthesize and secret excessive collagenⅣ,ⅠandⅢ, resulting in excessive deposition of extracellular matrix. Reconstruction of lung tissue is invalid, leading to the final formation of pulmonary fibrosis. MMP-12 is expected to become a new target on the mechanism of radiation-induced pulmonary fibrosis, and Pirfenidone may become a new drug for the prevention and treatment of pulmonary fibrosis. This study provides new therpeutic bases and approaches for clinically curing radiation-induced fibrosis effectively.