| 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 colla -gen typeⅣand the initiation process of pulmonary fibrosis in early radiation- induc -ed lung injury.The aim of this study was to explore the effect of collagen type IV, matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs(TIMPs) in early remodeling ,and the interlinkages between remodeling and motivation of fibrosis in early phase,in order to clarify the starting mechanism of radiation pulmonary fibrosis; Furthermore, to investigate the intervention of two peroxisome proliferator-activated receptor (PPAR)-γligands, Rosiglitazone and Pioglitazone , on early remodeling after radiation-induced lung injury.Materials and Methods Gene chip was used to screen early lung injury-related gene after irradiation; Quantitative analysis was performed to pulmonary collagen type IV, MMP-2, MMP-9, TIMP-2, TIMP-1 at the level of gene expression and prot -ein synthesis using Real-Time PCR and immunohistochemistry with image analysis respectively.Gelatinase(MMP-9 and MMP -2)activity was detected in the supernatant of alveoli macrophages (AM) stimulated byⅣcollagen by zymography analysis; Pro -liferation of human lung fibroblast (Fb) was determined by MTT stimulated by irrad -iation with 60Coγray of 1~10Gy, serum of irradiated rat and condition medium of alveolar macrophage (CMAM),respectively; Synthesis of collagen type IV and MMP -9 Were measured by ELISA and immunohistochemical staining; The activity of MMP-2,-9 in the above supernatants was determined by zymography. MTT assay was performed to evaluate the antagonism of Pioglitazone on Fb proliferation stimulated by 60 Coγ-ray and TGF-β1 ,respectively; Expression ofⅣcollagen andα-SMA in Fb were determined using immunocytochemistry;The activity of MMP-2,-9 in the above supernatants were analysised by zymography,while apoptosis bodies were detected by Hochest dyeing.Rat alveolar typeⅡcells (AT-Ⅱ) , interstitial cells (AM and Fb) we re separated from the irradiated and Rosiglitazone treatment group respectively; Ma -crophage(AM) was isolated from alveolar lavage solution (BALF); Activity of MMP -2,-9 were detected in supernatants from AT-Ⅱ,interstitial cells and AM cultured for 24～48h by zymography. Quantitative analysis was performed to pulmonary MMP-2, MMP-9,α-SMA and TIMP-1 at the level of gene expression and protein synthesis us -ing Reverse transcription-polymerase chainreaction(RT-PCR) and Western Blot with image analysis ,respectively.Results Gene chip Screening showed there was a significant increase in expression of IV collagen gene in 1～4 weeks after irradiation .Gene detection using Real-time PCR: gene expression of collagen type IV increased at 1 week and decreased 2 week after radiation; MMP-2 reached a high peak at 2 week in which an opposed alteration trend was displayed; MMP-9 appeared a significant trend of elevation, decrease and elevation which was similar to of collagen type IV; expression of TIMP-1 was lower, and there was no marked difference between all time points;TIMP-2 displayed a trend of slight elevation, decrease and elevation, which was opposed to MMP-2. Immunohis -tochemistry-Image analysis: Pulmonary collagen type IV obviously increaseed at 1 week, and began to decrease at 2 week; MMP-2 decreased at 2 week and then increa -sed;an opposed alteration trend to of collagen type IV was displayed; alteration trend of MMP-9 was similar to of collagen type IV but the extent was higher than that of later;gene expression of TIMP-1 slightly increased at 2 week and an opposed trend to of MMP-9 was displayedThe radiation of 1~7 Gy on pulmonary Fb could promote ce -llular proliferation and MMP-9 synthesis,but could not promote the collagen type IV synthesis.However,10% serum of irradiated rat and CMAM could not only promote Fb proliferation and MMP-9 synthesis but also promote collagen type IV synthesis and release, ,which is closely related to high level of TGF-β1 in the cell level. The dep -osition of collagen IV in lung tissue could be found one week after irradiation. Colla -gen typeⅣmay promote the synthesis and secretion of MMP-9和-2 by AM ; Piog -litazone inhibited Fb proliferation promoted by irradiation, and conversion into myo -fibroblasts (MFb) in dose-dependent; also induced Fb apoptosis; MMP-9, -2 activity in the culture supernatant treated by Pioglitazone were enhanced. In the Rosiglitazone treatment group, pathological changes in lung tissue were reduced significantly ; Rosi -glitazone inhibited lung Fb proliferation , reduced expression of collagen type IV and TIMP-1 and increased level of MMP-2, -9 after radiation-induced injury.Conclusion Collagen type IV, MMP-2, MMP-9 and their tissue inhibitors were invo -lved in ineffective remodeling in early radiation pulmonary injury;MMP-2 and MMP -9 play an important role in degradation of collagen type IV; disturbance of collagen type IV degradation is relation to the initiation of pulmonary fibrosis; AM plays an important role in in early lung remodeling. PPARγis expected to become a new tar -get on the pathomechanism of radiation-induced pulmonary fibrosis,and its artificial ligands Thiazolidinediones drug,Rosiglitazone and Pioglitazone, may have a protec -tive effect for the prevention and treatment of pulmonary fibrosis,and provide new ideas for clinical therapy on radiation fibrosis...
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