Role Of Ros In Proliferation And Collagen Synthesis Of Fibroblasts Induced By Bleomycin

Pulmonary fibrosis is a progressive and lethal lung disease characterized by excessive proliferation of fibroblasts and deposition of extracellular matrix (ECM) and the pathogenesis is unclear. BLM is the main drug used in study of pulmonary fibrosis at present, because the process of pulmonary fibrosis induced by BLM is similar to that of human idiopathic pulmonary fibrosis. BLM promoted the development of pulmonary fibrosis through type Ⅱ alveolar epithelial cells apoptosis, platelet aggregation and activation, collecting macrophages and the secretion of cytokines. But the directive effects of BLM on fibroblast cells are still needed to investigate. There many controversies exist about the direct effect of BLM on fibroblasts.Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen, including superoxide anion (O2-), hydrogen peroxide (H2O2), nitric oxide (NO), and the hydroxyl radical (OH-). ROS could cause DNA fracture, lipid peroxidation and carbohydrate oxidation. ROS is related to liver fibrosis, atherosclerosis and peritoneal fibrosis and so on. BLM caused fibrosis by attacking DNA directly. BLM induced DNA breaks through ROS mediated, and this effect could be decreased by superoxide dismutase. There are few studies on whether pulmonary fibrosis induced by BLM is mediated by ROS.Researches showed that physiology conentration of ROS play a role in the body by acting on receptor kinases, transcription factor, DNA and so on. Hypoxia-inducible factor-1(HIF-1) is a transcription factor, and it is one of the targets of ROS. ROS can affect (or influence) many activity through up or down regulated the HIF-1. But the relation between BLM and HIF-1is not clear.In this experiment, the proliferation and collagen synthesis of mouse fibroblast induced by BLM were observed. Regard that the reagent MnTBAP can offset the intracellular ROS; it was used to block the ROS to observe the role of ROS in pulmonary fibrosis induced by BLM. The roles of HIF-1in pulmonary fibrosis were also discussed. The purpose was to explore whether ROS were involved in the proliferation and collagen synthesis of mouse fibroblast induced by BLM through HIF-1.Methods:MTT assay and flow cytometry were used to determine the effect BLM had on mouse fibroblast proliferation. Meanwhile, the Sirius Red Staining and Real-time PCR were used to detect collagen protein expression of mouse fibroblast. We also used RT-PCR to detect the relative level of HIF-1mRNA.Result:1. Mouse fibroblast proliferation and collagen synthesis induced by BLM(1) The MTT assay showed that the optical density number of BLM group increased significantly than the control group in a dose dependent and time dependent (p<0.05or p<0.01).(2) We take the proliferation index (PrI) to reflect proliferation status. The flow cytometry assay shows that the percentage of cells in proliferation cycle of BLM group is obvious higher than the control group (p<0.05).(3) The SRS (sirius red staining) assay shows that mouse fibroblast collagen secrete increases (p<0.05) as the result of using BLM. But this effect is reduced as the dosage of BLM increased. The BLM groups show notable differences (p<0.01). (4) Real-time PCR assay shows that the relative expression level of type III procollagen mRNA in BLM groups is obviously higher than the control groups (p<0.05).2. Mouse fibroblast proliferation and collagen synthesis induced by bleomycin after blocking the ROS.(1) The flow cytometry assay shows that BLM+MnTBAP groups decrease obviously (p<0.05) than the BLM group after blocking the ROS.(2) Sirius red staining results shows that the MnTBAP+BLM groups see notable decrease compared to BLM groups(BLM1μg/L group p<0.01, BLM3μg/L group p<0.05).(3) Real-time PCR assay shows that the relative expression level of type III procollagen mRNA of BLM+MnTBAP group decrease obviously than the BLM group (p<0.05).3. The change of HIF-1mRNA level in mouse fibroblast induced by BLMThe HIF-1mRNA levels in BLM group cells were higher than that of control group obviously (p<0.01).Conclusion:1. BLM can promote the proliferation of mouse fibroblast and collagen synthesis directly. However, collagen synthesis has a downward trend with cell proliferation increasion.2. Mouse fibroblast proliferation and collagen synthesis induced by BLM are mediated by the ROS.3. BLM can up-regulated the expression of HIF-1mRNA in mouse fibroblast, and HIF-1may take part in the fibrosis caused by BLM.