A Study On The Regulating Mechanisms Of Pulmonary Fibrosis By ROS-mediated Antioxidative Defense System With Nrf2 As The Core

Pulmonary fibrosis (PF) is a chronic infla mma tory interstitial disease of lungwith potentia l fatal prognosis and poor response to available med ical therapy. It ischaracterized by an altered cellular composition of the alveolar region withexcessive deposition of colla gen. Although the pathogenesis of pulmonary fibrosishas not yet been well clarified, it has been indica ted by lots of evidences thatreactive oxygen species (ROS) play an important role in various kinds of lunginjury. Besides of infla mma tion stimula ted by ROS, it is more important thatoxidation-a ntioxidation bala nce in lung will be destroyed by ROS bursting whichmay lead to the loss of norma l regulating functions in body and therefore result inthe tissue injury and over repairment. Nrf2 is a recently found transcription factor,which can activate a lot of antioxida nt enzymes and phaseâ…¡detoxitic enzymes. Inthe occurrence of pulmonary fibrosis, whether the changes of Nrf2 andantioxida nt enzymes affect infla mma tion and colla gen metabolism and furtherresult in pathologic alteration has not yet been clarified. Will the drugs havetherapeutic effect on PF by this pathway? Therefore, the study on the changes ofthis pathway and the regulating mecha nisms is of great significa nce for discussingthe pathogenesis, prevention and cure of PF. In this study, through successful establishment of anima l model of PF in miceinduced by bleomycine(BLM), relative parameters of oxidative stress weredeterminate. The mRNA and protein level changes of Nrf2 and antioxida ntenzymes including CAT, SOD, GCS and HO-1 were observed by RT-PCR andWestern Blot. Lung fibroblasts were separated and cultured in vitro and theirviability and secretion of colla gen were assayed by MTT. H₂O₂, BLM and CAPEwere added to observe their independent or joint effects on the proliferation andamount of synthesized colla gen of L929 cells. The distribution of Nrf2 in cellswas observed by fluorescence immunocytochemistry and its binding activity toDNA was detected by EMSA. The mRNA and protein level changes of Nrf2 andantioxida nt enzymes including CAT, SOD, GCS and HO-1 were observed, at thesame time the production of cellular ROS was detected by flow cytometry.Common drugs DEX and antioxida nt CAPE were administered independently orjointly on PF anima l model to observe histomorphology indexes, amount of HYPand oxidative stress related indexes.The results are as follows. (1)The anima l model of PF on mice induced byBLM has been successfully established. Lung/body weight ratio, pathologica lfibrosis changes and the high level of HYP appearing in PF model has beenobserved. Increase of TBARs, decrease of GSH, gradua l reduction of SOD andCAT were also observed, which indica ted that oxidative stress existed in lung. Itwas found that the mRNA and protein expression of the key transcription factorNrf2 and antioxida nt enzymes changed obviously, which indica ted that thesegenes might regulate the oxida nt-antioxida nt bala nce in lung. The proliferationactivity of fibroblasts derived from the anima l model was greater than that of thecontrol group, and the MMP-1/TIMP-1 ratio related to colla gen metabolismchanged obviously, which indica ted that the changes of state and function of fibroblasts existed in PF. (2) Low dose of BLM and H2O2 was added to L929 cells.Increased proliferation and colla gen secretion were stimula ted. At the same time,the Nrf2 mRNA expression in cells was found up-regulated by both BLM andH2O2. However, the effect of BLM on Nrf2 protein level and activity was downregulation,while the effect of H2O2 was still up-regulation. The expressions ofimportant antioxida nt enzymes including CAT, SOD,γ-GCS and HO-1 were alsoaltered and their changing trends were the same as that of Nrf2. In addition, BLMand H2O2 can result in the increase of TBARs, the reduction of CAT and SODactivity, and the decrease of GSH. Nrf2 expression was up-regulated byantioxida nt, which affected the expressions of antioxida nt enzymes. It implicatedthat dose-effect relationship was involved in the regulation. Oxidative stressinduced by BLM and H2O2 can be improved by antioxida nt. As a result, theproliferation and colla gen secretion of fibroblasts can be controlled. After Nrf2was silenced by RNA interference, the weakened sensitivity of above mentionedantioxida nt enzymes to drugs, which indica ted that Nrf2 is the key factor of theirexpression and activity. (3) The changed expressions of Nrf2 and relatedantioxida nt enzymes have some relation to H2O2 concentration. Oxidants andantioxida nts both can affect cellular H2O2 concentration. The imcompatible ofCAT changes implicated there is a feedback regulation between Nrf2 and H2O2.Low concentration of H2O2 can stimula te cell proliferation, and cellular oxida ntantioxidant bala nce will be altered. (4) CAPE can regulate Nrf2 and relatedantioxida nt enzymes as well as the concentration of H2O2 at the norma l level.Oxidative stress induced by BLM and H2O2 can be improved by CAPE. It canmodulate the status of fibroblast by activating Nrf2, which due to its structure. Itwas showed that CAPE has some therapeutic effect on PF anima l model. (5) Thenew mecha nism was discussed that the adverse effect of BLM on pulmonary may due to its protein toxicity on Nrf2 or antioxida nt enzymes.This study discussed the regulating mecha nisms of pulmonary fibrosis byROS-mediated antioxidative defense system with Nrf2 as the core through PFanima l model, and gets severa l conclusions as followed. ROS can affect the statusand function of lung fibroblasts by regulating antioxida nt enzymes med iatedthrough Nrf2. H2O2 is the key molecular in regulating oxida nt-antioxida nt bala nce.The changes of ROS-mediated antioxidative defense system with Nrf2 as the coreare the ma in reason of oxida nt-antioxida nt unbala nce in vivo. At the same time, itwas also found that CAPE could have some therapeutic effects on PF throughactivating antioxidative defense system with Nrf2 as the core. In addition, newpossible mecha nism of PF induced by BLM may exist in its protein toxicity toNrf2 and antioxida nt enzymes. These findings will provide importantexperimental evidence for exploring the pathogenesis of PF as well as useful cluefor antioxida nt therapy in PF.