The Study Of Lysyl Oxidase In Epithelial-to-Mesenchymal Transition During Paraquat–Induced Pulmonary Fibrosis

Paraquat(PQ) is an effectively and widely used herbicide which is associated with high fatalities caused by accidental or voluntary ingestion in developing countries. Lung is the main target organ for the pathological effects of PQ. PQ rapidly accumulates in the lungs after administration, causing acute lung injury(ALI) or acute respiratory distress syndrome(ARDS), and could progress to extensive pulmonary fibrosis. Pulmonary fibrosis is a major manifestation and a leading cause of death in PQ poisoning. No specific antidote has been currently recommended which leads to high mortality of PQ poisoning. It has important significance to research molecular mechanism of PQ toxicity, especially PQ-induced pulmonary fibrosis.Pulmonary fibrosis is characterized by the loss of alveolar structures and functions following apoptosis of epithelial and endothelial cells, proliferation of fibroblasts and excessive deposition of extracellular matrix(ECM). The cells that are responsible for the deposition o f collagen are myofibroblasts, which have three sources: differentiation of resident fibroblasts, bone marrow-derived precursors, and Epithelial-to-Mesenchymal transition(EMT). Considerable attention has been given to the role of EMT in fibrosis recently. According to several lineage tracing studies in vivo, over 30% of the myofibroblasts are derived from epithelial cells by means of EMT. As fibrosis develops, collagen fibrils become permanently cross- linked as a result of the action of LO X, which is responsible for oxidation of specific lysine residues in collagen and elastin, leading to the formation of molecular crosslink essential for stabilization of the ECM. In addition, considerable attention has been given to the intracellular activity of LOX and proposed LOX might be involved in driving EMT. Our previous study demonstrated that EMT was strongly associated with PQ–induced pulmonary fibrosis.The objective of this study was to evaluate the potential involvement of LOX on EMT in the process of pulmonary fibrosis induced by PQ. To prove our postulation, we designed the following experiments.1. To observe the expression of LOX, pulmonary fibrosis and EMT after PQ poisoning(1) To establish in vivo experimental model, the animals were administered by lavage(50 mg/kg) with 20% PQ solution. In order to observe the lung pathological changes, the paraffin sections of lung were dyed by hematoxylin- eosin(HE) and Masson trichrome. The LOX and mesenchymal markers of EMT α-SMA, Vimentin and epithelial marker E-cadherin, ZO-1 were detected by immunohistochemistry and Western Blot.(2) To establish in vitro experiment model, RLE-6TN and A549 cells were treated with different concentrations of PQ. The cell morphological changes were observed under phase contrast microscopy, and Western Blot was used to detected the expressions of epithelial markers and mesenchymal markers.2. The Study of Lysyl O xidase in Epithelial-to-Mesenchymal Transition during Paraquat–Induced Pulmonary Fibrosis(1) To investigate the effect of LO X on EMT after PQ poisoning in vivo, BAPN was used to establish the experimental model. The paraffin sections of lung were dyed by hematoxylin- eosin(HE) and Masson trichrome. The LOX and mesenchymal markers of EMT α-SMA, Vimentin and epithelial marker E-cadherin, ZO-1 were detected by immunohistochemistry and Western Blot.(2) To investigate the effect of LOX in EMT after PQ poisoning in vitro, RLE-6TN and A549 cells were pretreated with BAPN, and then exposed to PQ. The cell morphological changes were observed under phase contrast microscopy, and Western Blot was used to detect the expressions of epithelial markers and mesenchymal markers.(3) To investigate the effect of LO X on EMT after PQ poisoning in vitro, A549 cells were pretreated with si RNA, and then exposed to PQ. The cell morphological changes were observed under phase contrast microscopy, and Western Blot and immunofluorescence were used to detect the expressions of epithelial markers and mesenchymal markers.The main results are as follows:1. HE and Masson staining showed inflammatory infiltration and collagen deposition with a tendency of exaggeration after PQ administration 72 h. The level of LOX was significantly increased after PQ administration. Expression of mesenchymal markers α-SMA and Vimentin were increased and epithelial marker E-cadherin and ZO-1 were decreased after PQ. Our results demonstrated that up-regulated expression of LOX and EMT was developed in PQ-induced pulmonary fibrosis.2. To study the relationship between LOX and EMT, we established in vitro model in RLE-6TN and A549 cells after PQ poisoning. The s urvival rate of the RLE-6TN cells and A549 cells were decreased after incubation in PQ solutions for 24 h as PQ concentration increased. We artificially set the PQ concentration of 80 μmol/L that the RLE-6TN cells survival rate was 47.88% and 800 μmol/L where the A549 cells survival rate was 54.96%, as the intervention point in this experiment to build in vitro model.3. After PQ poisoning for 24 h, the epithelial monolayer was disrupted and cells acquired a spindle-shaped morphology. In the PQ group, LOX e xpression was enhanced, mesenchymal marker α-SMA and Vimentin were up-regulated, while the expression of epithelial marker E-cadherin and ZO-1 were down-regulated. The loss of epithelial phenotypes and the acquisition of mesenchymal traits through the process of EMT have been shown.4. The manifestation of lung injury and lung fibrosis were reduced by inactivating LOX with BAPN. LOX protein expression was significantly up-regulated and collagen deposition was enhanced in rats after PQ poisoning.5. Inhibition of LOX protein expression reduced pulmonary fibrosis and EMT induced by PQ in A549 and RLE-6TN cel s.6. Knockdown of LOX inhibited PQ-induced EMT in A549 cell.7. RLE-6TN and A549 cells simply treated with inhibitor BAPN had no obvious change in cell shape, but the mesenchymal markers α-SMA and Vimentin of EMT expression levels were decreased while epithelial markers ZO-1, E-cadherin expression levels were increased. It demonstrated that MET has occurred.In summary, our study demonstrated that LOX could promote the progress of EMT and inactivating LO X may alleviate EMT in PQ-induced pulmonary fibrosis. The relationship between LOX and EMT could potentially be a newly identified candidate therapeutic target for pulmonary fibrosis induced by PQ.