Because PQ is highly toxic pesticide, and caused acute lung injury and subsequently develop pulmonary fibrosis, eventually it lead to respiratory failure and death(14). The main pathological changes associated with lung injury caused by PQ poisoning was that, Intranasal or oropharyngeal of PQ en-routed to lung casused oxidative damage imposed onto the alveolar cells thereby triggering the lung pathophysiology along with prodigious deposition of collagen onto the extracellular matrix, release of inflammatory cytokines, fibroblast proliferation etc(15, 16). However, the exact mechanisms of PQ-induced lung fibrosis remain largely unclear and no specific drugs for this disease have been approved. Thus, innovative treatment strategies are required to prevent, treat and even reverse pulmonary fibrosis which lied to PQ poisoning .
Importantly, this is the first study to report the preclinical vitro model of paraquat-induced pulmonary fibrosis and explore its potential mechanism though bioinformatics analysis. Pulmonary fibrosis is characterized by fibroblast proliferation and the abnormal accumulation of extracellular matrix (ECM) molecules, particularly fibrillar collagens(17, 18). Lung fibrosis-induced fibroblasts and myfibroblasts secrete more ECM, primarily collagen types I and III(19, 20), so the content of collagen in lung tissues can directly reflect the degree of pulmonary fibrosis. The extent of collagen deposition is reflected by the amount of Hyp content(21), and collagen deposition in local tissues can reflect the severity of pathology by collagen staining. Thus,in our study, we detected that in the PQ group after 200 mg/kg PQ poisoning lasting for 48 h, the collagen I and α-SMA were significantly upregulated expression. In another words, under PQ concentrations of 200 mg/kg, MRC-5 were severely pulmonary fibrosis.Thus, we found that PQ exposure significantly upregulated collage I,collage III and SAM expression in lung cells. These data indicated that PQ injury lung tissue, which activated the TNF signaling by recruiting inflammatory factor onto the lung cells, inline with the previous study(22).
Previous studies have demonstrated that TNF-α causes significant damages to lung tissues(23, 24), and are important regulators of the cell proliferation(25–27). Christopher et al. found that TNF-R2 can also independently activate JNK (c-jun N-terminal protein kinase) and ERK (extratracellular signal 2 regulated protein kinase) in lung tissue, indicating that TNF-R2 not only has "ligand transmission" Function, can also independently transmit signals(28).Paraquat can activate inflammatory cells such as macrophages and neutrophils to secrete a large number of inflammatory factors, and then participate in the occurrence of pulmonary fibrosis. Previous studies have suggested the levels of tumor necrosis factor α (TNF-α), nuclear factor (NF-κB), interleukin 1β (IL-1β) and IL-6 in experimental rats with acute lung injury and bronchoalveolar lavage fluid induced by paraquat(29). Nine hub genes (JUNB,FOSL2,SOCS3,DUSP1,CEBPB,CEBPD,ATF3,CCL2 and CXCL2) were identified as having the highest scores in the PPI network. JUNB,which is strongly dependent on the AP1 factor,could regulate collagen type 1 and collagen type II in pulmonary fibrosis(30).FOSL2 and SOCS3,that inhibited activation of signal transducers and activators of transcription 3 (STAT3) to promote fibroblast-to–myofibroblast transition, collagen release and fibrosis in vitro and in vivo(31).DUSP1,dual-specificity protein phosphatase-1, which aggravated the expression levels of collagen I, collagen IV, and fibronectin(32). CEBPB and CEBPD could act on bleomycin-induced fibrosis(33). ATF3 (activating transcription factor 3), a member of the integrated stress response (ISR), negatively regulates transcription of the PINK1 gene. ATF3 in type II lung epithelial cells accelerate mice from PQ-induced lung fibrosis(34). CCL2(chemokine ligand 2),which produced by AECs, promote fibrosis through CCR2 activation. CCR2 signaling is critical for the initiation and progression of pulmonary fibrosis, in part, through recruitment of pro-fibrotic bone marrow derived monocytes(35, 36).Chemokine receptor type 2 (CXCR2), which is a chemokine, which is highly expressed in the lung, and exhibiting inflammatory and fibrotic effects(37). Therefore, TNF modulators had possibly potential as therapeutic targets in PQ-induced pulmonary fibrosis. The results of the present study revealed that the expression levels of JUNB,FOSL2,SOCS3,DUSP1,CEBPB,CEBPD,ATF3,CCL2 and CXCL2 were significantly higher in the lung fibrosis, and promoting pulmonary fibrosis(38).Thus, our findings may provide new insights into gene therapeuty in regulating the PQ-induced pulmonary toxicity.