Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription activator that regulates the expression of target genes by binding antioxidant response elements (AREs) . NRF2 is important for the coordinated upregulation of genes in response to oxidative stress. Silica causes the significant accumulation of ROS and activates the antioxidative protein NRF2 and its downstream proteins in the early exposure to silica . Immune cells with anti-inflammatory properties, especially monocytes/macrophages are involved in the possibility of alternative mechanisms of silicosis fibrosis [38–40]. The present study focused on the lymphocyte oxidative state and found that the number of monocytes and the expression of NRF2 and NRF2-dependent antioxidative genes was significantly increased in PBMCs from patients with silicosis. Additionally, it was confirmed that respiratory function was significantly decreased in the patients with silicosis.
The respiratory dysfunction in patients with silicosis is closely related to the severity of pulmonary fibrosis. The TGF-β pathway can play an important direct inducer role in the process of collagen transcription in the development of silicosis , as TGF-βs are overexpressed in fibrosis . TGF-β signaling plays a key role in extracellular matrix remodeling, the epithelial-mesenchymal transition, and cell growth, migration, differentiation in fibrosis . Generally, TGF-β signaling is modulated by the phosphorylation of the cytoplasmic SMAD signaling molecules, which results in their translocation to the nucleus . TGF-β1 signaling molecules play a key role by promoting transdifferentiation the fibroblast into myofibroblasts, which promote collagen synthesis and ECM deposition in the pathology of silicosis .
The ECM, complex mixture of structural and functional macromolecules, has an important role in tissue fibrosis and in the maintenance of cell and tissue structure and function . Integrins and other transmembrane molecules mediate specific interactions between cells and the ECM [47, 48]. These interactions have the direct and indirect effects of cellular activities, lead to adhesion, proliferation, apoptosis, migration, and differentiation. In addition, integrins act as 'mechanoreceptors' that they would provide a specific physical link between the cytoskeleton and the ECM.
In the present study, the differential expression of mRNAs in the progression of silicosis was identified using RNA-Seq analysis. A total of 1158 dysregulated mRNAs were identified in PBMCs from patients with silicosis, including 475 upregulated and 683 downregulated mRNAs. GO analysis revealed that the functions of dysregulated mRNAs in PBMCs from patients with silicosis were related to the ECM, catalytic activity, oxidoreductase activity, transcription factor activity, metabolic processes, immune system processes, response to stimulus transcription, and biological adhesion. In KEGG pathway analysis, the dysregulated mRNAs were involved in regulating immune-related pathways, regulating metabolic processes, cellular community, cell adhesion molecules signaling pathway and the phagosome signaling pathway. Therefore, it is possible that the dysregulated mRNAs in these processes are involved in the pathogenesis of silicosis.
To confirm the results obtained by RNA-Seq analysis, seven differentially expressed mRNAs were selected to verify their expression in PBMCs from patients with silicosis using RT-qPCR. The results indicated that the expression of SMAD2, MAPK3, THBS1, SMAD3, ITGB3, and BMP4 was increased, while the expression of CD44 was significantly decreased in PBMCs from patients with silicosis compared to that of the control group, indicating consistent results with the RNA-Seq data. The TGF-β family of cytokines signals through receptor serine/threonine kinases to control cell behavior and fate. These signals are propagated through the transcription factors SMAD2 and SMAD3 downstream of TGFβ. SMAD2 and SMAD3 belong to the SMAD protein family that mediate multiple signaling pathways as transcriptional modulators . These findings indicate that dysregulation of the TGFβ1-SMAD signaling pathway may play an important role in the pathological process of silicosis, and are consistent with findings in animal experiments [51, 52]. SMAD proteins mediate signaling of TGF-β through its interaction with the SMAD anchor for receptor activation (SARA) protein. In response to a TGF-β signal, SMAD proteins are phosphorylated by TGF-β receptors. This study revealed that BMP4 was a differential gene hub in PBMCs from patients with silicosis. BMP4 encodes a secreted ligand of the TGF-β superfamily of proteins and activates SMAD family transcription factors that regulate gene expression. These results showed that SMAD2 and SMAD3 had different trends in the two groups. Recently, SMAD2 and SMAD3 have shown different roles in the TGF-β signaling pathway during embryonic development . The cell experiments revealed that BMP7 is associated with inhibiting silica-induced fibrosis through activated BMP7/SMAD and suppressed TGF-β/SMAD pathways [56, 57]. To date, BMP4 in the BMP protein family has not been reported in silicosis.
MAPK3 is especially involved in activation towards microtubule-associated protein-2 and the control of cell survival, proliferation and differentiation . In the present study, MAPK3 (ERK1) was significantly increased in PBMCs from patients with silicosis compared to that in the control group. Dysregulation of MAPK3 plays a significant role in the pathological processes of silicosis . Crystal compounds in silicosis activate ROS, which activate the inflammasome through MAPK3[59, 60]. In agreement with these findings, activation of MAPK3 (ERK1) and NF-κB in PBMCs is reported during oxidative stress .
The transcription factor thrombospondin-1 (THBS1) is an adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. THBS1 binds to cell surface receptors, including fibrinogen, fibronectin, laminin, type V collagen and integrins, such as ITGB3 (Calzada and Roberts, 2005). These studies suggest that THBS1 in human peripheral blood lymphocytes is involved in the regulation of pesticide-induced immune dysfunction .
The protein encoded by CD44 is a cell-surface glycoprotein involved in cell-cell interactions, cell migration and adhesion . It also interacts with other ligands, such as matrix metalloproteinases, and collagens . CD44 blockade alleviates silica-induced fibrosis and improves pulmonary function in vivo .
Nonetheless, the present study remains unclear that changes specific to different cell types present in PBMCs. Additionally, the effects of oxidative stress on the central signaling pathway, such as insulin signaling or glucose transport in silicosis patient should be addressed in subsequent investigations.