DsbA-L activates TGF-β1/SMAD3 signaling and M2 macrophage polarization by stimulating AKT1 and NLRP3 to promote pulmonary fibrosis.

Pulmonary fibrosis (PF) is a progressive and difficult-to-heal lung disease that poses a significant threat to human life and health. This study aimed to investigate the potential pathological mechanisms of PF and to identify new avenues for the treatment of PF. Clinical samples were collected to assess the effect of disulfide-bond A oxidoreductase-like protein (DsbA-L) on PF. TGF-β1-induced MLE-12 cell model and bleomycin (BLM)-induced mice model were established. Changes in physiological morphology and fibrosis were observed in the lung tissues. The degree of apoptosis and the mitochondrial function was analyzed. The expression of relative cytokines was examined. The CD68+/CD206+ ratio was determined to indicate M2 macrophage polarization. The expression of DsbA-L was upregulated in patients with PF and PF-like models. In vitro, DsbA-L overexpression exacerbated TGF-β1-induced the deposition of extracellular matrix (ECM), apoptosis, inflammation, and mitochondrial damage, whereas DsbA-L silencing exerted the opposite effects. DsbA-L silencing inhibited the activation of AKT1, NLRP3, and SMAD3 by TGF-β1. MLE-12 cells silencing DsbA-L limited the polarization of RAW264.7 cells towards the M2 phenotype. AKT1 agonist or NLRP3 agonist reversed the role of DsbA-L silencing in inhibiting the TGF-β1/SMAD3 pathway and M2 macrophage polarization. In vivo, DsbA-L knockout protected mice from PF-like pathological damage caused by BLM. DsbA-L exhibited a significant profibrotic effect in lung epithelial cells and mice, which increased the levels of AKT1 and NLRP3 to activate the TGF-β1/SMAD3 pathway and M2 macrophage polarization. These findings could shed light on new clues for comprehension and treatment of PF.

Wang J ，Xia Z ，Qing B ，Chen Y ，Gu L ，Chen H ，Ge Z ，Yuan Y ... -  《-》

Sinomenine attenuates pulmonary fibrosis by downregulating TGF-β1/Smad3, PI3K/Akt and NF-κB signaling pathways.

Since COVID-19 became a global epidemic disease in 2019, pulmonary fibrosis (PF) has become more prevalent among persons with severe infections, with IPF being the most prevalent form. In traditional Chinese medicine, various disorders are treated using Sinomenine (SIN). The SIN's strategy for PF defense is unclear. Bleomycin (BLM) was used to induce PF, after which inflammatory factors, lung histological alterations, and the TGF-/Smad signaling pathway were assessed. By administering various dosages of SIN and the TGF- receptor inhibitor SB-431,542 to human embryonic lung fibroblasts (HFL-1) and A549 cells, we were able to examine proliferation and migration as well as the signaling molecules implicated in Epithelial-Mesenchymal Transition (EMT) and Extra-Cellular Matrix (ECM). In vivo, SIN reduced the pathological changes in the lung tissue induced by BLM, reduced the abnormal expression of inflammatory cytokines, and improved the weight and survival rate of mice. In vitro, SIN inhibited the migration and proliferation by inhibiting TGF-β1/Smad3, PI3K/Akt, and NF-κB pathways, prevented the myofibroblasts (FMT) of HFL-1, reversed the EMT of A549 cells, restored the balance of matrix metalloenzymes, and reduced the expression of ECM proteins. SIN attenuated PF by down-regulating TGF-β/Smad3, PI3K/Akt, and NF-κB signaling pathways, being a potential effective drug in the treatment of PF.

Yao F ，Xu M ，Dong L ，Shen X ，Shen Y ，Jiang Y ，Zhu T ，Zhang C ，Yu G ... -  《BMC Pulmonary Medicine》

Pirfenidone modulates macrophage polarization and ameliorates radiation-induced lung fibrosis by inhibiting the TGF-β1/Smad3 pathway.

Radiation-induced lung injury (RILI) mainly contributes to the complications of thoracic radiotherapy. RILI can be divided into radiation pneumonia (RP) and radiation-induced lung fibrosis (RILF). Once RILF occurs, patients will eventually develop irreversible respiratory failure; thus, a new treatment strategy to prevent RILI is urgently needed. This study explored the therapeutic effect of pirfenidone (PFD), a Food and Drug Administration (FDA)-approved drug for (IPF) treatment, and its mechanism in the treatment of RILF. In vivo, C57BL/6 mice received a 50 Gy dose of X-ray radiation to the whole thorax with or without the administration of PFD. Collagen deposition and fibrosis in the lung were reversed by PFD treatment, which was associated with reduced M2 macrophage infiltration and inhibition of the transforming growth factor-β1 (TGF-β1)/Drosophila mothers against the decapentaplegic 3 (Smad3) signalling pathway. Moreover, PFD treatment decreased the radiation-induced expression of TGF-β1 and phosphorylation of Smad3 in alveolar epithelial cells (AECs) and vascular endothelial cells (VECs). Furthermore, IL-4-induced M2 macrophage polarization and IL-13-induced M2 macrophage polarization were suppressed by PFD treatment in vitro, resulting in reductions in the release of arginase-1 (ARG-1), chitinase 3-like 3 (YM-1) and TGF-β1. Notably, the PFD-induced inhibitory effects on M2 macrophage polarization were associated with downregulation of nuclear factor kappa-B (NF-κB) p50 activity. Additionally, PFD could significantly inhibit ionizing radiation-induced chemokine secretion in MLE-12 cells and consequently impair the migration of RAW264.7 cells. PFD could also eliminate TGF-β1 from M2 macrophages by attenuating the activation of TGF-β1/Smad3. In conclusion, PFD is a potential therapeutic agent to ameliorate fibrosis in RILF by reducing M2 macrophage infiltration and inhibiting the activation of TGF-β1/Smad3.

Ying H ，Fang M ，Hang QQ ，Chen Y ，Qian X ，Chen M ... -  《-》

USP7 Promotes TGF-β1 Signaling by De-Ubiquitinating Smad2/Smad3 in Pulmonary Fibrosis.

Tang F ，Gong H ，Ke T ，Yang W ，Yang Y ，Liu Z ... -  《-》

PGRN knockdown alleviates pulmonary fibrosis regulating the Akt/GSK3β signaling pathway.

Pulmonary fibrosis (PF) is a serious, chronic, and progressive disease with increased collagen deposition and the collapse of lung structures. Currently, the antifibrotic drugs for PF treatment, nintedanib and pirfenidone, have been proven to reduce the decline of pulmonary function in PF, but both have side effects, and to date, there is no significantly effective treatment to halt the progression of PF. The aim of this study was to investigate the molecular mechanism of pregranuloprotein (PGRN) in pulmonary fibrosis through in vitro and in vivo experiments. PF models was induced in animals using bleomycin (BLM) and treated MRC-5 cells with TGF-β1. The mRNA expression of PGRN in fasting peripheral blood samples was measured via RT-qPCR and ELSA. PGRN siRNAs were synthesized and transfected into MRC-5 cells. MAZ51, an activator of the Akt/GSK3β pathway, was applied in recovery experiment. The proliferation and apoptosis of MRC-5 cells were determined using the CCK8 kit, MTT kit, and Muse® Cell Analyzer. H&E and Masson staining were applied to evaluate the inflammatory and fibrosis in mouse lung tissue. Levels of PGRN, inflammatory factors (IL-6 and IL-1β), fibrosis markers (α-SMA, COL-I and COL-III), and Akt/GSK3β pathway-related proteins (AKT, GSK-3β and β-catenin) were determined in tissues or cells by ELISA, RT-qPCR, western blot, or Immunofluorescence. PGRN mRNA expression was elevated in the plasma of PF patients. In TGF-β1 induced MRC-5 cells, PGRN knockdown reduced the levels of IL-6, IL-1β, α-SMA, COL-I and COL-III, and suppressed the phosphorylation of AKT and GSK-β. Treatment with MAZ51 partially reversed the effect of PGRN knockdown on TGF-β1-induced PF. Moreover, PGRN knockdown mitigated BLM-induced alveolar destruction and wall thickening, inflammatory cell infiltration, and collagen deposition in mice. It also reduced the expression of α-SMA, TGF-β1, COL-I, COL-III, β-catenin, and the phosphorylation of AKT and GSK-3β in BLM-treated mice. PGRN knockdown alleviates PF in vitro and in vivo by modulating the Akt/GSK3β signaling pathway, proposing that PGRN could serve as a potential therapy or adjuvant therapy for lung fibrosis.

Xie T ，Zheng Y ，Zhang L ，Zhao J ，Wu H ，Li Y ... -  《-》