Proteomics Analysis Revealed the Importance of Inflammation-Mediated Downstream Pathways and the Protective Role of Curcumin in Bleomycin-Induced Pulmonary Fibrosis in C57BL/6 Mice

Abstract

Bleomycin (BLM)-induced pulmonary fibrosis is characterized by inflammation in the alveoli, subsequent deposition of extracellular matrix (ECM) and myofibroblasts, and an impaired fibrinolytic system. Here, we describe major hematological changes, the IL-17A-mediated p53-fibrinolytic pathway, and the high throughput hits of liquid chromatography−mass spectrometry (LC−MS) analysis during the progression of pulmonary fibrosis and the therapeutic potential of curcumin against disease progression. C57BL/6 mice were exposed to BLM, followed by curcumin intervention after 24 and 48 h. Mice were sacrificed after 7 days to validate the hematological parameters, molecular pathways, and proteomics. Various techniques such as western blotting, immunofluorescence, reverse transcriptase polymerase chain reaction (RT-PCR), hematoxylin and eosin staining, Masson's trichrome staining, and immunohistochemistry were used to validate the proposed theory. LC−MS analysis was performed using a Q-Orbitrap mass spectrometer. The Schrodinger approach was used to perform the in silico molecular docking studies. BLM-exposed mice exhibited gradual weight loss and altered lung morphology; however, these were reversed by curcumin treatment. Significant changes in the hematological parameters confirmed the severity of BLM exposure in the mice, and expression of IL-17A-mediated p53-fibrinolytic system components and alveolar epithelial cell (AEC) apoptosis further confirmed the pathophysiology of pulmonary fibrosis. Differentially expressed proteins were characterized and mapped using the proteomics approach. A strong interaction of curcumin is observed with p53, uPA, and PAI-I proteins. The key role of IL-17A-mediated inflammation in the impairment of the p53-fibrinolytic system and AEC apoptosis was confirmed during BLM-induced pulmonary fibrosis. Therapeutic efficacy of curcumin exhibited a protective role against the progression of pulmonary fibrosis, which promises potent therapeutic modality to target the IL-17A-mediated p53-fibrinolytic system during pulmonary fibrosis.

A comprehensive network map of IL-17A signaling pathway

“…The BLM stimulates the expression of of IL‐17A, induces the phosphorylation of tumor suppressor protein (Tp53), Mapk1/3, and upregulation of mechanistic target of rapamycin ( Mtor ), plasminogen activator, urokinase ( Plau ), PLAU receptor ( Plaur ), serpin family E member 1 ( Serpine 1), smooth muscle aortic alpha‐actin 2 ( Acta 2), cleaved cysteine‐aspartic acid protease 3 (cleaved Casp 3), marker of proliferation Ki‐67 ( MKI 67), and proteins including annexin A6 (Anxa6), proteasome 26S Subunit, ATPase 6 (Psmc6), alcohol dehydrogenase (Adh1), copine 1 (Cpne1), hemoglobin subunit beta‐H1 (Hbb‐bh1), UDP‐Glucose glycoprotein glucosyltransferase 1 (Uggt1), growth associated protein 43 (Gap43), superoxide dismutase 2 (Sod2), heat shock protein family 1B (Hspa1b), protein phosphatase 1 regulatory inhibitor subunit 14B (Ppp1r14b), bleomycin hydrolase (Blmh), antioxidant 1 copper chaperone (Atox1), hemoglobin subunit‐beta‐2 (Hbb‐b2), Ras‐related protein Rab‐10 (Rab10) and downregulation of Hbb‐b1, transcription factor p65 (Rela), neutrophilic granule protein (Ngp), paraoxonase 3 (Pon3), high mobility group box protein 1 (Hmgb1), and hypoxia up‐Regulated 1 (Hyou1), which are involved in response to a stimulus; increased expression of inflammation‐related proteins such as Acta2, Ras‐related protein R‐Ras2 (Rras2), actin related protein 2/3 complex subunit 2 (Arpc2), G Protein subunit gamma 12 (Gng12), Ras homolog family member A (RhoA), phospholipase C eta 1 (Plch1), signal transducer and activator of transcription 1 (Stat1), Rac family small GTPase 1 (Rac1), Arpc3, P21 (RAC1) activated kinase 3 (Pak3), myosin light chain kinase (Mylk), cell division control protein 42 homolog (Cdc42), Arpc5, protein kinase CAMP‐activated catalytic subunit beta (Prkacb) and downregulation of Rela and complement component C7 (C7) (Gouda et al 2020).…”

“…In recent years, IL-17A research has led to the discovery of its potential as a therapeutic target for various forms of inflammation and autoimmune disorders (Kuwabara et al 2017;McGeachy et al 2019). In the current study, we developed a resource of signaling events mediated by IL-17A/ IL-17RA signaling by excerpting all research articles from the literature and compiling them in similar lines to earlier reports on comprehensive signaling maps including IL-33, IL-18 and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), C-C motif chemokine ligand 18 (CCL18) (Pinto et al 2018;Rex et al 2020Rex et al , 2021Aravind et al 2022).…”

“…The lung is one of the most susceptible organ and it is prone to be affected by regular immune reactions as it encounters various pathogens and promotes various immune cells, tumour necrosis factor-α (TNF-α), chemokines, cytokines, and interleukins including IL-17A (Gouda and Bhandary 2018;Gouda et al 2018a). Recent studies show that the role of IL-17A is not only limited to promoting inflammation, but also plays a key mediator for various downstream pathways such as Akt, MAPKs, transforming growth factor beta (TGF-β), p53-fibrinolytic system, suppressor of mothers against decapentaplegic (Smad), and signal transducer and activator of transcription 3 (Stat3) signaling pathways (Gouda et al 2018b(Gouda et al , 2020. Besides, dysregulated IL-17A downstream signaling mechanism could result in severe tissue damage, followed by cell death and subsequent development of tissue fibrosis (Gouda et al 2020).…”

“…Recent studies show that the role of IL-17A is not only limited to promoting inflammation, but also plays a key mediator for various downstream pathways such as Akt, MAPKs, transforming growth factor beta (TGF-β), p53-fibrinolytic system, suppressor of mothers against decapentaplegic (Smad), and signal transducer and activator of transcription 3 (Stat3) signaling pathways (Gouda et al 2018b(Gouda et al , 2020. Besides, dysregulated IL-17A downstream signaling mechanism could result in severe tissue damage, followed by cell death and subsequent development of tissue fibrosis (Gouda et al 2020). IL-17A role in tumorigenesis is expressed by myeloid-derived suppressor cells (MDSCs) recruitment to regulate anti-tumour immunity, and IL-17A also could enhance tumour growth by promoting IL-6 resulting in the activation of transcription factor STAT3 and overexpressed pro-angiogenic genes in tumour (Wang et al 2009;He et al 2010;Chang et al 2014).…”

Protective effects of Qing-Re-Huo-Xue formula on bleomycin-induced pulmonary fibrosis through the p53/IGFBP3 pathway

“…The study by Wu Q et al suggests that p53 may be an important gene in the occurrence and progression of PF by regulating EMT, senescence, apoptosis, and other cellular processes [ 48 ]. Some researchers believe that the expression of p53 is upregulated in IPF [ 49 , 50 ]. However, Wang m et al found that the p53 protein expression was significantly downregulated in BLM-induced PF mice and could be upregulated by astaxanthin intervention [ 51 ].…”