Recent Advances (2015-2020) in Drug Discovery for Attenuation of Pulmonary Fibrosis and COPD.

A condition of scarring of lung tissue due to a wide range of causes (such as environmental pollution, cigarette smoking (CS), lung diseases, some medications, etc.) has been reported as pulmonary fibrosis (PF). This has become a serious problem all over the world due to the lack of efficient drugs for treatment or cure. To date, no drug has been designed that could inhibit fibrosis. However, few medications have been reported to reduce the rate of fibrosis. Meanwhile, ongoing research indicates pulmonary fibrosis can be treated in its initial stages when symptoms are mild. Here, an attempt is made to summarize the recent studies on the effects of various chemical drugs that attenuate PF and increase patients' quality of life. The review is classified based on the nature of the drug molecules, e.g., natural/biomolecule-based, synthetic-molecule-based PF inhibitors, etc. Here, the mechanisms through which the drug molecules attenuate PF are discussed. It is shown that inhibitory molecules can significantly decrease the TGF-ß1, profibrotic factors, proteins responsible for inflammation, pro-fibrogenic cytokines, etc., thereby ameliorating the progress of PF. This review may be useful in designing better drugs that could reduce the fibrosis process drastically or even cure the disease to some extent.

Epithelial Responses in Radiation-Induced Lung Injury (RILI) Allow Chronic Inflammation and Fibrogenesis.

Radiation models, such as whole thorax lung irradiation (WTLI) or partial-body irradiation (PBI) with bone-marrow sparing, have shown that affected lung tissue displays a continual progression of injury, often for months after the initial insult. Undoubtably, a variety of resident and infiltrating cell types either contribute to or fail to resolve this type of progressive injury, which in lung tissue, often develops into lethal and irreversible radiation-induced pulmonary fibrosis (RIPF), indicating a failure of the lung to return to a homeostatic state. Resident pulmonary epithelium, which are present at the time of irradiation and persist long after the initial insult, play a key role in the maintenance of homeostatic conditions in the lung and have often been described as contributing to the progression of radiation-induced lung injury (RILI). In this study, we took an unbiased approach through RNA sequencing to determine the in vivo response of the lung epithelium in the progression of RIPF. In our methodology, we isolated CD326+ epithelium from the lungs of 12.5 Gy WTLI C57BL/6J female mice (aged 8-10 weeks and sacrificed at regular intervals) and compared irradiated and non-irradiated CD326+ cells and whole lung tissue. We subsequently verified our findings by qPCR and immunohistochemistry. Transcripts associated with epithelial regulation of immune responses and fibroblast activation were significantly reduced in irradiated animals at 4 weeks postirradiation. Additionally, alveolar type-2 epithelial cells (AEC2) appeared to be significantly reduced in number at 4 weeks and thereafter based on the diminished expression of pro-surfactant protein C (pro-SPC). This change is associated with a reduction of Cd200 and cyclooxygenase 2 (COX2), which are expressed within the CD326 populations of cells and function to suppress macrophage and fibroblast activation under steady-state conditions, respectively. These data indicate that either preventing epithelial cell loss that occurs after irradiation or replacing important mediators of immune and fibroblast activity produced by the epithelium are potentially important strategies for preventing or treating this unique injury.

Activation of the NLRP3 inflammasome by HMGB1 through inhibition of the Nrf2/HO-1 pathway promotes bleomycin-induced pulmonary fibrosis after acute lung injury in rats

Objective: Acute lung injury (ALI) is a common complication of critical diseases with high morbidity and mortality. This study explored the regulatory role and mechanism of high mobility histone box 1 protein (HMGB1) on pulmonary fibrosis (PF) after ALI in rats through nucleotide oligomerization domain-like receptor protein-3 (NLRP3) inflammasome. Methods: PF rat models after ALI were established by induction of bleomycin. Degree of fibrosis was assessed by Masson staining and Ashcroft scoring. Hydroxyproline (Hyp) contents in lung tissues and rat lung tissue morphology were detected by enzyme-linked-immunosorbent serologic assay (ELISA) and hematoxylin and eosin staining. The levels of NLRP3, major proteins of NLRP3 inflammasome (NLRP3/ASC/caspase-1), and downstream inflammatory cytokines interleukin (IL)-1 and IL-18 were determined using immunohistochemistry, Western blotting analysis, and ELISA. The nuclear/cytoplasmic nuclear factor erythroid 2-related factor 2 (Nrf2) levels and HO-1 levels were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting analysis. Rats was injected with lentivirus carrying short hairpin (sh)-HMGB1 and zinc protoporphyria (ZNPP) (HO-1 inhibitor) to assess the effects of HMGB1 and HO-1 on PF and NLRP3 inflammasome activation. Results: Bleomycin induced PF after ALI in rats, manifested as patchy fibrosis, atelectasis, and excessive expansion, and increased Aschcroft score and Hyp content. Bleomycin treatment enhanced levels of NLRP3, ASC, caspase-1, IL-1, and IL-18 in rat lung tissues, which promoted activation of NLRP3 inflammasome. HMGB1 was up-regulated in bleomycin-induced rats. HMGB1 knockdown partially reversed NLRP3 inflammasome activation and PF progression (AU)

The alcohol extracts of Sceptridium ternatum (Thunb.) Lyon exert anti-pulmonary fibrosis effect through targeting SETDB1/STAT3/p-STAT3 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Pulmonary fibrosis (PF) is a pathological process of irreversible scarring of lung tissues, with limited treatment means. Sceptridium ternatum (Thunb.) Lyon (STE) is a traditional Chinese herbal medicine that has a traditional use in relieving cough and asthma, resolving phlegm, clearing heat, and detoxicating in China. However, its role in PF has not been reported. AIM OF THE STUDY: This study aims to investigate the protective role of STE in PF and the underlying mechanisms. MATERIALS AND METHODS: Sprague-Dawley (SD) rats were divided into control group, PF model group, positive drug (pirfenidone) group and STE group. After 28 days of STE administration in bleomycin (BLM)-induced PF rats, living Nuclear Magnetic Resonance Imaging (NMRI) was used to observe the structural changes of lung tissues. H&E and Masson's trichrome staining were used to observe PF-associated pathological alteration, and immunohistochemistry (IHC) staining, western blotting, and qRT-PCR were used to detect the expression of PF-related marker proteins in the lung tissues. ELISA was used to detect PF-associated biochemical criteria in the lung tissue homogenates. The proteomics technology was used to screen the different proteins. Co-immunoprecipitation, western blotting, and IHC staining were used to confirm the underlying targets of STE as well as its downstream signaling. UPLC-Triple-TOF/MS assay was used to explore the effective components in the alcohol extracts of STE. Autodock vina was used to detect the potential binding between the above effective components and SETDB1. RESULTS: STE prevented PF by inhibiting the activation of lung fibroblasts and ECM deposition in BLM-induced PF rats. Mechanism analyses demonstrated that STE could inhibit the up-regulation of SETDB1 induced by BLM and TGF-ß1, which further blocked the binding of SETDB1 and STAT3 as well as the phosphorylation of STAT3, ultimately preventing the activation and proliferation of lung fibroblasts. CONCLUSION: STE played a preventive role in PF by targeting the SETBD1/STAT3/p-STAT3 pathway, which may be a potential therapeutic agent for PF.

Reversal of Pulmonary Fibrosis: Human Umbilical Mesenchymal Stem Cells from Wharton's Jelly versus Human-Adipose-Derived Mesenchymal Stem Cells.

Pulmonary fibrosis (PF) is a progressive, non-reversible illness with various etiologies. Currently, effective treatments for fibrotic lungs are still lacking. Here, we compared the effectiveness of transplantation of human mesenchymal stem cells from umbilical cord Wharton's jelly (HUMSCs) versus those from adipose tissue (ADMSCs) in reversing pulmonary fibrosis in rats. Bleomycin 5 mg was intratracheally injected to establish a severe, stable, single left lung animal model with PF. On Day 21 post-BLM administration, one single transplantation of 2.5 × 107 HUMSCs or ADMSCs was performed. Lung function examination of Injury and Injury+ADMSCs rats displayed significantly decreased blood oxygen saturation and increased respiratory rates, while Injury+HUMSCs rats showed statistical amelioration in blood oxygen saturation and significant alleviation in respiratory rates. Reduced cell number in the bronchoalveolar lavage and lower myofibroblast activation appeared in the rats transplanted with either ADMSCs or HUMSCS than that in the Injury group. However, ADMSC transplantation stimulated more adipogenesis. Furthermore, matrix-metallopeptidase-9 over-expression for collagen degradation, and the elevation of Toll-like receptor-4 expression for alveolar regeneration were observed only in the Injury+HUMSCs. In comparison with the transplantation of ADMSCs, transplantation of HUMSCs exhibited a much more effective therapeutic effect on PF, with significantly better results in alveolar volume and lung function.

Neurotrophins: Expression of Brain-Lung Axis Development.

Neurotrophins (NTs) are a group of soluble growth factors with analogous structures and functions, identified initially as critical mediators of neuronal survival during development. Recently, the relevance of NTs has been confirmed by emerging clinical data showing that impaired NTs levels and functions are involved in the onset of neurological and pulmonary diseases. The alteration in NTs expression at the central and peripheral nervous system has been linked to neurodevelopmental disorders with an early onset and severe clinical manifestations, often named "synaptopathies" because of structural and functional synaptic plasticity abnormalities. NTs appear to be also involved in the physiology and pathophysiology of several airway diseases, neonatal lung diseases, allergic and inflammatory diseases, lung fibrosis, and even lung cancer. Moreover, they have also been detected in other peripheral tissues, including immune cells, epithelium, smooth muscle, fibroblasts, and vascular endothelium. This review aims to provide a comprehensive description of the NTs as important physiological and pathophysiological players in brain and lung development.

G protein coupled receptor 41 regulates fibroblast activation in pulmonary fibrosis via Gαi/o and downstream Smad2/3 and ERK1/2 phosphorylation.

Pulmonary fibrosis is a progressive and fatal fibrotic lung disease with mysterious pathogenesis and limited effective therapies. G protein-coupled receptors (GPRs) participate in a variety of physiologic functions, and several GPRs have critical fibrosis-promoting or -inhibiting roles in pulmonary fibrosis. Here, we explored the role of GPR41 in the pathobiology of pulmonary fibrosis. We found that GPR41 expression was elevated in lung tissues of mice with bleomycin-induced pulmonary fibrosis and lung fibroblasts treated with transforming growth factor-ß1 (TGF-ß1). Knockout of GPR41 attenuated pulmonary fibrosis in mice, as evidenced by improved lung morphology, decreased lung weight and collagen secretion, and down-regulated α-SMA, collagen type I alpha and fibronectin expression in lungs. Additionally, GPR41 knockout inhibited the differentiation of fibroblasts to myofibroblasts, and decreased myofibroblast migration. By further mechanistic analysis, we demonstrated that GPR41 regulated TGF-ß1-induced fibroblast-to-myofibroblast differentiation and Smad2/3 and ERK1/2 phosphorylation via its Gαi/o subunit but not Gßγ subunit. Together, our data indicate that GPR41 is involved in pulmonary fibroblast activation and fibrosis, and GPR41 represents a potential therapeutic target for pulmonary fibrosis.

Single-cell sequencing analysis fibrosis provides insights into the pathobiological cell types and cytokines of radiation-induced pulmonary fibrosis.

BACKGROUND: Radiotherapy is an essential treatment for chest cancer. Radiation-induced pulmonary fibrosis (RIPF) is an almost irreversible interstitial lung disease; however, its pathogenesis remains unclear. METHODS: We analyzed specific changes in cell populations and potential markers by using single-cell sequencing datasets from the Sequence Read Archive database, PERFORMED from control (0 Gy) and thoracic irradiated (20 Gy) mouse lungs at day 150 post-radiation. We performed IHC and ELISA on lung tissue and cells to validate the potential marker cytokines identified by the analysis on rat thoracic irradiated molds (30 Gy). RESULTS: Single-cell sequencing analysis showed changes in abundance across cell types and at the single-cell level, with B and T cells showing the most significant changes in abundance. And four cytokines, CCL5, ICAM1, PF4, and TNF, were significantly upregulated in lung tissues of RIPF rats and cell supernatants after ionizing radiation. CONCLUSION: Cytokines CCL5, ICAM1, PF4, and TNF may play essential roles in radiation pulmonary fibrosis. They are potential targets for the treatment of radiation pulmonary fibrosis.

Elevated expression of macrophage MERTK exhibits profibrotic effects and results in defective regulation of efferocytosis function in pulmonary fibrosis.

Increased apoptosis of alveolar epithelial cells is a prominent feature of pulmonary fibrosis. Macrophage efferocytosis, phagocytosis of apoptotic cells by macrophages, is crucial for maintaining tissue homeostasis. Expression of Mer tyrosine kinase (MERTK, an important recognition receptor in efferocytosis) in macrophages is thought to be associated with fibrosis. However, how macrophage MERTK affects pulmonary fibrosis and whether it depends on efferocytosis are not yet clear. Here, we found elevated MERTK expression in lung macrophages from IPF patients and mice with bleomycin-induced pulmonary fibrosis. In vitro experiments showed that macrophages overexpressing MERTK exhibit profibrotic effects and that macrophage efferocytosis abrogates the profibrotic effect of MERTK by downregulating MERTK, forming a negative regulatory loop. In pulmonary fibrosis, this negative regulation is defective, and MERTK mainly exhibits profibrotic effects. Our study reveals a previously unsuspected profibrotic effect of elevated macrophage MERTK in pulmonary fibrosis and defective regulation of efferocytosis function as a result of that elevation, suggesting that targeting MERTK in macrophages may help to attenuate pulmonary fibrosis.

Qilongtian ameliorate bleomycin-induced pulmonary fibrosis in mice via inhibiting IL-17 signal pathway.

Pulmonary fibrosis (PF) is a special type of pulmonary parenchymal disease, with chronic, progressive, fibrosis, and high mortality. There is a lack of safe, effective, and affordable treatment methods. Qilongtian (QLT) is a traditional Chinese prescription that is composed of Panax notoginseng, Earthworm, and Rhodiola, and shows the remarkable clinical curative effect of PF. However, the mechanism of QLT remains to be clarified. Therefore, we studied the effectivity of QLT in treating Bleomycin (BLM) induced PF mice. 36 C57BL/6 J mice were randomized into the control group, the model group, the low-, medium- and high-dose QLT group, and Pirfenidone group. After establishing a model of pulmonary fibrosis in mice, the control and model groups were infused with a normal saline solution, and the delivery group was infused with QLT. Pulmonary function in the mice from each group was detected. Pulmonary tissue morphologies and collagen deposition were stained by HE and Masson. The content of hydroxyproline (HYP) was detected by alkaline hydrolysis and the mRNA and protein expression of related genes in pulmonary tissues were detected by using q-PCR, ELISA, and Western blot. Our studies have shown that QLT significantly reduced the inflammatory injury, hydroxy-proline content, and collagen deposition of pulmonary tissue in BLM-induced PF mice and down-regulated the cytokine related to inflammation and fibrosis and PF expression on the mRNA and protein level in PF mice. To identify the mechanism of QLT, the Transcriptome was measured and the IL-17 signal pathway was screened out for further research. Further studies indicated that QLT reduced the mRNAs and protein levels of interleukin 17 (IL-17), c-c motif chemokine ligand 12 (CCL12), c-x-c motif chemokine ligand 5 (CXCL5), fos-like antigen 1 (FOSL1), matrix metalloproteinase-9 (MMP9), and amphiregulin (AREG), which are inflammation and fibrosis-related genes in the IL-17 signal pathway. The results indicated that the potential mechanism for QLT in the prevention of PF progression was by inhibiting inflammation resulting in the IL-17 signal pathway. Our study provides the novel scientific basis of QLT and represents new therapeutics for PF in clinical.

Derrone Targeting the TGF Type 1 Receptor Kinase Improves Bleomycin-Mediated Pulmonary Fibrosis through Inhibition of Smad Signaling Pathway.

Transforming growth factor-ß (TGF-ß) has a strong impact on the pathogenesis of pulmonary fibrosis. Therefore, in this study, we investigated whether derrone promotes anti-fibrotic effects on TGF-ß1-stimulated MRC-5 lung fibroblast cells and bleomycin-induced lung fibrosis. Long-term treatment with high concentrations of derrone increased the cytotoxicity of MRC-5 cells; however, substantial cell death was not observed at low concentrations of derrone (below 0.05 µg/mL) during a three-day treatment. In addition, derrone significantly decreased the expressions of TGF-ß1, fibronectin, elastin, and collagen1α1, and these decreases were accompanied by downregulation of α-SMA expression in TGF-ß1-stimulated MRC-5 cells. Severe fibrotic histopathological changes in infiltration, alveolar congestion, and alveolar wall thickness were observed in bleomycin-treated mice; however, derrone supplementation significantly reduced these histological deformations. In addition, intratracheal administration of bleomycin resulted in lung collagen accumulation and high expression of α-SMA and fibrotic genes-including TGF-ß1, fibronectin, elastin, and collagen1α1-in the lungs. However, fibrotic severity in intranasal derrone-administrated mice was significantly less than that of bleomycin-administered mice. Molecular docking predicted that derrone potently fits into the ATP-binding pocket of the TGF-ß receptor type 1 kinase domain with stronger binding scores than ATP. Additionally, derrone inhibited TGF-ß1-induced phosphorylation and nuclear translocations of Smad2/3. Overall, derrone significantly attenuated TGF-ß1-stimulated lung inflammation in vitro and bleomycin-induced lung fibrosis in a murine model, indicating that derrone may be a promising candidate for preventing pulmonary fibrosis.

Identification of a broadly fibrogenic macrophage subset induced by type 3 inflammation.

Macrophages are central orchestrators of the tissue response to injury, with distinct macrophage activation states playing key roles in fibrosis progression and resolution. Identifying key macrophage populations found in human fibrotic tissues could lead to new treatments for fibrosis. Here, we used human liver and lung single-cell RNA sequencing datasets to identify a subset of CD9+TREM2+ macrophages that express SPP1, GPNMB, FABP5, and CD63. In both human and murine hepatic and pulmonary fibrosis, these macrophages were enriched at the outside edges of scarring and adjacent to activated mesenchymal cells. Neutrophils expressing MMP9, which participates in the activation of TGF-ß1, and the type 3 cytokines GM-CSF and IL-17A coclustered with these macrophages. In vitro, GM-CSF, IL-17A, and TGF-ß1 drive the differentiation of human monocytes into macrophages expressing scar-associated markers. Such differentiated cells could degrade collagen IV but not collagen I and promote TGF-ß1-induced collagen I deposition by activated mesenchymal cells. In murine models blocking GM-CSF, IL-17A or TGF-ß1 reduced scar-associated macrophage expansion and hepatic or pulmonary fibrosis. Our work identifies a highly specific macrophage population to which we assign a profibrotic role across species and tissues. It further provides a strategy for unbiased discovery, triage, and preclinical validation of therapeutic targets based on this fibrogenic macrophage population.

Dysregulation of Immunity in Pulmonary Fibrosis is Associated with Increased Myeloid-specific Triggering Receptor-1 and Transforming Growth Factor-beta1 Expression.

Fibrosing pneumonia (FP) is classified into usual interstitial pneumonia (UIP) and nonspecific interstitial pneumonia (NSIP), each having its own etiology and prognosis. Both types of FP are progressive and chronic conditions with distinct etiologies. Cytokines and inflammatory mediators play critical roles in the pathogenesis of FP. Among them, the role of transforming growth factor beta-1 (TGF-ß1) and modulators triggering fibrosis are not well understood. In this study, the expression of triggering receptor expressed on myeloid cells-1 (TREM-1) as a stimulator for the production of TGF-ß1 and also CD4+CD25+Foxp3+ regulatory cells were investigted in FP patients. Sixteen UIP, 14 NSIP and 4 pulmonary fibrosis following Mycobacterium tuberculosis (TB) infection patients, were compared with 12 healthy controls. The frequency of blood CD14+TGF-ß1+ and CD14+TREM1+-gated monocytes and CD4+CD25+Foxp3+ regulatory T cells (Treg), as well as the plasma levels of TGF-ß1 and IL­10 were measured. Fibrosis patients compared to healthy controls had a greater frequency of CD14+TGF-ß1+ [15.9 (0.2-88.2) vs. 0.6 (0.2-11.0)] and CD14+TREM1+ [21.1 (2.3-91.2) vs. 10.3 (3.1-28.6)]-gated monocytes, and CD4+CD25+Foxp3+ [1.2 (0.3-3.6) vs. 0.2 (0.1-0.4)]-gated lymphocytes. Plasma TGF-ß1 were also significantly increased in patients with fibrosis compared to healthy controls [9316.2 (±5554.4) vs. 3787.5 (±2255.6)]. These results confirm the importance of TGF-ß1 and TREM1 in pulmonary fibrosis. It seems that this reciprocal cycle in healthy people is modulated by the production of IL­10 by Treg cells, thus limiting fibrosis, as observed in patients following TB infection. Further investigations are recommended to evaluate possible immunomodulatory mechanisms defects in pulmonary fibrosis.

Targeting TGF-ß/periostin signaling by sesamol ameliorates pulmonary fibrosis and improves lung function and survival.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disorder that severely impairs lung function, by increasing lung stiffness. Sesamol, a phenolic Phyto-molecule isolated from sesame seeds, possess a rich source of protein and is known to have extensive nutritional and health effects. Here we investigated the effect of sesamol on TGF-ß/periostin-induced fibroblast differentiation in in vitro and bleomycin-induced pulmonary fibrosis in an in vivo model. Our results demonstrated that activation of (DHLF, LL29, NHLF and A549) cells with TGF-ß, elevates the epithelial to mesenchymal transition, extracellular matrix, and collagen deposition and periostin signaling marker's expression, further treatment with sesamol attenuated these markers significantly. In addition, sesamol treatment improved the TGF-ß-induced contraction and migration of cells. Mechanistic studies showed that activation of IPF cells with periostin increased the TGF-ß signaling and treatment with sesamol significantly abrogated the periostin-induced TGF-ß activation and its downstream fibrotic marker's expression. In in vivo, sesamol treatment attenuated the lung inflammation, infiltration of cells, wall thickening and the formation of fibrous bands significantly in BLM-induced fibrosis rats. Molecular studies revealed that sesamol treatment reduced the bleomycin-induced fibrotic, inflammatory, apoptotic marker's expression by modulating the TGF-ß/periostin crosstalk signaling in a dose-dependent manner. Further, treatment with sesamol dramatically improved lung function and decreased mortality. Our study first time reports the sesamol's inhibitory effects on periostin signalling. Collectively, our study demonstrated that periostin and TGF-ß seem to work in a positive-feedback loop, inducing the other, therefore, targeting TGF-ß/periostin signaling may provide a better therapeutic approach against IPF and other fibrotic disorders.

Sparganii Rhizoma alleviates pulmonary fibrosis by inhibiting fibroblasts differentiation and epithelial-mesenchymal transition mediated by TGF-ß1/ Smad2/3 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Pulmonary fibrosis (PF), a lethal lung disease, can lead to structural destruction of the alveoli until death. Sparganii Rhizoma (SR), primarily distributed in East Asia, has been used clinically for hundreds of years against organ fibrosis and inflammation. AIM OF THE STUDY: We intended to verify the effect of SR alleviate PF and further explore mechanisms. METHODS: Murine model of PF was established by endotracheal infusion of bleomycin. We detected the anti-PF effect of SR through lung coefficient, hydroxyproline content, lung function and pathological staining. Then, we used Western Blot and RT-PCR to verify the mechanism. In vitro experiments, MRC-5 and BEAS-2B were induced to phenotypic transformation by TGF-ß1 and then RT-PCR, WB and IF were conducted to verify the effect of SR. RESULTS: SR significantly reduced BLM-induced PF in mice, improved lung function, slowed the degree of lung tissue lesions, and reduced collagen deposition. SR alleviated PF by inhibiting fibroblasts differentiation and epithelial-mesenchymal transition. In vivo studies explored the mechanism and found that it was related to TGF-ß1/Smad2/3 pathway. CONCLUSIONS: Our research proved SR could effectively treat PF, providing a fresh idea and approach for the treatment of PF with traditional Chinese medicine.

Functional roles of CD26/DPP4 in bleomycin-induced pulmonary fibrosis.

The pathogenesis of pulmonary fibrosis involves complex interplay between cell types and signaling pathways. Recurrent alveolar epithelial injury can occur during pulmonary inflammation, causing dysregulation of epithelial repair. Dysregulated repair interacts with mesenchymal, inflammatory, and endothelial cells to trigger fibroblast-to-myofibroblast activation. CD26/dipeptidyl peptidase-4 (DPP4) is a type II membrane protein mediating pleiotropic effect. However, the mechanistic role of CD26/DPP4 in pulmonary fibrosis remains unclear. In this study, we aimed to characterize Dpp4 deficiency in a mouse bleomycin (BLM)-induced pulmonary fibrosis model and in cell culture systems of human lung fibroblasts (HLFs). Dpp4 knockout (Dpp4 KO) mouse lungs exhibited lower Ashcroft scale indices, collagen content, and numbers of fibroblasts and myofibroblasts compared with those in C57BL/6 wild-type (WT) mice. Upregulation of Tgfb1 and Tgfb2 mRNA levels in the lungs after BLM treatment was lower in Dpp4 KO mice compared with those in WT mice. Although TGF-ß-driven endothelial-to-mesenchymal transition (EndMT) has been implicated as one of the mechanisms of pulmonary fibrosis, a number of partial EndMT cells in lungs did not differ between Dpp4 KO mice and WT mice. The proliferation capacity and mRNA levels of COL1A1, a collagen deposition-related gene, in cultured HLFs were suppressed in DPP4 small interfering RNA-treated cells. This study indicates that the genetic deficiency of DPP4 has protective effects against BLM-induced pulmonary fibrosis, partly through the reduction in TGF-ß expression and inhibition of fibroblast activation in the lung. Our study suggests that CD26/DPP4 inhibition is a potential therapeutic strategy for pulmonary fibrosis.

Improved targeting delivery of WED-load immunoliposomes modified with SP-A mAb for the treatment of pulmonary fibrosis.

The epithelial-mesenchymal transition (EMT) of type Ⅱ alveolar epithelial cells (AECS Ⅱ) induced by transforming growth factor (TGF-ß1) is a primary pathogenesis of pulmonary fibrosis (PF). To augment the therapeutic potency of wedelolactone (WED) for PF, herein, pulmonary surfactant protein A (SP-A) specifically expressed on AECS Ⅱ was selected as the targeted receptor. Immunoliposomes modified with SP-A monoclonal antibody (SP-A mAb), novel anti-PF drug delivery systems, were developed and investigated in vivo and in vitro. In vivo fluorescence imaging technique was performed to evaluate the pulmonary-targeting effects of immunoliposomes. The result showed that immunoliposomes accumulated more in the lung, compared with non-modified nanoliposomes. Fluorescence detection methods and flow cytometry were used to investigate the function of SP-A mAb and the cellular uptake efficiency of WED-ILP in vitro. SP-A mAb enabled the immunoliposomes to specifically target the A549 cells and increased uptake more effectively. The mean fluorescence intensity (MFI) of cells treated with the targeted immunoliposomes was about 1.4-fold higher than that of cells treated with regular nanoliposomes. The cytotoxicity of nanoliposomes was assessed by the MTT assay, which demonstrated that blank nanoliposomes have no significant effect on A549 cell proliferation even at the SPC concentration of 1000 µg/mL. Additionally, in vitro pulmonary fibrosis model was established to further investigate the anti-pulmonary fibrosis effect of WED-ILP. WED-ILP significantly (**P < 0.01) inhibited the proliferation of A549 cells stimulated by TGF-ß1 indicating that WED-ILP has great potential for the clinical treatment of PF.

Ovatodiolide inhibits SARS-CoV-2 replication and ameliorates pulmonary fibrosis through suppression of the TGF-ß/TßRs signaling pathway.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to pose threats to public health. The clinical manifestations of lung pathology in COVID-19 patients include sustained inflammation and pulmonary fibrosis. The macrocyclic diterpenoid ovatodiolide (OVA) has been reported to have anti-inflammatory, anti-cancer, anti-allergic, and analgesic activities. Here, we investigated the pharmacological mechanism of OVA in suppressing SARS-CoV-2 infection and pulmonary fibrosis in vitro and in vivo. Our results revealed that OVA was an effective SARS-CoV-2 3CLpro inhibitor and showed remarkable inhibitory activity against SARS-CoV-2 infection. On the other hand, OVA ameliorated pulmonary fibrosis in bleomycin (BLM)-induced mice, reducing inflammatory cell infiltration and collagen deposition in the lung. OVA decreased the levels of pulmonary hydroxyproline and myeloperoxidase, as well as lung and serum TNF-ɑ, IL-1ß, IL-6, and TGF-ß in BLM-induced pulmonary fibrotic mice. Meanwhile, OVA reduced the migration and fibroblast-to-myofibroblast conversion of TGF-ß1-induced fibrotic human lung fibroblasts. Consistently, OVA downregulated TGF-ß/TßRs signaling. In computational analysis, OVA resembles the chemical structures of the kinase inhibitors TßRI and TßRII and was shown to interact with the key pharmacophores and putative ATP-binding domains of TßRI and TßRII, showing the potential of OVA as an inhibitor of TßRI and TßRII kinase. In conclusion, the dual function of OVA highlights its potential for not only fighting SARS-CoV-2 infection but also managing injury-induced pulmonary fibrosis.

LncRNA TUG1 promotes pulmonary fibrosis progression via up-regulating CDC27 and activating PI3K/Akt/mTOR pathway.

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease with an unclear pathogenesis. This study aimed to elucidate the function and potential mechanisms of TUG1 in IPF progression. Cell viability and migration were detected by CCK-8 and transwell assays. Autophagy, fibrosis, or EMT-related proteins were measured by Western blotting. Pro-inflammatory cytokine levels were assessed by ELISA kits. The subcellular localization of TUG1 was observed by FISH assay. RIP assay detected the interaction between TUG1 and CDC27. TUG1 and CDC27 was up-regulated in TGF-ß1-induced RLE-6TN cells. TUG1 depletion suppressed pulmonary fibrosis via attenuating inflammation, EMT, inducing autophagy and inactivating PI3K/Akt/mTOR pathway in vitro and in vivo. TUG1 knockdown prevented CDC27 expression. TUG1 silencing ameliorated pulmonary fibrosis by reducing CDC27 expression and inhibiting PI3K/Akt/mTOR pathway.

CCL17 drives fibroblast activation in the progression of pulmonary fibrosis by enhancing the TGF-ß/Smad signaling.

Pulmonary fibrosis (PF) is a type of fatal respiratory diseases with limited therapeutic options and poor prognosis. The chemokine CCL17 plays crucial roles in the pathogenesis of immune diseases. Bronchoalveolar lavage fluid (BALF) CCL17 levels are significantly higher in patients with idiopathic PF (IPF) than in healthy volunteers. However, the source and function of CCL17 in PF remain unclear. Here, we demonstrated that the levels of CCL17 were increased in the lungs of IPF patients and mice with bleomycin (BLM)-induced PF. In particular, CCL17 were upregulated in alveolar macrophages (AMs) and antibody blockade of CCL17 protected mice against BLM-induced fibrosis and significantly reduced fibroblast activation. Mechanistic studies revealed that CCL17 interacted with its receptor CCR4 on fibroblasts, thereby activating the TGF-ß/Smad signaling pathway to promote fibroblast activation and tissue fibrosis. Moreover, the knockdown of CCR4 by CCR4-siRNA or blockade by CCR4 antagonist C-021 was able to ameliorate PF pathology in mice. In summary, the CCL17-CCR4 axis is involved in the progression of PF, and targeting of CCL17 or CCR4 inhibits fibroblast activation and tissue fibrosis and may benefit patients with fibroproliferative lung diseases.