Pulmonary Fibrosis Model Research Articles

Silencing FHL2 inhibits bleomycin-induced pulmonary fibrosis through the TGF-β1/Smad signaling pathway

ObjectiveThis study aimed to investigate the inhibiting effects of FHL2 and Arbutin on cell fibrosis and their possible mechanisms. MethodsThe mRNA expression of FHL2 in pulmonary fibrosis tissues was analyzed by bioinformatics. TGF⁃β1 induced fibrosis of mouse lung fibroblast (Mlg) and mouse primary pulmonary fibroblast (PPF) in rat's lung fibroblasts. FHL2 siRNA was transfected into Mlg and mouse PPF cells to inhibit FHL2. FHL2, α-smooth muscle actin (α-SMA), collagen 1 (Col I), and Fibronectin (Fn) were detected by qRT-PCR. Western blot expression levels of Smad3, p-Smad3, Smad2, and p-Smad2 proteins in cells. High-throughput drug screening for FHL2 inhibitors and the inhibitory effect of Arbutin on pulmonary fibrosis were validated in cellular and animal models of pulmonary fibrosis. ResultsThe mRNA expression of FHL2 in lung fiber tissue was increased. Meanwhile, the decrease of FHL2 expression significantly inhibited the cellular fibrosis morphological changes of rat's lung fibroblasts (Mlgs) and primary lung fibroblasts (PPFs). The expression levels of α⁃SMA, Col I, and Fn were decreased. High-throughput screening showed that Arbutin targeted FHL2. Arbutin alleviated bleomycin (BLM)-induced pulmonary fibrosis in rats by inhibiting FHL2 and then the TGF-β1/Smad signaling pathway. ConclusionInhibition of FHL2 can effectively reduce the fibrosis process induced by TGF⁃β1 and bleomycin, and then inhibit the fibrosis.

Myofibroblast-specific inhibition of the Rho kinase-MRTF-SRF pathway using nanotechnology for the prevention of pulmonary fibrosis.

Pulmonary fibrosis is characterized by the accumulation of myofibroblasts in the lung and progressive tissue scarring. Fibroblasts exist across a spectrum of states, from quiescence in health to activated myofibroblasts in the setting of injury. Highly activated myofibroblasts have a critical role in the establishment of fibrosis as the predominant source of type 1 collagen and profibrotic mediators. Myofibroblasts are also highly contractile cells and can alter lung biomechanical properties through tissue contraction. Inhibiting signaling pathways involved in myofibroblast activation could therefore have significant therapeutic value. One of the ways myofibroblast activation occurs is through activation of the Rho/myocardin-related transcription factor (MRTF)/serum response factor (SRF) pathway, which signals through intracellular actin polymerization. However, concerns surrounding the pleiotropic and ubiquitous nature of these signaling pathways have limited the translation of inhibitory drugs. Herein, we demonstrate a novel therapeutic antifibrotic strategy using myofibroblast-targeted nanoparticles containing a MTRF/SRF pathway inhibitor (CCG-1423), which has been shown to block myofibroblast activation in vitro. Myofibroblasts were preferentially targeted via the angiotensin 2 receptor, which has been shown to be selectively upregulated in animal and human studies. These nanoparticles were nontoxic and accumulated in lung myofibroblasts in the bleomycin-induced mouse model of pulmonary fibrosis, reducing the number of these activated cells and their production of profibrotic mediators. Ultimately, in a murine model of lung fibrosis, a single injection of these drugs containing targeted nanoagents reduced fibrosis as compared with control mice. This approach has the potential to deliver personalized therapy by precisely targeting signaling pathways in a cell-specific manner, allowing increased efficacy with reduced deleterious off-target effects.

The miR-15b-Smurf2-HSP27 axis promotes pulmonary fibrosis

BackgroundHeat shock protein 27 (HSP27) is overexpressed during pulmonary fibrosis (PF) and exacerbates PF; however, the upregulation of HSP27 during PF and the therapeutic strategy of HSP27 inhibition is not well elucidated.MethodsWe have developed a mouse model simulating clinical stereotactic body radiotherapy (SBRT) with focal irradiation and validated the induction of RIPF. HSP25 (murine form of HSP27) transgenic (TG) and LLC1-derived orthotropic lung tumor models were also used. Lung tissues of patients with RIPF and idiopathic pulmonary fibrosis, and lung tissues from various fibrotic mouse models, as well as appropriated cell line systems were used. Public available gene expression datasets were used for therapeutic response rate analysis. A synthetic small molecule HSP27 inhibitor, J2 was also used.ResultsHSP27 expression with its phosphorylated form (pHSP27) increased during PF. Decreased mRNA expression of SMAD-specific E3 ubiquitin-protein ligase 2 (Smurf2), which is involved in ubiquitin degradation of HSP27, was responsible for the increased expression of pHSP27. In addition, increased expression of miRNA15b was identified with decreased expression of Smurf2 mRNA in PF models. Inverse correlation between pHSP27 and Smurf2 was observed in the lung tissues of PF animals, an irradiated orthotropic lung cancer models, and PF tissues from patients. Moreover, a HSP27 inhibitor cross-linked with HSP27 protein to ameliorate PF, which was more effective when targeting the epithelial to mesenchymal transition (EMT) stage of PF.ConclusionsOur findings identify upregulation mechanisms of HSP27 during PF and provide a therapeutic strategy for HSP27 inhibition for overcoming PF.

Ginseng saponin metabolite 20(S)-protopanaxadiol relieves pulmonary fibrosis by multiple-targets signaling pathways

BackgroundPanax ginseng Meyer is a representative Chinese herbal medicine with antioxidant and anti-inflammatory activity. 20(S)-Protopanaxadiol (PPD) has been isolated from ginseng and shown to have promising pharmacological activities. However, effects of PDD on pulmonary fibrosis (PF) have not been reported. We hypothesize that PDD may reverse inflammation-induced PF and be a novel therapeutic strategy. MethodsAdult male C57BL/6 mice were used to establish a model of PF induced by bleomycin (BLM). The pulmonary index was measured, and histological and immunohistochemical examinations were made. Cell cultures of mouse alveolar epithelial cells were analyzed with Western blotting, co-immunoprecipitation, immunofluorescence, immunohistochemistry, siRNA transfection, cellular thermal shift assay and qRT-PCR. ResultsThe survival rate of PPD-treated mice was higher than that of untreated BLM-challenged mice. Expression of fibrotic hallmarks, including α-SMA, TGF-β1 and collagen I, was reduced by PPD treatment, indicating attenuation of PF. Mice exposed to BLM had higher STING levels in lung tissue, and this was reduced by phosphorylated AMPK after activation by PPD. The role of phosphorylated AMPK in suppressing STING was confirmed in TGF-β1-incubated cells. Both in vivo and in vitro analyses indicated that PPD treatment attenuated BLM-induced PF by modulating the AMPK/STING signaling pathway. ConclusionPPD ameliorated BLM-induced PF by multi-target regulation. The current study may help develop new therapeutic strategies for preventing PF.

Promising Therapeutic Effects of Embryonic Stem Cells-Origin Mesenchymal Stem Cells in Experimental Pulmonary Fibrosis Models: Immunomodulatory and Anti-Apoptotic Mechanisms.

Interstitial lung disease (ILD) involves persistent inflammation and fibrosis, leading to respiratory failure and even death. Adult tissue-derived mesenchymal stem cells (MSCs) show potential in ILD therapeutics but obtaining an adequate quantity of cells for drug application is difficult. Daewoong Pharmaceutical's MSCs (DW-MSCs) derived from embryonic stem cells sustain a high proliferative capacity following long-term culture and expansion. The aim of this study was to investigate the therapeutic potential of DW-MSCs in experimental mouse models of ILD. DW-MSCs were expanded up to 12 passages for in vivo application in bleomycin-induced pulmonary fibrosis and collagen-induced connective tissue disease-ILD mouse models. We assessed lung inflammation and fibrosis, lung tissue immune cells, fibrosis-related gene/protein expression, apoptosis and mitochondrial function of alveolar epithelial cells, and mitochondrial transfer ability. Intravenous administration of DW-MSCs consistently improved lung fibrosis and reduced inflammatory and fibrotic markers expression in both models across various disease stages. The therapeutic effect of DW-MSCs was comparable to that following daily oral administration of nintedanib or pirfenidone. Mechanistically, DW-MSCs exhibited immunomodulatory effects by reducing the number of B cells during the early phase and increasing the ratio of Tregs to Th17 cells during the late phase of bleomycin-induced pulmonary fibrosis. Furthermore, DW-MSCs exhibited anti-apoptotic effects, increased cell viability, and improved mitochondrial respiration in alveolar epithelial cells by transferring their mitochondria to alveolar epithelial cells. Our findings indicate the strong potential of DW-MSCs in the treatment of ILD owing to their high efficacy and immunomodulatory and anti-apoptotic effects.

Evaluation of Brilaroxazine (RP5063) in a Bleomycin-Induced Rodent Model of Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis pathology involves serotonin (5-HT), with an increased expression of 5-HT2A/2B/7 receptors in the lungs. This study tests the hypothesis that brilaroxazine (RP5063), an agent with a potent binding affinity for serotonin 5-HT1A/2A/2B/7 and dopamine D2/3/4 receptors, and moderate affinity for the 5-HT transporter, dosed at 15 mg twice daily (b.i.d.) shows efficacy as compared with placebo in a bleomycin (BLM)-induced model using Sprague Dawley rats. On Day 0, four groups received BLM-induction, and one received placebo. On Day 1, one group started on brilaroxazine (RPT). On Day 10, two groups started on brilaroxazine (RPI) and one continued on the vehicle (BLM). All interventions continued until Day 20. Compared with BLM, RPT and RPI sustained survival at 90.5% and 89.5%, respectively (P<0.05) and maintained weight (P<0.01). RPT normalized pulse pressure and cardiac output. It also lowered respiratory resistance, hydroxyproline, lung weight, bronchoalveolar lavage fluid cell counts, and total protein (P<0.05). RPI decreased hydroxyproline and reduced cell counts (P<0.01). Brilaroxazine lowered Ashcroft scores and Masson’s Trichome staining (P<0.001). Both brilaroxazine groups reduced proinflammatory and fibrotic cytokines (P<0.05). Brilaroxazine attenuated BLM-induced pulmonary fibrosis, inflammation, and extracellular deposition and improved cardiac and pulmonary functions in rodents.

Autotaxin facilitates selective LPA receptor signaling

Autotaxin (ATX; ENPP2) produces the lipid mediator lysophosphatidic acid (LPA) that signals through disparate EDG (LPA1-3) and P2Y (LPA4-6) G protein-coupled receptors. ATX/LPA promotes several (patho)physiological processes, including in pulmonary fibrosis, thus serving as an attractive drug target. However, it remains unclear if clinical outcome depends on how different types of ATX inhibitors modulate the ATX/LPA signaling axis. Here, we show that the ATX "tunnel" is crucial for conferring key aspects of ATX/LPA signaling and dictates cellular responses independent of ATX catalytic activity, with a preference for activation of P2Y LPA receptors. The efficacy of the ATX/LPA signaling responses are abrogated more efficiently by tunnel-binding inhibitors, such as ziritaxestat (GLPG1690), compared with inhibitors that exclusively target the active site, as shown in primary lung fibroblasts and a murine model of radiation-induced pulmonary fibrosis. Our results uncover a receptor-selective signaling mechanism for ATX, implying clinical benefit for tunnel-targeting ATX inhibitors.

Translational Studies Reveal the Divergent Effects of Simtuzumab Targeting LOXL2 in Idiopathic Pulmonary Fibrosis.

The composition of extracellular matrix (ECM) is altered during pathologic scarring in damaged organs including the lung. One major change in the ECM involves the cross-linking of collagen, which promotes fibroblast to myofibroblast differentiation. We examined the role of lysyl oxidase (LOX)-like 2 in lung progenitors and fibroblasts cultured from normal or IPF lung samples and in a humanized mouse model of IPF using a monoclonal antibody (Simtuzumab). Primary lung fibroblasts from normal donor lungs and IPF lung explants were examined for expression of LOXL2. Targeting LOXL2 with Simtuzumab on normal and IPF fibroblasts was examined both in vitro and in vivo for synthetic, functional, and profibrotic properties. LOXL2 was increased at transcript and protein level in IPF compared with normal lung samples. In a dose-dependent manner, Simtuzumab enhanced differentiation of fibroblasts into myofibroblasts. Inhibition of LOXL2 also enhanced fibroblast invasion and accelerated the outgrowth of fibroblasts from dissociated human lung cell preparations. Finally, preventative or delayed delivery of Simtuzumab enhanced lung fibrosis in a humanized mouse model of pulmonary fibrosis. Consistent with its failure in a Phase 2 clinical trial, Simtuzumab exhibited no therapeutic efficacy in translational in vitro and in vivo assays.

Ephedrine alleviates bleomycin-induced pulmonary fibrosis by inhibiting epithelial-mesenchymal transition and restraining NF-κB signaling.

Pulmonary fibrosis is a lethal and progressive pulmonary disorder in human beings. Ephedrine is a compound isolated from Ephedra and plays a regulatory role in inflammatory response. This study focused on the anti-pulmonary fibrosis effect of ephedrine and its potential molecular mechanism. After a mouse model of pulmonary fibrosis was established through bleomycin (BLM) induction, the survival percentage, body weight, and pulmonary index were measured. Hematoxylin-eosin staining and Masson's trichrome staining for lung tissues were performed to observe the pathological alterations. The viability of lung epithelial BEAS-2B cells, intracellular production of reactive oxygen species, and the levels of pro-inflammatory cytokines were examined by cell counting kit-8 assays, 2',7'-dichlorofluorescein diacetate (DCF-DA) staining, and enzyme-linked immunosorbent assay, respectively. Immunofluorescence staining was performed to determine E-cadherin and vimentin expression after BLM or ephedrine treatment. The mRNA and protein levels of cytokeratin-8, E-cadherin, α-SMA, and vimentin were subjected to quantitative polymerase chain reaction and immunoblotting. Experimental results revealed that ephedrine treatment rescued the repressive impact of BLM on BEAS-2B cell viability, and ephedrine inhibited BLM-induced overproduction of reactive oxygen species and inflammatory response in BEAS-2B cells. Additionally, ephedrine suppressed epithelial-mesenchymal transition (EMT) process stimulated by BLM treatment, as demonstrated by the reduced α-SMA and vimentin levels together with the increased cytokeratin-8 and E-cadherin levels in BLM + Ephedrine group. In addition, ephedrine inhibited NF-κB and activated Nrf-2 signaling in BLM-treated BEAS-2B cells. Moreover, ephedrine ameliorated pulmonary fibrosis in BLM-induced mice and improved the survival of model mice. In conclusion, ephedrine attenuates BLM-evoked pulmonary fibrosis by repressing EMT process via blocking NF-κB signaling and activating Nrf-2 signaling, suggesting that ephedrine might become a potential anti-pulmonary fibrosis agent in the future.

Vinpocetine's immunomodulating, anti-oxidant, anti-inflammatory, ant-ifibrotic, and PDE inhibiting potencies ameliorate bleomycin-induced pulmonary fibrosis.

Pulmonary fibrosis (PF) is a global health problem with a high economic burden. Intratracheal administration of bleomycin is the best model that resembles the pathogenesis of PF in humans. Recently, vinpocetine proved to have neuroprotective, cardioprotective, hepatoprotective, anti-aging, and antifibrotic effects through its anti-oxidant, immunomodulating, and anti-inflammatory activities. The present study investigated the antifibrotic potentiality of vinpocetine in a rat model of PF induced by intratracheal bleomycin administration. PF induced by a single intratracheal instillation of 5 mg/kg bleomycin in nine-week-old Wister rats. Oral vinpocetine was used at doses of 5, 10, or 20 mg/kg to treat PF for 21 days immediately after the bleomycin instillation. Vinpocetine dose-dependently ameliorates PF induced by bleomycin administration since vinpocetine effectively restored the normal body weight gain rates, pulmonary architecture, and collagen fiber distribution and suppressed the elevated BALF cell count, lymphocytes and neutrophils percentage, BALF, IL-6, TNF-α, and TGF-β1 levels and LDH activity, lung tissue MDA level, PDE activity, hydroxyproline content, immunohistochemical expression of α-SMA and CD68 positive macrophage, and fibrosis score. Meanwhile, it efficiently augmented the reduced BALF macrophage percentage, IL-10 level, lung tissue GSH level, CAT, and SOD activities. Vinpocetine may propose a new promising agent to manage PF.

Effect of Trichostatin A on Bleomycin Induced Pulmonary Fibrosis in Mice and its Mechanism

To investigate the effect and mechanism of trichostatin A on bleomycin induced pulmonary fibrosis in mice compared with dexamethasone and pirfenidone is the objective of the study. C57 black 6 mice were instilled intranasal bleomycin to establish pulmonary fibrosis model. They were divided into five groups: Control group, model group, dexamethasone group, pirfenidone group and trichostatin A group. Hematoxylin and eosin staining and Masson’s staining was conducted; the expression of related proteins in each group was observed by immunohistochemistry and Western blotting. Hematoxylin and eosin staining and Masson’s staining showed that the lung tissue was damaged in varying degrees, interstitial protein deposition, basal cell hyperplasia and a large number of macrophage infiltration in the alveolar cavity. The lung tissue injury in trichostatin A group was the least. The expression of vimentin, collagen-I, alpha smooth muscle actin and other interstitial proteins in the model group increased significantly, and the expression of above interstitial proteins in each intervention treatment group decreased in varying degrees, especially in trichostatin A group. The expression of suppressor of mothers against decapentaplegic homolog 7 protein decreased significantly in the model group and increased significantly in the trichostatin A group; there was no significant difference in the expression of p38-mitogen-activated protein kinase protein in each group. The expression of interstitial protein messenger RNA such as vimentin, collagen-I and alpha smooth muscle actin peaked on the 14th d after modeling. Trichostatin A could significantly inhibit the progression of pulmonary fibrosis by inhibiting the activity of histone deacetylase 1, while histone deacetylase 1 could activate transforming growth factor beta-1/suppressor of mothers against decapentaplegic pathway, involved in the progression of pulmonary fibrosis. Dexamethasone could not delay or improve pulmonary fibrosis, or even aggravate the progression of pulmonary fibrosis.

Acute exacerbation of idiopathic pulmonary fibrosis model in the rats using bleomycin and lipopolysaccharides.

This study was conducted to establish a rat model of acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) using the combination of bleomycin (BLM) and lipopolysaccharides (LPS). Twenty-four male Sprague Dawley rats were allocated into two equal groups: the sham or the bleomycin and lipopolysaccharides-induced AE-IPF group (BLM-LPS). On Day 7, BLM intratracheally and LPS intraperitoneally were both used to administer AE-IPF. The BLM-LPS group and its respective sham group were terminated on Days 8, 14, or 21. Samples of bronchoalveolar lavage fluid (BALF) and lungs were taken and investigated for cell count and histopathology. On Day 8, histological analysis revealed inflammatory cell infiltration with edema and hyaline membrane, and the BALF differential cell count revealed high neutrophil counts. By having a higher collagen density area and Ashcroft modified score than the sham group on Day 14, the BLM-LPS group displayed significantly lower oxygen saturation, alveolar air area, and a fibrotic appearance. However, there was a spontaneous resolution in inflammation and fibrotic appearance on Day 21 after the BLM administration. By combining BLM and LPS, it was possible to create a successful rat model of AE-IPF. The present model showed the peak exacerbation on Day 8 and the fibrotic peak on Day 14, which gradually improved. The optimal time for the new AE-IPF therapeutic intervention was determined to be between Days 8 and 14.

Mutual promotion of mitochondrial fission and oxidative stress contributes to mitochondrial-DNA-mediated inflammation and epithelial-mesenchymal transition in paraquat-induced pulmonary fibrosis.

Pulmonary fibrosis (PF) is one of the main causes of death in patients with paraquat (PQ) poisoning. This study aimed to evaluate the relationship between mitochondrial fission and oxidative stress in PQ-induced epithelial-mesenchymal transition (EMT) and PF. C57BL/6 mice and MLE-12 cells were exposed to PQ to construct a PF model in vivo and in vitro. Histological changes in the lungs were examined by hematoxylin and eosin (H&E) staining. Mitochondrial morphology was detected by MitoTracker® Deep Red FM or transmission electron microscopy (TEM). Western blotting and immunofluorescence were used to determine the expression of protein. The migration ability of the cells was detected by the cell scratch test. Mitochondrial DNA (mtDNA) levels were assessed by real-time polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) was applied to detect cytokine levels. Superoxide dismutase (SOD) activity and the levels of glutathione (GSH) and malondialdehyde (MDA) were detected by chemichromatometry. PQ exposure caused EMT and PF in vivo and in vitro. PQ destroyed mitochondrial structure and enhanced the expression of dynamin-related protein 1 (Drp1), which were accompanied by oxidative stress. Inhibiting mitochondrial fission using mitochondrial division inhibitor-1 (Mdivi-1), a selective inhibitor of Drp1, attenuated PQ-induced EMT and oxidative damage. Treatment with N-acetyl-L-cysteine (NAC), an antioxidant, reduced Drp1 expression, attenuated mitochondrial structure damage and inhibited PQ-induced EMT and PF. Both Mdivi-1 and NAC treatment markedly suppressed mtDNA release, the expression of Toll-like receptor 9 (TLR9) and phosphorylation (P)-NF-κB p65 as well as cytokines (interleukin 6 [IL-6], interleukin-1β [IL-1β], and tumor necrosis factor-α [TNF-α]) production. Mutual promotion of mitochondrial fission and oxidative stress contributes to EMT in PQ-induced PF, which is associated with the mtDNA/TLR9/NF-κB pathway.

Retrospektivna procjena učinka nintedaniba u mišjem modelu plućne fibroze

Pulmonary function tests (PFTs) routinely implemented in clinics are the first step in the diagnosis of idiopathic pulmonary fibrosis. Evaluation of PFTs in the mouse model of pulmonary fibrosis accompanied by histological readouts may improve the clinical predictability of new therapeutic candidates. Forced vital capacity (FVC) is considered the most predictive of restrictive pulmonary disorders. This study aimed to test the improvement of PFT in mice lung fibrosis induced by treatment with an approved substance nintedanib, considered the gold standard. The hypothesis that treatment in animal models will demonstrate similar effects as in humans in the most relevant clinical outcomes was tested. Two experimental designs were enrolled in this study, a preventive regimen, with treatment initiation from the day of the challenge; and a therapeutic regimen, starting on day 7 postchallenge when fibrotic changes are present in the lungs. Experiments were terminated at two different time points, at 14 and 21 days postchallenge. C57BL/6 mice were administered with bleomycin (BLM) intranasally and treated with nintedanib from day 0 to day 14 or from day 7 until day 21. Fourteen or 21 days after the BLM challenge, PFTs were assessed using the in vivo invasive lung function measurement system Buxco® Pulmonary Function Test (PFT) (DSI™, New Brighton, USA). Histological evaluation was performed as a modified Ashcroft score. The bleomycin challenge induced a significant decrease of FVC in both experiments. However, nintedanib treatment given in both regimens significantly improved lung functionality. These findings were confirmed with histological analysis of the Ashcroft scoring system, modified by Matsuse. In conclusion, a good correlation between functional test parameters and the clinical effect of nintedanib was shown in both experiments: the preventive regimen was sampled 14 days post-challenge and the therapeutic regimen 21 days post-challenge. Based on these findings, the implementation of PFTs could be a good platform to increase the translational value of the model and potential new treatments.

ML216 Prevents DNA Damage-Induced Senescence by Modulating DBC1-BLM Interaction.

DNA damage is the major cause of senescence and apoptosis; however, the manner by which DNA-damaged cells become senescent remains unclear. We demonstrate that DNA damage leads to a greater level of senescence rather than apoptosis in DBC1-deficient cells. In addition, we show that BLM becomes degraded during DNA damage, which induces p21 expression and senescence. DBC1 binds to and shields BLM from degradation, thus suppressing senescence. ML216 promotes DBC1-BLM interaction, which aids in the preservation of BLM following DNA damage and suppresses senescence. ML216 enhances pulmonary function by lowering the levels of senescence and fibrosis in both aged mice and a mouse model of bleomycin-induced idiopathic pulmonary fibrosis. Our data reveal a unique mechanism preventing DNA-damaged cells from becoming senescent, which may be regulated by the use of ML216 as a potential treatment for senescence-related diseases.

UHRF1-mediated ferroptosis promotes pulmonary fibrosis via epigenetic repression of GPX4 and FSP1 genes

Pulmonary fibrosis (PF), as an end-stage clinical phenotype of interstitial lung diseases (ILDs), is frequently initiated after alveolar injury, in which ferroptosis has been identified as a critical event aggravating the pathophysiological progression of this disease. Here in, a comprehensive analysis of two mouse models of pulmonary fibrosis developed in our lab demonstrated that lung damage-induced ferroptosis of alveolar epithelial Type2 cells (AEC2) significantly accumulates during the development of pulmonary fibrosis while ferroptosis suppressor genes GPX4 and FSP1 are dramatically inactivated. Mechanistically, upregulation of de novo methylation regulator Uhrf1 sensitively elevates CpG site methylation levels in promoters of both GPX4 and FSP1 genes and induces the epigenetic repression of both genes, subsequently leading to ferroptosis in chemically interfered AEC2 cells. Meanwhile, specific inhibition of UHRF1 highly arrests the ferroptosis formation and blocks the progression of pulmonary fibrosis in both of our research models. This study first, to our knowledge, identified the involvement of Uhrf1 in mediating the ferroptosis of chemically injured AEC2s via de novo promoter-specific methylation of both GPX4 and FSP1 genes, which consequently accelerates the process of pulmonary fibrosis. The above findings also strongly suggested Uhrf1 as a novel potential target in the treatment of pulmonary fibrosis.

IL-22 inhibits bleomycin-induced pulmonary fibrosis in association with inhibition of IL-17A in mice

BackgroundInterstitial lung disease, a common extra-articular complication of connective tissue disease, is characterized by progressive and irreversible pulmonary inflammation and fibrosis, which causes significant mortality. IL-22 shows a potential in regulating chronic inflammation and possibly plays an anti-fibrotic role by protecting epithelial cells. However, the detailed effects and underlying mechanisms are still unclear. In this study, we explored the impact of IL-22 on pulmonary fibrosis both in vivo and in vitro.MethodsTo induce pulmonary fibrosis, wild-type mice and IL-22 knockout mice were intratracheally injected with bleomycin followed by treatments with recombinant IL-22 or IL-17A neutralizing antibody. We investigated the role of IL-22 on bleomycin-induced pulmonary fibrosis and the mechanism in the possible interaction between IL-22 and IL-17A. Fibrosis-related genes were detected using RT-qPCR, western blot, and immunofluorescence. Inflammatory and fibrotic changes were assessed based on histological features. We also used A549 human alveolar epithelial cells, NIH/3T3 mouse fibroblast cells, and primary mouse lung fibroblasts to study the impact of IL-22 on fibrosis in vitro.ResultsIL-22 knockout mice showed aggravated pulmonary fibrosis compared with wild-type mice, and injection of recombinant IL-22 decreased the severe fibrotic manifestations in IL-22 knockout mice. In cell culture assays, IL-22 decreased protein levels of Collagen I in A549 cells, NIH/3T3 cells, and primary mouse lung fibroblasts. IL-22 also reduced the protein level of Collagen I in NIH/3T3 cells which were co-cultured with T cells. Mechanistically, IL-22 reduced the Th17 cell proportion and IL-17A mRNA level in lung tissues, and treatment with an IL-17A neutralizing antibody alleviated the severe pulmonary fibrosis in IL-22 knockout mice. The IL-17A neutralizing antibody also reduced Collagen I expression in NIH/3T3 cells in vitro. Knockdown of IL-17A with siRNAs or administration of IL-22 in NIH/3T3 cells and MLFs decreased expression of Collagen I, an effect blocked by concurrent use of recombinant IL-17A.ConclusionsIL-22 mediated an anti-fibrogenesis effect in the bleomycin-induced pulmonary fibrosis model and this effect was associated with inhibition of IL-17A.

Inhibitors of the Sialidase NEU3 as Potential Therapeutics for Fibrosis.

Fibrosing diseases are a major medical problem, and are associated with more deaths per year than cancer in the US. Sialidases are enzymes that remove the sugar sialic acid from glycoconjugates. In this review, we describe efforts to inhibit fibrosis by inhibiting sialidases, and describe the following rationale for considering sialidases to be a potential target to inhibit fibrosis. First, sialidases are upregulated in fibrotic lesions in humans and in a mouse model of pulmonary fibrosis. Second, the extracellular sialidase NEU3 appears to be both necessary and sufficient for pulmonary fibrosis in mice. Third, there exist at least three mechanistic ways in which NEU3 potentiates fibrosis, with two of them being positive feedback loops where a profibrotic cytokine upregulates NEU3, and the upregulated NEU3 then upregulates the profibrotic cytokine. Fourth, a variety of NEU3 inhibitors block pulmonary fibrosis in a mouse model. Finally, the high sialidase levels in a fibrotic lesion cause an easily observed desialylation of serum proteins, and in a mouse model, sialidase inhibitors that stop fibrosis reverse the serum protein desialylation. This then indicates that serum protein sialylation is a potential surrogate biomarker for the effect of sialidase inhibitors, which would facilitate clinical trials to test the exciting possibility that sialidase inhibitors could be used as therapeutics for fibrosis.

3D pulmonary fibrosis model for anti-fibrotic drug discovery by inkjet-bioprinting

Pulmonary fibrosis (PF) is known as a chronic and irreversible disease characterized by excessive extracellular matrix accumulation and lung architecture changes. Large efforts have been made to develop prospective treatments and study the etiology of pulmonary fibrotic diseases utilizing animal models and spherical organoids. As part of these efforts, we created an all-inkjet-printed three-dimensional (3D) alveolar barrier model that can be used for anti-fibrotic drug discovery. Then, we developed a PF model by treating the 3D alveolar barrier with pro-fibrotic cytokine and confirmed that it is suitable for the fibrosis model by observing changes in structural deposition, pulmonary function, epithelial–mesenchymal transition, and fibrosis markers. The model was tested with two approved anti-fibrotic drugs, and we could observe that the symptoms in the disease model were alleviated. Consequently, structural abnormalities and changes in mRNA expression were found in the induced fibrosis model, which were shown to be recovered in all drug treatment groups. The all-inkjet-printed alveolar barrier model was reproducible for disease onset and therapeutic effects in the human body. This finding emphasized that the in vitro artificial tissue with faithfully implemented 3D microstructures using bioprinting technology may be employed as a novel testing platform and disease model to evaluate potential drug efficacy.

The Restoring Effect of Human Umbilical Cord-Derived Mesenchymal Cell-Conditioned Medium (hMSC-CM) against Carbon Tetrachloride-Induced Pulmonary Fibrosis in Male Wistar Rats.

Pulmonary toxicity induced by CCl4, a model of idiopathic pulmonary fibrosis (IPF), leads to tissue remodeling and inflammation. Human umbilical cord mesenchymal cell-conditioned medium (hMSC-CM) is a potent anti-inflammatory, antioxidative, and antifibrotic agent. Forty male Wistar rats were assigned to the control (C), olive oil control (C.O) (hMSC-CM), control (C.Ms), fibrosis (fb), and fibrosis with hMSC-CM (f.Ms) treatment groups. The groups C, C.O, and C.Ms received PBS (200 µl), olive oil (1 ml/kg), and hMSC-CM (100 μg protein/kg), respectively. The fibrosis group was administered with only CCl4 (1 ml/kg). The last group, f.Ms was treated with CCl4 (1 ml/kg) and 100 μg protein/kg IV hMSC-CM. While the treatment with olive oil and CCl4 was performed for 2 days/week from the first week for 12 weeks, the treatment with PBS and hMSC-CM was carried out 2 days/week from week 4th to week 12th. The effect of the UC-MSC culture medium treatment on the lung was evaluated by assessing lysyl oxidase (LOX), tumor necrosis factor-alpha (TNF-α), and transforming growth factor-β1 (TGF-β1) genes, and proteins expression by real-time RCR and western blotting, respectively. Lysyl oxidase (LOX), tumor necrosis factor-alpha (TNF-α), transforming growth factor-b1 (TGF-β1), malondialdehyde (MDA), and oxidative stress levels were markedly higher in the fibrosis group than in the control groups (p ≤ 0.001). Additionally, glutathione (GSH) in the fibrosis group was markedly lower than those in the control groups (p ≤ 0.001). Fibrosis in the UC-MSC treatment group had milder histopathological injuries than in the fibrosis group. hMSC-MSC as a strong anti-inflammatory, antioxidative, and antifibrotic decreases the level of oxidative stress, proinflammatory cytokines, and MDA causing a restoring effect against CCl4-induced pulmonary fibrosis.