Fibrosis Model Research Articles

In Vitro Modeling of Idiopathic Pulmonary Fibrosis: Lung-on-a-Chip Systems and Other 3D Cultures.

Idiopathic pulmonary fibrosis (IPF) is a lethal disorder characterized by relentless progression of lung fibrosis that causes respiratory failure and early death. Currently, no curative treatments are available, and existing therapies include a limited selection of antifibrotic agents that only slow disease progression. The development of novel therapeutics has been hindered by a limited understanding of the disease's etiology and pathogenesis. A significant challenge in developing new treatments and understanding IPF is the lack of in vitro models that accurately replicate crucial microenvironments. In response, three-dimensional (3D) in vitro models have emerged as powerful tools for replicating organ-level microenvironments seen in vivo. This review summarizes the state of the art in advanced 3D lung models that mimic many physiological and pathological processes observed in IPF. We begin with a brief overview of conventional models, such as 2D cell cultures and animal models, and then explore more advanced 3D models, focusing on lung-on-a-chip systems. We discuss the current challenges and future research opportunities in this field, aiming to advance the understanding of the disease and the development of novel devices to assess the effectiveness of new IPF treatments.

Compression-induced apoptosis of fibroblasts and myofibroblasts in an in vitro model of pulmonary fibrosis by alginate/gelatin scaffold

Pulmonary fibrosis leads to increased mortality but is poorly understood. Fibrotic progression is associated with abnormal wound repair and an increase in myofibroblast cell populations. Here we investigate how the myofibroblast population is impacted by unique compression-induced apoptosis derived from mechanical strain characteristic of asthma. Using a mechanical device, both static and dynamic mechanical strains were applied to alginate/gelatin/CaCl2 scaffolds containing fibroblasts and myofibroblasts. As cell groups were stimulated with 30 % static strain for 12 h, fibroblast and myofibroblast cell groups showed increased cell apoptosis by 5.55 % and 19.56 %, respectively, compared to control groups. Additionally, myofibroblasts exhibited higher susceptibility to apoptosis induction than did fibroblasts. Comparing dynamic and static loading modes, dynamic loading resulted in a higher apoptosis rate of fibroblast and myofibroblast cells, indicating its potential to induce apoptosis effectively. These findings suggest that mechanical stimulation can be considered a promising approach to induce apoptosis in myofibroblasts, thus offering the potential for future approaches to treating pulmonary fibrosis. Moreover, mechanical loads can be designed for other diseases, selectively reducing or increasing apoptosis in either hard or soft cell groups, based on specific application needs.

Dual-Ligand-Functionalized Nanostructured Lipid Carriers as a novel Dehydrocavidine delivery system for Liver fibrosis Therapy

BackgroundLiver fibrosis is a common stage of various chronic liver diseases, often developing into liver cirrhosis, and even liver cancer. Activated hepatic stellate cells (aHSCs) have been shown to promote the development of liver fibrosis. Therefore, dual-targeted combination therapy for liver may be an effective strategy for the treatment of liver fibrosis. PurposeIn this study, the novel nanostructured lipid carriers (GA&GalNH2-DC-NLCs) were prepared for Dehydrocavidine (DC), glycyrrhetinic acid (GA) and galactose-PEG2000-NH2 (GalNH2) were selected as targeted ligand-modified nanostructured lipid carriers (NLCs), which enables dual-targeting to the liver for the treatment of liver fibrosis. Study designTo study the targeting effect of GA&GalNH2-DC-NLCs on liver and its therapeutic effect on liver fibrosis, we established aHSC-T6 cell model and rat model of liver fibrosis for study. ResultsGA&GalNH2-DC-NLCs promoted drug liver targeting efficiency and apoptosis rate by upregulating the expression of Bax. It showed that compared with no and/or GA-modified NLCs and GalNH2-modified NLCs, GA&GalNH2-DC-NLCs exhibited less extracellular matrix (ECM) deposition, induced apoptosis of aHSCs, and stronger anti-fibrosis effects in vivo. This may be due the fact that GA or GalNH2-modifified NLCs simultaneously block HSCs activation and inhibit the IL-6/STAT3 pathway. ConclusionGA&GalNH2-DC-NLCs is thus a potential strategy for liver fibrosis treatment.

Development of fully automated models for staging liver fibrosis using non-contrast MRI and artificial intelligence: a retrospective multicenter study

Accurate staging of liver fibrosis (LF) is essential for clinical management in chronic liver disease. While non-contrast MRI (NC-MRI) yields valuable information for liver assessment, its effectiveness in predicting LF remains underexplored. This study aimed to develop and validate artificial intelligence (AI)-powered models utilizing NC-MRI for staging LF. A total of 1726 patients from Shengjing Hospital of China Medical University, registered between October 2003 and October 2022, were retrospectively collected, and divided into development (n=1208) and internal test (n=518) cohorts. An external test cohort consisting of 337 individuals from six centers, registered between June 2015 and November 2022, were also included. All participants underwent NC-MRI (T1-weighted imaging, T1WI; and T2-fat-suppressed imaging, T2FS) and liver biopsies. Two classification models (CMs), named T1 and T2FS, were trained on respective image types using 3D contextual transformer networks and evaluated on both test cohorts. Additionally, three CMs-Clinic, Image, and Fusion-were developed using clinical features, T1 and T2FS scores, and their integration via logistic regression. Classification effectiveness of CMs was assessed using the area under the receiver operating characteristic curve (AUC). A comparison was conducted between the optimal models (OMs) with highest AUC and other methods (transient elastography, five serum biomarkers, and six radiologists). Fusion models (i.e., OM) yielded the highest AUC among the CMs, achieving AUCs of 0.810 for significant fibrosis, 0.881 for advanced fibrosis, and 0.918 for cirrhosis in the internal test cohort, and 0.808, 0.868, and 0.925, respectively, in the external test cohort. The OMs demonstrated superior performance in AUC, significantly surpassing transient elastography (only for staging≥F2 and ≥ F3 grades), serum biomarkers, and three junior radiologists for staging LF. Radiologists, with the aid of the OMs, can achieve a higher AUC in LF assessment. AI-powered models utilizing NC-MRI, including T1WI and T2FS, accurately stage LF. National Natural Science Foundation of China (No. 82071885); General Program of the Liaoning Provincial Department of Education (LJKMZ20221160); Liaoning Province Science and Technology Joint Plan (2023JH2/101700127); the Leading Young Talent Program of Xingliao Yingcai in Liaoning Province (XLYC2203037).

Validation of the Integrated Palliative Care Outcome Scale (IPOS) in adults with cystic fibrosis.

A primary palliative care model for cystic fibrosis (CF) recommends using the Integrated Palliative Care Outcome Scale (IPOS) for screening. Validation of the IPOS is needed. This secondary analysis utilized baseline data from a multisite trial of the palliative care model, Improving Life with CF. Adults with CF completed the IPOS, the Memorial Symptom Assessment Scale-CF (MSAS-CF), the CF Questionnaire-Revised (CFQ-R), the Patient Health Questionnaire (PHQ-8), the Generalized Anxiety Disorder (GAD-7), and the Perceived Stress Scale (PSS). IPOS structure was assessed using Cronbach α coefficients and a factor analysis. Construct validity was evaluated through bivariate relationships between IPOS scores and other questionnaire scores, and linear regressions assessing the extent to which the IPOS explains variance in quality-of-life domains. The sample comprised 256 adults with complete IPOS data. α coefficients were .86 for the IPOS total score, .81 for the Physical Symptoms subscale, .79 for the Emotional Symptoms subscale, and .63 for the Communication/Practical Issues subscale. A two-component factor structure best aligned with the current subscales. IPOS scores were significantly associated with other measures; associations with MSAS-CF and CFQ-R subscales differentiated the IPOS Physical and Emotional subscales. The IPOS total score provided unique information about the variance in the CFQ-R Physical Functioning and Respiratory Symptoms domain scores. In adults with CF, the IPOS has acceptable internal consistency and there is evidence of construct validity. These findings support adoption of the IPOS in the primary palliative care model for CF.

Assessment and integration of multiparametric MRI for liver fibrosis staging in rat non-alcoholic steatohepatitis: Evaluation of diagnostic efficiency and model interpretation

ObjectivesMulti-parametric MRI techniques, including intravoxel incoherence motion (IVIM), iterative decomposition of water and fat with echo asymmetry and least squares estimation (IDEAL IQ), T2* mapping and T2 mapping, were employed to develop and validate a predictive model for non-alcoholic steatohepatitis (NASH) diagnosis and liver fibrosis (LF) staging in rats. The combined model was interpreted using SHapley Additive exPlanations (SHAP) values for model understanding. Materials and methods160 healthy Sprague-Dawley (SD) rats were divided into control (n = 24) and experimental (n = 136) groups, and the 12-week and 16-week groups were injected intraperitoneally with carbon tetrachloride (CCl4) for 4 weeks, one month before the final feeding period. All rats were subjected to pathological examination to determine LF stage. Upon the study’s completion, 147 SD rats were assessed for liver fibrosis. Results84 SD rats were diagnosed with NASH and 31, 10, and 43 rats were histologically diagnosed with no fibrosis (F0), early LF (F1-F2), and late LF (F3-F4). For diagnosis of NASH and staging of liver fibrosis associated with NASH, a combined multi-parameter magnetic resonance imaging prediction model has a higher area under the ROC curve (AUC) than individual diffusion parameters, especially in advanced stages of fibrosis, with an AUC of 0.929 for the combined model. In SHAP, the free fluid(FF) value contributes most to the model for diagnosing NASH and advanced liver fibrosis, while the T2 value contributes most for diagnosing liver fibrosis and the apparent diffusion coefficient (ADC) value contributes most for diagnosing liver cirrhosis. ConclusionsThe integration of several magnetic resonance imaging methods along with SHAP value analysis provides a more comprehensive and theoretical investigation into the mechanisms and factors contributing to the progression of liver fibrosis in NASH, offering insights from various angles.

Loss of interferon regulatory factor-1 prevents lung fibrosis by upregulation of pon1 expression

BackgroundInterferon regulatory factor-1 (IRF1) is a transcription factor that plays a significant role in various biological processes, including inflammatory injury, viral infection, cell death, and immune responses, and it has been extensively studied in the context of different lung diseases. However, the mechanism underlying its involvement in lung fibrosis remains largely unknown.MethodsWild type (WT) mice, IRF1 global-null mice (Irf1−/−) were subjected to a bleomycin-induced lung fibrosis model to enable examination of the role of IRF1 in lung fibrosis. Proteomic analysis of lung tissue from WT and Irf1−/− mice treated with saline or bleomycin was performed to explore the mechanism of IRF1 in regulating lung fibrosis.ResultsIn the bleomycin-induced fibrosis mouse model, increased expression of IRF1 was observed. Irf1 knockout mice displayed decreased lung fibrosis relative to WT mice following treatment with bleomycin. The protein expression of fibronectin, as assessed by the Western blot analysis of lung tissues, was downregulated in Irf1−/− mice. We observed a similar reduction in collagen content using hydroxyproline detection. Histologically, there was less collagen deposition in the lungs of Irf1−/− mice compared with WT mice. Proteomics data revealed that IRF1 may be involved in lung fibrosis via the regulation of ferroptosis. We determined that paraoxonase 1(PON1), a poorly characterized protein in lung fibrosis, was upregulated in Irf1−/− mice following exposure to bleomycin. In vitro experiments revealed that IRF1 could regulate the level of GSH and MDA through PON1. We also determined that PON1 levels were lower in the plasma of IPF patients compared with healthy controls.ConclusionOur data highlight the importance of IRF1 in the fibrotic process, and PON1 may be a potential mediator of IRF1 in the progression of lung fibrosis.

Efficient and selective kidney targeting by chemically modified carbohydrate conjugates.

We investigated a renal tubule-targeting carbohydrate (RENTAC) that can selectively deliver small-molecule and nucleic acid analogs to the proximal convoluted tubules of the kidney following systemic delivery in mice. We comprehensively evaluated anti-miR-21-peptide nucleic acid-RENTAC, and fluorophore-RENTAC conjugates in cell culture and invivo. We established that RENTAC conjugates showed megalin- and cubilin-dependent endocytic uptake in the immortalized kidney cell line. Invivo biodistribution studies confirmed the retention of RENTAC conjugates in the kidneys for several days compared with other organs. Immunofluorescence staining confirmed the selective distribution of the RENTAC conjugates in proximal convoluted tubules. We further demonstrated proximal convoluted tubule targeting features of RENTAC conjugates in a folic acid-induced kidney fibrosis mouse model. As a biological readout, we targeted miR-33 using antisense peptide nucleic acid (PNA) 33-RENTAC conjugates in the fibrotic kidney disease model. The targeted delivery of PNA 33-RENTAC resulted in slower fibrosis progression and decreased collagen deposition. We also confirmed that the RENTAC ligand did not exert any adverse reactions. Thus, we established that the RENTAC ligand can be used for broad clinical applications targeting the kidneys selectively.

Siling Decoction Ameliorates Adenine-Induced Renal Fibrosis in Rats by the AKT/IKKβ/NFκB Signaling Pathway

ObjectiveInvestigating how Siling decoction (SLD) mitigates fibrosis in rats with chronic kidney disease CKD (chronic kidney disease) through network pharmacology analysis and experimental verification. MethodsInitially, the primary active components and their target actions of SLD (Fuling, Zhuling, Zexie, and Baizhu) were identified by the TCMSP database and liquid chromatography mass spectrometry (LC-MS). Treatment targets for renal fibrosis were screened through databases such as GeneCard, OMIM, PharmGkb, and GEO. Subsequently, a drug-disease-target network was constructed and subjected to PPI analysis. Intersecting targets underwent GO and KEGG pathway enrichment analyses. Renal fibrosis model was induced by adenine gavage, then treat with SLD. Masson, Sirius red, immunohistochemistry, and Western blot were used to detect renal function and fibrosis-related indicators. The mechanism was further validated in vitro experiments. ResultsNetwork pharmacology analysis identified 100 common targets associated with the active components of SLD, including core genes such as AKT1 and CCND1. GO enrichment analysis revealed that the top three biological processes impacted include response to xenobiotic stimulus, response to nutrient levels and response to oxidative stress. These processes involved cellular components such as membrane raft, membrane microdomain and synaptic membrane, with molecular functions predominantly associated with ubiquitin-like protein ligase binding, ubiquitin protein ligase binding, DNA-binding transcription factor binding, and RNA polymerase II-specific DNA binding transcription factor binding. KEGG pathway enrichment analysis indicated potential involvement of pathways like Lipid and atherosclerosis, PI3K-AKT signaling pathway, and prostate cancer are likely involved in the anti-fibrotic effect of SLD. Notably, the highlighted was the AKT/IKKβ/NFκB signaling pathway as a key mechanism. These findings were further confirmed in vivo and in vitro. ConclusionThe SLD effectively ameliorates adenine-induced chronic kidney disease fibrosis in rats, potentially by inhibiting the AKT/IKKβ/NFκB signaling pathway.

Hemin attenuates bleomycin-induced lung fibrosis in mice by regulating the TGF-β1/MAPK and AMPK/SIRT1/PGC-1α/HO-1/NF-κB pathways.

The objective of this study was to investigate the protective effect and potential mechanism of action of hemin on bleomycin-induced pulmonary fibrosis in mice. Male C57BL/6 mice were randomly divided into control, bleomycin and bleomycin + hemin groups. Mice in the bleomycin and bleomycin + hemin groups were injected intratracheally with bleomycin to establish the pulmonary fibrosis model. The bleomycin + hemin group mice were injected intraperitoneally with hemin starting 7 days before modeling until the end of Day 21 after modeling. Pathological changes in lung tissue were assessed by HE and Masson staining. Malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) levels were determined in lung tissue. Immunohistochemistry was performed to assess the expression of α-SMA and collagen I. The serum levels of IL-6 and TNF-α were measured via ELISA. Western blotting was used to determine the expression of TGF-β1, SIRT1, PGC-1α and HO-1 and the phosphorylation levels of p38, ERK1/2, JNK, AMPK and NF-κB p65 in lung tissue. Hemin significantly reduced lung indices, increased terminal body weight. It also significantly increased SOD and CAT activities; decreased MDA, IL-6 and TNF-α levels; reduced the levels of α-SMA and collagen I-positive cells; upregulated SIRT1, PGC-1α and HO-1 expression; promoted AMPK phosphorylation; and downregulated TGF-β1 expression and p38, ERK1/2, JNK and NF-κB p65 phosphorylation. Hemin might attenuate oxidative damage and inflammatory responses and reduces extracellular matrix deposition by regulating the expression and phosphorylation of proteins associated with the TGF-β1/MAPK and AMPK/SIRT1/PGC-1α/HO-1/NF-κB pathways, thereby alleviating bleomycin-induced pulmonary fibrosis.

Yohimbine treatment improves pulmonary fibrosis by attenuating the inflammation and oxidative stress via modulating the MAPK pathway

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disorder characterized by the accumulation of extracellular matrix and collagen, resulting in significant parenchymal scarring and respiratory failure that leads to mortality. Yohimbine (YBH) is an α-2 adrenergic receptor antagonist with anti-oxidant and anti-inflammatory properties. In the current study, we aimed to investigate the anti-inflammatory, anti-oxidant and anti-fibrotic activity of YBH against LPS/TGF-β-induced differentiation in BEAS-2B/LL29 cells and bleomycin (BLMN) induced pulmonary fibrosis model in rats. Network pharmacology, gene expression, Western-blot analysis, immune-cytochemistry/immunohistochemistry, lung function and histology techniques were used to assess the fibrotic marker expression/levels in cells or rat lung tissues. YBH treatment significantly attenuated the LPS-induced pro-inflammatory (identified through a network-pharmacology approach) and oxidative stress markers expression in lung epithelial cells. TGF-β stimulation significantly elevated the fibrotic cascade of markers and treatment with YBH attenuated these markers’ expression/levels. Intra-tracheal administration of BLMN caused a significant elevation of various inflammatory/oxidative stress and fibrotic markers expression in lung tissues and treatment with YBH significantly mitigated the same. Ashcroft score analysis revealed that BLMN exhibited severe distortion of the lungs, elevation of thickness of the alveolar walls and accumulation of collagen in tissues, further treatment with YBH significantly suppressed these events and improved the lung architecture. Lung functional parameters demonstrated that BLMN-induced stiffness and resistance were reduced considerably upon YBH treatment and restored lung function dose-dependently. Overall, this study reveals that YBH treatment significantly attenuated the BLMN-induced fibrosis by regulating the MAPK pathway and provided insightful information for progressing towards translational outcomes.

Ginsenoside compound K restrains hepatic fibrotic response by dual-inhibition of GLS1 and LDHA

BackgroundLiver fibrosis is a dynamic process marked by the accumulation of extracellular matrix due to hepatic stellate cells (HSCs) activation. Ginsenoside compound K (CK), a rare derivative of its parent ginsenosides, is known to significantly ameliorate metabolic disorders. PurposeThe aim of this study was to elucidate the protective effects of CK against liver fibrosis with a focus on metabolic regulation. MethodsWe established liver fibrosis models in mice using carbon tetrachloride (CCl4) challenge, bile duct ligation, or a methionine-choline deficient diet, with continuous oral administration of CK at specified doses and intervals. Simultaneously, we examined the impact of CK on metabolic regulation in cultured HSCs and investigated the associated mechanisms. ResultsCK was found to alleviate liver injury and curb fibrotic responses in mouse models, as well as decrease elevated levels of liver enzyme. Metabolomic analysis in vitro highlighted the crucial roles of pyruvate and glutamine metabolism in metabolic remodeling. Immunohistochemical staining indicated significantly elevated expressions of lactate dehydrogenase A (LDHA) (p = 0.014) and glutaminase 1 (GLS1) (p = 0.024) in liver cirrhosis patients. Comparable alterations were noted in the liver of model mice and in cultured HSCs. Molecular docking and bio-layer interferometry demonstrated that CK interacts with and inhibits the activities of LDHA and GLS1. As expected, CK attenuated glycolysis and glutaminolysis, reducing HSC growth dependently on lactate and α-ketoglutarate (α-KG). Upon HSC activation, metabolism is reprogrammed with Myc as a key regulator, transcriptionally controlling LDHA, GLS1, and glutamine transporters SLC1A5 and SLC38A5. CK inhibited Myc induction, integrating glycolysis and glutaminolysis regulation to counteract the fibrotic response. ConclusionCK inhibited LDHA and GLS1 activities, thereby inhibiting hepatic fibrosis. These findings offer new insights into the role of ginsenosides in liver protection, especially regarding metabolic disorders.

Longitudinal evaluation of liver stiffness reveals hepatic cholesterol as the determinant of fibrosis progression in mice

AimsThe metabolic dysfunction-associated steatotic liver disease (MASLD) affects approximately 30 % of the global population. While excessive consumption of dietary fat induces steatosis, it does not develop fibrosis, indicating that additional factors are required as “second hits” for further progression of MASLD. Here, based on shear wave elastography, we compared the longitudinal patterns of fibrogenesis induced by different diets and show the crucial role of cholesterol accumulation in fibrosis progression. Materials and methodsMice were fed chow, high-fat (HFD), high-fat high-cholesterol (HFHCD), choline-deficient, L-amino acid-defined high-fat (CDAHFD), or 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine diets over 12 weeks. Key findingsMice fed with HFD gained significant amounts of body weight but did not show an increase in liver stiffness. In contrast, the addition of cholesterol in the same diet robustly induced liver stiffening starting from the first week, which was comparable to the CDAHFD-induced fibrosis model. Longitudinal tracking of liver stiffness revealed a two-step progression of fibrosis after prolonged feeding of HFHCD and CDAHFD, likely due to cellular cholesterol accumulation over a certain threshold after the transition point. Biochemical analyses suggested the critical role of both total and hepatic cholesterol accumulation in liver fibrosis development. SignificanceCollectively, our results underscore the significance of cholesterol in liver fibrosis development, also highlighting the benefit of monitoring liver stiffness to understand the pathogenesis of liver fibrosis.

Double Braking Effects of Nanomedicine on Mitochondrial Permeability Transition Pore for Treating Idiopathic Pulmonary Fibrosis.

Mitochondrial permeability transition pore (mPTP) opening is a key hallmark of injured type II alveolar epithelial cells (AECIIs) in idiopathic pulmonary fibrosis (IPF). Inhibiting mPTP opening in AECIIs is considered a potential IPF treatment. Herein, a "double braking" strategy on mPTP by cyclosporin A (CsA) derived ionizable lipid with 3D structure (3D-lipid) binding cyclophilin D (CypD) and siRNA downregulating mitochondrial calcium uniporter (MCU) expression is proposed for treating IPF. 3D-lipid and MCU targeting siRNA (siMCU) are co-assembled to form stable 3D-LNP/siMCU nanoparticles (NPs), along with helper lipids. In vitro results demonstrated that these NPs effectively inhibit mPTP opening by 3D-lipid binding with CypD and siRNA downregulating MCU expression, thereby decreasing damage-associated molecular patterns (DAMPs) release and suppressing epithelial-to-mesenchymal transition (EMT) process in bleomycin-induced A549 cells. In vivo results revealed that 3D-LNP/siMCU NPs effectively ameliorated collagen deposition, pro-fibrotic factors secretion, and fibroblast activation in bleomycin-induced pulmonary fibrosis (PF) mouse models. Moreover, compared to the commercial MC3-based formulation, optimized Opt-MC3/siRNA NPs with incorporating 3D-lipid as the fifth component, showed superior therapeutic efficacy against PF due to their enhanced stability and higher gene silencing efficiency. Overall, the nanomedicine containing 3D-lipid and siMCU will be a promising and potential approach for IPF treatment.

Formononetin Induces Ferroptosis in Activated Hepatic Stellate Cells to Attenuate Liver Fibrosis by Targeting NADPH Oxidase 4.

Ferroptosis is a newly discovered type of cell death that exerts a crucial role in hepatic fibrosis. Formononetin (FMN), a natural isoflavone compound mainly isolated from Spatholobus suberectus Dunn, shows multiple biological activities, including antioxidant, anti-inflammatory, and hepatoprotection. This research aims to explore the regulatory mechanism of FMN in liver fibrosis and the relationship between NADPH oxidase 4 (NOX4) and ferroptosis. The effects of FMN on HSC ferroptosis were evaluated in rat model of CCl4-induced hepatic fibrosis. In vitro, N-acetyl-L-cysteine (NAC) and deferoxamine (DFO) were used to block ferroptosis and then explored the anti-fibrotic effect of FMN. The target protein of FMN was identified by bio-orthogonal click chemistry reaction as well as drug affinity responsive target stability (DARTS), cellular thermal shift (CETSA), surface plasmon resonance (SPR) assays, and isothermal titration calorimetry (ITC) analysis. Here, we found that FMN exerted anti-fibrotic effects via inducing ferroptosis in activated HSCs. NAC and DFO prevented FMN-induced ferroptotic cell death and collagen reduction. Furthermore, FMN bound directly to NOX4 through possible active amino acid residues sites, and increased NOX4-based NADPH oxidase activity to enhance levels of NADP+/NADPH, thus promoting ferroptosis of activated HSCs and relieving liver fibrosis. These results demonstrate that the direct target and mechanism by which FMN improves liver fibrosis, suggesting that FMN may be a natural candidate for further development of liver fibrosis therapy.

The effect of adrenalectomy on bleomycin-induced pulmonary fibrosis in mice.

Progressive lung fibrosis is often fatal and has limited treatment options. Though the mechanisms are poorly understood, fibrosis is increasingly linked with catecholamines such as adrenaline (AD) and noradrenaline (NA), and hormones such as aldosterone (ALD). The essential functions of the adrenal glands include the production of catecholamines and numerous hormones, but the contribution of adrenal glands to lung fibrosis remains less well studied. Here, we characterized the impact of surgical adrenal ablation in the bleomycin model of lung fibrosis. Wild type mice underwent surgical adrenalectomy or sham surgery followed by bleomycin administration. We found that while bleomycin-induced collagen over-deposition in the lung was not affected by adrenalectomy, histologic indices of lung remodeling were ameliorated. These findings were accompanied by a decrease of lymphocytes in bronchoalveolar lavage (BAL) and macrophages in lung tissues, along with concomitant reductions in alpha smooth muscle actin (⍺SMA) and fibronectin. Surgical adrenalectomy completely abrogated AD, not NA, detection in all compartments. Systemic ALD levels were reduced after adrenalectomy while ALD levels in lung tissues remained unaffected. Taken together, these results support the presence of a pulmonary-adrenal axis in lung fibrosis and suggest that adrenalectomy is protective in this disease. Further investigation will be needed to better understand this observation and aid in the development of novel therapeutic strategies.

A novel Non-rodent animal model of hydrochloric acid-induced acute and chronic lung injury

Hydrochloric acid is one of the most prevalent and hazardous chemicals. Accidental spills occur in industrial plants or during transportation. Exposure to HCl can induce severe health impairment, including acute and chronic pulmonary diseases. We have previously described the molecular, structural, and functional aspects of the development of chronic lung injury and pulmonary fibrosis caused by intratracheal instillation of HCl in mice. Although mouse models of human disease have many advantages, rodents are evolutionary far from human and exhibit significant anatomical and physiological differences. Genetic and anatomic similarities between rabbits and humans are significantly higher. Rabbit models of HCl-induced lung injury have been used sparsely to evaluate acute lung injury. In this study, for the first time, we utilized rabbits as a model of HCl-induced pulmonary fibrosis and chronic lung injury. We present molecular, histological, and functional evidence that demonstrate the utility of using this model for studying new pharmaceutics against pulmonary fibrosis.

Modulation of Notch signaling pathway in activated hepatic stellate cells does not ameliorate the outcome of liver fibrosis in carbon tetrachloride and DDC-feeding models.

Recent research suggests a possible role of Notch signaling pathway in development of liver fibrosis, but exact cellular and molecular mechanisms are still not well defined. Methods: We modulated Notch signaling in activated hepatic stellate cells/myofibroblasts using the model of inducible activation or inhibition of Notch signaling selective for αSMA positive cells in murine models of toxic fibrosis induced by CCl4 and cholestatic fibrosis induced by DDC supplemented feeding. Our results confirm that Notch signaling pathway is activated in both CCL4 and DDC model of liver fibrosis and that αSMA positive myofibroblasts are of activated hepatic stellate cells origin. However, neither the inhibition of canonical Notch signaling (in tamoxifen treated αSMACreER/RBP-Jfl/fl mice) nor its overactivation (in tamoxifen treated αSMACreER/NICD1 mice) changed the degree of liver fibrosis in comparison to the control groups in either of the investigated models. Furthermore, after the withdrawal of the fibrogenic treatment the degree of resolution of fibrosis was similar between the animals with Notch overactivation and controls. In addition to genetic manipulation, we investigated the effect of antibodies against NOTCH1 and NOTCH2 on the development of liver fibrosis. Treatment with antibodies had effects on thymus and spleen respectively, but failed to ameliorate liver fibrosis. In conclusion, our data demonstrate that modulation of Notch activity in activated HSC is not sufficient to change the outcome of liver fibrosis. The results obtained with inhibitory antibodies further demonstrate limitations of targeting Notch 1 and 2 receptors as antifibrotic therapy. Notch pathway remains a potential target for the treatment of liver fibrosis, but future studies should be directed to Notch 3 signaling and/or targeting different populations of cells.

Tanshinone IIA Regulates NRF2/NLRP3 Signal Pathway to Restrain Oxidative Stress and Inflammation in Uric Acid-Induced HK-2 Fibrotic Models.

This study aims to investigate the function and potential mechanism of Tanshinone IIA in uric acid-induced HK-2 fibrosis models. An in vitro model of fibrosis was constructed using uric acid stimulation. RT-qPCR and Western blot were used to evaluate the levels of inflammatory cytokines. The detection of ROS and ELISA assay were used to analyze the changes in oxidative stress. Tanshinone IIA inhibited the increase in inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-18 and the formation of NLRP3 inflammasome induced by uric acid stimulation. In addition, Tanshinone IIA treatment reduced the production of ROS and MDA, promoting the expression of SOD and CAT, thereby protecting HK-2 cells from oxidative stress damage. Besides, the expression of TGF-β, FN, and COL-1 was significantly reduced by the treatment of Tanshinone IIA. Mechanistically, Tanshinone IIA inhibited the expression of inflammatory cytokines and the formation of the NLRP3 inflammasome by targeting NRF2. Tanshinone IIA exerts a protective role in uric acid-induced HK-2 fibrosis models by targeting the NRF2-NLRP3 signaling pathway to reduce the occurrence of inflammation and oxidative stress.

Odoratin balances ROS/NO through EZH2/PPARγ signalling to improve myocardial fibrosis.

Myocardial fibrosis is a critical concern in clinical medicine. This study explores the potential of odoratin as a treatment for myocardial fibrosis and investigates its underlying mechanisms. In vitro experiments involved stimulating primary mouse cardiomyocytes with TGF-β1, followed by odoratin treatment, to assess levels of reactive oxygen species (ROS) and nitric oxide (NO). In vivo, a mouse model of myocardial fibrosis was established using abdominal aortic constriction (AAC) and treated with odoratin. ROS and NO levels in myocardial tissue were then evaluated. Immunofluorescence and Western blotting analysis showed that odoratin reduced excess ROS, enhanced NO production and decreased fibrosis-related protein expression in vitro. In vivo, odoratin significantly improved cardiac function, reduced ROS, increased NO levels and mitigated fibrosis in AAC-induced mice. Both in vitro and in vivo, odoratin inhibited the expression of NADPH oxidase 4 and EZH2, while promoting the expression of phosphorylated endothelial nitric oxide synthase (p-eNOS) and PPARγ. The anti-fibrotic effects of odoratin were reversed by PPARγ antagonism, and EZH2 overexpression diminished PPARγ activation by odoratin. These findings suggest that odoratin may combat myocardial fibrosis by balancing ROS and NO through PPARγ activation, with EZH2 inhibition likely playing a key regulatory role.