Modulation by 17,20S(OH)2pD of Fibrosis-Related Mediators in Dermal Fibroblast Lines from Healthy Donors and from Patients with Systemic Sclerosis

We previously demonstrated that the non-calcemic pregnacalciferol (pD) analog 17,20S (OH)₂pD suppressed TGF-β1-induced type I collagen production in cultured normal human dermal fibroblasts. In the present studies, we examined fibroblasts cultured from the lesional skin of patients with systemic sclerosis (scleroderma (SSc)) and assessed the effects of 17,20S(OH)₂pD on fibrosis-related mediators. Dermal fibroblast lines were established from skin biopsies from patients with SSc and healthy controls. Fibroblasts were cultured with either 17,20S(OH)₂pD or 1,25(OH)₂D₃ (positive control) with/without TGF-β1 stimulation and extracted for protein and/or mRNA for collagen synthesis and mediators of fibrosis (MMP-1, TIMP-1, PAI-1, BMP-7, PGES, GLI1, and GLI2). 1 7,20S(OH)₂pD (similar to 1,25(OH)₂D₃) significantly suppressed net total collagen production in TGF-β1-stimulated normal donor fibroblast cultures and in cultures of SSc dermal fibroblasts. 17,20S(OH)₂pD (similar to 1,25(OH)₂D₃) also increased MMP-1, BMP-7, and PGES and decreased TIMP-1 and PAI1 expression in SSc fibroblasts. Although 17,20S(OH)₂pD had no effect on Gli1 or Gli2 in SSc fibroblasts, it increased Gli2 expression when cultured with TGF-β1 in normal fibroblasts. These studies demonstrated that 17,20S(OH)₂pD modulates mediators of fibrosis to favor the reduction of fibrosis and may offer new noncalcemic secosteroidal therapeutic approaches for treating SSc and fibrosis.     

WNT/β-Catenin Signaling Promotes TGF-β-Mediated Activation of Human Cardiac Fibroblasts by Enhancing IL-11 Production

Cardiac fibrosis is a pathological process associated with the development of heart failure. TGF-β and WNT signaling have been implicated in pathogenesis of cardiac fibrosis, however, little is known about molecular cross-talk between these two pathways. The aim of this study was to examine the effect of exogenous canonical WNT3a and non-canonical WNT5a in TGF-β-activated human cardiac fibroblasts. We found that WNT3a and TGF-β induced a β-catenin-dependent response, whereas WNT5a prompted AP-1 activity. TGF-β triggered profibrotic signatures in cardiac fibroblasts, and co-stimulation with WNT3a or co-activation of the β-catenin pathway with the GSK3β inhibitor CHIR99021 enhanced collagen I and fibronectin production and development of active contractile stress fibers. In the absence of TGF-β, neither WNT3a nor CHIR99021 exerted profibrotic responses. On a molecular level, in TGF-β-activated fibroblasts, WNT3a enhanced phosphorylation of TAK1 and production and secretion of IL-11 but showed no effect on the Smad pathway. Neutralization of IL-11 activity with the blocking anti-IL-11 antibody effectively reduced the profibrotic response of cardiac fibroblasts activated with TGF-β and WNT3a. In contrast to canonical WNT3a, co-activation with non-canonical WNT5a suppressed TGF-β-induced production of collagen I. In conclusion, WNT/β-catenin signaling promotes TGF-β-mediated fibroblast-to-myofibroblast transition by enhancing IL-11 production. Thus, the uncovered mechanism broadens our knowledge on a molecular basis of cardiac fibrogenesis and defines novel therapeutic targets for fibrotic heart diseases.     

The Chinese Herbal Medicine Formula mKG Suppresses Pulmonary Fibrosis of Mice Induced by Bleomycin

Pulmonary fibrosis (PF) is a serious progressive lung disease and it originates from inflammation-induced parenchymal injury with excessive extracellular matrix deposition to result in the destruction of the normal lung architecture. Modified Kushen Gancao Formula (mKG), derived from traditional Chinese herbal medicine, has a prominent anti-inflammatory effect. The present study is to explore the inhibitory effects of mKG on bleomycin (BLM)-induced pulmonary fibrosis in mice. mKG significantly decreased pulmonary alveolitis, fibrosis scores, and interleukin-6 (IL-6), interleukin-17 (IL-17), transforming growth factor-β (TGF-β) and hydroxyproline (HYP) levels in lung tissue of mice compared with BLM treatment. It markedly alleviated the increase of HYP content in the lung tissues and pathologic changes of pulmonary fibrosis caused by BLM instillation. In conclusion, mKG has an anti-fibrotic effect and might be employed as a therapeutic candidate agent for attenuating pulmonary fibrosis.     

SB203580—A Potent p38 MAPK Inhibitor Reduces the Profibrotic Bronchial Fibroblasts Transition Associated with Asthma

Subepithelial fibrosis is a component of the remodeling observed in the bronchial wall of patients diagnosed with asthma. In this process, human bronchial fibroblasts (HBFs) drive the fibroblast-to-myofibroblast transition (FMT) in response to transforming growth factor-β₁ (TGF-β₁), which activates the canonical Smad-dependent signaling. However, the pleiotropic properties of TGF-β₁ also promote the activation of non-canonical signaling pathways which can affect the FMT. In this study we investigated the effect of p38 mitogen-activated protein kinase (MAPK) inhibition by SB203580 on the FMT potential of HBFs derived from asthmatic patients using immunocytofluorescence, real-time PCR and Western blotting methods. Our results demonstrate for the first time the strong effect of p38 MAPK inhibition on the TGF-β₁-induced FMT potential throughout the strong attenuation of myofibroblast-related markers: α-smooth muscle actin (α-SMA), collagen I, fibronectin and connexin 43 in HBFs. We suggest the pleiotropic mechanism of SB203580 on FMT impairment in HBF populations by the diminishing of TGF-β/Smad signaling activation and disturbances in the actin cytoskeleton architecture along with the maturation of focal adhesion sites. These observations justify future research on the role of p38 kinase in FMT efficiency and bronchial wall remodeling in asthma.     

Paricalcitol Improves Hypoxia-Induced and TGF-β1-Induced Injury in Kidney Pericytes

Recently, the role of kidney pericytes in kidney fibrosis has been investigated. This study aims to evaluate the effect of paricalcitol on hypoxia-induced and TGF-β1-induced injury in kidney pericytes. The primary cultured pericytes were pretreated with paricalcitol (20 ng/mL) for 90 min before inducing injury, and then they were exposed to TGF-β1 (5 ng/mL) or hypoxia (1% O₂ and 5% CO₂). TGF-β1 increased α-SMA and other fibrosis markers but reduced PDGFRβ expression in pericytes, whereas paricalcitol reversed the changes. Paricalcitol inhibited the TGF-β1-induced cell migration of pericytes. Hypoxia increased TGF-β1, α-SMA and other fibrosis markers but reduced PDGFRβ expression in pericyte, whereas paricalcitol reversed them. Hypoxia activated the HIF-1α and downstream molecules including prolyl hydroxylase 3 and glucose transporter-1, whereas paricalcitol attenuated the activation of the HIF-1α-dependent molecules and TGF-β1/Smad signaling pathways in hypoxic pericytes. The gene silencing of HIF-1α vanished the hypoxia-induced TGF-β1, α-SMA upregulation, and PDGFRβ downregulation. The effect of paricalcitol on the HIF-1α-dependent changes of fibrosis markers was not significant after the gene silencing of HIF-1α. In addition, hypoxia aggravated the oxidative stress in pericytes, whereas paricalcitol reversed the oxidative stress by increasing the antioxidant enzymes in an HIF-1α-independent manner. In conclusion, paricalcitol improved the phenotype changes of pericyte to myofibroblast in TGF-β1-stimulated pericytes. In addition, paricalcitol improved the expression of fibrosis markers in hypoxia-exposed pericytes both in an HIF-1α-dependent and independent manner.     

Ghrelin Attenuates Liver Fibrosis through Regulation of TGF-β1 Expression and Autophagy

Ghrelin is a stomach-derived growth hormone secretagogue that promotes various physiological effects, including energy metabolism and amelioration of inflammation. The purpose of this study was to investigate the protective mechanism of ghrelin against liver fibrosis. Liver fibrosis was induced in C57BL/6 mice by intraperitoneal injection of CCl₄ (2.0 mL/kg of 10% CCl₄ v/v solution in peanut oil) two times per week for eight weeks. Ghrelin (10 μg/kg) was intraperitoneally injected two times per week for eight weeks. A second murine liver fibrosis model was induced by bile duct ligation (BDL) and concurrent ghrelin administration for four weeks. Hematoxylin eosin (H&E), and Masson’s trichrome were used to detect pathological changes to liver tissue. Western blotting was used to detect protein levels of transforming growth factor (TGF)-β1, phosphorylated Smad3 (p-Smad3), I-collage, α-smooth muscle actin (α-SMA), matrix metalloproteinases (MMPs) 2, tissue inhibitor of matrix metalloproteinases (TIMPs) 1, phosphorylated NF-κB (p-NF-κB), and microtubule-associated protein light chain 3 (LC3). In addition, qRT-PCR was used to detect mRNA levels of TGF-β1, I-collage, α-SMA, MMP2, TIMP1 and LC3, while levels of TGF-β1, p-Smad3, I-collage, α-SMA, and LC3 were detected immunohistochemically. Levels of aspartate aminotransferase and alanine aminotransferase were significantly decreased by ghrelin treatment. Ghrelin administration also significantly reduced the extent of pathological changes in both murine liver fibrosis models. Expression levels of I-collage and α-SMA in both models were clearly reduced by ghrelin administration. Furthermore, ghrelin treatment decreased protein expression of TGF-β1 and p-Smad3. The protein levels of NF-κB and LC3 were increased in the CCl₄- and BDL-treatment groups but were significantly reduced following ghrelin treatment. In addition, ghrelin inhibited extracellular matrix formation by decreasing NF-κB expression and maintaining the balance between MMP2 and TIMP1. Our results demonstrated that ghrelin attenuates liver fibrosis via inhibition of the TGF-β1/Smad3 and NF-κB signaling pathways, as well as autophagy suppression.     

Chrysin Ameliorates Cyclosporine-A-Induced Renal Fibrosis by Inhibiting TGF-β1-Induced Epithelial–Mesenchymal Transition

Cyclosporine A (CsA) is a nephrotoxicant that causes fibrosis via induction of epithelial–mesenchymal transition (EMT). The flavonoid chrysin has been reported to have anti-fibrotic activity and inhibit signaling pathways that are activated during EMT. This study investigated the nephroprotective role of chrysin in the prevention of CsA-induced renal fibrosis and elucidated a mechanism of inhibition against CsA-induced EMT in proximal tubule cells. Treatment with chrysin prevented CsA-induced renal dysfunction in Sprague Dawley rats measured by blood urea nitrogen (BUN), serum creatinine and creatinine clearance. Chrysin inhibited CsA-induced tubulointerstitial fibrosis, characterized by reduced tubular damage and collagen deposition. In vitro, chrysin significantly inhibited EMT in LLC-PK₁ cells, evidenced by inhibition of cell migration, decreased collagen expression, reduced presence of mesenchymal markers and elevated epithelial junction proteins. Furthermore, chrysin co-treatment diminished CsA-induced TGF-β₁ signaling pathways, decreasing Smad 3 phosphorylation which lead to a subsequent reduction in Snail expression. Chrysin also inhibited activation of the Akt/ GSK-3β pathway. Inhibition of both pathways diminished the cytosolic accumulation of β-catenin, a known trigger for EMT. In conclusion, flavonoids such as chrysin offer protection against CsA-induced renal dysfunction and interstitial fibrosis. Chrysin was shown to inhibit CsA-induced TGF-β₁-dependent EMT in proximal tubule cells by modulation of Smad-dependent and independent signaling pathways.     

COVID-19: Immunohistochemical Analysis of TGF-β Signaling Pathways in Pulmonary Fibrosis

Acute respiratory distress syndrome (ARDS) followed by repair with lung remodeling is observed in COVID-19. These findings can lead to pulmonary terminal fibrosis, a form of irreversible sequelae. There is evidence that TGF-β is intimately involved in the fibrogenic process. When activated, TGF-β promotes the differentiation of fibroblasts into myofibroblasts and regulates the remodeling of the extracellular matrix (ECM). In this sense, the present study evaluated the histopathological features and immunohistochemical biomarkers (ACE-2, AKT-1, Caveolin-1, CD44v6, IL-4, MMP-9, α-SMA, Sphingosine-1, and TGF-β1 tissue expression) involved in the TGF-β1 signaling pathways and pulmonary fibrosis. The study consisted of 24 paraffin lung samples from patients who died of COVID-19 (COVID-19 group), compared to 10 lung samples from patients who died of H1N1pdm09 (H1N1 group) and 11 lung samples from patients who died of different causes, with no lung injury (CONTROL group). In addition to the presence of alveolar septal fibrosis, diffuse alveolar damage (DAD) was found to be significantly increased in the COVID-19 group, associated with a higher density of Collagen I (mature) and III (immature). There was also a significant increase observed in the immunoexpression of tissue biomarkers ACE-2, AKT-1, CD44v6, IL-4, MMP-9, α-SMA, Sphingosine-1, and TGF-β1 in the COVID-19 group. A significantly lower expression of Caveolin-1 was also found in this group. The results suggest the participation of TGF-β pathways in the development process of pulmonary fibrosis. Thus, it would be plausible to consider therapy with TGF-β inhibitors in those patients recovered from COVID-19 to mitigate a possible development of pulmonary fibrosis and its consequences for post-COVID-19 life quality.     

Exercise Training Alleviates Cardiac Fibrosis through Increasing Fibroblast Growth Factor 21 and Regulating TGF-β1-Smad2/3-MMP2/9 Signaling in Mice with Myocardial Infarction

Exercise training has been reported to alleviate cardiac fibrosis and ameliorate heart dysfunction after myocardial infarction (MI), but the molecular mechanism is still not fully clarified. Fibroblast growth factor 21 (FGF21) exerts a protective effect on the infarcted heart. This study investigates whether exercise training could increase FGF21 protein expression and regulate the transforming growth factor-β1 (TGF-β1)-Smad2/3-MMP2/9 signaling pathway to alleviate cardiac fibrosis following MI. Male wild type (WT) C57BL/6J mice and Fgf21 knockout (Fgf21 KO) mice were used to establish the MI model and subjected to five weeks of different types of exercise training. Both aerobic exercise training (AET) and resistance exercise training (RET) significantly alleviated cardiac dysfunction and fibrosis, up-regulated FGF21 protein expression, inhibited the activation of TGF-β1-Smad2/3-MMP2/9 signaling pathway and collagen production, and meanwhile, enhanced antioxidant capacity and reduced cell apoptosis in the infarcted heart. In contrast, knockout of Fgf21 weakened the cardioprotective effects of AET after MI. In vitro, cardiac fibroblasts (CFs) were isolated from neonatal mice hearts and treated with H₂O₂ (100 μM, 6 h). Recombinant human FGF21 (rhFGF21, 100 ng/mL, 15 h) and/or 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR, 1 mM, 15 h) inhibited H₂O₂-induced activation of the TGF-β1-Smad2/3-MMP2/9 signaling pathway, promoted CFs apoptosis and reduced collagen production. In conclusion, exercise training increases FGF21 protein expression, inactivates the TGF-β1-Smad2/3-MMP2/9 signaling pathway, alleviates cardiac fibrosis, oxidative stress, and cell apoptosis, and finally improves cardiac function in mice with MI. FGF21 plays an important role in the anti-fibrosis effect of exercise training.     

AP2M1 Supports TGF-β Signals to Promote Collagen Expression by Inhibiting Caveolin Expression

The extracellular matrix (ECM) is important for normal development and disease states, including inflammation and fibrosis. To understand the complex regulation of ECM, we performed a suppressor screening using Caenorhabditis elegans expressing the mutant ROL-6 collagen protein. One cuticle mutant has a mutation in dpy-23 that encodes the μ2 adaptin (AP2M1) of clathrin-associated protein complex II (AP-2). The subsequent suppressor screening for dpy-23 revealed the lon-2 mutation. LON-2 functions to regulate body size through negative regulation of the tumor growth factor-beta (TGF-β) signaling pathway responsible for ECM production. RNA-seq analysis showed a dominant change in the expression of collagen genes and cuticle components. We noted an increase in the cav-1 gene encoding caveolin-1, which functions in clathrin-independent endocytosis. By knockdown of cav-1, the reduced TGF-β signal was significantly restored in the dpy-23 mutant. In conclusion, the dpy-23 mutation upregulated cav-1 expression in the hypodermis, and increased CAV-1 resulted in a decrease of TβRI. Finally, the reduction of collagen expression including rol-6 by the reduced TGF-β signal influenced the cuticle formation of the dpy-23 mutant. These findings could help us to understand the complex process of ECM regulation in organism development and disease conditions.     

Neoagarooligosaccharide Protects against Hepatic Fibrosis via Inhibition of TGF-β/Smad Signaling Pathway

Hepatic fibrosis occurs when liver tissue becomes scarred from repetitive liver injury and inflammatory responses; it can progress to cirrhosis and eventually to hepatocellular carcinoma. Previously, we reported that neoagarooligosaccharides (NAOs), produced by the hydrolysis of agar by β-agarases, have hepatoprotective effects against acetaminophen overdose-induced acute liver injury. However, the effect of NAOs on chronic liver injury, including hepatic fibrosis, has not yet been elucidated. Therefore, we examined whether NAOs protect against fibrogenesis in vitro and in vivo. NAOs ameliorated PAI-1, α-SMA, CTGF and fibronectin protein expression and decreased mRNA levels of fibrogenic genes in TGF-β-treated LX-2 cells. Furthermore, downstream of TGF-β, the Smad signaling pathway was inhibited by NAOs in LX-2 cells. Treatment with NAOs diminished the severity of hepatic injury, as evidenced by reduction in serum alanine aminotransferase and aspartate aminotransferase levels, in carbon tetrachloride (CCl₄)-induced liver fibrosis mouse models. Moreover, NAOs markedly blocked histopathological changes and collagen accumulation, as shown by H&E and Sirius red staining, respectively. Finally, NAOs antagonized the CCl₄-induced upregulation of the protein and mRNA levels of fibrogenic genes in the liver. In conclusion, our findings suggest that NAOs may be a promising candidate for the prevention and treatment of chronic liver injury via inhibition of the TGF-β/Smad signaling pathway.     

Curcumin Suppresses TGF-β1-Induced Myofibroblast Differentiation and Attenuates Angiogenic Activity of Orbital Fibroblasts

Orbital fibrosis, a hallmark of tissue remodeling in Graves’ ophthalmopathy (GO), is a chronic, progressive orbitopathy with few effective treatments. Orbital fibroblasts are effector cells, and transforming growth factor β1 (TGF-β1) acts as a critical inducer to promote myofibroblast differentiation and subsequent tissue fibrosis. Curcumin is a natural compound with anti-fibrotic activity. This study aims to investigate the effects of curcumin on TGF-β1-induced myofibroblast differentiation and on the pro-angiogenic activities of orbital fibroblasts. Orbital fibroblasts from one healthy donor and three patients with GO were collected for primary cell culture and subjected to myofibroblast differentiation under the administration of 1 or 5 ng/mL TGF-β1 for 24 h. The effects of curcumin on TGF-β1-induced orbital fibroblasts were assessed by measuring the cellular viability and detecting the expression of myofibroblast differentiation markers, including connective tissue growth factor (CTGF) and α-smooth muscle actin (α-SMA). The pro-angiogenic potential of curcumin-treated orbital fibroblasts was evaluated by examining the transwell migration and tube-forming capacities of fibroblast-conditioned EA.hy926 and HMEC-1 endothelial cells. Treatment of orbital fibroblasts with curcumin inhibited the TGF-β1 signaling pathway and attenuated the expression of CTGF and α-SMA induced by TGF-β1. Curcumin, at the concentration of 5 μg/mL, suppressed 5 ng/mL TGF-β1-induced pro-angiogenic activities of orbital fibroblast-conditioned EA hy926 and HMEC-1 endothelial cells. Our findings suggest that curcumin reduces the TGF-β1-induced myofibroblast differentiation and pro-angiogenic activity in orbital fibroblasts. The results support the potential application of curcumin for the treatment of GO.     

Loss of SP-A in the Lung Exacerbates Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating and common chronic lung disease that is pathologically characterized by the destruction of lung architecture and the accumulation of extracellular matrix in the lung. Previous studies have shown an association between lung surfactant protein (SP) and the pathogenesis of IPF, as demonstrated by mutations and the altered expression of SP in patients with IPF. However, the role of SP in the development of lung fibrosis is poorly understood. In this study, the role of surfactant protein A (SP-A) was explored in experimental lung fibrosis induced with a low or high dose of bleomycin (BLM) and CRISPR/Cas9-mediated genetic deletion of SP-A. Our results showed that lung SP-A deficiency in mice promoted the development of fibrotic damage and exacerbated inflammatory responses to the BLM challenge. In vitro experiments with murine lung epithelial LA-4 cells demonstrated that in response to transforming growth factor-β1 (TGF-β1), LA-4 cells had a decreased protein expression of SP-A. Furthermore, exogenous SP administration to LA-4 cells inhibited the TGF-β1-induced upregulation of fibrotic markers. Overall, these findings suggest a novel antifibrotic mechanism of SP-A in the development of lung fibrosis, which indicates the therapeutic potential of the lung SP-A in preventing the development of IPF.     

Oncostatin M Counteracts the Fibrotic Effects of TGF-β1 and IL-4 on Nasal-Polyp-Derived Fibroblasts: A Control of Fibrosis in Chronic Rhinosinusitis with Nasal Polyps?

Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with inflammation and tissue remodeling including myofibroblasts differentiation and extracellular matrix (ECM) deposition mediated by TGF-β1 and IL-4. Oncostatin M (OSM) is a cytokine involved in fibrotic processes in other cellular subtypes. We investigated the mechanisms of action of OSM in the fibrosis process associated with CRSwNP. The expression of IL-4, OSM and TGF-β1 was assessed by RT-qPCR. Primary human cultures of nasal-polyp-derived fibroblasts were established and stimulated by TGF-β1 and/or IL-4 and/or OSM. The expression of ECM components and αSMA was determined by RT-qPCR and Western blot. TGF-β1-Smad3 signaling was investigated by immunofluorescence. TGF-β1, IL-4 and OSM as well as αSMA were overexpressed in nasal polyps when compared to noninflammatory nasal mucosa. In TGF-β1-stimulated nasal-polyp-derived fibroblasts, ECM genes and αSMA gene and protein were overexpressed, as well as αSMA in IL-4-stimulated fibroblasts. OSM counteracted the profibrotic effect of TGF-β1 on ECM components and αSMA. TGF-β1-induced nuclear translocation of Smad3 was completely reversed by OSM. OSM counteracts the profibrotic effect of IL-4 and also TGF-β1, by inhibiting the nuclear translocation of Smad3. We suggest OSM could be an efficient tool to protect against fibrosis in CRSwNP.     

Blocking the Function of Inflammatory Cytokines and Mediators by Using IL-10 and TGF-β: A Potential Biological Immunotherapy for Intervertebral Disc Degeneration in a Beagle Model

The debilitating effects of lower back pain are a major health issue worldwide. A variety of factors contribute to this, and oftentimes intervertebral disk degeneration (IDD) is an underlying cause of this disorder. Inflammation contributes to IDD, and inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, play key roles in the pathology of IDD. Therefore, the development of treatments that inhibit the expression and/or effects of TNF-α and IL-1β in IDD patients should be a promising therapeutic approach to consider. This study characterized the potential to suppress inflammatory cytokine production in degenerative intervertebral disc (NP) cells by treatment with IL-10 and TGF-β in a canine model of IDD. IDD was induced surgically in six male beagles, and degenerative NP cells were isolated and cultured for in vitro studies on cytokine production. Cultured degenerative NP cells were divided into four experimental treatment groups: untreated control, IL-10-treated, TGF-β-treated, and IL-10- plus TGF-β-treated cells. Cultured normal NP cells served as a control group. TNF-α expression was evaluated by fluorescence activated cell sorting (FACS) analysis and enzyme-linked immunosorbent assay (ELISA); moreover, ELISA and real-time PCR were also performed to evaluate the effect of IL-10 and TGF-β on NP cell cytokine expression in vitro. Our results demonstrated that IL-10 and TGF-β treatment suppressed the expression of IL-1β and TNF-α and inhibited the development of inflammatory responses. These data suggest that IL-10 and TGF-β should be evaluated as therapeutic approaches for the treatment of lower back pain mediated by IDD.     

Honokiol Acts as a Potent Anti-Fibrotic Agent in the Liver through Inhibition of TGF-β1/SMAD Signaling and Autophagy in Hepatic Stellate Cells

Chronic liver injury may result in hepatic fibrosis, which can progress to cirrhosis and eventually liver failure. There are no drugs that are specifically approved for treating hepatic fibrosis. The natural product honokiol (HNK), a bioactive compound extracted from Magnolia grandiflora, represents a potential tool in the management of hepatic fibrosis. Though HNK has been reported to exhibit suppressive effects in a rat fibrosis model, the mechanisms accounting for this suppression remain unclear. In the present study, the anti-fibrotic effects of HNK on the liver were evaluated in vivo and in vitro. In vivo studies utilized a murine liver fibrosis model, in which fibrosis is induced by treatment with carbon tetrachloride (CCl₄). For in vitro studies, LX-2 human hepatic stellate cells (HSCs) were treated with HNK, and expression of markers of fibrosis, cell viability, the transforming growth factor-β (TGF-β1)/SMAD signaling pathway, and autophagy were analyzed. HNK was well tolerated and significantly attenuated CCl₄-induced liver fibrosis in vivo. Moreover, HNK decreased HSC activation and collagen expression by downregulating the TGF-β1/SMAD signaling pathway and autophagy. These results suggest that HNK is a new potential candidate for the treatment of hepatic fibrosis through suppressing both TGF-β1/SMAD signaling and autophagy in HSCs.     

Effect of Fiber Length on Carbon Nanotube-Induced Fibrogenesis

Given their extremely small size and light weight, carbon nanotubes (CNTs) can be readily inhaled by human lungs resulting in increased rates of pulmonary disorders, particularly fibrosis. Although the fibrogenic potential of CNTs is well established, there is a lack of consensus regarding the contribution of physicochemical attributes of CNTs on the underlying fibrotic outcome. We designed an experimentally validated in vitro fibroblast culture model aimed at investigating the effect of fiber length on single-walled CNT (SWCNT)-induced pulmonary fibrosis. The fibrogenic response to short and long SWCNTs was assessed via oxidative stress generation, collagen expression and transforming growth factor-beta (TGF-β) production as potential fibrosis biomarkers. Long SWCNTs were significantly more potent than short SWCNTs in terms of reactive oxygen species (ROS) response, collagen production and TGF-β release. Furthermore, our finding on the length-dependent in vitro fibrogenic response was validated by the in vivo lung fibrosis outcome, thus supporting the predictive value of the in vitro model. Our results also demonstrated the key role of ROS in SWCNT-induced collagen expression and TGF-β activation, indicating the potential mechanisms of length-dependent SWCNT-induced fibrosis. Together, our study provides new evidence for the role of fiber length in SWCNT-induced lung fibrosis and offers a rapid cell-based assay for fibrogenicity testing of nanomaterials with the ability to predict pulmonary fibrogenic response in vivo.     

Role of Human Primary Renal Fibroblast in TGF-β1-Mediated Fibrosis-Mimicking Devices

Renal fibrosis is a progressive chronic kidney disease that ultimately leads to end-stage renal failure. Despite several approaches to combat renal fibrosis, an experimental model to evaluate currently available drugs is not ideal. We developed fibrosis-mimicking models using three-dimensional (3D) co-culture devices designed with three separate layers of tubule interstitium, namely, epithelial, fibroblastic, and endothelial layers. We introduced human renal proximal tubular epithelial cells (HK-2), human umbilical-vein endothelial cells, and patient-derived renal fibroblasts, and evaluated the effects of transforming growth factor-β (TGF-β) and TGF-β inhibitor treatment on this renal fibrosis model. The expression of the fibrosis marker alpha smooth muscle actin upon TGF-β1 treatment was augmented in monolayer-cultured HK-2 cells in a 3D disease model. In the vascular compartment of renal fibrosis models, the density of vessels was increased and decreased in the TGF-β-treated group and TGF-β-inhibitor treatment group, respectively. Multiplex ELISA using supernatants in the TGF-β-stimulating 3D models showed that pro-inflammatory cytokine and growth factor levels including interleukin-1 beta, tumor necrosis factor alpha, basic fibroblast growth factor, and TGF-β1, TGF-β2, and TGF-β3 were increased, which mimicked the fibrotic microenvironments of human kidneys. This study may enable the construction of a human renal fibrosis-mimicking device model beyond traditional culture experiments.     

Oxidative Stress Enhances the TGF-β2-RhoA-MRTF-A/B Axis in Cells Entering Endothelial-Mesenchymal Transition

Around 45% of deaths in the EU and the US are due to fibrotic diseases. Although myofibroblasts are detected in various fibrotic tissues, they are mostly transdifferentiated from endothelial cells during the endothelial-mesenchymal transition (EndMT) induced by tumor growth factor-beta (TGF-β) family members. Growing evidence indicates that oxidative stress might enhance the sensitivity and the effects of TGF-β stimulation; however, the molecular mechanisms involved in the coordination of oxidative stress and TGF-β inductions remain poorly understood. Our findings indicate for the first time that oxidative stress enhances mesenchymal trans-differentiation of human microvascular endothelial cells (HMEC-1 cells) and that the oxidative stress-dependent TGF-β2-RhoA/Rac1-MRTF-A axis is critical for the induction of later stages of EndMT. This additive effect was manifested in TGF-β1-stimulated and Snail-overexpressed cells, where it caused higher cell elongation and faster migration on collagen I layers. Additionally, Western blot assay indicated the presence of alterations in cell contraction and EndMT markers. We conclude that complex anti-fibrotic therapies based on the inhibition of MRTF activities and oxidative stress might be an attractive target for fibrosis treatment.     

Corilagin Attenuates Aerosol Bleomycin-Induced Experimental Lung Injury

Idiopathic pulmonary fibrosis (IPF) is a progressing lethal disease with few clinically effective therapies. Corilagin is a tannin derivative which shows anti-inflammatory and antifibrotics properties and is potentiated in treating IPF. Here, we investigated the effect of corilagin on lung injury following bleomycin exposure in an animal model of pulmonary fibrosis. Corilagin abrogated bleomycin-induced lung fibrosis as assessed by H&E; Masson’s trichrome staining and lung hydroxyproline content in lung tissue. Corilagin reduced the number of apoptotic lung cells and prevented lung epithelial cells from membrane breakdown, effluence of lamellar bodies and thickening of the respiratory membrane. Bleomycin exposure induced expression of MDA, IKKα, phosphorylated IKKα (p-IKKα), NF-κB P65, TNF-α and IL-1β, and reduced I-κB expression in mice lung tissue or in BALF. These changes were reversed by high-dose corilagin (100 mg/kg i.p) more dramatically than by low dose (10 mg/kg i.p). Last, corilagin inhibits TGF-β1 production and α-SMA expression in lung tissue samples. Taken together, these findings confirmed that corilagin attenuates bleomycin-induced epithelial injury and fibrosis via inactivation of oxidative stress, proinflammatory cytokine release and NF-κB and TGF-β1 signaling. Corilagin may serve as a promising therapeutic agent for pulmonary fibrosis.