Proliferation Of Pulmonary Fibroblasts Research Articles

Toosendanin Restrains Idiopathic Pulmonary Fibrosis by Inhibiting ZEB1/CTBP1 Interaction.

Extensive deposition of extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) is due to hyperactivation and proliferation of pulmonary fibroblasts. However, the exact mechanism is not clear. This study focused on the role of CTBP1 in lung fibroblast function, elaborated its regulation mechanism, and analyzed the relationship between CTBP1 and ZEB1. Meanwhile, the antipulmonary fibrosis effect and its molecular mechanism of Toosendanin were studied. Human IPF fibroblast cell lines (LL-97A and LL-29) and normal fibroblast cell lines (LL-24) were cultured in vitro. The cells were stimulated with FCS, PDGF-BB, IGF-1, and TGF-β1, respectively. BrdU detected cell proliferation. The mRNA expression of CTBP1 and ZEB1 was detected by QRT-PCR. Western blotting was used to detect the expression of COL1A1, COL3A1, LN, FN, and α-SMA proteins. An animal model of pulmonary fibrosis was established to analyze the effects of CTBP1 silencing on pulmonary fibrosis and lung function in mice. CTBP1 was up-regulated in IPF lung fibroblasts. Silencing CTBP1 inhibits growth factor-driven proliferation and activation of lung fibroblasts. Overexpression of CTBP1 promotes growth factor-driven proliferation and activation of lung fibroblasts. Silencing CTBP1 reduced the degree of pulmonary fibrosis in mice with pulmonary fibrosis. Western blot, CO-IP, and BrdU assays confirmed that CTBP1 interacts with ZEB1 and promotes the activation of lung fibroblasts. Toosendanin can inhibit the ZEB1/CTBP1protein interaction and further inhibit the progression of pulmonary fibrosis. CTBP1 can promote the activation and proliferation of lung fibroblasts through ZEB1. CTBP1 promotes lung fibroblast activation through ZEB1, thereby increasing excessive deposition of ECM and aggravating IPF. Toosendanin may be a potential treatment for pulmonary fibrosis. The results of this study provide a new basis for clarifying the molecular mechanism of pulmonary fibrosis and developing new therapeutic targets.

Overexpression of hsa_circ_0001861 inhibits pulmonary fibrosis through targeting miR-296-5p/BCL-2 binding component 3 axis.

Pulmonary fibrosis is a progressive lung disorder. Evidence has shown that hsa_circular (circ)RNA_0001861 is dysregulated in pulmonary fibrosis. However, the detailed function of hsa_circRNA_0001861 in pulmonary fibrosis remains unexplored. To investigate the function of hsa_circRNA_0001861 in pulmonary fibrosis, human pulmonary fibroblasts in vitro were used, and cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) staining were performed to assess cell viability and proliferation, respectively. Western blot analysis and reverse transcription-quantitative PCR (RT-qPCR) were used to evaluate protein and mRNA levels. Meanwhile, the relationship among hsa_circRNA_0001861, miR-296-5p and BCL-2 binding component 3 (BBC3) was investigated by RNA pull-down assays. Furthermore, an in vivo model of lung fibrosis was constructed to assess the function of hsa_circRNA_0001861 in lung fibrosis. The data revealed that TGF‑β1 significantly increased the proliferation of pulmonary fibroblasts, while this phenomenon was markedly abolished by hsa_circRNA_0001861 overexpression. hsa_circRNA_0001861 overexpression markedly inhibited TGF‑β1‑induced fibrosis in pulmonary fibroblasts through the mediation of α-smooth muscle actin, E-cadherin, collagen III and fibronectin 1. Meanwhile, hsa_circRNA_0001861 could bind with miR-296-5p, and BBC3 was identified to be the downstream mRNA of miR-296-5p. In addition, the upregulation of hsa_circRNA_0001861 clearly reversed TGF‑β1‑induced fibrosis and proliferation in pulmonary fibroblasts through the upregulation of BBC3. Furthermore, hsa_circRNA_0001861 upregulation markedly alleviated pulmonary fibrosis in vivo. Hsa_circRNA_0001861 upregulation attenuated pulmonary fibrosis by modulating the miR-296-5p/BBC3 axis. Hence, the present study may provide some insights for the discovery of new methods against pulmonary fibrosis.

Fibroblast growth factor 2 acts as an upstream regulator of inhibition of pulmonary fibroblast activation.

Fibroblast growth factor (FGF) signaling plays a crucial role in lung development and repair. Fibroblast growth factor 2 (FGF2) can inhibit fibrotic gene expression and suppress the differentiation of pulmonary fibroblasts (PFs) into myofibroblasts in vitro, suggesting that FGF2 is a potential target for inhibiting pulmonary fibrosis. To gain deeper insights into the molecular mechanism underlying FGF2-mediated regulation of PFs, we performed mRNA sequencing analysis to systematically and globally uncover the regulated genes and biological functions of FGF2 in PFs. Gene Ontology analysis revealed that the differentially expressed genes regulated by FGF2 were enriched in multiple cellular functions including extracellular matrix (ECM) organization, cytoskeleton formation, β-catenin-independent Wnt signaling pathway, supramolecular fiber organization, epithelial cell proliferation, and cell adhesion. Gene Set Enrichment Analysis and cellular experiments confirmed that FGF2 can suppress ECM and actin filament organization and increase PFs proliferation. Taken together, these findings indicate that FGF2 acts as an upstream regulator of the inhibition of PFs activation and may play a regulatory role in pulmonary fibrosis.

IL33-mediated NPM1 promotes fibroblast-to-myofibroblast transition via ERK/AP-1 signaling in silica-induced pulmonary fibrosis.

Silicosis is a global occupational pulmonary disease due to the accumulation of silica dust in the lung. Lacking effective clinical drugs makes the treatment of this disease quite challenging in clinics largely because the pathogenic mechanisms remain obscure. Interleukin 33 (IL33), a pleiotropic cytokine, could promote wound healing and tissue repair via the receptor ST2. However, the mechanisms governing the involvement of IL33 in silicosis progression remain to be further explored. Here, we demonstrated that the IL33 levels in the lung sections were significantly overexpressed after bleomycin and silica treatment. Chromatin immunoprecipitation assay, knockdown, and reverse experiments were performed in lung fibroblasts to prove gene interaction following exogenous IL33 treatment or cocultured with silica-treated lung epithelial cells. Mechanistically, we illustrated that silica-stimulated lung epithelial cells secreted IL33 and further promoted the activation, proliferation, and migration of pulmonary fibroblasts by activating the ERK/AP-1/NPM1 signaling pathway in vitro. And more, treatment with NPM1 siRNA-loaded liposomes markedly protected mice from silica-induced pulmonary fibrosis in vivo. In conclusion, the involvement of NPM1 in the progression of silicosis is regulated by the IL33/ERK/AP-1 signaling axis, which is the potential therapeutic target candidate in developing novel antifibrotic strategies for pulmonary fibrosis.

Eosinophils promote proliferation of pulmonary structural cells by stimulating extracellular matrix production in asthma

Introduction. Eosinophilic asthma can be characterized by intense infiltration of eosinophils into the airways, where they abundantly release various cytokines, chemokines and growth factors. Airway remodeling is closely related to increased airway smooth muscle (ASM) mass; however, another important feature could be changes in extracellular matrix (ECM) homeostasis, driven by the production of ECM proteins and metalloproteinases by lung structural cells. Aim. To evaluate and compare eosinophil adhesion to ASM cells and pulmonary fibroblasts or their secreted ECM and the effect on pulmonary structural cell autocrine proliferation adjustment in asthma. Methods. A total of 12 allergic asthma (AA), 8 severe eosinophilic asthma (SEA) patients and 11 healthy subjects (HS) were examined. Blood eosinophils were isolated using high density Ficoll centrifugation and magnetic separation. For each study individual combined cell cultures (co-cultures) between isolated eosinophils and pulmonary structural cells or their secreted ECM were prepared. ECM purification was performed using ammonium hydroxide (NH4OH) based cell lysis. Eosinophils adhesion was evaluated by measuring eosinophils peroxidase activity in co-cultures; pulmonary structural cell proliferation was measured by AlamarBlue assay. Results. In all investigated groups eosinophil adhesion to ECM was significantly increased compared to eosinophil adhesion to ASM cells and pulmonary fibroblasts (p < 0.01). Eosinophil adhesion to ASM cells and pulmonary fibroblasts or their secreted ECM in asthma groups was significantly enhanced compared to HS group (p < 0.05). Isolated ASM cell or pulmonary fibroblast’ produced ECM after co-cultures with AA, SEA and HS eosinophils promoted newly seeded ASM cell and pulmonary fibroblast proliferation (p < 0.01) with stronger effect in both asthma groups. ECM, isolated after combined cultures with AA, SEA or HS eosinophils and the blood serum of the study patient also significantly promoted the proliferation of ASM cells and pulmonary fibroblasts (p < 0.01). Isolated ASM cell or pulmonary fibroblast’ produced ECM after co-cultures with AA and SEA eosinophils had a stronger effect to newly seeded pulmonary structural cell proliferation than after co-cultures with HS eosinophils. Conclusions. Eosinophils demonstrate enhanced adhesion to ECM components than to ASM cells or pulmonary fibroblasts, and adhesion further increases in asthma. Pulmonary structural cells promote their proliferation in autocrine manner via released ECM components after incubation with eosinophils in asthma.

RNF2 mediates pulmonary fibroblasts activation and proliferation by regulating mTOR and p16-CDK4-Rb1 signaling pathway.

Pulmonary fibrosis (PF) is a chronic, progressive interstitial lung disease with unknown etiology, associated with increasing morbidity and pessimistic prognosis. Pulmonary fibroblasts (PFbs) are the key effector cells of PF, in which abnormal activation and proliferation is an important pathogenesis of PF. Ring finger protein 2 (RNF2), is identified as the catalytic subunit of poly-comb repressive complex 1, which is closely related to occurrence and development of lung cancer, but its function in PF has not been revealed. In this paper, we sought to identify the regulatory role of RNF2 in lung fibrogenesis and its underlying mechanisms. The expression of RNF2 in lung fibrosis tissue (human and Bleomycin-induced mouse) and cell model (TGF-β1-induced HFL1 cells) was examined by immunoblotting analysis and immunofluorescence. Western blot, qRT-PCR were performed to evaluate the expression of pro-fibrogenic cytokines (including α-SMA, ECM and MMPs/ TIMPs) induced by TGF-β1 in HFL1 cells. Cell proliferation, cycle progression and apoptosis were examined by fow cytometric. Molecular interactions were tested by Co-IP assays. RNF2 expression was elevated in PF tissues compared to normal adjacent tissues and in PFbs (HFL1) induced by TGF-β1. Furthermore, knockdown of RNF2 could evidently inhibit the abnormal expression of pro-fibrogenic cytokines (including α-SMA, ECM and MMPs/TIMPs) induced by TGF-β1 in HFL1 cells. Functionally, RNF2 silencing could significantly suppress TGF-β1-induced anomalous proliferation, cell cycle progression, apoptosis and autophagy in HFL1 cells. Mechanistically, RNF2 deficiency could effectively inhibit the abnormal activation of mTOR signaling pathway in TGF-β1-induced HFL1 cells, and mTOR pathway had feedback regulation on the expression of RNF2. Further studies RNF2 could regulate the phosphorylation level of RB1 through interacting with p16 to destroy the binding of p16 and CDK4 competitively. Simultaneously, overexpression of RNF2 could show the opposite results. These results indicated that RNF2 is a potent pro-fibrogenic molecule for PFbs activation and proliferation through mTOR and p16-CDK4-Rb signaling pathways, and RNF2 inhibition will be a potential therapeutic avenue for treating PF.

Expression and Related Mechanisms of miR-100 and TRIB2 in COPD Patients.

Background Chronic obstructive pulmonary disease (COPD) is one of the most common chronic respiratory diseases in the world. COPD is a general term for a class of lung diseases, including emphysema, chronic bronchitis, and refractory asthma. It is characterized by irreversible airflow obstruction and chronic tracheal inflammation. Objective This study aimed to investigate the expression and related mechanisms of miR-100 and TRIB2 in patients with COPD. Methods We collected the serum of patients admitted to our hospital and healthy volunteers undergoing physical examination at the same time, pulmonary fibroblasts were purchased for the experiments, miR-100 was overexpressed, and TRIB2 expression was inhibited in cells. The miR-100 and TRIB2 expression levels in serum and cells were detected by qRT-PCR and Western blot, cell proliferation and apoptosis were detected by CCK-8 and flow cytometry, and the relationship between miR-100 and TRIB2 was explored by the dual-luciferase report. Results The miR-100 expression in the serum of the COPD group was expressed normally, while the TRIB2 expression was expressed abnormally (p < 0.05). The AUC of serum miR-146a and TRIB2 for COPD diagnosis were 0.965 and 0.954, respectively. Overexpressing miR-100 and inhibiting the TRIB2 expression could decrease cell proliferation and increase apoptosis rate. According to the dual-luciferase report, miR-100 and TRIB2 had a targeted regulatory relationship. Rescue experiments showed that overexpressing TRIB2 could reverse the changes of cell proliferation and apoptosis caused by overexpression of miR-100. Conclusion miR-100 and TRIB2 were expressed abnormally in serum of COPD patients, and miR-100 could inhibit proliferation of pulmonary fibroblasts and promote their apoptosis.

In vitro and vivo study of tranilast protects from acute respiratory distress syndrome and early pulmonary fibrosis induced by smoke inhalation

BackgroundTranilast (N-[3＇,4＇-dimethoxycinnamoyl]-anthranilic acid) is an analog of a tryptophan metabolite. It was identified with anti-inflammatory and antifibrotic activities, and used in the treatment of a variety of diseases, such as anti - allergy, bronchial asthma, and hypertrophic scars. As a drug with few adverse reactions, tranilast has attracted great attention, but its application is limited due to the uncertainty of dosages and mechanisms. In this study, the protection effects of different doses of tranilast on smoke inhalation mediated lung injury on rats, and on the damage of three kinds of lung cells in vitro were investigated. MethodIn vivo, Sprague-Dawley rats were randomly divided into sham group, smoke group (rats were exposed to pine sawdust smoke three times, each time for 5 min), different doses of tranilast treatment group (doses were 100 mg/kg, 200 mg/kg and 300 mg/kg, ip.) and placebo group. After 1, 3 and 7 days, pulmonary function, pathologic injury by HE staining, cytokines and oxidative stress level by kits were determined. At 7days, lung fibrosis was assessed by Masson’s trichrome staining and the level of hydroxyproline (HYP). In vitro, three kinds of lung cells from normal rats were isolated: type II alveolar epithelial cells (AT-II), pulmonary microvascular endothelial cells (PMVECs) and pulmonary fibroblasts (PFs). To investigate the potential effects of tranilast on cell proliferation, cell cycle and cytokine production of three kinds of lung cells exposed to smoke. ResultsCompared with smoke group and placebo group, tranilast treatment significantly reduced histopathological changes (such as pulmonary hemorrhage, edema and inflammatory cell infiltration, etc.), significantly reduced histopathological score (p < 0.05), increased arterial oxygen partial pressure, and decreased the levels of IL-1β, TNF-α, TGF-β1 (p < 0.05), oxidative stress and the expression of nuclear transcription factor κB (NF-κB) smoke exposed rats (p < 0.01). In particular, the effect of 200 mg/kg dose was more prominent. In vitro, smoke induced AT-II and PMVECs apoptosis, improved PFs proliferation (p < 0.01), activity of SOD and decreased the content of MDA (p < 0.01). However, tranilast seems to be turning this trend well. The inflammatory factor IL-11β, TNF-α and TGF-β1, and the expression of NF-κB were significantly lower in the tranilast treatment than in the smoke group (p < 0.01). ConclusionThis study indicates that tranilast had a protective effect on acute respiratory distress syndrome and early pulmonary fibrosis of rats in vivo. In addition, tranilast promotes proliferation of AT-II and PMVECs but inhibits PFs proliferation, down-regulates secretion of inflammatory cytokines and alleviates oxidative stress of AT-II, PMVECs and PFs after smoke stimuli in vitro.

C-MYC induces idiopathic pulmonary fibrosis via modulation of miR-9-5p-mediated TBPL1

We sought to pinpoint the potential role of C-MYC in pulmonary fibroblast proliferation in idiopathic pulmonary fibrosis (IPF) and its mechanism. A mouse model of IPF was established by injection of bleomycin. C-MYC and miR-9-5p expression was determined by RT-qPCR and Western blot analysis. The interaction among C-MYC, miR-9-5p, and TBPL1 was detected by ChIP assay and dual luciferase reporter gene assay. After alteration of C-MYC, miR-9-5p, and TBPL1, their roles in pulmonary fibrosis and collagen fiber deposition in mice as well as proliferation and differentiation of pulmonary fibroblasts were assessed. Upregulated C-MYC expression was seen in the lung tissues of IPF mice and its silencing retarded IPF in mice. C-MYC could activate miR-9-5p that negatively regulated TBPL1 expression. Up-regulated C-MYC promoted proliferation and differentiation of pulmonary fibroblasts by inhibiting TBPL1 via activation of miR-9-5p, thus triggering IPF. Moreover, in the lung tissues-derived cells of IPF mice, C-MYC inhibitor, 10,058-F4, was observed to inhibit miR-9-5p expression, thereby repressing pulmonary fibrosis by up-regulating TBPL1. Our data provided evidence pinpointed the aggravative role of C-MYC in IPF by activating miR-9-5p to regulate TBPL1 expression

Mesenchymal stromal cells can repair radiation-induced pulmonary fibrosis via a DKK-1-mediated Wnt/β-catenin pathway.

Pulmonary injury occurring after thoracic radiotherapy is a main factor limiting the curative effect of radiotherapy. Robust activation of the Wnt signalling pathway induced by ionizing radiation stress plays a critical role in epithelial-mesenchymal transition (EMT) in irradiated type II alveolar epithelial cells and in the proliferation of pulmonary fibroblasts, which contributes to the formation of fibrotic lesions in irradiated lungs. The pathogenesis of radiation-induced pulmonary fibrosis could be restricted by systemic delivery of human adipose-derived mesenchymal stromal cells (Ad-MSCs), as evidenced by the inhibitory effects of Ad-MSCs on EMT in irradiated type II alveolar epithelial cells. The purpose of this study is to observe the effects of mesenchymal stromal cells (MSCs) on repairing fibrosis caused by radiation. We used western blotting and real-time PCR to observe the expression of DKK-1 in MSCs of different origins and passages. After the successful establishment of a radiation-induced lung injury model, we investigated the potency of the supernatant from stromal cells to reduce pro-fibrotic events, including EMT and fibroblast activation. To study the mechanism, we evaluated the levels of active β-catenin, TCF4 and the target genes Snail, Twistand c-Myc. After the injection of Ad-MSCs into mice via the tail vein, proteins related to EMT, fibroblasts and Wnt/β-catenin signalling were investigated. The TGF-β and IL-10 protein concentrations in peripheral blood were measured by ELISA. Ad-MSC-derived supernatant effectively reversed the decrease in E-cadherin expression and inhibited the increase in vimentin expression induced by ionizing radiation in epithelial cells and suppressed the expression of α-SMA, a mediator of fibroblast proliferation. The canonical Wnt pathway may be activated by irradiation but the nuclear localization of active β-catenin was reduced in the presence of the supernatant from Ad-MSCs. In addition, the expression of target genes involved in EMT was downregulated. Additionally, when DKK-1 in the supernatant was neutralized, all these effects were reversed. Changes in the levels of proteins related to EMT and fibroblast activation, as well as those of active β-catenin and TCF4, were similar in vivo and in vitro. The serum level of the immunosuppressive factor IL-10 was increased after radiation and was further enhanced after Ad-MSC interference for one month. In conclusion, Ad-MSCs medium can contain DKK-1 and inhibit the induction of EMT via Wnt/β-catenin signalling in vitro and in vivo.

Epigenetic Regulation of Pulmonary Arterial Hypertension-Induced Vascular and Right Ventricular Remodeling: New Opportunities?

Pulmonary artery hypertension (PAH) is a rare chronic disease with high impact on patients’ quality of life and currently no available cure. PAH is characterized by constant remodeling of the pulmonary artery by increased proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), fibroblasts (FBs) and endothelial cells (ECs). This remodeling eventually leads to increased pressure in the right ventricle (RV) and subsequent right ventricle hypertrophy (RVH) which, when left untreated, progresses into right ventricle failure (RVF). PAH can not only originate from heritable mutations, but also develop as a consequence of congenital heart disease, exposure to drugs or toxins, HIV, connective tissue disease or be idiopathic. While much attention was drawn into investigating and developing therapies related to the most well understood signaling pathways in PAH, in the last decade, a shift towards understanding the epigenetic mechanisms driving the disease occurred. In this review, we reflect on the different epigenetic regulatory factors that are associated with the pathology of RV remodeling, and on their relevance towards a better understanding of the disease and subsequently, the development of new and more efficient therapeutic strategies.

Exoantigens of Paracoccidioides spp. Promote Proliferation and Modulation of Human and Mouse Pulmonary Fibroblasts.

Paracoccidioidomycosis (PCM) is a systemic granulomatous fungal infection caused by thermally dimorphic fungi of the genus Paracoccidioides. Endemic in Latin America, PCM presents with high incidence in Brazil, Colombia, and Venezuela, especially among rural workers. The main clinical types are acute/subacute (AF) form and chronic form (CF). Even after effective antifungal treatment, patients with CF usually present sequelae, such as pulmonary fibrosis. In general, pulmonary fibrosis is associated with dysregulation wound healing and abnormal fibroblast activation. Although fibrogenesis is recognized as an early process in PCM, its mechanisms remain unknown. In the current study, we addressed the role of Paracoccidioides spp. exoantigens in pulmonary fibroblast proliferation and responsiveness. Human pulmonary fibroblasts (MRC-5) and pulmonary fibroblasts isolated from BALB/c mice were cultivated with 2.5, 5, 10, 100, and 250 µg/ml of exoantigens produced from P. brasiliensis (Pb18 and Pb326) and P. lutzii (Pb01, Pb8334, and Pb66) isolates. Purified gp43, the immunodominant protein of P. brasiliensis exoantigens, was also evaluated at concentrations of 5 and 10 µg/ml. After 24 h, proliferation and production of cytokines and growth factors by pulmonary fibroblasts were evaluated. Each exoantigen concentration promoted a different level of interference of the pulmonary fibroblasts. In general, exoantigens induced significant proliferation of both murine and human pulmonary fibroblasts (p < 0.05). All concentrations of exoantigens promoted decreased levels of IL-6 (p < 0.05) and VEGF (p < 0.05) in murine fibroblasts. Interestingly, decreased levels of bFGF (p < 0.05) and increased levels of TGF-β1 (p < 0.05) and pro-collagen I (p < 0.05) were observed in human fibroblasts. The gp43 protein induced increased TGF-β1 production by human cells (p = 0.02). In conclusion, our findings showed for the first time that components of P. brasiliensis and P. lutzii interfered in fibrogenesis by directly acting on the biology of pulmonary fibroblasts.

The study on the role of extracellular histones in the pathogenesis of coal worker's pneumoconiosis

Objective: To explore the role of extracellular histones in the pathogenesis of coal worker's pneumoconiosis (CWP) , the relationship of extracellular histones in plasma with pulmonary fibrosis caused by coal mine dust was analyzed, and the stimulating effect of extracellular histones on fibroblast proliferation was studied. Methods: In May 2019, a total of 220 coal mine dust exposure workers (including coal miners and CWP patients) who visited the occupational disease outpatient department of Peking University Third Hospital from 2012 to 2015 were enrolled in the study. According to the classification of small opacity profusion (SOP) in chest radiograph for pneumoconiosis diagnosis (category 0, 1, 2, 3) , 61 coal miners were in category 0 SOP, 65 coal miners were in category 1 SOP, 56 coal miners were in category 2 SOP and 38 coal miners were in category 3 SOP. The plasma levels of extracellular histone H4 and platelet-derived growth factor (PDGF) were measured by enzyme-linked immunosorbent assay (ELISA) kit. The stimulating effects of CWP patients' plasma and calf thymus histones (CTHs) on fibroblast and the antagonizing effect of anti-H4 antibody were investigated by fibroblast proliferation experiment in vitro. Results: Among the study subjects, there were 195 males (88.6%, 195/220) and 25 females (11.4%, 25/220) , age (55.1±7.2) years, coal mine dust exposure time (16.3±4.4) years. The plasma concentrations of histone H4 in the coal miners with category 0, 1, 2 and 3 SOP were (3.92±1.75) 、(9.84±4.17) 、(14.35±5.52) and (17.83±7.69) μg/ml, respectively. There were significant differences among the four groups (P<0.01) . The plasma level of histone H4 was positively correlated with the plasma level of PDGF in the coal miners (r=0.769, P<0.01) . Compared with healthy control plasma group, the cell proliferation percentages of patients' plasma group (272%±87%) and CTH group (283%±84%) were significantly increased (P<0.05) . Compared with patients' plasma group, the cell proliferation percentage of patients' plasma+anti-H4 antibody group (185%±66%) was significantly decreased (P<0.05) . Compared with CTH group, the cell proliferation percentage of CTH+anti-H4 antibody group (167%±59%) was significantly decreased (P<0.05) . Conclusion: Extracellular histones in plasma are associated with pulmonary fibrosis in patients with CWP. Studies in vitro have shown that extracellular histones can promote proliferation of pulmonary fibroblasts. It is suggested that extracellular histones can be important biomarkers for pulmonary fibrosis caused by coal mine dust.

Connexin 43 plays a role in proliferation and migration of pulmonary arterial fibroblasts in response to hypoxia.

Pulmonary hypertension (PH) is a disease associated with vasoconstriction and remodelling of the pulmonary vasculature. Pulmonary artery fibroblasts (PAFs) play an important role in hypoxic-induced remodelling. Connexin 43 (Cx43) is involved in cellular communication and regulation of the pulmonary vasculature. Using both in vitro and in vivo models of PH, the aims of this study were to (i) investigate the role of Cx43 in hypoxic-induced proliferation and migration of rat PAFs (rPAFs) and rat pulmonary artery smooth muscle cells (rPASMCs) and (ii) determine whether Cx43 expression is dysregulated in the rat sugen5416/hypoxic model of PH. The role of Cx43 in hypoxic-induced proliferation and migration was investigated using Gap27 (a pharmacological inhibitor of Cx43) or genetic knockdown of Cx43 using siRNA. Cx43 protein expression was increased by hypoxia in rPAFs but not rPASMCs. Hypoxic exposure, in the presence of serum, resulted in an increase in proliferation of rPAFs but not rPASMCs. Hypoxic exposure caused migration of rPAFs but not rPASMCs. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) and ERK1/2 were increased by hypoxia in rPAFs. The effects of hypoxia on proliferation, migration and MAPK phosphorylation in rPAFs were attenuated in the presence of Gap27 or Cx43 siRNA. Cx43 protein expression was increased in sugen5416/hypoxic rat lung; this increased expression was not observed in sugen5416/hypoxic rats treated with the MAPK pathway inhibitor GS-444217. In conclusion, Cx43 is involved in the proliferation and migration of rPAFs in response to hypoxia via the MAPK signalling pathway.

Mesenchymal stem cell-derived extracellular vesicles suppress the fibroblast proliferation by downregulating FZD6 expression in fibroblasts via micrRNA-29b-3p in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF), a progressive and fatal lung disease, usually leads to an irreversible distortion of the pulmonary structure. The functional roles of bone marrow-derived mesenchymal stem cells (BMSC)-secreted extracellular vesicles (EVs) in fibroblasts have been implicated, yet their actions in the treatment of IPF are not fully understood. This study investigated the roles of BMSC-derived EVs expressing miR-29b-3p in fibroblasts in IPF treatment. EVs derived from BMSCs were successfully isolated and could be internalized by pulmonary fibroblasts, and Cell Counting Kit-8 (CCK-8) and Transwell assay results identified that EVs inhibited the activation of fibroblast in IPF. miR-29b-3p, frizzled 6 (FZD6), α-skeletal muscle actin (α-SMA), and Collagen I expressions were examined, which revealed that miR-29b-3p was poorly expressed and FZD6, α-SMA, and Collagen I were overexpressed in pulmonary tissues. Dual-luciferase reporter assay results demonstrated that miR-29b-3p could inversely target FZD6 expression. The gain- and loss-of-function assays were conducted to determine regulatory effects of FZD6 and miR-29b-3p on IPF. CCK-8 and Transwell assays results displayed that BMSCs-derived EVs overexpressing miR-29b-3p contributed to inhibited pulmonary interstitial fibroblast proliferation, migration, invasion, and differentiation. Furthermore, the effects of BMSCs-derived EVs overexpressing miR-29b-3p on IPF progression were assessed in vivo, which confirmed the repressive effects of BMSCs-derived EVs overexpressing miR-29b-3p on IPF progression. Collectively, BMSCs-derived EVs overexpressing miR-29b-3p relieve IPF through FZD6.

Baicalin alleviates bleomycin‑induced pulmonary fibrosis and fibroblast proliferation in rats via the PI3K/AKT signaling pathway.

Baicalin is an important flavonoid compound THAT is isolated from the Scutellaria baicalensis Georgi Chinese herb and plays a critical role in anti-oxidative, anti-inflammatory, anti-infection and anti-tumor functions. Although baicalin can suppress the proliferation of tumor cells, the underlying mechanisms of baicalin in bleomycin (BLM)-induced pulmonary fibrosis remain to be elucidated. Thus, the aim of the present study was to determine the role of baicalin in pulmonary fibrosis and fibroblast proliferation in rats. Hematoxylin and eosin (H&E) and Masson staining were used to measure the morphology of pulmonary fibrosis, ELIASA kits were used to test the ROS and inflammation, and western blotting and TUNEL were performed to study the apoptosis proteins. In vitro, MTT assay, flow cytometry, western blotting and immunofluorescence were performed to investigate the effects of baicalin on proliferation of fibroblasts. The most significantly fibrotic changes were identified in the lungs of model rats at day 28. Baicalin (50 mg/kg) attenuated the degree of pulmonary fibrosis, and the hydroxyproline content of the lung tissues was decreased in the baicalin group, compared with the BLM group. Further investigation revealed that baicalin significantly increased glutathione peroxidase (GSH-px), total-superoxide dismutase (T-SOD) and glutathione (GSH) levels, whilst decreasing that of serum malondialdehyde (MDA). TUNEL-positive cells were significantly decreased in rats treated with baicalin group, compared with the model group. Furthermore, it was found that BLM promoted fibroblasts viability in a dose-dependent manner in vivo, which was restricted following treatment with different concentrations of baicalin. Moreover, BLM promoted the expression levels of cyclin A, D and E, proliferating cell nuclear antigen, phosphorylated (p)-AKT and p-calcium/calmodulin-dependent protein kinase type. BLM also promoted the transition of cells from the G0/G1 phase to the G2/M and S phases, and increased the intracellular Ca2+ concentration, which was subsequently suppressed by baicalin. Collectively, the results of the present study suggested that baicalin exerted a suppressive effect on BLM-induced pulmonary fibrosis and fibroblast proliferation.

Protective effects of intermedin/adrenomedullin-2 in a cellular model of human pulmonary arterial hypertension

Proliferation of pulmonary fibroblasts (PF) and distal migration of smooth muscle cells (PSM) are hallmarks of pulmonary arterial hypertension (PAH). Intermedin/adrenomedullin-2 (IMD/AM2) belongs to the Calcitonin Gene-Related Peptide (CGRP)/Adrenomedullin (AM) superfamily. These peptides act via Calcitonin-Like Receptors (CLR) combined with one of three Receptor activity-modifying proteins (RAMPs). IMD/AM2 is a potent pulmonary vasodilator in animal studies. The aim was to describe expression of IMD/AM2, AM and receptor components in human pulmonary vascular cells and to elucidate effects of IMD/AM2 on human PSM migration and PF proliferation. Gene expression was detected by immunofluorescence, immunoblotting and qRT-PCR. Normotension and hypertension were simulated by applying pulsatile mechanical stretch (Flexcell® apparatus). Viable cell numbers were determined by dye exclusion. PSM chemotaxis was measured via Dunn chamber. IMD/AM2 protein was co-expressed with AM and their receptor components in pulmonary artery and microvascular endothelial (PAEC, PMVEC) and non-endothelial cells (PF, PSM), and localised to vesicles. IMD/AM2 was secreted under basal conditions, most abundantly from PF and PMVEC. Secretion from PF and PSM was enhanced by stretch. IMD/AM2 mRNA expression increased in response to hypertensive stretch of PSM. IMD/AM2 inhibited PDGF (10−7 M)-mediated PSM migration maximally at 3 × 10-10 M and PF proliferation maximally at 3 × 10-9 M. Angiotensin II (5 × 10-8 M), normotensive and hypertensive stretch augmented PF proliferation. IMD/AM2 (10-9 M) abolished the proliferative effects of Angiotensin II and normotensive stretch and attenuated the proliferative effect of hypertensive stretch alone and combined with angiotensin II. These findings indicate an important counter-regulatory role for IMD/AM2 in PAH.

The antifibrotic effect and mechanism of a novel tyrosine kinase inhibitor, ZSP1603, in preclinical models of pulmonary fibrosis.

This study aimed to investigate the efficacy and molecular mechanisms of ZSP1603 as a novel anti-fibrotic compound. The unilateral left pulmonary fibrosis model was established in the Sprague Dawley (SD) rats. The bilateral pulmonary fibrosis model was established in the C57BL/6J mice. The therapeutic treatment regimen began after the induction of pulmonary fibrosis. The preventive treatment regimen began on the first day of bleomycin administration. Animals were randomly divided into the sham, model, Nintedanib, and ZSP1603 treatment groups. Haematoxylin and eosin (H&E) and Masson's trichrome staining were performed to evaluate pulmonary injury, inflammation, and fibrosis. Cell Counting Kit-8 (CCK-8) assay and Western blot were used to investigate the effects and mechanisms of ZSP1603 on the proliferation of primary human pulmonary fibroblasts (pHPFs). The messenger ribonucleic acid (mRNA) expression of transforming growth factor (TGF)-β1, tissue inhibitor of metalloproteinase 1 (TIMP-1), and collagen 1A1 (COL1A1) in pHPFs was detected by quantitative Real Time-Polymerase Chain Reaction (PCR). ZSP1603 inhibited the proliferation of pHPFs in vitro by blocking the platelet-derived growth factor receptor-β (PDGF-Rβ) and extracellular signal-regulated kinase (ERK) signalling pathway. ZSP1603 also inhibited the differentiation of pHPFs by reducing the expression of TGF-β1, TIMP-1, and COL1A1. ZSP1603 significantly attenuated pulmonary injury, inflammation, and fibrosis in vivo in four independent animal studies of pulmonary fibrosis. ZSP1603 is an effective anti-fibrotic compound with clear mechanisms.

Necrostatin-1 Alleviates Bleomycin-Induced Pulmonary Fibrosis and Extracellular Matrix Expression in Interstitial Pulmonary Fibrosis.

BackgroundInterstitial pulmonary fibrosis (IPF) is harmful for patients’ life and health. The effective treatment of IPF is lacking because of unclear pathogenesis. Necrostatin-1 has protective effects on lung injury and can suppress the fibrosis development. I this study we investigated whether necrostatin-1 could decrease the proliferation of pulmonary fibroblasts, pulmonary fibrosis and expression of extracellular matrix (ECM) in IPF.Material/MethodsThe IPF mice model was conducted by intra-tracheal injection of bleomycin (BLM) (2 mg/kg) for C57BL/6N mice. Necrostatin-1 treatment was performed with 1 mg/kg necrostatin-1 by an intravenous injection for C57BL/6N mice. Lung tissue structures and collagen deposition were observed by hematoxylin and eosin staining and Masson staining. IPF in vitro model was constructed by MRC-5 cells induced by transforming growth factor beta 1 (TGF-β1). And, 20 μM necrostatin-1 was used to treat the TGF-β1 induced MRC-5 cells. Cell Counting Kit-8 (CCK-8) assay detected the viability of MRC-5 cells. The expression of receptor-interacting protein kinase-1 and -3 (RIPK1 and RIPK3), α smooth muscle actin (α-SMA), collagen IV, collagen I, fibronectin (FN), and transforming growth factor-β (TGF-β) in lung tissues and MRC-5 cells was measured by western blot analysis. The α-SMA expression in lung tissues was also analyzed by immunohistochemistry.ResultsThe expression of RIPK1 and RIPK3 in lung tissues of BLM induced mice was increased. The degree of pulmonary fibrosis and expression of α-SMA, collagen IV, collagen I, FN, and TGF-β in lung tissues of BLM induced mice was enhanced. The proliferation of MRC-5 cells was increased when MRC-5 cells were induced by TGF-β. The expression of RIPK1, RIPK3, α-SMA, collagen IV, collagen I, and FN was increased in TGF-β induced MRC-5 cells. And, necrostatin-1 could effectively reverse the changes of pulmonary fibrosis, RIPK1, RIPK3, and ECM in vivo and in vitro experiments.ConclusionsNecrostatin-1 attenuated pulmonary fibrosis in lung tissues of BLM induced mice and inhibited the fibroblast proliferation. And, necrostatin-1 also decreased the expression of RIPK1, RIPK3, and ECM in lung tissues of BLM induced mice and TGF-β induced fibroblasts. Necrostatin-1 could be a new effective drug for the treatment of IPF.

CXCL16/CXCR6 axis promotes bleomycin-induced fibrotic process in MRC-5 cells via the PI3K/AKT/FOXO3a pathway

ObjectiveInterstitial lung disease (ILD) is a progressive and irreversible lung disease with very limited therapeutic options. Previous studies have found that chemokine ligands CXCL16 and CXCR6 play critical roles in organ fibrosis. However, whether CXCL16 and CXCR6 are also involved in the pathogenesis of ILD, as well as their regulatory role in pulmonary fibrosis, has not been reported. MethodsIn this study, we detected CXCL16 levels in patients with rheumatoid arthritis-associated ILD (RA-ILD) and examined the critical role of the CXCL16/CXCR6 axis in the proliferation and collagen production of human pulmonary fibroblasts (MRC-5 cells). The effect of anti-CXCL16 antibody on the bleomycin-induced fibrogenesis in cultured MRC-5 cells was also evaluated. ResultsOur results indicated that serum soluble CXCL16 was significantly higher in RA-ILD patients and also associated with the severity of lung fibrosis. CXCL16 facilitates fibrosis by enhancing proliferation, migration, and collagen production of MRC-5 cells. Furthermore, a synergistic fibrogenic effect of CXCL16 and bleomycin has been found. CXCL16 stimulated the activation of PI3K/AKT/FOXO3a signaling pathway in MRC-5 cells, and the inhibition by specific inhibitors Wortmannin and LY294002, or knockdown of CXCR6 by siRNA also suppressed the biological functions of MRC-5 cells mediated by CXCL16. Similarly, down-regulation of CXCR6 also partly blocked BLM-induced fibrogenesis in MRC-5 cells. ConclusionsCXCL16/CXCR6 axis promotes proliferation and collagen production of MRC-5 cells by the PI3K/AKT/FOXO3a signaling pathway, and inhibition of the CXCL16/CXCR6 axis may provide a new therapeutic strategy targeting pulmonary fibrosis.