Rat Lung Fibroblasts Research Articles

Prolonged Particulate Hexavalent Chromium Exposure Induces DNA Double-Strand Breaks and Inhibits Homologous Recombination Repair in Primary Rodent Lung Cells.

Hexavalent chromium [Cr(VI)] is a known lung carcinogen and a driving mechanism in human lung cells for Cr(VI)-induced lung cancer is chromosome instability, caused by prolonged Cr(VI) exposure inducing DNA double-strand breaks, while simultaneously inhibiting the repair of these breaks. In North Atlantic right whales, Cr(VI) induces breaks but does not inhibit repair. It is unclear if this repair inhibition is specific to human lung cells or occurs in other species, as it has only been considered in humans and North Atlantic right whales. We evaluated these outcomes in rodent cells, as rodents are an experimental model for metal-induced lung carcinogenesis. We used a guinea pig lung fibroblast cell line, JH4 Clone 1, and rat lung fibroblasts. Cells were exposed to two different particulate Cr(VI) compounds, ranging from 0 to 0.5 ug/cm2, for 24 or 120h and assessed for cytotoxicity, DNA double-strand breaks, and DNA double-strand break repair. Both particulate Cr(VI) compounds induced a concentration-dependent increase in cytotoxicity and DNA double-strand breaks after acute and prolonged exposures. Notably, while the repair of Cr(VI)-induced DNA double-strand breaks increased after acute exposure, the repair of these breaks was inhibited after prolonged exposure. These results are consistent with outcomes in human lung cells indicating rodent cells respond like human cells, while whale cells have a markedly different response.

The mechanism of Shenlong Jianji treatment of idiopathic pulmonary fibrosis inhibits fibroblast-to-myofibroblast transformation via the TGF-β1/smads signaling pathway

Ethnopharmacological relevanceShenlong Jianji (SLJJ) is a Chinese herbal compound composed of traditional medicines for supplementing Qi, nourishing Yin, promoting blood circulation, and removing obstruction in channels. It is widely used to treat idiopathic pulmonary fibrosis (IPF) in China. However, the underlying mechanism of SLJJ remains unclear. Aim of this studyTo elucidate the efficacy and mechanisms of SLJJ in the treatment of IPF through in vivo and in vitro experiments. Material and methods84 Wistar rats were randomly and equally divided into 7 groups: the control group (CTRL), the SHAM operation group (SHAM), the model group (IPF), the low dose of SLJJ group (L-SLJJ), the middle dose of SLJJ group (M-SLJJ), the high dose of SLJJ group (H-SLJJ), and the pirfenidone group (PFD). The rats in the CTRL, SHAM, and IPF groups were given normal saline each time for 28 days; the SLJJ groups were given Shenlong Jianji (9 g kg-1·d-1, 18 g kg-1·d-1, 36 g kg-1·d-1), and pirfenidone was administered as a sequential dose. After 28 days, the general condition of the rats was evaluated, and samples were collected. The lung coefficient was measured. The pathological changes of lung in each group were observed by H&E staining and Masson staining. α-SMA, collagen 1, and E-cadherin proteins were detected by immunohistochemistry. α-SMA, collagen 1, vimentin, E-cadherin, N-cadherin, TGF-β1, smad2, and smad3 proteins were detected by WB in vivo.In vitro, A scratch test was used to assess the ratio of cell migration. α-SMA, vimentin, E-cadherin, and N-cadherin protein levels were evaluated by a cellular immunofluorescence assay. TGF-β1/smads signaling pathway was detected by WB. HPLC-Q-TOF/MS analysis was used to identify the active compounds in the SLJJ. Molecular docking determined the free binding energy of the compound with the TGF-β1 protein. ResultsSLJJ significantly improved the respiratory symptoms, heart rate, mental state, and food intake of IPF group rats and decreased the lung coefficient. In the IPF group, inflammatory cells were infiltrated, and the thickened alveoli wall and alveoli collapse were shown, while significantly alleviating pathological changes in the SLJJ and PFD groups. Masson staining showed that SLJJ and PFD decreased the collagen expression. Immunohistochemical results showed that the expressions of α-SMA, collagen 1, and N-cadherin decreased in the SLJJ and PFD groups, while E-cadherin increased significantly compared with the IPF group. SLJJ regulates TGF-β1/smads signaling pathway proteins in vivo. SLJJ decreased the ratio of migration in HFL-1 cells; SLJJ reduced the fluorescence intensity of α-SMA, vimentin, and N-cadherin and increased the fluorescence intensity of E-cadherin in primary rat lung (PRL) fibroblast cells and HFL-1 cells. WB results showed that SLJJ significantly down-regulated α-SMA, Vimentin, N-cadherin, TGF-β1, smad2, and p-smad2/3 proteins expression and up-regulated E-cadherin protein expression in vitro, whereas SRI-011381 (a TGF-β1 agonist) antagonized the effects of SLJJ. ConclusionSLJJ inhibits idiopathic pulmonary fibrosis. The TGF- β1/Smads signaling pathway can be the target of SLJJ, which inhibits fibroblast-to-myofibroblast transformation and is expected to be a new drug for the treatment of IPF.

Ethanol-exposed lung fibroblasts cause airway epithelial barrier dysfunction.

Chronic alcohol ingestion predisposes to lung injury and disrepair during sepsis. Our previous studies outlined roles for transforming growth factor-beta 1 (TGFβ1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in epithelial barrier homeostasis and how alcohol perturbs their expression and signaling. Here we hypothesize that ethanol-exposed lung fibroblasts (LF) are a source of dysregulated TGFβ1 and GM-CSF and thereby alter airway epithelial barrier function. Human or rat LF were cultured ± ethanol for 2 weeks and then co-cultured with human or rat airway epithelial cells (AEC) seeded on Transwell permeable supports. In selected groups, a TGFβ1 receptor type 1 (TGFβR1) inhibitor (SB431542) or a TGFβ1 neutralizing antibody was applied. Transepithelial electrical resistance (TER) was measured prior to co-culture and on day 5 of co-culture. AEC were then analyzed for the expression of selected tight junction and mesenchymal proteins, and transwell membranes were analyzed by immunofluorescence microscopy for ZO-1 expression and localization. TGFβ1 and GM-CSF levels in conditioned media from the co-cultures were quantified by ELISA. AEC co-cultured with ethanol-exposed LF (ELF) showed a significant reduction in TER and corresponding decreases in ZO-1 expression, whereas collagen type 1A1 and α-smooth muscle actin protein expression were increased. In parallel, in conditioned media from the ELF + AEC co-cultures, activated TGFβ1 levels increased and GM-CSF levels decreased. Notably, all the effects of ELF on the AEC were prevented by blocking TGFβ1 activity. Prior ethanol exposure to LF induces barrier dysfunction in naive AEC in a paracrine fashion through activation of TGFβ1 signaling and suppression of GM-CSF. These experimental findings provide a potential mechanism by which chronic alcohol ingestion impairs airway epithelial integrity and renders individuals susceptible to lung injury.

P53 regulates CCAAT/Enhancer binding protein β gene expression

BackgroundThe transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) is implicated in diverse processes and diseases. Its two isoforms, namely liver-enriched activator protein (LAP) and liver-enriched inhibitor protein (LIP) are translated from the same mRNA. They share the same C-terminal DNA binding domain except LAP has an extra N-terminal activation domain. Probably due to its higher affinity for its DNA cognate sequences, LIP can inhibit LAP transcriptional activity even at substoichiometric levels. However, the regulatory mechanism of C/EBPβ gene expression and the LAP: LIP ratio is unclear. MethodsIn this study, the C/EBPβ promoter sequence was scanned for conserved P53 response element (P53RE), and binding of P53 to the C/EBPβ promoter was tested by Electrophoretic Mobility Shift Assay (EMSA) and chromatin immunoprecipitation assay. P53 over-expression and dominant negative P53 expression plasmids were transfected into rat lung fibroblasts and tested for C/EBPβ gene transcription and expression. Western blot analysis was used to test the regulation of C/EBPβ LAP and LIP isoforms. Constructs containing the LAP 5′untranslated region (5′UTR) or the LIP 5′UTR region were used to test the importance of 5′UTR in the control of C/EBPβ LAP and LIP translation. ResultsThe C/EBPβ promoter sequence was found to contain a conserved P53 response element (P53RE), which binds P53 as demonstrated by Electrophoresis Mobility Shift Assay and chromatin immunoprecipitation assays. P53 over-expression suppressed while dominant negative P53 stimulated C/EBPβ gene transcription and expression. Western blot analysis showed that P53 differentially regulated the translation of the C/EBPβ LAP and LIP isoforms through the regulation of eIF4E and eIF4E-BP1. Further studies with constructs containing the LAP 5′untranslated region (5′UTR) or the LIP 5′UTR region showed that the 5′UTR is important in differential control of C/EBPβ LAP and LIP translation. ConclusionAnalysis of the effects of P53 on C/EBPβ expression revealed a novel mechanism by which P53 could antagonize the effects of C/EBPβ on its target gene expression. For the first time, P53 is shown to be a repressor of C/EBPβ gene expression at both transcriptional and translational levels, with a differential effect in the magnitude of the effect on LAP vs. LIP isoforms.

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

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

The effect of cerium dioxide nanocrystals on the prooxidant status of rat lung fibroblasts in vitro under γ-irradiation conditions

Redox-active nanocrystals of cerium dioxide (CeO2) are of interest as antioxidants; therefore, it is relevant to study the potential ability of these nanocrystals to correct prooxidative markers in irradiated cell culture in vitro. The purpose of the study was to evaluate the effect of redox-active spherical nanocrystals of cerium dioxide on markers of oxidative stress induced by γ-irradiation in a two-dimensional culture of rat lung fibroblasts. Materials and methods. The study was performed on a monolayer of lung fibroblasts of Wistar rats. Nanocrystals were added to the nutrient medium one hour before irradiation until their final concentration in the medium of 2.5 μg/L. The cells were incubated with nanocrystals for one hour and then irradiated. The radiation dose was 0.75 Gy. The concentration of 8-isoprostane was determined by spectrophotometry. The concentration of free oxygen species and the degree of lipid peroxidation in living cells were determined by fluorimetry. Visualization and measurement of fluorescence intensity were performed by confocal laser scanning microscopy. Results. 3 hours after irradiation under the conditions of pre-incubation with CeO2 nanocrystals, the content of reactive oxygen species, the level of lipid peroxidation, and the content of 8-isoprostane were significantly decreased in rat lung fibroblast culture. At the same time, incubation with cerium dioxide nanocrystals much more effectively reduced the content of reactive oxygen species in the cytoplasm of cells than in their nuclei and nucleoli in particular. Conclusion. Preincubation of rat lung fibroblasts in vitro with СeО2 nanocrystals can significantly reduce the oxidizing effects of ionizing radiation.

PEDF is an antifibrosis factor that inhibits the activation of fibroblasts in a bleomycin-induced pulmonary fibrosis rat model

BackgroundIdiopathic pulmonary fibrosis (IPF) is a highly heterogeneous and fatal lung disease. In addition to dense fibrous tissue, abnormal angiogenesis is also an important feature of IPF. Pigment epithelium-derived factor (PEDF) is an angiogenesis inhibitor and a potential anti-fibrous factor. The purpose of this experiment is to observe the effect of PEDF on bleomycin (BLM)-induced pulmonary fibrosis in rats.MethodsIn vivo, pathological examination and detection of related factors were performed on pulmonary fibrosis induced by BLM in rats, and the temporal and spatial distribution of PEDF was investigated. Furthermore, lung gene delivery (PEDF-adeno-associated virus) was performed to investigate the effect of PEDF on pulmonary fibrosis. In vitro, lentiviral vectors were used to construct PEDF over-expression or knock out primary rat lung (PRL) fibroblasts. The effect of PEDF on fibroblast activation under TGF-β1 stimulation was evaluated, and the activation of TGF-β1/smad pathway and PPAR-γ expression (in the presence or absence of PPAR-γ inhibitors) were analyzed.ResultsIn vivo results showed that PEDF expression decreased during the inflammatory phase and increased during the fibrotic phase. PEDF could inhibit the progression of pulmonary fibrosis in rats. In vitro results showed that PEDF could effectively inhibit TGF-β1-stimulated fibroblast activation and reduce the production of α-SMA and collagen-I. PEDF could inhibit the TGF-β1/smad pathway by up-regulating the activity of PPAR-γ.ConclusionsPEDF can act as an anti-fibrotic factor, inhibit fibroblast activation by upregulating PPAR-γ activity and reduce BLM-induced pulmonary fibrosis in rats.

A Promising transwell co-culture cell model for silicosis

Silicosis, one of the most ancient occupational diseases, has not been elucidated and addressed until now. Although some medicines were partly effective in the clinical treatment and certain plausible mechanisms were partially ascertained, it is still urgent and necessary to explore its real and specific pathogenesis. With the development of the cell co-culture technique, the transwell co-culture system has been applied in several scientific fields, which is able to simulate the pathogenic process in vivo to the largest extent. In this sense, a transwell co-culture cell model for silicosis including RFL-6 (lung fibroblasts of rats), RLE-6TN (type II alveolar epithelial cells of rats) and NR8383 (alveolar macrophages of rats) cells was successfully established and the developmental process of silicosis in this model could be roughly divided into three stages: inflammatory stage, progressive stage and fibrotic stage, as evidenced by the morphological features and the inflammatory cytokines and fibrotic proteins of supernatants and the cells in this system. Results of this project will pave the way for clarifying the mechanism of silicosis and building a practical platform for the further exploration of molecular initiating event (MIE) and adverse outcome pathway (AOP) of silicosis.

Overexpression of bone morphogenetic protein receptor type 2 suppresses transforming growth factor β-induced profibrotic responses in lung fibroblasts

Purpose of the study: Idiopathic pulmonary fibrosis (IPF) is a progressive disease with a poor prognosis. As the efficacy of currently available antifibrotics is limited, development of new therapies is warranted. Transforming growth factor (TGF)-β plays a central role in the pathogenesis of IPF through mechanisms such as promoting the production of extracellular matrix by fibroblasts. Conversely, bone morphogenetic proteins (BMPs) are known to be antifibrotic and may counterbalance TGF-β signaling via BMP receptor type 2 (BMPR2). However, little is known about the expression status of BMPR2 and its function in pulmonary fibrosis, and manipulation of BMPR2 expression has never been attempted. In this study, we aimed at evaluating the effectiveness of BMPR2 upregulation for modulating the imbalance of the TGF-β/BMP axis and reduce the profibrotic changes in lung fibroblasts. Materials and Methods We investigated BMPR2 expression in pulmonary fibrosis and TGF-β/BMP signaling in lung fibroblasts. Then we evaluated the impact of BMPR2 upregulation using adenoviral transduction on TGF-β-induced Smad2/3 phosphorylation and fibronectin production in lung fibroblasts. Results BMPR2 was distributed in airway epithelium and alveolar walls in rat lungs. BMPR2 expression was decreased in fibrotic lesions in the lungs of rats with bleomycin-induced pulmonary fibrosis and in human lung fibroblasts (HLFs) stimulated with TGF-β. Although Smad2/3 phosphorylation and fibronectin production were not suppressed solely by BMPs, phosphorylated Smad2/3 was decreased in BMPR2-transduced cells even without BMP stimulation. Fibronectin was decreased only when BMPR2-transduced HLFs were stimulated with BMP7 (but not BMP4). Similar results were also observed in IPF patient HLFs and rat lung fibroblasts. Conclusions BMPR2 expression was reduced in fibrotic lungs and lung fibroblasts stimulated with TGF-β. BMPR2 transduction to lung fibroblasts reduced Smad2/3 phosphorylation, and reduced fibronectin production when treated with BMP7. Upregulation of BMPR2 may be a possible strategy for treating pulmonary fibrosis.

Protectin DX promotes the inflammatory resolution via activating COX-2/L-PGDS-PGD2 and DP1 receptor in acute respiratory distress syndrome

PurposeAcute respiratory distress syndrome (ARDS) is characterized by uncontrollable inflammation. Cyclooxygenase-2(COX-2) and its metabolite prostaglandins are known to promote the inflammatory resolution of ARDS. Recently, a newly discovered endogenous lipid mediator, Protectin DX (PDX), was also shown to mediate the resolution of inflammation. However, the regulatory of PDX on the pro-resolving COX-2 in ARDS remains unknown. Material and MethodsPDX (5 μg/kg) was injected into rats intravenously 12 h after the lipopolysaccharide (LPS, 3 mg/kg) challenge. Primary rat lung fibroblasts were incubated with LPS (1 μg/ml) and/or PDX (100 nM). Lung pathological changes examined using H&E staining. Protein levels of COX-2, PGDS and PGES were evaluated using western blot. Inflammatory cytokines were tested by qPCR, and the concentration of prostaglandins measured by using ELISA. ResultsOur study revealed that, COX-2 and L-PGDS has biphasic activation characteristics that LPS could induce induced by LPS both in vivo and in vitro.. The secondary peak of COX-2, L-PGDS-PGD2 promoted the inflammatory resolution in ARDS model with the DP1 receptor being activated and PDX up-regulated the inflammatory resolutionvia enhancing the secondary peak of COX-2/L-PGDS-PGD2 and activating the DP1 receptor. ConclusionPDX promoted the resolution of inflammation of ARDS model via enhancing the expression of secondary peak of COX-2/L-PGDS-PGD2 and activating the DP1 receptor. PDX shows promising therapeutic potential in the clinical management of ARDS.

Role of inner mitochondrial protein OPA1 in mitochondrial dysfunction by tobacco smoking and in the pathogenesis of COPD

BackgroundChronic lung diseases, such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are linked to several mitochondrial alterations. Cigarette smoke (CS) alters the structure and function of mitochondria. OPA1 is the main inner mitochondrial GTPase responsible for the fusion events. OPA1 undergoes proteolytic cleavage from long to short forms during acute stress and mitophagy. However, the exact role of OPA1 isoforms and related proteins during CS-induced mitophagy and COPD is not clear. MethodsLung tissues from non-smokers, smokers, COPD and IPF were used to determine the relative expression of OPA1 and related proteins. Additionally, we used mouse lungs from chronic (6 months) CS exposure to evaluate the status of OPA1. Primary lung fibroblasts from normal and COPD patients and naked mole rat (NMR) lung fibroblasts, human fetal lung fibroblast (HFL1), mouse embryonic fibroblast from wild type (WT), OPA1−/−, MFN1 and MFN2−/− were used to determine the effect of CS on OPA1 isoforms. Various mitochondrial fusion promoters/activators (BGP-15, leflunomide, M1) and fission inhibitor (DRP1) were used to determine their effect on OPA1 status and cigarette smoke extract (CSE)-induced lung epithelial (BEAS2B) cell damage, respectively. Seahorse flux analyzer was used to determine the effect of these compounds in BEAS2B cells with and without CSE exposure. FindingsShort OPA1 isoforms were predominantly detected and significantly increased in COPD subjects. Acute CSE treatment in various cell lines except NMR was found to increase the conversion of long to short OPA1 isoforms. CSE treatment significantly increased mitochondrial stress-related protein SLP2 in all the cells used. OPA1 interacting partners like prohibitins (PHB1 and 2) were also altered depending on the CS exposure. Finally, BGP-15 and leflunomide treatment were able to preserve the long OPA1 isoform in cells treated with CSE. Interpretation/conclusionThe long OPA1 isoform along with SLP2 and prohibitins play a crucial role in CS-induced lung damage, causing mitophagy/mitochondrial dysfunction in COPD, which may be used as a novel therapeutic target in COPD.

Protectin DX promotes epithelial injury repair and inhibits fibroproliferation partly via ALX/PI3K signalling pathway

Acute respiratory distress syndrome/acute lung injury (ARDS/ALI) is histologically characterized by extensive alveolar barrier disruption and excessive fibroproliferation responses. Protectin DX (PDX) displays anti‐inflammatory and potent inflammation pro‐resolving actions. We sought to investigate whether PDX attenuates LPS (lipopolysaccharide)‐induced lung injury via modulating epithelial cell injury repair, apoptosis and fibroblasts activation. In vivo, PDX was administered intraperitoneally (IP) with 200 ng/per mouse after intratracheal injection of LPS, which remarkedly stimulated proliferation of type II alveolar epithelial cells (AT II cells), reduced the apoptosis of AT II cells, which attenuated lung injury induced by LPS. Moreover, primary type II alveolar cells were isolated and cultured to assess the effects of PDX on wound repair, apoptosis, proliferation and transdifferentiation in vitro. We also investigated the effects of PDX on primary rat lung fibroblast proliferation and myofibroblast differentiation. Our result suggests PDX promotes primary AT II cells wound closure by inducing the proliferation of AT II cells and reducing the apoptosis of AT II cells induced by LPS, and promotes AT II cells transdifferentiation. Furthermore, PDX inhibits transforming growth factor‐β1 (TGF‐β1) induced fibroproliferation, fibroblast collagen production and myofibroblast transformation. Furthermore, the effects of PDX on epithelial wound healing and proliferation, fibroblast proliferation and activation partly via the ALX/ PI3K signalling pathway. These data present identify a new mechanism of PDX which targets the airway epithelial cell and fibroproliferation are potential for treatment of ARDS/ALI.

ERK1/2 Signaling Pathway Activated by EGF Promotes Proliferation, Transdifferentiation, and Migration of Cultured Primary Newborn Rat Lung Fibroblasts.

Background Bronchopulmonary dysplasia (BPD) is a common and serious complication in premature infants. Lung fibroblasts (LFs) are present in the extracellular matrix and participate in pulmonary development in response to BPD. The aim of this study was to investigate the effect of extracellular signal-regulated kinase (ERK) on LFs cultured from newborn rats. Material and Methods. Primary LFs were isolated and treated with epidermal growth factor (EGF, 20 ng/mL) in the presence or absence of an ERK inhibitor, PD98059 (10 μmol/L). Phosphorylated ERK1/2 (p-ERK1/2) protein levels were determined using immunocytochemistry, western blotting, and real-time reverse transcription quantitative (RT–q)PCR. LF proliferation was examined by flow cytometry and a cell counting kit-8 assay. LF transdifferentiation was examined by protein and mRNA expression of α-smooth muscle actin (α-SMA) by immunocytochemistry, western blotting, and RT–qPCR. LF migration was examined by the transwell method. Results Phosphorylated ERK1/2, which was activated by EGF, promoted LF proliferation by accelerating cell-cycle progression from the G1 to S phase. After treatment with PD98059, the expression of p-ERK1/2 in LFs, cellular proliferation, and the percentage of cells in S phase were significantly decreased. Phosphorylated ERK1/2 also promoted the differentiation of LFs into myofibroblasts through increased α-SMA synthesis and migration. Conclusion The activation of ERK promotes proliferation, transdifferentiation, and migration of lung fibroblasts from newborn rats.

Differential expression of lncRNAs during silicosis and the role of LOC103691771 in myofibroblast differentiation induced by TGF-β1

ObjectiveThe role and molecular mechanism of long non-coding RNA (lncRNA)-related pathways in silicosis have not been elucidated clearly. The aims of this study were to evaluate the expression of lncRNAs during silica-induced pulmonary fibrosis and verify the function and molecular mechanism of LOC103691771 in myofibroblast differentiation induced by transforming growth factor-β1 (TGF-β1). MethodsRNA-sequencing was performed to assess differential expression of lncRNAs in control and silicotic rat lungs. Differential expression of lncRNAs was analyzed by Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes to identify their biological roles. LOC103691771, LOC102549714, LOC102550137, LOC103693125, and LOC103692016 were selected to verify their expression by real-time PCR of silicotic rat lung tissue and lung fibroblasts stimulated by TGF-β1. Specific small interfering RNA and an LOC103691771 overexpression plasmid were used to analyze the molecular mechanism in myofibroblast differentiation induced by TGF-β1. ResultA total of 306 lncRNAs were expressed differentially in silicotic rat lungs, including 224 upregulated and 82 downregulated lncRNAs. The expression of LOC103691771, LOC102549714 and LOC102550137 was upregulated, while the expression of LOC103693125 and LOC103692016 was downregulated in silicotic rat lungs and TGF-β1-induced fibroblast, which was consistent with the results of RNA-sequencing. Furthermore, LOC103691771 gene silencing attenuated myofibroblast differentiation, whereas LOC103691771 overexpression promoted myofibroblast differentiation via regulation of the TGF-β1-Smad2/3 signaling pathway. ConclusionOur findings revealed that differential expression of lncRNAs was related to the development of silicosis, and LOC103691771 played a major role in myofibroblast differentiation induced by TGF-β1, which may serve as a potential therapeutic target for silicosis.

Tspan8-Tumor Extracellular Vesicle-Induced Endothelial Cell and Fibroblast Remodeling Relies on the Target Cell-Selective Response.

Tumor cell-derived extracellular vesicles (TEX) expressing tetraspanin Tspan8-alpha4/beta1 support angiogenesis. Tspan8-alpha6/beta4 facilitates lung premetastatic niche establishment. TEX-promoted target reprogramming is still being disputed, we explored rat endothelial cell (EC) and lung fibroblast (Fb) mRNA and miRNA profile changes after coculture with TEX. TEX were derived from non-metastatic BSp73AS (AS) or metastatic BSp73ASML (ASML) rat tumor lines transfected with Tspan8 (AS-Tspan8) or Tspan8-shRNA (ASML-Tspan8kd). mRNA was analyzed by deep sequencing and miRNA by array analysis of EC and Fb before and after coculture with TEX. EC and Fb responded more vigorously to AS-Tspan8- than AS-TEX. Though EC and Fb responses differed, both cell lines predominantly responded to membrane receptor activation with upregulation and activation of signaling molecules and transcription factors. Minor TEX-initiated changes in the miRNA profile relied, at least partly, on long noncoding RNA (lncRNA) that also affected chromosome organization and mRNA processing. These analyses uncovered three important points. TEX activate target cell autonomous programs. Responses are initiated by TEX targeting units and are target cell-specific. The strong TEX-promoted lncRNA impact reflects lncRNA shuttling and location-dependent distinct activities. These informations urge for an in depth exploration on the mode of TEX-initiated target cell-specific remodeling including, as a major factor, lncRNA.

Roles of the mammalian target of rapamycin (mTOR) signaling pathway in the repair of hyperoxia-induced acute lung injury.

Rapamycin inhibits the mammalian target of rapamycin (mTOR) activity and has been proven effective for the treatment of lung injury. The objective of this study was to investigate the roles of the mTOR pathway and its inhibitor rapamycin in the repair of hyperoxia-induced acute lung injury (ALI). Firstly, premature rat lung fibroblast L929 cells were cultured under different oxygen concentrations (40%, 60%, and 90%). At day 3, 7 and 14 after exposure, MTT assay and flow cytometry were used to evaluate the effect of oxygen stress on cell viability and apoptosis of L929 cells, respectively. Secondly, microscopy, MTT assay and flow cytometry was used to investigate the effect of 10 nM rapamycin on 90% O2 exposed L929 cells. We also used small interfering RNAs (siRNAs) to abrogate the expression of mTOR in 90% O2 exposed L929 cells, and then evaluated the apoptosis and cell viability using flow cytometry and the MTT assay, respectively. In addition, western blot was used to detect the protein expression of Bcl-2, p53, TGF-β and connective tissue growth factor (CTGF). A hyperoxia-induced lung injury model was established in Sprague Dawley (SD) rats in order to evaluate the histopathological changes in lung tissues and expression of the mTOR pathway and fibrosis related factors. Exposure to 40%, 60% or 90% oxygen all significantly inhibited the growth of L929 cells. Application of 10 nM rapamycin was found to effectively promote apoptosis of 90% O2 exposed L929 cells. In addition, mTOR siRNA promoted the apoptosis and inhibited the growth of L929 cells. Rapamycin inhibited the activation of the mTOR signaling pathway, down-regulated the expression of downstream proteins p70S6K and 4EBP1, reduced the collagen deposition and the production of fibrosis-inducing factors, including TGF-β and CTGF in hyperoxia-induced lung injury rats. Rapamycin may be useful for the treatment of hyperoxia-induced acute lung injury (ALI) by inhibiting the activation of mTOR signaling pathway.

Dynamic Variation of RAS on Silicotic Fibrosis Pathogenesis in Rats

The dynamic variation of renin-angiotensin system (RAS) in silicosis remains unclear. Seventy Wistar rats were divided into 7 groups including control group, silicosis groups (inhaling SiO2 for 2, 4, 8, 16 and 24 weeks, respectively) and Captopril (Cap) group. Rat lung primary fibroblasts were divided into control group, SiO2-stimulated group (0, 0.5, 1, 3, 6, 12, 24 and 48 h) and Cap group. The silicotic nodules were formed and collagens were deposited gradually in silicosis group observed by haematoxylin and eosin (HE) staining and Van Gieson (VG) staining. Cap relieved the lung fibrosis and collagen deposition. Immunohistochemistry indicated the positive expression of α-smooth muscle actin (α-SMA) was increased gradually in silicotic rat lung tissue. Western blotting revealed the expression of collagen type I (Col I) and α-SMA was up-regulated in silicotic rat lung tissue and fibroblasts stimulated by SiO2. Cap decreased the expression of Col I and α-SMA in silicotic rat lung tissue and fibroblasts stimulated by SiO2. Western blotting also demonstrated the expression of angiotensin-converting enzyme (ACE) and angiotensin II type 1 receptor (AT1) was increased, and the expression of ACE2 and Mas was decreased gradually in silicotic rat lung tissue and fibroblasts stimulated by SiO2. ELISA showed the serum levels of ACE and angiotensin II (Ang II) were also increased and ACE2 and Ang (1-7) were decreased in the silicosis group. Treatment with Cap decreased the expression levels of ACE, Ang II and AT1, and increased the expression levels of ACE2, Ang (1-7) and Mas. These findings suggested that an imbalance between ACE-Ang II-AT1 axis and ACE2-Ang (1-7)-Mas axis may participate in the development of silicosis.

Protective Effect of Angiotensin (1-7) on Silicotic Fibrosis in Rats

ObjectiveSilicosis, caused by inhalation of silica dust, is the most serious occupational disease in China and the aim of present study was to explore the protective effect of Ang (1-7) on silicotic fibrosis and myofibroblast differentiation induced by Ang II. MethodsHOPE-MED 8050 exposure control apparatus was used to establish the rat silicosis model. Pathological changes and collagen deposition of the lung tissue were examined by H.E. and VG staining, respectively. The localizations of ACE2 and α-smooth muscle actin (α-SMA) in the lung were detected by immunohistochemistry. Expression levels of collagen type I, α-SMA, ACE2, and Mas in the lung tissue and fibroblasts were examined by western blot. Levels of ACE2, Ang (1-7), and Ang II in serum were determined by ELISA. Co-localization of ACE2 and α-SMA in fibroblasts was detected by immunofluorescence. ResultsAng (1-7) induced pathological changes and enhanced collagen deposition in vivo. Ang (1-7) decreased the expressions of collagen type I and α-SMA and increased the expressions of ACE2 and Mas in the silicotic rat lung tissue and fibroblasts stimulated by Ang II. Ang (1-7) increased the levels of ACE2 and Ang (1-7) and decreased the level of Ang II in silicotic rat serum. A779 enhanced the protective effect of Ang (1-7) in fibroblasts stimulated by Ang II. ConclusionAng (1-7) exerted protective effect on silicotic fibrosis and myofibroblast differentiation induced by Ang II by regulating ACE2-Ang (1-7)-Mas axis.

Identification of Betamethasone-Regulated Target Genes and Cell Pathways in Fetal Rat Lung Mesenchymal Fibroblasts.

Preterm birth is characterized by severe lung immaturity that is frequently treated antenatally or postnatally with the synthetic steroid betamethasone. The underlying cellular targets and pathways stimulated by betamethasone in the fetal lung are poorly defined. In this study, betamethasone was compared with corticosterone in steroid-treated primary cultures of fetal rat lung fibroblasts stimulated for 6 hours and analyzed by whole-cell transcriptome sequencing and glucocorticoid (GC) receptor (GR) chromatin immunoprecipitation sequencing (ChIP-Seq) analysis. Strikingly, betamethasone stimulated a much stronger transcriptional response compared with corticosterone for both induced and repressed genes. A total of 483 genes were significantly stimulated by betamethasone or corticosterone, with 476 stimulated by both steroids, indicating a strong overlap in regulation. Changes in mRNA levels were confirmed by quantitative PCR for eight induced and repressed target genes. Pathway analysis identified cell proliferation and cytoskeletal/cell matrix remodeling pathways as key processes regulated by both steroids. One target, transglutaminase 2 (Tgm2), was localized to fetal lung mesenchymal cells. Tgm2 mRNA and protein levels were strongly increased in fibroblasts by both steroids. Whole-genome GR ChIP-Seq analysis with betamethasone identified GC response element-binding sites close to the previously characterized GR target genes Per1, Dusp1, Fkbp5, and Sgk1 and near the genes identified by transcriptome sequencing encoding Crispld2, Tgm2, Hif3α, and Kdr, defining direct genomic induction of expression in fetal lung fibroblasts via the GR. These results demonstrate that betamethasone stimulates specific genes and cellular pathways controlling cell proliferation and extracellular matrix remodeling in lung mesenchymal fibroblasts, providing a basis for betamethasone's treatment efficacy in preterm birth.

Anti-senescence role of heterozygous fumarate hydratase gene knockout in rat lung fibroblasts in vitro.

Abnormalities in tricarboxylic acid (TCA) cycle function were related to a variety of pathological processes. Fumarate hydratase (FH) is a required enzyme in the TCA cycle. To explore the general influence of FH knockout, we isolated FH+/– rat and normal rat lung fibroblasts and cultured these cells in vitro. The isolated fibroblasts with the current method were rather homogeneous and were confirmed spindle in morphology, positive for vimentin and negative for α-SMA (α-smooth muscle actin). Sequencing of the PCR (polymerase chain reaction) products flanking the FH gene mutation verified the FH+/– status, and the FH gene and protein expression were confirmed to be reduced in the FH+/– cells. No sign of ageing for the FH+/– cells after 61 passages was observed, but the controls died out at this stage. Flow cytometry revealed increased S-phase and decreased G1/G0 proportions with significantly less early apoptosis in FH+/– cells compared to that in control cells. At the same time, increased glucose consumption, intracellular fumarate production and extracellular lactate secretion were verified in the FH+/– cells. Correspondingly, FH+/– cells showed a lower basal oxygen consumption rate (OCR) but a higher level of reactive oxygen species (ROS) production. Single cell cloning and cell line establishment were successfully performed with the FH+/– cells at the 84th passage. All the above results indicate an important role for FH+/– in the longevity or immortality of the FH+/– cells, in which increased p53 and TERT (telomerase reverse transcriptase) protein expression, decreased p21 and p16 protein expression and negative SA-β-Gal (senescence-associated beta-galactosidase) were verified along with metabolic reprogramming.