Human Pulmonary Fibroblast Research Articles

Reusable glassware for routine cell culture—a sterile, sustainable and affordable alternative to single-use plastics

Cell culture is a cornerstone of in vitro biological research. Whilst glassware was once commonplace in tissue culture facilities, in recent decades laboratories have moved towards a heavy reliance on single use plastics for routine procedures. Single use plastics allow for accessible, sterile, and often affordable equipment that comes at a high environmental cost. We developed a glassware preparation and cleaning process that allowed the comparison of “traditional” plastic-heavy, and adapted “sustainable,” cell culture practices, to empirically compare the sterility, viability, and proliferative capacity of cells cultured with differing techniques, by observing IL-6 production, morphology, and proliferation rate of cultured human pulmonary fibroblast cells. During which, we calculated the carbon footprint of traditional versus sustainable methods. We additionally endeavored to provide a realistic overview of the steps required to transition to more sustainable cell culture practices and make suggestions to ease the cost, labor, and time required to uptake similar practices in other laboratories. Cells cultured using reusable glassware did not show signs of contamination or stress compared to cells grown solely with plasticware, and glassware baked at 180°C for 120 min was sufficiently decontaminated and depyrogenated for culturing these cells. An individual researcher adopting the same methodology could reduce their carbon footprint by 105.92 kg of Carbon dioxide equivalent (CO2e) whilst also saving money (£408.78) over a 10-year period. We predict that these benefits would be greater if more researchers were to uptake these adapted practices. We intend for this paper to reassure researchers that viable, sterile, and sustainable routine cell culture can be achieved with little upfront cost to the researcher, with the prospective benefit of greatly reducing the cost to the environment. We additionally hope that increased uptake, and thus demand of more sustainable practices, encourages suppliers, policy makers, and funding bodies to make sustainable practices more accessible to individual researchers and institutions worldwide.

Identification of anti-fibrotic and pro-apoptotic bioactive compounds from Ganoderma formosanum and their possible mechanisms in modulating TGF-β1-induced lung fibrosis

Ethnopharmacological relevanceThe Compendium of Materia Medica and the Classic of Materia Medica, the two most prominent records of traditional Chinese medicine, documented the therapeutic benefits of Ganoderma sinense particularly in addressing pulmonary-related ailments. Ganoderma formosanum, an indigenous subspecies of G. sinense from Taiwan, has demonstrated the same therapeutic properties. Aim of the studyThe aim of this study is to identify bioactive compounds and evaluate the potential of G. formosanum extracts as a novel treatment to alleviate pulmonary fibrosis (PF). Using an in-house drug screening platform, two-stage screening was performed to determine their anti-fibrotic efficacy. Methods and materialsG. formosanum was fractionated into four partitions by solvents of different polarities. To determine their antifibrotic and pro-apoptotic properties, the fractions were analyzed using two TGF-β1-induced pulmonary fibrosis cell models (NIH-3T3) and human pulmonary fibroblast cell lines, immunoblot, qRT-PCR, and annexin V assays. Subsequently, transcriptomic analysis was conducted to validate the findings and explore possible molecular pathways. The identification of potential bioactive compounds was achieved through UHPLC-MS/MS analysis, while molecular interaction study was investigated by multiple ligands docking and molecular dynamic simulations. ResultsThe ethyl acetate fraction (EAF) extracted from G. formosanum demonstrated substantial anti-fibrotic and pro-apoptotic effects on TGF-β1-induced fibrotic models. Moreover, the EAF exhibited no discernible cytotoxicity. Untargeted UHPLC-MS/MS analysis identified potential bioactive compounds in EAF, including stearic acid, palmitic acid, and pentadecanoic acid. Multiple ligands docking and molecular dynamic simulations further confirmed that those bioactive compounds possess the ability to inhibit TGF-β receptor 1. ConclusionPotential bioactive compounds in G. formosanum were successfully extracted and identified in the EAF, whose anti-fibrotic and pro-apoptotic properties could potentially modulate pulmonary fibrosis. This finding not only highlights the EAF's potential as a promising therapeutic candidate to treat pulmonary fibrosis, but it also elucidates how Ganoderma confers pulmonary health benefits as described in the ancient texts.

Propyl gallate induces cell death in human pulmonary fibroblast through increasing reactive oxygen species levels and depleting glutathione

Propyl gallate (PG) exhibits an anti-growth effect on various cell types. The present study investigated the impact of PG on the levels of reactive oxygen species (ROS) and glutathione (GSH) in primary human pulmonary fibroblast (HPF) cells. Moreover, the effects of N-acetyl cysteine (NAC, an antioxidant), l-buthionine sulfoximine (BSO, a GSH synthesis inhibitor), and small interfering RNA (siRNAs) against various antioxidant genes on ROS and GSH levels and cell death were examined in PG-treated HPF cells. PG (100–800 μM) increased the levels of total ROS and O2·− at early time points of 30–180 min and 24 h, whereas PG (800–1600 μM) increased GSH-depleted cell number at 24 h and reduced GSH levels at 30–180 min. PG downregulated the activity of superoxide dismutase (SOD) and upregulated the activity of catalase in HPF cells. Treatment with 800 μM PG increased the number of apoptotic cells and cells that lost mitochondrial membrane potential (MMP; ΔΨm). NAC treatment attenuated HPF cell death and MMP (ΔΨm) loss induced by PG, accompanied by a decrease in GSH depletion, whereas BSO exacerbated the cell death and MMP (ΔΨm) loss without altering ROS and GSH depletion levels. Furthermore, siRNA against SOD1, SOD2, or catalase attenuated cell death in PG-treated HPF cells, whereas siRNA against GSH peroxidase enhanced cell death. In conclusion, PG induced cell death in HPF cells by increasing ROS levels and depleting GSH. NAC was found to decrease HPF cell death induced by PG, while BSO enhanced cell death. The findings shed light on how manipulating the antioxidant system influence the cytotoxic effects of PG in HPF cells.

Propyl gallate induces human pulmonary fibroblast cell death through the regulation of Bax and caspase-3

Propyl gallate (PG) has been found to exert an inhibitory effect on the growth of different cell types, including lung cancer cells. However, little is known about the cytotoxicological effects of PG specifically on normal primary lung cells. The current study examined the cellular effects and cell death resulting from PG treatment in human pulmonary fibroblast (HPF) cells. DNA flow cytometry results demonstrated that PG (100–1,600 μM) had a significant impact on the cell cycle, leading to G1 phase arrest. Notably, 1,600 μM PG slightly increased the number of sub-G1 cells. Additionally, PG (400–1,600 μM) resulted in the initiation of cell death, a process that coincided with a loss of mitochondrial membrane potential (MMP; ΔΨm). This loss of MMP (ΔΨm) was evaluated using a FACS cytometer. In PG-treated HPF cells, inhibitors targeting pan-caspase, caspase-3, caspase-8, and caspase-9 showed no significant impact on the quantity of annexin V-positive and MMP (ΔΨm) loss cells. The administration of siRNA targeting Bax or caspase-3 demonstrated a significant attenuation of PG-induced cell death in HPF cells. However, the use of siRNAs targeting p53, Bcl-2, or caspase-8 did not exhibit any notable effect on cell death. Furthermore, none of the tested MAPK inhibitors, including MEK, c-Jun N-terminal kinase (JNK), and p38, showed any impact on PG-induced cell death or the loss of MMP (ΔΨm) in HPF cells. In conclusion, PG induces G1 phase arrest of the cell cycle and cell death in HPF cells through apoptosis and/or necrosis. The observed HPF cell death is mediated by the modulation of Bax and caspase-3. These findings offer insights into the cytotoxic and molecular effects of PG on normal HPF cells.

Ebselen Inhibits the Growth of Lung Cancer Cells via Cell Cycle Arrest and Cell Death Accompanied by Glutathione Depletion.

Ebselen is a glutathione (GSH) peroxidase (GPx) mimic originally developed to reduce reactive oxygen species (ROS). However, little is known about its cytotoxicological effects on lung cells. Therefore, this study aimed to investigate the effects of Ebselen on the cell growth and cell death of A549 lung cancer cells, Calu-6 lung cancer cells, and primary normal human pulmonary fibroblast (HPF) cells in relation to redox status. The results showed that Ebselen inhibited the growth of A549, Calu-6, and HPF cells with IC50 values of approximately 12.5 μM, 10 μM, and 20 μM, respectively, at 24 h. After exposure to 15 μM Ebselen, the proportions of annexin V-positive cells were approximately 25%, 65%, and 10% in A549, Calu-6, and HPF cells, respectively. In addition, Ebselen induced arrest at the S phase of the cell cycle in A549 cells and induced G2/M phase arrest in Calu-6 cells. Treatment with Ebselen induced mitochondrial membrane potential (MMP; ΔΨm) loss in A549 and Calu-6 cells. Z-VAD, a pan-caspase inhibitor, did not decrease the number of annexin V-positive cells in Ebselen-treated A549 and Calu-6 cells. Intracellular ROS levels were not significantly changed in the Ebselen-treated cancer cells at 24 h, but GSH depletion was efficiently induced in these cells. Z-VAD did not affect ROS levels or GSH depletion in Ebselen-treated A549 or Ebselen-treated Calu-6 cells. In conclusion, Ebselen inhibited the growth of lung cancer and normal fibroblast cells and induced cell cycle arrest and cell death in lung cancer cells with GSH depletion.

Construction of Human Three-Dimensional Lung Model Using Layer-by-Layer Method.

The respiratory tract is one of the frontline barriers for biological defense. Lung epithelial intercellular adhesions provide protection from bacterial and viral infections and prevent invasion into deep tissues by pathogens. Dysfunction of lung epithelial intercellular adhesion caused by pathogens is associated with development of several diseases, such as acute respiratory distress syndrome, pneumonia, and asthma. To elucidate the pathological mechanism of respiratory infections, two-dimensional cell cultures and animal models are commonly used, although are not useful for evaluating host specificity or human biological response. With the rapid progression and worldwide spread of severe acute respiratory syndrome coronavirus-2, there is increasing interest in the development of a three-dimensional (3D) in vitro lung model for analyzing interactions between pathogens and hosts. However, some models possess unclear epithelial polarity or insufficient barrier functions and need the use of complex technologies, have high cost, and long cultivation terms. We previously reported about the fabrication of 3D cellular multilayers using a layer-by-layer (LbL) cell coating technique with extracellular matrix protein, fibronectin (FN), and gelatin (G). In the present study, such a LbL cell coating technique was utilized to construct a human 3D lung model in which a monolayer of the human lower airway epithelial adenocarcinoma cell line Calu-3 cells was placed on 3D-cellular multilayers composed of FN-G-coated human primary pulmonary fibroblast cells. The 3D lung model thus constructed demonstrated an epithelial-fibroblast layer that maintained uniform thickness until 7 days of incubation. Moreover, expressions of E-cadherin, ZO-1, and mucin in the epithelial layer were observed by immunohistochemical staining. Epithelial barrier integrity was evaluated using transepithelial electrical resistance values. The results indicate that the present constructed human 3D lung model is similar to human lung tissues and also features epithelial polarity and a barrier function, thus is considered useful for evaluating infection and pathological mechanisms related to pneumonia and several pathogens. Impact statement A novel in vitro model of lung tissue was established. Using a layer-by-layer cell coating technique, a three-dimensional cultured lung model was constructed. The present novel model was shown to have epithelial polarity and chemical barrier functions. This model may be useful for investigating interaction pathogens and human biology.

Human epididymis protein 4 aggravates airway inflammation and remodeling in chronic obstructive pulmonary disease

BackgroundChronic obstructive pulmonary disease (COPD) is a progressive disease characterized by chronic inflammation and airway remodeling. Human epididymis protein 4 (HE4) plays a critical role in various inflammatory or fibrotic diseases. However, the role of HE4 in COPD remains unidentified.MethodsHE4 expression was determined in the lung tissues from COPD patients and cigarette smoke (CS)-exposed mice using immunohistochemical staining, qPCR, or western blot. The plasma level of HE4 was detected by ELISA. The regulations of HE4 in the expressions of CS extract (CSE)-induced inflammatory cytokines in human bronchial epithelial cells (HBE) were investigated through knockdown or overexpression of HE4. The role of secretory HE4 (sHE4) in the differentiation and proliferation in human pulmonary fibroblast cells (HPF) was explored via qPCR, western blot, CCK8 assay or 5-ethynyl-2′-deoxyuridine (EdU) staining. The probe of related mechanism in CSE-induced HE4 increase in HBE was conducted by administrating N-acetylcysteine (NAC).ResultsHE4 was up-regulated in both the lung tissue and plasma of COPD patients relative to controls, and the plasma HE4 was negatively associated with lung function in COPD patients. The same enhanced HE4 expression was verified in CS-exposed mice and CSE-induced HBE, but CSE failed to increase HE4 expression in HPF. In vitro experiments showed that reducing HE4 expression in HBE alleviated CSE-induced IL-6 release while overexpressing HE4 facilitated IL-6 expression, mechanistically through affecting phosphorylation of NFκB-p65, whereas intervening HE4 expression had no distinctive influence on IL-8 secretion. Furthermore, we confirmed that sHE4 promoted fibroblast-myofibroblast transition, as indicated by promoting the expression of fibronectin, collagen I and α-SMA via phosphorylation of Smad2. EdU staining and CCK-8 assay demonstrated the pro-proliferative role of sHE4 in HPF, which was further confirmed by enhanced expression of survivin and PCNA. Pretreatment of NAC in CSE or H2O2-induced HBE mitigated HE4 expression.ConclusionsOur study indicates that HE4 may participate in airway inflammation and remodeling of COPD. Cigarette smoke enhances HE4 expression and secretion in bronchial epithelium mediated by oxidative stress. Increased HE4 promotes IL-6 release in HBE via phosphorylation of NFκB-p65, and sHE4 promotes fibroblastic differentiation and proliferation.

Integrated multicomponent analysis based on UHPLC-Q-Exactive Orbitrap-MS and network pharmacology to elucidate the potential mechanism of Baoyuan decoction against idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a serious lung disease with a high mortality rate. Baoyuan decoction (BYD), a classic medicinal food homology recipe, has anti-apoptotic effects, enhances immune function, and alleviates fibrosis, suggesting that it may be a potential therapeutic drug for IPF. We aimed to identify the main active ingredients of BYD, determine the basis of its efficacy, prove its anti-IPF effects, and explore the mechanisms underlying its anti-IPF effects. In this study, the active components of BYD were detected and analysed by ultra-high-performance liquid chromatography coupled with hybrid quadrupole Orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS). A network pharmacology analysis was performed to determine the potential targets and relevant pathways of BYD in treating IPF. Western blotting and quantitative real-time polymerase chain reaction (qPCR) were conducted to verify the efficacy of BYD against IPF. Finally, molecular docking and qPCR were performed to identify the central targets of BYD. A total of 39 components of BYD were identified. After performing the network pharmacology analysis, 35 active components and eight presumptive targets of BYD were found to play a central role in its anti-IPF effects. The molecular docking results indicated that most of the active components of BYD exhibited good binding activity with these eight central target proteins. In addition, the expression of collagen, α-SMA, and these eight targets in human pulmonary fibroblast (HPF) cells was suppressed from treatment with BYD. This study determined the efficacy of BYD against IPF and clarified its multiple-target and multiple-pathway mechanisms. Furthermore, the study also provides a new method for exploring the chemical and pharmacological bases of other traditional Chinese medicine (TCM).

HGF-Modified Dental Pulp Stem Cells Mitigate the Inflammatory and Fibrotic Responses in Paraquat-Induced Acute Respiratory Distress Syndrome.

Paraquat (PQ) poisoning can cause acute lung injury and progress to pulmonary fibrosis and eventually death without effective therapy. Mesenchymal stem cells (MSCs) and hepatocyte growth factor (HGF) have been shown to partially reverse this damage. MSCs can be derived from bone marrow (BM-MSCs), adipose tissue (AD-MSCs), umbilical cord (UC-MSCs), dental pulp (DPSCs), and other sources. The biological characteristics of MSCs are specific to the tissue source. To develop an effective treatment for PQ poisoning, we compared the anti-inflammatory and antifibrotic effects of UC-MSCs and DPSCs and chose and modified a suitable source with HGF to investigate their therapeutic effects in vitro and in vivo. In this study, MSCs' supernatant was beneficial to the viability and proliferation of human lung epithelial cell BEAS-2B. Inflammatory and fibrosis-related cytokines were analyzed by real-time PCR. The results showed that MSCs' supernatant could suppress the expression of proinflammatory and profibrotic cytokines and increase the expression of anti-inflammatory and antifibrotic cytokines in BEAS-2B cells and human pulmonary fibroblast MRC-5. Extracellular vesicles (EVs) derived from MSCs performed more effectively than MSCs' supernatant. The effect of DPSCs was stronger than that of UC-MSCs and was further strengthened by HGF modification. PQ-poisoned mice were established, and UC-MSCs, DPSCs, and DPSCs-HGF were administered. Histopathological assessments revealed that DPSCs-HGF mitigated lung inflammation and collagen accumulation more effectively than the other treatments. DPSCs-HGF reduced lung permeability and increased the survival rate of PQ mice from 20% to 50%. Taken together, these results indicated that DPSCs can suppress inflammation and fibrosis in human lung cells better than UC-MSCs. The anti-inflammatory and antifibrotic effects were significantly enhanced by HGF modification. DPSCs-HGF ameliorated pulmonitis and pulmonary fibrosis in PQ mice, effectively improving the survival rate, which might be mediated by paracrine mechanisms. The results suggested that DPSCs-HGF transplantation was a potential therapeutic approach for PQ poisoning.

Dehydrobufotenin extracted from the Amazonian toad Rhinella marina (Anura: Bufonidae) as a prototype molecule for the development of antiplasmodial drugs.

Background:The resistance against antimalarial drugs represents a global challenge in the fight and control of malaria. The Brazilian biodiversity can be an important tool for research and development of new medicinal products. In this context, toxinology is a multidisciplinary approach on the development of new drugs, including the isolation, purification, and evaluation of the pharmacological activities of natural toxins. The present study aimed to evaluate the cytotoxicity, as well as the antimalarial activity in silico and in vitro of four compounds isolated from Rhinella marina venom as potential oral drug prototypes.Methods: Four compounds were challenged against 35 target proteins from P. falciparum and screened to evaluate their physicochemical properties using docking assay in Brazilian Malaria Molecular Targets (BraMMT) software and in silico assay in OCTOPUS® software. The in vitro antimalarial activity of the compounds against the 3D7 Plasmodium falciparum clones were assessed using the SYBR Green I based assay (IC50). For the cytotoxic tests, the LD50 was determined in human pulmonary fibroblast cell line using the [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay. Results: All compounds presented a ligand-receptor interaction with ten Plasmodium falciparum-related protein targets, as well as antimalarial activity against chloroquine resistant strain (IC50 = 3.44 μM to 19.11 μM). Three of them (dehydrobufotenine, marinobufagin, and bufalin) showed adequate conditions for oral drug prototypes, with satisfactory prediction of absorption, permeability, and absence of toxicity. In the cell viability assay, only dehydrobufotenin was selective for the parasite. Conclusions: Dehydrobufotenin revealed to be a potential oral drug prototype presenting adequate antimalarial activity and absence of cytotoxicity, therefore should be subjected to further studies.

A bis-indazolic ruthenium(II) complex: Reactivity and biological studies on cancer cells

Ru(II)-based complexes have been widely investigated as potential anticancer agents, leading cell death of tumor mass. In this work, a metal complex, cis-[RuII(Hind)2(bpy)2](PF6)2 (Hind = indazole and bpy = 2,2′-bipyridine), was synthesized and characterized by elemental analysis, spectroscopy and electrochemistry methods, validating its formulation. This complex can act as a Brønsted-Lowry acid with pKa close to physiological pH. Photosubstitution reaction was noticed upon blue light irradiation with release of indazol molecules in acetonitrile, a behavior also noticed, indicating its photosensitivity. This metal complex was screened in vitro against distinct human cancer cell lines. Hepatocellular carcinoma cells (HepG2) were the most sensitive, with the half maximal inhibitory concentration (IC50) at 7.2 µmol L−1. Further biological studies showed DNA fragmentation induced by this compound, which resulted in cell cycle arrest in S phase and HepG2 cell death by apoptosis. Moreover, there was no significant cytotoxicity against healthy human pulmonary fibroblast cells (MRC-5), and therefore the compound exhibited a high selectivity index, indeed a better in vitro anticancer profile than some structurally related compounds (e.g. NAMI-A and KP1039). Our results indicate exciting potential pharmacological applications of this metal complex in liver cancer treatment, which might be supported by further studies.

Stimulatory effect of gastroesophageal reflux disease (GERD) on pulmonary fibroblast differentiation

Epidemiological studies indicate that prolonged micro-aspiration of gastric fluid is associated in gastroesophageal reflux disease with the development of chronic respiratory diseases, possibly caused by inflammation-related immunomodulation. Therefore, we sought to ascertain the effect of gastric fluid exposure on pulmonary residential cells. The expression of α-smooth muscle actin as a fibrotic marker was increased in both normal human pulmonary fibroblast cells and mouse macrophages. Gastric fluid enhanced the proliferation and migration of HFL-1 cells and stimulated the expression of inflammatory cytokines in an antibody assay. Elevated expression of the Rho signaling pathway was noted in fibroblast cells stimulated with gastric fluid or conditioned media. These results indicate that gastric fluid alone, or the mixture of proinflammatory mediators induced by gastric fluid in the pulmonary context, can stimulate pulmonary fibroblast cell inflammation, migration, and differentiation, suggesting that a wound healing process is initiated. Subsequent aberrant repair in pulmonary residential cells may lead to pulmonary fibroblast differentiation and fibrotic progression. The results point to a stimulatory effect of chronic GERD on pulmonary fibroblast differentiation, and this may promote the development of chronic pulmonary diseases in the long term.

Cytotoxic effects of alkaline tetrasodium EDTA irrigating solutions.

The aim of this study is to determine the cytotoxic effects of tetrasodium ethylenediaminetetraacetic acid (EDTANa4) when used alone or when combined with sodium hypochlorite (NaOCl), with and without the addition of cetrimide (CTR). Human pulmonary fibroblast cell line was exposed to the following irrigating solutions: group 1, 2.5% NaOCl; group 2, 10% EDTANa4; group 3, 20% EDTANa4; group 4, 2.5% NaOCl/5% EDTANa4; group 5, 2.5% NaOCl/10% EDTANa4; group 6, 2.5% NaOCl/5% EDTANa4/0.2% CTR; group 7, 2.5% NaOCl/10% EDTANa4/0.2% CTR; group 8, control, cells in Dulbecco's modified Eagle's medium. Methyl thiazol tetrazolium assay was used to determine the viability of cells after 1 and 24 h. Viability percentages were analyzed for global comparison using the Welch test followed by the Games-Howell test to determine groups with similar viability, and the Student's t test was used to compare the two times. The lowest viability was obtained with a 2.5% NaOCl solution at both time periods. The association of NaOCl with EDTANa4 increased the cellular viability in direct relation with the concentration of the chelating agent. Globally, after 24 h of exposure, cell viability reduced. The solutions of EDTANa4 showed moderate cytotoxic effects when compared with NaOCl alone.

Ambient particulate matter attenuates Sirtuin1 and augments SREBP1-PIR axis to induce human pulmonary fibroblast inflammation: molecular mechanism of microenvironment associated with COPD.

Evidences have shown a strong link between particulate matter (PM) and increased risk in human mortality and morbidity, including asthma, chronic obstructive pulmonary disease (COPD), respiratory infection, and lung cancer. However, the underlying toxicologic mechanisms remain largely unknown. Utilizing PM-treated human pulmonary fibroblasts (HPF) models, we analyzed gene expression microarray data and Ingenuity Pathway Analysis (IPA) to identify that the transcription factor sterol regulatory element-binding protein 1 (SREBP1) was the main downstream regulator of Sirtuin1 (SIRT1). Quantitative PCR and western blot results showed that SIRT1 inhibited SREBP1 and further downregulated Pirin (PIR) and Nod-like receptor protein 3 (NLRP3) inflammasome after PM exposure. Inhibitors of SIRT1, SREBP1, and PIR could reverse PM-induced inflammation. An in silico analysis revealed that PIR correlated with smoke exposure and early COPD. Immunohistochemical analysis of tissue microarrays from PM-fed mouse models was used to determine the association of PIR with PM. These data demonstrate that the SIRT1-SREBP1-PIR/ NLRP3 inflammasome axis may be associated with PM-induced adverse health issues. SIRT1 functions as a protector from PM exposure, whereas PIR acts as a predictor of PM-induced pulmonary disease. The SIRT1-SREBP1-PIR/ NLRP3 inflammasome axis may present several potential therapeutic targets for PM-related adverse health events.

Carbon nanotubes and crystalline silica stimulate robust ROS production, inflammasome activation, and IL-1β secretion in macrophages to induce myofibroblast transformation.

Pulmonary exposure to inhaled particulates elicits complex inflammatory and fibrotic responses that may progress to chronic fibrosis. The fibrogenic potentials of respirable particulates are influenced by their physicochemical properties and their interactions with major pathways to drive fibrotic development in the lung. Macrophages were exposed to six carbon nanotubes (CNTs) of varying dimensions, crystalline silica, or carbon black (CB), with lipopolysaccharide (LPS) and transforming growth factor (TGF)-β1 as positive controls. Macrophage-conditioned media was collected and applied to cultures of human pulmonary fibroblast line WI38-VA13 to induce myofibroblast transformation. Multi-walled and single-walled CNTs (MWCNTs and SWCNTs, respectively) and silica, but not CB, stimulated robust myofibroblast transformation through macrophage-conditioned media. On an equal weight basis, MWCNTs induced higher induction than SWCNTs. High induction was observed for MWCNTs with a long and slender or a short and rigid shape, and silica, atlevelscomparable to those by LPS and TGF-β1. Fibrogenic particulates induced high levels of IL-1β mRNA expression and protein secretion that are required for macrophage-guided myofibroblast transformation. Induction of IL-1β is dependent on the activation of the NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome and ROS (reactive oxygen species) production in macrophages, as inhibition of NLRP3 by MCC950 and reduction of ROS production by N-acetylcysteine blocked NLRP3 activation,IL-1β induction, and fibroblast activation anddifferentiation. Therefore, fibrogenic CNTs and silica, but not CB, elicit robust macrophage-guided myofibroblast transformation, which depends on the induction of IL-1β through the NLRP3 inflammasome pathway andthe increased production of ROS in macrophages.

Pulmonary activation of vitamin D3 and preventive effect against interstitial pneumonia.

Calcitriol [1,25(OH)2D3] is usually investigated in studies on the preventive effect of activated vitamin D against interstitial pneumonia. Although cholecalciferol (vitamin D3) can be easily obtained in the diet and has a longer half-life than calcitriol, there have been few investigations of its effect on interstitial pneumonia. We used human pulmonary fibroblast cell lines (HPFCs) and a mouse model of bleomycin-induced pulmonary fibrosis to evaluate whether vitamin D3 was activated in the lungs and had a preventive effect against interstitial pneumonia. Expression of the vitamin D receptor gene and genes for enzymes metabolizing vitamin D was evaluated in two HPFCs, and the suppressive effect of vitamin D3 on induction of inflammatory cytokines was also assessed. Gene expression of the vitamin D receptor and vitamin D-metabolizing enzymes was observed in both human pulmonary fibroblast cell lines. Vitamin D3 suppressed bleomycin-induced expression of inflammatory cytokines and fibrosis markers by the HPFCs. In mice, symptoms of bleomycin-induced pulmonary fibrosis were improved and expression of fibrosis markers/fibrosis inducers was decreased by a high vitamin D3 diet. Vitamin D3 is activated locally in lung tissues, suggesting that high dietary intake of vitamin D3 may have a preventive effect against interstitial pneumonia.

PM2.5 induced pulmonary fibrosis in vivo and in vitro

Epidemiological studies have revealed positive correlation between particulate matter with an aerodynamic diameter of < 2.5 µm (PM2.5) and pulmonary fibrosis (PF). As etiology and pathogenesis of PF have not been fully elucidated, this study was to investigate the potential mechanism by which PM2.5 exposure adversely induced PF in vivo and in vitro. In the present study, 6-week-old C57/BL6J mice were intranasally administrated with PM2.5 (100 μg/day) for 4 weeks. Micro-CT and hematoxylin-eosin (HE) staining analysis showed that lung inflammation and incipient fibrosis symptoms were induced after PM2.5 exposure. The expression of Transforming growth factor-β1 (TGF-β1), α-Smooth muscle actin (α-SMA), and Collagen type I (COL1) in mice lung was increased. Upregulation of TGF-β1 in mice serum was also detected by ELISA after exposure to PM2.5. Moreover, chronic PM2.5 exposure on human bronchial epithelial cell line BEAS-2B cells led to activation of TGF-β1/SMAD3 pathway, TGF-β1 excretion and epithelial-mesenchymal transition (EMT), while PM2.5 also triggered the activation of TGF-β1/SMAD3 pathway, TGF-β1 excretion as well as differentiation of human pulmonary fibroblast cell line HFL-1 cells, and TGF-β1 production in mouse macrophage cell line RAW264.7 cells. Furthermore, cell culture medium of PM2.5-treated BEAS-2B and RAW264.7 cells could both activate TGF-β1/SMAD3 signaling, α-SMA and COL1 upregulation in HFL-1 cells. Therefore, we concluded that PM2.5 could induce PF by targeting pulmonary epithelium, macrophages and fibroblasts, suggesting that PM2.5 was a potent initiator of PF.

A novel molecular mechanism of microRNA-21 inducing pulmonary fibrosis and human pulmonary fibroblast extracellular matrix through transforming growth factor β1-mediated SMADs activation.

Pulmonary fibrosis (PF), characterized by the destruction of lung tissue architecture and the abnormal deposition of extracellular matrix (ECM) proteins, currently has no satisfactory treatment. The role of microRNA (miR)-21 in PF has been reported; the current study attempted to investigate a novel molecular mechanism by which miR-21 exerted its function. Consistent with previous studies, miR-21 inhibition reduced ECM protein levels in bleomycin (BLM)-induced mouse model of PF. In human pulmonary fibroblast (IMR-90), miR-21 inhibition reduced transforming growth factor β1 (TGFβ1)-induced ECM protein expression. Regarding a novel molecular mechanism, TGFβ1 combined withTGFβ1 receptor 1 (TGFβ1RI) to activate SMAD2/3, promote SMAD4 nucleus transformation, and thus regulate miR-21 expression and ECM. SMAD3 and SMADs complex could bind to the promoter region of miR-21 to promote miR-21 expression. In conclusion, miR-21 exerts promotive effects on BLM-induced PF and TGFβ1-induced ECM in IMR-90; TGFβ1 combines with TGFβ1RI to activate SMAD2/3, promote SMAD4 nucleus transformation, promote miR-21 expression, and thus to promote BLM-induced PF and TGFβ1-induced ECM in IMR-90 cells.

DNA Topoisomerase IB as a Potential Ionizing Radiation Exposure and Dose Biomarker.

In radiation exposure scenarios where physical dosimetry is absent or inefficient, dose estimation must rely on biological markers. A reliable biomarker is of utmost importance in correlating biological system changes with radiation exposure. Human DNA topoisomerase ІB (topo І) is a ubiquitous nuclear enzyme, which is involved in essential cellular processes, including transcription, DNA replication and DNA repair, and is the target of anti-cancer drugs. It has been shown that the cellular activity of this enzyme is significantly sensitive to various DNA lesions, including radiation-induced DNA damages. Therefore, we investigated the potential of topo I as a biomarker of radiation exposure and dose. We examined the effect of exposure of different human cells to beta, X-ray and gamma radiation on the cellular catalytic activity of topo I. The results demonstrate a significant reduction in the DNA relaxation activity of topo I after irradiation and the level of the reduction was correlated with radiation dose. In normal human peripheral blood lymphocytes, exposure for 3 h to an integral dose of 0.065 mGy from tritium reduced the enzyme activity to less than 25%. In MG-63 osteoblast-like cells and in human pulmonary fibroblast (HPF) cells exposed to gamma radiation from a 60Co source (up to 2 Gy) or to X rays (up to 2.8 Gy), a significant decrease in topo I catalytic activity was also observed. We observed that the enzyme-protein level was not altered but was partially posttranslational modified by ADP-ribosylation of the enzyme protein that is known to reduce topo I activity. The results of this study suggest that the decrease in the cellular topo I catalytic activity after low-dose exposure to different radiation types may be considered as a novel biomarker of ionizing radiation exposure and dose. For this purpose, a suitable ELISA-based method for large-scale analysis of radiation-induced topo I modification is under development.

Prodigiosin Alleviates Pulmonary Fibrosis Through Inhibiting miRNA-410 and TGF-β1/ADAMTS-1 Signaling Pathway

Background/Aims: Pulmonary fibrosis is a common outcome of various interstitial lung diseases. Prodigiosin (PG) is a series of red pigment with methoxypyrrole ring. This studyinvestigates therole of prodigiosin in pulmonary fibrosis and its underlying mechanisms. Methods: A pulmonary fibrosis rat model was established by intra-trachealinjection ofbleomycin A5. Rats were divided into 4 groups: Normal group, pulmonary fibrosis Model group, Prodigiosin treatment group and hydrocortisone treatmentgroup. HE and Masson staining were carried outto evaluate histopathological changes. The content of hydroxyproline in lung tissue was determined by alkaline hydrolysis. The expression of PICP and PIIINP was examined by ELISA. The mRNA expression of miR-410, TGF-β1 and ADAMTS1 in lung homogenate were detected by RT-PCR. The bronchoalveolar lavage fluid (BALF) and lung tissues of rats were collected and analyzed. Human embryonic pulmonary fibroblast (HEPF) was used for study in vitro. A dual-luciferase reporter assay was conducted to examine the effect of miR-410 on ADAMTS1 expression. Cell transfection was conducted to inhibit miR-410. MTT assay was performed to investigate cell proliferation. The expressions of miR-410, TGF-β1, ADAMTS1and other fibrosis related biomarkers (Col I, Col III, and α-SMA) wereexamined by RT-PCR and Western Blot. Results: HE and Masson staining showed thickened alveolar septum, hyperplasticcapillaries, and large areas of collagen fiber deposition in pulmonary fibrosis model rats. Rats in prodigiosin and hydrocortisone treatment groups had alleviated symptoms. There was high hydroxyproline expression in model rats, whereas the expression of hydroxyproline reduced after prodigiosin or hydrocortisone treatments. RT-PCR results showed high miR-410,high TGF-β1 and low ADAMTS1 in lung tissue of model rats. The expression of PICP and PIIINP werehigher in BALF of model group than in treatment groups. Prodigiosin and hydrocortisone treatment significantly reduced PICP and PIIINP content. RT-PCR and Western Blot analysis showed that prodigiosin inhibited expression of miR-410 and TGF-β1, but up-regulated ADAMTS1 expression. MTT assay indicated that prodigiosin inhibited HEPF proliferation induced by miR-410 overexpression. Conclusion: Prodigiosin down-regulates the expression of miR-410 and TGF-β1, up-regulates ADAMTS1, leading to decrease accumulation of fibrotic proteins. It could be used in alleviating pulmonary fibrosis.