RESUMO
Polyhexamethylene guanidine phosphate (PHMG-p), used as a humidifier disinfectant, causes interstitial lung disease, obliterative bronchiolitis, and lung fibrosis; however, little is known about its effect on intercellular interactions. Extracellular vesicles (EVs), which carry diverse compounds including proteins, RNA, and DNA to mediate cell-to-cell communication through their paracrine effects, have been highlighted as novel factors in lung fibrogenesis. This study aimed to identify the effect of proteins on small EVs (sEVs) from bronchoalveolar lavage fluid (BALF) of the recipient cells after PHMG-p exposure. A week after intratracheal administration of PHMG-p, sEVs were isolated from BALF of tissue showing overexpressed inflammatory and fibrosis markers. To investigate the role of sEVs in inflammation, naïve macrophages were cultured with sEVs, which induced their activation. To identify sEV proteins that are associated with these responses, proteomics analysis was performed. In the gene ontology analysis, coagulation, fibrinolysis, and hemostasis were associated with the upregulated proteins in sEVs. The highest increase was observed in fibrinogen levels, which was also related to those gene ontologies. We validated role of exosomal fibrinogen in inflammation using recombinant fibrinogen and an inhibitor of the integrin, which is the binding receptor for fibrinogen. Overall, we elucidated that increased fibrinogen levels in the early sEVs-PHMG activated inflammatory response during early fibrosis. These results suggest that sEVs from the BALF of PHMG-p-exposed mice could aggravate fibrogenesis by activating naïve macrophages via various proteins in the sEVs, Furthermore, this finding will be broadening the spectrum of communicating mediators.
Assuntos
Vesículas Extracelulares , Fibrose Pulmonar , Camundongos , Animais , Fibrinogênio , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Guanidinas/toxicidade , Inflamação/induzido quimicamente , Vesículas Extracelulares/metabolismoRESUMO
The detection of high levels of microplastics in indoor and outdoor air has increased concerns regarding its toxic effects on the respiratory system. They are not easily degradable and can be deposited deep in the lungs. Although several studies have reported inhalation toxicities of microplastics, they are still controversial due to a lack of evidence. Herein, we evaluated the inhalation toxicities of three differently charged polystyrene microplastics (PS-MPs), the most abundant microplastics in the air. Cytotoxicity and ROS generation were evaluated using WST-1 and DCF-DA assays, respectively. To evaluate the toxic effects on the lung, inflammatory responses were analyzed after repeated exposure to the PS-MPs through intratracheal instillation. To explore the mechanism of toxicity, autophagy and ER stress-associated proteins were analyzed. Only the positively charged PS-MPs (NH2 -PS-MPs) showed cytotoxicity and increased ROS generation in BEAS-2B cells. Similarly, only NH2 -PS-MPs significantly increased the expression and secretion of the pro-inflammatory cytokine IL-ß in the animal experiments. The expression of ER stress proteins indicated that NH2 -PS-MPs increased ER stress via PERK-EIF2α and ATF4-CHOP pathways. Moreover, accumulation of NH2 -PS-MPs in lysosomes and deformity of the nucleus were observed in BEAS-2B cells with autophagy induction. Taken together, our results demonstrated that NH2 -PS-MPs induced autophagic cell death in bronchial epithelial cells, leading to inflammatory responses in the lungs. These results suggest that repeated inhalation of microplastics can result in inflammatory responses in the lung through cellular damage of lung epithelial cells, and that inhalation microplastics should be monitored to reduce inhalation health risks.
Assuntos
Morte Celular Autofágica , Poliestirenos , Animais , Humanos , Poliestirenos/toxicidade , Microplásticos/toxicidade , Plásticos/toxicidade , Espécies Reativas de Oxigênio , Células Epiteliais/metabolismoRESUMO
Extracellular vesicles (EVs) play novel roles in homeostasis through cell-to-cell communication in human airways via transferring miRNAs. However, the contribution of EV miRNAs to pulmonary phenotypic homeostasis is not clearly understood. Hence, the aim of this study was to elucidate the functional role of miRNAs obtained from epithelium-derived EVs in lung fibrogenesis. Pulmonary fibrosis was induced by exposure of polyhexamethylene guanidine phosphate (PHMG-p)-instilled mice. In histopathological changes, a clear phenotypic change was observed in bronchial epithelium. For figuring out the role of EVs derived from conditioned media of untreated cells (EV-Con) and PHMG-p-treated BEAS-2B (EV-PHMG), significant increase in EVs released from PHMG-p-treated BEAS-2B was detected. Functional analysis with targets of differentially expressed miRNAs in EVs was annotated to epithelial-mesenchymal transition (EMT). Especially, the most abundant miRNA, miR-451a, was downregulated in EV of PHMG-p-treated BEAS-2B cells. We found that odd-skipped related 1 (OSR1) was a putative target for miR-451a, which had been known as a transcription factor of several fibrosis-associated genes. Transfer of decreased miR-451a via EV-PHMG upregulated OSR1 and induced EMT compared to Con-EV-treated cells. In pulmonary fibrosis mice, miR-451a levels were significantly reduced in EV derived from bronchoalveolar lavage fluid and OSR1 expression was increased in lung tissues of mice with PHMG-p exposure. MiR-451a-transfected EVs markedly alleviated fibrogenesis in the PHMG-p-exposed lungs. Low level of miR-451a in EVs modulated EMT and fibrogenesis in recipient cells by increasing OSR1 levels in vitro and in vivo. Our results suggest that transferring EV miR-451a induces anti-fibrotic autocrine effect by downregulating its target, OSR1 maintaining pulmonary homeostasis disrupted by PHMG-p exposure, which can be a potential therapeutic target.
Assuntos
Vesículas Extracelulares , MicroRNAs , Fibrose Pulmonar , Animais , Meios de Cultivo Condicionados/metabolismo , Células Epiteliais/metabolismo , Vesículas Extracelulares/genética , Humanos , Pulmão/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Fatores de Transcrição/genéticaRESUMO
Hepatic fibrosis is a chronic liver disease characterized by the accumulation of extracellular matrix (ECM). Activation of hepatic stellate cells (HSCs) after repetitive liver damage is a key event in hepatic fibrogenesis. As part of ongoing research projects to identify pharmacologically effective natural products, the phytochemical investigation of a MeOH extract of Centipeda minima led to the isolation of a sesquiterpene lactone, brevilin A, which was explored to elucidate potential anti-fibrotic effects by reversing HSC activation. First, we observed that transforming growth factor (TGF)-ß1 treatment significantly increased the expression levels of HSC activation marker, α-smooth muscle actin (α-SMA), and ECM protein such as collagen and fibronectin. Then, we demonstrated that brevilin A reversed the TGF-ß1-induced increase in protein and mRNA expression levels of α-SMA and collagen. To investigate the underlying molecular mechanism of brevilin A, we evaluated the effects of brevilin A on the STAT3 signaling pathway. STAT3 phosphorylation, increased by TGF-ß1 treatment, was strongly inhibited by brevilin A; the expression levels of fibronectin and connective tissue growth factor were also significantly decreased by brevilin A. The present study indicated that brevilin A has a preventive and therapeutic potential against hepatic fibrosis.
Assuntos
Crotonatos/farmacologia , Desenho de Fármacos , Cirrose Hepática/tratamento farmacológico , Fator de Transcrição STAT3/antagonistas & inibidores , Sesquiterpenos/farmacologia , Crotonatos/química , Relação Dose-Resposta a Droga , Células Estreladas do Fígado/efeitos dos fármacos , Humanos , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Estrutura Molecular , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Sesquiterpenos/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Cigarette smoking is a major cause of lung cancer. Although tobacco smoking-induced genotoxicity has been well established, there is apparent lack of abundance functional epigenetic effects reported On cigarette smoke-induced lung carcinogenesis. The aim of this study was to determine effects of intratracheal administration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) utilizing target gene expression DNA methylation patterns in lung tissues of mice following twice weekly for 8 weeks treatment. An unbiased approach where genomic regions was undertaken to assess early methylation changes within mouse pulmonary tissues. A methylated-CpG island recovery assay (MIRA) was performed to map the DNA methylome in lung tissues, with the position of methylated DNA determined using a Genome Analyzer (MIRA-SEQ). Alterations in epigenetic-regulated target genes were confirmed with quantitative reverse transcription-PCR, which revealed 35 differentially hypermethylated genes including Cdkn1C, Hsf4, Hnf1a, Cdx1, and Hoxa5 and 30 differentially hypomethylated genes including Ddx4, Piwi1, Mdm2, and Pce1 in NNK-exposed lung tissue compared with controls. The main pathway of these genes for mediating biological information was analyzed using the Kyoto Encyclopedia of Genes and Genomes database. Among them, Rssf1 and Mdm2 were closely associated with NNK-induced lung carcinogenesis. Taken together, our data provide valuable resources for detecting cigarette smoke-induced lung carcinogenesis.
Assuntos
Carcinogênese/induzido quimicamente , Carcinógenos/toxicidade , Epigênese Genética/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Nitrosaminas/toxicidade , Animais , Biomarcadores Tumorais/genética , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinógenos/análise , Metilação de DNA/efeitos dos fármacos , Epigenoma/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Nitrosaminas/análise , Fumar Tabaco/efeitos adversosRESUMO
Rhubarb is a well-known herb worldwide and includes approximately 60 species of the Rheum genus. One of the representative plants is Rheum palmatum, which is prescribed as official rhubarb due to its pharmacological potential in the Korean and Chinese pharmacopoeia. In our bioactive screening, we found out that the EtOH extract of R. palmatum inhibited hepatic stellate cell (HSC) activation by transforming growth factor ß1 (TGF-ß1). Chemical investigation of the EtOH extract led to the isolation of chrysophanol 8-O-glucoside, which was determined by structural analysis using NMR spectroscopic techniques and electrospray ionization mass spectrometry (ESIMS). To elucidate the effects of chrysophanol 8-O-glucoside on HSC activation, activated LX-2 cells were treated for 48 h with chrysophanol 8-O-glucoside, and α-SMA and collagen, HSC activation markers, were measured by comparative quantitative real-time PCR (qPCR) and western blotting analysis. Chrysophanol 8-O-glucoside significantly inhibited the protein and mRNA expression of α-SMA and collagen compared with that in TGF-ß1-treated LX-2 cells. Next, the expression of phosphorylated SMAD2 (p-SMAD2) and p-STAT3 was measured and the translocation of p-STAT3 to the nucleus was analyzed by western blotting analysis. The expression of p-SMAD2 and p-STAT3 showed that chrysophanol 8-O-glucoside strongly downregulated STAT3 phosphorylation by inhibiting the nuclear translocation of p-STAT3, which is an important mechanism in HSC activation. Moreover, chrysophanol 8-O-glucoside suppressed the expression of p-p38, not that of p-JNK or p-Erk, which can activate STAT3 phosphorylation and inhibit MMP2 expression, the downstream target of STAT3 signaling. These findings provided experimental evidence concerning the hepatoprotective effects of chrysophanol 8-O-glucoside against liver damage and revealed the molecular basis underlying its anti-fibrotic effects through the blocking of HSC activation.
Assuntos
Antraquinonas/farmacologia , Glucosídeos/farmacologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Substâncias Protetoras/farmacologia , Rheum/química , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Antraquinonas/química , Etanol , Glucosídeos/química , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Fosforilação , Extratos Vegetais/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Extracts of Peperomia pellucida [L.] Kunth have previously been demonstrated to have in vivo estrogenic-like effects, thereby functioning as an anti-osteoporotic agent. However, the compounds responsible for these effects have not yet been determined. Therefore, the aim of this study is to isolate and elucidate potential compounds with estrogenic activity. The structures of the isolated compounds were identified using 1D 1H and 13C-NMR and confirmed by 2D FT-NMR. The estrogenic activity was evaluated using the E-SCREEN assay, and a molecular docking study was performed to predict the binding affinity of the isolated compounds to estrogen receptors. In this experiment, we successfully isolated three phenylpropanoids and two lignan derivatives, namely, 6-allyl-5-methoxy-1,3-benzodioxol-4-ol (1), pachypostaudin B (2), pellucidin A (3), dillapiole (4), and apiol (5). Among these compounds, the isolation of 1 and 2 from P. pellucida is reported for the first time in this study. Activity assays clearly showed that the ethyl acetate extract and its fractions, subfractions, and isolated compounds exerted estrogenic activity. Methanol fraction of the ethyl acetate extract produced the highest estrogenic activity, while 1 and 2 had partial agonist activity. Some compounds (derivates of dillapiole and pellucidin A) also had, in addition, anti-estrogenic activity. In the docking study, the estrogenic activities of 1-5 appeared to be mediated by a classical ligand-dependent mechanism as suggested by the binding interaction between the compounds and estrogen receptors; binding occurred on Arg 394 and His 524 of the alpha receptor and Arg 346 and His 475 of the beta receptor. In summary, we reveal that P. pellucida is a promising anti-osteoporotic agent due to its estrogenic activity, and the compounds responsible for this activity were found to be lignan and phenylpropanoid derivatives. The presence of other compounds in either the extract or fraction may contribute to a synergistic effect, as suggested by the higher estrogenic activity of the methanol fraction. Hence, we suggest further research on the osteoporotic activity and safety of the identified compounds, especially regarding their effects on estrogen-responsive organs.
Assuntos
Lignanas/isolamento & purificação , Lignanas/farmacologia , Peperomia/química , Fitoestrógenos/isolamento & purificação , Fitoestrógenos/farmacologia , Propanóis/isolamento & purificação , Propanóis/farmacologia , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Humanos , Lignanas/metabolismo , Células MCF-7 , Modelos Moleculares , Simulação de Acoplamento Molecular , Fitoestrógenos/metabolismo , Propanóis/químicaRESUMO
Polyhexamethylene guanidine phosphate (PHMG-p), an antimicrobial additive, was used as a humidifier disinfectant in Korea and caused severe lung injuries, including lung fibrosis, in hundreds of victims. As PHMG-p-induced lung fibrosis is different from that induced by known fibrogenic agents such as bleomycin, it is important to understand the molecular mechanisms underlying this effect. A recent study showed that epithelial-mesenchymal transition (EMT) could play key roles in PHMG-p-induced pulmonary fibrosis. Therefore, we aimed to characterize the molecular mechanisms associated with PHMG-p-induced EMT. We observed EMT, macrophage infiltration, and fibrosis in mouse lung tissues after intratracheal instillation of PHMG-p. Furthermore, PHMG-p-induced EMT was observed in A549 cells by the evaluation of cell morphology and quantitation of mRNA and protein expression. The use of EMT inhibitors revealed that PHMG-p induced EMT through the activation of Akt and Notch signaling. Moreover, the transcription factor ZEB2 was observed in PHMG-p-treated A549 cells and mouse lungs. The results indicated that upstream regulators, including Akt and Notch 1, acted as intracellular effectors that triggered ZEB2 expression after exposure to PHMG-p. Attenuation of PHMG-p-induced EMT following inhibition or silencing of Akt and Notch signaling or ZEB2 implied that PHMG-p-induced EMT was a result of Akt, Notch, and ZEB2 activation. Our findings showed that PHMG-p induced EMT through Akt/Notch signaling pathways and that ZEB2 played an important role in PHMG-p-induced lung toxicity. This study will help to understand the mechanisms of action of PHMG-p associated with lung fibrogenesis.
Assuntos
Desinfetantes/toxicidade , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Guanidinas/toxicidade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fibrose Pulmonar/metabolismo , Receptores Notch/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Células A549 , Animais , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/genética , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Receptores Notch/genética , Transdução de Sinais/efeitos dos fármacos , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genéticaRESUMO
Abstracts Objective: The major active ingredient of humidifier disinfectant, polyhexamethylene guanidine-phosphate (PHMG-P), caused hundreds of deaths with pulmonary fibrosis. However, structurally similar guanidine-based disinfectants are still in use in various fields. Moreover, as they are precursors of excellent antimicrobial compounds, new chemicals with guanidine-based structures have been synthesized and introduced. In this study, we evaluated pulmonary fibrotic responses induced by PHMG-P, polyhexamethylene biguanide (PHMB), and oligo(2-(2-ethoxy)ethoxyethyl guanidinium chloride (PGH) and their toxicity mechanisms in type II alveolar epithelial A549 cells. Materials and methods: Cellular damage was compared by using the cytotoxicity test (WST-1 assay) and plasma membrane toxicity tests (Lactate dehydrogenase leakage detection assay and plasma membrane staining). As a measure of fibrotic response, induction of the epithelial-mesenchymal transition (EMT) was evaluated by measuring E-cadherin and α-smooth muscle actin (α-SMA) protein expression (epithelial and mesenchymal marker, respectively). Results: All tested compounds showed membrane damage; PHMG-P and PGH induced the highest and lowest damage, respectively. Moreover, they induced EMT when the test chemicals were treated with similar cytotoxic concentrations. Conclusions: Our study indicates that three guanidine-based disinfectants are potential fibrosis-inducing chemicals that induce EMT through cellular damage. Therefore, use of guanidine-based polymers should be strictly regulated by considering their potential adverse effects on the lungs.
Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Biguanidas/toxicidade , Desinfetantes/toxicidade , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Guanidinas/toxicidade , Polímeros/toxicidade , Células A549 , Actinas/metabolismo , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Antígenos CD/metabolismo , Caderinas/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/patologia , Sobrevivência Celular/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/metabolismo , Testes de ToxicidadeRESUMO
Epithelial-to-mesenchymal transition (EMT) is increasingly recognized as contributing to the pathogenesis of idiopathic pulmonary fibrosis. Therefore, novel plant-based natural, active compounds have been sought for the treatment of fibrotic EMT. The aim of the present study was to investigate the inhibitory effects of Astilbe rubra on TGF-ß1-induced EMT in lung alveolar epithelial cells (A549). A. rubra was subjected to extraction using 70% ethanol (ARE), and ethanol extracts of the aerial part and that of the rhizome were further partitioned using various solvents. Protein expression and cell motility were investigated to evaluate the inhibitory effects of ARE on EMT. EMT occurred in A549 cells treated with TGF-ß1, but was prevented by co-treatment with ARE. The dichloromethane fractions showed the strongest inhibitory effect on TGF-ß1-induced EMT. ß-Peltoboykinolic acid was isolated from the dichloromethane fractions of A. rubra by activity-oriented isolation. ß-Peltoboykinolic acid not only attenuated TGF-ß1-induced EMT, but also the overproduction of extracellular matrix components including type I collagen and fibronectin. The Smad pathway activated by TGF-ß1 was inhibited by co-treatment with ß-peltoboykinolic acid. Taken together, these results indicate that ß-peltoboykinolic acid from A. rubra and dichloromethane fractions shows potential as an antifibrotic agent in A549 cells treated with TGF-ß1.
Assuntos
Células Epiteliais Alveolares/citologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Cloreto de Metileno/farmacologia , Saxifragaceae/química , Fator de Crescimento Transformador beta1/efeitos adversos , Células A549 , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Movimento Celular/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Fibronectinas/metabolismo , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Cloreto de Metileno/química , Componentes Aéreos da Planta/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Rizoma/química , Transdução de Sinais/efeitos dos fármacosRESUMO
Pulmonary fibrosis (PF) is a chronic and fatal lung disease with few treatment options. Although the pathogenesis of PF is not clear, a chronic inflammatory response to continuous damage is considered the cause of pulmonary fibrosis. PF is characterized by excessive accumulation of extracellular matrix (ECM), therefore, inhibition of myofibroblast differentiation is a good therapeutic target for PF. As part of our continuing endeavor to explore biologically active metabolites from insect-associated microbes, we found that the MeOH extract of the culture broth from the entomopathogenic fungus Beauveria bassiana inhibited collagen induction and E-cadherin down-regulation. In order to identify active compounds, we carried out chemical analysis of the MeOH extract with the assistance of LC/MS-guided isolation approach, which led to the successful identification of four cyclodepsipeptides 1â»4. Among the isolates, compound 2 showed inhibitory effects on myofibroblast differentiation induced by TGF-ß1. Compound 2 inhibited induction of α-SMA and N-cadherin, which are myofibroblast markers, and blocked the accumulation of ECM proteins such as collagen and fibronectin. Overall these findings demonstrate that compound 2 can be used to attenuate pulmonary fibrosis by targeting myo- fibroblast differentiation.
Assuntos
Beauveria/química , Diferenciação Celular/efeitos dos fármacos , Depsipeptídeos/farmacologia , Fibrose Pulmonar/tratamento farmacológico , Células A549 , Actinas/genética , Células Epiteliais Alveolares/efeitos dos fármacos , Caderinas/genética , Colágeno/genética , Depsipeptídeos/química , Depsipeptídeos/isolamento & purificação , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Miofibroblastos/citologia , Miofibroblastos/efeitos dos fármacos , Fibrose Pulmonar/genética , Fator de Crescimento Transformador beta1/genéticaRESUMO
The first total synthesis and biological evaluation of penchinone A and its structural analogues are described. The key steps for the preparation of penchinone A derivatives involve the oxime-directed palladium(II)-catalyzed oxidative acylation, Claisen rearrangement, and base-mediated olefin migration. This transformation efficiently provides a range of allyl-substituted biaryl ketones with site-selectivity and functional group compatibility. In addition, all synthetic compounds were screened for anti-inflammatory activity against nitric oxide (NO), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6) with lipopolysaccharide (LPS)-induced RAW264.7 cells. Generally, a range of penchinone A derivatives potently inhibited NO, TNF-α, and IL-6 productions, compared to dexamethasone as a positive control. Notably, penchinone A (8g) and its derivatives (8e and 8f) were found to exhibit anti-inflammatory activity stronger than that of dexamethasone.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Interleucina-6/antagonistas & inibidores , Lignanas/farmacologia , Óxido Nítrico/antagonistas & inibidores , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Interleucina-6/biossíntese , Lignanas/síntese química , Lignanas/química , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Estrutura Molecular , Óxido Nítrico/biossíntese , Células RAW 264.7 , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Pharbitidis Semen, the seed of Morning glory (Pharbitis nil), is a medicinal agent that has traditionally been used as a purgative in Korea. Pharbilignan C (PLC) is a dihydro[b]-benzofuran-type neolignan from Pharbitidis Semen, which reportedly exhibited the most potent cytotoxicity against human tumor cells. To further study the antiproliferative activity of PLC, its molecular mechanisms of action in two breast adenocarcinoma cells, MCF-7 and MDA-MB 231 cells were investigated. PLC inhibited the proliferation of MDA-MB 231 and MCF-7 cells, in order of sensitivity (IC50 of MDA-MB 231 cells: 7.0±2.0µM). PLC induced apoptosis in MDA-MB 231 cells with down regulation of Bcl-2 and up-regulation of Bax expression. It also decreased mitochondrial membrane potential accompanied with increasing initiator caspase, caspase-9 activation and executioner caspase, caspase-3 activation. This study demonstrates that PLC inhibited proliferation of MDA-MB 231 cells by inducing apoptosis via the mitochondria-mediated intrinsic pathway.
Assuntos
Apoptose/efeitos dos fármacos , Benzofuranos/farmacologia , Neoplasias da Mama/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , HumanosRESUMO
Polyhexamethylene guanidine (PHMG) phosphate was used as a disinfectant for the prevention of microorganism growth in humidifiers, without recognizing that a change of exposure route might cause significant health effects. Epidemiological studies reported that the use of humidifier disinfectant containing PHMG-phosphate can provoke pulmonary fibrosis. However, the pulmonary toxicity of PHMG-phosphate aerosol particles is unknown yet. This study aimed to elucidate the toxicological relationship between PHMG-phosphate aerosol particles and pulmonary fibrosis. An in vivo nose-only exposure system and an in vitro air-liquid interface (ALI) co-culture model were applied to confirm whether PHMG-phosphate induces inflammatory and fibrotic responses in the respiratory tract. Seven-week-old male Sprague-Dawley rats were exposed to PHMG-phosphate aerosol particles for 3 weeks and recovered for 3 weeks in a nose-only exposure chamber. In addition, three human lung cells (Calu-3, differentiated THP-1 and HMC-1 cells) were cultured at ALI condition for 12 days and were treated with PHMG-phosphate at set concentrations and times. The reactive oxygen species (ROS) generation, airway barrier injuries and inflammatory and fibrotic responses were evaluated in vivo and in vitro. The rats exposed to PHMG-phosphate aerosol particles in nanometer size showed pulmonary inflammation and fibrosis including inflammatory cytokines and fibronectin mRNA increase, as well as histopathological changes. In addition, PHMG-phosphate triggered the ROS generation, airway barrier injuries and inflammatory responses in a bronchial ALI co-culture model. Those results demonstrated that PHMG-phosphate aerosol particles cause pulmonary inflammatory and fibrotic responses. All features of fibrogenesis by PHMG-phosphate aerosol particles closely resembled the pathology of fibrosis that was reported in epidemiological studies. Finally, we expected that PHMG-phosphate infiltrated into the lungs in the form of aerosol particles would induce an airway barrier injury via ROS, release fibrotic inflammatory cytokines, and trigger a wound-healing response, leading to pulmonary fibrosis. A simultaneous state of tissue destruction and inflammation caused by PHMG-phosphate had whipped up a "perfect storm" in the respiratory tract.
Assuntos
Aerossóis/toxicidade , Guanidinas/toxicidade , Pneumonia/induzido quimicamente , Fibrose Pulmonar/induzido quimicamente , Administração por Inalação , Aerossóis/química , Animais , Linhagem Celular , Técnicas de Cocultura , Desinfetantes , Guanidinas/química , Humanos , Pulmão/citologia , Masculino , Pneumonia/patologia , Fibrose Pulmonar/patologia , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Testes de Toxicidade/métodosRESUMO
Phytochemical investigation of an extract of the aerial part of Barleria lupulina resulted in the identification of four new iridoid glycosides (1-4), together with 14 known analogues (5-18). The structures of 1-4 were determined through 1D and 2D NMR spectroscopic data analysis, HRMS, and acid hydrolysis. This is the first report of iridoid glycosides with a formate group. The free-radical scavenging activity of compounds 9, 12, and 15-17 was assessed using the DPPH assay. Compounds 16 and 17 scavenged DPPH radicals weakly with IC50 values of 97.5 and 78.6 µg/mL, respectively.
Assuntos
Acanthaceae/química , Sequestradores de Radicais Livres/isolamento & purificação , Glicosídeos Iridoides/isolamento & purificação , Compostos de Bifenilo/farmacologia , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/farmacologia , Concentração Inibidora 50 , Glicosídeos Iridoides/química , Glicosídeos Iridoides/farmacologia , Estrutura Molecular , Picratos/farmacologia , Componentes Aéreos da Planta/química , Extratos Vegetais/química , VietnãRESUMO
Next-generation risk assessment (NGRA) has emerged as a promising alternative to non-animal studies owing to the increasing demand for the risk assessment of inhaled toxicants. In this study, NGRA was used to assess the inhalation risks of two biocides commonly used as humidifier disinfectants: polyhexamethylene guanidine phosphate (PHMG-p) and chloromethylisothiazolinone/methylisothiazolinone (CMIT/MIT). Human bronchial epithelial cell transcriptomic data were processed based on adverse outcome pathways and used to establish transcriptome-based points of departure (tPODs) for each biocide. tPOD values were 0.00500-0.0510 µg/cm2 and 0.0342-0.0544 µg/cm2 for PHMG-p and CMIT/MIT, respectively. tPODs may provide predictive power comparable to that of traditional animal-based PODs (aPODs). The tPOD-based NGRA determined that both PHMG-p and CMIT/MIT present a high inhalation risk. Moreover, the identified PHMG-p posed a higher risk than CMIT/MIT, and children were identified as more susceptible population compared to adults. This finding is consistent with observations from actual exposure events. Our findings suggest that NGRA with transcriptomics offers a reliable approach for risk assessment of specific humidifier disinfectant biocides, while acknowledging the limitations of current models and in vitro systems, particularly regarding uncertainties in pharmacokinetics (PK) and pharmacodynamics (PD).
Assuntos
Desinfetantes , Guanidinas , Tiazóis , Desinfetantes/toxicidade , Medição de Risco , Humanos , Tiazóis/toxicidade , Guanidinas/toxicidade , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/genética , Transcriptoma/efeitos dos fármacos , Perfilação da Expressão Gênica , Exposição por Inalação/efeitos adversos , Células Epiteliais/efeitos dos fármacos , UmidificadoresRESUMO
Dosimetry modeling and point of departure (POD) estimation using in vitro data are essential for mechanism-based hazard identification and risk assessment. This study aimed to develop a putative adverse outcome pathway (AOP) for humidifier disinfectant (HD) substances used in South Korea through a systematic review and benchmark dose (BMD) modeling. We collected in vitro toxicological studies on HD substances, including polyhexamethylene guanidine hydrochloride (PHMG-HCl), PHMG phosphate (PHMG-p), a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (CMIT/MIT), CMIT, and MIT from scientific databases. A total of 193 sets of dose-response data were extracted from 34 articles reporting in vitro experimental results of HD toxicity. The risk of bias (RoB) in each study was assessed following the office of health assessment and translation (OHAT) guideline. The BMD of each HD substance at different toxicity endpoints was estimated using the US Environmental Protection Agency (EPA) BMD software (BMDS). Interspecies- or interorgan differences or most critical effects in the toxicity of the HD substances were analyzed using a 95% lower confidence limit of the BMD (BMDL). We found a critical molecular event and cells susceptible to each HD substance and constructed an AOP of PHMG-p- or CMIT/MIT-induced damage. Notably, PHMG-p induced ATP depletion at the lowest in vitro concentration, endoplasmic reticulum (ER) stress, epithelial-to-mesenchymal transition (EMT), inflammation, leading to fibrosis. CMIT/MIT enhanced mitochondrial reactive oxygen species (ROS) production, oxidative stress, mitochondrial dysfunction, resulting in cell death. Our approach will increase the current understanding of the effects of HD substances on human health and contribute to evidence-based risk assessment of these compounds.
RESUMO
Objectives: Exposure to humidifier disinfectants has been linked to respiratory diseases, including interstitial lung disease, asthma, and pneumonia. Consequently, numerous toxicological studies have explored respiratory damage as both a necessary and sufficient condition for these diseases. We systematically reviewed and integrated evidence from toxicological studies by applying the evidence integration method established in previous research to confirm the biological plausibility of the association between exposure and disease. Methods: We conducted a literature search focusing on polyhexamethylene guanidine phosphate (PHMG) and chloromethylisothiazolinone/methylisothiazolinone (CMIT/MIT), the primary ingredients in humidifier disinfectants. We selected relevant studies based on their quality and the population, exposure, comparator, outcome (PECO) statements. These studies were categorized into 3 lines of evidence: hazard information, animal studies, and mechanistic studies. Based on a systematic review, we integrated the evidence to develop an aggregate exposure pathway-adverse outcome pathway (AEP-AOP) model for respiratory damage. The reliability and relevance of our findings were assessed by comparing them with the hypothesized pathogenic mechanisms of respiratory diseases. Results: The integration of each AEP-AOP component for PHMG and CMIT/MIT led to the development of an AEP-AOP model, wherein disinfectants released from humidifiers in aerosol or gaseous form reached target sites, causing respiratory damage through molecular initiating events and key events. The model demonstrated high reliability and relevance to the pathogenesis of respiratory diseases. Conclusion: The AEP-AOP model developed in this study provides strong evidence that exposure to humidifier disinfectants causes respiratory diseases. This model demonstrates the pathways leading to respiratory damage, a hallmark of these conditions.
RESUMO
In bony fish or other aquatic vertebrates, the aryl hydrocarbon receptor (AhR) signaling pathway is initiated by exposure to polycyclic (or/and halogenated) aromatic hydrocarbons (PAHs, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), which subsequently induces the up-regulated expression of a series of related genes (such as cytochrome P4501A (CYP1A)). However, a lack of applicable protein reagents hinders our further understanding of the AhR signaling pathway, which focuses only on gene-based investigations. The goldfish (Carassius auratus) is an ideal model for a study of environmental pollution in whole-Asian fresh water. Here, three sensitive and specific polyclonal antisera against goldfish AhR1, AhR2, and CYP1A proteins were developed. These antisera not only bound the in-vitro synthesized target proteins, but recognized the real proteins expressed in goldfish tissues, with minimal cross-reactivity to non-specific proteins. Together with the analysis of semi-quantitative RT-PCR and polyclonal-antibody-based sandwich ELISA, we confirmed that goldfish AhRs differed in the expression (mRNA and protein levels) patterns among test tissues. Importantly, the relative abundance of each AhR mRNA levels from the different tissues showed no obvious consistency with their protein levels. After exposure to TCDD, goldfish AhR2 showed a more sensitivity than AhR1, and stimulated CYP1A expression directly, similar with the other reported fish models. Overall, development of these antibodies in this study will allow valuable and versatile investigations to further understand the AhR signaling pathway, and different expression (mRNA and protein) patterns represent the first step in determining the regulatory mechanisms underlying the TCDD-exposed aquatic environment.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Regulação da Expressão Gênica , Carpa Dourada/genética , Carpa Dourada/metabolismo , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/genética , Poluentes Químicos da Água/toxicidade , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Ensaio de Imunoadsorção Enzimática/veterinária , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Injeções Intraperitoneais/veterinária , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , Reação em Cadeia da Polimerase/veterinária , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismoRESUMO
An in vitro steroidogenesis assay using H295R human adenocarcinoma cells is a useful tool for the fast identification of compounds that affect the production of testosterone and 17ß-estradiol. Selective and sensitive hormone measurement by liquid chromatography-tandem mass spectrometry (LC-MS/MS) can make this assay more reliable. Therefore, in the present study, a sensitive and selective method for the quantification of testosterone and 17ß-estradiol in the H295R steroidogenesis assay was developed and fully validated using LC-MS/MS coupled with an online sample enrichment technique. To prove its usefulness, the method developed was applied to investigate the effect of sildenafil on steroidogenesis. Cell medium samples were diluted and prepared using solid-phase extraction. The samples were prepared on ice and were not kept for more than 30 min to prevent degradation of hormones. The extracts were dried, reconstituted, filtered, and analyzed by LC-MS/MS with polarity switching electrospray ionization. The validation results for selectivity, matrix effect, recovery, linearity, precision, and accuracy were satisfactory. The limits of detection for testosterone and 17ß-estradiol were 5 and 10 pg/mL, respectively, and the limit of quantification for both testosterone and 17ß-estradiol was 10 pg/mL, which was in accordance with the OECD guideline. No degradation was observed under the storage conditions for 7 and 14 days at -80 °C as well as after three freeze-thaw cycles, whereas 17ß-estradiol was degraded after 1 h on ice during sample processing. The method developed was successfully used for the investigation of the effect of sildenafil on steroidogenesis. This method can be very useful for the initial selection of drugs with androgenic and/or estrogenic effects for specific purposes, e.g., in the selection of drugs that are used to reverse the effects of chemical castration.