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1.
Zhonghua Shao Shang Za Zhi ; 35(8): 580-586, 2019 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-31474037

RESUMO

Objective: To investigate the role and mechanism of nonreceptor tyrosine kinase Tec in the production of pro-inflammatory cytokine interleukin-8 (IL-8) induced by endotoxin/lipopolysaccharide (LPS) in human alveolar epithelial cells A549. Methods: Human alveolar epithelial cells A549 were routinely cultured and passaged in Roswell Park Memorial Institute-1640 medium containing 10% fetal bovine serum. The second or third passage of cells were collected for subsequent experiments. (1) Cells were collected and divided into 6 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in simple LPS group were routinely cultured for 1 h and then stimulated by 1 µg/mL LPS for 1 h. Cells in simple LFM-A13 group were cultured with conventional culture medium adding 75 µmol/L LFM-A13 for 1 h and then cultured with replaced conventional culture medium for 1 h. Cells in 25 µmol/L LFM-A13+ LPS group, 75 µmol/L LFM-A13+ LPS group, and 100 µmol/L LFM-A13+ LPS group were cultured with conventional culture medium adding 25, 75, and 100 µmol/L LFM-A13 respectively for 1 h and then all stimulated by 1 µg/mL LPS added into the replaced conventional culture medium for 1 h. The protein expression of Tec in cells of each group was detected by Western blotting, and the content of IL-8 in cell culture supernatant of each group was determined by enzyme-linked immunosorbent assay. (2) Cells were collected and divided into 5 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in small interfering RNA (siRNA) control+ LPS group were transfected with empty lentivirus for 10 h and then stimulated by 1 µg/mL LPS added into the conventional culture medium for 2 h. Cells in Tec mus-298 RNA interference (RNAi)+ LPS group, Tec mus-299 RNAi+ LPS group, and Tec mus-300 RNAi+ LPS group were transfected with lentivirus loaded with Tec mus-298 RNAi, Tec mus-299 RNAi, and Tec mus-300 RNAi respectively for 10 h and then stimulated by 1 µg/mL LPS added into the conventional culture medium for 2 h. The protein expression of Tec in cells of each group was detected by Western blotting to screen Tec-siRNA with the best silencing effect on Tec gene. (3) Cells were collected and divided into 4 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in virus control group were transfected with empty lentivirus for 10 h and then routinely cultured for 2 h. Cells in simple LPS group were stimulated by 1 µg/mL LPS added into the conventional culture medium for 2 h. Cells in Tec-siRNA+ LPS group were transfected with lentivirus loaded with Tec-siRNA with the best silencing effect on Tec gene for 10 h and then stimulated by 1 µg/mL LPS added into the conventional culture medium for 2 h. The protein expressions of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) MAPK of cells in each group were detected by Western blotting. Data were processed with one-way analysis of variance and the least significant difference-t test. Results: (1) Compared with that of blank control group, the protein expression of Tec of cells in simple LPS group was obviously increased (t=9.72, P<0.05), but the protein expression of Tec of cells in simple LFM-A13 group was not obviously changed (t=4.31, P=0.05). Compared with that of simple LPS group, the protein expression of Tec of cells in 25 µmol/L LFM-A13+ LPS group, 75 µmol/L LFM-A13+ LPS group, or 100 µmol/L LFM-A13+ LPS group was obviously decreased (t=9.72, 9.07, 16.33, P<0.05 or P<0.01). Compared with (189±22) pg/mL of blank control group, the content of IL-8 in culture supernatant of cells in simple LPS group was obviously increased [(214±10) pg/mL, t=2.18, P<0.05], but the content of IL-8 in culture supernatant of cells in simple LFM-A13 group was not obviously changed [(173±43) pg/mL, t=0.64, P>0.05]. Compared with that of simple LPS group, the content of IL-8 in culture supernatant of cells in 25 µmol/L LFM-A13+ LPS group was not obviously changed [(204±38) pg/mL, t=0.54, P>0.05], but the content of IL-8 in culture supernatant of cells in 75 µmol/L LFM-A13+ LPS group and 100 µmol/L LFM-A13+ LPS group was obviously decreased [(144±44), (137±51) pg/mL, t=3.63, 2.55, P<0.05 or P<0.01]. (2) Compared with that of blank control group, the protein expression of Tec of cells in siRNA control+ LPS group was obviously increased (t=14.24, P<0.01). Compared with that of siRNA control+ LPS group, the protein expression of Tec of cells in Tec mus-298 RNAi+ LPS group or Tec mus-299 RNAi+ LPS group was obviously decreased (t=36.03, 18.23, P<0.01), but the protein expression of Tec of cells in Tec mus-300 RNAi+ LPS group was not obviously changed (t=4.08, P>0.05). The protein expression of Tec was the lowest in cells of Tec mus-298 RNAi+ LPS group, so Tec mus-298 RNAi was used in subsequent experiment. (3) Compared with 1.16±0.16 and 0.78±0.11 of blank control group, the protein expressions of p38 MAPK and ERK MAPK of cells in virus control group were not obviously changed (1.66±0.13, 0.89±0.11, t=11.09, 3.60, P>0.05), but the protein expressions of p38 MAPK and ERK MAPK of cells in simple LPS group were obviously increased (2.83±0.29, 1.86±0.37, t=9.70, 7.23, P<0.05). Compared with those of simple LPS group, the protein expression of p38 MAPK and protein expression of ERK MAPK of cells in Tec-siRNA+ LPS group were obviously decreased (0.69±0.16, 1.03±0.24, t=13.78, 4.12, P<0.05 or P<0.01). Conclusions: Tec may mediate the production and release of pro-inflammatory cytokine IL-8 from human alveolar epithelial cells A549 induced by LPS via the p38 MAPK and ERK MAPK signal pathways.


Assuntos
Células Epiteliais Alveolares/metabolismo , Interleucina-8/metabolismo , Proteínas Tirosina Quinases/metabolismo , Células A549 , Humanos , Lipopolissacarídeos , Transdução de Sinais
2.
EBioMedicine ; 46: 305-316, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31383554

RESUMO

BACKGROUND: Cigarette smoke is the main risk factor of pulmonary emphysema development, which is characterized by alveolar wall destruction. Mitochondria are important for alveolar type II (ATII) cell metabolism due to ATP generation. METHODS: We isolated ATII cells from control non-smoker and smoker organ donors, and after lung transplant of patients with emphysema to determine mitochondrial function, dynamics and mitochondrial (mt) DNA damage. FINDINGS: We found high mitochondrial superoxide generation and mtDNA damage in ATII cells in emphysema. This correlated with decreased mtDNA amount. We also detected high TOP1-cc and low TDP1 levels in mitochondria in ATII cells in emphysema. This contributed to the decreased resolution of TOP1-cc leading to accumulation of mtDNA damage and mitochondrial dysfunction. Moreover, we used lung tissue obtained from areas with mild and severe emphysema from the same patients. We found a correlation between the impaired fusion and fission as indicated by low MFN1, OPA1, FIS1, and p-DRP1 levels and this disease severity. We detected lower TDP1 expression in severe compared to mild emphysema. INTERPRETATION: We found high DNA damage and impairment of DNA damage repair in mitochondria in ATII cells isolated from emphysema patients, which contribute to abnormal mitochondrial dynamics. Our findings provide molecular mechanisms of mitochondrial dysfunction in this disease. FUND: This work was supported by National Institutes of Health (NIH) grant R01 HL118171 (B.K.) and the Catalyst Award from the American Lung Association (K.B.).


Assuntos
Células Epiteliais Alveolares/metabolismo , Mitocôndrias/metabolismo , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/metabolismo , Trifosfato de Adenosina/biossíntese , Dano ao DNA , DNA Mitocondrial , Progressão da Doença , Metabolismo Energético , Humanos , Mitocôndrias/genética , Estresse Oxidativo , Diester Fosfórico Hidrolases/metabolismo , Transporte Proteico , Enfisema Pulmonar/patologia , Espécies Reativas de Oxigênio/metabolismo , Fumaça/efeitos adversos , Superóxidos/metabolismo
3.
Toxicol Lett ; 315: 47-54, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31449845

RESUMO

Particulate matter with a diameter of less than 2.5 µm (PM2.5) easily deposits on lung alveoli and degrades human health. Surfactant protein A (SP-A) is the most abundant pulmonary surfactant protein stored in lamellar bodies (LBs) of alveolar epithelial type II cells. The impacts of PM2.5 on SP-A are multifaceted and intractable, and the underlying mechanism remains unclear. In this study, the expression and distribution of SP-A in Balb/c mice and A549 cells under PM2.5 exposure were investigated. The results showed that the low and medium concentration of PM2.5 gradually enhanced SP-A protein and mRNA expression, whereas the high concentration of PM2.5 conspicuously decreased SP-A protein but not its mRNA compared with the control. The trafficking of SP-A to LBs was gradually disturbed, and concomitantly, the lesions of LBs responsible for the transport and storage of SP-A protein were exacerbated with increased PM2.5 concentration. Reactive oxygen species production abundantly increased upon PM2.5 exposure, and it was antagonized by the oxidant inhibitor N-acetylcysteine. Subsequently, the injured LBs and the decrease in SP-A expression under exposure to the high concentration of PM2.5 were well rescued. The present study provides a new perspective to investigate the adverse effects of PM2.5 or diesel exhaust particles on other proteins transported to and stored in LBs.


Assuntos
Células Epiteliais Alveolares/metabolismo , Material Particulado/toxicidade , Alvéolos Pulmonares/fisiopatologia , Proteína A Associada a Surfactante Pulmonar/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Emissões de Veículos/toxicidade , Animais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Tamanho da Partícula
4.
Sheng Li Xue Bao ; 71(4): 575-580, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31440754

RESUMO

The aim of the present study was to investigate the effect of salidroside (Sal) on inflammatory activation induced by lipopolysaccharide (LPS) in the co-culture of rat alveolar macrophages (AM) NR 8383 and type II alveolar epithelial cells (AEC II) RLE-6TN. CCK-8 colorimetric method was used to detect cell proliferation percentage. The enzyme-linked immunosorbent assay (ELISA) was used to determine the content of tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2) and interleukin-10 (IL-10) in the supernatant. Western blot was used to examine the expression levels of phosphorylated AKT (p-AKT) and total AKT protein. The results showed that pretreatment of RLE-6TN cells or co-culture of RLE-6TN and NR 8383 cells with 32 and 128 µg/mL Sal for 1 h, followed by continuous culture for 24 h, significantly increased the cell proliferation (P < 0.05). Compared with control group, 32 and 128 µg/mL Sal pretreatment significantly increased the ratio of p-AKT/AKT in RLE-6TN cells (P < 0.05). Pretreatment of 32 µg/mL Sal not only inhibited the secretion of TNF-α and MIP-2 by NR 8383 cells induced by LPS (P < 0.05), but also enhanced the inhibitory effect of RLE-6TN and NR 8383 cells co-culture on the secretion of TNF-α and MIP-2 by NR 8383 cells induced by LPS (P < 0.05). In addition, 32 µg/mL Sal pretreatment promoted LPS-induced IL-10 secretion by NR 8383 cells (P < 0.05), and enhanced the promoting effect of co-culture of RLE-6TN and NR 8383 cells on the IL-10 secretion by LPS-induced NR 8383 cells (P < 0.05). In conclusion, Sal may directly inhibit LPS-induced inflammatory activation of AM (NR 8383), promote the proliferation of AEC II (RLE-6TN) through PI3K/AKT signaling pathway, and enhance the regulatory effect of AEC II on LPS-induced inflammatory activation of AM.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Glucosídeos/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Fenóis/farmacologia , Transdução de Sinais , Células Epiteliais Alveolares/metabolismo , Animais , Linhagem Celular , Quimiocina CXCL2/metabolismo , Técnicas de Cocultura , Interleucina-10/metabolismo , Lipopolissacarídeos , Macrófagos Alveolares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Fator de Necrose Tumoral alfa/metabolismo
5.
Biomed Pharmacother ; 117: 109193, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31387171

RESUMO

Accumulating evidence indicates that angiotensin (1-7) [Ang-(1-7)] protects against idiopathic pulmonary fibrosis (IPF) in animal experiments. However, whether Ang-(1-7) effectively inhibits epithelial-mesenchymal transition (EMT) induced by transforming growth factor-ß1 (TGF-ß1) remains unclear. The aim of this study is to examine the eff ;ects of Ang-(1-7) on TGF-ß1-induced EMT in human alveolar epithelial cells. We found that angiotensin-converting enzyme 2 (ACE2) /Ang-(1-7)/MasR were decreased in the lungs of mice with IPF induced by bleomycin, and were negatively correlated with Tgfb1 mRNA expression. In vitro, our data showed that exogenous Ang-(1-7) restored the expression of E-cadherin and decreased the expressions of α-SMA and Vimentin induced by TGF-ß1 in A549 cells. Ang-(1-7) also reduced TGF-ß1-induced migration and synthesis of the extracellular matrix, such as collagen Ⅰ and collagen Ⅲ. Mechanistically, we observed that Ang-(1-7) directly inhibited TGF-ß1-induced phosphorylation of Smad2 and Smad3, and suppressed the expression of the downstream target gene of TGF-ß1-Smad signaling, including ZEB1, ZEB2, TWIST, and SNAIL1. Additionally, phosphorylation of mTOR induced by TGF-ß1 also been suppressed by Ang-(1-7) treatment in A549 cells. Interestingly, we found that TGF-ß1 strongly suppressed the expression of ACE2 in A549 cells through inhibiting SIRT1. In conclusion, our findings indicate that Ang-(1-7) directly inhibits TGF-ß1-induced EMT in alveolar epithelial cells via disruption of TGF-ß1-Smad signaling pathway, contributing to the protective effect against IPF.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Angiotensina I/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Células A549 , Células Epiteliais Alveolares/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
6.
J Agric Food Chem ; 67(35): 9789-9795, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31373816

RESUMO

Pulmonary fibrosis is a chronic lung disease characterized by abnormal accumulation of the extracellular matrix (ECM). Chronic damage of the alveolar epithelium leads to a process called "epithelial-mesenchymal transition" (EMT) and increases synthesis and deposition of ECM proteins. Therefore, inhibition of EMT might be a promising therapeutic approach for the treatment of pulmonary fibrosis. ß-Sitosterol is one of the most abundant phytosterols in the plant kingdom and the major constituent in corn silk, which is derived from the stigma and style of maize (Zea mays). In this study, we elucidated that ß-sitosterol inhibited transforming growth factor-ß1 (TGF-ß1)-induced EMT and consequently had an antifibrotic effect. ß-Sitosterol (1-10 µg/mL) significantly downregulated the TGF-ß1-induced fibrotic proteins, such as collagen, fibronectin, and α-smooth muscle actin in human alveolar epithelial cells (p < 0.01). After 24 h, relative wound density (RWD) was increased in TGF-ß1 treated group (82.16 ± 5.70) compare to the control group (64.63 ± 2.21), but RWD was decreased in ß-sitosterol cotreated group (10 µg/mL: 71.54 ± 7.39; 20 µg/mL: 65.69 ± 6.42). In addition, the changes of the TGF-ß1-induced morphological shape and protein expression of EMT markers, N-cadherin, vimentin, and E-cadherin, were significantly blocked by ß-sitosterol treatment (p < 0.01). The effects of ß-sitosterol on EMT were found to be associated with the TGF-ß1/Snail pathway, which is regulated by Smad and non-Smad signaling pathways. Taken together, these findings suggest that ß-sitosterol can be used to attenuate pulmonary fibrosis through suppression of EMT by inhibiting the TGF-ß1/Snail pathway.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Extratos Vegetais/farmacologia , Alvéolos Pulmonares/efeitos dos fármacos , Fibrose Pulmonar/fisiopatologia , Sitosteroides/farmacologia , Zea mays/química , Actinas/genética , Actinas/metabolismo , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Extratos Vegetais/química , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/fisiopatologia , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
8.
Mol Med Rep ; 20(3): 2419-2425, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322172

RESUMO

The present study aimed to explore the role of endoplasmic reticulum calcium (ER Ca2+) in the apoptosis of human lung type II alveolar epithelial A549 cells induced by paraquat (PQ) in vitro. PQ significantly elevated the intracellular Ca2+ concentration. Treatment with the Ca2+­ATPase inhibitor thapsigargin significantly increased PQ­induced cytotoxicity, elevated the intracellular level of Ca2+, and increased the apoptosis rate, the protein expression of glucose­regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP), and the activities of caspase­7 and caspase­12 in PQ­treated cells. By contrast, treatment with heparin, an inositol 1,4,5­triphosphate receptor inhibitor, remarkably attenuated cytotoxicity and decreased the intracellular level of Ca2+, the apoptosis rate and the expression levels of GRP78, CHOP and Caspases. In conclusion, PQ impaired the regulating function of ER Ca2+ and resulted in an excessive increase of intracellular Ca2+. Therefore, influencing the Ca2+ signaling in the ER influenced the apoptosis of A549 cells via the ER stress pathway.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Herbicidas/efeitos adversos , Paraquat/efeitos adversos , Células A549 , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos
9.
Monoclon Antib Immunodiagn Immunother ; 38(4): 171-174, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31313968

RESUMO

Podoplanin (PDPN) is utilized as a specific marker of type I alveolar cells of lung and lymphatic endothelial cells of every tissue. Therefore, sensitive and specific monoclonal antibodies detecting PDPN are necessary for immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for Western blot, flow cytometry, and immunohistochemical analyses. In this study, immunohistochemical analyses showed that PMab-247 strongly detected bPDPN, which is expressed in type I alveolar cells and lymphatic endothelial cells of bear lung and podocytes of bear kidney. These findings suggest that PMab-247 could be useful for pathophysiological analyses using immunohistochemistry.


Assuntos
Células Epiteliais Alveolares/metabolismo , Anticorpos Monoclonais/imunologia , Células Endoteliais/metabolismo , Rim/metabolismo , Pulmão/metabolismo , Glicoproteínas de Membrana/imunologia , Células Epiteliais Alveolares/imunologia , Animais , Células Endoteliais/imunologia , Imuno-Histoquímica/métodos , Rim/imunologia , Pulmão/imunologia , Ursidae
10.
Molecules ; 24(14)2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31311194

RESUMO

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 fármacos
11.
Nutrients ; 11(7)2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261654

RESUMO

The alveolar epithelia of the lungs require manganese (Mn) as an essential nutrient, but also provide an entry route for airborne Mn that can cause neurotoxicity. Transporters involved in Mn uptake by alveolar epithelial cells are unknown. Recently, two members of the Zrt- and Irt-like protein (ZIP) family of metal transporters, ZIP8 and ZIP14, have been identified as crucial Mn importers in vivo. ZIP8 is by far most abundantly expressed in the lungs, whereas ZIP14 expression in the lungs is low compared to other tissues. We hypothesized that Mn uptake by alveolar epithelial cells is primarily mediated by ZIP8. To test our hypothesis, we used A549 cells, a type II alveolar cell line. Mirroring the in vivo situation, A549 cells expressed higher levels of ZIP8 than cell models for the liver, intestines, and kidney. Quantification of ZIP8 and ZIP14 revealed a strong enrichment of ZIP8 over ZIP14 in A549 cells. Using siRNA technology, we identified ZIP8 and ZIP14 as the major transporters mediating Mn uptake by A549 cells. To our surprise, knockdown of either ZIP8 or ZIP14 impaired Mn accumulation to a similar extent, which we traced back to similar amounts of ZIP8 and ZIP14 at the plasma membrane. Our study highlights the importance of both ZIP8 and ZIP14 in Mn metabolism of alveolar epithelial cells.


Assuntos
Células Epiteliais Alveolares/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Neoplasias Pulmonares/metabolismo , Manganês/metabolismo , Células A549 , Células CACO-2 , Proteínas de Transporte de Cátions/genética , Células HEK293 , Células Hep G2 , Humanos , Transporte de Íons , Cinética , Neoplasias Pulmonares/genética
12.
Nat Commun ; 10(1): 3390, 2019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31358769

RESUMO

Accumulating evidence illustrates a fundamental role for mitochondria in lung alveolar type 2 epithelial cell (AEC2) dysfunction in the pathogenesis of idiopathic pulmonary fibrosis. However, the role of mitochondrial fusion in AEC2 function and lung fibrosis development remains unknown. Here we report that the absence of the mitochondrial fusion proteins mitofusin1 (MFN1) and mitofusin2 (MFN2) in murine AEC2 cells leads to morbidity and mortality associated with spontaneous lung fibrosis. We uncover a crucial role for MFN1 and MFN2 in the production of surfactant lipids with MFN1 and MFN2 regulating the synthesis of phospholipids and cholesterol in AEC2 cells. Loss of MFN1, MFN2 or inhibiting lipid synthesis via fatty acid synthase deficiency in AEC2 cells exacerbates bleomycin-induced lung fibrosis. We propose a tenet that mitochondrial fusion and lipid metabolism are tightly linked to regulate AEC2 cell injury and subsequent fibrotic remodeling in the lung.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Metabolismo dos Lipídeos , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Células Cultivadas , Colesterol/metabolismo , GTP Fosfo-Hidrolases/genética , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Camundongos Knockout , Camundongos Transgênicos , Dinâmica Mitocondrial/efeitos dos fármacos , Dinâmica Mitocondrial/genética , Fosfolipídeos/biossíntese
13.
Cell Mol Biol Lett ; 24: 35, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31160894

RESUMO

Background: Pulmonary edema is one of the pathological characteristics of acute respiratory distress syndrome (ARDS). The epithelial sodium channel (ENaC) is thought to be the rate-limiting factor for alveolar fluid clearance (AFC) during pulmonary edema. The peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone was shown to stimulate ENaC-mediated salt absorption in the kidney. However, its role in the lung remains unclear. Here, we investigated the role of the PPARγ agonist in the lung to find out whether it can regulate AFC during acute lung injury (ALI). We also attempted to elucidate the mechanism for this. Methods: Our ALI model was established through intratracheal instillation of lipopolysaccharide (LPS) in C57BL/6 J mice. The mice were randomly divided into 4 groups of 10. The control group underwent a sham operation and received an equal quantity of saline. The three experimental groups underwent intratracheal instillation of 5 mg/kg LPS, followed by intraperitoneal injection of 4 mg/kg rosiglitazone, 4 mg/kg rosiglitazone plus 1 mg/kg GW9662, or only equal quantity of saline. The histological morphology of the lung, the levels of TNF-α and IL-1ß in the bronchoalveolar lavage fluid (BALF), the level of AFC, and the expressions of αENaC and serum and glucocorticoid-induced kinase-1 (SGK1) were determined. Type 2 alveolar (AT II) cells were incubated with rosiglitazone (15 µM) with or without GW9662 (10 µM). The expressions of αENaC and SGK1 were determined 24 h later. Results: A mouse model of ALI was successfully established. Rosiglitazone significantly ameliorated the lung injury, decreasing the TNF-α and IL-1ß levels in the BALF, enhancing AFC, and promoting the expressions of αENaC and SGK1 in ALI mice, which were abolished by the specific PPARγ blocker GW9662. In vitro, rosiglitazone increased the expressions of αENaC and SGK1. This increase was prevented by GW9662. Conclusions: Rosiglitazone ameliorated the lung injury and promoted ENaC-mediated AFC via a PPARγ/SGK1-dependent signaling pathway, alleviating pulmonary edema in a mouse model of ALI.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Líquido da Lavagem Broncoalveolar/química , Canais Epiteliais de Sódio/metabolismo , PPAR gama/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Rosiglitazona/farmacologia , Transdução de Sinais , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Animais , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
14.
Monoclon Antib Immunodiagn Immunother ; 38(3): 104-107, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31161964

RESUMO

Podoplanin (PDPN)/T1 alpha is known as a specific marker of lymphatic endothelial cells and type I alveolar cells. Sensitive and specific monoclonal antibodies (mAbs) for PDPN are needed for immunohistochemical analyses. Recently, we developed an anticetacean PDPN mAb, PMab-237. Herein, immunohistochemical analyses showed that PMab-237 strongly detected pulmonary type I alveolar cells, renal podocytes, and lymphatic endothelial cells of the harbor porpoise. These findings suggest that PMab-237 may be useful for immunohistochemical analyses for cetacean tissues.


Assuntos
Células Epiteliais Alveolares/metabolismo , Anticorpos Monoclonais/imunologia , Células Endoteliais/metabolismo , Epitopos/imunologia , Glicoproteínas de Membrana/imunologia , Phocoena/metabolismo , Podócitos/metabolismo , Células Epiteliais Alveolares/imunologia , Animais , Especificidade de Anticorpos/imunologia , Células Endoteliais/imunologia , Humanos , Imuno-Histoquímica , Phocoena/imunologia , Podócitos/imunologia
15.
Respir Res ; 20(1): 118, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186017

RESUMO

OBJECTIVE: The mechanisms of lung injury in acute respiratory distress syndrome (ARDS) are not well understood.Piezo1 was recently identified as a mechanotransduction protein. The present study found the expression of Piezo1 in type II pneumocytes and investigated its role in mediating ARDS-related lung injury. METHODS: Sprague-Dawley rats were used to establish an ARDS model, the expression of Piezo1,lung injuries, apoptosis as well as calcium influx were assessed. RESULTS: Piezo1 was expressed in type II pneumocytes as shown by immunofluorescence staining and expression was increased in the ARDS model. Knockdown of Piezo1 reduced apoptosis which was related to the elevation of Bcl-2.Calcium influx played a vital role in Piezo1-induced apoptosis. CONCLUSION: Piezo1 was expressed in type II pneumocytes. Mechanical stretch of alveoli during ARDS induced activation of the Piezo1 channel,which resulted in calcium influx. The increased intracellular Ca2+ induced the apoptosis of type II pneumocytes, which may be related to the Bcl-2 pathway.


Assuntos
Células Epiteliais Alveolares/metabolismo , Apoptose/fisiologia , Proteínas de Membrana/biossíntese , Síndrome do Desconforto Respiratório do Adulto/metabolismo , Estresse Mecânico , Células A549 , Células Epiteliais Alveolares/patologia , Animais , Humanos , Ratos , Ratos Sprague-Dawley , Síndrome do Desconforto Respiratório do Adulto/patologia
16.
Cells ; 8(7)2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31247991

RESUMO

The epithelial-mesenchymal transition (EMT) is important in organ fibrosis. We hypothesized that growth arrest-specific protein 6 (Gas6) and its underlying mechanisms play roles in the prevention of EMT in alveolar epithelial cells (ECs). In this study, to determine whether Gas6 prevents TGF-ß1-induced EMT in LA-4 and primary alveolar type II ECs, real-time PCR and immunoblotting in cell lysates and ELISA in culture supernatants were performed. Migration and invasion assays were performed using Transwell chambers. Pretreatment of ECs with Gas6 inhibited TGF-ß1-induced EMT based on cell morphology, changes in EMT marker expression, and induction of EMT-activating transcription factors. Gas6 enhanced the levels of cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) and PGD2 as well as of their receptors. COX-2 inhibitors and antagonists of PGE2 and PGD2 receptors reversed the inhibition of TGF-ß1-induced EMT, migration, and invasion by Gas6. Moreover, knockdown of Axl or Mer reversed the enhancement of PGE2 and PGD2 and suppression of EMT, migration and invasion by Gas6. Our data suggest Gas6-Axl or -Mer signalling events may reprogram ECs to resist EMT via the production of PGE2, PGD2, and their receptors.


Assuntos
Células Epiteliais Alveolares/metabolismo , Transição Epitelial-Mesenquimal , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células A549 , Animais , Movimento Celular , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/biossíntese , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Cultura Primária de Células , Prostaglandina D2/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Receptores de Prostaglandina E/metabolismo , Proteínas Recombinantes/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismo
17.
Oxid Med Cell Longev ; 2019: 2654910, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31217836

RESUMO

The oxidative stresses are a major insult in pulmonary injury such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), two clinical manifestations of acute respiratory failure with substantially high morbidity and mortality. Mesenchymal stem cells (MSCs) hold a promise in treatments of many human diseases, mainly owing to their capacities of immunoregulation and antioxidative activity. The strong immunoregulatory role of human placental MSCs of fetal origin (hfPMSCs) has been previously demonstrated; their antioxidant activity, however, has yet been interrogated. In this report, we examined the antioxidative activity of hfPMSCs by accessing the ability to scavenge oxidants and radicals and to protect alveolar epithelial cells from antioxidative injury using both a cell coculture model and a conditioned culture medium (CM) of hfPMSCs. Results showed a comparable antioxidative capacity of the CM with 100 µM of vitamin C (VC) in terms of the total antioxidant capacity (T-AOC), scavenging abilities of free radicals DPPH, hydroxyl radical (·OH), and superoxide anion radical (O2 -), as well as activities of antioxidant enzymes of SOD and GSH-PX. Importantly, both of the CM alone and cocultures of hfPMSCs displayed a protection of A549 alveolar epithelial cells from oxidative injury of 600 µM hydrogen peroxide (H2O2) exposure, as determined in monolayer and transwell coculture models, respectively. Mechanistically, hfPMSCs and their CM could significantly reduce the apoptotic cell fraction of alveolar epithelial A549 cells exposed to H2O2, accompanied with an increased expression of antiapoptotic proteins Bcl-2, Mcl-1, Nrf-2, and HO-1 and decreased proapoptotic proteins Bax, caspase 3, and Keap1, in comparison with naïve controls. Furthermore, hfPMSCs-CM (passage 3) collected from cultures exposed an inhibition of the Nrf2/Keap1/ARE signaling pathway which led to a significant reduction in caspase 3 expression in A549 cells, although the addition of Nrf2 inhibitor ML385 had no effect on the antioxidative activity of hfPMSCs-CM. These data clearly suggested that hfPMSCs protected the H2O2-induced cell oxidative injury at least in part by regulating the Nrf2-Keap1-ARE signaling-mediated cell apoptosis. Our study thus provided a new insight into the antioxidative mechanism and novel functions of hfPMSCs as antioxidants in disease treatments, which is warranted for further investigations.


Assuntos
Células Epiteliais Alveolares/metabolismo , Antioxidantes/metabolismo , Células-Tronco Mesenquimais/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Placenta/metabolismo , Feminino , Humanos , Estresse Oxidativo , Gravidez , Transdução de Sinais
18.
Toxicol Lett ; 313: 60-65, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31226396

RESUMO

The use of waterpipes in the United States is increasing in a largely unregulated market. The shisha smoked in a waterpipe is a complex matrix of tobacco, flavorings, and humectant with smoke generated by an external heat source. This study explored the relationship between shisha components and the particulate matter size distributions and toxicity of smoke generated with heating. Standard waterpipe puff topography of charcoal- or electronic- heated whole shisha and shisha components generated smoke particulate matter that was characterized using a TSI Engine Exhaust Particle Sizer. Relative toxicity of the whole smoke was determined via measurement of lysosomal integrity and measures of membrane integrity following acute exposure of type II alveolar cells at the air-liquid interface. All waterpipe aerosols exhibited a unimodal particle size distribution, the peak and concentration of which varied depending upon the shisha components present. Acute exposure to charcoal-heated whole shisha, flavoring syrup, or humectant smoke, or electronic-heated whole shisha smoke caused significant alveolar cell damage and death, indicating neither tobacco nor charcoal are needed for these cytotoxic effects to occur.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Carvão Vegetal/toxicidade , Aromatizantes/análise , Higroscópicos/toxicidade , Fumaça/efeitos adversos , Tabaco para Cachimbos de Água/toxicidade , Fumar Cachimbo de Água/efeitos adversos , Aerossóis , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Carvão Vegetal/análise , Aromatizantes/efeitos adversos , Higroscópicos/análise , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/patologia , Tamanho da Partícula , Ratos , Medição de Risco , Fumaça/análise , Tabaco para Cachimbos de Água/análise
19.
Int J Mol Med ; 44(1): 227-239, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115492

RESUMO

Endoplasmic reticulum (ER) stress in alveolar epithelial cells (AECs) is associated with the pathogenesis of pulmonary fibrosis. Bone marrow­derived mesenchymal stromal cells (BM­MSCs) can exert protective effects on ER­stressed AECs via paracrine signaling. In the present study, mouse lung epithelial (MLE)­12 cells were directly stimulated with various concentrations of bleomycin (BLM). MLE­12 cell apoptosis was detected by flow cytometry, and Ki67 expression was detected by immunofluorescence to reflect cell proliferation. The results revealed that BLM increased the protein expression levels of X­box binding protein 1 and immunoglobulin heavy chain­binding protein, thus inducing ER stress, and caused cell dysfunction by inhibiting proliferation and promoting apoptosis. In addition, MSC­derived conditioned medium (MSC­CM) protected MLE­12 cells from BLM­induced injury, by reducing ER stress, promoting cell proliferation and inhibiting cell apoptosis. Our previous studies reported that N­methyl­D­aspartate (NMDA) receptor activation partially inhibits the antifibrotic effect of BM­MSCs on BLM­induced pulmonary fibrosis through downregulating the paracrine factor hepatocyte growth factor (HGF). In the present study, the synthesis and secretion of HGF were detected by western blotting and ELISA, respectively. Results further demonstrated that NMDA inhibited the synthesis and secretion of HGF in BM­MSCs, and NMDA­preconditioned MSC­CM had no protective effects on BLM­induced injury in MLE­12 cells. In addition, activation of the NMDA receptor decreased the phosphorylation levels of extracellular signal­regulated kinase (ERK)1/2 in BM­MSCs. Using Honokiol and FR180204, the activator and inhibitor of ERK1/2, respectively, it was then revealed that Honokiol partially eliminated the decrease in HGF expression, whereas FR180204 further promoted the reduction in HGF caused by NMDA. Collectively, these findings suggested that NMDA receptor activation may downregulate HGF by inhibiting ERK signaling in BM­MSCs, thus weakening their protective effects on BLM­induced lung epithelial cell damage.


Assuntos
Células Epiteliais Alveolares/metabolismo , Bleomicina/efeitos adversos , Células da Medula Óssea/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Sistema de Sinalização das MAP Quinases , Células-Tronco Mesenquimais/metabolismo , Comunicação Parácrina , Fibrose Pulmonar/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Bleomicina/farmacologia , Células da Medula Óssea/patologia , Linhagem Celular , Feminino , Células-Tronco Mesenquimais/patologia , Camundongos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Fibrose Pulmonar/prevenção & controle
20.
Int J Mol Sci ; 20(9)2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31075901

RESUMO

The alveolar epithelial cells represent an important part of the alveolar barrier, which is maintained by tight junction proteins, particularly JAM-A, occludin, and claudin-18, which regulate paracellular permeability. In this study, we report on a strong increase in epithelial JAM-A expression in P2X7 receptor knockout mice when compared to the wildtype. Precision-cut lung slices of wildtype and knockout lungs and immortal epithelial lung E10 cells were treated with bleomycin, the P2X7 receptor inhibitor oxATP, and the agonist BzATP, respectively, to evaluate early changes in JAM-A expression. Biochemical and immunohistochemical data showed evidence for P2X7 receptor-dependent JAM-A expression in vitro. Inhibition of the P2X7 receptor using oxATP increased JAM-A, whereas activation of the receptor decreased the JAM-A protein level. In order to examine the role of GSK-3ß in the expression of JAM-A in alveolar epithelial cells, we used lithium chloride for GSK-3ß inhibiting experiments, which showed a modulating effect on bleomycin-induced alterations in JAM-A levels. Our data suggest that an increased constitutive JAM-A protein level in P2X7 receptor knockout mice may have a protective effect against bleomycin-induced lung injury. Bleomycin-treated precision-cut lung slices from P2X7 receptor knockout mice responded with a lower increase in mRNA expression of JAM-A than bleomycin-treated precision-cut lung slices from wildtype mice.


Assuntos
Moléculas de Adesão Celular/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Células Epiteliais Alveolares/metabolismo , Animais , Bleomicina , Moléculas de Adesão Celular/genética , Camundongos , Agonistas do Receptor Purinérgico P2X/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Receptores Purinérgicos P2X7/deficiência
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