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1.
Neurochem Res ; 49(8): 2197-2214, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38834846

RESUMEN

Neuroinflammation and endothelial cell apoptosis are prominent features of blood-brain barrier (BBB) disruption, which have been described in Alzheimer's disease (AD) and can predict cognitive decline. Recent reports revealed vascular ß-amyloid (Aß) deposits, Muller cell degeneration and microglial dysfunction in the retina of AD patients. However, there has been no in-depth research on the roles of inflammation, retinal endothelial cell apoptosis, and blood-retinal barrier (BRB) damage in AD retinopathy. We found that Raddeanin A (RDA) could improve pathological and cognitive deficits in a mouse model of Alzheimer's disease by targeting ß-amyloidosis, However, the effects of RDA on AD retinal function require further study. To clarify whether RDA inhibits inflammation and apoptosis and thus improves BRB function in AD-related retinopathy. In vitro we used Aß-treated HRECs and MIO-M1 cells, and in vivo we used 3×Tg-AD mice to investigate the effect of RDA on BRB in AD-related retinopathy. We found that RDA could improve BRB function in AD-related retinopathy by inhibiting NLRP3-mediated inflammation and suppressing Wnt/ß-catenin pathway-mediated apoptosis, which is expected to improve the pathological changes in AD-related retinopathy and the quality of life of AD patients.


Asunto(s)
Enfermedad de Alzheimer , Apoptosis , Barrera Hematorretinal , Ratones Transgénicos , Retina , Animales , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Apoptosis/efectos de los fármacos , Barrera Hematorretinal/efectos de los fármacos , Barrera Hematorretinal/metabolismo , Retina/efectos de los fármacos , Retina/metabolismo , Retina/patología , Ratones , Inflamación/metabolismo , Inflamación/tratamiento farmacológico , Ratones Endogámicos C57BL , Humanos , Péptidos beta-Amiloides/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/fisiología , Masculino
2.
Int J Biol Macromol ; 264(Pt 2): 130690, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38458297

RESUMEN

Nowadays, non-small cell lung cancer (NSCLC) is still one of the most life-threatening diseases in the world. In previous studies, a fungal protein PFAP with anti-NSCLC properties was isolated and identified from Pleurotus ferulae lanzi. In this study, the amino acid sequence of PFAP was analyzed and found to be highly homologous to the aegerolysin family. PFAP, like other members of the aegerolysin family, specifically recognizes lipid raft domains rich in cholesterol and sphingomyelin, which is probably its specific anti-tumor mechanism. Previous studies have shown that PFAP can induce AMPK-mediated autophagy and G1-phase cell cycle arrest in A549 lung cancer cells. This study further revealed that PFAP can also induce paraptosis and endoplasmic reticulum stress (ERS) in A549 cells in vitro by targeting AMPK. PFAP induces multi-pathway death of A549 cells, and thus demonstrates its potential value for developing new drugs for NSCLC.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Células A549 , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Línea Celular Tumoral , Apoptosis , Paraptosis , Proteínas Quinasas Activadas por AMP , Estrés del Retículo Endoplásmico
3.
Nat Commun ; 14(1): 4394, 2023 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-37474626

RESUMEN

The incidence of rheumatoid arthritis (RA) is increasing with age. DNA fragments is known to accumulate in certain autoimmune diseases, but the mechanistic relationship among ageing, DNA fragments and RA pathogenesis remain unexplored. Here we show that the accumulation of DNA fragments, increasing with age and regulated by the exonuclease TREX1, promotes abnormal activation of the immune system in an adjuvant-induced arthritis (AIA) rat model. Local overexpression of TREX1 suppresses synovial inflammation in rats, while conditional genomic deletion of TREX1 in AIA rats result in higher levels of circulating free (cf) DNA and hence abnormal immune activation, leading to more severe symptoms. The dysregulation of the heterodimeric transcription factor AP-1, formed by c-Jun and c-Fos, appear to regulate both TREX1 expression and SASP induction. Thus, our results confirm that DNA fragments are inflammatory mediators, and TREX1, downstream of AP-1, may serve as regulator of cellular immunity in health and in RA.


Asunto(s)
Artritis Experimental , Artritis Reumatoide , Humanos , Ratas , Animales , Proteínas Proto-Oncogénicas c-fos/genética , Inflamación , Factor de Transcripción AP-1/metabolismo
4.
Int J Biol Macromol ; 244: 125453, 2023 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-37330099

RESUMEN

A new protein, designated PFAP, with activity against non-small cell lung cancer (NSCLC), was isolated from Pleurotus ferulae lanzi, a medicinal and edible mushroom. The purification method involved hydrophobic interaction chromatography on a HiTrap Octyl FF column and gel filtration on a Superdex 75 column. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single band with a molecular weight of 14.68 kDa. Following de novo sequencing and liquid chromatography-tandem mass spectrometry, PFAP was identified as a protein consisting of 135 amino acid residues, with a theoretical molecular weight of 14.81 kDa. Tandem mass tag (TMT)™-based quantitative proteomic analysis and western blotting revealed that AMP-activated protein kinase (AMPK) was significantly upregulated in NSCLC A549 cells, following PFAP treatment. The downstream regulatory factor mammalian target of rapamycin (mTOR) was suppressed, resulting in the activation of autophagy and upregulated expressions of P62, LC3 II/I, and other related proteins. PFAP blocked NSCLC A549 cells in the G1 phase of the cell cycle via upregulating P53 and P21, while subsequently downregulating the expression of cyclin-dependent kinases. PFAP suppresses tumour growth via the same mechanism in a xenograft mouse model in vivo. These results demonstrate that PFAP is a multifunctional protein with anti-NSCLC properties.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Pleurotus , Humanos , Animales , Ratones , Neoplasias Pulmonares/patología , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas Quinasas Activadas por AMP/metabolismo , Células A549 , Pleurotus/química , Proteómica , Autofagia , Proteínas Fúngicas , Puntos de Control del Ciclo Celular , Proliferación Celular , Mamíferos/metabolismo
5.
Anal Chem ; 95(12): 5443-5453, 2023 03 28.
Artículo en Inglés | MEDLINE | ID: mdl-36930753

RESUMEN

The detection of hydrogen sulfide (H2S), the third gas signaling molecule, is a promising strategy for identifying the occurrence of certain diseases. However, the conventional single- or dual-signal detection can introduce false-positive or false-negative results, which ultimately decreases the diagnostic accuracy. To address this limitation, we developed a luminescent, photothermal, and electrochemical triple-signal detection platform by optically trapping the synthetic highly doped upconversion coupled SiO2 microbeads coated with metal-organic frameworks H-UCNP-SiO2@HKUST-1 (H-USH) to detect the concentration of H2S. The H-USH was first synthesized and proved to have stable structure and excellent luminescent, photothermal, and electrochemical properties. Under 980 nm optical trapping and 808 nm irradiation, H-USH showed great detection linearity, a low limit of detection, and high specificity for H2S quantification via triple-signal detection. Moreover, H-USH was captured by optical tweezers to realize quantitative detection of H2S content in serum of acute pancreatitis and spontaneously hypertensive rats. Finally, by analyzing the receiver operating characteristic (ROC) curve, we concluded that triple-signal detection of H2S was more accurate than single- or dual-signal detection, which overcame the problem of false-negative/positive results in the detection of H2S in actual serum samples.


Asunto(s)
Sulfuro de Hidrógeno , Pancreatitis , Ratas , Animales , Sulfuro de Hidrógeno/química , Luminiscencia , Electroquímica , Enfermedad Aguda , Dióxido de Silicio , Microesferas
6.
Redox Biol ; 61: 102637, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36821955

RESUMEN

Alzheimer's disease is a neurodegenerative disorder characterized by a decline in cognitive function. The ß-amyloid (Aß) hypothesis suggests that Aß peptides can spontaneously aggregate into ß-fragment-containing oligomers and protofibrils, and this activation of the amyloid pathway alters Ca2+ signaling in neurons, leading to neurotoxicity and thus apoptosis of neuronal cells. In our study, a blood-brain barrier crossing flavonol glycoside hyperoside was identified with anti-Aß aggregation, BACE inhibitory, and neuroprotective effect in cellular or APP/PSEN1 double transgenic Alzheimer's disease mice model. While our pharmacokinetic data confirmed that intranasal administration of hyperoside resulted in a higher bio-availability in mice brain, further in vivo studies revealed that it improved motor deficit, spatial memory and learning ability of APP/PSEN1 mice with reducing level of Aß plaques and GFAP in the cortex and hippocampus. Bioinformatics, computational docking and in vitro assay results suggested that hyperoside bind to Aß and interacted with ryanodine receptors, then regulated cellular apoptosis via endoplasmic reticulum-mitochondrial calcium (Ca2+) signaling pathway. Consistently, it was confirmed that hyperoside increased Bcl2, decreased Bax and cyto-c protein levels, and ameliorated neuronal cell death in both in vitro and in vivo model. By regulating Aß-induced cell death via regulation on Ca2+ signaling cascade and mitochondrial membrane potential, our study suggested that hyperoside may work as a potential therapeutic agent or preventive remedy for Alzheimer's disease.


Asunto(s)
Enfermedad de Alzheimer , Ratones , Animales , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Ratones Transgénicos , Calcio/metabolismo , Transducción de Señal , Retículo Endoplásmico/metabolismo , Modelos Animales de Enfermedad
7.
Neoplasma ; 69(5): 1108-1118, 2022 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-35951456

RESUMEN

Sperm-associated antigen 1 (SPAG1) is considered to be associated with infertility and tumorigenesis. However, its function in acute myeloid leukemia (AML) remains unclear. In this study, we evaluated the expression level of SPAG1 and explored its clinical prognostic value in patients with AML, as well as its biological function in AML cells. SPAG1 is widely expressed in AML patients, resulting in a poor prognosis. However, its expression was not associated with Fms-related receptor tyrosine kinase 3 (FLT3) mutations. Utilizing the RNA interference knockdown tests, we found that SPAG1 could promote the proliferation and survival of AML cells and regulate the expression of structural maintenance of chromosomes protein 3 (SMC3), activating the ERK/MAPK signaling pathway. Furthermore, we discovered that inhibiting SPAG1 impacted AML cell susceptibility to venetoclax. In conclusion, SPAG1 may serve as a potential therapeutic target in AML.


Asunto(s)
Antígenos de Superficie , Compuestos Bicíclicos Heterocíclicos con Puentes , Proteínas de Unión al GTP , Leucemia Mieloide Aguda , Sulfonamidas , Antígenos de Superficie/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Mutación , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Sulfonamidas/farmacología , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismo
8.
Oxid Med Cell Longev ; 2022: 6584631, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35663201

RESUMEN

By reason of surgical demand, the majority of cardiovascular procedures still depend on the use of cardiopulmonary bypass (CPB). Due to the nonphysiological state of CPB, it can cause complex and unpredictable inflammatory response, which may lead to significant morbidity and mortality. Unfortunately, the pharmacological and mechanical strategies that currently exist do not offer significant advantages in controlling inflammatory response and improving patient outcomes. The best strategy to reduce inflammation in CPB is still uncertain. In recent years, adsorptive blood purification techniques (BPTs) have emerged, among which CytoSorb is the latest representative device. Currently, the primary application area of adsorptive BPTs is in the control and treatment of systemic hyperinflammatory states, such as refractory septic shock patients. However, the evidences on efficacy and safety of adsorptive BPTs application during CPB surgery are still inconclusive, so we summarize the relevant evidences here and suggest future potential research areas.


Asunto(s)
Procedimientos Quirúrgicos Cardíacos , Puente Cardiopulmonar , Procedimientos Quirúrgicos Cardíacos/métodos , Humanos , Inflamación
9.
ACS Sens ; 7(5): 1572-1580, 2022 05 27.
Artículo en Inglés | MEDLINE | ID: mdl-35482449

RESUMEN

Although great headway has been made in DNAzyme-based detection of Pb2+, its adaptability, sensitivity, and accessibility in complex media still need to be improved. For this, we introduce new ways to surmount these hurdles. First, a spherical nucleic acid (SNA) fluorescence probe (Au nanoparticles-DNAzyme probe) is utilized to specifically identify Pb2+ and its suitability for precise detection of Pb2+ in complex samples due to its excellent nuclease resistance. Second, the sensitivity of Pb2+ detection is greatly enhanced via the use of a clustered regularly interspaced short palindromic repeats-Cas12a with target recognition accuracy to amplify the fluorescent signal upon the trans cleavage of the SNA (signal probe), and the limit of detection reaches as low as 86 fM. Third, we boost the fluorescence on photonic crystal chips with a bionic periodic arrangement by employing a straightforward detection device (smartphone and portable UV lamp) to achieve on-site detection of Pb2+ with the limit of detection as low as 24 pM. Based on the abovementioned efforts, the modified Pb2+ fluorescence sensor has the advantages of higher sensitivity, better specificity, accessibility, less sample consumption, and so forth. Moreover, it can be applied to accurately detect Pb2+ in complex biological or environmental samples, which is of great promise for widespread applications.


Asunto(s)
ADN Catalítico , Nanopartículas del Metal , Sistemas CRISPR-Cas , Oro , Plomo
10.
Appl Opt ; 59(16): 4790-4795, 2020 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-32543471

RESUMEN

In this paper, the self-absorption of InGaN quantum wells at high photon density is studied based on a rectangular ridge structure. The ridge structure was fabricated based on a standard GaN-based blue LED wafer grown on (0001) patterned sapphire substrate. The high-density photons were obtained by a high-power femtosecond laser with high excitation of 42kW/cm2 at room temperature. Based on the analysis of the photoluminescence intensities of the InGaN quantum wells, we found that the absorption coefficient of the InGaN quantum wells varies with the background photon density. The results revealed that the final absorption coefficient of the InGaN quantum well decreases with the increase of photon density, which can be 48.7% lower than its normal value under our experimental conditions.

11.
J Exp Clin Cancer Res ; 39(1): 17, 2020 Jan 17.
Artículo en Inglés | MEDLINE | ID: mdl-31952546

RESUMEN

BACKGROUND: STAT5 plays an important role in the transformation of hematopoietic cells by BCR-ABL. However, the downstream target genes activated by STAT5 in chronic myeloid leukemia (CML) cells remain largely unclear. Here, we investigated the mechanistic functional relationship between STAT5A-regulated microRNA and CML cell apoptosis. METHODS: The expression of USP15, Caspase-6, STAT5A-regulated miR-202-5p and STAT5A was detected by qRT-PCR and Western blotting in CML cell lines and PBMCs of CML patients. Cell apoptosis was evaluated by flow cytometry. Both gain- and loss-of-function experiments were used to investigate the roles of USP15, miR-202-5p and STAT5A in CML. Luciferase reporter assay detected the effect of miR-202-5p on USP15 expression. Xenograft animal model was used to test the effect of anti-miR-202-5p and pimozide on K562 cell xenograft growth. RESULTS: USP15 expression was significantly downregulated in CML cell lines and PBMCs of CML patients. Depletion of USP15 increased, whereas overexpression of USP15 reduced the resistance of CML cells to Imatinib. Further, decreased deubiquitinating activity of USP15 by USP15 downregulation led to reduced caspase-6 level, thus attenuating CML cell apoptosis. Mechanistically, miR-202-5p was upregulated in K562G cells and negatively regulated USP15 expression by directly targeting USP15 3'-UTR. Correspondingly, upregulation of miR-202-5p enhanced the resistance of CML cells to Imatinib by inhibiting cell apoptosis. Importantly, STAT5A was upregulated in CML cells and directly activated miR-202-5p transcription by binding to the pre-miR-202 promoter. Pimozide induced CML cell apoptosis and significantly reduced K562 cell xenograft growth in vivo by blocking STAT5A/miR-202-5p/USP15/Caspase-6 regulatory axis. CONCLUSIONS: we provide the first evidence that de-regulated STAT5A/miR-202-5p/USP15/Caspase-6 regulatory axis suppresses the apoptosis of CML cells, targeting this pathway might be a promising therapeutic approach for the treatment of CML.


Asunto(s)
Caspasa 6/metabolismo , Mesilato de Imatinib/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , MicroARNs/metabolismo , Factor de Transcripción STAT5/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Estudios de Casos y Controles , Regulación hacia Abajo , Resistencia a Antineoplásicos , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Transducción de Señal , Proteasas Ubiquitina-Específicas/biosíntesis
12.
Biosci Rep ; 39(4)2019 04 30.
Artículo en Inglés | MEDLINE | ID: mdl-30962263

RESUMEN

microRNAs (miRNA), as tumor suppressors or oncogenes, are involved in modulating cancer cell behavior, including cell proliferation and apoptosis. The miR-140-5p acts as a tumor suppressor in several tumors, but the role of miR-140-5p in chronic myeloid leukemia (CML) remains unclear. Here, we investigated the suppression of miR-140-5p in CML patients and CML cell lines using quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH). Overexpression miR-140-5p in CML cells significantly inhibited cell proliferation as revealed by the CCK-8 assay and promoted cell apoptosis as revealed by flow cytometry. Moreover, the sine oculis homeobox 1 (SIX1) gene had been confirmed as a direct target of miR-140-5p using bioinformatics analysis and luciferase reporter assays. Overexpression of miR-140-5p decreased the SIX1 protein level in CML cells. SIX1 mRNA and protein levels were significantly up-regulated in CML patients and CML cell lines. Knockdown of SIX1 expression significantly inhibited CML cell proliferation and promoted cell apoptosis. Furthermore, SIX1 as a transcriptional factor positively regulated pyruvate kinase isozyme type M2 (PKM2) expression and played an important role in the Warburg effect. In addition, these findings indicated that miR-140-5p functions as a tumor suppressor and plays a critical role in CML cell apoptosis and metabolism by targeting SIX1. Moreover, the miR-140-5p/SIX1 axis may be a potential therapeutic target in CML.


Asunto(s)
Proteínas de Homeodominio/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , MicroARNs/genética , Adulto , Anciano , Apoptosis/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Femenino , Regulación Leucémica de la Expresión Génica , Humanos , Células K562 , Leucocitos Mononucleares/patología , Leucocitos Mononucleares/fisiología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , MicroARNs/metabolismo , Persona de Mediana Edad , Hormonas Tiroideas/genética , Hormonas Tiroideas/metabolismo , Proteínas de Unión a Hormona Tiroide
13.
Fitoterapia ; 129: 179-184, 2018 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-29981396

RESUMEN

The fruits of Swietenia macrophylla (skyfruits) are commercially used as healthcare products to improve blood circulation. An investigation of active ingredients of skyfruits led to the isolation of four new limonoids, swietemacrolides A-D (1-4), together with ten known limonoids (5-14) and one proto-limonoid (15). Their structures were elucidated on the basis of MS and NMR data analysis. Swietemacrolide C (3) at the concentration of 10 µM showed significant protective effect on H2O2-induced apoptosis in human umbilical vascular endothelial cells (HUVECs), while swieteliacate D (5) displayed moderate anti-apoptotic activity.


Asunto(s)
Apoptosis/efectos de los fármacos , Frutas/química , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Limoninas/farmacología , Meliaceae/química , Humanos , Peróxido de Hidrógeno , Limoninas/aislamiento & purificación , Melanesia , Estructura Molecular
14.
Front Physiol ; 9: 1817, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30618827

RESUMEN

In smokers with chronic obstructive pulmonary disease, more severe lung inflammation is associated with menthol cigarette smoking compared to non-menthol cigarette smoking. However, the mechanisms remain unclear. Menthol is an activator of transient receptor potential melastatin-8 (TRPM8), which is also sensitive to reactive oxygen species (ROS). Our recent in vitro study demonstrated that the extracts of menthol cigarette smoke (M-CS) can induce greater ROS-sensitive, TRPM8-mediated, mitogen-activated protein kinase (MAPK)-dependent inflammatory responses in lung epithelial cells than the extracts of non-menthol cigarette smoke (Non-M-CS) can. In this study, we tested the hypothesis that M-CS can induce more severe lung inflammation than Non-M-CS can via the additional action of menthol in M-CS on epithelial and lung TRPM8 in mice. Compared with Non-M-CS exposure, subchronic M-CS exposure for 7 days up-regulated the epithelial and lung expression of TRPM8, induced more vigorous activation of epithelial and lung MAPKs, and caused more severe lung inflammation. The MAPK activation was evidenced by the increased expression of phosphor-extracellular signal-regulated and phosphor-c-Jun N-terminal kinases. The lung inflammation was evidenced by pathohistological findings and increases in several inflammatory indices. Moreover, treatment with a TRPM8 antagonist (N-(3-aminopropyl)-2-{[(3-methylphenyl)methyl]oxy}-N-(2-thienylmethyl)benzamide; AMTB) greatly suppressed the MAPK activation and lung inflammation induced by Non-M-CS and M-CS, and the residual responses to these two types of CS did not differ. Conversely, the levels of biomarkers of acute CS exposure (20 min), including carboxyhemoglobin and cotinine (a nicotine metabolite) in blood plasma, and superoxide and hydrogen peroxide (two major types of ROS) in bronchoalveolar lavage fluid, did not show significant differences in the mice with Non-M-CS and M-CS exposure. We concluded that M-CS could induce greater TRPM8-mediated activation of MAPKs and lung inflammation than Non-M-CS could in mice with subchronic exposure. The augmented inflammatory effects of M-CS are unlikely due to a larger total amount of CS inhaled, but may be caused by an additional stimulation of epithelial and lung TRPM8 by menthol in M-CS. A common stimulant (presumably ROS) generated by both CS types may also stimulate TRPM8, activate MAPKs, and induce lung inflammation because treatment with AMTB could reduce these responses to Non-M-CS.

15.
Front Physiol ; 8: 263, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28496415

RESUMEN

Clinical studies suggest that smokers with chronic obstructive pulmonary disease who use menthol cigarettes may display more severe lung inflammation than those who smoke non-menthol cigarette. However, the mechanisms for this difference remain unclear. Menthol is a ligand of transient receptor potential melastatin-8 (TRPM8), a Ca2+-permeant channel sensitive to reactive oxygen species (ROS). We previously reported that exposure of human bronchial epithelial cells (HBECs) to non-menthol cigarette smoke extract (Non-M-CSE) triggers a cascade of inflammatory signaling leading to IL-8 induction. In this study, we used this in vitro model to compare the inflammatory effects of menthol cigarette smoke extract (M-CSE) and Non-M-CSE and delineate the mechanisms underlying the differences in their impacts. Compared with Non-M-CSE, M-CSE initially increased a similar level of extracellular ROS, suggesting the equivalent oxidant potency. However, M-CSE subsequently produced more remarkable elevations in intracellular Ca2+, activation of the mitogen-activated protein kinases (MAPKs)/nuclear factor-κB (NF-κB) signaling, and IL-8 induction. The extracellular ROS responses to both CSE types were totally inhibited by N-acetyl-cysteine (NAC; a ROS scavenger). The intracellular Ca2+ responses to both CSE types were also totally prevented by NAC, AMTB (a TRPM8 antagonist), or EGTA (an extracellular Ca2+ chelator). The activation of the MAPK/NF-κB signaling and induction of IL-8 to both CSE types were suppressed to similar levels by NAC, AMTB, or EGTA. These results suggest that, in addition to ROS generated by both CSE types, the menthol in M-CSE may act as another stimulus to further activate TRPM8 and induce the observed responses. We also found that menthol combined with Non-M-CSE induced greater responses of intracellular Ca2+ and IL-8 compared with Non-M-CSE alone. Moreover, we confirmed the essential role of TRPM8 in these responses to Non-M-CSE or M-CSE and the difference in these responses between the both CSE types using HBECs with TRPM8 knockdown and TRPM8 knockout, and using HEK293 cells transfected with hTRPM8. Thus, compared with exposure to Non-M-CSE, exposure to M-CSE induced greater TRPM8-mediated inflammatory responses in HBECs. These augmented effects may be due to a double-hit on lung epithelial TRPM8 by ROS generated from CSE and the menthol in M-CSE.

16.
Front Physiol ; 8: 193, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28408888

RESUMEN

Pulmonary fibrosis is a severe and progressive disease that is characterized by an abnormal deposition of extracellular matrix, such as collagens. The pathogenesis of this disease may be initiated by oxidative damage of lung epithelial cells by fibrogenic stimuli, leading to lung inflammation, which in turn promotes various lung fibrotic responses. The profibrogenic effect of transforming growth factor-ß1 (TGF-ß1) on lung fibroblasts is crucial for the pathogenesis of this disease. Paeonol, the main phenolic compound present in the Chinese herb Paeonia suffruticosa, has antioxidant and anti-inflammatory properties. However, whether paeonol has therapeutic effects against pulmonary fibrosis remains unclear. Using a murine model, we showed that 21 days after the insult, intratracheal bleomycin caused pulmonary inflammation and fibrosis, as evidenced by lung histopathological manifestations and increase in various indices. The inflammatory indices included an increase in total cell count, differential cell count, and total protein concentration in bronchoalveolar lavage fluid. The fibrotic indices included an increase in lung levels of TGF-ß1, total collagen, type 1α1 collagen (COL1A1), and α-smooth muscle actin (α-SMA; a marker of myofibroblasts). Bleomycin also was found to cause an increase in oxidative stress as reflected by increased levels of malondialdehyde and 4-hydroxynonenal in the lungs. Importantly, all these pathophysiological events were suppressed by daily treatment with paeonol. Using human lung fibroblasts, we further demonstrated that exposure of human lung fibroblasts to TGF-ß1 increased productions of α-SMA and COL1A1, both of which were inhibited by inhibitors of Jun N-terminal kinase (JNK), p38, and Smad3. JNK and p38 are two subfamily members of mitogen-activated protein kinases (MAPKs), whereas Smad3 is a transcription factor. TGF-ß1 exposure also increased the phosphorylation of JNK, p38, and Smad3 prior to the induction of α-SMA and COL1A1. Notably, all these TGF-ß1-induced cellular events were suppressed by paeonol treatment. Our findings suggest that paeonol has antioxidant, anti-inflammatory, and anti-fibrotic functions against bleomycin-induced pulmonary fibrosis in mice. The beneficial effect of paeonol may be, at least in part, mediated through the inhibition of the MAPKs/Smad3 signaling.

17.
Sci Rep ; 7: 39720, 2017 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-28045089

RESUMEN

We study theoretically the bio-sensing capabilities of metal nanowire surface plasmons. As a specific example, we couple the nanowire to specific sites (bacteriochlorophyll) of the Fenna-Matthews-Olson (FMO) photosynthetic pigment protein complex. In this hybrid system, we find that when certain sites of the FMO complex are subject to either the suppression of inter-site transitions or are entirely disconnected from the complex, the resulting variations in the excitation transfer rates through the complex can be monitored through the corresponding changes in the scattering spectra of the incident nanowire surface plasmons. We also find that these changes can be further enhanced by changing the ratio of plasmon-site couplings. The change of the Fano lineshape in the scattering spectra further reveals that "site 5" in the FMO complex plays a distinct role from other sites. Our results provide a feasible way, using single photons, to detect mutation-induced, or bleaching-induced, local defects or modifications of the FMO complex, and allows access to both the local and global properties of the excitation transfer in such systems.


Asunto(s)
Proteínas Bacterianas/química , Técnicas Biosensibles/métodos , Complejos de Proteína Captadores de Luz/química , Resonancia por Plasmón de Superficie/métodos , Proteínas Bacterianas/genética , Transferencia de Energía , Complejos de Proteína Captadores de Luz/genética , Metales/química , Modelos Teóricos , Nanoestructuras/química
18.
Mediators Inflamm ; 2015: 148367, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26504357

RESUMEN

The mechanism underlying the inflammatory role of TRPA1 in lung epithelial cells (LECs) remains unclear. Here, we show that cigarette smoke extract (CSE) sequentially induced several events in LECs. The Ca(2+) influx was prevented by decreasing extracellular reactive oxygen species (ROS) with the scavenger N-acetyl-cysteine, removing extracellular Ca(2+) with the chelator EGTA, or treating with the TRPA1 antagonist HC030031. NADPH oxidase activation was abolished by its inhibitor apocynin, EGTA, or HC030031. The increased intracellular ROS was halted by apocynin, N-acetyl-cysteine, or HC030031. The activation of the MAPKs/NF-κB signaling was suppressed by EGTA, N-acetyl-cysteine, or HC030031. IL-8 induction was inhibited by HC030031 or TRPA1 siRNA. Additionally, chronic cigarette smoke (CS) exposure in wild-type mice induced TRPA1 expression in LECs and lung tissues. In CS-exposure trpa1 (-/-) mice, the increased BALF level of ROS was similar to that of CS-exposure wild-type mice; yet lung inflammation was lessened. Thus, in LECs, CSE may initially increase extracellular ROS, which activate TRPA1 leading to an increase in Ca(2+) influx. The increased intracellular Ca(2+) contributes to activation of NADPH oxidase, resulting in increased intracellular ROS, which activate the MAPKs/NF-κB signaling leading to IL-8 induction. This mechanism may possibly be at work in mice chronically exposed to CS.


Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Pulmón/patología , Proteínas del Tejido Nervioso/metabolismo , Humo/efectos adversos , Canales de Potencial de Receptor Transitorio/metabolismo , Acetanilidas/química , Acetofenonas/química , Animales , Biomarcadores/metabolismo , Líquido del Lavado Bronquioalveolar , Canales de Calcio/genética , Quelantes/química , Quimiocina CXCL2/metabolismo , Ácido Egtácico/química , Activación Enzimática , Regulación Neoplásica de la Expresión Génica , Humanos , Inflamación , Interleucina-8/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , NADPH Oxidasas/metabolismo , Proteínas del Tejido Nervioso/genética , Estrés Oxidativo , Purinas/química , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Canal Catiónico TRPA1 , Canales de Potencial de Receptor Transitorio/genética
19.
Appl Biochem Biotechnol ; 176(5): 1335-50, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26041055

RESUMEN

Due to its systemic arginine degradation, arginine deiminase (ADI) has attracted attentions as an anti-tumor drug. Its low activity at physiological conditions among other limitations has necessitated its engineering for improved properties. The present study describes the hydrophobic mutagenesis and semi-rational engineering of ADI from Pseudomonas plecoglossicida (PpADI). Using an improved ADI variant M13 (D38H/A128T/E296K/H404R/I410L) as parent, site saturation mutagenesis at position 162 resulted in an over 20 % increase in protein solubility. Compared with M13 (15.23 U/mg), mutants M13-2 (M13+S245D) and M13-5 (M13+R243L) exhibited enhanced specific activity of 21.19 and 31.20 U/mg at physiological conditions. M13-5 displayed enhanced substrate specificity with a dramatic reduction in its K m value (from 0.52 to 0.16 mM). It is speculated that the improvements in M13-5 could mainly be attributed to the enhanced structural stability due to an R243L substitution. The hydrophobic contribution of Leu 243 was supported by mutant M13-9 (M13+A276W) generated based on the hydrophobic mutagenesis concept. M13-9 showed a specific activity of 18.68 U/mg, as well as remarkable thermal and pH stability. It retained over 90 % activity over pH range from 4.5 to 8.5. At 60 °C, the half-life of M13-9 was enhanced from 4 to 17.5 min in comparison with M13, and its specific activity at 62 °C (93.0 U/mg) was approximately fivefold of that determined at 37 °C. Our results suggest that the increased hydrophobicity around the active regions of PpADI might be crucial in improving its structural stability and ultimately catalytic efficiency.


Asunto(s)
Biocatálisis , Hidrolasas/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Mutagénesis , Ingeniería de Proteínas/métodos , Rotación , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Hidrolasas/química , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Pseudomonas/enzimología , Alineación de Secuencia , Solubilidad , Temperatura
20.
J Cell Physiol ; 230(8): 1781-93, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25503516

RESUMEN

Inhaled cigarette smoke (CS) causes persistent lung inflammation in smokers. Interleukin 8 (IL-8) released from macrophages is a key chemokine during initiation and progression of CS-induced lung inflammation, yet its regulation is largely unknown. AMP-activated protein kinase (AMPK), a crucial energy homeostasis regulator, may modulate inflammation. Here we report that CS extract (CSE) increased the level of intracellular reactive oxygen species (ROS), activating AMPK, mitogen-activated protein kinases (MAPKs), and NF-κB, as well as inducing IL-8, in human macrophages. N-acetyl-cysteine (ROS scavenger) or hexamethonium [nicotinic acetylcholine receptor (nAChR) antagonist] attenuated the CSE-induced increase in intracellular ROS, activation of AMPK and NF-κB, as well as IL-8 induction, which suggests that nAChRs and ROS are important. Prevention of AMPK activation by compound C or AMPK siRNA reduced CSE-induced IL-8 production, confirming the role of AMPK. Compound C or AMPK siRNA also inhibited the activation of MAPKs and NF-κB by CSE, which suggests that these molecules are downstream of AMPK. Additionally, exposure of human macrophages to nicotine activated AMPK and induced IL-8 and that these effects were lessened by hexamethonium or compound C, implying that nicotine in CS may be causative. Furthermore, chronic CS exposure in mice promoted AMPK phosphorylation and expression of MIP-2 (an IL-8 homolog) in infiltrated macrophages and in lung tissues, as well as induced lung inflammation, all of which were reduced by compound C treatment. Thus, we identified a novel nAChRs-dependent, ROS-sensitive, AMPK/MAPKs/NF-κB signaling pathway, which seems to be important to CS-induced macrophage IL-8 production and possibly to lung inflammation.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Interleucina-8/biosíntesis , Macrófagos/metabolismo , Transducción de Señal/efectos de los fármacos , Humo/efectos adversos , Productos de Tabaco/efectos adversos , Animales , Western Blotting , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Inmunohistoquímica , Ratones , FN-kappa B/metabolismo , Neumonía/metabolismo , ARN Interferente Pequeño , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección
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