RESUMEN
BACKGROUND: To explore the effect and mechanism of action of miR-210 on postmenopausal osteoporosis (PMPO) in ovariectomized rats in vivo. METHODS: An ovariectomized (OVX) rat model was established by ovariectomy. Tail vein injection was performed to overexpress and knock down miR-210 in OVX rats, followed by the collection of blood and femoral tissues from each group of rats. And quantitative real-time polymerase chain reaction (qRT-PCR) was applied to assess the expression level of miR-210 in femoral tissues of each group. Micro computed tomography (Micro CT) was adopted to scan the microstructure of the femoral trabecula in each group to obtain relevant data like bone mineral density (BMD), bone mineral content (BMC), trabecular bone volume fraction (BV/TV), trabecular thickness (Tb.Th), bone surface-to-volume ratio (BS/BV), and trabecular separation (Tb.Sp). ELISA was used for determining the level of bone alkaline phosphatase (BALP), amino-terminal propeptide of type I procollagen (PINP), osteocalcin (OCN), and C-terminal telopeptide of type I collagen (CTX-1) in serum; and Western blot for the protein level of Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and collagen type I alpha 1 (COL1A1) in femoral tissues. RESULTS: MiR-210 expression was significantly decreased in femoral tissues of OVX rats. Overexpression of miR-210 could obviously increase BMD, BMC, BV/TV and Tb.Th, whereas significantly decrease BS/BV and Tb.Sp in femurs of OVX rats. Moreover, miR-210 also downregulated BALP and CTX-1 level, upregulated PINP and OCN level in the serum of OVX rats promoted the expression of osteogenesis-related markers (Runx2, OPN and COL1A1) in the femur of OVX rats. Additionally, further pathway analysis revealed that high expression of miR-210 activated the vascular endothelial growth factor (VEGF)/Notch1 signaling pathway in the femur of OVX rats. CONCLUSION: High expression of miR-210 may improve the micromorphology of bone tissue and modulate bone formation and resorption in OVX rats by activating the VEGF/Notch1 signaling pathway, thereby alleviating osteoporosis. Consequently, miR-210 can serve as a biomarker for the diagnosis and treatment of osteoporosis in postmenopausal rats.
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MicroARNs , Osteoporosis Posmenopáusica , Osteoporosis , Animales , Femenino , Ratas , Densidad Ósea , Subunidad alfa 1 del Factor de Unión al Sitio Principal/farmacología , Osteoporosis/metabolismo , Osteoporosis Posmenopáusica/diagnóstico por imagen , Osteoporosis Posmenopáusica/genética , Ovariectomía , Ratas Sprague-Dawley , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/farmacología , Microtomografía por Rayos XRESUMEN
Papillary thyroid cancer (PTC) is a common malignancy. MicroRNAs (miRNAs) may act as oncogenes or tumor suppressor genes. However, the role of miR-451a in PTC is not fully understood. Hence, the objective of the study was to research the effect and mechanism of miR-451a in PTC. Differentially expressed miRNAs between GSE113629 and GSE103996 databases were assessed by Venn diagram. miR-451a and its downstream target genes were assessed by RT-PCR and Western blot. The proliferation, invasion, and apoptosis were determined by CCK-8, EdU, transwell, and flow cytometry assays. Dual-luciferase reporter assay were used to evaluated the target of miR-451a. Xenografted tumors was used to explore the function of miR-451a in vivo. Pathological changes and related protein expression were measured by HE staining and immunohistochemistry. MiR-451a was downregulated in PTC tissues and blood, and low expression of miR-451a was related to short overall survival, serious lymph node metastasis and high TNM grade in PTC patients. Moreover, increase of miR-451a restrained the proliferation and invasion and accelerated the apoptosis. Furthermore, miR-451a repressed VEGF signaling pathway. Importantly, miR-451a was demonstrated to target DCBLD2 and AKT1. Overexpression of DCBLD2 and AKT1 could restore the effect of miR-451a on PTC cells. In addition, miR-451a reduced the growth of xenografted tumors in vivo. The data suggested that miR-451a attenuated the proliferation, invasion and promoted apoptosis in PTC cells via inhibiting DCBLD2 and AKT1.
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Carcinoma Papilar , MicroARNs , Neoplasias de la Tiroides , Humanos , Apoptosis/genética , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Proteínas de la Membrana/genética , MicroARNs/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Cáncer Papilar Tiroideo/genética , Cáncer Papilar Tiroideo/patología , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismoRESUMEN
Irreversible pulmonary fibrosis induced by paraquat (PQ) poisoning is the major cause of death in patients with PQ poisoning. The epithelial-mesenchymal transition (EMT) is postulated to be one of the main mechanisms of pulmonary fibrosis. Here, we investigated the role of miR-210 in PQ-induced EMT and its relationship with hypoxia-inducible factor-1α (HIF-1α). Western blotting, immunofluorescence, immunoprecipitation and other methods were used in this study. We found that miR-210 expression was significantly increased after PQ poisoning, and it may be regulated by HIF-1α. Overexpression of miR-210 further increased the HIF-1α protein level and promoted EMT. Moreover, miR-210 knock-down reduced the HIF-1α protein level and decreased the degree of EMT. Runt-related transcription factor-3 (RUNX3), a direct target of miR-210, was inhibited by miR-210 in response to PQ poisoning. RUNX3 increased the hydroxylation ability of prolyl hydroxylase domain-containing protein 2 (PHD2), a key enzyme that promotes HIF-1α degradation. PHD2 immunoprecipitated with RUNX3 and its level changed similarly to that of RUNX3. The expression of the HIF-1α protein was significantly reduced when RUNX3 was overexpressed. HIF-1α protein levels were markedly increased when RUNX3 was silenced. Based on these results, a positive feedback loop may exist between miR-210 and HIF-1α. The mechanism may function through miR-210-mediated repression of RUNX3, which further decreases the hydroxylation activity of PHD2, enhances the stability of HIF-1α, and promotes PQ-induced EMT, aggravating the progression of pulmonary fibrosis. This study further elucidates the mechanism of PQ-induced pulmonary fibrosis and may provide a new perspective for the future development of therapies.
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Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Transición Epitelial-Mesenquimal/efectos de los fármacos , Retroalimentación Fisiológica , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , MicroARNs/genética , Paraquat/envenenamiento , Fibrosis Pulmonar/genética , Células A549 , Animales , Línea Celular , Subunidad alfa 3 del Factor de Unión al Sitio Principal/antagonistas & inhibidores , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Regulación de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Masculino , MicroARNs/metabolismo , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Estabilidad Proteica , Proteolisis , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
Paraquat (PQ) poisoning-induced pulmonary fibrosis is one of the primary causes of death in patients with PQ poisoning. Hypoxia-inducible factor-1α (HIF-1α) and epithelial-mesenchymal transition (EMT) are involved in the progression of pulmonary fibrosis. Snail and ß-catenin are two other factors involved in promoting EMT. However, the relationship among HIF-1α, Snail and ß-catenin in PQ poisoning-induced pulmonary fibrosis is not clear. Our research aimed to determine whether the regulation of HIF-1α in EMT occurs via the Snail and ß-catenin pathways in PQ poisoning-induced pulmonary fibrosis. Sixty-six Sprague-Dawley rats were randomly and evenly divided into a control group and a PQ group. The PQ group was treated with an intragastric infusion of a 20% PQ solution (50 mg/kg) for 2, 6, 12, 24, 48 and 72 hrs. A549 and RLE-6TN cell lines were transfected with HIF-1α siRNA for 48 hrs before being exposed to PQ. Western blotting, real-time quantitative PCR, immunofluorescence, immunohistochemistry and other assays were used in our research. In vivo, the protein levels of HIF-1α and α-SMA were increased at 2 hrs and the level of ZO-1 (Zonula Occluden-1) was reduced at 12 hrs. In vitro, the transient transfection of HIF-1α siRNA resulted in a decrease in the degree of EMT. The expression levels of Snail and ß-catenin were significantly reduced when HIF-α was silenced. These data demonstrate that EMT may be involved in PQ poisoning-induced pulmonary fibrosis and regulated by HIF-1α via the Snail and ß-catenin pathways. Hypoxia-inducible factor-1α may be a therapeutic target for the treatment of PQ poisoning-induced pulmonary fibrosis.
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Transición Epitelial-Mesenquimal/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Fibrosis Pulmonar/genética , Factores de Transcripción de la Familia Snail/genética , beta Catenina/genética , Células A549 , Animales , Línea Celular , Progresión de la Enfermedad , Regulación de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Paraquat , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Factores de Transcripción de la Familia Snail/metabolismo , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismo , beta Catenina/metabolismoRESUMEN
PURPOSE: In osteoporosis, the balance between osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs) is disrupted. The osteogenic differentiation of bone marrow MSCs (BMSCs) is important for improving osteoporosis. The aim of this study was to explore the role and molecular mechanism of miR-210 in the balance of osteogenic/adipogenic differentiation of BMSCs in postmenopausal osteoporosis. METHODS: Postmenopausal osteoporosis rat models were constructed by ovariectomy (OVX). BMSCs were isolated from the femur in rats of Sham and OVX groups. MiR-210 was overexpressed and suppressed by miR-210 mimics and inhibitor, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the relative mRNA expression of miR-210, ephrin type-A receptor 2 (EPHA2), alkaline phosphatase (ALP), osterix (OSX), osteocalcin (Bglap), Runt-related transcription factor 2 (Runx2), peroxisome proliferator activated receptor gamma, and fatty acid binding protein 4 (FABP4) in each group of rat femoral tissues or BMSCs. Western blot was applied to detect the protein expression level of EPHA2 in rat femoral tissues and cells. Alizarin red S staining and oil red O staining were performed to assess the osteogenic and adipogenic differentiation of BMSCs, respectively. In addition, the targeting relationship between miR-210 and EPHA2 was verified by a dual luciferase gene reporter assay. RESULTS: The expression of miR-210 was significantly reduced in femoral tissues and BMSCs of OVX rats, and its low expression was associated with reduced bone formation. The osteogenic differentiation was enhanced in OVX rats treated with miR-210 mimic. Overexpression of miR-210 in transfected BMSCs was also found to significantly promote osteogenic differentiation and even inhibit adipogenic differentiation in BMSCs, while knockdown of miR-210 did the opposite. Further mechanistic studies showed that miR-210 could target and inhibit the expression of EPHA2 in BMSCs, thus promoting osteogenic differentiation and inhibiting adipogenic differentiation of BMSCs. CONCLUSION: MiR-210 promotes osteogenic differentiation and inhibits adipogenic differentiation of BMSCs by down-regulating EPHA2 expression. As it plays an important role in the osteogenic/adipogenic differentiation of osteoporosis, miR-210 can serve as a potential miRNA biomarker for osteoporosis.
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Células Madre Mesenquimatosas , MicroARNs , Osteoporosis Posmenopáusica , Osteoporosis , Animales , Femenino , Ratas , Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Regulación hacia Abajo , Células Madre Mesenquimatosas/metabolismo , MicroARNs/metabolismo , Osteogénesis/genética , Osteoporosis/genética , Osteoporosis Posmenopáusica/genética , Osteoporosis Posmenopáusica/metabolismo , Receptor EphA2/metabolismoRESUMEN
Lysyl oxidase (LOX) is a copper-dependent amine oxidase that plays a critical role in pulmonary fibrosis. Our previous study demonstrated that epithelial-to-mesenchymal transition (EMT) was strongly associated with paraquat (PQ) induced pulmonary fibrosis. This present study was aimed to evaluate the potential involvement of LOX on EMT in the process of pulmonary fibrosis induced by PQ. We established an in vivo rat model and an in vitro cell model induced by PQ treatment and found that LOX protein expression was significantly up-regulated and collagen deposition was enhanced in rats. The EMT process was strongly found in A549 and RLE-6TN cells after PQ exposure. After inactivating LOX with an inhibitor, pulmonary fibrosis was significantly reduced and EMT was also suppressed. Additionally, small interfering RNA (siRNA) targeting LOX was used to silence LOX expression to observe EMT in A549 cells. As a result, LOX could promote the progress of EMT, and inactivating LOX alleviated the EMT process in PQ-induced pulmonary fibrosis and mesenchymal-to-epithelial transition (MET) occurred after inactivating LOX in vitro and in vivo. In conclusion, LOX could promote the progress of EMT and inactivating LOX alleviated EMT in PQ-induced pulmonary fibrosis. Therefore, LOX could potentially be a new candidate therapeutic target for pulmonary fibrosis induced by PQ by regulating the balance between EMT and MET.
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Transición Epitelial-Mesenquimal , Proteína-Lisina 6-Oxidasa/fisiología , Fibrosis Pulmonar/enzimología , Animales , Línea Celular Tumoral , Movimiento Celular , Humanos , Masculino , Paraquat , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , Ratas Sprague-DawleyRESUMEN
Previous studies have shown that lung recruitment maneuvers are important means of treating ARDS. Although computed tomography (CT) scans and pressure-volume curves are the most common ways to evaluate lung recruitment, there are still many disadvantages. Not only do the scans have to take place in a CT room, but the patient is exposed to large doses of radiation through the multiple scans necessary to define the optimal PEEP. Pressure-volume curves require deep sedation and muscle relaxation. Thus, bedside lung ultrasound may be considered to be a safer and easier alternative to CT scans or pressure-volume curves. In our case, we evaluated the effectiveness of lung recruitment with a bedside ultrasound on a patient who was suffering from life-threatening hypoxemia. Bedside ultrasound is a faster and more convenient imaging method because it reduces the need for patient transport compared with CT scan and requires no muscle relaxation. This case supports that ultrasound may become an alternative imaging tool to guide and evaluate alveolar recruitment in patients with ARDS. Additionally, we have also included a brief review of lung recruitment evaluation by ultrasound to supplement this case study.
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Pulmón/diagnóstico por imagen , Sistemas de Atención de Punto , Atelectasia Pulmonar/diagnóstico por imagen , Síndrome de Dificultad Respiratoria/complicaciones , Ultrasonografía Intervencional , Anciano , Humanos , Masculino , Neumonía/complicaciones , Neumonía/diagnóstico , Respiración con Presión Positiva/métodos , Atelectasia Pulmonar/etiologíaRESUMEN
OBJECTIVE: To investigate the relationship between pulmonary fibrosis and endoplasmic reticulum stress (ERS) in rats with paraquat poisoning. METHODS: One hundred male Sprague-Dawley (SD) rats were randomly divided into control group (n=10) and paraquat poisoning group (n=90). The animals were sacrificed by exsanguination at 2, 6, 12, 24, 48, 72, 96, 168, 336 hours after administration with 20% parquat solution. The paraffin sections of lung tissue were stained with hematoxylin-eosin (HE) and Masson trichrome to observe the pathological changes. Glucose-regulated protein 78 (GRP78) was determined by immunohistochemistry, and malondialdehyde (MDA) of lung tissue was measured. The total protein of tissue was abstracted, and the α-smooth muscle actin (α-SMA) and GRP78 was detected by Western blotting. RESULTS: HE and Masson staining demonstrated inflammatory infiltration and collagen deposition in the lung after paraquat administration with a tendency of exaggeration with time, and finally resulted in fibrosis. The expressions of MDA, α-SMA and GRP78 in the lung tissue were significantly increased 2 hours after paraquat administration compared with those of control group (MDA: 1.38 ± 0.18 nmol/mg vs. 0.85 ± 0.05 nmol/mg, α-SMA: 0.23 ± 0.01 vs. 0.14 ± 0.03, GRP78: 0.72 ± 0.02 vs. 0.37 ± 0.06, P<0.05 or P<0.01), and the expressions of MDA and α-SMA were gradually increased with time. GRP78 protein expression was decreased at 72 hours after paraquat administration. CONCLUSIONS: The results reveal that the expressions of α-SMA and GRP78 in paraquat poisoning group are up-regulated, suggesting ERS plays an important role in paraquat induced-pulmonary fibrosis.
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Estrés del Retículo Endoplásmico , Pulmón/patología , Paraquat/envenenamiento , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Actinas/metabolismo , Animales , Proteínas de Choque Térmico/metabolismo , Pulmón/metabolismo , Masculino , Malondialdehído/metabolismo , Fibrosis Pulmonar/inducido químicamente , Ratas , Ratas Sprague-DawleyRESUMEN
It is commonly accepted that epithelial-mesenchymal transition contributes to fibrotic remodeling, but the molecular pathways involved in paraquat (PQ)-induced epithelial-mesenchymal transition remain uncharacterized. The objective of this study was to evaluate the potential involvement of HIF-1α in TGF-ß1/ß-Catenin and Snail pathway after PQ poisoning. In our study, 86 Spragne-Dawley rats were randomly divided into control group and PQ group, which received intragastric infusion of 20% PQ solution 50 mg/kg. Rats in the PQ group were subsequently divided into eight subgroups (10 for each subgroup) and samples were collected at different predetermined time points (2, 6, 12, 24, 48, 72, 96 h and 7 d). Fibrosis markers, including ß-catenin, snail and α-SMA, were measured by western blot. The activity of HIF-1α was determined by western blot and immunofluorescence. We found that in PQ-induced pulmonary fibrosis, the level of PaO2 was significantly reduced in the 6-h subgroup, when compared to the control group (P < 0.01). Interestingly, between 6 and 72 h, there was no significant difference in PaO2. On the other hand, the level of PaCO2 started to increase from 72-h subgroup (P < 0.01). Fibrosis markers including ß-catenin, snail and α-SMA, measured by western blot, were significantly increased at 2 h, while the level of p-GSK-3ß was increased at 6 h. And the level of GSK-3ß showed significant reduction beginning at 24 h. The activity of HIF-1α measured by western blot assays was significantly increased starting from 2 h with sustained expression. The result of Pearson coefficient analysis showed that HIF-1α was positively correlated with Snail (r = 0.935, P < 0.01) and ß-catenin (r = 0.761, P < 0.05). Meanwhile, immunofluorescent analysis of HIF-1α revealed partial staining appearing from 2 h. Our data illustrated a positive correlation between Snail, ß-catenin signaling and HIF-1α, suggesting a potential synergistic role of HIF-1α in PQ-induced pulmonary fibrosis, which may be independent of GSK-3ß. It might also represent a potential therapeutic window for treatment of paraquat poisoning.
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Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Fibrosis Pulmonar/metabolismo , Actinas/metabolismo , Actinas/fisiología , Animales , Dióxido de Carbono/sangre , Transición Epitelial-Mesenquimal , Oxígeno/sangre , Paraquat , Fibrosis Pulmonar/inducido químicamente , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Factores de Transcripción de la Familia Snail , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/fisiología , beta Catenina/metabolismo , beta Catenina/fisiologíaRESUMEN
BACKGROUND: Ulinastatin (UTI) is a urinary trypsin inhibitor extracted and purified from urine of males. This study aimed to explore the effects of UTI on paraquat-induced-oxidative stress in human type II alveolar epithelial cells. METHODS: The human type II alveolar epithelial cells, A549 cells, were cultured in vitro. The A549 cells were treated with different concentrations of paraquat (200, 400, 600, 800, 1 000, 1 200 µmol/L) and ulinastatin(0, 2 000, 4 000, 6 000, 8 000 U/mL) for 24 hours, the cell viability was measured by cell counting kit-8 and the median lethal concentration was selected. In order to establish an in vitro model of paraquat intoxication and to determine the safe dose of ulinastatin, we calculated LD50 using cell counting kit-8 to determine the survival rate of the cells. A549 cells were divided into normal control group, paraquat group and paraquat+ulinastatin group. The levels of malondialdehyde (MDA) and myeloperoxidase (MPO) were detected by biochemistry colorimetry, while the level of reactive oxygen spies (ROS) was detected by DCFH-DA assay. RESULTS: The survival rate of A549 cells treated with different concentrations of paraquat decreased in a concentration-dependent manner. Whereas there was no decrease in the survival rate of cells treated with 0-4 000 U/mL ulinastatin. The levels of MDA, MPO, and ROS were significantly higher in the paraquat group than in the normal control group after 24-hour-exposure. And the survival rate of the paraquat+ulinastatin group was higher than that of the paraquat group, but lower than that of the normal control group. The levels of MDA, MPO, and ROS were lower than those of the paraquat group. CONCLUSION: Ulinastatin can alleviate the paraquat-induced A549 cell damage by reducing oxidative stress.