RESUMEN
BACKGROUND: Pulmonary fibrosis is a progressive diffuse parenchymal lung disorder with a high mortality rate. Studies have indicated that injured lung tissues release various pro-inflammatory factors, and produce a large amount of nitric oxide. There is also accumulation of collagen and oxidative stress-induced injury, collectively leading to pulmonary fibrosis. Antrodia cinnamomea is an endemic fungal growth in Taiwan, and its fermented extracts exert anti-inflammatory effects to alleviate liver damages. Hence, we hypothesized and tested the feasibility of using A. cinnamomea extracts for treatment of pulmonary fibrosis. METHODS: The TGF-ß1-induced human lung fibroblast cells (MRC-5) in vitro cell assay were used to evaluate the effects of A. cinnamomea extracts on the collagen production in MRC-5. Eight-week-old ICR mice were intratracheally administered bleomycin and then fed with an A. cinnamomea extract on day 3 post-administration of bleomycin. At day 21 post-bleomycin administration, the pulmonary functional test, the expression level of inflammation- and fibrosis-related genes in the lung tissue, and the histopathological change were examined. RESULTS: The A. cinnamomea extract significantly attenuated the expression level of collagen in the TGF-ß1-induced MRC-5â¯cells. In the A. cinnamome-treated bleomycin-induced lung fibrotic mice, the bodyweight increased, pulmonary functions improved, the lung tissues expression level of inflammatory factor and the fibrotic indicator were decreased, and the histopathological results showed the reduction of thickening of the inter-alveolar septa. CONCLUSIONS: The Antrodia cinnamomea extract significant protects mice against bleomycin-induced lung injuries through improvement of body weight gain and lung functions, and attenuation of expression of inflammatory and fibrotic indicators.
RESUMEN
BACKGROUND: The development of alcohol-associated liver disease (ALD) is influenced by the amount and duration of alcohol consumption. The resulting liver damage can range from reversible stages, such as steatosis, steatohepatitis and alcoholic fibrosis, to the advanced and irreversible stage of cirrhosis. Aldo-keto reductase family 1 member A1 (AKR1A1) is a member of the aldo-keto reductase family that catalyzes the reduction of aldehyde groups to their corresponding alcohols in an NADPH-dependent manner. AKR1A1 was found to be downregulated in patients diagnosed with ALD. This study aims to interpret the protective effects of AKR1A1 on the development of ALD. METHODS: A 5% alcohol-fed (AF) Akr1a1 knockout (Akr1a1-/-) mouse model and an AML12 hepatocyte model were used. The effects of AKR1A1 on liver function, inflammation, oxidative stress, lipid accumulation, and fibrosis were assessed by ELISA, western blotting, RTâPCR, and a variety of histological staining methods in AF-induced wild-type (WT) and Akr1a1-/- mice compared to control liquid diet-fed (PF) WT and Akr1a1-/- mice. RESULTS: The results demonstrated that AF-WT mice expressed higher levels of AKR1A1 than WT mice fed a control diet, and they did not show any noticeable liver steatosis. However, AF-Akr1a1-/- mice displayed a lower survival rate and more severe liver injury than AF-WT mice, as demonstrated by increased proinflammatory cytokines, oxidative stress, lipid accumulation, fibrosis, and reduced antioxidant enzymes in their livers. Additionally, elevated levels of 4-HNE and p53 phosphorylation were observed in AF-Akr1a1-/- mice, suggesting that the loss of AKR1A1 led to increased 4-HNE accumulation and subsequent activation of p53, which contributed to the progression of ALD. Furthermore, in AML12 hepatocytes, Akr1a1 knockdown aggravated oxidative stress and steatosis induced by palmitic acid/oleic acid (P/O) inflammation induced by lipopolysaccharide (LPS), and fibrosis induced by TGF-ß1. CONCLUSIONS: This loss-of-function study suggests that AKR1A1 plays a liver-protective role during chronic alcohol consumption by reducing the accumulation of 4-HNE and inhibiting 4-HNE-mediated p53 activation.
RESUMEN
Lactoferrin (LF) stands as one of the extensively investigated iron-binding glycoproteins within milk, exhibiting diverse biological functionalities. The global demand for LF has experienced consistent growth. Biotechnological strategies aimed at enhancing LF productivity through microbial expression systems offer substantial cost-effective advantages and exhibit fewer constraints compared to traditional animal bioreactor technologies. This study devised a novel recombinant plasmid, wherein the AOX1 promoter was replaced with a glucose-inducible G1 promoter (PG1) to govern the expression of recombinant porcine LF (rpLF) in Pichia pastoris GS115. High-copy-number PG1-rpLF yeast clones were meticulously selected, and subsequent induction with 0.05 g/L glucose demonstrated robust secretion of rpLF. Scaling up production transpired in a 5 L fermenter, yielding an estimated rpLF productivity of approximately 2.8 g/L by the conclusion of glycerol-fed fermentation. A three-step purification process involving tangential-flow ultrafiltration yielded approximately 6.55 g of rpLF crude (approximately 85% purity). Notably, exceptional purity of rpLF was achieved through sequential heparin and size-exclusion column purification. Comparatively, the present glucose-inducible system outperformed our previous methanol-induced system, which yielded a level of 87 mg/L of extracellular rpLF secretion. Furthermore, yeast-produced rpLF demonstrated affinity for ferric ions (Fe3+) and exhibited growth inhibition against various pathogenic microbes (E. coli, S. aureus, and C. albicans) and human cancer cells (A549, MDA-MB-231, and Hep3B), similar to commercial bovine LF (bLF). Intriguingly, the hydrolysate of rpLF (rpLFH) manifested heightened antimicrobial and anticancer effects compared to its intact form. In conclusion, this study presents an efficient glucose-inducible yeast expression system for large-scale production and purification of active rpLF protein with the potential for veterinary or medical applications.
Asunto(s)
Antiinfecciosos , Lactoferrina , Proteínas Recombinantes , Animales , Bovinos , Humanos , Antiinfecciosos/farmacología , Escherichia coli/metabolismo , Fermentación , Glucosa/metabolismo , Lactoferrina/biosíntesis , Lactoferrina/genética , Lactoferrina/farmacología , Pichia/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Saccharomycetales , Staphylococcus aureus/efectos de los fármacos , PorcinosRESUMEN
MicroRNA (miRNA) 107 expression is downregulated but Wnt3a protein and ß-catenin are upregulated in degenerated intervertebral disc (IVD). We investigated mir-107/Wnt3a-ß-catenin signaling in vitro and in vivo following hyperbaric oxygen (HBO) intervention. Our results showed 96 miRNAs were upregulated and 66 downregulated in degenerated nucleus pulposus cells (NPCs) following HBO treatment. The 3' untranslated region (UTR) of the Wnt3a mRNA contained the "seed-matched-sequence" for miR-107. MiR-107 was upregulated and a marked suppression of Wnt3a was observed simultaneously in degenerated NPCs following HBO intervention. Knockdown of miR-107 upregulated Wnt3a expression in hyperoxic cells. HBO downregulated the protein expression of Wnt3a, phosphorylated LRP6, and cyclin D1. There was decreased TOP flash activity following HBO intervention, whereas the FOP flash activity was not affected. HBO decreased the nuclear translocation of ß-catenin and decreased the secretion of MMP-3 and -9 in degenerated NPCs. Moreover, rabbit serum KS levels and the stained area for Wnt3a and ß-catenin in repaired cartilage tended to be lower in the HBO group. We observed that HBO inhibits Wnt3a/ß-catenin signaling-related pathways by upregulating miR-107 expression in degenerated NPCs. HBO may play a protective role against IVD degeneration and could be used as a future therapeutic treatment.
Asunto(s)
Oxigenoterapia Hiperbárica , MicroARNs , Núcleo Pulposo , Animales , Conejos , beta Catenina , Oxígeno , Modelos Animales , Regiones no Traducidas 3' , MicroARNs/genéticaRESUMEN
E7050 is an inhibitor of VEGFR2 with anti-tumor activity; however, its therapeutic mechanism remains incompletely understood. In the present study, we aim to evaluate the anti-angiogenic activity of E7050 in vitro and in vivo and define the underlying molecular mechanism. It was observed that treatment with E7050 markedly inhibited proliferation, migration, and capillary-like tube formation in cultured human umbilical vein endothelial cells (HUVECs). E7050 exposure in the chick embryo chorioallantoic membrane (CAM) also reduced the amount of neovessel formation in chick embryos. To understand the molecular basis, E7050 was found to suppress the phosphorylation of VEGFR2 and its downstream signaling pathway components, including PLCγ1, FAK, Src, Akt, JNK, and p38 MAPK in VEGF-stimulated HUVECs. Moreover, E7050 suppressed the phosphorylation of VEGFR2, FAK, Src, Akt, JNK, and p38 MAPK in HUVECs exposed to MES-SA/Dx5 cells-derived conditioned medium (CM). The multidrug-resistant human uterine sarcoma xenograft study revealed that E7050 significantly attenuated the growth of MES-SA/Dx5 tumor xenografts, which was associated with inhibition of tumor angiogenesis. E7050 treatment also decreased the expression of CD31 and p-VEGFR2 in MES-SA/Dx5 tumor tissue sections in comparison with the vehicle control. Collectively, E7050 may serve as a potential agent for the treatment of cancer and angiogenesis-related disorders.
Asunto(s)
Sarcoma , Transducción de Señal , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Animales , Embrión de Pollo , Humanos , Inhibidores de la Angiogénesis/uso terapéutico , Movimiento Celular , Proliferación Celular , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sarcoma/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
E7050 is a potent inhibitor of c-Met receptor tyrosine kinase and has potential for cancer therapy. However, the underlying molecular mechanism involved in the anti-cancer property of E7050 has not been fully elucidated. The main objective of this study was to investigate the anti-tumor activity of E7050 in multidrug-resistant human uterine sarcoma MES-SA/Dx5 cells in vitro and in vivo, and to define its mechanisms. Our results revealed that E7050 reduced cell viability of MES-SA/Dx5 cells, which was associated with the induction of apoptosis and S phase cell cycle arrest. Additionally, E7050 treatment significantly upregulated the expression of Bax, cleaved PARP, cleaved caspase-3, p21, p53 and cyclin D1, while it downregulated the expression of survivin and cyclin A. On the other hand, the mechanistic study demonstrated that E7050 inhibited the phosphorylation of c-Met, Src, Akt and p38 in HGF-stimulated MES-SA/Dx5 cells. Further in vivo experiments showed that treatment of athymic nude mice carrying MES-SA/Dx5 xenograft tumors with E7050 remarkably suppressed tumor growth. E7050 treatment also decreased the expression of Ki-67 and p-Met, and increased the expression of cleaved caspase-3 in MES-SA/Dx5 tumor sections. Therefore, E7050 is a promising drug that can be developed for the treatment of multidrug-resistant uterine sarcoma.
Asunto(s)
Sarcoma , Neoplasias de los Tejidos Blandos , Neoplasias Uterinas , Ratones , Femenino , Animales , Humanos , Proteínas Proto-Oncogénicas c-met/metabolismo , Caspasa 3/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos , Ratones Desnudos , Apoptosis , Sarcoma/metabolismo , Neoplasias Uterinas/patología , Transducción de SeñalRESUMEN
2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-Glucoside (THSG) is the main active ingredient extracted from Polygonum multiflorum Thunb. (PMT), which has been reported to possess extensive pharmacological properties. Nevertheless, the exact role of THSG in pulmonary fibrosis has not been demonstrated yet. The main purpose of this study was to investigate the protective effect of THSG against bleomycin (BLM)-induced lung fibrosis in a murine model, and explore the underlying mechanisms of THSG in transforming growth factor-beta 1 (TGF-ß1)-induced fibrogenesis using MRC-5 human lung fibroblast cells. We found that THSG significantly attenuated lung injury by reducing fibrosis and extracellular matrix deposition. THSG treatment significantly downregulated the expression levels of TGF-ß1, fibronectin, α-SMA, CTGF, and TGFBR2, however, upregulated the expression levels of antioxidants (SOD-1 and catalase) and LC3B in the lungs of BLM-treated mice. THSG treatment decreased the expression levels of fibronectin, α-SMA, and CTGF in TGF-ß1-stimulated MRC-5 cells. Conversely, THSG increased the expression levels of SOD-1 and catalase. Furthermore, treatment of THSG profoundly reduced the TGF-ß1-induced generation of reactive oxygen species (ROS). In addition, THSG restored TGF-ß1-induced impaired autophagy, accompany by increasing the protein levels of LC3B-II and Beclin 1. Mechanism study indicated that THSG significantly reduced TGF-ß1-induced increase of TGFBR2 expression and phosphorylation of Smad2/3, Akt, mTOR, and ERK1/2 in MRC-5 cells. These findings suggest that THSG may be considered as an anti-fibrotic drug for the treatment of pulmonary fibrosis.
RESUMEN
Platelet concentrates (PCs) are widely used in regenerative medicine; as it is produced from freeze-thawing PC, platelet lysate (PL) has a longer shelf life. The thrombotic risk of PL therapy needs to be explored since PL and PC contain cytokines that contribute to platelet aggregation and thrombus formation. Whole blood samples of 20 healthy subjects were collected; PL was produced from PCs with expired shelf life through freeze-thawing. The direct mixing of PL with platelet-rich plasma (PRP) or whole blood was performed. In addition, rotational thromboelastometry (ROTEM) was used to investigate whether PL enhanced coagulation in vitro; the effects of fibrinogen depletion and anticoagulants were evaluated to prevent hypercoagulation. The results showed that PL induced platelet aggregation in both PRP and whole blood. In ROTEM assays, PL was shown to cause a significantly lower clotting onset time (COT) and clot formation time (CFT), and a significantly greater α angle and maximum clot firmness (MCF). Compared with the controls, which were 1:1 mixtures of normal saline and whole blood, fibrinogen depletion of PL showed no significant difference in CFT, α angle and MCF. Moreover, heparin- and rivaroxaban-added PL groups demonstrated no clot formation in ROTEM assays. Platelet lysate-induced hypercoagulability was demonstrated in vitro in the present study, which could be prevented by fibrinogen depletion or the addition of an anticoagulant.
RESUMEN
End-stage renal disease (ESRD) patients experience oxidative stress due to excess exogenous or endogenous oxidants and insufficient antioxidants. Hence, oxidative stress and inflammation cause endothelial damage, contributing to vascular dysfunction and atherosclerosis. Therefore, ESRD patients suffer more cardiovascular and hospitalization events than healthy people. This study aims to test the correlations between ROS, SOD3, IL-2, IL-6, and IL-18 and the first kidney disease-related hospitalization or death events in ESRD patients undergoing regular hemodialysis. A total of 212 participants was enrolled, including 45 normal healthy adults and 167 ESRD patients on regular dialysis. Blood samples from all participants were collected for ROS, SOD3, IL-2, IL-6, and IL-18 measurement at the beginning of the study, and every kidney disease-related admission or death was recorded for the next year. Multivariate analysis was conducted by fitting a linear regression model, logistic regression model, and Cox proportional hazards model to estimate the adjusted effects of risk factors, prognostic factors, or predictors on continuous, binary, and survival outcome data. The results showed that plasma SOD3 and serum IL-18 were two strong predictors of the first kidney disease-related hospitalization or death. In the Cox proportional hazards models (run in R), higher IL-18 concentration (>69.054 pg/mL) was associated with a hazard ratio of 3.376 for the first kidney disease-related hospitalization or death (95% CI: 1.2644 to 9.012), while log(SOD3) < 4.723 and dialysis clearance (Kt/V; 1.11 < value < 1.869) had a hazard ratio = 0.2730 (95% CI: 0.1133 to 0.6576) for reducing future kidney disease-related hospitalization or death. Other markers, including body mass index (BMI), transferrin saturation, total iron binding capacity, and sodium and alkaline phosphate, were also found to be significant in our study. These results reveal the new predictors SOD3 and IL-18 for the medical care of end-stage renal disease patients.
RESUMEN
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a malignant cancer and chemotherapy ineffectively treats PDAC, leading to the requirement for alternative tumor-targeted treatment. Human amniotic fluid mesenchymal stem cells (hAFMSCs) have been revealed to suppress tumor growth in various cancers and they are a strong candidate for treating PDAC. METHODS: To evaluate the effects of hAFMSCs on human pancreatic carcinoma cells (PANC1, AsPC1 and BxPC3 cell lines) and the possible mechanism involved, an in vitro cell coculture system was used. A PANC1 orthotopic xenograft mouse model was established and hAFMSCs were injected intravenously at 4 weeks post-xenograft. RESULTS: An in vitro coculture assay showed that hAFMSCs inhibited PANC1 cell proliferation by inducing S phase cell cycle arrest and increased cell apoptosis in a time-dependent manner. In PANC1 cells, hAFMSCs caused the downregulation of Cyclin A and Cyclin B1 as well as the upregulation of p21 (CDKN1A) at 24 h post coculture. The upregulation of pro-apoptotic factors Caspase-3/-8 and Bax at 24 h post coculture reduced the migration and invasion ability of PANC1 cells through inhibiting the epithelial-mesenchymal transition (EMT) process. In a PANC1 orthotopic xenograft mouse model, a single injection of hAFMSCs showed significant tumor growth inhibition with evidence of the modulation of cell cycle and pro-apoptotic regulatory genes and various genes involved in matrix metallopeptidase 7 (MMP7) signaling-triggered EMT process. Histopathological staining showed lower Ki67 levels in tumors from hAFMSCs-treated mice. CONCLUSIONS: Our data demonstrated that hAFMSCs strongly inhibit PDAC cell proliferation, tumor growth and invasion, possibly by altering cell cycle arrest and MMP7 signaling-triggered EMT.
Asunto(s)
Carcinoma Ductal Pancreático , Células Madre Mesenquimatosas , Neoplasias Pancreáticas , Líquido Amniótico , Animales , Apoptosis , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Metaloproteinasa 7 de la Matriz/genética , Metaloproteinasa 7 de la Matriz/metabolismo , Metaloproteinasa 7 de la Matriz/farmacología , Células Madre Mesenquimatosas/metabolismo , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/terapia , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias PancreáticasRESUMEN
Arthritis is a disorder that is characterized by joint inflammation and other symptoms. Rheumatoid arthritis (RA), an autoimmune disease, is one of the most common arthritis in worldwide. Inflammation of the synovium is the main factor that triggers bone erosion in the joints in RA, but the pathogenesis of RA is not clearly understood. Kefir grain-fermented products have been demonstrated to enhance immune function and exhibit immune-modulating bioactivities. This study aims to explore the role of kefir peptides (KPs) on the regulation of dendritic cell, which are found in RA synovial fluid, and the protection effects of KPs on mice with collagen-induced arthritis (CIA). Immature mouse bone marrow-derived dendritic cells (BMDCs) were treated with KPs (2.2 and 4.4 mg/ml) and then exposed to lipopolysaccharide (LPS) to study the immune regulation function of KPs in dendritic cells. Mice with CIA (n = 5 per group) were orally administrated KPs (3.75 and 7.5 mg/day/kg) for 21 days and therapeutic effect of KPs on mice with arthritis were assessed. In this study, we found that KPs could inhibit surface molecule expression, reduce inflammatory cytokine release, and repress NF-κB and MAPK signaling in LPS-stimulated mouse BMDCs. In addition, a high dose of KPs (7.5 mg/kg) significantly alleviated arthritis symptoms, decreased inflammatory cytokine expression, suppressed splenic DC maturation and decrease the percentage of Th1 and Th17 in the spleens on mice with CIA. Our findings demonstrated that KPs ameliorate CIA in mice through the mechanism of suppressing DC maturation and inflammatory cytokine releases.
RESUMEN
Lung cancer remains a challenge in daily practice. Chemotherapy is first considered for advanced lung adenocarcinoma bearing no active driver mutations. Maintaining drug efficacy and overcoming drug resistance are essential. This study aimed to explore the real-world use of anti-diabetic agent metformin in combination with pemetrexed-based platinum doublets in a first-line setting. We retrospectively collected data during 2004~2013 from TaiwaN's National Health Insurance Research Database to access the survival benefit of metformin combined with pemetrexed-based platinum doublets as a first-line therapy for diabetic patients with advanced lung adenocarcinoma. Demographic data and information regarding platinum reagents, diabetes medications, and metformin doses were gathered, and overall survival status regarding metformin use was analyzed. Overall survival status based on the daily dose and the calculated cumulative defined daily dose (DDD) of metformin prescribed during the first 3 months after lung cancer was diagnosed was also assessed. A total of 495 patients were enrolled with a mean age of 67 years old, and the majority of the patients were male. After adjusting for age, sex, diabetes medication, and platinum reagents used, the adjusted hazard ratio (HR) for the metformin-user group was 0.61 (95% confidence interval (CI); 0.46~0.79; p < 0.001). The metformin-user group had a survival benefit (log-rank p < 0.001). We analyzed metformin dosing during the first 3 months after lung cancer diagnosis, and for a daily dose ≥ 1500 mg, the adjusted hazard ratio (aHR) was 0.42 (95% CI; 0.27~0.65; p < 0.001). Regarding the cumulative DDD of metformin, a DDD equal to or exceeding 21 resulted in aHR of 0.48 (95% CI; 0.34~0.69; p < 0.001). In this study, we found that the combination of metformin and pemetrexed-based platinum doublets provides a robust survival benefit as a first-line therapy for diabetic patients with advanced lung adenocarcinoma. It is worth conducting a large and randomized clinical trial to further investigate the antitumor effects of metformin on advanced lung adenocarcinoma when used as a first-ling therapy, including in non-diabetic patients.
Asunto(s)
Adenocarcinoma del Pulmón/tratamiento farmacológico , Complicaciones de la Diabetes/tratamiento farmacológico , Diabetes Mellitus/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Metformina/administración & dosificación , Pemetrexed/administración & dosificación , Platino (Metal)/administración & dosificación , Adenocarcinoma del Pulmón/complicaciones , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica , Supervivencia sin Enfermedad , Quimioterapia Combinada , Femenino , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/complicaciones , Masculino , Persona de Mediana Edad , Mutación , Probabilidad , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Taiwán , Resultado del TratamientoRESUMEN
Osteoporosis is a rising health threat in the increasingly aging world population. It is a common skeletal disease strongly linked to genetic predisposition. We aim to identify the effects of the anti-inflammatory TGF-ß1- and IL-10-specific single-nucleotide polymorphism (SNP) combination on the risk for osteoporosis. We investigated and analyzed the relationships between three TGF-ß1 SNPs (-509C/T, +869 T/C and +29T/C), one IL-10 SNP (+1927A/C) and the level of bone mineral density (BMD), as well as the risk of osteoporosis in Taiwanese osteoporotic patients. A total of 217 subjects were recruited, including 88 osteoporotic patients and 129 healthy controls, for SNPs, BMD and clinical characteristics statistical analyses. Females with TGF-ß1 SNP (-509 C/C) and IL-10 SNP (+1927 C/C) genotypes showed a great benefit for femoral neck T-scores. However, the combination of TGF-ß1 SNP (-509 T/T) and IL-10 SNP (+1927 A/A) genotypes in all subjects showed a significant decrease in total hip BMD T-scores. The TGF-ß1 SNP (-509 C/T) genotype in all subjects and TGF-ß1 SNP (-509 T/T) and IL-10 SNP (+1927 A/C) genotypes in males showed positive effects on body height. The combination of the many SNPs in the anti-inflammatory TGF-ß1 and IL-10 genes may be cooperatively involved in the development of osteoporosis. Our data suggested that the specific SNP combination of TGF-ß1 (-509) and IL-10 (+1927) may act as a predictive factor for postmenopausal osteoporosis in Taiwanese women.
Asunto(s)
Interleucina-10/genética , Osteoporosis/genética , Polimorfismo de Nucleótido Simple , Factor de Crecimiento Transformador beta1/genética , Anciano , Anciano de 80 o más Años , Densidad Ósea , Femenino , Humanos , Masculino , Persona de Mediana Edad , TaiwánRESUMEN
Hypoxia is a nonphysiological oxygen tension which is common in most malignant tumors. Hypoxia stimulates complicated cell signaling networks in cancer cells, e.g., the HIF, PI3K, MAPK, and NFκB pathways. Then, cells release a number of cytokines such as VEGFA to promote the growth of peripheral blood vessels and lead to metastasis. In the current work, understanding of the internal hypoxic environment in solid tumor tissue was attempted by developing a folding paper system. A paper-based solid tumor was constructed by folding a filter paper cultured with cancer cells. The cellular response in each layer could be analyzed by disassembling the folded paper after the culture course. The result showed that an internal hypoxic environment was successfully reproduced in the paper-based solid tumor. The cells in the inner layer expressed high levels of HIF1-α and VEGFA. Hence, proliferation and migration of endothelial cells were shown to be induced by the cells located in the internal hypoxic environment. Moreover, the paper-based solid tumor was transplanted into nude mice for the study of hypoxic response and angiogenesis. The crosstalk between internal and external parts of solid tumor tissue could be analyzed by sectioning each layer of the paper-based solid tumor. This approach provides a favorable analytical method for the discovery of the interaction between cancer cells, hypoxia, and peripheral angiogenesis.
Asunto(s)
Xenoinjertos , Hipoxia/fisiopatología , Neoplasias/fisiopatología , Papel , Animales , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , Neovascularización Patológica/fisiopatología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
A co-culture model of mesenchymal stem cells (MSCs) and fibroblasts is an efficient and rapid method to evaluate the anti-fibrotic effects of MSCs-based cell therapy. Transforming growth factor (TGF)-ß1 plays a key role in promotion of fibroblast activation and differentiation which can induce collagen deposition, increase ECM production in lung tissue, eventually resulted in pulmonary fibrosis. Here, we use this co-culture system and examine the ECM production in activated fibroblasts by western blot and quantitative real-time analysis to understand the therapeutic effects of MSCs.
Asunto(s)
Fibroblastos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Fibrosis Pulmonar/metabolismo , Factor de Crecimiento Transformador beta1/efectos adversos , Animales , Línea Celular , Técnicas de Cocultivo , Fibroblastos/patología , Células Madre Mesenquimatosas/patología , Ratones , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (-1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (-1369~+28 nt), Δ2-pGzmg (-939~+28 nt), Δ3-pGzmg (-711~+28 nt) and Δ4-pGzmg (-417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the -417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.
Asunto(s)
Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica/genética , Granzimas/genética , Factor de Transcripción STAT3/genética , Animales , Blastocisto/fisiología , Núcleo Celular/genética , Femenino , Proteínas Fluorescentes Verdes/genética , Masculino , Ratones , Ratones Endogámicos ICR , Embarazo , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética , Activación Transcripcional/genética , Cigoto/fisiologíaRESUMEN
BACKGROUND: MicroRNA (miRNA) plays a vital role in the intervertebral disc (IVD) degeneration. The expression level of miR-573 was downregulated whereas Bax was upregulated notably in human degenerative nucleus pulposus cells. In this study, we aimed to investigate the role of miR-573 in human degenerative nucleus pulposus (NP) cells following hyperbaric oxygen (HBO) treatment. METHODS: NP cells were separated from human degenerated IVD tissues. The control cells were maintained in 5% CO2/95% air and the hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute. MiRNA expression profiling was performed via microarray and confirmed by real-time PCR, and miRNA target genes were identified using bioinformatics and luciferase reporter assays. The mRNA and protein levels of Bax were measured. The proliferation of NPCs was detected using MTT assay. The protein expression levels of Bax, cleaved caspase 9, cleaved caspase 3, pro-caspase 9, and pro-caspase 3 were examined. RESULTS: Bioinformatics analysis indicated that the 3' untranslated region (UTR) of the Bax mRNA contained the "seed-matched-sequence" for hsa-miR-573, which was validated via reporter assays. MiR-573 was induced by HBO and simultaneous suppression of Bax was observed in NP cells. Knockdown of miR-573 resulted in upregulation of Bax expression in HBO-treated cells. In addition, overexpression of miR-573 by HBO increased cell proliferation and coupled with inhibition of cell apoptosis. The cleavage of pro-caspase 9 and pro-caspase 3 was suppressed while the levels of cleaved caspase 9 and caspase 3 were decreased in HBO-treated cells. Transfection with anti-miR-573 partly suppressed the effects of HBO. CONCLUSION: Mir-573 regulates cell proliferation and apoptosis by targeting Bax in human degenerative NP cells following HBO treatment.
Asunto(s)
Apoptosis/genética , Proliferación Celular/genética , Oxigenoterapia Hiperbárica , MicroARNs/fisiología , Núcleo Pulposo/citología , Proteína X Asociada a bcl-2/metabolismo , Anciano , Células Cultivadas , Femenino , Expresión Génica/genética , Humanos , Degeneración del Disco Intervertebral/metabolismo , Degeneración del Disco Intervertebral/patología , Masculino , Persona de Mediana Edad , Núcleo Pulposo/metabolismo , Proteína X Asociada a bcl-2/genéticaRESUMEN
PURPOSE: High levels of oxygen are usually used in ventilatory support and extracorporeal membrane oxygenation (ECMO) in the intensive care unit of hospitals. Hyperoxia may induce the production of reactive oxygen species (ROS) that can cause lung damage and even systemic injury. In this study, the NF-κB/luciferase transgenic mouse model with non-invasive real-time in vivo imaging was established to test the functions of lactoferrin (LF) in antioxidant and anti-inflammation. PROCEDURES: The NF-κB/luciferase transgenic mice were used to assess the effects of oral administration of LF on attenuation of the systemic inflammatory response and organ damage after 72 h of hyperoxia (FiO2 > 95 %) exposure via monitoring using an in vivo imaging system (IVIS). RESULTS: Using luciferase IVIS imaging, we found that the lungs and kidneys were the most evidently affected organs after hyperoxia treatment. The groups treated with low dose (150 mg/kg) or high dose (300 mg/kg) of LF had lower luciferase expression and less injury, with a dose-dependent effect on the lungs and kidneys. Moreover, ROS, mitogen-activated protein kinases (MAPK), and pro-inflammatory cytokine (TNF-α, IL-1ß, and IL-6) expression levels were all significantly decreased (P < 0.01), and the protein level of IκB was statistically increased (P < 0.01) after LF treatment. CONCLUSIONS: Our results suggest that hyperoxia can induce systemic inflammation, and the oral administration of LF as a natural antioxidant decreases the production of ROS, attenuates inflammation, and lessens kidney and lung injuries from hyperoxia via the use of live image monitoring of the response in NF-kB/luciferase transgenic mice.
Asunto(s)
Hiperoxia/metabolismo , Enfermedades Renales/prevención & control , Lactoferrina/farmacología , Luciferasas/metabolismo , FN-kappa B/metabolismo , Neumonía/prevención & control , Animales , Antiinfecciosos/farmacología , Modelos Animales de Enfermedad , Femenino , Enfermedades Renales/etiología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Masculino , Ratones , Ratones Transgénicos , FN-kappa B/genética , Consumo de Oxígeno , Neumonía/metabolismo , Neumonía/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Kefir peptides, generated by kefir grain fermentation of milk proteins, showed positive antioxidant effects, lowered blood pressure and modulated the immune response. In this study, kefir peptide was evaluated regarding their anti-inflammatory effects on particulate matter <4 µm (PM4.0)-induced lung inflammation in NF-κB-luciferase+/+ transgenic mice. The lungs of mice under 20 mg/kg or 10 mg/kg PM4.0 treatments, both increased significantly the generation of reactive oxygen species (ROS) and inflammatory cytokines; increased the protein expression levels of p-NF-κB, NLRP3, caspase-1, IL-1ß, TNF-α, IL-6, IL-4 and α-SMA. Thus, we choose the 10 mg/kg of PM4.0 for animal trials; the mice were assigned to four treatment groups, including control group (saline treatment), PM4.0 + Mock group (only PM4.0 administration), PM4.0 + KL group (PM4.0 + 150 mg/kg low-dose kefir peptide) and PM4.0 + KH group (PM4.0 + 500 mg/kg high-dose kefir peptide). Data showed that treatment with both doses of kefir peptides decreased the PM4.0-induced inflammatory cell infiltration and the expression of the inflammatory mediators IL-lß, IL-4 and TNF-α in lung tissue by inactivating NF-κB signaling. The oral administrations of kefir peptides decrease the PM4.0-induced lung inflammation process through the inhibition of NF-κB pathway in transgenic luciferase mice, proposing a new clinical application to particulate matter air pollution-induced pulmonary inflammation.
Asunto(s)
Kéfir , Material Particulado/efectos adversos , Péptidos/farmacología , Neumonía/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Humanos , Interleucina-1beta/genética , Interleucina-4/genética , Pulmón/efectos de los fármacos , Pulmón/patología , Ratones , Ratones Transgénicos , FN-kappa B/genética , Péptidos/química , Neumonía/inducido químicamente , Neumonía/genética , Neumonía/patología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
INTRODUCTION: Pulmonary emphysema is a major component of chronic obstructive pulmonary disease (COPD). Emphysema progression attributed not only to alveolar structure loss and pulmonary regeneration impairment, but also to excessive inflammatory response, proteolytic and anti-proteolytic activity imbalance, lung epithelial cells apoptosis, and abnormal lung remodeling. To ameliorate lung damage with higher efficiency in lung tissue engineering and cell therapy, pre-differentiating graft cells into more restricted cell types before transplantation could enhance their ability to anatomically and functionally integrate into damaged lung. In this study, we aimed to evaluate the regenerative and repair ability of lung alveolar epithelium in emphysema model by using lung epithelial progenitors which pre-differentiated from amniotic fluid mesenchymal stem cells (AFMSCs). METHODS: Pre-differentiation of eGFP-expressing AFMSCs to lung epithelial progenitor-like cells (LEPLCs) was established under a modified small airway growth media (mSAGM) for 7-day induction. Pre-differentiated AFMSCs were intratracheally injected into porcine pancreatic elastase (PPE)-induced emphysema mice at day 14, and then inflammatory-, fibrotic-, and emphysema-related indices and pathological changes were assessed at 6 weeks after PPE administration. RESULTS: An optimal LEPLCs pre-differentiation condition has been achieved, which resulted in a yield of approximately 20% lung epithelial progenitors-like cells from AFMSCs in a 7-day period. In PPE-induced emphysema mice, transplantation of LEPLCs significantly improved regeneration of lung tissues through integrating into the lung alveolar structure, relieved airway inflammation, increased expression of growth factors such as vascular endothelial growth factor (VEGF), and reduced matrix metalloproteinases and lung remodeling factors when compared with mice injected with AFMSCs. Histopathologic examination observed a significant amelioration in DNA damage in alveolar cells, detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL), the mean linear intercept, and the collagen deposition in the LEPLC-transplanted groups. CONCLUSION: Transplantation of predifferentiated AFMSCs through intratracheal injection showed better alveolar regeneration and reverse elastase-induced pulmonary emphysema in PPE-induced pulmonary emphysema mice.