RESUMEN
Alginate oligosaccharides (AOS), products of alginate degradation by endotype alginate lyases, possess favorable biological activities and have broad applications. Although many have been reported, alginate lyases with homogeneous AOS products and secretory production by an engineered host are scarce. Herein, the alginate lyase AlyC7 from Vibrio sp. C42 was characterized as a trisaccharide-producing lyase exhibiting high activity and broad substrate specificity. With PelB as the signal peptide and 500 mM glycine as the additive, the extracellular production of AlyC7 in Escherichia coli reached 1122.8 U/mL after 27 h cultivation in Luria-Bertani medium. The yield of trisaccharides from sodium alginate degradation by the produced AlyC7 reached 758.6 mg/g, with a purity of 85.1%. The prepared AOS at 20 µg/mL increased the root length of lettuce, tomato, wheat, and maize by 27.5%, 25.7%, 9.7%, and 11.1%, respectively. This study establishes a robust foundation for the industrial and agricultural applications of AlyC7.
Asunto(s)
Escherichia coli , Polisacárido Liasas , Trisacáridos , Vibrio , Polisacárido Liasas/metabolismo , Trisacáridos/biosíntesis , Vibrio/enzimología , Especificidad por Sustrato , Alginatos , Zea mays , OligosacáridosRESUMEN
BACKGROUND: Alginate oligosaccharides (AOs) are the degradation products of alginate, a natural polysaccharide abundant in brown algae. AOs generated by enzymatic hydrolysis have diverse bioactivities and show broad application potentials. AOs production via enzymolysis is now generally with sodium alginate as the raw material, which is chemically extracted from brown algae. In contrast, AOs production by direct degradation of brown algae is more advantageous on account of its cost reduction and is more eco-friendly. However, there have been only a few attempts reported in AOs production from direct degradation of brown algae. RESULTS: In this study, an efficient Laminaria japonica-decomposing strain Pseudoalteromonas agarivorans A3 was screened. Based on the secretome and mass spectrum analyses, strain A3 showed the potential as a cell factory for AOs production by secreting alginate lyases to directly degrade L. japonica. By using the L. japonica roots, which are normally discarded in the food industry, as the raw material for both fermentation and enzymatic hydrolysis, AOs were produced by the fermentation broth supernatant of strain A3 after optimization of the alginate lyase production and hydrolysis parameters. The generated AOs mainly ranged from dimers to tetramers, among which trimers and tetramers were predominant. The degradation efficiency of the roots reached 54.58%, the AOs production was 33.11%, and the AOs purity was 85.03%. CONCLUSION: An efficient, cost-effective and green process for AOs production directly from the underutilized L. japonica roots by using strain A3 was set up, which differed from the reported processes in terms of the substrate and strain used for fermentation and the AOs composition. This study provides a promising platform for scalable production of AOs, which may have application potentials in industry and agriculture.
Asunto(s)
Alginatos , Laminaria , Análisis Costo-Beneficio , OligosacáridosRESUMEN
Alginate is abundant in the cell walls of brown algae. Alginate lyases can degrade alginate, and thus play an important role in the marine carbon cycle and industrial production. Currently, most reported alginate lyases contain only one functional alginate lyase domain. AlyC8 is a putative alginate lyase with two alginate lyase domains (CD1 and CD2) from the marine alginate-degrading strain Vibrio sp. C42. To characterize AlyC8 and its two catalytic domains, AlyC8 and its two catalytic domain-deleted mutants, AlyC8-CD1 and AlyC8-CD2, were expressed in Escherichia coli. All three proteins have noticeable activity toward sodium alginate and exhibit optimal activities at pH 8.0-9.0 and at 30-40 °C, demonstrating that both CD1 and CD2 are functional. However, CD1 and CD2 showed opposite substrate specificity. The differences in substrate specificity and degradation products of alginate between the mutants and AlyC8 demonstrate that CD1 and CD2 can act synergistically to enable AlyC8 to degrade various alginate substrates into smaller oligomeric products. Moreover, kinetic analysis indicated that AlyC8-CD1 plays a major role in the degradation of alginate by AlyC8. These results demonstrate that AlyC8 is a novel alginate lyase with two functional catalytic domains that are synergistic in alginate degradation, which is helpful for a better understanding of alginate lyases and alginate degradation.
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Proteínas Bacterianas , Polisacárido Liasas , Vibrio , Alginatos/química , Concentración de Iones de Hidrógeno , Cinética , Polisacárido Liasas/química , Polisacárido Liasas/genética , Polisacárido Liasas/aislamiento & purificación , Especificidad por Sustrato , Vibrio/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Mutación , Dominio CatalíticoRESUMEN
Alginate oligosaccharides (AOS) have many biological activities and significant applications in prebiotics, nutritional supplements, and plant growth development. Alginate lyases have unique advantages in the preparation of AOS. However, only a limited number of alginate lyases have been so far reported to have potentials in the preparation of AOS with specific degrees of polymerization. Here, an alginate-degrading strain Pseudoalteromonasarctica M9 was isolated from Sargassum, and five alginate lyases were predicted in its genome. These putative alginate lyases were expressed and their degradation products towards sodium alginate were analyzed. Among them, AlyM2 mainly generated trisaccharides, which accounted for 79.9% in the products. AlyM2 is a PL6 lyase with low sequence identity (≤28.3%) to the characterized alginate lyases and may adopt a distinct catalytic mechanism from the other PL6 alginate lyases based on sequence alignment. AlyM2 is a bifunctional endotype lyase, exhibiting the highest activity at 30 °C, pH 8.0, and 0.5 M NaCl. AlyM2 predominantly produces trisaccharides from homopolymeric M block (PM), homopolymeric G block (PG), or sodium alginate, with a trisaccharide production of 588.4 mg/g from sodium alginate, indicating its promising potential in preparing trisaccharides from these polysaccharides.
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Alginatos/química , Proteínas Bacterianas , Polisacárido Liasas , Pseudoalteromonas/enzimología , Sargassum/microbiología , Trisacáridos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Genoma Bacteriano , Polisacárido Liasas/química , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Pseudoalteromonas/genética , Pseudoalteromonas/aislamiento & purificación , ARN Ribosómico 16SRESUMEN
The Mariana Trench is the deepest site on earth with diverse extreme conditions such as high hydrostatic pressure, low temperature and lack of light. Organisms surviving in this extreme environment and their life strategies have been largely uninvestigated. Here, we report the complete genome of Marinomonas profundi M1K-6T, isolated from the Mariana Trench deep seawater. The assembled genome comprised 3,648,059 bp without any plasmid. Gene annotation showed that strain M1K-6T possesses a series of genes encoding cold-shock proteins, DEAD box RNA helicase and enzymes for biosynthesis of unsaturated fatty acids, implying its high cold tolerance. Abundant genes responsible for transports of ion, branched-chain amino acids and organic compatible solutes were detected, which could maintain cellular osmotic balance disturbed by high hydrostatic pressure. In addition, detected genes (related to storage carbon, transport systems and two-component regulatory systems) could help strain M1K-6T to improve its ecological fitness in the deep-sea microaerobic and nutrient-limiting environments. Genomic information on M. profundi M1K-6T, provides insights into the adaptation strategies of Marinomonas spp. in the extreme deep-sea environment of the Mariana Trench.
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Marinomonas , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Genómica , Marinomonas/genética , Océano Pacífico , Filogenia , ARN Ribosómico 16S/genética , Agua de MarRESUMEN
Disordered hepatic glucagon response contributes to hyperglycemia in diabetes. The regulators involved in glucagon response are less understood. This work aims to investigate the roles of mitochondrial ß-oxidation enzyme HADHA and its downstream ketone bodies in hepatic glucagon response. Here we show that glucagon challenge impairs expression of HADHA. Liver-specific HADHA overexpression reversed hepatic gluconeogenesis in mice, while HADHA knockdown augmented glucagon response. Stable isotope tracing shows that HADHA promotes ketone body production via ß-oxidation. The ketone body ß-hydroxybutyrate (BHB) but not acetoacetate suppresses gluconeogenesis by selectively inhibiting HDAC7 activity via interaction with Glu543 site to facilitate FOXO1 nuclear exclusion. In HFD-fed mice, HADHA overexpression improved metabolic disorders, and these effects are abrogated by knockdown of BHB-producing enzyme. In conclusion, BHB is responsible for the inhibitory effect of HADHA on hepatic glucagon response, suggesting that HADHA activation or BHB elevation by pharmacological intervention hold promise in treating diabetes.
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Ácido 3-Hidroxibutírico/biosíntesis , Glucagón/metabolismo , Hígado/metabolismo , Mitocondrias/metabolismo , Subunidad alfa de la Proteína Trifuncional Mitocondrial/metabolismo , Ácido 3-Hidroxibutírico/metabolismo , Acetilación , Animales , Glucemia/metabolismo , Dieta Alta en Grasa , Proteína Forkhead Box O1/metabolismo , Gluconeogénesis , Células HEK293 , Histona Desacetilasas/metabolismo , Humanos , Hidroxibutirato Deshidrogenasa , Marcaje Isotópico , Cuerpos Cetónicos/metabolismo , Luciferasas/metabolismo , Masculino , Ratones Endogámicos C57BL , Modelos Biológicos , Oxidación-Reducción , Unión ProteicaRESUMEN
BACKGROUND: A plethora of prognostic biomarkers for esophageal squamous cell carcinoma (ESCC) that have hitherto been reported are challenged with low reproducibility due to high molecular heterogeneity of ESCC. The purpose of this study was to identify the optimal biomarkers for ESCC using machine learning algorithms. METHODS: Biomarkers related to clinical survival, recurrence or therapeutic response of patients with ESCC were determined through literature database searching. Forty-eight biomarkers linked to recurrence or prognosis of ESCC were used to construct a molecular interaction network based on NetBox and then to identify the functional modules. Publicably available mRNA transcriptome data of ESCC downloaded from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets included GSE53625 and TCGA-ESCC. Five machine learning algorithms, including logical regression (LR), support vector machine (SVM), artificial neural network (ANN), random forest (RF) and XGBoost, were used to develop classifiers for prognostic classification for feature selection. The area under ROC curve (AUC) was used to evaluate the performance of the prognostic classifiers. The importances of identified molecules were ranked by their occurrence frequencies in the prognostic classifiers. Kaplan-Meier survival analysis and log-rank test were performed to determine the statistical significance of overall survival. RESULTS: A total of 48 clinically proven molecules associated with ESCC progression were used to construct a molecular interaction network with 3 functional modules comprising 17 component molecules. The 131,071 prognostic classifiers using these 17 molecules were built for each machine learning algorithm. Using the occurrence frequencies in the prognostic classifiers with AUCs greater than the mean value of all 131,071 AUCs to rank importances of these 17 molecules, stratifin encoded by SFN was identified as the optimal prognostic biomarker for ESCC, whose performance was further validated in another 2 independent cohorts. CONCLUSION: The occurrence frequencies across various feature selection approaches reflect the degree of clinical importance and stratifin is an optimal prognostic biomarker for ESCC.
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Biomarcadores de Tumor , Carcinoma de Células Escamosas de Esófago/diagnóstico , Carcinoma de Células Escamosas de Esófago/etiología , Aprendizaje Automático , Algoritmos , Biología Computacional , Perfilación de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Pronóstico , Reproducibilidad de los Resultados , TranscriptomaRESUMEN
A Gram-stain-negative, aerobic, polarly flagellated, straight or curved rod-shaped bacterium, designated strain M1K-6T, was isolated from deep seawater samples collected from the Mariana Trench. The strain grew at -4 to 37 °C (optimum, 25-30 °C), at pH 5.5-10.0 (optimum, pH 7.0) and with 0.5-14.0ââ% (w/v) NaCl (optimum, 2.0â%). It did not reduce nitrate to nitrite nor hydrolyse gelatin or starch. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain M1K-6T was affiliated with the genus Marinomonas, sharing 93.1-97.0ââ% sequence similarity with the type strains of recognized Marinomonas species. The major cellular fatty acids were summed feature 3 (C16â:â1 ω6c/C16â:â1 ω7c), summed feature 8 (C18â:â1 ω7c/C18â:â1 ω6c), C16â:â0, C10â:â0 3-OH and C18â:â0. The predominant respiratory quinone was ubiquinone-8. Polar lipids of strain M1K-6T included phosphatidylethanolamine, phosphatidylglycerol and two unidentified lipids. The genomic G+C content of strain M1K-6T was 46.0 mol%. Based on data from the present polyphasic study, strain M1K-6T was considered to represent a novel species within the genus Marinomonas, for which the name Marinomonas profundi sp. nov. is proposed. The type strain is M1K-6T (=KCTC 72501T=MCCC 1K03890T).
Asunto(s)
Marinomonas/clasificación , Filogenia , Agua de Mar/microbiología , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/química , Marinomonas/aislamiento & purificación , Hibridación de Ácido Nucleico , Océano Pacífico , Fosfolípidos/química , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Ubiquinona/químicaRESUMEN
A Gram-stain-negative, oxidase- and catalase-positive, facultative anaerobic and rod-shaped bacterium, designated strain SM1977T, was isolated from the surface of coralline algae collected from the intertidal zone at Qingdao, PR China. The strain grew at 10-35 °C, pH 4.5-8.5 and with 1-8.5% (w/v) NaCl. It reduced nitrate to nitrite and hydrolysed Tween 20 and DNA. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SM1977T was affiliated with the genus Vibrio, having the highest sequence similarity (97.6â%) to the type strain of Vibrio casei, followed by those of another five species (95.6-97.6â%) in the Rumoiensis clade of the genus Vibrio. However, the in silico DNA-DNA hybridization (75.3-75.9â%) and average nucleotide identity (21.6-22.8â%) values of SM1977T against these close relatives were all below the corresponding thresholds to discriminate bacterial species. The major fatty acids were summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), C16:0 and summed feature 8 (C18:1 ω6c and /or C18:1 ω7c). The predominant polar lipids were phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The sole respiratory quinone was Q-8. The genomic DNA G+C content of strain SM1977T, determined from the obtained whole genomic sequence, was 42.3 mol%. On the basis of the polyphasic results obtained in this study, strain SM1977T is considered to represent a novel species within the genus Vibrio, for which the name Vibrio algicola sp. nov. is proposed. The type strain is SM1977T (=MCCC 1K04351T=KCTC 72847T).
Asunto(s)
Antozoos/microbiología , Chlorophyta/microbiología , Filogenia , Vibrio/clasificación , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , China , ADN Bacteriano/genética , Ácidos Grasos/química , Hibridación de Ácido Nucleico , Fosfolípidos/química , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Ubiquinona/química , Vibrio/aislamiento & purificaciónRESUMEN
AIMS: Our aim was to search for clinical predictors of good glycemic control in patients starting or intensifying oral hypoglycemic pharmacological therapy. METHODS: A multicenter, prospective cohort of 499 diabetic subjects was enrolled in this study: patients with newly diagnosed diabetes (NDM group) or poor glycemic control with oral antidiabetic drugs (OADs) (PDM group). All subjects then started or intensified OADs therapy and followed up for 91â¯days. Glycemic control was determined according to HbA1c at day 91 with HbA1c <7% considered good. RESULTS: The proportions of patients with good glycemic control after follow up for 91â¯days were 66.9% and 34.8% in NDM group and PDM group respectively. Logistic regression analysis showed that the change in GA at 28â¯days was the only predictor of good glycemic control in NDM patients (ORâ¯=â¯1.630, 95% CI 1.300-2.044, Pâ¯<â¯0.001). In PDM patients, changes in GA at 28â¯days, CPI, baseline HbA1c, diabetic duration, and BMI were all independent predictors of good glycemic control (All Pâ¯<â¯0.05). CONCLUSIONS: GA decline is a good predictor of future success in newly diagnosed patients. In patients intensifying therapy, beside GA decline, other individualized clinical characteristics should also be considered.
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Diabetes Mellitus Tipo 2/sangre , Control Glucémico , Hipoglucemiantes/uso terapéutico , Albúmina Sérica/análisis , Administración Oral , Adulto , Biomarcadores/sangre , Glucemia/análisis , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Femenino , Hemoglobina Glucada/análisis , Productos Finales de Glicación Avanzada , Humanos , Hiperglucemia/sangre , Hiperglucemia/diagnóstico , Hiperglucemia/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Resultado del Tratamiento , Albúmina Sérica GlicadaRESUMEN
BACKGROUND AND GOALS: Combined transcutaneous neuromodulation (TN) at acupoint ST36 (Zusanli) and TN at the posterior tibial nerve (PTN) has been reported effective in treating functional constipation. This study was designed to compare the effectiveness of TN between these 2 points. MATERIALS AND METHODS: Eighteen functional constipation patients (M/F: 9/9) were recruited to participate in a cross-over study with a 2-week TN at ST36 and a 2-week TN at PTN. A bowel movement diary, and the questionnaires of Patient Assessment of Constipation Symptom (PAC-SYM) and Constipation Quality of Life (PAC-QoL) were completed; anorectal manometry and spectral analysis of heart rate variability for assessing the autonomic function were performed. RESULTS: (1) Both TN at ST36 and TN at PTN improved constipation-related symptoms (PAC-SYM scores on pre-TN vs. post-TN: 1.4±0.1 vs. 0.6±0.1 for ST36, 1.4±0.1 vs. 0.9±0.1 for PTN, both P≤0.001). (2) TN at ST36, but not TN at PTN, increased the number of weekly spontaneous bowel movements (0.9±0.2 pre-TN vs. 3.5±0.7 post-TN, P<0.001) and decreased the total PAC-QoL score. TN at ST36 was more potent than TN at PTN in improving the PAC-SYM score (decrement 0.8±0.1 vs. 0.5±0.1, P<0.05). (3) TN at ST36 rather than TN at PTN resulted in a reduction in sensation thresholds, including rectal distention for urge (134.1±14.3 mL pre-TN vs. 85.6±6.5 mL post-TN, P<0.01) and maximum tolerance (P<0.05). (4) Both TN at ST36 and TN at PTN significantly increased vagal activity and decreased sympathetic activity (P<0.05). CONCLUSIONS: TN at ST36 is more potent than TN at PTN in treating constipation and improving constipation-related symptoms and rectal sensation.
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Estreñimiento , Calidad de Vida , Estreñimiento/terapia , Estudios Cruzados , Humanos , Nervio Tibial , Resultado del TratamientoRESUMEN
Two Gram-stain-negative, aerobic, rod-shaped bacteria, polar flagellated, designated strains SM2066T and SM1966T, were respectively isolated from the surfaces of Colpomenia sinuosa and Ulva pertusa macroalgae collected off the coastal areas of Rongcheng, PR China. Strain SM2066T grew at 8-37 °C and with 0.5-7.0â% (w/v) NaCl, while strain SM1966T grew at 5-30 °C and with 0.5-8.5% (w/v) NaCl. Both of them reduced nitrate to nitrite and required Na+ for growth but neither of them hydrolysed starch and DNA. Phylogenetic analysis based on 16S rRNA gene and single-copy orthologous cluster sequences revealed that both strains SM2066T and SM1966T were affiliated with the genus Marinomonas but formed distinct phylogenetic branches from known Marinomonas species, respectively sharing the highest 16S rRNA gene sequence similarities with type strains of Marinomonas ushuaiensis (97.9â%) and Marinomonas blandensis (96.7â%). The digital DNA-DNA hybridization and average nucleotide identity values between strains SM2066T and SM1966T and type strains of closely related Marinomonas species were all below 22.9 and 79.9âmol%, respectively. The major fatty acids of the two strains were summed feature 3 (C16â:â1 ω6c/C16â:â1 ω7c), summed feature 8 (C18â:â1 ω7c) and C16â:â0, with their predominant polar lipids being phosphatidylethanolamine and phosphatidylglycerol, and their sole respiratory quinone being Q-8. The genomic DNA G+C contents of strains SM2066T and SM1966T determined from genomic sequences were 40.3 and 41.6âmol%, respectively. On the basis of the polyphasic evidence presented in this study, strains SM2066T and SM1966T are considered to represent two novel species within the genus Marinomonas, for which the names Marinomonas colpomeniae sp. nov. and Marinomonas algicola sp. nov. are proposed. The type strains are SM2066T (=MCCC 1K04390T= KCTC 82372T) and SM1966T (=MCCC 1K04387T= KCTC 72848T), respectively.
RESUMEN
The present study aimed to evaluate the anti-diabetic property of peanut shell polyphenol extracts (PSPEs). Diabetic rats were oral-administrated with PSPE at doses of 50, 100, and 200 mg/kg body weight (BW) per day for 28 consecutive days, with metformin (Met) as a positive control. The results showed that, similar to the Met treatment, administration of PSPE caused significant decreases in food intake, water intake, fasting blood glucose, total cholesterol, triglyceride, low-density lipoprotein cholesterol, and methane dicarboxylic aldehyde in serum, and significant increases in BW, insulin level, high-density lipoprotein cholesterol, superoxide dismutase, glutathione, and liver glycogen. Further, glucose tolerance was markedly improved in the PSPE-treated diabetic groups. Histopathological results showed that PSPE improved cellular structural and pathological changes in liver, kidney, and pancreatic islets. Collectively, the results indicated that the hypoglycemic effects of PSPE on high-fat diet/streptozotocin (HFD/STZ)-induced diabetes are comparable to Met, though their exact mechanism actions are still under investigation. Therefore, the current study suggests that PSPE could be a potential health-care food supplement in the management of diabetes.
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Arachis/química , Diabetes Mellitus Experimental/tratamiento farmacológico , Hipoglucemiantes/farmacología , Extractos Vegetales/farmacología , Polifenoles/farmacología , Animales , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Lípidos/sangre , Hígado/patología , Masculino , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Wistar , EstreptozocinaRESUMEN
AIM: To investigate whether there is a link between diabetes mellitus (DM) and gastroesophageal reflux disease (GERD). METHODS: We conducted a systematic search of PubMed and Web of Science databases, from their respective inceptions until December 31, 2013, for articles evaluating the relationship between DM and GERD. Studies were selected for analysis based on certain inclusion and exclusion criteria. Data were extracted from each study on the basis of predefined items. A meta-analysis was performed to compare the odds ratio (OR) in DM between individuals with and without GERD using a fixed effect or random effect model, depending on the absence or presence of significant heterogeneity. Subgroup analyses were used to identify sources of heterogeneity. Publication bias was assessed by Begg's test. To evaluate the results, we also performed a sensitivity analysis. RESULTS: When the electronic database and hand searches were combined, a total of nine eligible articles involving 9067 cases and 81 968 controls were included in our meta-analysis. Based on the random-effects model, these studies identified a significant association between DM and the risk of GERD (overall OR = 1.61; 95%CI: 1.36-1.91; P = 0.003). Subgroup analyses indicated that this result persisted in studies on populations from Eastern countries (OR = 1.71; 95%CI: 1.38-2.12; P = 0.003) and in younger patients (mean age < 50 years) (OR = 1.70; 95%CI: 1.22-2.37; P = 0.001). No significant publication bias was observed in this meta-analysis using Begg(')s test (P = 0.175). The sensitivity analysis also confirmed the stability of our results. CONCLUSION: This meta-analysis suggests that patients with DM are at greater risk of GERD than those who do not have DM.
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Diabetes Mellitus/epidemiología , Reflujo Gastroesofágico/epidemiología , Adulto , Diabetes Mellitus/diagnóstico , Esofagitis/epidemiología , Femenino , Reflujo Gastroesofágico/diagnóstico , Humanos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Prevalencia , Pronóstico , Factores de RiesgoRESUMEN
OBJECTIVE: To investigate the role of store-operated calcium channels (SOCs) in primary hepatocytes under conditions of calcium overload and ethanol-induced injury. METHODS: The in vitro model of chronic ethanol-induced hepatocyte injury was established using primary hepatocytes isolated from Sprague-Dawley rats. Ethanol-induced changes (24, 48 and 72 h; 50, 100, 200, 400 and 800 mmol/L) in expression of the SOCs proteins stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (Oria1) were detected by qualitative PCR analysis (mRNA) and western blotting (protein). The possible role of these two SOCs proteins in the ethanol-induced extracellular calcium influx and related liver cell injury was determined by treating the cell system with various channel blockers (EGTA, La3+, and 2-APB). Cell viability was determined by MTT assay and cytosolic free calcium ion concentration was determined by flow cytometry. RESULTS: After 24 h of exposure to 0 (untreated) to 800 mM/L ethanol, the cell viability was reduced in a concentration-dependent manner. The 400 mmol/L concentration of ethanol decreased cell viability by 57.34% +/- 2.34%. and was chosen for use in subsequent experiments. Compared with the untreated control cells, the ethanol-treated cells showed significantly up-regulated mRNA and protein expression of both STIM1 and Orai1 at all times examined, suggesting that the ethanol-stimulated expression of STIM1 and Orai1 could persist for at least 72 h. The ethanol treatment induced increase in cytoplasmic calcium levels was significantly (and similarly) reduced by co-treatment with any of the three channel blockers. CONCLUSION: Chronic ethanol exposure can increase the expression of STIM1 and Orai1 in primary liver cells, suggesting that ethanol may increase extracellular calcium influx by up-regulating expression of these SOCs protein molecules, ultimately aggravating liver cell damage.
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Canales de Calcio/metabolismo , Calcio/metabolismo , Etanol/efectos adversos , Hepatocitos/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacología , Proteínas de Unión al Calcio/metabolismo , Supervivencia Celular , Células Cultivadas , Hepatocitos/efectos de los fármacos , Masculino , Glicoproteínas de Membrana/metabolismo , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Molécula de Interacción Estromal 1RESUMEN
OBJECTIVE: To investigate the mechanism of ethanol-induced calcium overload in hepatocytes and the related role of store-operated calcium channels (SOCs). METHODS: HepG2 cells were treated an ethanol concentration gradient with or without intervention treatment with the extracellular calcium chelator EGTA or the SOCs inhibitor 2-aminoethoxydiphenyl borate (2-APB). Effects on cell viability were assessed by the CCK8 assay. Effects on leakage of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by automatic biochemical analyzer measurements of the culture supernatants. Effects on cytoplasmic free Ca2+ concentration ([Ca2+]i) were accessed by detecting fluorescence intensity of the calcium indicator Fluo-3/AM with a flow cytometer. Effects on mRNA and protein expression levels of SOCs, stromal interacting factor 1 (STIM1), and calcium release-activated calcium channel protein 1 (Orai1) were evaluated by qPCR and western blotting. RESULTS: The ethanol treatment produced dose-dependent reduction in cell viability (r = -0.985, P less than 0.01) and increases in leakage of ALT (F = 15.286, P less than 0.01) and AST (F = 39.674, P less than 0.01). Compared to untreated controls, the ethanol treatments of 25, 50, 100, 200 and 400 mM induced significant increases in [Ca2+]i level (1.25+/-0.36, 1.31+/-0.15, 1.41+/-0.18, 2.29+/-0.25, 2.58+/-0.19; F = 15.286, P less than 0.01). Both intervention treatments, EGTA and 2-APB, significantly reduced the 200 mM ethanol treatment-induced [Ca2+]i increase (2.32+/-0.08 reduced to 1.79+/-0.15 (t = 7.201, P less than 0.01) and 1.86+/-0.09 (t = 8.183, P less than 0.01) respectively). EGTA and 2-APB also increased the ethanol-treated cells' viability and reduced the ALT and AST leakage. The 200 mM ethanol treatment stimulated both gene and protein expression of STIM1 and Orai1, and the up-regulation effect lasted at least 72 h after treatment. CONCLUSION: Ethanol-induced dysregulation of SOCs may be an important molecular mechanism of ethanol-induced [Ca2+]i rise in hepatocytes and the related liver cell injury.