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Objective:To observe the effects of Xiaoqinglong Decoction and Qingqi Huatan Pills on interleukin-1β(IL-1β)-induced mucushypersecretion model of human airway epithelial H292 cellsand related molecules of nuclear factor-κB/microRNA-494 (NF-κB/miR-494) signaling pathway, and to explore the mechanism of the two medicines in improving pathological airway mucus.Methods:Methyl thiazolyl tetrazolium (MTT) colorimetric method was used to detect the effects of different concentrations of Xiaoqinglong Decoction and Qingqi Huatan Pills on the activity of H292 cellsinduced by IL-1β, and the appropriate concentration was selected for subsequent experiments. Cells were randomly divided into blank group, IL-1β model group (5 μg/L IL-1β), NF-κB inhibitor pyrrolidinedithiocarbamate (PDTC) group (5 μg/L IL-1β+100 μmol/L PDTC), Xiaoqinglong Decoction (5 μg/L IL-1β+1 000 mg/L Xiaoqinglong Decoction) and Qingqi Huatan Pill group (5 μg/L IL-1β+1 000 mg/L Qingqi Huatan Pills). 5 μg/L IL-1β was used to induce H292 cells for 24 hours to establish a model of airway epithelial mucus hypersecretion. Enzyme linked immunosorbent assay (ELISA) method was used to detect the levels of mucin 5AC (MUC5AC), tumor necrosis factor-α (TNF-α) and IL-8 and the synthesis of intracellular MUC5AC and cystic fibrosis transmembrane conductance regulator (CFTR). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression levels of MUC5AC mRNA, CFTR mRNA, miR-494. Western blotting was used to detect protein expression of key proteins (p65) and NF-κB inhibitors (IκB) in NF-κB signaling pathway.Results:Xiaoqinglong Decoction and Qingqi Huatan Pills with the concentration of 1 000 mg/L were selected for the follow-up experiment. Compared with the blank group, the levels of MUC5AC, TNF-α and IL-8 were significantly increased in the model group, intracellular MUC5AC protein content and mRNA expression were also significantly increased, intracellular CFTR protein content and mRNA expression were significantly decreased, and intracellular p65 protein expression was significantly up-regulated, the expression of IκB protein was significantly down-regulated, and the expression of miR-494 was significantly increased. Compared with the model group, the levels of MUC5AC, TNF-α and IL-8 were significantly reduced in PDTC group, Xiaoqinglong Decoction group and Qingqi Huatan Pill group, intracellular MUC5AC protein content and mRNA expression were also significantly decreased, and intracellular p65 protein expression was significantly down-regulated, and IκB protein expression was significantly up-regulated, miR-494 expression was significantly reduced. Intracellular CFTR protein content and mRNA expression were significantly increased in both PDTC group and Qingqi Huatan Pill group. Compared with the PDTC group, the level of TNF-α in the Xiaoqinglong Decoction group was significantly increased (ng/L: 22.77±3.14 vs. 11.09±3.37, P < 0.05), the content and mRNA expression of CFTR and IκB protein expression was significantly decreased [CFTR protein (ng/L): 97.38±6.62 vs. 227.04±19.48, CFTR mRNA (2 -ΔΔCt): 0.99±0.08 vs. 1.21±0.08, IκB/β-actin: 1.69±0.11 vs. 2.00±0.18, all P < 0.05], the level of TNF-α in Qingqi Huatan Pill group was significantly higher (ng/L: 19.08±3.71 vs. 11.09±3.37, P < 0.05). Compared with Xiaoqinglong Decoction group, the protein content and mRNA expression of CFTR and IκB protein expression in Qingqi Huatan Pill group were significantly increased [CFTR protein (ng/L) : 235.01±22.71 vs. 97.38±6.62, CFTR mRNA(2 -ΔΔCt): 1.32±0.15 vs. 0.99±0.08, IκB/β-actin: 1.94±0.16 vs. 1.69±0.11, all P < 0.05]. Conclusions:The effect of Xiaoqinglong Decoctionin improving the hypersecretion of mucus in the airway epithelium may be related to the inhibition of NF-κB/miR-494 inflammatory signal-mediated MUC5AC hypersecretion, while the effect of Qingqi Huatan Pills may be related to the inhibition of NF-κB/miR-494 inflammatory signal-mediated MUC5AC hypersecretion and CFTR dysfunction. Therefore, the difference in the mechanism of the two treatments of airway pathological mucus is mainly in the regulation of CFTR mRNA and protein.
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Objective@#To explore the value of serum microRNA-494 (miR-494) expression in predicting the prognosis of acute renal injury (AKI) after cardiac surgery in children.@*Methods@#116 children with AKI after cardiopulmonary bypass for congenital heart disease admitted to Sanya People's Hospital from January 2016 to March 2019 were enrolled. The expression of miR-494 in serum was detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), and the levels of serum neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) were detected by enzyme linked immunosorbent assay (ELISA) of all the children. The children were divided into survival group and death group according to 28-day survival. Serum levels of miR-494, NGAL and KIM-1 were measured in two groups. Multivariate Logistic regression was used to analyze the risk factors of death in children with AKI after cardiac surgery. The receiver operating characteristic (ROC) curve analysis of serum levels of miR-494, NGAL and KIM-1 in predicting prognosis of children with AKI after cardiac surgery was performed. Pearson correlation analysis was used to analyze the correlation between serum levels of miR-494 and NGAL, KIM-1.@*Results@#After cardiopulmonary bypass in 116 children with AKI, 27 cases died and 89 cases survived during the 28-day observation. Compared with the survival group, the proportion of cyanosis in the death group was significantly increased, the proportion of blood perfusion was significantly decreased, the time of cardiopulmonary bypass and postoperative mechanical ventilation were significantly prolonged, and the blood glucose level was significantly increased after operation. There was no significant difference in other general data. The serum levels of miR-494, NGAL and KIM-1 in the death group were significantly higher than those in the survival group [miR-494 (2-ΔΔCt): 3.75±1.28 vs. 1.48±0.71, NGAL (mg/L): 583.60±52.72 vs. 320.52±31.84, KIM-1 (μg/L): 30.53±6.38 vs. 17.40±3.72, all P < 0.01]. Multivariate Logistic regression analysis showed cyanosis [odds ratio (OR) = 1.716, 95% confidence interval (95%CI) was 1.184-2.982, P = 0.039], postoperative blood glucose (OR = 1.925, 95%CI was 1.262-3.387, P = 0.005), serum miR-494 (OR = 2.527, 95%CI was 1.706-5.148, P < 0.001), NGAL (OR = 2.473, 95%CI was 1.620-4.935, P < 0.001) and KIM-1 (OR = 1.805, 95%CI was 1.213-3.106, P < 0.001) were independent risk factors for death in children with AKI after cardiac surgery. ROC curve analysis showed the area under the curve (AUC) to predict the death of children with postoperative AKI was 0.868, 0.857 and 0.819 respectively, AUC of serum miR-494, NGAL and KIM-1 levels combination to predict the death of children with postoperative AKI was the largest (0.964, 95%CI was 0.908-0.997), with a high sensitivity and specificity of 97.0% and 91.8%. The correlation analysis showed the expression level of serum miR-494 was positively correlated with NGAL and KIM-1 in the death group (r1 = 0.902, r2 = 0.873, both P < 0.01).@*Conclusion@#Serum levels of miR-494 increased significantly in children with AKI after cardiac surgery, which is an independent risk factor for death in children with AKI after cardiac surgery, and the combination of NGAL and KIM-1 levels had a high value in predicting the prognosis of children with AKI after cardiac surgery.
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Objective To explore the value of serum microRNA-494 (miR-494) expression in predicting the prognosis of acute renal injury (AKI) after cardiac surgery in children. Methods 116 children with AKI after cardiopulmonary bypass for congenital heart disease admitted to Sanya People's Hospital from January 2016 to March 2019 were enrolled. The expression of miR-494 in serum was detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), and the levels of serum neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) were detected by enzyme linked immunosorbent assay (ELISA) of all the children. The children were divided into survival group and death group according to 28-day survival. Serum levels of miR-494, NGAL and KIM-1 were measured in two groups. Multivariate Logistic regression was used to analyze the risk factors of death in children with AKI after cardiac surgery. The receiver operating characteristic (ROC) curve analysis of serum levels of miR-494, NGAL and KIM-1 in predicting prognosis of children with AKI after cardiac surgery was performed. Pearson correlation analysis was used to analyze the correlation between serum levels of miR-494 and NGAL, KIM-1. Results After cardiopulmonary bypass in 116 children with AKI, 27 cases died and 89 cases survived during the 28-day observation. Compared with the survival group, the proportion of cyanosis in the death group was significantly increased, the proportion of blood perfusion was significantly decreased, the time of cardiopulmonary bypass and postoperative mechanical ventilation were significantly prolonged, and the blood glucose level was significantly increasedafter operation. There was no significant difference in other general data. The serum levels of miR-494, NGAL and KIM-1 in the death group were significantly higher than those in the survival group [miR-494 (2-ΔΔCt): 3.75±1.28 vs. 1.48±0.71, NGAL (mg/L): 583.60±52.72 vs. 320.52±31.84, KIM-1 (μg/L): 30.53±6.38 vs. 17.40±3.72, all P < 0.01]. Multivariate Logistic regression analysis showed cyanosis [odds ratio (OR) = 1.716, 95％ confidence interval (95％CI) was 1.184-2.982, P = 0.039], postoperative blood glucose (OR = 1.925, 95％CI was 1.262-3.387, P = 0.005), serum miR-494 (OR = 2.527, 95％CI was 1.706-5.148, P < 0.001), NGAL (OR = 2.473, 95％CI was 1.620-4.935, P < 0.001) and KIM-1 (OR = 1.805, 95％CI was 1.213-3.106, P < 0.001) were independent risk factors for death in children with AKI after cardiac surgery. ROC curve analysis showed the area under the curve (AUC) to predict the death of children with postoperative AKI was 0.868, 0.857 and 0.819 respectively, AUC of serum miR-494, NGAL and KIM-1 levels combination to predict the death of children with postoperative AKI was the largest (0.964, 95％CI was 0.908-0.997), with a high sensitivity and specificity of 97.0％ and 91.8％. The correlation analysis showed the expression level of serum miR-494 was positively correlated with NGAL and KIM-1 in the death group (r1 = 0.902, r2 = 0.873, both P < 0.01). Conclusion Serum levels of miR-494 increased significantly in children with AKI after cardiac surgery, which is an independent risk factor for death in children with AKI after cardiac surgery, and the combination of NGAL and KIM-1 levels had a high value in predicting the prognosis of children with AKI after cardiac surgery.
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MicroRNA-494 (miR-494) is one of the microRNAs from 14q32.31 miR-gene cluster.Recently, miR-494 was found to closely relate with tumors and other diseases .This article reviews the expression changes , roles and possible regulatory mechanisms of miR-494 in multiple tumors and other hypoxia/ischemia diseases .Recent studies demon-strate that the expression of miR-494 is affected by many factors , and miR-494 could be a biomarker of diagnosis , staging and prognosis in tumors and other diseases , and a target of therapy in future .
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Objective To optimize the concentration of a microRNA-21 (miR-21) inhibitor and a miR-494 mimic for the transfection of A375 human melanoma cells,and to estimate the effect of the miR-21 inbihitor and miR-494 mimic on the proliferation of A375 cells.Methods A miR-21 inbihitor and a miR-494 mimic were designed and constructed.To optimize the concentration of the miR-21 inbihitor and miR-494 mimic for transfection,six concentrations (70-250 nmol/L) of the inbihitor and mimic were transfected into A375 cells separately by using LipofectamineTM2000.Then,quantitative fluorescence-based PCR was performed to determine the expression of miR-21 and miR-494 in A375 cells.Some A375 cells were classified into five groups:Mock blank control group remaining untransfected,miR-21 inhibitor group transfected with the miR-21 inhibitor,miR-21 control group transfected with the miR-21 inhibitor negative control,miR-494 mimic group transfected with the miR-494 mimic,and miR-494 control group transfected with the miR-494 mimic negative control.Mter another 48-hour culture,the cells were collected for the analysis of cell apoptosis and cycle by using flow cytometry.Meanwhile,Cy5-labelled miR-494 mimic negative control was transfected into A375 cells for the evaluation of the transfection efficiency by using an inverted fluorescence microscope.Results miRNAs were successfully extracted from A375 cells.As quantitative PCR revealed,the A375 cells transfected with the miR-21 inhibitor at 120 nmol/L showed the lowest expression level (2-△△Ct) of miR-21 (average:0.80; range:0.65-0.92),and those transfected with the miR494 mimic at 250 nmol/L displayed the highest expression level of miR-494 (average:126.82; range:111.52-144.22).The transfection efficiency in A375 cells was higher than 90％.Compared with the corresponding negative control groups,the miR-21 inhibitor group and miR-494 mimic group showed increased apoptosis rate ((27.74 ± 1.39)％ vs.(12.93 ± 0.65)％,(34.30 ± 2.35)％ vs.(15.54 ± 1.02)％,both P ＜ 0.01),percentage of G1-phase cells ((61.61 ± 3.25)％ vs.(50.34 ± 5.62)％,(61.05 ± 3.17)％ vs.(49.95 ± 2.58)％,both P＜ 0.05),but decreased proliferation index ((38.39 ± 3.25)％ vs.(49.66 ± 5.62) ％,(38.95 ± 3.17)％ vs.(50.05 ± 2.58)％,both P ＜ 0.05).Conclusions Both the miR-21 inhibitor and miR-494 mimic can promote the G1-phase arrest and apoptosis in A375 cells,and miR-21 may act as a protooncogene accelerating the proliferation of A375 cells,while miR-494 may founction as a tumor suppressor inhibiting the proliferation of A375 cells.