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ObjectiveTo investigate the mechanism of Biejiajian Wan in the intervention of primary liver cancer based on long non-coding RNA SNHG5 （lncRNA SNHG5）/micro RNA-26a-5p （miRNA-26a-5p）/glycogen synthase kinase-3β （GSK-3β） signal axis. MethodDouble luciferase reporting assay was used to verify the targeted interaction between lncRNA SNHG5 and miRNA-26a-5p， miRNA-26a-5p， and GSK-3β in HepG2 cells. Nude-mouse transplanted tumor model of human HepG2 were established and randomly divided into model group， Biejiajian Wan low-dose group （0.5 g·kg-1）， medium-dose group （1.0 g·kg-1）， and high-dose group （2.0 g·kg-1）， and sorafenib group （100 mg·kg-1）， with 10 mice in each group. The mice were given intragastric administration of normal saline or drug for 28 days， and the tumor volume was measured at different time. Hematoxylin-eosin （HE） staining was used to observe the histological changes of tumors. The nucleic acid levels of lncRNA SNHG5， miRNA-26a-5p， GSK-3β， and β-catenin mPNA in tumor tissue were detected by real-time quantitative polymerase chain reaction （Real-time PCR）. The protein expression levels of GSK-3β and β-catenin in tumor tissue were detected by western blot. ResultCompared with the SNHG5-WT （wild type） + miRNA NC （negative control） group， the relative luciferase activities of the SNHG5-WT + miRNA-26a-5p mimic group were decreased （P<0.05）. Compared with the GSK-3β-WT + miRNA NC group， the relative luciferase activity of the GSK-3β-WT + miRNA-26a-5p mimic group was decreased （P<0.05）. Compared with the model group， the tumor volume of Biejiajian Wan low-dose， medium-dose， and high-dose groups was significantly decreased （P<0.05， P<0.01）. Compared with the model group， the cells in the tumor tissue of nude mice in each dose group of Biejiajian Wan were sparsely arranged with necrocytosis， which showed concentration-dependent changes. Compared with the model group， the expression levels of lncRNA SNHG5， GSK-3β， and β-catenin were decreased （P<0.05， P<0.01）， while the expression of miRNA-26a-5p was increased in each dose group of Biejiajian Wan （P<0.05， P<0.01）. Compared with the model group， the protein expression levels of GSK-3β and β-catenin were decreased in each dose group of Biejiajian Wan （P<0.05， P<0.01）. ConclusionBiejiajian Wan may affect the necrosis of liver cancer cells through lncRNA SNHG5/miRNA-26a-5p/GSK-3β signal axis and thus play an anti-tumor role. This research will provide more theoretical basis for the clinical application of Biejiajian Wan.
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OBJECTIVE To evaluate the cost-effectiveness of sintilimab combined with chemotherapy than single-use chemotherapy in the first-line treatment of advanced， recurrent or metastatic esophageal squamous cell carcinoma （ESCC） from the perspective of health system of our country， and provide reference for rational use of drug in clinic. METHODS Based on ORIENT-15 study data， TreeAge Pro 2011 software was used to establish a three-state Markov model of non-progressive survival （PFS）， disease progression and death for cost-utility analysis. The model period was 3 weeks， the research time limit was 10 years， and the discount rate was 5%. The main outputs of the model were total cost， quality-adjusted life year （QALY） and incremental cost-effectiveness ratio （ICER）. The 1-3 times of China’s GDP per capita in 2021 was taken as the threshold of willing- ness to pay （WTP）. The uncertainty of the parameters was analyzed by single factor sensitivity analysis and probability sensitivity analysis， and the cost-effectiveness of the two schemes was discussed under three situations： different discount rates， comparison with other similar treatment schemes and charitable drug donation schemes. RESULTS The results of basic analysis showed that compared with chemotherapy plan alone， the ICER of sintilimab combined with chemotherapy was 64 208.75 yuan/QALY， which was less than WTP threshold. The results of single factor sensitivity analysis show that PFS state utility value， cycle cost of sintilimab and discount rate had relatively great influence on the results. Probability sensitivity analysis showed that when WTP≥120 000 yuan， the economic probability of sintilimab combined with chemotherapy plan was 100%. The results of situational analysis showed that sintilimab combined chemotherapy was more cost- effective than single-use chemotherapy. CONCLUSIONS Sintilimab combined with chemotherapy is more cost-effective than single-use chemotherapy in the first-line treatment of advanced， recurrent or metastatic ESCC.
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Objective@#To evaluate the efficacy of systemic glucocorticoid (steroid) combined with high dose inhaled steroid in the treatment of children with acute laryngitis.@*Methods@#A total of 78 children with acute laryngitis were randomly divided into study group(n=40) and control group(n=38) between November 2016 and April 2017. In addition to routine treatment of anti infection and symptomatic treatment, Dexamethasone injection(0.3-0.5 mg/kg, 1-3 d, according to the patient′s condition) was provided to each group. In addition to the treatment mentioned above, the study group were assigned to receive 1.0 mg Budesonide suspension for inhalation, oxygen-driven atomizing inhalation, every/30 minutes, 2 times in a row, after that every 12 hours. The improvement of inspiratory dyspnea, hoarseness, barking cough and wheezing of both groups was evaluated at 30 min, 1 h, 2 h, 6 h, 12 h, 24 h and 72 h after treatment.Sigmaplot 11.5 software was used to analyze the data.@*Results@#No significant difference was detected in terms of inspiratory dyspnea, hoarseness, barking cough or stridor score before treatment between the two groups(P>0.05). Compared with those before treatment, symptoms of inspiratory dyspnea, hoarseness, barking cough and stridor score of both groups improved markedly at 12 h and 24 h after treatment(P<0.05). While there was no significant difference regarding inspiratory dyspnea, hoarseness, barking cough or stridor score at each time point after treatment between the two groups(P>0.05). The effective rate was 92.50% and 92.11% in study group and control group, respectively, and no significant difference was noted (P>0.05).@*Conclusion@#Compared with single systemic glucocorticoid, systemic glucocorticoids combined with inhaled steroid possessed similar efficacy in treating acute laryngitis and relieving laryngeal obstruction of children.
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OBJECTIVE To develop a new method for detecting 22q11.2 deletion syndrome (22q11.2 DS) in clinical settings. METHODS Specific primers and fluorescence probes were designed to target the TBX1 gene within the 22q11.2 deletion region and a reference gene RPP30. Multiplexed droplet digital PCR (ddPCR) was run to detect the 22q11.2 microdeletion by calculating the ratio of positive droplet number of TBX1/RPP30. RESULTS Three cases of 22q11.2 microdeletion previously confirmed by array comparative genome hybridization were successfully identified. Subsequently, the ddPCR detected two further cases of 22q11.2 microdeletion among 14 children with congenital heart diseases. CONCLUSION The ddPCR technique has provided a rapid and cost-effective method for detecting 22q11.2 microdeletion in clinical settings.
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Objective To establish an effective DNA isolation method for neonatal disease screening,so as to explore its application to the methylation detection.Methods The 20 dried blood spots samples were randomly divided into 2 groups according to the gender:the traditional method group (n =10) and the improved kit method group(n =10).The DNA quality was evaluated based on its concentration,integrity and whether it could be used in polymerase chain reaction (PCR).These DNA samples with or without bisulfite treatment were used as template in the methylation-specific polymerase chain reaction (MSP).The methylation levels of Leptin and tumor necrosis factor-α (TNF-α) gene promoter region were detected.Results DNA concentration of the improved kit method [(5.70 ± 0.81) mg/L] was significantly higher than that of the traditional method [(3.50 ± 0.45) mg/L] (t =2.79,P ＜ 0.05),and biochemical analyzer analysis showed a better DNA integrity.Agarose gel electrophoresis revealed that 18S gene fragment could be successfully amplified by PCR method,suggesting its potential application to PCR study.MSP results showed different DNA methylation levels of Leptin and TNF-α genes promoter regions from various samples.Conclusions The improved kit method can effectively extract DNA from dried blood spots samples,and these DNA can be used in methylation research.The study can provide a new research direction and technical method to reveal the pathogenesis of disease from the perspective of DNA methylation.
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Objective To evaluate the role of extracellular signal-related kinase (ERK) 1/2 signal transduction pathway at the supraspinal level in maintenance of neuropathic pain in mice.Methods Sixty-four Kunming mice,aged 2 months,weighing 18-20 g,were randomly divided into 4 groups (n =16 each):sham operation group (group S),chronic constrictive injury (CCI) group; CCI + U0126 (MEK inhibitor) group; CCI + dimethyl sulfoxide (DMSO) group.Neuropathic pain was induced by CCI.The sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1mmintervals with 4-0 silk thread in CCI,CCI + U0126 and CCI +DMSO groups.On 5 days after CCI,5 μg U0126 (in 5 μl of 5％ DMSO) and 5％ DMSO 5 μl were injected into the lateral cerebral ventricle over 10 s in CCI + U0126 and CCI + DMSO groups,respectively,and the time of.needle retaining was 20 s.Paw withdrawal threshold to mechanical stimulation with yon Frey filament (MWT) and paw withdrawal latency to thermal stimulation (TWL) were measured before operation (baseline),before intracerebroventricular injection (T1),and at 30 min and 2,6,12 and 24h after intracerebroventricular injection (T2-6).Resuits Compared with group S,MWT was significantly decreased and TWL was shortened at T1-6 in CCI and CCI +DMSO groups,and at T1 in CCI + U0126 group (P ＜ 0.05),while no significant change in MWT and TWL was found at T2-6 in group CCI + U0126 (P ＞ 0.05).Compared with group CCI,MWT was significantly increased and TWL was prolonged at T2-6 in group CCI + U0126 (P ＜ 0.05),while no significant change in MWT and TWL was found in group CCI + DMSO (P ＞ 0.05).Conclusion ERK1/2 signaling transduction pathway at the supraspinal level is involved in maintenance of neuropathic pain in mice.
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Objective To evaluate the role of ERK1/2 signal transduction pathway in sevoflurane postconditioning-induced reduction of oxygen-glucose deprivation(OGD)injury in rat hippocampal slices.Methods Male adult SD rats weighing 80-100 g were anesthetized with ether and decapitated.The hippocampi were removed and sagittally sliced(400μm thick)and placed in artificial cerebrospinal fluid(aCSF)aerated with 95％ O2-5％CO2.Fifty hippoeampal slices were randomly divided into 5 groups(t =10 each):OGD group; 4％ sevoflurane postconditioning group(group Sevo); PD98059(specific inhibitor of ERK)group(group PD); dimethyl sulfoxide (DMSO)group; 4％ sevoflurane postconditioning + PD98059 group(group SPD).OGD was induced by incubating the slices in glucose-free aCSF aerated with 95％ N2-5％ CO2 for 15 min in group OGD.The hippocampal slices were perfused with aCSF saturated with 4％ sevoflurane for 30 min after OGD was induced in group Sevo.The hippocampal slices were perfused with aCSF containing PD98059 50 μmol/L for 10 min after OGD was induced in group PD.The hippocampal slices were perfused with aCSF containing DMSO 1 mol/L for 10 min after OGD was induced in group DMSO.The hippocampal slices were perfused with aCSF containing PD98059 50 μmol/L and aerated with 4％ sevoflurane for 30 min after OGD was induced in group SPD.The hippocampal slices were then perfused with plain aCSF for 1 h again in all the groups.The electrophysiological technique was used 1o record the amplitude of orthodromic population spike(OPS)in the stralum pyramidale of the CAI region.TTC staining was used to determine the degree of tissue injury.Results Compared with group OGD,the recovery amplitude and rate of OPS were significantly increased,and the degree of tissue injury was significantly decreased in group Sevo(P ＜0.01),while no significant change was found in each parameter in the other three groups(P ＞ 0.05).Compared with group Sevo,the recovery amplitude and tale of OPS were significandy decreased,and the degree of tissue injury was significantly increased in groups PD,DMSO and SPD(P ＜ 0.01).Conclusion ERK1/2 signal transduction pathway is involved in sevoflurane postcondilioning-induced reduction of OGD injury in tat hippocampal slices.
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ObjectiveTo investigate the effects of diazoxide pretreatment on expression of phosphatidylinositol 3-kinase(PI3K) mRNA and protein serine/threonine kinase(Akt) mRNA in rat myocardial microvascular endothelial cells exposed to hypoxia-reoxygenation (H/R).MethodsThe SD rat myocardial microvascular endothelial cells were cultured.The cells were seeded in 96-well plates ( 100μd/hole) or in 6 cm diameter dishes (2 ml/dish) with the density of 1 × 106/ml and randomly divided into 4 groups ( n =25 each):normal control group (group C),H/R group,diazoxide pretreatment group (group DZ) and diazoxide pretreatment + 5-hydroxydecanoate (5-HD,a mitochondrial ATP-sensitive potassium channel blocker) group (group DZ + 5-HD).The cells were exposed to 2 h hypoxia followed by 2 h reoxygenation.Diazoxide 100 μmol/L and diazoxide 100 μmol/L + 5-HD 100 μmol/L were added to the culture medium 2 h before hypoxia in groups DZ and DZ + 5-HD respectively.The cell viability,apoptotic rate and expression of PI3K mRNA and Akt mRNA were detected at the end of reoxygenation.ResultsCompared with group C,the cell viability was significantly decreased,while the apoptotic rate increased and expression of PI3K mRNA and Akt mRNA up-regulated in group H/R (P ＜0.05 or 0.01).Compared with group H/R,the cell viability was significantly increased,while the apoptotic rate decreased and expression of PI3K mRNA and Akt mRNA up-regulated in group DZ (P ＜ 0.05 or 0.01).5-HD could inhibit diazoxide pretreatment-induced changes mentioned above (P ＜ 0.05 or 0.01 ).ConclusionDiazoxide pretreatment can reduce H/R injury by promoting PI3K gene and Akt gene transcription through activation of mitochondrial ATP-sensitive potassium channels in rat myocardial microvascular endothelial cells.
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Objective To investigate the effects of diazoxide pretreatment on the expression of NF-κB mRNA and fractalkine (FKN) mRNA in rat myocardial microvascular endothelial cells exposed to hypoxia-reoxygenation (H/R). Methods The SD rat myocardial microvascular endothelial cells were cultured. The cells were seeded in 96-well plates (100μl/hole) or in 6 cm diameter dishes (2 ml/dish) with the density of 1 × 106/ml and randomly divided into4 groups (n = 24 each): Ⅰ normal control group (group C), Ⅱ H/R group, Ⅲ diazoxide pretreatment group (group DZ), Ⅳ diazoxide pretreatment + mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate (5-HD) group (group DZ + 5-HD). The cells were exposed to 2 h hypoxia followed by 2 h reoxygenation. Diazoxide 100 μmol/L and diazoxide 100 μmol/L + 5-HD 100μmol/L were added to the cultured medium 2 h before hypoxia in group DZ and DZ + 5-HD respectively. The cell vitality, apoptotic rate and expression of NF-κB mRNA and FKN mRNA were detected at end of reoxygenation. Results Compared with group C, the cell vitality was significantly decreased, apoptotic rate increased and the expression of NF-κB mRNA and FKN mRNA up-regulated in H/R group. Compared with group H/R, the cell vitality was significantly increased,apoptotic rate decreased and the expression of NF-κB mRNA and FKN mRNA down-regulated in group DZ. 5-HD could inhibit diazoxide pretreatment-induced changes mentioned above. Conclusion Diazoxide pretreatment can reduce H/R injury in rat myocardial microvascular endothelial cells through down-regulating the expression of NFκB and FKN, and the mechanism is related to activation of mitochondrial ATP-sensitive potassium channels.