Objective To establish an efficient HPLC method for the determination of uric acid in plasma of hyperuricemia model mice,and the evaluation of uric acid lowering effect of Lesinurad.Methods The Laballiance Series Ⅲ HPLC system was adopted with Kromasil C18 column (100 mm × 4.6 mm,5 μm).The mobile phase consisted of methanol-0.5％ acetic acid (10:90) for isocratic elution with a flow rate of 0.4 mL/min.The detection wavelength was set at 283 nm.The established HPLC method was used to detect the plasma uric acid level of mice at 0.5,1.0,and 2.0 h time points after which being ip injected with 250 and 500 mg/kg uric acid.Lesinurad of 250 and 500 mg/kg was ig given to mice,0.5 h later,mice were ip injected with 500 mg/kg uric acid to establish hyperuricemia model,and 1 h later,the established HPLC method was used to detect the plasma uric acid level of mice.Results There was a good linear relationship between peak area and the concentration of plasma uric acid in the range of 7.5-150 μg/mL (r =0.997).The specificity,repeatability,precision,stability,and recovery of the established HPLC method was in accordance with the guiding rules of biological sample determination.Compared with the endogenous serum uric acid concentration of control group mice,serum uric acid concentration of 250 mg/kg dose group was significantly increased 0.5 h after ip administration with uric acid (P ＜ 0.01),and serum uric acid concentration of 500 mg/kg dose group was significantly increased 0.5,1.0,and 2.0 h after ip administration with uric acid.Compared with model group,the concentration of uric acid in plasma decreased significantly in low dosage group administered with Lesinurad (P ＜ 0.05),while decreased more significantly in high dosage group (P ＜ 0.01).Conclusion This convenient,rapid,and accurate method can be applied to the determination of uric acid in mouse plasma and the evaluation of relative drugs,which provide an efficient analysis way for establishing hyperuricemia model and screening relative drugs.

AIM:To study the induction of apoptosis by c-myc antisense oligonucleotide in osteosarcoma cell(MG-63).METHODS:The designed c-myc antisense oligonucleotide fragment was transfected into human osteosarcoma MG-63 cells.The cell growth and apoptosis were measured by the methods of MTT,FCM,HE staining and transmission electron microscopy.RESULTS:The results showed that the proliferation of human osteosarcoma MG-63 cells was inhibited and apoptotic rate was 37.92% when treated with c-myc antisense oligonucleotide at the does of 10.0 ?mol/L for 48 h.c-myc antisense oligonucleotide(10.0 ?mol/L) also inhibited the expression of c-myc protein.CONCLUSION:c-myc antisense oligonucleotide is able to induce apoptosis in human osteosarcoma MG-63 cells.

Objective To investigate the electrophysiological characteristics of myocardium after hypothermic ischemia-reperfusion (I/R) in rats with different degrees of arrhythmia using an in vitro experiment.Methods Healthy clean-grade male Sprague-Dawley rats,aged 2-3 months,weighing 300-400 g,were used in this study.The rats were sacrificed after anesthesia,and their hearts were rapidly excised.Sixteen Langendorff-perfused hearts were prepared and divided into 2 groups (n=8 each) by a random number table method:control group (group C) and hypothermic I/R group (group I/R).The hearts were made globally ischemic for 60 min followed by 30-min hypothermic (4 ℃) reperfusion to establish the model of hypothermic I/R injury.The occurrence and duration of arrhythmia and time of recovery of spontaneous heartbeat were recorded during reperfusion.The rats in group I/R were further divided into low-risk group (I/R-L group,ventricular arrhythmia score≤3 points) and high-risk group (I/R-H group,ventricular arrhythmia score＞3 points) according to the arrhythmia score.Monophasic action potential amplitude (MAPA),monophasic action potential (MAP) duration at 50％ and 90％ repolarization (MAPDs0 and MAPD90) and maximum ascending velocity (Vmax) of phase 0 in the endocardium,myocardium and epicardium of the left ventricular anterior wall were recorded at 30 min of equilibration (T0) and 15 and 30 min of reperfusion (T1,2).The effective refractory period (ERP) and ventricular fibrillation threshold (VFT) of the left ventricle were measured by programmed electrical stimulation,and the ERP/MAPD90 ratio was calculated.Results Compared with the baseline at T0,MAPA in the three layers was significantly decreased,and MAPD50 and MAPD90 were prolonged at T1,2 in I/R-L and I/R-H groups,and V in the three layers was decreased at T1,2 in I/R-H group (P＜0.05).MAPD50 and MAPD90 in the three layers were significantly shorter at T2 than at T1 in I/R-L and I/R-H groups (P＜0.05).Compared with group C,MAPDs0,MAPDg0 and ERP in the three layers were significantly prolonged at T1,2,the ERP/MAPDg0 ratio was decreased,and VFT was increased in I/R-L and I/R-H groups (P ＜ 0.05).Compared with I/R-L group,the duration of arrhythmia and MAPD90 and ERP in the three layers were significantly prolonged at T2,the ERP/MAPDg0 ratio was decreased,and VFT was increased in group I/R-H (P＜0.05).Conclusion Myocardial depolarization is inhibited,repolarization duration is prolonged,and electrophysiological stability is decreased after hypothermic I/R in the rats with arrhythmia,and the prolongation of myocardial repolarization and decrease in electrophysiological stability are more obvious in the rats at high risk of arrhythmia.

Objective To evaluate the effect of sevoflurane on the electrophysiological stability of i-solated rat hearts subjected to hypothermic perfusion. Methods Clean-grade healthy adult male Sprague-Dawley rats, weighing 280-360 g, were heparinized and anesthetized with pentobarbital sodium. Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95％ O2-5％CO2 at 37℃. Twenty-four Langendorff-perfused hearts were divided into 4 groups ( n=6 each) using a ran-dom number table method: control group ( C group ) , sevoflurane group ( S group ) , 32 ℃ hypothermia group ( H group) and 32℃ hypothermia plus sevoflurane group ( HS group) . After 15 min of equilibration, the isolated hearts were continuously perfused for 30 min with K-H solution at 37℃, with K-H solution con-taining 2. 3％ sevoflurane at 37 ℃, with K-H solution at 32 ℃, and with K-H solution containing 2. 3％sevoflurane at 32℃ in C, S, H and HS groups, respectively. Heart rate and monophasic action potential in three layers of the left ventricular anterior wall were recorded at 15 min of equilibration ( T0 ) and 30 of con-tinuous perfusion ( T2 ) , the transmural dispersion of repolarization ( TDR) were calculated, and the occur-rence of arrhythmia was observed. Results Compared with C and S groups, the heart rate was significantly decreased and TDR was enlarged at T1 , and the incidence of arrhythmia was increased in H and HS groups ( P<0. 05) . Compared with H group, TDR was significantly reduced at T1 , and the incidence of arrhythmia was decreased in HS group ( P<0. 05) . Conclusion Sevoflurane can improve the electrophysiological in-stability of isolated rat hearts subjected to hypothermic perfusion, and thus decrease the development of ar-rhythmia.

Objective To study the effects of different concentrations of sevoflurane on monophasic action potentials (MAPs)of three-layer myocardium of ischemia reperfusion in isolated rat hearts.Methods Thirty-two healthy SD male rats,weighing 280-320 g,were randomly divided into four groups after successful preparation of langendorff isolated heart perfusion model and 1 5 min perfusion and balance of K-H fluid.In the ischemia-reperfusion group(group IR),K-H fluid perfusion was stopped and balanced for 15 min and cardiac arrest was induced for 60 min with the injection of Thomas solution (4℃,20 ml/kg)while the heart was protected by the low temperature Thomas so-lution (4℃)around it.Reperfusion of Thomas solution (4℃,10 ml/kg)was performed for 30 min and the heart was resuscitated by the perfusion of K-H fluid for 60 min.In the 0.5 MAC sevoflurane group (group Sev0.5 ),K-H fluid contained 0.5 MAC sevoflurane and other procedures were the same as in group IR.1.0 MAC sevoflurane group (group Sev1.0 ),K-H fluid contained 1.0 MAC sevoflurane and other procedures were the same as in group IR.2.0 MAC sevoflurane group (group Sev2.0),K-H fluid contained 2.0 MAC sevoflurane and other procedures were same as in group IR. HR,MAPs including time course (MAPD50,MAPD90)and MAP amplitude of endocardium,mid-layer myodardium and epicardium was recorded at the time of continuous balance perfusion for 1 5 min (T0),continuous perfusion for 15 min (T1),reperfusion for 15 min (T2)and 30 min (T3). Results Compared with T0and T1,HR was slower at T2and T3(P<0.05);Compared with group IR at T2and T3,HR in group Sev0.5 and group Sev1.0 was higher,that in group Sev2.0 was slower P<0.05);At T2,arrhythmia was observed in 6 rats in group IR,while arrhythmia was observed in 1 rats in group Sev0.5 ,and arrhythmia was observed in 2 rats in group Sev1.0 and arrhythmia was observed in 1 rats in group Sev2.0;Compared with group IR at T3,MAPD50in group Sev0.5 was shorter in three sites(P<0.05);Compared with group IR at T3,MAPD90in other three groups was shorter.Conclusion Different concentrations of sevoflurane can shorten MAPD90of MAPs,and the effects don＇t depend on the concertrations of sevoflurane when it changes from 0.5 MAC to 2.0 MAC;which may be the mechanism of decreased arrhythmias risk caused by sevoflurane.

Objective:To observe the changes in the expression of microRNAs in ventricular myocardium in a rat model of hypothermic ischemia-reperfusion (I/R).Methods:Healthy clean-grade male Sprague-Dawley rats, aged 2-3 months, weighing 300-400 g, were anesthetized with intraperitoneal chloral hydrate.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95%O 2-5%CO 2.Sixteen Langendorff-perfused hearts were prepared and divided into 2 groups ( n=8 each) by a random number table method: control group (group C) and hypothermic I/R group (group I/R). The hearts were made globally ischemic for 60 min followed by 30-min hypothermic (4 ℃) reperfusion to establish the model of hypothermic I/R injury.The type and duration of arrhythmia and time of recovery of spontaneous heartbeats were recorded during reperfusion.The rats in group I/R were further divided into low-risk group (I/R-L group) and high-risk group (I/R-H group). The left ventricular myocardium was collected after the end of perfusion for high throughput sequencing to screen the differentially expressed microRNAs, and the reliability of the sequencing results was verified by quantitative real-time polymerase chain reaction.Gene Ontology and KEGG databases were used to analyze the biological regulatory pathways of differentially expressed target genes. Results:Compared with group C, there were 437 up-regulated microRNAs and 242 down-regulated microRNAs in group I/R-L and 419 up-regulated microRNAs and 260 down-regulated microRNAs in group I/R-H.Compared with group I/R-L, 392 microRNAs were up-regulated, and 287 microRNAs were down-regulated in group I/R-H.There were 84 microRNAs with absolute value of fold change ≥2 and significantly differential expression ( P<0.01) among the three groups.Subsequently, 4 microRNAs were randomly selected for validation using quantitative real-time polymerase chain reaction, confirming that the sequencing results were reliable.These differentially expressed target genes were involved in 11 biological processes and 6 KEGG pathways which were related to reperfusion arrhythmia.Potassium ion transmembrane transport and the adrenergic receptor signaling pathway in cardiomyocytes were enriched by the largest number of target genes. Conclusion:The expression of microRNAs in ventricular myocardium changes significantly after heart hypothermic I/R.These differentially expressed microRNAs regulate potassium ion transmembrane transport probably and mainly through the adrenergic receptor signaling pathway in the cardiomyocytes and thus are involved in the occurrence and development of hypothermic I/R arrhythmias.

Objective:To evaluate the effect of electroacupuncture (EA) on electrophysiological characteristics of ventricular myocardium during myocardial ischemia-reperfusion (I/R) in rats.Methods:Twenty-four clean-grade healthy adult male Sprague-Dawley rats, weighing 280-320 g, were divided into 3 groups ( n=8 each) using a random number table method: sham operation group (group SH), I/R group and group EA.The model of myocardial I/R injury was established by ligating the left anterior descending branch of coronary artery for 30 min followed by 30-min reperfusion in anesthetized rats.Bilateral Neiguan acupoints in forelimbs were stimulated for 30 min during the period of reperfusion in group EA.Heart rate (HR), monophasic action potential amplitude (MAPA), maximum depolarization rate (V max), and monophasic action potential duration at 90% repolarization (MAPD 90) were recorded.The development of arrhythmias and arrhythmias score were recorded during reperfusion. Results:Compared with group SH, HR was significantly decreased, MAPA and V max were decreased, MAPD 90 was prolonged, and the incidence of ventricular premature beat, ventricular tachycardia and ventricular fibrillation and arrhythmias score were increased at T 1, 2 in I/R and EA groups ( P<0.05). Compared with group I/R, HR was significantly increased, MAPA and V max were increased, MAPD 90 was shortened, and the incidence of ventricular tachycardia and ventricular fibrillation and arrhythmias score were decreased at T 2 in EA group ( P<0.05). Conclusion:EA can accelerate myocardial depolarization and shorten repolarization, thus decreasing the occurrence of reperfusion arrhythmia in rats.

Objective:To evaluate the changes in the electrical conduction of ventricular myocardium during hypothermic ischemia-reperfusion (I/R) in rats with arrhythmia.Methods:Healthy clean-grade adult male Sprague-Dawley rats, aged 2-3 months, weighing 200-300 g, were studied.The hearts were removed and retrogradely perfused with oxygenated K-H solution in a Langendorff apparatus. Sixteen isolated hearts were divided into 2 groups ( n=8 each) using a random number table method: normal control group (group C) and hypothermic I/R group (group I/R). In group C, the heart was perfused with K-H solution at 37 ℃ for 120 min.In group I/R, the heart was perfused with K-H solution at 37 ℃ for 30 min, and then perfusion was stopped, cardiac arrest was induced through injecting Thomas solution (4 ℃), the area around the heart was protected with low temperature (4 ℃) Thomas solution, and hearts were perfused with 4 ℃ Thomas solution at 30 min after cardiac arrest and with 37 ℃ K-H solution for 30 min staring from 60 min after cardiac arrest.The rats in group I/R were further divided into high-risk subgroup (I/R-H subgroup) and low-risk subgroup (I/R-L subgroup). The time of spontaneous recovery of heart beat and development of arrhythmia were recorded.At the end of reperfusion, the atrioventricular conduction 2∶1 block point (2∶1B) and ventricular electrical conduction velocity (CV) were measured and recorded by program-controlled electrical stimulation. Results:Compared with group C, CV and 2∶1B were significantly decreased in IR-L and IR-H subgroups ( P<0.05). Compared with IR-L subgroup, the time for restoration of spontaneous heart beat was significantly prolonged, the incidence of ventricular fibrillation and arrhythmia score were increased, and CV and 2∶1B were decreased in IR-H subgroup ( P<0.05). Conclusion:The electrical CV of ventricular myocardium is decreased during hypothermic I/R, which may be the mechanism of reperfusion-induced ventricular arrhythmia in rats with arrhythmia.

Objective:To determine the changes in the expression of myocardial miRNA and the target genes in the rats with hypothermic ischemia-reperfusion (I/R) arrhythmia.Methods:Clean-grade healthy male Sprague-Dawley rats, aged 2-3 months, weighing 300-400 g, were anesthetized, the chest was opened, and the heart was taken to establish an isolated heart perfusion model.Six successfully perfused isolated hearts were divided into 2 groups ( n=3 each) using a random number table method: control group (group C) and heart I/R group (IR group). The model of hypothermic global I/R injury was established by interrupting perfusion for 60-min followed by 30-min reperfusion in chloral hydrate-anesthetized rats.The arrhythmia score was recorded during reperfusion.High-throughput sequencing was used to identify the differentially expressed miRNAs in two groups.The RNAhybrid and miRanda databases were used to predict the target genes of mRNA regulated by the differentially expressed miRNAs, and the enrichment for target genes was performed by Gene Ontology and KEGG databases, and the miRNAs closely related to arrhythmia and with higher expression were selected to carry out the real-time polymerase chain reaction detection. Results:The results of high-throughput sequencing showed that there were 7 differentially expressed miRNAs (novel-miR-17, novel-miR-19, novel-miR-30, novel-miR-43, rno-miR-122-5p, novel-miR-16 and rno-miR-429) in group IR as compared with group C. There were 4 miRNAs that were closely related to arrhythmia and had higher expression: the expression of novel-miR-17, novel-miR-30 and rno-miR-122-5p was significantly up-regulated, and the expression of rno-miR-429 was down-regulated in group IR when compared with group C ( P<0.05). The miRNA-mRNA correlation analysis revealed that GJA1 gene was the target of novel-miR-17. Conclusion:Myocardial novel-miR-17 is involved in the occurrence of hypothermic I/R arrhythmia probably by acting on GJA1 gene in rats.

Objective: To understand the prevalence of low vision among Tujia and Han children and adolescents in Tujia inhabited areas, and to provide reference for the prevention and control of myopia in children and adolescents in minority areas. Methods: A cluster sampling of Tujia and Han primary school students from two primary schools in Lichuan City, Enshi Tujia and Miao Autonomous Prefecture, Hubei Province (2 466 Tujia and 971 Han) were selected for visual acuity assessment. Univariate χ 2 test and multivariate Logistic analysis were used. Low vision and associated factors between Tujia and Han nationality were compared. Results: The overall detection rate of low vision among children and adolescents in Tujia inhabited areas was 44.9%. There were differences in the degree of low vision in the left and right eyes of individuals, and the detection rate of low vision varied significantly by ethnic, gender and grade ( χ 2=22.10, 18.43, 19.06, 17.97 for the left eye, 17.52, 20.44, 21.49, 18.61 for the right eye, P < 0.05). There were many factors affecting low vision among children and adolescents in Tujia inhabited areas, overweight and obesity were negatively associated with low vision ( OR=1.81, 1.70, 95%CI=1.76-1.92, 1.66-1.82, P <0.01). Conclusion Low vision is highly prevalent in Tujia children and adolescents. Effective intervention measures should be taken to treat and prevent myopia in children and adolescents.