AIM:To investigate the role of bcl-2 and bax expression in apoptosis of pulmonary artery smooth muscle cells(PASMC) of rats induced by Na +/H + exchanger isoform-1(NHE-1) inhibition in vitro. METHODS:Intracellular Ca 2+ concentration ([Ca 2+ ]i) was measured using Fura-2/AM. The expression of bcl-2 and bax mRNA in cells were detected by sqRT-PCR, and the expression of Bcl-2 and Bax protein were examined immunohistochemically. RESULTS:The expression of bax mRNA and Bax protein and [Ca 2+ ]i in cells transfected with NHE-1 ribozyme gene increased significantly compared with those of cells transfected with pLXSN and nontransfected control. Meanwhile, the expression of bcl-2 mRNA and Bcl-2 protein in cells transfected with NHE-1 ribozyme gene decreased significantly. CONCLUSION:Apoptosis of pulmonary artery smooth muscle cells induced by inhibiting NHE-1 may have relevance to the increase in [Ca 2+ ]i and bax expression and the decrease in bcl-2 expression.

Objective To study the anti-inflammatory effects of idazoxan (IDA) on endotoxin lipopolysaccharide (LPS) challenged mice in vivo and activated macrophages in vitro,and explore its potential molecular mechanisms.Methods To do the experiments in vivo,30 adult male C57BL/6 mice were randomly divided into control group,model group,and low,medium and high doses IDA groups (IDA-L,IDA-M,and IDA-H groups),n =6 in each group.The inflammatory model was reproduced by intraperitoneal injection of LPS 10 mg/kg,and the control group was injected with the same amount of normal saline.The IDA groups received LPS (10 mg/kg) and IDA 0.3,1.0 and 3.0 mg/kg,respectively.The blood samples of mice in each group were collected at 6 hours after the reproduction of the model.For the in vitro experiments,primary peritoneal macrophages were collected from 20 adult male C57BL/6 mouse cells and they were divided into control group,LPS group (10 mg/L) and LPS+IDA-L,IDA-M,IDA-H groups (10 mg/L LPS + 5,25,100 μmol/L IDA,respectively).Cell culture supernatants were collected at 24 hours after the reproduction of the model.Detection methods:enzyme linked immunosorbent assay (ELISA) was used to determine the levels of serum tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6),monocyte chemotactic protein-1 (MCP-1) and nitric oxide (NO).Western Blot was used to determine the effect of IDA on the expression levels of nuclear factor-κB (NF-κB) in macrophages.Results ① For the in vivo experiment,the serum levels of TNF-α and IL-6 were significantly elevated in the model group as compared with those in the control group [TNF-o (ng/L):403.96 ± 40.98 vs.17.50 ± 8.68;IL-6 (ng/L):61 400.31 ± 7 826.61 vs.2 436.30 ± 448.89;both P ＜ 0.01].IDA treatment could inhibit the elevation of inflammatory cytokines in a dose-dependent manner,with the most significant decrease in LPS+IDA-H group [TNF-α (ng/L):170.09 ± 28.53 vs.403.96 ± 40.98,IL-6 (ng/L):16 570.81 ± 1 083.65 vs.61 400.31± 7 826.61;both P ＜ 0.01].② For the in vitro experiment,the levels of TNF-α,IL-6,MCP-1,and NO secreted by LPS-stimulated macrophages were distinctly higher in the LPS group than those in the control group [TNF-α (ng/L):7 259.14 ± 320.70 vs.28.50±27.08,IL-6 (ng/L):14809.60±5852.73 vs.1 113.47±465.53,MCP-1 (ng/L):20847.37± 1 788.33 vs.447.37± 395.69,NO (μmol/L):1 900.00 ± 144.31 vs.603.03 ± 102.18;all P ＜ 0.01].However,IDA intervention could lower the secretion of TNF-α,IL-6,MCP-1 and NO in a dose-dependent manner,with the most notable decrease in the LPS+IDA-H group [TNF-α (ng/L):784.40±281.90 vs.7259.14±320.70,IL-6 (ng/L):1 802.96± 1 534.18 vs.14 809.60± 5 852.73,MCP-1 (ng/L):2005.26± 1 534.28 vs.20847.37 ± 1 788.33,NO (μ mol/L):654.54± 150.21 vs.1 900.00 ± 144.31;all P ＜ 0.05].In addition,IDA at the concentration of 100 μmol/L could promote the translocation of NF-κBp65 in macrophages into the nucleus 15 minutes early and lead to increased NF-κBp65 expression (gray value:18.70 ± 2.29 vs.1.09 ± 0.36,P ＜ 0.05),hut significantly reduce the expression levels of NF-κBp50 in the nucleus at 45 minutes after treatment (gray value:1.99 ± 0.14 vs.2.94 ± 0.54,P ＜ 0.05).Conclusions IDA could significantly reduce inflammation of mice challenged with LPS and inhibit inflammatory cytokines and mediators secreted by macrophage in a dose-dependent manner.High concentration of IDA (100 μmol/L) exhibited the greatest anti-inflammatory effects.The anti-inflammatory effect of IDA may be worked through NF-κB signaling pathway.

Objective To explore the effects of hammerhead ribozyme on the expression and activity of NHE 1 and pHi in pulmonary artery smooth muscle cells (PASMCs) of rats and its role in PASMCs proliferation in vitro . Methods According to the secondary structure of NHE 1 mRNA in rats, NHE 1 specific hammerhead ribozyme was designed with the assistance of computer. The recombinant vector of retroviral plasmid pLXSN and hammerhead ribozyme, PRZ, was transfected into the cultured PASMCs. G418 resistant cell clones were screened with 100 ?g/ml G418. Then, the expression of NHE 1 mRNA was detected by RT PCR, intracellular pH(pHi) value and recovery rate of pHi after intracellular acid loading were measured by fluorescent probe BCECF. 22 Na uptake and 3H TdR incorporation were determined, respectively. Results Compared with the cells transfected with pLXSN and non transfected cells, NHE 1 mRNA, pHi value, pHi recovery rate, 22 Na uptake and 3H TdR incorporation decreased significantly in cells transfected with recombinant vector PRZ. No significance was found between the pLXSN transfected group and non transfected group. Conclusion Hammerhead ribozyme can cleave the target NHE 1 mRNA specifically, reduce the expression of NHE 1 mRNA, induce intracellular acidosis and consequently suppress the proliferation of PASMCs.

Objective To detect the strain features of regional wall abnormalities in patients with coronary artery disease during each diastolic period, and its possibility to evaluate regional left ventricular cardiocyte viability and diastolic function.Methods 54 patients with anterior myocardial infarction (MI group) and 78 normal subjects (NOR group) underwent Doppler tissue imaging, which were performed in 2-chamber-view by strain curves synchronously.Results In the NOR group, strain value of 66 cases (84.62%) showed an gradually increasing negative value from the apex to base to middle of left ventricle, while in the MI group, there were 9 cases (16.67%) with such a trend. IR phase: in the NOR group, 564 segments (90.38%) were upward wave bands, but in the MI group, there were 123 segments (28.47%) having such waves ( P<0.05). RF phase: in the NOR group, 576 segments (92.31%) were upward and steep wave bands, but in the MI group, the number of upward waves were obviously less (102 segments,23.61%) ( P<0.01). SF period: compared with the NOR group, which was horizontal, the MI group had upward wave bands ( P<0.05).Conclusion Regional myocardial ischemia and infarction can cause significant regional diastolic wall abnormalities of strain value in active diastolic phase. Regional diastolic wall motion abnormalities can be evaluated quantitatively and synchronously with high sensitivity by strain curve which has the potential value in cardiocyte viability and diastolic function.

Objectives To identify the COL7A1 gene mutation in a recessive dystrophic epidermolysis bullosa (RDEB) family, and to perform prenatal diagnosis in the patient's offspring. Methods The genomic DNA, obtained from the patient and his wife, was used to screen all 118 exons of the type VII collagen gene (COL7A1) via polymerase chain reaction (PCR) followed by direct DNA sequencing of the PCR products. Fetal DNA was extracted from amniotic fluid of the patient's wife at the 15th week of gestation. PCR, direct DNA sequencing and restriction fragment length polymorphisms (RFLPs) were performed for prenatal diagnosis. Results The patient in this study was a compound heterozygote for a S48P missense mutation in exon 2 and an 11 base pair deletion (3625del11) leading to a premature termination codon (PTC) in exon 27, which are a novel combination of COL7A1 mutations in RDEB. The COL7A1 genotype of his wife was normal. In the fetus, the same deletion of 11 base pair (3625del11) was found in exon 27, but no mutation was found in exon 2. Thus, the fetus was predicted to be a clinically normal child with a carrier genotype. Seven months later, a clinically unaffected male infant was born and the prediction was confirmed. Conclusion We successfully performed the first DNA-based prenatal diagnosis in China in a family with Hallopeau-Siemens RDEB.

Objective To study mRNA expression of neuroglobin (NGB) in thalamus of DAI rats and explore the relation between NGB and heed injury. Method 46 male SD rats were randomly divided into four groups. The DAI model was established according to modi-fied Mamamu method. The mRNA expression of NGB in thalamus at different time point after injury was detected by RT-PCR method. All data were analyzed with statistical software. Result The mRNA expression of NGB in thalamus increased obviously from 30 minutes after in-jury while decreased at the 24th hour after injury. But the expression increased again and reached peak at the 48th hour after injury. Conclu-sion The mRNA expression of NGB in thalamus increased after DM. Those changes suggested NGB might participate in the response mech-anism of stress, secondary ischemia and hypoxic brain injury after neuron injury.

Objective To evaluate the clinical efficacy of universal self-locking anatomical plate for acetabulum and pelvis (USAPAP) in the treatment of posterior acetabular fractures.Methods Data of 55 patients with posterior acetabular fractures who were treated with the USAPAP from January 2014 to January 2016 were retrospectively analyzed.There were 39 males and 16 females with an average age of 38.5 years old (range,17-82 years).There were 35 fractures on the left side while the other 20 fractures were on the right side,including 52 fresh cases and 3 old cases.According to Letournel-Judet classification,there were 32 cases of posterior wall,9 cases of transverse and posterior wall,5 cases of anterior column and posterior wall,2 cases of posterior column,2 cases of transverse,2 cases of two columns,1 case of anterior column and posterior hemitransverse,2 cases of Pipkin type Ⅳ.All patients were managed operatively by the USAPAP,which allows simultaneous fixation for two columns and quadrilateral surface fractures through a single Kocher-Langenback approach.The quality of reduction was assessed by Matta's score system.The mean follow-up period was 18.5 months (range,12-24 months).Average operation time was 95 min,and average blood loss was 350 ml.Average operation time of plate and screws fixation was 19 min,and average fluoroscopy times in the surgery was 2 times.The mean time of bony union was 4.3 months.According to the criteria described by Matta,the excellent and good rate of radiological score was 92.7％ (51/55),including 39 cases excellent,12 cases good and 4 cases poor.The excellent and good rate of Merle d'Aubigne-Postel score was 87.3％ (48/55),including 35 cases of excellent,13 cases of good,5 cases of fair,and 2 cases of poor.Heterotopic ossification occurred in 1 patient (Brooker type Ⅱ) at three months postoperatively.One case with Pipkin type Ⅳ fracture underwent total hip arthroplasty due to femoral head necrosis at seven months postoperatively.Complications including wound infection,sciatic nerve injury,screw invade joint,redisplacement of the fracture,avascular necrosis of bone fragment,hardware failure,nonunion or malunion was not found in any case at the latest follow-up.Conclusion The USAPAP through the single posterior Kocher-Langenback approach provides strong and stable fixation for complex acetabular fractures associated with posterior region including posterior wall,both columns and quadrilateral surface.Satisfactory clinical results can be achieved by the use of this method.

Objective To investigate the effect of propofol on rat′s limb ischemia reperfusion injury .Methods Sixty healthy SD rats were randomly divided into sham operate group ,ischemia-reperfusion group and propofol group (n= 20) ,each group was divided into 4 subgroups according to the different reperfusion time .To copy the right lower limb ischemia reperfusion model ,5 min before reperfusion ,use propofol injection (50 mg/kg ,intraperitoneal inject) ,various subjects in the corresponding time points (3 ,6 , 9 ,12 h) were sacrificed .TNF-α ,NF-κB of blood and MDA ,SOD of Skeletal muscle were measured ,calculate muscle wet dry weight ratio .Results Compared with ischemia reperfusion group ,propofol could significantly reduce expression of TNF-alpha ,NF-κB lev-els in serum (P< 0 .05) ,inhibit the increase of the MDA level and decrease of the SOD level in muscle (P< 0 .05) ,also reduce the extent of skeletal muscle cell edema(P< 0 .05) .Conclusion Propofol can attenuate limb ischemia reperfusion injury by inhibiting inflammation response and reducing the oxygen free radicals′ damage .

Objective To discuss the clinical effects of transverse screw fixation of double columns in crescent pelvic fractures.Methods Twenty patients with crescent pelvic fractures hospitalized from December 2012 to December 2015 were reviewed retrospectively.There were thirteen male and seven female patients with the age ranging from 22 to 72 years (mean,39.5 years).Causes of injury were traffic accidents in fifteen patients,falling from high places in three and hitting by heavy objects in two.Time interval between injury and operation was 3-14 d.According to the Orthopedic Trauma Association (OTA) classification,all were classified as 61-B2 type.Reconstruct plate was used to stabilize the supperior pubic ramus fractures,and transverse double-column fixation with anterograde or retrograde screws was used for the posterior iliac crescent fractures.Duration of inserting screws,operation time,intraoperative blood loss and frequency of C-arm X-ray were recorded.Radiological and clinical outcomes were evaluated after operation.Results Duration of inserting screws ranged from 3 to 16 min (mean,5 min).Operation time ranged from 50 to 130 min (mean,80 min) and the intraoperative blood loss ranged from 200 to 550 ml (mean,280 ml).Frequency of C-arm X-ray in the surgery ranged from 1 to 5 times (mean,2 times).All screws were in the expected location,without any piercing out of the iliac bone dependent on the confirmation of X-ray and CT-scan after operation.According to the Matta and Tornetta radiological evaluation,the reduction was rated excellent in sixteen patients and good in four,with the excellent-good rate of 100％.There was no death,wound infection,sciatic nerve or superior gluteal nerve injury or deep venous thrombosis of lower extremities.At the followup,no nonunion,loss of reduction,and breakage of internal fixation occurred.Nineteen patients were followed up,and mean follow-up time was 19.6 months (range,3 to 36 months).Union was obtained in all patients in a period of 8-17 months (mean,11.2 months).According to the Majeed functional evaluation at the final follow-up,the outcome was rated excellent in seventeen patients and good in two,with the excellent-good rate of 100％.Conclusion Transverse screw fixation of double columns in crescent pelvic fracture of 61-B2 type can reduce the operation injury,decrease complications,and have good clinical results.

OBJECTIVE:To establish a method for the determination of related substances in lorcaserin hydrochloride.METHODS:HPLC method was adopted.The determination was performed on Welch ultimate XB-ODS column with mobile phase consisted of Sodium dihydrogen phosphate (pH =6.5)-acetonitrile (gradient elution) at the flow rate of 1.0 mL/min.The detection wavelength was set at 220 nm,and column temperature was 35 ℃.The sample size was 20 μL.RESULTS:The linear ranges of impurity 1,2,3 were 0.075 60-7.560 μg/mL(r=0.999 9),0.081 40-8.140 μg/mL(r=0.999 9),0.099 24-9.924 μg/mL(r=0.999 9),respectively.The limits of quantification were 0.075 60,0.081 40,0.099 25 μg/mL.The limits of detection were 0.022 68,0.024 42,0.029 77 μg/mL.RSD of precision test was lower than 2.0％.Impurity 1 was found in stability test and reproducibility test,RSD＜2.0％.The recoveries were 98.53％-102.45％ (RSD=1.06％,n=9),98.26％-101.64％ (RSD=1.03 ％,n=9),100.08％-102.10％ (RSD=0.70％,n=9),respectively.CONCLUSIONS:The method is sensitive,rapid,accurate and reliable,which can be used to determine the related substances of lorcaserin hydrochloride.