OBJECTIVE:To develop a high performance liquid chromatography-mass spectrography method (LC-MS/MS) for determination of huperzine A in human serum. METHODS:The clonidine was used as the internal standard,and the test serum samples after being liquid-liquid extracted by ethyl acetate,dried in organic phase and reconstituted in mobile phase were analyzed on a Phenomenex Luna CN column interfaced with LC-MS with Methanol-0.08% formic acid (46∶54 containing 0.2% ammonium acetate). Positive ion SRM detection was performed for ESI iron source. Target ions were at m/z 243. 2→210. 9 for Huperzine A with an impact energy of 30V and m/z 229. 9→212. 9 for the internal standard with an impact energy of 32V during 0.5 s (scan time). RESULTS:The assay was proved to be free from the interference from endogenous substance,and the linear concentration range of Huperzine A was 0.08～8 ng?mL-1(r=0.993 0～0.996 0). The intra-and inter-day precision over the entire concentration range were less than 15%. At low,middle and high concentrations,the mean extraction recoveries of Huperzine A were 69.9%,63.7% and 63.7%,respectively. CONCLUSION:The LC-MS/MS method is rapid,sensitive,simple,accurate and in which little amount of mediator is needed,thus meeting the requirement for the detection of biological specimen and applicable for the treatment of large batch of serum samples in clinical pharmacokinetic study.

Objective To discuss the prognostic impact of atrial fibrillation (AF) in patients with chronic heart failure(CHF).Methods From January,2005 through December,2006 we studied 461 patients with a discharge diagnosis of CHF.Patients were divided by baseline rhythm in sinus rhythm(SR) or AF groups.The main endpoints were all cause death within 3 years and readmission to the hospital for heart failure.Results AF group were more likely to be older and female and to have a history of valvular disease and have a higher left ventricular ejection fraction (LVEF).However,SR group were more likely to have a history of ischemic heart disease.During 3 year follow-up,the main endpoints was higher in AF group than in those with SR group (P ＜0.01).After adjusting for other covariables,AF and age were related to increased the risk of main endpoints during 3 years follow-up (RR =1.311,95％ CI:1.002-1.715,P ＜0.05 ; RR =1.014,95％ CI:1.004-1.024,P ＜ 0.01).Conclusion AF and age were the main risk factors of increased adverse forward prognosis in patients with CHF.

AIM:To investigate the effect of activation of retinoid X receptor (RXR) on transforming growth factor β1 (TGF-β1) induced collagen synthesis under hypoxic environment in rat cardiac fibroblasts (CFs) and underlying molecular mechanisms .METHODS: CFs were cultured using myocardial tissue with dry method .Hypoxic environment was established for CFs by continuous nitrogen supplement .Type I and type III collagens in supernatants were detected by ELISA.Nuclear and cytoplasmic extractions were prepared using NE-PER nuclear and cytoplasmic extraction reagents .The protein levels of Smad2 and p-Smad2 were determined by Western blot and immunocytochemical staining .RESULTS:Un-der hypoxic condition , TGF-β1 (0.01~10 μg/L) increased the synthesis of type I and type III collagens in a dose-de-pendent manner in the CFs .At the concentration of 5μg/L, the synthesis of collagen I and III was significantly increased as compared with control group (P<0.01).RXR agonist 9-cis-retinoic acid (9-cis-RA;10 -9 ~10 -6 mol/L) decreased TGF-β1 (5μg/L)-induced synthesis of type I and III collagens in a dose-dependent manner in the CFs under hypoxic con-dition.The synthesis of type I and type III collagens was significantly inhibited by 9-cis-RA (P<0.01).Smad2 inhibitor ( 20 nmol/L) showed similar inhibitory effect on the synthesis of type I and III collagens induced by TGF -β1 under hypoxic condition.Compared with TGF-β1 intervention group, the cytoplasmic level of p-Smad2 in the CFs was significantly in-creased in TGF-β1+9-cis-RA group, but the nuclear p-Smad2 level was significantly decreased (P<0.05).CONCLU-SION:Retinoid X receptor agonist 9-cis-RA inhibits TGF-β1-induced synthesis of type I and type III collagens in the CFs by repressing p-Smad2 nuclear translocation under hypoxic condition .

Objective:To observe the effect of Radix Paeoniae Bubra(RPB)on NADPH oxidase p22phox,monocyte chemotactic protein-1(MCP-1)mRNA expression and neointimal hyperplasia after carotid artery balloon injury in cholesterol-fed rabbits.Methods:The rabbit model of carotid balloon injury was established adopting Clowes method,and treated with extract of RPB.Component of neointima and expression of proliferating cell nuclear antigen(PCNA)and macrophage was determined by immunochemical stain.The collagen of typeⅠwas detected by special staining for blood vessels and the area of neointima was measured by image assay system.Expression of NADPH oxidase p22phox mRNA,MCP-1 mRNA was detected by in situ hybridization and transcription-polymerase chain reaction.Results:RPB can attenuate the neointima area and proliferation of collagen typeⅠinduced by balloon-injury,remarkable prevent the formation of restenosi,and down-regulate expression of NADPH oxidase p22 and MCP-1mRNA significantly and decrease the degree of macrophages infiltration especially in vessel wall of injuring carotid artery.Conclusions:RPB inhibited NADPH oxidase P22Phox and MCP-1 mRNA expression,and modestly reduced neointimal hyperplasia,which might be partly attributed to its antioxidant and inflammatory effects.

Objective:To observe the effect of Radix Paeoniae Rubra on intimal proliferation,activating of angiotensin Ⅱ(AgⅡ) and expression of Mitogen-activated protein kinase phosphatase-1(MKP-1) after carotid artery balloon injury in cholesterol-fed rabbits.Methods: Male rabbits were randomly divided into Radix Paeoniae Rubra(PPR) groups: high dose group(75、50、25g.kg-1.d-1;n=8),middle dose group(2g.kg-1.d-1),low dose group(1g.kg-1.d-1;n=8) and control group.Both groups received high fat forage(2% cholesterol + 5% lard).Balloon injury of carotid artery was performed.Carotid artery were harvested at the end of 10 weeks.Expression level of AgⅡwas measured by radioimmunoassay.MKP-1 expression was determined by RT-PCR.Immunohistochemical staining and morphological detection were adopted.Results: Compare with control group,expression of AgⅡ decreased obviously(P

Objective To establish a high speed and effective real-time PCR assay to analyze apolipoprotein E(apoE)genotyping in Chinese population using hybridization probes and melting curves. Methods Lightcycler was used to analyzed two codons'polymorphism after condition was optimized. The persons elder than 60 years including 133 patients with abdominal fat and 108 healthy elder were selected. The detection probes were labeled with L-Cred 640 and LC-Red 705 at 5'end covers codons 112 and 158 with the corresponding anchor probes labeled with fluorescein at 3'ends.A 265-bp fragment of the apoE gene was amplified from human genomic DNA to produce FRET.Depending on the various types of base-pair mismatch in the heteroduplex,wild type and mutant type were differentiated.Results The peaks represented the sequence-specific melting points(Tm) and each genetype showed perfect peak.E2/3 and E3/4 in abdominal fat group were much more common allele than health persons(x2=4.210.P＜0.004,x2=6·328,P＜0.012).The frequencies of abdominal fat group was E2/3(27.8%),E3/4(24.8%),E3/3(42.1%),E2/4(2.3%),E4/4(2.2%)and E2/2(0.8%).The frequencies of healthy controls were E2/3(16.7%),E3/4(12%),E3/3(68.5%),E2/4(1.9%),E4/4(O%)and E2/2(0.9%).It showed high agreement as compared with DNA sequencing analysis The expression of apoE in abdominal fat group (101.5±73.6)was up-regulated than the healthy group(50.6±27.1,P＜0.01).Conclusions Apolipoprotein E genotyping method by melting curve is faster and simpler than other technique. It can prevent the cross-contaminated and is suitable to be applied in clinical diagnosis.There was significant difference between the two groups.There was positive relationship between the elder's abdominal fat and apoE gene polymorphism. The genotyping of E3/4.E2/3 or E4/4 had the important role in the elder's abdominal fat on genetic susceptibility.

AIM:To explore the effects of atorvastatin (Atorv) on atherosclerosis in streptozotocin (STZ)-in-duced diabetic apolipoprotein E knockout ( ApoE-/-) mice with fat-rich diet and the possible mechanism .METHODS:C57 mice served as control.ApoE-/-mice (n=34) fed with high-fat diet were randomly divided into ApoE-/-group, STZ-ApoE-/-group and STZ-ApoE-/-+Atorv group.Intraperitoneal injection of streptozotocin was performed to create di-abetic animal model .Blood glucose was determined by glucose oxidase method .Blood lipid levels were detected by enzymic method or selective homogeneous method .The plaque area in the thoracic aorta was measured by HE staining .The protein level of nicotinamide-adenine dinucleotide phosphate ( NADPH) oxidase subunit gp91phox in the thoracic aorta was deter-mined by Western blotting .The levels of reactive oxygen species ( ROS) in blood and thoracic aorta homogenates were de-tected by Fenton reaction and Griess reagent .Human umbilical vein endothelial cells ( HUVECs ) were isolated from healthy umbilical cords by collagenase I and cultured .ROS production was detected by flow cytometry .NADPH oxidase ac-tivity was measured using lucigenin assay .Effects of retinoid X receptor α( RXRα) on inhibition of oxidative stress by ator-vastatin were evaluated by RNA interference and plasmid transfection .RESULTS: (1) Compared with C57 group, the plaque areas of the thoracic aorta in ApoE-/-group were increased .No difference of the fasting glucose between the 2 groups was observed.The levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), thoracic aorta gp91phox protein and ROS in blood and thoracic aorta homogenates were higher in ApoE-/-group than those in C57 group.(2) Compared with ApoE-/-group, the plaque areas of the thoracic aorta in STZ-ApoE-/-group were further enlarged [(314.13 ±35.72) μm2 vs (215.88 ±34.19) μm2, P<0.05].The levels of blood glucose, TG, TC and LDL-C, thoracic aorta gp91phox protein and ROS in blood and thoracic aorta homogenates were higher in STZ-ApoE-/-group than those in ApoE-/-group (P<0.05).(3) Compared with STZ-ApoE-/-group, the plaque areas of the thoracic aorta in STZ-ApoE-/-+Atorv group were reduced [(217.47 ±24.56) μm2 vs (314.13 ±35.72) μm2, P<0.05].The levels of blood glucose , LDL-C, TC, HDL-C and TG showed no significant difference between the 2 groups.Thoracic aorta gp91phox protein level and ROS production in blood and thoracic aorta homogenates were lower in STZ -ApoE-/-+Atorv group than those in STZ-ApoE-/-group (P<0.05).(4) High glucose-induced increases in NADPH oxidase activity and gp91phox expression were significantly inhibited by atorvastatin (10-6 mol/L) in HUVECs.The inhibitory effects of atorvasta-tin on high glucose-induced ROS production and NADPH oxidase activation were largely impaired when the cells were trans -fected with RXRαsiRNA.However , the effect of atorvastatin was significantly strengthened when RXRαwas over-expressed in the HUVECs transfected with RXRαplasmid.CONCLUSION: Atorvastatin inhibits atherogenesis by depressing high glucose-induced oxidative stress in diabetic ApoE-/-mice with fat-rich diet.The anti-oxidative stress effect of atorvastatin is mediated by RXRα.

Objective To compare the importance of heart murmurs and transcutaneous oxygen saturation (SpO2)in screening neonatal congenital heart diseases(CHD),and to explore a reliable and easy-to-use method for the screening of CHD. Methods All infants (16 070 cases) born in Changzhou Maternal and Child Health Hospital from May 1 st 2011 to April 30 th 2013 were screened of CHD for heart murmurs and SpO2 . The screen-positive infants were divided into 3 groups:heart murmurs group,abnormal SpO2 group and mixed group. All screen-positive infants re-ceived cardiac color ultrasound to make a definite diagnosis of CHD,so as to compare the values of heart murmurs and SpO2 . Results Eight thousand seven hundred and ninety-nine boys and 7 271 girls were screened,in which the ratio was 1. 21:1. 00. One hundred and eighteen infants were screened positive,including 76 murmurs of which 45 were confirmed as CHD,28 with abnormal SpO2 of which 18 were confirmed and 14 mixed of which 13 were confirmed,so the confirmation rates were 59. 21%,64. 29% and 92. 86%,respectively. The diagnostic rate of CHD in mixed group was higher than other 2 groups,which had statistical significance(P=0. 045). The top three formation types of CHD were ventricular septal defect (VSD) (29/76 cases,38. 16%),patent ductus arteriosus (PDA) (13/76 cases, 17. 11%) and atrial septal defect (ASD) (11/76 cases,14. 48%). Conclusions The combination of heart murmurs and SpO2 was useful in increasing detection and decreasing misdiagnosis of CHD.

Purpose:To study the significance of p53 protein, p53mRNA, and C erbB 2 protein expressions in Africa endemic and AIDS related Kaposi's sarcoma (KS).Methods:Immunohistochemistry and hybridization in situ were performed with p53 and C erbB 2 in 30 cases with endemic and AIDS related KS.The degree of pasitivity of p53 and C erbB 2 in KS tissues were measured with image analysis system. KS was classified into three types, i.e., angiomatous type, spindle cell type, and mixed type according to the proportion of blood vessel components and spindle cells in the lesions.Results:The degree of positivity of p53 protein was stronger in spindle cell type than that in angiomatous type ( P

Aim To investigate the influence of total fl avones of buckwheat flower (TFBF) on the productivity of the non-enzymatic advanced glycation end products (AGEs) in vivoand vitro. Methods TFBF in different dosages (0.1 g?kg -1?d -1,0.2 g?kg -1?d -1,0.4 g?kg -1?d -1) was taken orally by streptozotocin-induced diabetic rats for 12 wk. After the treatment, blood glucose, fructosamine and AGEs in plasma and kidney were measured. Meanwhile, glucose and bovine serum albumin (BSA) were incubated with TFBF at different concentrations (0.01 mg?L -1,0.05 mg?L -1,0.10 mg?L -1) respectively for 4,8,12 wk.The fluorescence intensity of glycated BSA was detected by a spectrophotometer BSA was detected spectrophotometer.Results TFBF significantly lowered the level of blood glucose in diabetic rats (P