BACKGROUND: Previous studies have indicated that hyperhomocysteinemia can induce atherosclerosis by enhancing oxidative stress, whereas Ginkgo biloba extract (GbE) can scavenge oxygen-derived free radicals.OBJECTIVE: To observe the role of reactive oxygen species (ROS) and nuclear factor-kappa B (NF-κB) in the expression of cell adhesion molecules (CAMs) induced by homocysteine (Hcy), and investigate the effect of GbE.DESIGN: A randomized controlled animal trial.SETTING: Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERrALS: Twenty-four healthy male rabbits of 6 months old. Dl-methionine (Sigma Chemical, Co.,Ltd.); GbE (Guizhou Yibai Pharmaceutical Company; powder).METHODS: The experiment was accomplished in the Laboratory of Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology from March 2003 to April 2004. ① After adaptive feeding for 2 weeks, the rabbits were randomly divided into three groups: Model group (n =12): The rabbits were treated with subcutaneous injection of dl-methionine (80 mg/kg per day); GbE group (n =8): The rabbits were administrated with GbE (mixed with feed, 50 m/kg per day) at 1 hour before the subcutaneous injection of dl-methionine; Control group (n=4): The rabbits were injected with equivalent sodium chloride. They were administrated for 7 weeks continuously. ②Histological changes were observed under light and electron microscopes: ROS level was determined with colorimetries (721 visible spectrophotometer); The expressions of CAMs and NF-κB in endothelial cells were detected with immunohistochemical methods; The concentration of plasma Hcy was measured with high-performance liquid chromatography.MAIN OUTCOME MEASURES: Histological changes, ROS level and expressions of CAMs and NF-κB in endothelial cells.RESULTS: All the 24 rabbits were involved in the analysis of results. ① ROS level: After administration, the ROS level in the model group was obviously increased (2.92±0.20,2.48±0.26, P ＜ 0.05), whereas those in the GbE group and control group (2.41±0.23, 2.43±0.20) had no obvious differences as compared with those before administration (2.31±0.27,2.47±0.32, P ＞ 0.05). ② Histological changes: Aortas of rabbits in the model group presented initial changes of atherosclerosis, including shedding or necrosis of endothelial cells and nuclear pyknosis or standing in a clutter of smooth muscle cells. There were scarcely any changes in the GbE group and control group. ③ Expressions of CAMs and NF-κB in endothelial cells: After treatments, the expressions of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and NF-κB in the model group were obviously higher than those in the control group (P ＜0.05), and there were no significant differences between the GbE group and control group (P ＞ 0.05). ④ Concentration of plasma Hcy: After 7 weeks, the concentration of plasma Hcy was higher in the model group and GbE group than in the control group [(25.01±6.80), (26.71±2.36), (16.85± 1.64) μmol/L, P ＜ 0.05].CONCLUSION: Hcy-induced oxidative stress plays an important role in NF-κB activation. GbE might suppress the activation of NF-κB and expression of CAMs by reducing ROS.

Histocytochemistry ; methods ; Humans ; Pathology, Clinical ; methods ; Specimen Handling ; methods ; Staining and Labeling ; methods

OBJECTIVETo explore the effect of peimine on excision repair cross-complementation 1 (ERCC1) mRNA and lung resistant protein (LRP) expressions in A549/cisplatin (DDP) multidrug resistance (MDR) cell line.

METHODSLung cancer A549/DDP cells were cultured in vitro.Cells at logarithmic growth phase were divided into 4 groups, i.e., the blank control group, the DDP group, the ligustrazine group (DDP+ligustrazine), the peimine group (DDP + peimine). After 48-h drug action, ERCC1 mRNA expression was detected by RT-PCR and LRP expression detected by cell immunofluorescence.

RESULTSThere was no statistical difference in expression levels of ERCC1 mRNA and LRP between the DDP group and the blank control group (P > 0.05). Compared with the DDP group, expression levels of ERCC1 mRNA and LRP obviously decreased in the ligustrazine group and the peimine group (P < 0.05). They were obviously lower in the peimine group than in the ligustrazine group (P < 0.05).

CONCLUSIONSPeimine could reverse MDR of A549/DDP cell line. Its mechanism might be associated with down-regulating ERCC1 mRNA and LRP expression levels.

Cell Line, Tumor ; Cevanes ; pharmacology ; Cisplatin ; DNA-Binding Proteins ; genetics ; Down-Regulation ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; drug effects ; Endonucleases ; genetics ; Humans ; Low Density Lipoprotein Receptor-Related Protein-1 ; genetics ; Lung Neoplasms ; RNA, Messenger ; metabolism

Adolescent ; Adult ; Aged ; Child ; Endoscopes ; Female ; Humans ; Male ; Middle Aged ; Otorhinolaryngologic Surgical Procedures ; Smell ; Turbinates ; surgery ; Young Adult

Objective To establish a method for culturing neonatal mice cardiomyocytes, and construct an in vitro model of cardiomyocytes for assessing the cardiac toxicity of chemicals. Methods Hearts of neonatal mice of 1 day old were digested with enzyme mixture of trypsin/collagenase type Ⅱ/dispase and the cell suspensions were pre-plated to flask for a short time and then seeded on coated dishes. The cultured cells were treated with 5-fiuorine-deoxy-uridine(15 μg/ml)and uridine(35 μg/ml)to enrich cardiomyocytes that were identified according to the morphology and immunocytochemistry of α-actin antigen. Cardiac myocytes were incubated with 0-64 μg/ml of a fusarium mycotoxin butenolide(BUT) for 12 h. Cell viability was then evaluated by MTT assay. Microscopic observation showed that BUT induced significant morphological changes including cellular swelling, vacuolation and breakage of muscle fibers. Results It was found that 96% of the cultured cells were cardiomyocytes and the myocytes kept beating after 90 days of culture. Concentration-dependent decreases in cell viability following exposure of cardiac myocytes to BUT were observed. Conclusion The results indicated that relatively pure primary culture of neonatal mice cardiomyocytes is successfully established. BUT possesses the potential to induce myocardial toxicity.

Objective To explore the characteristics of dipyridamole 201 Tl myocardial perfusion imaging (MPI) SPECT in patients with dilated cardiomyopathy. Methods Thirty patients with dilated cardiomyopathy underwent pharmacological stress 201Tl MPI SPECT after intravenous infusion of dipyridamole (0. 56 mg/kg) for 4 min. The early and delayed SPECT images were acquired respectively at 10 and 240 min after 201Tl injection. The images were analyzed and reported by two or three experienced nuclear medicine physicians. Results All patients were found to have abnormal perfusion patterns at delay imaging, however 90.00% (27/30) were also abnormal at early images. Six patients had reverse redistribution. Conclusion Dipyridamole 201Tl MPI SPECT imaging may be of some value for the assessment of patients with dilated cardiomyopathy.

Objective To investigate the effect of intra-articular carboxymethylated ehitosan(CM- CTS)injection on inducible nitric oxide synthase(iNOS)expression in cartilage at the early stage of os- teoarthfitis(OA).Methods Thirty-two white rabbits were underwent unilateral anterior cruciate ligament transection(ACLT)and were randomly divided into 4 groups 5 weeks after transection.Rabbits of group A re- ceived 0.3 ml of 2% high molecular weight CMCTS(H-CMCTS)once every two weeks.Rabbits in group B were treated using 2% low molecular weight(L-CMCTS)CMCTS at:the same intervals.Group C rabbits were injected intra-articularly with 0.3 ml of 1% sodium hyaluronate(Na-HA)once a week.Animals of group D were not injected.At sacrifice,11 weeks following surgery,the expression of iNOS in cartilages was analyzed by immunohistochemistry and reverse transcription-polymerase chain reaction(RT-PCR)methods.Results Both immunohistochemistry and RT-PCR showed that the level of iNOS expression of cartilage in CMCTS in- jection groups was lower than that in Na-HA injection group and the untreated group.There was no significant difference in iNOS expression between the two different molecular weight CMCTS injection groups. No signifi- cant difference of iNOS expression in cartilage was found between Na-HA injection group and the untreated group.Conclusion CMCTS suppresses iNOS expression in cartilage during the early stage of OA.Na-HA treatment has no effect on iNOS expression in cartilage.

Immunohistochemical technique was an essential tool of conventional diagnosis,therefore,the application of immunohisto- chemistry in reformation of pathology laboratory teaching would boost pathological experimental teaching standards to a higher level.

Objective To establish a specific,sensitive,simple human respiratory adenovirus detection method to put a good experimental foundation for developing universal and specific diagnostic reagents of human respiratory adenovirus.Methods The nucleotide sequences of 10 serotype of human respiratory adenovirus were obtained from GenBank.Highly consewed five pairs of universal and specific adenovirus primers were designed on the evaluation of multiple sequence alignment of the 10 full genomic sequences with the software DNAMAN 5.2.2,Gene Runner 3.05,BLAST,and to ensure that the polymerase chain reaction(PCR)products were type-specific.NP-40 sample lytic method was employed to prepare the template.The effectiveness,specificity and sensitivity of primers were evaluated.And PCR was carried out to test 64 samples of throat swabs of acute respiratory infection children.The positive PCR products were sequenced directly to identify the adenovirus serotypes.The positive specimens was inoculated on HeLa cells to observe cytopathie effect(CPE)under light microscope,and virus morphology were observed under electron microscope.Results BLAST results indicated that the five pairs of primers were specific adenovirus primers with low homology to the others.The primers were identified as PCR positive fragments obtained by using five pairs of primers to amplify the human adenovirus type 3 DNA,which did not react to the respiratory syncytial virus and Coxsackie virus.the effectiveness and specificity of the primer were thus indicated.PCR sensitive results showed 10-5 dilutin of adenovirus culture and DNA sample could be deteeted which meant the method was sensitive and stable.Two PCR positive specimens were detected in 64 clinical samples.the positive rate was 3.13%(2/64).Using the two PCR positive specimens to inoculate the HeLa cells,the typical adenovirus CPEs of rounded and aggregated,detatched cells were observed under light microscope.And a large number of adenovirus typical particles with characteristic lattice arrangement were observed in the infected cells under electron microscope.PCR product sequencing results showed that these two isolated adenoviruses were typed in human adenovims group B.Conclusions Universal and specific adenovirus PCR primers with the serotype specificity of the PCR products were designed successfully.The PCR primers was sensitive and specific and could be routinely applied in clinical adenovirus diagnosis for respiratory specimens.

Objective Previous studies indicated that activation of Toll-like receptor4 (TLR4) was involved in the progression and instability of atherosclerotic plaque.Anti-inflammatory effects were shown in statins. However,the mechanisms underlying these effects have not been well explored.We test the hypothesis that a por- tion of these anti-inflammatory effects are mediated by regulation of TLR4 expression.Methods One hundred twenty-one subjects (22 normal persons,17 patients with stable angina and 82 patients with ACS) were recruited. 41 patients with ACS were randomized to atorvastatin 10 mg/d or atorvastatin 40 mg/d on top of routine anti-anginal treatment.Serum level of hsCRP,blood lipids,TLR4 expression on CD_(14)~+ monocytes were measuered before and after one month treatment.TLR4 expression on CD_(14)~+ monocytes were quantified via flow-cytometry.Results hsCRP and TLR4 expression on CD_(14)~+ monocytes in patients with ACS were higher than patients with stable angina and normal persons(hsCRP,ACS:11.1?14.3 vs stable angina:2.5?2.7 mg/L vs normal:2.3?4.2 mg/L,P