As a classic fluorescent detect technique, fluorescence resonance energy transfer (FRET) has been widely used in biological researches. Researchers have developed a series of fluorescence detect probes which were based on FRET. Caspase family plays an important role in apoptosis pathway, especially Caspase-8 which located, at the initial of death receptor mediated apoptosis pathway, whose its activation can trigger subsequent precaspases' activation and lead to apoptosis. So it is of great significance to detect the activation of Caspase-8 in apoptosis assay. In this study, a fluorescent probe based on FRET has been designed which can detect the activity change of Caspase-8 in cells. To identify the effectiveness and specificity of the probe, we measure the Caspase-8 activity under the Caspase-8 specifically activated apoptosis inducer RGD-TRAIL with the flow cytometry FRET detection platform. The results show that the probe can respond to the activity change of Caspase-8 in apoptotic cells, and the change can be quantified rapidly by flow cytometry. The study provides a more efficient and convenient detection method of Caspase-8 activity in living cells.
Apoptosis ; Caspase 8 ; metabolism ; Flow Cytometry ; Fluorescence Resonance Energy Transfer ; Fluorescent Dyes ; Humans

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Humans ; Injections, Intravenous ; Male ; Middle Aged ; Motor Neuron Disease ; drug therapy ; Phytotherapy ; Treatment Outcome

OBJECTIVETo evaluate the curative effect of Danxiaoling Pill (DXLP), a Chinese herbal preparation, in treating intrahepatic cholestasis in pregnancy (ICP).

METHODSFifty-eight cases of ICP were divided randomly into two groups and treated by DXLP and Composite Yiganling as control respectively with the other identical conventional treatment. The changes of clinical symptoms, related laboratory parameters after treatment and the condition of labor were observed.

RESULTSThe total effective rate in both groups was 100%, but the markedly effective rate in the DXLP treated group was higher than that in control group (P < 0.01). Levels of blood cholyglycine acid (CGA), alanine transaminase (ALT), aspartate transaminase (AST), total bilirubin, total cholesterol and low density lipoprotein were all decreased in both groups after treatment, but DXL showed a better efficacy in decreasing CGA, ALT and AST than that of Yiganling. Moreover, the amniotic fluid meconium contaminated rate, premature delivery occurrence in the DXLP group were lower than those in the control group, while the weight of newborn baby was higher in the former than in the latter.

CONCLUSIONDXLP could effectively lower the serum bile acid and improve liver function in treating ICP.

Adult ; Alanine Transaminase ; blood ; Aspartate Aminotransferases ; blood ; Cholestasis, Intrahepatic ; blood ; drug therapy ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Glycocholic Acid ; blood ; Humans ; Phytotherapy ; Pregnancy ; Pregnancy Complications ; blood ; drug therapy

AIMTo establish a solid phase extraction-high performance liquid chromatographic (SPE-HPLC) method for determining plasma scutellarin concentration, and study its pharmacokinetics after i.v. breviscapine in rabbits.

METHODSMethanol-water-phosphoric acid (50:50:0.5) mixture was used as mobile phase, Nucleosil C18 column (250 mm x 4.6 mm ID) was selected. The wavelength of UV detection was 335 nm. Fifteen rabbits, randomized into 3 groups, were given breviscapine i.v. at the dose of 10, 20 and 40 mg.kg-1. Scutellarin in plasma was determined by SPE-HPLC method.

RESULTSLinearity was obtained over the range of 0.02-10.0 mg.L-1 of scutellarin. The method recovery was 96.15%-99.31%; the within-day and between-day RSDs were all below 10%. After i.v. 10, 20 and 40 mg.kg-1 of breviscapine to rabbits, the concentration-time curve of scutellarin fitted to a three compartment model. The main pharmacokinetic parameters showed no significant difference between low and medium doses, but the difference was significant between high dose and other doses.

CONCLUSIONThis assay method was accurate, sensitive, simple and suitable for the measurement of plasma scutellarin concentration. The pharmacokinetic characteristics were found to fit a three-compartment model following i.v. injection of breviscapine to rabbits. The changes of drug concentration in vivo exhibited linear kinetics ove the dosage range of 10-20 mg.kg-1, but when the dosage was 40 mg.kg-1, the linear kinetic properties disappeared.

Animals ; Apigenin ; Area Under Curve ; Chromatography, High Pressure Liquid ; methods ; Flavonoids ; blood ; pharmacokinetics ; Male ; Rabbits

OBJECTIVETo evaluate the curative effect of Wuling pill (WLP), a traditional Chinese patent medicine, in treating gestation period intrahepatic cholestasis (GPI).

METHODSIn the clinical study, 90 GPI patients were divided into the treated group treated by conventional therapy plus WLP and the control group treated by conventional therapy plus compound Yiganling (YGL) with a ratio of 2:1. Clinical symptoms and accouchement condition were observed. Levels of cholyglycine acid (CGA), total and direct bilirubin (TB and DB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were detected before and after treatment. For the experimental study, GPI rat model was induced by injecting estradiol benzoate to pregnant Wistar rats. The model rats in the treated group were administrated with WLP by gastro-perfusion and those in the control group, were administrated with YGL. Levels of CGA,TB,DB, ALT, AST and ALP in the mother and fetal rats, as well as in the amnionic fluid were determined. Besides, the volume of bile excreted by the mother rats was observed.

RESULTSIn clinical trials, the markedly effective rate in the treated group (47 cases, 78.3% ) was higher than that in the control group (15 cases, 50%, chi2 = 7.17286, P < 0.01). Levels of blood CGA, TB, ALT and AST were all decreased in both groups after treatment, but WLP showed a better efficacy than YGL (P < 0.05) in lowering CGA, ALT and AST. Moreover, the occurrence of meconium contaminated amnionic fluid and premature delivery were lower, while weight and Apgar grade of newborn babies were higher in the treated group than those in the control group. In animal experiment, WLP showed significant effects in decreasing CGA level in amniotic fluid, and in blood of the mother and fetal rats. In addition, it could also decrease the levels of bilirubin, ALT and AST, and promote the bile excretion to reduce CGA concentration in bile. All the above effects showed a dose-dependent pattern.

CONCLUSIONWLP could effectively lower the serum bile acid, improve the hepatic function and better the pregnant outcome in treating GPI.

Adult ; Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Bilirubin ; blood ; Cholestasis, Intrahepatic ; drug therapy ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Glycocholic Acid ; blood ; Humans ; Phytotherapy ; Pregnancy ; Pregnancy Complications ; drug therapy ; Pregnancy Outcome ; Rats ; Rats, Wistar

BACKGROUNDLung cancer is the leading cause of cancer-related death in China. Mutation analysis reveals that LKB1 inactivation is present in 30% of non-small-cell lung cancer (NSCLC), indicating its role as a tumor suppressor. However, the molecular mechanism is still not clear. Our study attempted to establish LKB1 stable knockdown NSCLC cell line, detect alterations in gene expression and identify the genes regulated by LKB1.

METHODSLKB1 stable knockdown H1299 cell line was established using a lentiviral short hairpin RNA. To identify the knockdown effect, LKB1 mRNA and protein expression level were evaluated with quantitative real-time PCR and Western blotting. We treated the cell lines with 2-deoxyglucose to determine if LKB1 protein function was impacted. Gene microarray analysis was performed to detect the gene expression alterations in LKB1 stable knockdown H1299 cells.

RESULTSLKB1 mRNA and protein expression were significantly suppressed in LKB1 stable knockdown H1299 cell line. 2-DG treatment had little impact on the phosphorylation of AMPK, which is the downstream target of LKB1, indicating the loss of function of LKB1. The microarray data showed that LKB1 knockdown resulted in expression alterations of 1243 kinds of genes, including those involved in cell migration, cell proliferation and cell apoptosis.

CONCLUSIONSThe establishment of LKB1 stable knockdown H1299 cell line provides us with a great tool to investigate various genes regulated by LKB1 through microarray. The discovery of cell proliferation and migration-related genes regulated by LKB1 is critical for unraveling molecular mechanisms of LKB1's role in the development and metastasis of lung cancer.

Blotting, Western ; Cell Line, Tumor ; Gene Expression Profiling ; methods ; Humans ; Lung Neoplasms ; genetics ; metabolism ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction

AIMTo clarify the ultrastructural changes of penile tunica albuginea (TA) in streptozotocin (STZ)-induced diabetic rats.

METHODSIntraperitoneal injection of STZ was used to induce diabetes mellitus (DM) in 12 Sprague Dawley rats. Ten rats (age and weight-matched) were used as control. Blood samples from the tail snips of the rats were used for the determination of serum glucose levels with SureStep Plus Blood Meter. At week 4 and 10 after the injection, half of the rats in each group were sacrificed and penile samples were obtained from the middle third of the penile shaft for the examination of TA under scanning electron microscopy.

RESULTSIn the diabetic group, the serum glucose levels were higher (P<0.01 at both time points) and the TA were thinner (P<0.05) than those of the controls. In the control group, the fibers of TA were rich and arranged regularly and undulated, while in the diabetic group, the fibers were diminished, lost the undulations and were arranged irregularly.

CONCLUSIONIn rats, DM appeared to impair the penile TA ultrastructures and this impairment could contribute to diabetic erectile dysfunction in part by impairing the veno-occlusive function.

Animals ; Blood Glucose ; metabolism ; Diabetes Mellitus, Experimental ; pathology ; Male ; Microscopy, Electron, Scanning ; Penis ; pathology ; ultrastructure ; Rats ; Rats, Sprague-Dawley

Adolescent ; Adult ; Aged ; Ascites ; enzymology ; Ascitic Fluid ; enzymology ; pathology ; Diagnosis, Differential ; Female ; Glucuronidase ; biosynthesis ; genetics ; Humans ; Liver Cirrhosis ; diagnosis ; Male ; Middle Aged ; Peritoneal Neoplasms ; diagnosis ; secondary ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo investigate the knowledge and attitudes of healthcare professionals (doctors, nurses and administrators) to adverse drug reactions (ADR) in Wuhan city and to identify the reasons for under-reporting.

METHODSStructured interviews were carried out in Wuhan, Hubei province. Questionnaire survey to approximately 15% of the medical practitioners selected from 16 hospitals, was conducted during the period from February to March 2003.

RESULTSOnly 2.7% of the interviewees knew the definition of adverse drug reactions. 61.7% of the doctors, 62.7% of the nurses and 61.1% of the administrators had ever encountered an ADR during their practices, but did not report to the national monitoring center or other centers. The major reasons for not reporting included: ignorant about the requirement and the reporting process of ADR (71.4%); address of the reporting agency and Forms unavailable (67.9%, 60.4%); unaware of the existence of a national ADR reporting system (52.2%); needless to report as the ADR being too well known (44.1%). They mainly reported an ADR to the hospital pharmacy or other departments, or to the pharmaceutical administration. Education, training and developing new institutions were ways to improve the reporting system.

CONCLUSIONSOur results showed that healthcare professionals had little knowledge on the basic ADR knowledge. The main reasons for underreporting were related to factors on reporting process, address of related centers and unavailable of the Forms. Education and training to doctors and nurses to enhance the awareness of administrators were the ways to improve the reporting system.

Adverse Drug Reaction Reporting Systems ; Attitude of Health Personnel ; China ; Drug-Related Side Effects and Adverse Reactions ; Female ; Health Knowledge, Attitudes, Practice ; Humans ; Male ; Practice Patterns, Physicians' ; Surveys and Questionnaires

BACKGROUNDA voluntary procedure for reporting adverse drug reactions (ADRs) was formally put in place in 1989. However, only a small proportion of ADR reports are actually forwarded to the national monitoring center. To identify the reasons for underreporting, the authors investigated the awareness and attitudes of healthcare professionals (doctors, nurses, and administrators) toward the ADR system in China. In addition, the authors sought to formulate approaches to improve the current ADR reporting system.

METHODSStructured interviews were carried out in 16 hospitals selected from 27 municipal hospitals in Wuhan, Hubei Province, China. A questionnaire survey of a stratified random sample of approximately 15% of healthcare professionals in each selected hospital was conducted during February to March 2003.

RESULTSThe response rate of this survey was 85%. One thousand six hundred and fifty-three questionnaires were used in the final analysis. Only 2.7% of the healthcare professionals had a correct understanding to the definition of ADR. Eighty-nine point two percent of the healthcare professionals had encountered ADRs. Ninety-four percent of them were aware of the need to report these to the ADR monitoring center. However, only 28.5% of doctors, 22.8% of nurses, and 29.7% of administrators actually submitted a report. For the most part, they reported ADRs to the hospital pharmacy (66.0%), to other departments in the hospital (72.5%), and to the pharmaceutical industry (23.0%), rather than to the national monitoring center (2.9%) or regional monitoring center (9.5%). Severe or rare ADRs and ADRs to new products were generally perceived to be significant enough to report. Sixty-two point one percent of the healthcare professionals had encountered ADRs, yet not reported them to anybody. The major reasons for not reporting included no knowledge of the reporting procedure (71.4%), unavailability of the reporting center mailing address (67.9%), unavailability of the ADR report form (60.4%), lack of knowledge of the existence of a national ADR reporting system (52.2%), and belief that the ADR in question was already well known (44.1%).

CONCLUSIONSHealthcare professionals in Wuhan, China have little basic knowledge of ADR and of the voluntary reporting system. The main reasons for underreporting were lack of basic knowledge about ADRs and the voluntary reporting procedure. Education and training of healthcare professionals is needed to improve the current ADR reporting system.

Adverse Drug Reaction Reporting Systems ; trends ; Attitude of Health Personnel ; China ; Health Knowledge, Attitudes, Practice ; Hospital Administrators ; Humans ; Interviews as Topic ; Nurses ; Physicians ; Surveys and Questionnaires