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

AIM To establish the HPLC fingerprints for Tanacetum tatsienense(Bureau & Franchet)K.Bremer & Humphries and to determine the contents of chlorogenic acid,quercetin-3-O-glucoside,luteolin-7-O-glucoside,luteolin-7-O-glucuronide,apigenin-7-O-glucuronide,luteolin and apigenin.METHODS The analysis was performed on a 35 ℃ thermostatic Agilent ZORBAX Extend C18 column(4.6 mm×250 mm,0.5 μm),with the mobile phase comprising of 0.5％phosphoric acid-acetonitrile flowing at 1.0 mL/min,and the detection wavelength was set at 350 nm.Subsequently,principal component analysis,partial least squares discriminant analysis and cluster analysis were carried out.RESULTS There were twelve common peaks in the fingerprints for fourteen batches of samples with the similarities of 0.761-0.975.Seven constituents showed good linear relationships within their own ranges(R2≥0.999 1),whose average recoveries were 84.00％-105.11％with the RSDs of 1.28％-2.86％.Various batches of samples were clustered into three types,and seven differential constituents were observable,containing peaks 4(luteolin-7-O-glucoside),12(apigenin),9(apigenin-7-O-glucuronide),8,11(luteolin),5(luteolin-7-O-glucuronide)and 2.CONCLUSION This precise,stable,specific and reproducible method can be used for the quality control of T.tatsienense.

OBJECTIVETo assess objectively the efficacy of migraine without aura treated by comprehensive auricular acupuncture.

METHODSNinty patients of migraine without aura from three centers were selected as the observation subjects. Thirty cases from each center were treated with the comprehensive auricular acupuncture. This therapy included the retroauricular venous bleeding on the ears, the autologous blood injection at Fengchi (GB 20) and Yanglingquan (GB 34) and bleeding at the Nie (temporal), Zhen (occiput), Yidan (pancreas), Shenmen, Pizhixia (subcortex) and Neifenmi (endocrine) of auricular point. The therapy was applied once every 7-10 days. The continuous 3 treatments made one session.

RESULTSAt the end of one session for the patients in three centers, the headache scores were reduced apparently as compared with those before treatment (all P< 0.05). There was no statistically significant difference in comparison among three centers (all P>0.05). The clinical total effective rates were 93.3% (28/30), 90.0% (27/30) and 93.3% (28/30) in three centers separately. There was no obvious difference in the efficacy in comparison among three centers (P>0.05).

CONCLUSIONThe comprehensive auricular acupuncture reduces apparently the headache score for migraine without aura and relieves the clinical symptoms of migraine. It is the simple and effective therapy for migraine without aura.

Acupuncture, Ear ; Adolescent ; Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Migraine without Aura ; therapy ; Treatment Outcome ; Young Adult

OBJECTIVETo study the incidence and spatiotemporal dynamic variation of hemorrhagic fever with renal syndrome (HFRS) in Shandong province.

METHODSAccording to surveillance data on HFRS epidemics and host animals, a 'contour area multifractal model' was estimated on the HFRS' incidence and multi-analysis model was applied to study spatiotemporal dynamic variation.

RESULTSThe process could be classified into 5 periods: 1st period (1974-1981) when HFRS was in completely natural focal state in Shandong, and the nature of focus was typical Apodemus type. 2nd period (1982-1986) indicated the process of expanding and merging of the Apodemus type focus in the southeastern part of Linyi district and the Rattus type focus was in the southern part of Jining city. 3rd period (1987-1990) indicated that through the expanding and merging of the two epidemic focuses,one mixed focus dominated by the Apodemus type had been formed in the hilly area of the southern and middle part of Shandong while another one dominated by the Rattus type in the Yellow River valley of the northwestern part of Shandong. 4th period (1991-1993) showed that the process of the spatial pattern of the mixed focus dominated by the Rattus type in Shandong. 5th period (1994-2004) referred to the spatial pattern of the mixed focus dominated by the Rattus became stabilized.

CONCLUSIONEvolution of the characteristics of HFRS focus in Shandong province experienced the following three processes: the simple Apodemus type and the simple Rattus type were seen separately to the mixed foci with the Apodemus type dominant and the Rattus dominant type coexisted and merged to the stable state of the mixed focus with Rattus as the dominant one.

China ; epidemiology ; Hemorrhagic Fever with Renal Syndrome ; epidemiology ; Humans ; Incidence