Objective To observe the effect of apigenin on the recovery of neurological function following cerebral ischemia-reperfusion and investigate its mechanism. Methods Ninety male Sprague-Dawley rats were randomized into a sham-operated group, a model group and an apigenin-treated group. A transient ( 1.5 h) focal cerebral ischemia-reperfu-sion model was established in the rats of the model and apigenin-treated groups. In the sham-operated rats the middle cere-bral artery was not occluded. The rats in the apigenin-treated group received an intra-abdominal injection of apigenin, and the rats in the other two groups received injections of normal saline solution. Neurological behavior scores were assessed in accordance with the Zea Longa method at the 24th, 48th and 72nd hour and the 7th day after reperfusion. Cellular and sub-cellular morphology were observed with an optical microscope and an electron microscope, and the levels of TNF-α and IL-1β were measured using ELISA. Results Neurological function improved by the 7th day after reperfusion in the model group, but improved significantly by the 72nd hour after reperfusion in the apigenin-treated group. Average TNF-α and IL-1β levels in the model group and the apigenin-treated group were significantly higher than in the sham-operated group. Av-erage TNF-α and IL-1β levels in the apigenin-treated group were significantly lower than in the model group at the 48th and 72nd hour after reperfusion. Neurological behavior scores had a positive correlation with the IL-1β and TNF-α levels. In the model group, obvious intracellular and intercellular edema and vacuolization were observed in the ischemic cortices and hippocampuses, with remarkable karyopycnosis and organelle broadening and dissolution and vacuolization in glial cells and neurons. In the apigenin-treated group, similar but significantly milder morphological changes were observed. Conclusion Apigenin can promote the recovery of neurological function in rats by downregulating the expression of TNF-α and IL-1βfollowing focal cerebral ischemia-reperfusion.

Objective To establish an early cognitive disorder model in rats and investigate the early cognitive functioning after ischemic hypoxic brain injury during the neonatal period. Methods Forty-six newborn Sprague-Dawley rats were randomized into a 21-d-old group and a 31-d-old group. These 2 groups were then subdivided into model and sham-operated subgroups (M21, n=12; SH21, n=11; M31, n=12; SH31, n=11). A model of neonatal early cognitive disorder was established in the rats of the M21 and M31 groups using a modification of Rice's method. Rats in the SH21 and SH31 groups received skin incisions and common carotid artery separation without ligation or hypoxia. Each group was tested with a Morris water maze. The rats were sacrificed after testing, and brain tissue was examined under the electron microscope. Nissl staining allowed Nissl body quantification and neurocyte acin the M21 group was significantly longer than in the SH21 group. The 31-d-old subgroups had shorter average escaping latencies than the corresponding 21-d-old subgroups. (b) Spatial memory: The average platform times, Ⅰ region times and Ⅰ region distances showed no significant differences among groups. ②Brain pathology (a) Gross appearance: Obvious ischemic hemisphere atrophy was observed in the M group, and no abnormality was observed in the SH group. (b) Electron microscopic observation: In the SH group cell ultrastructures in the ischemic hippocampus were normal. Karyopyknosis and dilated endoplasmic reticulums were found in the M group. More mitochondria were found in the presynaptic membranes of the ischemic hippocampus in the M group than that in the SH group. (c) Nissl body quantification and neurocyte activity analysis: Significantly less activity in the ischemic cortex was found in the M21 group compared to the SH21 group. More activity was observed in the 31-d-old subgroups than in the corresponding 21-d-old subgroups. Conclusions ①The neonatal rats with ischemic hypoxic brain injury had prolonged average escaping latency and depressed neuronal activity. ②The 31-d-old rats had better spatial localization learning ability than the 21-d-old rats.

OBJECTIVETo construct a noninvasive model to predict histological liver cirrhosis in patients with chronic hepatitis B.

METHODS275 patients with chronic hepatitis B were divided into a training group (206 cases) and a validation group (69 cases). The constituent ratios of patients in the fibrosis stages S0-S3, fibrosis stage S4 (early cirrhosis) and active cirrhosis stage were calculated according to the liver biopsy results. 30 noninvasive variables, including age-platelet index (API), aspartate aminotransferase to platelet ratio index (APRI), spleen-platelet ratio index (SRPI) and age-spleen-platelet ratio index (ASPRI), were analyzed by univariate analysis and multivariate logistic regression. Variables that were significantly different between patients with and without cirrhosis were used to construct a noninvasive prediction model, and the model was then tested in the validation group.

RESULTS(1) Of the 275 patients with chronic hepatitis B, 193 (70.2%) were in the fibrosis stages S0-S3, 42 (15.3%) in fibrosis stage S4, 40 (14.5%) in active cirrhosis stage. (2) There were 23 variables that are significantly different between patients with and without cirrhosis by univariate analysis. The 23 variables were further analyzed by multivariate logistic regression, and 4 independent factors, including international normalized ratio (INR), gamma glutamyltranspeptidase (GGT), ASPRI, hepatitis B e antigen (HBeAg) were used to construct a noninvasive prediction model. (3) By receiver operating characteristic curves (ROC) analysis, to discriminate patients in stages S0-S3 from patients in stage S4 and patients in active cirrhosis stage, the area under ROC (AUROC), cut-off value, sensitivity, specificity and accuracy of the model were 0.871, 0.458, 84.4%, 75.7%, and 79.7% respectively. To discriminate patients in active cirrhosis stage from patients in other stages, the AUROC, cut-off value, sensitivity, specificity and accuracy were 0.753, 0.526, 81.8%, 62.9%, and 67.4% respectively. There was no significant difference in AUROC between the training group and the validation group (P less than 0.05).

CONCLUSIONINR, GGT, ASPRI and HBeAg are associated with early cirrhosis and active cirrhosis. Our model can be used to predict early cirrhosis and active cirrhosis.

Adolescent ; Adult ; Biopsy ; Female ; Hepatitis B e Antigens ; blood ; Hepatitis B, Chronic ; blood ; complications ; diagnosis ; Humans ; Liver ; pathology ; Liver Cirrhosis ; blood ; diagnosis ; etiology ; Male ; Middle Aged ; Models, Biological ; Platelet Count ; Predictive Value of Tests ; Prognosis ; ROC Curve ; Sensitivity and Specificity ; Severity of Illness Index ; Young Adult ; gamma-Glutamyltransferase ; blood

To explore a new method to identify Moutan Cortex to guarantee its safe use, internal transcribed spacer 2 (ITS2) sequence was used to identify Moutan Cortex and its adulterants. DNA was extracted and target fragments were amplified. Sequences were analyzed and assembled by CodonCode Aligner V3.7.1. Genetic distances were computed and phylogenetic tree was constructed based on kimura 2-parameter (K2P) model by MEGA 5.0. The length of the 20 ITS2 sequences of Moutan Cortex from nine different places is 227 bp, and no variation site was detected. The maximum inter-specificK2P distance of Moutan Cortex is 0, the minimum intra-specific K2P distance is 0.041, the average intra-specific K2P distance is 0.222. According to NJ analysis, Moutan Cortex from different places can get together as one branch with bootstrap support values 99%, which indicates Moutan Cortex can be easily distinguished from its adulterants. Using ITS2 sequence can accurately identify Moutan Cortex and its adulterants, it is an effective supplementary to traditional identification methods.
Base Sequence ; China ; DNA Barcoding, Taxonomic ; methods ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Drug Contamination ; prevention & control ; Drugs, Chinese Herbal ; chemistry ; classification ; Molecular Sequence Data ; Paeonia ; classification ; genetics ; Phylogeny ; Quality Control

Classical swine fever virus (CSFV), an enveloped positive-stranded RNA virus in the genus Pestivirus of the Flaviviridae family, is the causative agent of a highly contagious swine disease characterized by symptoms of hemorrhagic fever and immune depression, usually leading to substantial economic losses. The serological methods for detection of CSFV antibody such as ELISA are important means for the diagnosis of CSFV and immune surveillance. It is difficult to obtain CSFV antigen with high quality using traditional method because its titration titer is low in cell culture. CSFV has four structural protein named C, E0, El and E2. The E2 protein contains major antigenic determinants that are conserved between different CSFV strains and involved in neutralization by antibodies. So recombinant E2 protein can be developed as an alternative to the intact viral antigen. So far, CSFV E2 have not been expressed in E. coli with high level. Many factors, such as the secondary structure, the stability of 5' and 3' terminus of gene, the location of SD sequence and the bias of codes, are involved in the expressing level of foreign gene in E. coli . In this study, two sites of the E2 gene sequence were confirmed to be detrimental to its expression efficiency in E. coli through the computer-aided analysis. So they were mutated using recombinant PCR without changing the amino acids sequence of CSFV E2 gene. A plasmid was constructed by inserting the mutated E2 gene into the prokaryotic expression vector pET-28a(+) and named pETE2. The E. coli competent host BL21 (DE3)lysS transformed with pETE2 could express the E2 gene at high level, amounting to 28% of the total protein of the induced recombinant bacteria at the presence of IPTG. Except the hydrophobic transmembrane domain at C terminus, the recombinant E2 protein includes the total aa sequence. So it contains all the potential linear antigen epitopes of E2 protein because hydrophobic aa region can not form epitope. The recombinant E2 protein was CSFV-specific as proved by Western blotting and indirect ELISA. The rabbits immunized with the recombinant E2 can be protect from the challenge of hog cholera lapinized virus. This is the first report that E2 gene is expressed with high level expression in E. coli. In conclusion, it is an effective measure that mutate the CSFV E2 gene to increase its expression level in E. coli. The recombinant CSFV E2 protein possess fine immunonicity and can be used the antigen for the detection of CSFV antibody.
Antigens, Viral ; genetics ; immunology ; metabolism ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Mutagenesis, Site-Directed ; methods ; Polymerase Chain Reaction ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Viral Envelope Proteins ; genetics ; immunology ; metabolism

A comprehensively comparison of the chemical profiles between sun-drying BJ (NBJ) and sulfur-fumigated BJ (SBJ) was conducted by HPLC analysis and the discrepant peaks were identified or tentatively assigned by HPLC-ESI-MSn. A total of 32 chemical components were used for qualitative comparison. Meanwhile, a quantitative comparison of BJwere conducted by HPLC analysis and determining seven compounds from 3 NBJ and 3 SBJ samples dramatic chemical changes were found. After sulfur fumigation, the contents of flavonoids glycosides and phenolic acids were remarkably reduced, but the contents of flavonoids aglycones were significantly increased. Multivariate statistics, including principle component analysis (PCA) and partial least squares discriminate analysis (PLS-DA) were used to investigate the potential damaging effect of sulfur-fumigating process. The PCA score plots showed six samples were clearly classified into the sun-drying and sulfur-fumigating groups. And according to VIP >1, the most important chemical markers were apigenin, luteolin and 3,5-dicaffeoylquninic acid which could be used to distinguish NBJ and SBJ samples. Combining the results of qualitative and quantitative analysis, it showed that the sulfur fumigation has a significant effect on BJ.
Chromatography, High Pressure Liquid ; Chrysanthemum ; chemistry ; Fumigation ; Least-Squares Analysis ; Principal Component Analysis ; Sulfur

OBJECTIVETo study the related proteins of apoptosis initiation induced by homoharringtonine (HHT) in HL-60 cells.

METHODSAfter establishment of an apoptosis initiation model induced by HHT in HL-60 cells, proteins of untreated and HHT treated HL-60 cells were extracted, and the two-dimensional polyacrylamide gel electrophoresis (2-DE) maps of the extracted proteins were established by using the immobilized pH gradient (IPG) two-dimensional electrophoresis respectively. The alteration protein spots were identified with assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching.

RESULTSProteomics analysis showed that proteins including MHC class I antigen, calbindin D-28K, chloride channel protein 6, oncoprotein 18, zinc finger protein Helios and apoptosis inhibitor like protein 2 were involved in apoptosis initiation induced by HHT.

CONCLUSIONThe present study might conduce to the researches of HL-60 cells carcinogenesis and pave the way to exploit drug precursor related to HHT and initiation of apoptosis in HL-60 cells.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Calbindins ; Chloride Channels ; analysis ; DNA-Binding Proteins ; analysis ; Electrophoresis, Gel, Two-Dimensional ; methods ; HL-60 Cells ; Harringtonines ; pharmacology ; Histocompatibility Antigens Class I ; analysis ; Humans ; Ikaros Transcription Factor ; Inhibitor of Apoptosis Proteins ; Microtubule Proteins ; Phosphoproteins ; analysis ; Proteins ; analysis ; Proteome ; analysis ; S100 Calcium Binding Protein G ; analysis ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Stathmin ; Transcription Factors ; analysis

In order to evaluate the efficiency of ITS2 and psbA-trnH sequences used as DNA barcodes to distinguish Plantaginis Semen from its adulterants, we collected 71 samples of Plantaginis Semen and its adulterants. The ITS2 and psbA-trnH sequences were aligned through Clustal W, and the genetic distances were calculated by kimura 2-parameter (K2P) model and the Neighbor-Joining (NJ) phylogenetic trees were constructed using MEGA 5.1. The results indicated that the ITS2 sequence lengths of Plantago asiatica and P. depressa were 199 bp and 200 bp, respectively; the maximum intra-specific K2P distance were lower than the minimum inter-specific K2P distance; the NJ tree based on ITS2 sequence indicated that Plantaginis Semen and its adulterants could be distinguished clearly. The sequence lengths of psbA-trnH of both P. asiatica and P. depressa were 340 bp; the maximum intra-specific K2P distances were lower than the minimum inter-specific K2P distance; the NJ tree based on psbA-trnH sequence showed that Plantaginis Semen can be distinguished clearly from its adulterants except for P. major. Therefore, ITS2 sequences can be used as an ideal DNA barcode to distinguish Plantaginis Semen from its adulterants.
Base Sequence ; DNA Barcoding, Taxonomic ; methods ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Drug Contamination ; prevention & control ; Drugs, Chinese Herbal ; chemistry ; classification ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; genetics ; Plantago ; classification ; genetics ; Quality Control ; Seeds ; classification ; genetics

Adenine ; analogs & derivatives ; therapeutic use ; Adult ; Antiviral Agents ; therapeutic use ; Drug Resistance, Viral ; Female ; Hepatitis B, Chronic ; drug therapy ; Humans ; Lamivudine ; therapeutic use ; Male ; Organophosphonates ; therapeutic use ; Treatment Failure ; Young Adult

To explore the alternative material for the stems of Evodia lepta used in clinic, the leaves extract of E. lepta was chemically investigated by silica gel, Sephadex LH-20, ODS column chromatographies, and preparative HPLC and the structures of the compounds were identified mainly by spectroscopic methods. Ten known compounds 4-hydroxy-4, 7-dimethyl-1-tetralone (1), (6R, 7E) -4, 7-megastigmadien-3, 9-dione (2), 4-megastigmen-3, 9-dione (3), formononetin (4), daidzein (5), oroxylin A (6), wogonin (7), 5, 7-dihydroxy-3, 4'-dimethoxyflavone (8), N-trans-coumaroyltyranine (9) and (E) -p-hydroxycinnamic acid (10), have been obtained and identified. All these compounds were isolated from this species for the first time. The results revealed that there is a considerate chemical difference between the stems and leaves of E. lepta.
Evodia ; chemistry ; Magnetic Resonance Spectroscopy ; Plant Extracts ; chemistry ; isolation & purification ; Plant Leaves ; chemistry