To study on the effects of Achyranthes bidentata on Tongsaimai pellets main active ingredients chlorogenic acid, isoliquiritin, harpagoside and glycyrrhizin in rats in vivo pharmacokinetic behaviors, a method for the simultaneous determination of chlorogenic acid, isoliquiritin, harpagoside and liquiritigenin in rat plasma was established by UPLC-MS/MS. The analysis was performed on a waters Acquity BEH C18 column (2.1 mm x 100 mm, 1.7 microm) with the mixture of acetonitrile and 0.1% formic acid/water as mobile phase, and the gradient elution at a flow rate of 0.3 mL x min(-1). The analytes were detected by tandem mass spectrometry with the electrospray ionization (ESI) source and in the multiple reaction monitoring (MRM) mode. It turned out that the analytes of Tongsaimai pellets groups C(max) and AUC(Q-infinity) values were higher than that with A. bidentata group, and the C(max) values of chlorogenic acid had significantly difference (P < 0.05), the AUC(0-infinity) values of chlorogenic acid and glycyrrhizin had significantly difference (P < 0.05); The T(max) and CL values of two groups had no significantly difference. Results showed that the established method was specific, rapid, accurate and sensitive for the studies of Tongsaimai pellets four main active ingredients in rat in vivo pharmacokinetic, and A. bidentata have varying degrees of effects on Tongsaimai pellets four main active ingredients in rat in vivo pharmacokinetic behaviors.
Achyranthes ; chemistry ; Animals ; Chalcone ; administration & dosage ; analogs & derivatives ; blood ; pharmacokinetics ; Chlorogenic Acid ; administration & dosage ; blood ; pharmacokinetics ; Drugs, Chinese Herbal ; administration & dosage ; pharmacokinetics ; Glucosides ; administration & dosage ; blood ; pharmacokinetics ; Glycosides ; administration & dosage ; blood ; pharmacokinetics ; Glycyrrhizic Acid ; administration & dosage ; pharmacokinetics ; Herb-Drug Interactions ; Male ; Pyrans ; administration & dosage ; blood ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry

OBJECTIVETo investigate the different expressions of cytoskeletal organizer ezrin and cytoskeleton protein beta- and gamma-actin in hepatocellular carcinoma (HCC) cell lines with different metastatic potentials and to explore the role of ezrin in cell growth and metastasis in HCC cell lines SF7721 and MHCC97-H.

METHODSImmunofluorescence, RT-PCR and Western blot were used to detect the gene and protein expressions of ezrin and actin in hepatocellular carcinoma cell lines with different metastatic potentials. RNA interference (RNAi) was applied to down-regulate the ezrin expression in SF7721 and MHCC97-H. Changes of the cell growth and metastasis potentials after the RNAi treatment were studied. MTT assay was used to detect cell proliferation changes and Transwell assay was applied to observe the changes of cell motility and invasiveness.

RESULTSBoth ezrin and cytoskeleton protein were demonstrated in the cytoplasma of the cells at the same time. The expression of them in cell lines with high metastatic potential, such as SF7721, MHCC-1 and MHCC97-H was obviously higher than in those with low metastatic potentials, such as SMMC-7721, Hep3B and HepG2 (chi2= 13.277, P = 0.010; chi2= 21.815). The mRNA and ezrin and cytoskeleton protein gamma-actin were over-expressed in HCC cell lines with high metastatic potentials. The expressions of beta-actin of cell lines with different metastatic potentials showed no differences. Ezrin protein was successfully down-regulated and the proliferation and the invasiveness of the cells decreased with low ezrin protein level in SF7721 and MHCC97-H.

CONCLUSIONOver-expression of ezrin and cytoskeleton protein gamma-actin are associated with the process of metastasis of hepatocellular carcinoma cells. The growth and invasiveness of SF7721 and MHCC97-H cells can be inhibited by down-regulating ezrin expression.

Actins ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cytoskeletal Proteins ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Neoplasm Invasiveness

Abstract: Objective　To investigate the distribution characteristics of HCV genotypes and subtypes in patients with HIV (human immunodeficiency virus, HIV)/HCV co-infection in Kunming based on the nucleocapsid protein gene sequence of HCV (hepatitis C virus). Methods　Serum was collected from HIV/HCV co-infected patients with household registration in 14 county-level cities, districts and counties under the jurisdiction of Kunming, who admitted to Yunnan Provincial Infectious Disease Hospital from March to August 2019. The viral RNA was extracted from the serum, reverse transcribed to synthesize cDNA, and the HCV nucleocapsid protein gene-specific primers were used for nested PCR amplification. The positive amplification products were sequenced, bioinformatics software such as DNAstar and MEGAX were used for sequence analysis. Results　A total of 64 samples from co-infected patients with clinical diagnosis of suspected HIV/HCV were collected and amplified by HCV nucleocapsid protein gene-specific primers, of which 17 samples were amplified positively. The results of sequence analysis showed that the sequences of 9 cases were located in the same evolutionary branch as the HCV 3b subtype sequence, and the nucleotide homology was 93.3%-95.2%; the sequences of 5 cases were located in the same evolutionary branch as the HCV 1b subtype sequence, and the nucleotide homology was 96.8%-97.6%; the sequence of one case and the subtype sequence of HCV 3a gene were located in the same evolutionary branch, and the nucleotide homology was 95.2%; the sequence of one case and HCV 6n gene subtype sequence were located in the same evolutionary branch, and the nucleotide homology was 97.9%; One case was located in the same evolutionary branch as the HCV 6u gene subtype sequence, and the nucleotide homology was 98.4%. Conclusions　HCV 1b, HCV 3a, HCV 3b, HCV 6n and HCV 6u genotypes or subtypes of HCV are prevalent in Kunming, and HCV 3b is the most prevalent genotype.

OBJECTIVEThe study was to describe the breastfeeding status of children under the age of three in counties of western China and to provide evidence to the government for decision-making on intervention.

METHODSA cross-sectional study with probability-proportional-to-size (PPS) sampling method was used. The information on breastfeeding was obtained through memory of the mothers. Fourteen thousand and seventy-seven children were studied. Data on breastfed status in counties of western China was compared with those of the children from the survey of the counties of western China in 2001.

RESULTSThe breastfeeding rate of children under 3 years old in western China was 96.5%. However, the overall breastfed rate of children under 6 months were only 33.4%, with rates of 11.4% and 22.0% on exclusively and predominantly breastfed groups respectively. Timely first-suckling rate was 43.5% with the continued breastfeeding rate (1 year) as 64.9%, but the continued breastfeeding rate (2 year) was only 9.7%. Reasons causing mothers to wean would include according to her own intention and to be able to attend the physical labor while exclusive breastfeeding under 6 months was for the growth and development of children, which might reduce the two-week prevalence of diarrhea. Major risks of exclusive breastfeeding of children under 6 months were seen as: level of education of the mothers, economic depression of the counties and mother's nationality (if as minority).

CONCLUSIONMost of the children were ever or being breastfed at the time of interview with timely first-suckling took place earlier than in 2001. However continued breastfeeding did not last long. During these five years, the exclusive breastfeeding rate had been at low level, especially at the economic depression and the minority area.

Breast Feeding ; epidemiology ; Child, Preschool ; China ; epidemiology ; ethnology ; Humans ; Infant ; Infant, Newborn ; Weaning

OBJECTIVETo investigate the efficiency of damage control surgery (DCS) and predictors of mortality in critically multiple trauma patients.

METHODSFrom May 1998 to February 2007, DCS were carried out in 27 patients with critically multiple trauma. Of the patients 15 cases survived (survival group) and 12 cases died (dead group). The surgical complications, causes of death, demographic, physiologic and medical parameters were collected and compared between the two groups. Multiple logistic regression analysis were performed to identify possible predictors of mortality.

RESULTSThe incidence of surgical complications was 37.0 percent, and the intra-abdominal infections was the most frequent (18.5%). The overall mortality rate was 44.4 percent. The most common causes of death was multiple organ dysfunction syndrome (50.0%). With respect to predicting mortality, statistically significant differences was found in parameters as age, injury severity score (ISS), initial temperature and base excess (BE), estimated blood loss, initial ICU temperature and length of hospital stay. Older age, increased absolute value of initial BE and lower initial ICU temperature were determined as independent predictors of mortality on multiple logistic regression analysis.

CONCLUSIONSThere is a comparable high morbidity and mortality rate in severely injured patients managed with DCS. Increased age, a larger absolute value of initial BE and lower initial ICU temperature could independently predict death of the patients.

Adolescent ; Adult ; Age Factors ; Aged ; Female ; Humans ; Injury Severity Score ; Logistic Models ; Male ; Middle Aged ; Multiple Trauma ; mortality ; surgery ; Multivariate Analysis ; Postoperative Complications ; Prognosis ; Temperature ; Young Adult

Background: Daratumumab is a human monoclonal antibody targeting CD38 used widely in various related conditions. Caution is advised when interpreting the pretransfusion tests in daratumumab-treated patients because they may show nonspecific reactions with red blood cells. This paper provides experimental evidence for the false-positive interference phenomena induced by daratumumab in in-vitro and ex-vivo experiments and experimental support for resolving it using dithiothreitol (DTT). Methods: Fifteen crossmatching pairs, four cardiac amyloidosis (CA) patients treated with daratumumab, and three healthy individuals were included. The flow cytometry crossmatch (FCMXM) was conducted with negatively selected T and B cells. After spiking the sera with 500 μg/mL daratumumab, the T and B cells were treated with DTT. The prospective FCMXM was conducted with the sera of CA patients treated with daratumumab. The CD38 expression levels in T, B, and NK cells were measured without and with a DTT or pronase treatment. Results: Five hundred μg/mL of daratumumab spiking was sufficient to elicit a false positive effect in T cell FCMXM. In particular, the administration of 0.1 M DTT efficiently resolved the induced false positivity in flow cytometry. Moreover, DTT caused a decrease in the CD38 expression levels in T, B, and NK cells. Conclusion A typical therapeutic dose of daratumumab causes false-positive FCMXM, which was effectively addressed by a DTT treatment. Therefore, information about the patient’s medical condition and the use of immunotherapeutics, such as daratumumab, is needed, given its impact on diverse CD38-expressing cells.

OBJECTIVETo know genotypes and serotypes of hepatitis B virus (HBV) detected from hepatitis B infected people in Yunnan Province.

METHODSSerum samples were collected from HBsAg carriers detected from people who had a physical examination at Yunnan Provincial Center for Disease Control and Prevention. The S genes of HBV were amplified by nested PCR and the PCR products were sequenced. The viral genotype was identified by phylogenetic analysis. 27 reference sequences corresponding to HBV genotype A to I were obtained from GenBank. According to the amino acid sequences deduced from the nucleotide sequences of S gene, the dominant serotype of HBV detected from these people were confirmed.

RESULTS39 HBsAg positive serum samples were detected from 2216 people who had a physical examination. The results shows that 76.9% were C genotype; 15.4% were B genotype; 5.1% were D genotype; 2.5% were I genotype. Three serotypes were found. The rates of adw2, adrq+ and ayr serotypes are 71.8%, 17.9% and 10.3% respectively. All of adw2 subtype specimens are C genotype. Among the serum specimens in which both HBsAg and HBeAg are positive, 75% were C genotype and adw2 subtype.

CONCLUSIONIt is determined that the main genotype and subtype of HBV prevailed in Yunnan province is C genotype and adw2 subtype.

Antibodies, Viral ; immunology ; China ; Female ; Hepatitis B ; virology ; Hepatitis B Surface Antigens ; genetics ; immunology ; Hepatitis B virus ; classification ; genetics ; immunology ; isolation & purification ; Humans ; Male ; Physical Examination ; Population Surveillance

The aim of study was to set up a suitable method of isolation, culture and identification of endothelial progenitor cells (EPC) derived from rabbit bone marrow. Density gradient centrifugation was used to isolate mononuclear cells from bone marrow, the isolated mononuclear cells were cultured with specific culture medium for EPCs. EPCs were identified by cellular morphologic observation, immunohistochemistry testing, flow cytometry and the function test of taking up Dil-ac-LDL and FITC-UEA-1. The results indicated that the newly isolated bone marrow-derived mononuclear cells exhibited a round appearance, following culture for 48 hours, adherent cells grew in colony cluster, presenting with round or irregular appearance, and nuclear division was obvious. On day 7, flaky cell colonies mutually connected together, presenting with spindle-shaped cells. Immunohistochemistry testing in the EPCs showed CD133(+), CD34(+), VIII factor(++), KDR(++); flow cytometry testing showed that the positive rate of CD133 was (18.23+/-7.12)%, the positive rate of CD34 was 47.71+/-14.85%, the positive rate of CD31 was (71.61+/-13.51)%, the positive rate of KDR was (87.24+/-11.40)%. And more than 80% EPC could take up both Dil-acLDL and FITC-UEA-1. It is concluded that the mononuclear cells isolated from bone marrow by density gradient centrifugation can differentiate into EPCs under special culture situation.
Animals ; Bone Marrow Cells ; cytology ; Cell Culture Techniques ; methods ; Cell Differentiation ; Cells, Cultured ; Endothelial Cells ; cytology ; Rabbits ; Stem Cells ; cytology

OBJECTIVETo study the radiosensitizing effect of gefitinib on nasopharyngeal carcinoma cell line CNE2 in vitro.

METHODSNasopharyngeal carcinoma cell line CNE2 was cultured in RP2MI 1640. MTT assay was performed to evaluate the cell proliferation changes in response to gefitinib treatment and the radiosensitizing effect of gefitinib. The cell survival curves and sensitive enhancement ratio (SERs) were obtained with a clonogenic assay. Flow cytometry analysis was applied to detect the cell cycle changes and cell apoptosis.

RESULTSMTT assay showed that cells exposed to gefitinib and radiation had a significantly lower survival ratio compared to the cells with radiation exposure only (0.582∓0.012 vs 0.398∓0.016, P=0.002), with a SER of 1.535∓0.134. The S phase cell percentage was significantly decreased and G(2)-M phase cells increased in gefitinib plus radiation group (P=0.000), suggesting a synergistic effect of gefitinib and radiation.

CONCLUSIONGefitinib can enhance the radiosensitivity of nasopharyngeal carcinoma CNE2 cells in vitro possibly by inhibiting cell proliferation, inducing cell apoptosis, and causing changes in the cell cycle distribution.

Apoptosis ; drug effects ; Carcinoma ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Flow Cytometry ; Humans ; Nasopharyngeal Neoplasms ; pathology ; Quinazolines ; pharmacology ; Radiation Tolerance ; drug effects

OBJECTIVETo study the effect of hydroquinone on apoptosis of bone marrow mononuclear cells, and to evaluate the toxic effect of benzene on stem cells.

METHODSCell morphology was observed by HT fluorescent stain method, and DNA fragments were analyzed by agarose gel electrophoresis. Anti-Annexin V FITC plus PI staining for apoptotic and necrotic rate was examined by flow cytometer.

RESULTSAfter adding different concentrations of hydroquinone to the cells for 6 h culture, the fluorescent intensity of nucleus increased, the color of nucleus became deep and inhomogeneous, and the chromatin was condensed and distributed around the neucleus. DNA ladder was detected in all samples. Cell apoptotic rate in different concentration of hydroquinone groups was significantly higher than that in blank control group (P < 0.05). With the increase of the concentration of hydroquinone, the apoptotic and necrotic rate also increased. The optimal concentration of hydroquinone was 50 micro mol/L. When it was >or= 75 micro mol/L, the necrotic rate increased significantly. Hydroquinone-induced apoptosis was associated with culture time at the concentration of 50 micro mol/L, and the peak apoptotic time was 10 h, then the apoptotic rate decreased and necrotic rate increased.

CONCLUSIONHydroquinone can induce apoptosis of bone marrow mononuclear cells in vitro with dose-effect and time-effect relationship.

Apoptosis ; drug effects ; Bone Marrow Cells ; cytology ; Cells, Cultured ; Dose-Response Relationship, Drug ; Humans ; Hydroquinones ; pharmacology ; Leukocytes, Mononuclear ; cytology ; drug effects ; Mutagens ; pharmacology