Background Ginkgolide B (GB) has been proved to have neuroprotective and anti-apoptotic effects and can effectively inhibit apoptosis of retinal photoreceptor cells.But the high hydrophobic feature and low bioavailability of GB limit its clinical application.Self microemulsifying drug delivery system (SMEDDS) can effectively improve the infusibility drug dissolution and bioavailability in the retina.Objective This study was to investigate the pharmacokinetics and drug-time change of GB-loaded SMEDDS in retina.Methods Eighty SD rats were randomized into 2 groups,2.5％ GB(40 mg/kg) of SMEDDS or GB suspension(0.1％ DMSO dissolve) were gastrically given respectively in two groups.The rats were sacrificed and retinas were isolated 15,30,45 minutes and 1 hour,2,4,8,12 hours to prepare the retinal suspension.The content of GB in retina was assayed with high performance liquid chromatography-electrospray ionization-(1) (1)ss spectrum (HPLC-ESI-MS) and contrasted with standard curve.Practical drug dynamics program 3p87 was used to detect the pharmacokinetics parameters.The maximal content(Cmax,mg/g),time to peak (Tmax,h),clearance ratio (Ke/h),high-life period (t1/2) and area under the concentration-time curve(AUC0-∞,mg/(g · h)) of GB in various time points in retina after a single oral dose were calculated and compared between two groups.Results The standard curve was obtained over the concentration range of 1-32 mg/L with a linear regression equation,Y =0.0732X + 0.056 (r =0.992).A similar content-time curve was seen between GB suspension group and GB-SMEDDS group.The GB content was higher in GB-SMEDDS group than that in GB suspension group from 30 minutes through 12 hours after administration of drugs.The Cmax of GB-SMEDDS group and GB suspension group were(15.83±1.84) mg/g and(2.65±0.10) mg/g,the AUC0-∞ were(15.30±0.11)mg/(g· h)and(6.42±0.19)mg/(g · h).Conclusions HPLC-ESI-MS is proved to be a rapid,accurate,sensitive and suitable method for pharmocokinetic study of GB.SMEDDS can raise the concent of GB in retina,and it probably improve the bioavailability of GB.

Adolescent ; Female ; Humans ; Mucocele ; Nose Diseases ; pathology ; Turbinates ; pathology

OBJECTIVE Liver cancer is one of the most common causes of cancer related deaths worldwide, specially, in China. Hinesol, extracted from Atractylodeslance a(Thunb.) DC. has been proved that has anti-cancer effect in leukemia in vitro and in vivo.However,it has been not well under-stood in liver cancer cells.METHODS Cell proliferation,apoptosis,cell cycle and invasion were performed to investigate the anti-liver cancer effect of hinesol in SMMC-7721 and LM3 by MTT assay,flow cytometry and scratch assay.Western blot was used to research the potential mechanism.RESULTS We revealed that hinesol suppresses cell proliferation and invasion,prompts population of G1 phase,induces apop-tosis in dose-dependent manner in SMMC-7721 and LM3 cells.Western blot data showed that hinesol could inhibits the expression of cyclin-D1, Bcl-2 and Bax, and inhibited phosphorylation of MEK and ERK, down-regulated the expressions of NF-κB p65 and phosphor-p65 in nucleus. The results indicated that hinesol reduces cell proliferation via arresting cell cycle at G1 phase and induces apoptosis.Further-more,western blot showed that hinesol inhibited phosphorylation of MEK and ERK,down-regulated the expressions of NF-κB p65 and phosphor-p65 in nucleus.CONCLUSION Our results demonstrate that hinesolreduces cell proliferation via arresting cell cycle at G1 phase and induces apoptosis, it has potent anti-cancer effect against liver cancer cells via down-regulation of MEK/ERK and NF-κB pathway,and indicate that hinesol is a potential liver cancer drug for further research.

Objective:To methodologically establish the lentivirus granule packaging, concentration and infection against CD34~+ cells from umbilical blood. Methods:The lentivirus system of the 3~(rd) generation was used to produce the virus. Ultrafiltration and ultracentrifugation were employed to concentrate virus. Several treatments were used to improve virus infection including in vitro amplification culture, facilitation of rest cells into cell cycle, promotion of cell adhesion and immobilization during infection, and repeat infection methods. Results:CD34~+ cells were not obviously changed by checking the expression level of CD34 marker on the cell surface after 48 h culture. After two-step concentration, virus titer was increased up to 5.06×10~7/ml, and the infection rate against CD34~+ cells from umbilical blood was increased up to 37.7%.Conclusion:Lentivirus supernatant with over 10~7/ml titer can be obtained using the above methods. Efficient infection against CD34~+ cells from umbilical blood can be achieved.

Adult ; Aged ; Animals ; Female ; Humans ; Kidney Calculi ; surgery ; Male ; Middle Aged ; Nephrostomy, Percutaneous ; instrumentation ; methods ; Pyonephrosis ; surgery ; Swine ; Swine, Miniature ; Ultrasonography, Interventional

Adolescent ; Arrhythmias, Cardiac ; etiology ; Cardiac Catheterization ; Child ; Child, Preschool ; Electrocardiography ; Female ; Heart Septal Defects, Atrial ; therapy ; Humans ; Infant ; Male

This study is to report the study of protective effects of myricitrin against oxidative stress-induced apoptosis of vascular endothelial cells and the investigation of the possible mechanisms of action of myricitrin. The model of H2O2-induced apoptosis of vascular endothelial cells was used to determine the protective effects of myricitrin. The levels of LDH, MDA and the activities of SOD, NO were measured using the respective kits. The H2O2-induced apoptosis of vascular endothelial cells was detected using MTT reduction, TUNEL assay, JC-1 and ROS staining. The activation of Caspase-3 was also measured by fluorometry. The expression of apoptosis-related proteins was determined with Western blotting assay. Myricitrin had significant protective effects against H2O2-induced apoptosis of vascular endothelial cells in a time- and dose-dependent manner. The results show that myricitrin could attenuate H2O2-induced decrease in the activities of SOD (P < 0.01). Myricitrin could decrease the levels of LDH, MDA and increase cell viability and the mitochondrial membrane potential (P < 0.01). Myricitrin had protective effects in a dose-dependent manner between 32 micromol x L(-1) to 64 micromol x L(-1). Myricitrin pretreatment could attenuate H2O2-induced increase of p-ERK. Moreover, myricitrin pretreatment could up-regulate the expression of the anti-apoptotic protein Bcl-2, down-regulate the expression of the pro-apoptotic protein Bax, and decrease the expression of Caspase-3, 9. In conclusion, myricitrin had significant protective effects against H2O2-induced apoptosis of vascular endothelial cells. Myricitrin can enhance the activities of anti-oxidative enzymes and decrease the production of free radicals. The possible mechanisms of action of myricitrin are due to myricitrin-mediated inhibition of phosphorylation of the apoptosis signaling pathways-related kinase ERK, up-regulation of the expression of the anti-apoptotic protein, and down-regulation of the expression of the pro-apoptotic protein.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Survival ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Endothelial Cells ; cytology ; Flavonoids ; administration & dosage ; pharmacology ; Humans ; Hydrogen Peroxide ; toxicity ; L-Lactate Dehydrogenase ; metabolism ; Malondialdehyde ; metabolism ; Membrane Potential, Mitochondrial ; drug effects ; Nitric Oxide ; metabolism ; Oxidative Stress ; drug effects ; Protective Agents ; administration & dosage ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; metabolism ; bcl-2-Associated X Protein ; metabolism

BACKGROUNDRenal transplantation in sensitized candidates remains a highly significant challenge worldwide. The production of panel reactive antibody (PRA) against human leukocyte antigen (HLA) is a major risk factor in presensitized recipients. The aim of this study was to evaluate the impact of HLA matching and recipients' PRA on two-year outcome in presensitized renal allograft recipients.

METHODSWe determined the percentage of panel reactivity and specificity of anti-HLA immunoglobulin (Ig) G antibodies in 73 presensitized renal allograft recipients compared with 81 unsensitized recipients (control group). HLA genotyping of both recipients and corresponding donors was performed by PCR with sequence-specific primers (PCR-SSP). We analyzed the factors influencing the early graft outcome (two-year rejection rates and survival rates of the grafts), including HLA mismatching, class and degree of panel reactivity, and target antigen of donors.

RESULTSPresensitized recipients had a worse two-year outcome than unsensitized recipients (P = 0.019 for rejection rate, P = 0.01 for survival rate). The difference in number of HLA-mismatched alleles with either 6-antigen matching (Ag M) standard or amino acid residue matching (Res M) standard was not significant between the rejection and non-rejection groups of presensitized recipients or between the graft survival group and graft loss group. Compared with the control group, recipients with both PRA-I and PRA-II antibodies had a significantly worse two-year outcome (P = 0.001 for rejection rate, P = 0.002 for survival rate). The two-year outcomes of the peak PRA >/= 50% group and its subgroup, at-transplant PRA > or = 50% group, were significantly worse compared with the control group (P = 0.025 and P = 0.001 for rejection rate, P = 0.043 and P = 0.024 for survival rate). The rejection rates of the at-transplant target antigen positive group and its subgroup, HLA-I target antigen positive group, were significantly higher than the control group (P = 0.001 and P = 0.001), target antigen negative group (P = 0.003 and P = 0.001), and peak target antigen positive with negative at-transplant target antigen group (P = 0.024 and P = 0.002). Two-year graft survival rates of the target antigen positive group and HLA-I target antigen positive group were significantly lower than the control group (P = 0.012 and P = 0.001). The two-year outcome of target antigen unknown group was similar to that of the target antigen positive group. Presensitized recipients with pre-transplant plasmapheresis or immunoadsorption (PRA prepared group) had a better but non-significant two-year outcome than the control group. However, the PRA unprepared presensitized recipients were different to the control group (P = 0.004 for rejection rate and P = 0.005 for survival rate). Hyperacute rejection (HR) occurred in three recipients with positive HLA-I target antigen and without mismatch according to Res M and in one case with positive PRA-II (for an unknown target antigen). No HR occurred in eight cases with positive HLA-II target antigens.

CONCLUSIONSPre-transplant PRA preparations might improve the access of presensitized patients to renal donors. Avoiding antigen-positive donors remains a fundamental measure in preventing HR and early rejections.

Adult ; Enzyme-Linked Immunosorbent Assay ; Female ; Graft Rejection ; immunology ; Graft Survival ; immunology ; HLA Antigens ; immunology ; Histocompatibility Testing ; Humans ; Isoantibodies ; blood ; Kidney Transplantation ; adverse effects ; immunology ; mortality ; Male ; Middle Aged ; Transplantation, Homologous ; immunology ; Treatment Outcome

OBJECTIVETo observe the role of tumor necrosis factor α (TNF-α) in endothelial-mesenchymal transition (EnMT), and to explore the mechanism of fibrosis disease.

METHODSHuman umbilical vein endothelial cells (HUVEC) from umbilical cord of healthy fetus were isolated by enzymatic digestion and identified by immunofluorescence assay. The third to fifth generations of cultured HUVEC in logarithmic phase were harvested and seeded in 12-well plates and 6-well plates, and they were divided into control group (ordinary culture without any stimulation), 5, 10, 25, 50, and 100 ng/mL TNF-α groups (5, 10, 25, 50, 100 ng/mL of TNF-α was respectively added into the nutrient solution) according to the random number table, with three samples in each group. After being cultured for 72 hours, the cell morphology was observed under inverted phase-contrast microscope; the expression levels of coagulation factor VIII and α smooth muscle actin (α-SMA) were detected by immunofluorescence assay, and the ratios of numbers (absorbance values) of cells with expression of both factors were calculated. The mRNA expression levels of cadherin, α-SMA, and type I collagen were detected by RT-PCR (denoted as gray value ratio). Data were processed with one-way analysis of variance and LSD test.

RESULTS(1) The shape of primary HUVEC was round, short-spindle, or flat, and cells grew vigorously in cobblestone appearance after passages. After being subcultured for 1, 2, 3, 4, 5 passage (s), the positive rate of coagulation factor VIII of HUVEC was respectively (85.5 ± 1.8)%, (88.1 ± 5.0)%, (93.6 ± 3.7)%, (92.9 ± 4.8)%, (89.5 ± 1.1)%, and they were significantly higher than that of primary HUVEC [(81.4 ± 3.8)%, with F values all equal to 7.481, P values all below 0.05]. (2) As compared with that in control group, the appearance of cells in 5, 10, 25, 50, and 100 ng/mL TNF-α groups was gradually transformed from round, short-spindle, or flat shape to long-spindle shape with reduced intercellular junction and larger intercellular gap along with the increase in the concentration of TNF-α. (3) The ratios of numbers and the absorbance values of coagulation factor VIII and α-SMA double positive cells in control group (0.055 ± 0.015, 0.078 ± 0.017) were significantly lower than those in 5, 10, 25, 50, and 100 ng/mL TNF-α groups (0.257 ± 0.106, 0.280 ± 0.129, 0.505 ± 0.059, 0.817 ± 0.035, 0.929 ± 0.101 and 0.437 ± 0.040, 0.456 ± 0.097, 0.496 ± 0.082, 0.787 ± 0.131, 0.885 ± 0.087, with F value respectively 45.009, 50.099, P values all below 0.01). (4) The expression levels of cadherin mRNA in 5, 10, 25, 50, and 100 ng/mL TNF-α groups were 0.70 ± 0.05, 0.63 ± 0.06, 0.60 ± 0.10, 0.45 ± 0.16, and 0.26 ± 0.14, and it was significantly lower in the latter four groups than in control group (0.83 ± 0.03, with F values all equal to 11.593, P < 0.05 or P < 0.01). The mRNA expression levels of α-SMA and collagen I in 5, 10, 25, 50, and 100 ng/mL TNF-α groups were 0.45 ± 0.10, 0.51 ± 0.16, 0.49 ± 0.12, 0.60 ± 0.09, 0.76 ± 0.03 and 0.38 ± 0.18, 0.45 ± 0.15, 0.52 ± 0.12, 0.66 ± 0.17, 0.76 ± 0.20, and they were significantly higher in the latter three groups than in control group (0.37 ± 0.14, 0.31 ± 0.12, with F value respectively 7.839, 2.898, P < 0.05 or P < 0.01).

CONCLUSIONSTNF-α can obviously promote EnMT in a dose-dependent manner. EnMT may be another significant source of myofibroblasts that contributes to fibrotic tissue in scar formation.

Cell Differentiation ; Cells, Cultured ; Epithelial-Mesenchymal Transition ; drug effects ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Stromal Cells ; cytology ; Tumor Necrosis Factor-alpha ; pharmacology

Volatile oil components and the contents and types of amino acid in spica of Prunella vulgaris were analysed by GC-MS and amino acid analyzer. Esters, fatty acids, aromatic hydrocarbon, ketone and several alcohol compounds were identified by mass spectrum comparison. In these ingredients, beta-ionone smelled aroma of cedar, raspberry, nerolidol showed weak sweet soft orange blossom flavor, neroli tasted sweet and fresh, nerolidol tasted sweet with light aroma of wood, hexadecanal showed a weak aroma of flowers and wax, alpha-sinensal had rich and fresh sweet orange flavor. To some extent, these types of aromatic substances can affect the taste of herbal tea or decoction made of Spica Prunellae. Among amino acids detected, natural amino acids accounted for a larger proportion, and those natural amino acids showed bitterness, slight bitterness, sourness (freshness), sweetness, slight sweetness, sourness (slight freshness). The results indicated that bitter and slightly bitter amino acids have the greatest impacts on the sense of Spica Prunellae.
Amino Acids ; analysis ; Gas Chromatography-Mass Spectrometry ; Oils, Volatile ; analysis ; Prunella ; chemistry ; Taste