Background Proliferation of the human Tenon capsule fibroblasts(HTFs) is a main cause of failure of filtering surgery.To search the drug of inhibiting the growth of the HTFs is essential for the improvement of successful rate of filtering surgery.Objective The aim of this study was to investigate the inhibitory effect of apigenin on HTFs and its mechanism.Methods Human Tenon capsular tissue was obtained during the strabismus correction surgery.HTFs was primarily cultured using explant method and identified using vimentin by immunochemistry.The 3-5 generation of cells were incubated to 96-well plate.Apigenin of 0,20,40,80,160 μmol/L was added into the medium,respectively,for 24,48,72 hours,and the proliferation of HTFs was detected by sulfonyl chloride (SRB) at the wavelength of 560 nm (A560).Bromodeoxyuridine (BrdU) of 10 μg/L was added to culture the cells for 48 hours to calculate the labeling rate of BrdU.The morphology of the cells was observed using Hoechst 33258 staining,and apoptosis and cells cycle were evaluated by flow cytometry.Results Cultured cells grew well with the positive response for vimentin,showing the green fluorescence in cytoplasm.SRB assay showed that the A560 value was gradually declined with the increase of the dosage of apigenin and prolong of time (Fgroup =480.306,P =0.000 ; Ftime =555.144,P =0.000).The labeling rate after 0,40,80 μmol/L apigenin acted for 48 hours was (87.860 ±0.632)％,(61.520±4.306)％ and (23.480±4.472)％,showing a significant difference among the three groups (F =299.347,P =0.000).The labeling rate of HTFs for BrdU was significantly decreased in the 40 and 80 μmol/L apigenin groups compared with the 0 μmol/L apigenin group (P＜0.05).Hoechse 33258 staining found that the number of the HTFs was gradually decreased and the cell number of karyopyknosis and nuclear deformation was increased with the increase of apigenin dosage.Percentage of cells in G0/G1 phase were raised and that in S and G2/M phase were declined in the higher dosage apigenin group,with a significant difference among the different groups (FG0/G1 =58.621,P=0.000;Fs =32.357,P=0.001 ;FG2/M =83.998,P=0.000).In the 72nd hour after acted by 0,40,80,160 μmol/L apigenin,the apoptosis rate of HTFs was (4.77±0.21) ％,(13.24±1.35)％,(18.33±1.86) ％,(31.58 ± 2.77) ％,respectively,with a statistically significant difference among the four groups (F =204.791,P＜0.05).Conclusions Apigenin restrains the growth of HTFs by evoking G0/G1 cell cycle arrest and inducing apoptosis in a dosage-and time-dependent manner.

Child ; Female ; Humans ; Menisci, Tibial ; abnormalities ; surgery

Prolyl-4-hydroxylase domain (PHDs) family is one of the most important regulatory factors in hypoxic stress. PHD2 plays a critical role in cells and tissues adaptation to the low oxygen environment. Its hydroxylation activity regulates the stability and transcriptional activity of the hypoxia-inducible factor 1 (HIF-1), which is the key factor in response to hypoxic stress. Subsequently, PHD2 acts as an important factor in oxygen homeostasis. Studies have shown that PHD2, through its regulation on HIF-1, plays an important role in the post-ischemic neovascularization. Furthermore, under hypoxic condition, PHD2 also regulates other pathways that positively regulate angiogenesis factors HIF-1 independently. Moreover, recently, several evidences have also shown that PHD2 also affects tumor growth and metastasis in a tumor microenvironment. Based on these facts, PHD2 have been considered as a potential therapeutic target both in treating ischemic diseases and tumors. Here, we review the molecular regulation mechanism of PHD2 and its physiological and pathological functions. We focus on the role of PHD2 in both therapeutic angiogenesis for ischemic disease and tumor angiogenesis, and the current progress in utilizing PHD2 as a therapeutic target.
Animals ; Humans ; Hydroxylation ; Hypoxia-Inducible Factor 1 ; metabolism ; Hypoxia-Inducible Factor-Proline Dioxygenases ; antagonists & inhibitors ; physiology ; Neoplasms ; blood supply ; metabolism ; pathology ; therapy ; Neovascularization, Pathologic ; metabolism ; pathology ; Tumor Microenvironment ; Vascular Diseases ; pathology ; therapy

Aged ; Aged, 80 and over ; Anti-Bacterial Agents ; pharmacology ; Coal Mining ; Cross Infection ; complications ; microbiology ; Drug Therapy, Combination ; therapeutic use ; Humans ; Male ; Methicillin ; pharmacology ; Methicillin Resistance ; Microbial Sensitivity Tests ; Middle Aged ; Pneumonia ; drug therapy ; etiology ; Silicosis ; classification ; complications ; Staphylococcus aureus ; drug effects ; Treatment Outcome

0.05).Compared with the CSS,GMFM and WV before treatment,there were statistically difference after 6 and 12 weeks treatment in two groups(Pa

Objective To investigate the effect and mechanism of transplantation of marrow multi- function stem ceils in treating femoral head necrosis.Methods Sixty japanese rabbits were divided into A,B and C groups randomly.After creation of the models of hormone induced necrosis of femoral head;A group was designated as the treatment,B as the control and C as the normal groups.The bone marrow of A group was extracted and isolated and then injected into the left femoral head and the right femoral head was decompressed by drilling only.The rabbits were killed at 8 weeks after the treatment and changes in various parameters were observed,including imaging data of molybdenum target films,CT and MRI;routine pathology with HE staining and ultrastructural alteration by election microscopy.Results Eight weeks after the treatment of transplantation of marrow multifunction stem cells,the X-ray showed only a little change but the typical appearances were revealed by CT and MRI.Pathohistologic manifestation demonstrated decrease of empty bone lacuna,increase of osteoblast and new bone formation.Election microscopy displayed abundant organelles in osteoblasts with few empty bone lacuna,in addition the tansplantation of marrow multifunction stem cells could obtain better reconstraction for the involved femoral head.Conclusions The treatment of transplantation of marrow muhifunction stem cells in femoral head necrosis could accelerate the process of repairing,worthy to be acknowledged as a good and valuable management in rabbits.(J Intervent Radial,2007,16:122-126)

Objective: To observe the therapeutic efficacy of acupoint application at different groups of acupoints in treating bronchial asthma in remission stage. Methods:A total of 120 patients with bronchial asthma in remission stage were recruited and divided by the random number table method into acupoint application group 1, acupoint application group 2 and acupoint application group 3, with 40 cases in each group. In all the three groups, Tiantu (CV 22), Dazhui (GV 14) and Feishu (BL 13) were selected, with Dingchuan (EX-B 1) added in acupoint application group 1, Shenshu (BL 23) added in acupoint application group 2, and Gaohuang (BL 43) added in acupoint application group 3. Before intervention, one month and 3 months after intervention, clinical symptoms, peak expiratory flow (PEF) andforced expiratory volume in 1 second percentage of predicted value (FEV1％) of the three groups were observed, and their clinical efficacies were evaluated. Results: Comparing the therapeutic efficacy regarding traditional Chinese medicine symptoms and signs, after 1-month treatment, the total effective rate was 87.5％ in acupoint application group 1, versus 62.5％ in acupoint application group 2 and 55.0％ in acupoint application group 3, and the between-group differences were statistically significant. After 3-month treatment, the total effective rate was 95.0％ in acupoint application group 1, versus 70.0％ in acupoint application group 2 and 65.0％ in acupoint application group 3, and the between-group differences were statistically significant. After intervention, the three groups all showed significant improvements in pulmonary function with statistical significance; among the three groups, the improvement in acupoint application group 1 was more significant than that in the other two groups. Conclusion: Tiantu (CV 22), Dazhui (GV 14) and Feishu (BL 13) as basic prescription plus Dingchuan (EX-B 1) can improve symptoms of bronchial asthma in remission stage, and it works better in improving pulmonary function than the basic prescription plus Shenshu (BL 23) or Gaohuang (BL 43).

To establish a UPLC-MS/MS method for the simultaneous determination of geniposide, genipin 1-O-beta-D-gentiobioside and geniposidic acid in rat brains and study the brain pharmacokinetics of the three iridoid glycosides in stroke rat after the oral administration of Xingnaojing. In this experiment, brain samples were precipitated with protein for twice. Acquity BEH C18 column was adopted, with acetonitrile-0.1% formic acid-water as the mobile phase for gradient elution. ESI source was adopted for mass spectra; multiple reaction monitoring (MRM) was conducted to detect negative ions. The time for sample analysis was 3.5 min. the results showed good linear relations among the three iridoid glycosides, with the extraction recovery between 99.6% and 114.3%, good intra- and inter-day precisions and accuracies and stability in line with the requirements. The t1/2 and MRT in the three components were similar in brains of stroke rats. Geniposide and genipin 1-O-beta-D-gentiobioside showed double peaks; where as geniposidic acid showed a single peak. In conclusion, the method is so specific, sensitive, accurate and reliable that it can be used to study the brain pharmacokinetics of Xingnaojing oral preparation.
Animals ; Brain ; metabolism ; Brain Chemistry ; Chromatography, High Pressure Liquid ; methods ; Drug Stability ; Drugs, Chinese Herbal ; chemistry ; pharmacokinetics ; Iridoids ; chemistry ; pharmacokinetics ; Male ; Rats ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry ; methods

The aim of the present study was to investigate the role of mitogen-activated protein kinase kinase 6 (MKK6)-P38 signaling pathway in cyclic mechanical stretch-induced high mobility group box 1 protein (HMGB1) expression in alveolar macrophages. In the study, Sprague-Dawley rats were anesthetized and then sacrificed by bloodletting. The lungs were lavaged six times with prechilled PBS. Alveolar macrophages were isolated from lavage samples. Recombinant plasmids were transfected into alveolar macrophages with liposome DOTAP. Alveolar macrophages transfected with P38(AF)/pGFP and MKK6b(E)/pGFP plasmids were taken as treated groups, while the groups that transfected with pcDNA3 plasmid and pGFP plasmid served as blank transfection group and control group, respectively. All the groups were then cultured in 6-well Bioflex cell culture plates and exposed to cyclic mechanical stretch at 20% elongation using Flexercell 4000T cell stretching unit. The results showed that the transfection of MKK6b(E) led to a marked increases in P38 kinase activity compared with control group. In contrast, the transfection of P38(AF) significantly inhibited P38 kinase activity. Compared with control group, HMGB1 protein and mRNA expression in MKK6b(E) transfected cells increased markedly, while HMGB1 expression in P38(AF) transfected cells decreased markedly. These results suggest that MKK6-P38 MAPK signaling pathway regulates the expression of HMGB1 induced by cyclic mechanical stretch in alveolar macrophages.
Animals ; Cells, Cultured ; HMGB1 Protein ; metabolism ; MAP Kinase Kinase 6 ; metabolism ; Macrophages, Alveolar ; enzymology ; Phosphorylation ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Stress, Mechanical ; p38 Mitogen-Activated Protein Kinases ; metabolism

Metabolic engineering is the analysis of metabolic pathway and designing rational genetic modification to optimize cellular properties by using principle of molecular biology. Aromatic metabolites such as tryptophan, phenylalanine, and tyrosine are essential amino acids for human and animals. In addition, phenylalanine is used in aspartame production. Escherichia coli and many other microoganism synthesize aromatic amino acids through the condensation reaction between phospho-enolpyruvate (PEP) and erythrose-4-phosphate(E4P) to form 3-deoxy-D-arabinoheptulosonate 7-phosphate(DAHP). But many enzymes compete for intracellular PEP, especially the phosphotransferase system which is responsible for glucose transport in E. coli. This system uses PEP as a phosphate donor and converts it to pyruvate, which is less likely to recycle back to PEP. To channel more carbon flux into the aromatic pathway, one has to overcome pathways competing for PEP. ppsA and tktA are the key genes in central metabolism of aromatic amino acids biosynthesis. ppsA encoding phosphoenolpyrucate synthetase A (PpsA) which catalyzes pyruvate into PEP; tktA encoding transketolase A which plays a major role in erythrose-4-phosphate (E4P) production of pentose pathway. We amplified ppsA and tktA from E. coli K-12 by PCR and constructed recombinant plasmids of them in pBV220 vector containing P(R)P(L) promoter. Because of each gene carrying P(L) promoter, four productions of ligation were obtained. The monoclonal host containing recombinant plasmids was routinely grown in Luria-Bertani (LB) medium added Ampicillin at 37 degrees C overnight, and then inoculated in LB (Apr) medium by 3%-5% in flasks on a rotary shaker at 30 degres C, induced at 42 degrees C for 4.5 hours when OD600 = 0.4, cells were obtained by centrifugation at 10,000 r/min at 4 degrees C. The results of SDS-PAGE demonstrated that the bands at 84kD and 73kD were more intensive than the same ones of the controls. The specific activity of PpsA in crude extracts was increased by 10.8-fold, and TktA, by 3.9-fold. When both genes were co-expressed in E. coli, the activity of PpsA varied from 2.1-9.1 fold comparing to control, but the activity of TktA was relatively stable(3.9-4.5 fold). Whatever the two genes were expressed respectively or cooperatively, both could promote the production of DAHP, the first intermediate of the common aromatic pathway, but co-expression was more effective on forming DAHP. The results demonstrate that co-expression of ppsA and tktA can improve the production of DAHP to near theoretical yield. This report details a different strategy based on co-expression of two genes in one vector in vivo to release the burden and paves the way for construction of genetic engineering bacteria for further research.
Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; enzymology ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; metabolism ; Plasmids ; genetics ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; genetics ; Pyruvate Synthase ; genetics ; metabolism ; Transketolase ; genetics ; metabolism