BACKGROUND:To date,no single material can completely meet the clinical requirements.However,the composite materials characterized by good biodegradability,biocompatibility and osteoconductivity have become a highlight of the artificial bone materials.OBJECTIVE:To synthesize the basic fibroblast growth factor (bFGF)-poly(lactic-co-glycolic acid) (PLGA) microspheres/hydroxyapatite (HA)/poly(I-lactic acid) (PLLA) porous bone scaffolds,and to observe the physicochemical properties and biocompatibility of the composite material.METHODS:The bFGF-PLGA microspheres were prepared by double emulsion method,and then six kinds of materials were made including PLLA,PLLNHA,PLLAJPLGA,PLLNHNPLGA,PLLNHA/bFGF,and bFGF-PLGA microspheres/PLLA/HA.The characterization of the materials were observed by particle size analyzer,transmission electron microscopy,X-ray diffraction,Fourier transform infrared spectrometer,microcomputer differential thermal balance,and scanning electron microscope.Toxicity of these materials and proliferation of bone marrow mesechymal stem cells seeded onto these materials were analyzed and compared.RESULTS AND CONCLUSION:The average particle size of bFGF-PLGA microspheres was about 250 nm,the average drug-loading capacity was (26.03±0.17)％,and the entrapment percentage was (90.65±2.68)％.The prepared bFGF-PLGA microspheres were spherical and had good dispersibility.In addition,all the six kinds of materials had a porous structure with similar pore diameter,in which the microspheres and particles exhibited a rational distribution.The toxic level of bFGF-PLGA microspheres/PLLNHA,bFGF/HNPLLA and HAP/PLLA was graded as 1 (with a relative survival rate ≥ 80％),indicating no obvious toxicity or slight toxicity.All these six kinds of composite materials can promote the proliferation of bone marrow mesenchymal stem cells,and the bFGF-PLGA microspheres/PLLNHA shows the best effects on cell proliferation and has good biocompatibility.

Animals ; Fatty Liver ; drug therapy ; metabolism ; prevention & control ; Liver ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism ; Taurine ; therapeutic use

Human Nestin (hNestin) has been found to express in melanoma,and its expression is positively correlated with the advanced stage of melanoma.However,the precise role of hNestin in the development of melanoma has not been fully understood.The present study aimed to explore the role of hNestin in the proliferation and invasion of melanoma cells.The lentivirus vector carrying a short hairpin RNAs (shRNAs) targeting hNestin (hNestin-shRNA-LV) was stably infected into human melanoma cells UACC903,which expressed high levels of hNestin.The effects of hNestin knockdown on the proliferation,apoptosis,migration of melanoma cells and the related signaling pathways were investigated by immunofluorence,Western blotting and reverse transcription polymerase chain reaction (RT-PCR),respectively.The results showed that hNestin was expressed in most melanoma specimens and the melanoma cells studied.Knockdown of hNestin expression significantly inhibited the proliferation of melanoma cells,blocked the formation of cell colony,arrested cell cycle at G1/S stage and suppressed the activation of Akt and GSK3β.hNestin-silent cells also showed a sheet-like appearance with tight cell-cell adhesion,decreased membrane expression of N-cadherin and β-catenin,and attenuated migration.Furthermore,hNestin silence resulted in the inhibition of tumor growth in vivo.Our study indicates that hNestin knockdown suppresses the proliferation of melanoma cells,which might be through affecting Akt-GSK3β-Rb pathway-mediated G1/S arrest,and hNestin silence inhibits the migration by selectively modulating the expression of cell adhesion molecules in the process of epithelial-mesenchymal transition.

Objective To develop a high-performance liquid chromatography (HPLC) method for the measurement of lecithin-cholesterol acyltransferase(LCAT) activity and analyze the relationships between LCAT activity and the traditional risk factors of atherosclerotic cardiovascular disease(CVD).Methods The liposome which contained 7-dehydrocholesterol and 1,2-didecanoyl-sn-glycero-3-phosphocholine (10∶0 PC)as the substrate of LCAT and LCAT activating peptide (LAP642)as LCAT activator was mixed with 10 microliters of serum sample(50∶ 1,V/V)in ice-water bath and subsequently incubated at 37 ℃ for 1 h.After extracting with hexane,the lipid was analyzed by HPLC and the LCAT activity was calculated as the ratio of 7-dehydrocholesterol ester to free 7-dehydroeholesterol.LCAT activities of 120 health volunteers were measured and its relationship with traditional risk factors of CVD was analyzed.Results The liposome composed of substrates(7-dehydrocholesterol and 10∶0 PC with ratio of amount 1∶ 8.5)and LAP642 was stable,efficient and easy for preparation.LCAT activity was a linear correction during 8 hours of incubation and was independent of the volume of serum added in the range from 0 to 20 microliters.The averages of intra-and total coefficients of variation(CV)were less than 1.76％ and 3.11％ respectively.The comparison of two methods showed that the results of the HPLC method were highly correlated with LCAT mass measured by commercial ELISA method and LCAT activity measured by endogenous substrate fractional esterification of high density lipoprotein cholesterol (FERHDL)(P ＜ 0.01).LCAT activity positively correlated with body mass index(BMI),triglyceride (TG) (P ＜ 0.05) and negatively correlated with apolipoprotein AI (apoAI) (P ＜ 0.05) and high density lipoprotein cholesterol (HDL-C) (P ＜ 0.01) in the volunteers.Conclusion A simple,precise and reliable HPLC method for determination of LCAT activity using artificial substrate has been established,and the results were not influenced by endogenous cholesterol levels in serum.The newly developed method could be a useful tool in the study of lipid metabolism and the assessment for risk factors of CVD.

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

OBJECTIVETo explore the possible mechanism(s) of taurine-inhibiting the proliferation of hepatic stellate cells (HSC), this study investigated the effect of taurine on the HSC cell cycle and its regulatory protein expression.

METHODSCell proliferation was assessed by MTT assay. Cell cycle was analyzed by flow cytometry. Cell cycle regulatory protein Cyclin D1 and P21waf1 expression were determined by immunocytochemistry and image-analysis system, and real-time quantitative PCR.

RESULTSHSC proliferation was markedly inhibited when HSC were treated with taurine at concentrations of 5, 10, 20, 30, 40 and 50 mmol/L for 48 hours, and the inhibition rates were 6.7%, 14.4%, 23.3%, 32.2%, 36.7% and 45.6% respectively (P < 0.05-0.01). In the flow cytometry analysis, it was found that taurine could block HSC in the G0/G1 phase from entering the S phase, resulting in more cells in the G0/G1 phase and fewer in the S phase. The percentage of the cells in the G0/G1 phase and the S phase at the dosage of 40 mmol/L were 68.2%+/-1.4% and 26.2+/-1.3% respectively, which was significantly different in comparison to the controls (56.2%+/-1.7% and 38.5%+/-0.8% respectively, P < 0.01). HSC expressed cyclin D1 and P21waf1. Taurine inhibited cyclin D1 expression and induced P21waf1 expression. The cyclin D1 protein and mRNA in the HSC treated with 40 mmol/L taurine were significantly reduced compared with the controls [protein (optical density value): 0.13+/-0.02 versus 0.18+/-0.02, P < 0.01; mRNA: 5776.7+/-3345.0 versus 18,400.6+/-1374.8 copies/10(6) GAPDH, P < 0.01]; and the P21waf1 protein and mRNA were markedly increased compared with the controls [protein (optical density value): 0.19+/-0.02 versus 0.14+/-0.01, P < 0.01; mRNA: 44,866.7+/-3910.7 versus 16,933.3+/-960.9 copies/10(6) GAPDH, P less than 0.05].

CONCLUSIONSCyclin D1 and P21waf1 were cell cycle regulatory proteins in HSC, and taurine can inhibit the HSC cyclin D1 expression and stimulate P21waf1 expression, facilitate arresting cells in G0/G1 phase, and suppress cell proliferation.

Animals ; Cell Cycle Proteins ; biosynthesis ; genetics ; Cell Line ; Cell Proliferation ; Cyclin D1 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Depression, Chemical ; Hepatocytes ; cytology ; Rats ; Taurine ; pharmacology

To establish a new high-throughput screening model for the agonist of PPARdelta, PPARdelta gene was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR), and subcloned to pGEM-T Vector for sequencing, then the PPARdelta fragment was excised by restriction enzymes, and inserted into pTARGET Vector to construct expression vector pTARGET-ppARdelta. Insert three copies of PPRE into pGl3-promoter vector to construct expression vector pGl3-PPRE x 3-luc. The vector pTARGET-ppARdelta was transiently cotransfected with pGl3-PPRE x 3-luc into different cell lines to assay the expression levels of luciferase. The PPARdelta agonist screening model was established and optimized. Bezafibrate and linoleic acid can induce the expression of luciferase significantly and in a dose-dependent manner. This method can be used for high throughput screening for the agonist of PPARdelta, which might become lead compounds for new anti-atheroscleriosis or anti-adiposity drugs.
3T3 Cells ; Animals ; Bezafibrate ; pharmacology ; Cell Line ; Dose-Response Relationship, Drug ; Drug Evaluation, Preclinical ; methods ; Genetic Vectors ; chemistry ; genetics ; HeLa Cells ; Humans ; Linoleic Acid ; pharmacology ; Lipids ; chemistry ; Luciferases ; genetics ; metabolism ; Mice ; PPAR delta ; agonists ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; methods

Objective To explore the influence of drug dosage, solvent and other main influencing factors on the successful establishment of alloxan-induced hyperglycemia mouse model and the effect on the stability of this model. Methods 160 6-8-week-old Kunming mice ofSPF grade, (male:female=1:1) were used in this study.The influences of different dosages of alloxan and solvent combinations on the successful establishment rate of the model, survival rate, body weight, fasting blood glucose, blood glucose area under curve, serum insulin level and their stabilities were dynamically observed for six weeks.Results By single intraperitoneal injection of 160 mg/kg bw alloxan ( pH 4.5 citrate sodium as solvent) , we were able to obtain a stable experimental hyperglycemic mouse model with higher levels of successful establishment rate (70%), survival rate (75%), fasting blood glucose (15-20 mmol/L), glucose area under the curve (55-65 mmol/L) and a lower but not loss of serum insulin levels (21 mIU/L).Conclusions In the present study we have carefully considered the influence of main factors such as drug dosages, solvent, etc., on the alloxan-induced experimental hyperglycemic mouse model, and successfully established this model after 6-week period observation of its stability.This model may provide a useful tool in the research of experimental diabetes and hypoglycemic functional studies.

Objective To investigate the activities of infraspinatus (IS) and posterior deltoid (PD) under shoulder external rotation at open kinetic chain (OKC) and closed kinetic chain (CKC) exercise with shoulder abduction 0° and 90° to determine the optimal external rotation rehabilitation exercise.Methods From April to June, 2018, 19 healthy adults finished the movement of 0° OKC, 0° CKC, 90° OKC and 90°CKC. The root mean square (RMS) of IS and PD was recorded with surface electromyography (sEMG), then the standardized RMS (RMS％), ratio of IS/PD and onset time of activation were calculated.Results RMS％ of PD was the minimal at 90° CKC, and was less than that of 0° CKC (P ＜ 0.05). IS/PD was the most at90° CKC, and was more than that of 90° OKC (P ＜ 0.05). The onset time of IS was the earliest in 90° CKC, and earlier than that of 90° OKC (P ＜ 0.05) and 0°OKC (P ＜ 0.05). The onset time of PD was the latest in 0° CKC, and latter than that of 90° OKC (P ＜ 0.05).Conclusion 90°CKC activates IS mostly and earliest, which can be used in early rehabilitation for rotator cuff injury.

The paper is to report the study of the effect of Shenfu injection on the enzyme activity of liver CYP450 and its mRNA level of rat liver. Microsome of rat liver was prepared after intravenous administration of Shenfu injection for 7 days. The enzyme activity was quantified by Cocktail method. Meanwhile, the mRNA expression of CYP1A2, CYP2B1/2, CYP2C11 and CYP3A1 in the liver was detected by RT-PCR. Shenfu injection obviously induced the enzyme activities of CYP2B and CYP2C. Meantime Shenfu injection decreased the enzyme activities of CYP1A2 and CYP3A. The mRNA levels of CYP2B and CYP2C were also induced in rats treated with Shenfu injection. But it obviously inhibited the mRNA level of CYP1A2 and CYP3A. Since the enzyme activity and mRNA level were obviously changed after administration, the potential effect of drug-drug interaction should be concerned.
Aconitum ; chemistry ; Animals ; Aryl Hydrocarbon Hydroxylases ; genetics ; metabolism ; Cytochrome P-450 CYP1A2 ; genetics ; metabolism ; Cytochrome P-450 CYP2B1 ; genetics ; metabolism ; Cytochrome P-450 CYP3A ; genetics ; metabolism ; Cytochrome P-450 Enzyme System ; genetics ; metabolism ; Cytochrome P450 Family 2 ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Injections ; Male ; Microsomes, Liver ; enzymology ; Panax ; chemistry ; Plants, Medicinal ; chemistry ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Steroid 16-alpha-Hydroxylase ; genetics ; metabolism