Objective To observe pathological process of intestinal epithelial cells subjected to ischemia,ischemia/reperfusion injury and inflammation simulated hypoxia/reoxygenation (H/R) and lipopolysaccharide (LPS) challenged human fetal normal colonic cell (FHC) line in vivo,and to observe the changes when the assaulted intestinal epithelial cells were treated with emodin,in order to explore the possible intervention targets of emodin.Methods Normoxia group:the FHC cells were cultured in 95％ air and 5％ CO2 at 37 ℃.Hypoxia (H) group:the cells were cultured with a mixed anaerobic gas of 1％ O2,5％ CO2 and 94％ N2 at 37 ℃ for 1,2,3,4 hours.H + LPS group:the cells were cultured in hypoxic condition as H group with simultaneous challenge of LPS (1 mg/L).H/R group:the cells were cultured in hypoxia for 3 hours followed by reoxygenation for 1,2,3 and 4 hours,respectively.H/R + LPS group:the cells were cultured in H/R as H/R group and LPS (1 mg/L) simultaneously.Emodin intervention group:the cells were cultured in H3 h/R2 h + LPS and emodin (20,40,60,80 μmol/L) simultaneously.The variation trends of phosphorylation nuclear factor-κB profilin-o (pIκB-α),phosphorylation NF-κBp65 (pNF-κBp65) and their downstream target gene cyclooxygenase-2 (COX-2),and hypoxia-inducible factor-1α (HIF-1 α) were determined by Western Blot.The morphological changes in intestinal epithelium in different groups were observed using light microscope.The effect of emodin on the proliferation of intestinal epithelial cell was measured by methyl thiazolyl tetrazolium (MTT) assay.Results ① H group:the expressions of pIκB-α,pNF-κBp65 and COX-2 were upregulated,peaking at H1 h (0.350 ± 0.018,1.083 ± 0.054,0.903 ± 0.045),and then they gradually lowered (F value was 3.011,7.247,5.754,P value was 0.013,0.000,0.005,respectively).The expression of HIF-1 α peaked at H3 h (1.511 ± 0.076),but there was no significant difference among different groups (F=1.881,P=0.062).H + LPS group:the expressions of pIκB-α,pNF-κBp65,COX-2,HIF-1α were increased with elongation of duration of hypoxia,and a maximal induction was observed at H3 h (0.504 ± 0.025,1.255 ± 0.063,0.812 ± 0.041,1.209 ± 0.075,F value was 2.683,8.774,9.765,2.432,and P value was 0.011,0.000,0.000,0.026,respectively).H/R group:with the prolonged duration of reoxygenation,the expressions of NF-κB signaling pathway proteins (pIκB-α,pNF-κBp65,COX-2) were decreased and dropped to nadir at H3 h/R4 h (0.712 ± 0.034,1.202 ± 0.048,0.691 ± 0.042,F value was 1.923,6.765,2.719,and P value was 0.063,0.000,0.016,respectively).Compared with H group,HIF-1α was decreased with a prolonged duration of reoxygenation in H/R group,but there was no significant difference in value among different time points (F=1.280,P=0.081).H/R + LPS group:pIκB-o,pNF-κBp65,COX-2,HIF-1α showed no sign of degradation with the prolonged duration of reoxygenation,and their expression increased to maximum analogously at R2-3 h (3.302 ± 0.061,2.315 ± 0.055,2.017 ± 0.043,2.413 ± 0.098,Fvalue was 4.614,1.652,5.970,2.076,and Pvalue was 0.001,0.067,0.000,0.037,respectively).Emodin group:emodin when co-treated with H/R + LPS inhibited the expression of HIF-1o and NF-κB pathways with a dose-effect relationship (P＜0.05 or P＜0.01).Emodin at the dose of 80 μmol/L showed most marked inhibition (2.599 ± 0.130,1.772 ± 0.089,2.590 ± 0.129,2.518 ± 0.125).However,after treatment of emodin did not show such effect.② After treatment with H/R + LPS,there were morphological changes in cells:vacuoles,deformation and fusion.The speed of cell growth became much slower compared with H group.③ Emodin (20-80 μmol/L) had no significant effect on cell proliferation.Although emodin produced biological effect in this concentration range,it had no cellular toxicity.Conclusions Both hypoxia and inflammation can activate the hypoxia pathway of HIF-1α and the pro-inflammatory pathway of NF-κB,but different stimuli cause varying degrees of activation in these two pathways.In H/R group,both pathways were weakened during reoxygenation.However,in H/R + LPS group,the proteins remained to show a relatively high expression during the process of reoxygenation.This may be related to the pathophysiological mechanism of intestinal ischemia/reperfusion injury:hypoxia/reperfusion injury and LPS act together to destroy the intestinal epithelial cells and induce gut-derived sepsis.Emodin may inhibit inflammation by blocking HIF-1α/NF-κB-COX-2 signaling pathways.

Objective To establish an HPLC method for the determination of related substances of rabeprazole sodium.Methods The determination was performed on a Xtimate C18 column.The mobile phase consisted of 2 g/L ammonium acetate-acetonitrile (95:5)and 2 g/L ammonium-methanol(15:85), with linear gradient elution and the flow rate of 1.0 mL/min.Detection wavelength was 290 nm.Results Related substances were completely separated from the main constituent.The limit of detection of rabeprazole was 0.25 ng and the limit of quantification was 0.75 ng,which were 0.01% and 0.03% of test sample and met the detection.With the selected solvents, principal component could be extracted efficiently and good stability.The sample solution was not stable at room temperature.Conclusion The method is simple, rapid and accurate, and can be used to control the quality of rabeprazole sodium enteric-coated pellets.

BACKGROUND:Neurotrophin-3 is found in the repair of spinal cord injury in the role of the strongest neurotrophic factor.It can effectively promote axonal regeneration through the glial scar tissue in repairing spinal cord injury.OBJECTIVE:To construct recombinant lentiviral vectors for gene delivery of homo sapiens neurotrophin-3(hNT3),and to investigate the expression of hNT3 gene in Schwann cells after transfection.DESIGN,TIME AND SETTING:Observational experiment was performed from June 2007 to March 2008 at the Central Laboratory of Changhai Hospital.MATERIALS:Bilateral sciatic nerves were harvested from 3-day-old Sprague Dawley rats for culture and identification of Schwann cells.Three-plasmid lentivirus systems:pGC-E1-EGFP,pHelper 1.0 and pHelper 2.0 were gained from Shanghai Chemical Technology Co.,Ltd.METHODS:pGC-E1-hNT3-EGFP plasmid was constructed by double restriction enzyme digestion and ligation,and then the plasmid was transformed into E.coli DH5?.Purified pGC-E1-hNT3-EGFP plasmids from the positive clones was confirmed by PCR and sequencing.293T cells were cotransfected with lentiviral vector pGC-E1-hNT3-EGFP,pHelper 1.0 and pHelper 2.0 by Lipofectamine 2000 to produce lentivirus.2 mL recombinant virus complete culture solution was added according to multiplicity of infection=1,4,8,10,12.The titer of virus was tested according to the expression level of enhanced green fluorescent protein.The control groups were Schwann cells and Schwann cells transfected by no-loaded lentivirus.MAIN OUTCOME MEASURES:The lentiviruses were transduced to Schwann cells,and the transfection efficiency was examined by flow cytometry,the overexpression of hNT3 was determined by Real-time PCR and Western blotting.RESULTS:The exogenous gene sequence of the recombinant hNT3 was completely in accordance with that of its open reading frame in GeneBank.The titer of concentrated virus was 5?107 TU/L.After recombinant LV-hNT3 infection,Schwann cells gave off strikingly bright green fluorescence,and the transfection efficiency amounted to 85%(multiplicity of infection=10).Real-time RCR test showed that the hNT3 mRNA were highly expressed in hNT3-Schwann cells,while did not express in the control group.Western blotting showed the hNT3 expression in Schwann cells.CONCLUSION:Construction of hNT3 gene lentiviral vector can transfect Schwann cells leading to an efficient overexpression of hNT3.

AIM: To investigate the role of nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC) and protein kinase C (PKC) signaling in tumor necrosis factor-? (TNF-?)-induced cardioprotection against hypoxia/reoxygenation (H/R) injury. METHODS: Neonatal rat ventricular myocytes were pretreated with TNF-? or sodium nitroprusside (SNP) or L-arginine (L-Arg), respectively, for 12 h and then subjected to continuous hypoxia for 12 h, followed by reoxygenation for 6 h. The manganese superoxide dismutase (Mn-SOD) activity of the cells was measured after H/R. Myocyte injury was determined by the release of lactic dehydrogenase (LDH). RESULTS: TNF-? (10~5 (U/L)) significantly increased the Mn-SOD activity and decreased release of LDH from ventricular myocytes. The cardioprotection against H/R injury was induced by the pretreatment with SNP (5 ?mol/L) or L-Arg (5 mmol/L), which was blocked by ODQ (10 ?mol/L), the specific sGC inhibitor, and Chel (5 ?mol/L), the specific PKC inhibitor. Pretreatment with L-NAME (100 ?mol/L), ODQ, Chel, antoxidant 2-MPG (400 ?mol/L) or tyrosine kinase inhibitor genistein (50 ?mol/L) attenuated the increased Mn-SOD activity and reduced LDH level induced by TNF-?. CONCLUSION: The results suggest that NO may play a role in TNF-?-induced cardioprotection, which is mediated by sGC and PKC. [

In the present study, packaging system composed of pAAV-CMV-GFP, pAAV-RC and pHelper were transfected into human embryonic kidney 293 cells (HEK293 cells) mediated by polyethyleneimine (PEI) to explore an optimal transfection condition. Different total plasmid DNA dosages (1, 2, 3, 4, 5, 6 μg) and different PEI/Plasmid ratios (1:1, 3:1, 5:1, 7:1) were tested with detection of green fluorescence protein (GFP) with ImagePro Plus6. 0 Software. Then transfection efficiency of the optimized transfection system was further observed for different time periods(12, 24, 36, 48, 60, 72 h). The results showed that total plasmid dosage of 4 μg/well with PEI/plasmid ratio of 3 : 1-5 : 1 was an efficient transfection condition. Transfection efficiency-time curve was an S-shaped curve. Transfection efficiency reached a plateau at 60 h after transfection. The optimized conditions for PEI-mediated transfection at the optimal time result in enhanced transfection efficiency of triple plasmid into HEK293 cells.
Green Fluorescent Proteins ; HEK293 Cells ; Humans ; Plasmids ; Polyethyleneimine ; Transfection ; methods

There are 250 species of Zanthoxylum (Rutaceae) in the world. This genus distributed in tropical and subtropical regions. Alkaloids are the major and representative ingredients in these plants including quinolines, isoquinolines, and amide alkaloids, with such biological activities as anti-tumor, anti-inflammatory, analgesic, anti-virus, anti-platelet aggregation, anti-bacteria and anti- oxidant. These species have been used for a long time to treat toothache, urinary and venereal diseases, lumbago and rheumatism. This review summarizes the chemical constituents and pharmacological activities from the Z. sppplants, in an effort to the systematic research and application of the alkaloids of this genus.
Alkaloids ; chemistry ; pharmacology ; Animals ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Humans ; Molecular Structure ; Zanthoxylum ; chemistry

OBJECTIVETo evaluate the effect of electro-acupuncture (EA) on gastric mucosal oxygenation and systemic inflammatory response in patients undergoing endoscopic sinus surgery with controlled hypotension (CH), and to explore its protective effect on gastric mucosa.

METHODSFifty-four patients, 18-65 years old, grade I-II of American Society of Anesthesiology (ASA), who were scheduled for endoscopic sinus surgery were randomly assigned to two groups, group A (general anesthesia group) and group B (general anesthesia combined EA anesthesia group), 27 in each group. Conrolled hypotension was executed during operation, and mean arterial pressure (MAP) was maintained at 55-65 mmHg. After tracheal intubation gastric tesiometer catheter was indwelled through nasal cavity or oral cavity. After successful indwelling, it was connected with gastric mucosa monitoring mode of multifunctional parameters monitor. Patients' MAP and heart rate (HR), pHi, partial pressure of carbon dioxide (PgCO2), arterial partial pressure of carbon dioxide (Pg-aCO2) and endtidal pressure of carbon dioxide (Pg-etCO2) were measured and recorded at T, (immediately before induced hypotension), T, (20 min following induced hypotension to target MAP), T2 (40 min following induced hypotension to target MAP), T3 (20 min after ending induced hypotension), and T4(40 min after ending induced hypotension). Blood samples were intravenously collected, TNF-alpha, IL-1, and IL-6 were detected by ELISA 24 h before operation, during operation (T3), and 24 h after operation.

RESULTSAfter hypotension was induced, Pg-CO2, Pg-aCO2 and Pg-etCO2 increased significantly (P < 0.01, P < 0.05), while pHi decreased significantly (P < 0.01) in both groups at T1-T4 than those at T0. During T1-T4, PgCO2, Pg-aCO2, and Pg-etCO2 were higher (P < 0.01, P < 0.05), while pHi was lower in group A than in group B (P < 0.01). Furthermore, TNF-alpha, IL-1, and IL-6 increased significantly in both groups during operation and 24 h after operation, when compared with those 24 h before operation (P < 0.01, P < 0.05). TNF-alpha and IL-1 in group A were higher than those in group B (P < 0.05) during operation and 24 h after operation, but with no significant difference in the plasma concentration of IL-6 (P > 0.05).

CONCLUSIONEA exerted obvious protective effect of gastric mucosal injury in endoscopic sinus surgery with controlled hypotension, which might be achieved by increasing gastric mucosal blood flow, maintaining oxygen supply and demand, inhibiting inflammatory response, and alleviating injury of gastric mucosal barrier.

Acupuncture Analgesia ; methods ; Adolescent ; Adult ; Aged ; Anesthesia, General ; Arteries ; Blood Pressure ; Electroacupuncture ; methods ; Endoscopy ; Female ; Gastric Mucosa ; surgery ; Heart Rate ; Humans ; Hypotension, Controlled ; Interleukin-1 ; Interleukin-6 ; Male ; Middle Aged ; Tumor Necrosis Factor-alpha ; Young Adult

OBJECTIVETo investigate the results of using the modified Jaslow technique to treat the recurent lumbar disc herniation.

METHODSFrom January 2002 to December 2008,62 patients with recurrent lumbar disc herniation were treated with a modified Jaslow technique. There were 42 males and 20 females with an average age of 53.6 years old, ranging from 36 to 70 years. The primary surgical procedures were enlarged fenestration in 20 cases, unilateral semi-laminectomy in 20 cases, bilateral semi-laminectomy in 8 cases and total-laminectomy in 14 cases. The procedures were performed at L3.4 level in 2 cases, L4.5 in 32 cases, L5S1 in 15 cases, L3.4-L4.5 in 3 cases and L4.5-L5S1 in 10 cases respectively. The clinical symptoms included low back pain and radicular pain. Pre-and postoperative JOA score (including subjective symptoms, self-care ability and pain), ratio of disc height and the fusion condition of the involved segments were applied to assess clinical outcome.

RESULTSAll patients were followed up for 1 to 5 years (averaged 3 years). The space height ratio increased from (62.5 +/- 10.4)% to (90.5 +/- 10.3)%, fusion rate was 96.8% (60/62) at the last follow-up. Mean JOA score was (10.42 +/- 2.50) preoperative, improved to (24.26 +/- 2.35) at last follow-up (P < 0.001). The clinic results was excellent in 39 cases, good in 14, fair in 9.

CONCLUSIONThe modified Jaslow technique was a good alternative method for the treantment of recurent lumbar disc herniation with complete decompression, solid interbody fusion and satisfactory clinical result.

Adult ; Aged ; Female ; Follow-Up Studies ; Humans ; Intervertebral Disc Displacement ; surgery ; Lumbar Vertebrae ; surgery ; Male ; Middle Aged ; Spinal Fusion ; methods

AIMTo study the cellular uptake of chitosan oligosaccharide nanoparticles by A549 cells and evaluate the possibility of chitosan oligosaccharide nanoparticles used as a potential drug carrier.

METHODSChitosan oligosaccharide (CSO) was obtained by ultrafiltration separation after regulation of the condition of chitosanase degradation. The molecular weight of CSO was determined by gel permeation chromatography (GPC). Chitosan oligosaccharide nanoparticles (CSO-NPs) were prepared by a novel solvent diffusion method in an oil system after the carrier material grafted fluorescein isothiocyanate (FITC) and the particle size distribution and zeta potential were determined by light scattering and electrophoretic mobility. The cytotoxicity and uptake of FITC-labeled CSO-NPs in A549 cells following various incubation periods were studied by the MTT method and fluorescence microscopy, flow cytometric analysis, respectively.

RESULTSThe molecular weight (MW) of CSO was 18,678 u and the particles sizes of CSO-NPs were 133.3 nm (number average) and 368.2 nm (volume average), respectively. The IC50 of CSO and CSO-NPs were 944.36 and 643.16 mg x L(-1), respectively, and the result showed low cytotoxicity. Cellular uptake of CSO and CSO-NPs were relative to the concentration and the incubation time. Internalization of CSO-NPs increased 0.49 - 13.9 times more than that of the CSO with the same incubation time.

CONCLUSIONCSO and CSO-NPs have low cytotoxicity. CSO-NPs can significantly improved the uptake of CSO-NPs by A549 cells compared to the same molecular weight of CSO.

Adenocarcinoma ; pathology ; Chitin ; analogs & derivatives ; metabolism ; toxicity ; Chitosan ; Dose-Response Relationship, Drug ; Drug Carriers ; metabolism ; toxicity ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Lung Neoplasms ; pathology ; Nanotechnology ; Oligosaccharides ; metabolism ; toxicity ; Particle Size ; Time Factors ; Tumor Cells, Cultured

AIMTo investigate the cellular uptake of monostearin solid lipid nanoparticles (MSLN) and the influence on the cellular uptake by MSLN modified with PEG2000 in human-type II cell alveolar epithelial cell line (A549) and murine macrophages cell line (J774A1).

METHODSMSLN were prepared by a novel solvent diffusion method. The particle size distribution and zeta potential of MSLN, measured by light scattering and electrophoretic mobility, were investigated. The cytotoxicity of MSLN and MTX-loaded MSLN in A549 cells were performed by the MTT method. Rhodamine B was incorporated into solid lipid nanoparticles as fluorescent marker, after PEG2000 integrating monostearin during preparation, the cellular uptake of MSLN by A549 and J774A1 cell lines were determined spectrofluorimetrically.

RESULTSThe IC50 of MSLN and MTX-loaded MSLN on A549 cells were 227.56 microg x mL(-1) and 71.37 microg x mL(-1), respectively. The percentage of cellular uptake showed a negative correlation to the concentration of MSLN in incubation medium and the internalization behaved rapidly. Contrary to situation in J774A1 cell line, internalization of solid lipid nanoparticles was promoted with increasing the content of PEG2000 incorporated into MSLN in A549 cell line.

CONCLUSIONAfter modifying MSLN with PEG2000, it represents relative lower phagocytosis by J774A1 cell line and higher uptake in A549 cell line.

Adenocarcinoma ; metabolism ; pathology ; Animals ; Cell Line ; Cell Line, Tumor ; Cell Survival ; drug effects ; Drug Carriers ; Drug Delivery Systems ; Glycerides ; chemistry ; metabolism ; toxicity ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Macrophages ; cytology ; Mice ; Nanotechnology ; Particle Size ; Phagocytosis