[Effects of cyclic stretch on expression of cytokines and intercellular adhesion molecule-1 in human pulmonary artery endothelial cell].

OBJECTIVE: To observe the effects of cyclic stretch on expression of cytokines and adhesion molecules in human pulmonary artery endothelial cells (HPAECs), herein to provide a theoretical basis to ventilator-induced lung injury (VILI). METHODS: HPAECs were subjected to cyclic stretch by the Flexcell FX-5000T system at 0.5 Hz of 10% or 20% elongation for 3, 6, 12, 24 hours respectively. The mRNA and protein expression of interleukin (IL-6, IL-8), monocyte chemotactic protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) was determined by fluorescent quantitation reverse transcription-polymerase chain reaction (qRT-PCR), enzyme linked immunosorbent assay (ELISA) or Western blotting. RESULTS: Increasing the stretch force, the mRNA and protein expression of IL-8, MCP-1, ICAM-1 were up regulated with increasing stretch time. Compared with the control (set 1), after 20% cyclic stretch for 24 hours, IL-8 mRNA expression was up regulated to 1.58±0.10, MCP-1 mRNA expression was up regulated to 2.85±0.52, and ICAM-1 mRNA expression was up regulated to 1.90±0.14 (all P<0.05). Compared with control group, after 20% cyclic stretch for 24 hours, the protein expression of IL-8 and MCP-1 in HPAEC was significantly increased (IL-8: 3401.08±439.60 ng/L vs. 1422.60±66.98 ng/L, MCP-1: 1117.64±237.54 ng/L vs. 307.88±80.84 ng/L, both P<0.05), ICAM-1 protein expression was up regulated to 2.15±0.40 (P<0.05), while the expression of IL-6 mRNA and protein had no statistic difference compared with control group. CONCLUSIONS: Cyclic stretch enhanced the expression of IL-8, MCP-1 and ICAM-1 in an intensity-dependent fashion, so it may be involved in the pathogenesis of lung injury induced by mechanical ventilation.

[Lipopolysaccharide induced expression of peroxiredoxin 1 in airway epithelial cell].

OBJECTIVE: To investigate the effect of lipopolysaccharide (LPS) on expression of peroxiredoxin 1 (prdx1) in airway epithelial cells. METHODS: The airway epithelium cell line BEAS-2B was cultivated, and the cells were stimulated with 0, 1, and 10 mg/L of LPS for 12 hours and 24 hours, and then were harvested for prdx1 expression detection. The mRNA expression of prdx1 was detected by reverse transcription-polymerase chain reaction (RT-PCR).The airway epithelium cells were stimulated with 0, 0.1 , 0.5, 1 , 5, and 10 mg/L of LPS for 12 hours, and were collected for determination of prdx 1 protein expression by Western blotting. RESULTS: RT-PCR results showed that the prdx1 mRNA expression was significantly increased within 12 hours of stimulation with elevation of the dosage of LPS. The prdx1 mRNA expression at 12 hours of stimulation by 10 mg/L LPS was significantly higher than that in control group (2.014 ± 0.197 vs. 0.644 ± 0.178, P<0.05). However, with prolongation of LPS stimulation time, the prdx1 mRNA expression at 24 hours was slightly declined. Western blotting results showed that the prdx1 protein expression was gradually increased with elevation of dosage of LPS. The prdx1 protein expression at 12 hours of stimulation with 5 mg/L LPS was significantly higher than that in control group (1.069 ± 0.175 vs. 0.328 ± 0.010, P<0.05), and the expression remained at high level at 12 hours of stimulation with 10 mg/L LPS (0.984 ± 0.220 ). CONCLUSION: 10 mg/Lof LPS can induce the mRNA and protein expression of prdx1 in BEAS-2B cell after 12 hours of stimulation.

[Effect of emodin lipid nano-microbubble on MAPK signal pathway and inflammation cytokine in AT-II cells by mechanical stretch].

OBJECTIVE: To investigate the effect of emodin lipid nano-microbubble on MAPK signaling pathway and the level of inflammation cytokine in AT-II cells induced by mechanical stretch and its mechanism. METHODS: Emodin nanofibers, prepared by electrospinning with lecithin and PVP as carrier, were put into a seal bottle full of perfluoropropane, after they mixed with water and turned into self-assembled nano-microbubble. AT-II cells, separated and purified from primary rat AT-II cells, suffered 20% mechanical stretch for 4, 8, 16, 24, 48 h, and its protein expressions of p-P38/P38, p-ERK/ERK, p-JNK/JNK and the inflammation cytokine release levels of TNF-alpha, IL-1beta, IL-6 were detected by Western-Blot and ELISA. And further oberved the intervention effect of emodin lipid nano-microbubble on VILI. RESULTS: The protein expressions of p-P38, p-ERK, p-JNK were significantly increased in AT-II cells induced by mechanical stretch, and continuously evaluated from the 4-16 h, but the protein expressions of p38, ERK, JNK had no significant difference. The release levels of TNF-alpha, IL-1beta, IL-6 in AT-II cells had no change during 8 h, and they were gradually increased significantly in followed 16 h. In AT-II cells induced by mechanical stretch, due to intervention effect of emodin lipid nano-microbubble, the the protein expressions of p-P38, p-ERK, p-JNK and the inflammation cytokine release levels of TNF-alpha, IL-1beta, IL-6 were significantly decreased. CONCLUSION: Emodin lipid nano-microbubble shows protective effect on AT-II cells induced by mechanical stretch (VILI), and its mechanism may be related to down-regulation of protein expression of MAPK signaling pathway to regulate the release levels of inflammatory cytokine.

[Isolation and characterization of human alveolar type II cells and phenotypes maintaining study].

OBJECTIVE: To establish a method of isolate, purify, primary culture and identify human alveolar type II cells (AT II ) in vitro, as well as its possible maintaining phenotype characteristics. METHODS: The marginal lung tissue was collected. AT II cells were isolated with trypsin and elastase, purified by a series of steps, such as, cell sieve filtration, differential adhesion, gradient separation and anti-CD14 beads separation. AT II cells were identified with immunofluorescence of human pro-surfactant-associated protein C (pro-SP-C), Green DND-26 probe and electron microscope. The purity of AT II cells was measured by immunofluorescence of human pro-SP-C and Green DND-26 probe. The viability of AT II cells was measured by trypan blue staining. The phenotypes (SP-A, SP-B,SP-C, SP-D) were monitored with reverse transcription-polymerase chain reaction (RT-PCR) at different time points. RESULTS: The output of AT II cells from lung tissue was (5-10) x 105/g, and the cell viability was (93 ± 2)% with trypan blue staining, the cell purity was about 98% with pro-SP-C immunofluorescence and Green DND-26 fluorescent probe, the lamellar bodies were clearly observed with transmission electron microscope. In the aspect of phenotypes maintaining, the time of surfactant expression was about 24 days [SP-A: 0.52 + 0.03 (day 16), 0.35 + 0.02 (day 20),0.26 ± 0.01 (day 24), 0.10 + 0.08 (day 28); SP-C: 0.68 0.16 (day l6), 0.31 + 0.04 (day 20), 0.18 + 0.06 (day 24), 0.14 + 0.09 (day 28)], and the longest one was more than 28 days [SP-B: 1.05 + 0.17 (day 16), 0.76 + 0.35(day 20), 0.55 0.15 (day 24), 0.36 0.19 (day 28); SP-D: 0.52 0.19 (day 16), 0.33 + 0.12 (day 20), 0.31 +0.04 (day 24), 0.23 ± 0.02 (day 28)). CONCLUSION: We successfully established a procedure to separate, purify,identify of AT II cells, which retain primary phenotypic characteristics over long period.

Electrospun drug-loaded core-sheath PVP/zein nanofibers for biphasic drug release.

Core-sheath nanofibers prepared using coaxial electrospinning were investigated for providing biphasic drug release profiles. With ketoprofen (KET) as the model drug, polyvinylpyrrolidone and zein as the sheath polymer and core matrix, respectively, the coaxial process could be carried out smoothly and continuously without any clogging of the spinneret. Scanning electron microscopy and transmission electron microscopy observations demonstrated that the nanofibers were linear with homogeneous structure and had a clear core-sheath structure with an average diameter of 730 ± 190 nm, in which the sheath had a thickness of ca. 90 nm. Differential scanning calorimetric and X-ray diffraction analyses verified that all the components in the core-sheath nanofibers were present in an amorphous state. Attenuated total reflectance Fourier transform infrared spectra demonstrated both the sheath and core matrix had good compatibility with KET owing to hydrogen bonding. In vitro dissolution tests showed that the nanofibers could provide an immediate release of 42.3% of the contained KET, followed by a sustained release over 10h of the remaining drug. The present study exhibited a simple and useful approach to systematically design and fabricate nanostructures using coaxial electrospinning for providing biphasic drug release profiles.

[Gene transfer system mediated by PEI-cholesterol lipopolymer with lipid microbubbles].

The properties of polyethyleneimine-cholesterol cationic lipopolymer (PEI-Chol) as gene carries and its gene transfer efficiency in vitro with lipid microbubbles were presented in this paper. PEI-Chol lipopolymer was synthesized by linking cholesterol chloroformate to the amino groups of branched poly(ethyleneimine) (PEI) of 1 800. The structure and molecular weight of PEI-Chol were confirmed by IR, 1H NMR and MADI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry), respectively. The average molecular weight of PEI-Chol was approximately 2 000. The gene delivery system of bubble/PEI-Chol/DNA was constructed by mixed PEI-Chol/pDNA (N/P 10:1) complexes with lipid microbubbles (2-8 microm) which were prepared by DPPC, DSPE-PEG2000 and perfluoropropane with the reverse phase evaporation technique. pEGFP-Cl (enhanced green fluorescent protein) was used as report gene to investigate the DNA condensing ability of PEI-Chol lipopolymer by agarose gel electrophoresis. And their cytotoxicity and in vitro transfer efficiency of different complexes were compared with each other in A549 and MCF-7. The results indicated PEI-Chol lipopolymer can condense plasmid DNA when N/P ratio upto 4, PEI-Chol complexes and bubble/PEI-Chol/DNA complexes were nontoxic to A549 and MCF-7 when formulated at the N/P ratio of 10/1 as determined by MTT assay. This bubble/PEI-Chol/DNA delivery system provided good transfer efficiency with other desirable characteristics such as against-precipitation of plasma proteins. In conclusion, bubble/PEI-Chol/DNA complex is a novel non-viral gene delivery system.

[Preparation of self-emulsifying soft capsule and its pharmacokinetic in rats for epimedium flavonoids].

OBJECTIVE: To study the formulation optimization of epimedium flavonoids self-emulsifying drug delivery system and evaluate its effect in vitro and in vivo. METHODS: Based on the degree of emulsification and emulsifying time, the formulation optimization (i.e., screening of suitable oil phases, nonionic surfactants and co-surfactants) was made by the use of determination of the solubility, orthogonal design and construction of tertiary phase diagram. The dissolution of SEDDS was measured and its pharmacokinetic in rats was measured. RESULTS: The experimental results revealed the optimized formulation of the self-emulsifying drug delivery system which consisted of oleic acid as oil phase, Tween-80 as nonionic surfactant, and PEG400 as co-surfactant in the proportion of 2:4:4. The dissolution of SEDDS in water was more than 85% in 25 minutes, while that of the epimedium flavonoids capsule was less than 50% in 60 minutes. CONCLUSION: Application of the optimized formulation of epimedium flavonoids self-emulsifying drug delivery system could significantly increase the solubility of epimedium flavonoids in water and improve its bioavailability.

[Study on the natural ligand(s) of triggering receptor expressed on myeloid cell-1 on bacteria cell wall].

OBJECTIVE: To look for the natural ligand(s) of human triggering receptor expressed on myeloid cell-1 (TREM-1), in order to provide the theoretical basis for elucidation of the pathogenesis of sepsis. METHODS: Neutrophils and monocytes isolated from human peripheral blood were treated with heat-inactivated Staphylococcus aureus, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Staphylococcus aureus L-form or Pseudomonas aeruginosa L-form respectively for 24 hours. The cell wall was extracted from Staphylococcus aureus, Pseudomonas aeruginosa and Mycobacterium tuberculosis by ultrasound. Neutrophils and monocytes were isolated and treated with the cell wall respectively for 24 hours. Neutrophils and monocytes were isolated and treated with three main components from bacterial cell wall (polysaccharides, lipids and proteins) respectively for 24 hours. The level of TREM-1 mRNA was measured with fluorescent quantitative polymerase chain reaction (PCR), and the concentrations of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) were measured with enzyme-linked immunosorbent assay (ELISA). RESULTS: The TREM-1 mRNA level and the concentrations of TNF-alpha and IL-1 beta in cell supernatant of neutrophils and monocytes were upgraded when treated with cell, cell wall and cell wall polysaccharides of Staphylococcus aureus and Pseudomonas aeruginosa. Compared with the blank control group, the TREM-1 mRNA level of neutrophils and monocytes was upgraded to (3.86+/-0.20)-fold and (5.15+/-0.56)-fold respectively when treated with cell wall polysaccharides of Staphylococcus aureus (both P<0.05); the TREM-1 mRNA level of neutrophils and monocytes was upgraded to (4.03+/-0.15)-fold and (7.22+/-0.73)-fold respectively when treated with cell wall polysaccharides of Pseudomonas aeruginosa (both P<0.05). The effect could be attenuated by the addition of LP17 which could bind TREM-1 ligand. This attenuating effect was not found when the cells were treated with cell, cell wall or cell wall polysaccharides of Mycobacterium tuberculosis. CONCLUSION: The study provides the evidence that TREM-1 natural ligand(s) is present on cell wall of bacteria including Staphylococcus aureus and Pseudomonas aeruginosa, and it might be polysaccharides.

[Effects of Tankejing dry powder inhaler on the inflammatory damage in the lungs of mice infected by FM1].

OBJECTIVE: To study the effect of Tankejing Dry Powder inhaler on inflammatory in lung injury of mice infected by influenza virus (FM1). METHODS: Model mice infected by influenza virus FM1 were randomly divided into six groups: the nomal group, model group, low dose, medium dose and high dose of Tankejing Dry Powder inhaler group,and Ribavirin group. The following indices including the change of NF-kappaB in lung tissue at 24 h, the changes of TNF-alpha, IL-1beta, IL-6 in lung tissue, the lung index and the death rate were observed. RESULTS: Compared with model group, medium and high dosages of Tankejing Dry Powder inhaler could modulate the expression of NF-kappaB, reduce the level of TNF-alpha, IL-beta, IL-6 in lung tissue (P < 0.05), and reduce the lung index and the death number of animals within 14 days (P < 0.05). CONCLUSION: Tankejing Dry Powder inhaler may reduce the inflammatory injury caused by influenza virus FM1 through regulating the expression level of NF-kappaB and cytokine.

[Anti-viral effects of Tankejing preparations against influenza virus A].

OBJECTIVE: To investigate the anti-viral effects of Tankejing preparations (including solutions, dry power inhalation and powder) against influenza virus A in vitro and the relationship between its anti-viral effect and preparations. METHODS: The inhibitive effect of different drug-added ways of Tankejing extracts against human influenza virus A (H3N2) in vitro were assayed by crystallized purple staining method with ribavirin as positive reference drug. Then we compared the anti-viral actions of different kinds of Tankejing preparations. At last, we carried on serum pharmacodynamics to test and verify the effect. RESULTS: Tankejing extracts had an effect on comprehensive inhibition and an inhibition on virus proliferation after adsorption. When the concentration was 0.062 g/mL, the inhibition rate of solution dry power inhalation and powder were 43.50%, 41.50% and 37.36%, respectively. The anti-viral effect of dry powder inhalation was better than power's in serum pharmacokinetis experiment. CONCLUSION: Tankejing preparations display an action against human influenza virus A in vitro. Those preparation with better solubility have greater anti-viral effect.

[Expression and significance of severe acute respiratory syndrome associated coronavirus (SARS-CoV)-X4 protein in lungs of SARS patients].

OBJECTIVE: To investigate the significance of severe acute respiratory syndrome associated coronavirus (SARS-CoV)-X4 protein expression in lungs of patients with SARS. METHODS: Pathological features of the lungs from 4 SARS patients were examined and the expression of SARS-CoV-X4 protein in the lungs was evaluated with immunohistochemical staining using specific antibodies against protein X4. RESULTS: Microscopically, all lungs from 4 cases showed edema, erythrocyte and fibrin exudates in the alveoli, hyperplasia of alveolar epithelium, necrosis, hyaline membrane formation and fibroblast foci. Immunohistochemical stains showed a strong positivity of X4 protein in denudation cells, vascular endothelial cells and also erythrocytes and neutrophils in the alveoli of the lung tissues from the 4 cases. CONCLUSIONS: Expression of SARS-CoV-X4 protein in the lungs may be involved in the pathogenesis and progression of SARS.

Evaluation by indirect immunofluorescent assay and enzyme linked immunosorbent assay of the dynamic changes of serum antibody responses against severe acute respiratory syndrome coronavirus.

BACKGROUND: Severe acute respiratory syndrome coronavirus (SARS-CoV) is a newly emerging virus that gives rise to SARS patients with high rates of infectivity and fatality. To study the humoral immune responses to SARS-CoV, the authors evaluated IgG and IgM specific antibodies in patients' sera. METHODS: Two methods, enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent assay (IFA), were used to detect specific serum IgG and IgM against SARS-CoV in 98 SARS patients and 250 controls consisting of patients with pneumonia, health-care professionals and healthy subjects. The serum antibody profiles were investigated at different times over one and a half years in 18 of the SARS patients. RESULTS: The sensitivity and specificity of ELISA for detecting IgG against SARS-CoV were 100.0% and 97.2% and for IgM 89.8% and 97.6% respectively; the figures using IFA for IgG were 100.0% and 100.0% and for IgM 81.8% and 100.0% respectively. During the first seven days of the antibodies trace test, no IgG and IgM were detected, but on day 15, IgG response increased dramatically, reaching a peak on day 60, remaining high up to day 180 and decreasing gradually until day 540. On day 15, IgM was detected, rapidly reached a peak, then declined gradually until day 180 when IgM was undetectable. CONCLUSION: The detection of antibodies against SARS virus is helpful in the clinical diagnosis of SARS.

[Protective effect of specific antibody in serum of convalescent patient with SARS].

OBJECTIVE: To investigate inhibitory effect of serum severe acute respiratory syndrome (SARS) -specific antibodies from convalescent patients after half an year of onset on SARS-CoV-mediated cytopathic response. METHODS: SARS-CoV immunoglobulin G (IgG) antibody was determined by enzyme linked immunoadsorbent assay (ELISA). Twelve serum samples from convalescent patients, diluted by 1:8 with maintenance medium, were mixed with the three dilution supernatants of SARS-CoV. SARS-CoV were isolated, cultured and identified by the Guangzhou Institute of Respiratory Disease, and cultured with Vero E6 cell suspension. The extent of cytopathic response was observed. RESULTS: The absorbance (A) value of SARS-CoV IgG antibody ranged from 0.81 to 2.06 in patients after half an year of SARS onset, and form 0.79 to 2.01 in patients before half an year of SARS onset. The extent of cytopathic response was decreased by more than 25% in all 12 convalescent patients, as compared with control serum. CONCLUSION: The A value of SARS-CoV IgG antibody in serum of convalescent patients tended to elevate in half an year after SARS onset. SARS-CoV IgG antibody could inhibit SARS-CoV-mediated cytopathic response, indicating it might be one of protective antibodies.

[Effects of epimedium total flavonoids phytosomes on preventing and treating bone-loss of ovariectomized rats].

OBJECTIVE: To observe the effects of Epimedium total Flavonoids Phytosomes on preventing and treating bone-loss of the castrate osteoporosis rat model. METHOD: The osteoporosis model was established with 4-month-odl panther's rats, their ovaries on both sides castrated. Dual energy X-ray scanning was used to determine the bone density, and immunity and ELASA were used to assay concentration of estradiol and IL-6 in serum respectively, then determine their effect. RESULT: The BMP and E2 of high dosage group nilestriol group and normal group are higher than those of model group (P < 0.01), while their content of IL-6 is apparently lower than that of model group(P < 0.01). CONCLUSION: The osteoporosis model was established successfully and the using of EFP can improve the bone density, enhance E2 level and decrease the IL-6 concentration in serum.