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ObjectiveTo study the effect of heat stress on the cytoskeleton and cell cycle of human umbilical vein endothelial cell (HUVEC) in vitro.Methods HUVEC was cultured in vitro in 5%CO2 medium at 37℃ (control group) or 43℃ (heat stress group) for 1 hour. Coomassie brilliant blue R-250 staining was used to determine the effect of heat stress on the cytoskeleton. The cells in heat stress group were subsequently cultured at 37℃in 5%CO2 medium after heat stress for 1 hour, and cell cycle of HUVEC was determined at 0, 6, 12, 18 and 24 hours with flow cytometry.Results Under light microscopy normal cytoskeleton was observed in control group, but thicker and shorter cytoskeleton was found after a rise of temperature, and stress fibers were found in heat stress group. The DNA content of HUVEC at all time points in G0/G1 stage was 38.07%-55.19% after heat stress. The DNA content in control group was 48.57%, and it was 54.06%, 55.19%, 48.23%, 38.07%, and 41.03% at 0, 6, 12, 18, 24 hours in G0/G1 stage in heat stress group. DNA content in S phase was 35.33%-48.18%. The DNA content in control group was 44.62%, and it was 35.33%, 39.50%, 42.50%, 48.18%, and 47.99% at 0, 6, 12, 18, 24 hours in S stage in heat stress group. DNA content in G2/M phase was 5.31%-13.75%. The DNA content in control group was 6.81, and it was 10.61%, 5.31%, 9.27%,13.75%, and 10.98% at 0, 6, 12, 18, 24 hours in G2/M stage in heat stress group. It was demonstrated that compared with control group, the DNA content in G0/G1 stage was significantly increased when the HUVEC were separated from heat stress within 6 hours, and it recovered at a similar level as control group at 12 hours.Conclusion Heat stress can change the cytoskeleton of HUVEC, and cause stagnation at G0/G1 stage in cell cycle.
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Objective To study the effect of heat stress on the permeability,cytoskeleton and cell cycle of human skeletal muscle cell (HSKMC).Methods The HSKMC membrane permeability was detected by calcium ion inflow with flow cytometer,the cytoskeleton was stained by CBB 250,and the cell cycle was determined by flow cytometer.Results After 1 h of heat stress on human HSKMC cells under different temperature gradient,the median level of calcium ion was 91.63 in 43 ℃ heat stress group compared with 22.98 in 37 ℃ control group.As temperature increased,thicker and shorter cytoskeleton and stress fiber were shown under the high power lens of microscope.The DNA expression of skeleton cells at G0/G1 stage was 44.13-62.98 in groups under heat stress.Compared with normal control group,DNA expression was much higher in heat stress group,when HSKMC was cultured under 37 ℃ temperature for another 18 h,it kept decreasing DNA expression to a similar level as control group.Conclusions Heat stress can cause calcium iron inflow resulting in intracellular calcium overload,and affect the cytoskeleton leading to loss of normal web ordered arrangement and increased gap in HSKMC cells,which give rise to blocking cell cycle into G0/G1 stage.
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Objective To investigate the mechanism of mastoparan-1 (MP-1) antagonizing lipopolysaecharide (LPS) in vitro. Methods The affinity of MP-1 for lipid A was assayed by biosensor, and the neutralization of MP-1 on LPS (2 μg/L) was detected by kinetic turbidimetric limulus test. After exposing fluorescin isothiecyanate (FITC) labeled LPS (FITC-LPS) to MP-1 at different concentrations (5, 10, 20, 40 μmol/L), the binding of FITC-LPS to murine RAW264.7 cells was analyzed by laser scanning confocal microscopy. The influence of MP-1 on TLR4 expression in RAW264.7 cells stimulated by LPS (100 μg/L) was detected by immunoeytochemieal staining. The expressions of TLR4, TNF-α and IL-6 at the gene and protein level were detected by RT-PCR and ELISA after exposing LPS (100 μg/ L) stimulated RAW264.7 cells to MP-1 at different concentrations. The effect of MP-1 on the viability of RAW264.7 cells was detected by MTT assay. Results MP-1 had high affinity to lipid A and could neutralize LPS. MP-1 at 10 μmol/L significantly inhibited not only binding of FITC-LPS to RAW264.7 (P < 0.05), but also protein and gene expressions of TLR4, TNF-α and IL-6 in LPS stimulated RAW264.7 cells in a dose-dependent manner (P < 0.05). No toxic effect of MP-1 on the viability of RAW264.7 cells was found (P > 0.05). Conclusions MP-1 inhibits cell viability mediated by LPS, which may be related to its neutralization of LPS and inhibition of binding of LPS to RAW264.7 cell membrane receptors.
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Objective To investigate the changes of collagen types Ⅰ and Ⅲ, the effects of Oleum Curcumae Aromaticae oil injection on injuried rat arteries and collagen types Ⅰ and Ⅲ after balloon angioplasty Methods The left common carotid artery in rats was denuded with a balloon catheter The rats were killed 14 days after injury Cross sections stained with picrosirius red from the injuried segments were processed for distribution, arrangememt, amount, proportion of collagen type Ⅰ and type Ⅲ by polarizing microscope, and those stained with hematoxylin eosin for histological and morphological changes studied by computer assisted picture analysis system Results Fourteen days after balloon injury, collagen type Ⅰ and type Ⅲ of the model group were found mainly in adventitia, much more, thicker, brighter and more disordered than those of the sham operation group Compared with the model group, collagen type Ⅰ and type Ⅲ of the E Zhu You group were found mainly in adventitia, less, thinner, dimmer and more orderly Lumen area, EEL perimeter, IEL perimeter of the model group were decreased and the areas of collagen types Ⅰ and Ⅲ increased more significantly than those of the sham operation group Compared with the model group, maximal intimal thickness, neointima area, the areas of collagen type Ⅰ and Ⅲ of the E Zhu You group were decreased significantly which was not different from those of the sham operation group Lumen area, EEL perimeter and IEL perimeter were increased markedly up to those of the sham operation group The ratio of collagen subtypes Ⅰ/Ⅲ was not significantly different between the two of the three groups Conclusion The accumulation and derangement of collagen types Ⅰ and Ⅲ may be more important in restenosis than the ratio of collagen subtypes Ⅰ/Ⅲ We concluded that Oleum Curcumae Aromaticae oil injection inhibits neointimal formation and vascular remodeling after arterial injury by mechanisms involving collagen types Ⅰ and Ⅲ production and their arrangement
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Objective To assess the protective effect of mastoparan-1(MP-1) on acute lung injury in mouse model with endotoxemia(ETM) induced by lipopolysaccharide(LPS),and to investigate the possible mechanism of protcetive effect.Methods The endotoxemia murine model was reproduced by tail vein injection of LPS(5mg/kg) in mice.Animals were randomly divided into normal control group(n=8),endotoxemia group(n=48) and MP-1-treatment group(MP-1 was injected in 3mg/kg at the same time of LPS injection,n=48).Animals of the latter two groups were sacrificed at 2,6,12,24,48 and 72 hours after injection,and then the blood and lung tissue samples were collected.Plasma LPS was assayed using kinetic turbidimetric limulus test,TNF-? and IL-6 were measured by appropriate ELISA kits,TLR4,TNF-? and IL-6 mRNA expressions in lung tissues were analyzed by real-time RT-PCR,myeloperoxidase(MPO) activity of lung tissues was determined by spectrophotometric method,and the pathological changes in lung tissues were observed under microscope.Result The plasma levels of LPS,TNF-? and IL-6 in the mice of endotoxemia group were increased at 2-48 hours after LPS injection(P
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To explore the effect of Kaixin Capsule (KC, composed of Radix Panacis Quinquefolii, Radix Astragali, Radix Ophiopogonis, Rhizoma Chuanxiong,Rhizoma Cyperi, etc.) in reconstructing the collagen of non infarct region of left ventricle in rats after myocardial infarction (MI) and to study its mechanism.Seventy rats were allocated to mimic operation group (Group Ⅰ), model group (Group Ⅱ), high dosage KC group (Group Ⅲ), low dosage KC group (Group Ⅳ) and positive control group (Group Ⅴ). Rat models of left ventricle reconstruction were established by the ligation of the main stem of left coronary artery. Three days after modeling, the rats were treated with KC for eight weeks. And then the total collagen content in the myocardium of non infarct region and angiotensin Ⅱ (AngⅡ) and aldosterone (ALD) levels in blood and myocardium were measured by oxyproline hydroxyproline method and radioimmunoassay. The total collagen content was higher in Group Ⅱ than that in Group Ⅰ (P0 05) . [Conclusion]KC can inhibit the production of collagen and increase myocardial compliance and its mechanism may be involved in the regulation of rennin angiotensin aldosterone system.