ObjectiveTo investigate the role of platelet in mouse pulmonary microvascular endothelial cell (PMVEC) injury caused by lipopolysaccharide( LPS)-activated neutrophil.MethodsPMVECs were obtained from pathogen-free C3H/HeN mice of both sexes aged 6-8 weeks weighing 18-25 g according to the method described by Lim YC et al.Platelets and neutrophils were isolated from mouse blood by twice centrifugation and denaity gradient centrifugation respectively.PMVECs were seeded into twelve- or six-well plates ( 1 or 2 ml/well) after 2-5 passages and were randomly divided into 4 groups (n =31 each): group LPS; group platelets (group P);group neutrophils (group N) and group platelets + neutrophils (group PN).Each well contained about 5 × 107/ml platelets and/or 5 × 105/ml neutrophils respectively.PMVECs were incubated with LPS1 μg/ml at 37 ℃ in a 5％ CO2 humidified atmosphere for 1,6,12,18 and 24 h respectively in all 4 groups.The cells were examined with phase contrast microscope for morphological changes and survival condition.Viability rate,apoptotic rate and activation rate of PMVECs were detected by flow cytometry at each time points.ResultsThere was no significant difference in morphology and number of endothelial cells (ECs) among the 4 groups,while the number of activated ECs was significantly increased but the number of living cells decreased in group PN compared with group LPS.The activation rate of ECs was significantly higher after being incubated with LPS for 6-12 h in groups P and N than in group LPS.The viability rate was significantly lower,while the apoptotic rate and activation rate were significantly higher after ECs were incubated with LPS in group PN than in groups LPS,P and N.ConclusionPlatelets play a decisive role in mouse PMVEC injury induced by LPS activated neutrophils.

Objective To optimize purification technology of total flavonoids in Callicarpa nudiflora Hook. et Arm. by macroporous adsorption resin. Methods Macroporous resin models including AB-8, D-101, HP-20, HP2MG, were optimized by static adsorption and desorption experiments regarding to adsorption rate and desorption rate of total flavonoids. Purification technology parameters of total flavonoids were optimized by single factor test. Results HP-20 macroporous resin presented the best purification efficiency,the optimum purification conditions were that taking 4. 46 mg·mL-1 of total flavonoidsat pH 3. 0, loading at 3 BV·h-1, washed with 3BV of water at 3 BV·h-1,then eluted with 4 BV 75% ethanol at 2 BV·h-1, finally obtaining the total flavonoids from the dry extract of Callicarpa nudiflora Hook. et Arn. with the purity of 47. 4%. Conclusion HP-20 macroporous resin is suitable for preliminary purification of total flavonoids in Callicarpa nudiflora Hook. et Arn.

BACKGROUND:Studies have shown that atorvastatin can up-regulate the expression of heme oxygenase-1 and enhance the anti-inflammation and anti-oxidative damage ability of cells.However,whether atorvastatin can regulate macrophage polarization,inhibit inflammation and reduce cholesterol accumulation by inducing heme oxygenase-1 expression remains unclear. OBJECTIVE:To investigate the effect of atorvastatin on polarization,inflammation and cholesterol content of oxidized low-density lipoprotein stimulated RAW264.7 macrophages by inducing heme oxygenase-1 expression and its related mechanism. METHODS:Firstly,RAW264.7 cells were randomly divided into six groups and incubated with different concentrations of atorvastatin for 24 hours.The expression of heme oxygenase-1 protein and cell activity were detected to explore the optimal dose of atorvastatin for subsequent studies.RAW264.7 cells were randomly divided into control group,atorvastatin group and heme oxygenase-1 inhibition group.Cells were preincubated with pure medium,atorvastatin 20 μmol/L and atorvastatin 20 μmol/L + zinc protoporphyrin IX 10 μmol/L for 24 hours,and then oxidized low-density lipoprotein 50 mg/L was added for 48 hours.The polarization of macrophages was detected by flow cytometry.The secretion of inflammatory factors such as transforming growth factor β,interleukin 10,interleukin 1β,and tumor necrosis factor α was detected by ELISA.The expression levels of heme oxygenase-1,LC3II,LC3I,P62,PPARγ and ABCA1 were detected by western blot assay.The intracellular cholesterol content was measured with the oxidose method and the accumulation degree of intracellular lipid droplets was evaluated by oil red O staining. RESULTS AND CONCLUSION:(1)Atorvastatin could induce the expression of heme oxygenase-1 protein in macrophages in a dose-dependent manner.(2)Oxidized low-density lipoprotein could induce macrophages to polarize towards M1,secrete proinflammatory factors,and increase the accumulation of intracellular cholesterol.(3)Compared with the control group,the heme oxygenase-1 protein expression of macrophages was increased after atorvastatin intervention,and the cells turned to M2-type polarization and mainly secreted anti-inflammatory factors such as transforming growth factor-β and interleukin-10.PPARγ,ABCA1,LC3II/I and other signal molecules reflecting cholesterol efflux and autophagy increased,and the contents of intracellular cholesterol and lipid droplets decreased significantly(P<0.05).(4)The heme oxygenase-1 inhibition group treated with zinc protoporphyrin IX significantly reversed the above changes in the atorvastatin group.(5)The results have shown that atorvastatin may promote the polarization of macrophages stimulated by oxidized low-density lipoprotein to M2 type and inhibit inflammation by up-regulating the expression of heme oxygenase-1 and by up-regulating PPARγ/ABCA1 signaling pathway and enhancing autophagy.Atorvastatin can increase the outflow of intracellular cholesterol and reduce the accumulation of intracellular lipids.