| ObjectiveTo observe the effects of polysaccharide sulfate on lipopolysaccharide-induced mice pulmonary microvascular endothelial cell in vitro and lipopolysaccharide-induced acute lung injury in mice, and explored the possible mechanism.Methods1. Mice pulmonary microvascular endothelial cell (PMVEC) was used as stable experiment objectives after the covery, training, passages of PMVEC. PMVEC were identified by CD34, lectin from Bandeiraea simplicifolia in the cultured cells were quantified by immunocytochemical stainning.2. The growth of cultured PMVEC were observed by means of MTT which were treated with PSS (final concentration:25,50,100,200,400μg-mL-1) or without PSS separately. The effect of PSS on the growth of LPS-induced PMVEC was observed by means of MTT.3. The fourth and/or fifth generation PMVEC was used in our research. The cells were divided into three groups:①Control group (C group), cells didn't received any intervention;②LPS group (L group), cells were incubated with LPS;③PSS+LPS group (LP group), cells firstly were incubated with PSS for one hour, and incubated with LPS. All cells were incubated for 24h before experiment.4. Adhesion between PMVEC and PMN was observed through measuring the PMN stained by rose Bengal.5. The concentrations of TNF-αin supernatant liquid were detected by the methods of ELISA. Expression change of ICAM-1 protein was observed by the method of immunocytochemical staining.6. The level of phosphorylating p38MAPK and NF-κB p65 were determined by Western Blotting.7. The concentrations of PAI-1 and t-PA in supernatant liquid were detected by the methods of ELISA. The mRNA of PAI-1 and t-PA were detected by RT-PCR.8. A mouse model of ALI was established by tail vein injection of LPS at a dose of 10mg·Kg-1. Forty mice were randomly divided into four groups:control group (C group), PSS group (P group), LPS group (L group), PSS+LPS group (LP group). After 8h injection, the artery blood and lung tissue were collected. The histological changes of lung tissue were observed under optical microscope and scored. The wet/dry ratio of lung tissue was measured. The expressions of TNF-αand ICAM-1 in homogenate of lung tissue were measured by the methods of ELISA. ELISA was also used to detect the concentrations of PAI-1 and t-PA in plasma. The positive expression change after nuclear translocation of actived NF-κB was observed by immunohistochemical staining. Twenty mice were randomly divided into four groups:C group, P group, L group, LP group. After 8h injection, the artery blood was collected, and the coagulation index (PT, APTT, and TT) was observed. Results1. The PMVEC showed in rhombus and polygon shapes, PMVEC was monolayer growth and showed a cobblestone appearance. The cultured cells had features of binding lectin from Bandeiraea simplicifolia and positive immunocytochemical staining with CD34 antibody.2. The growth of PMVEC in PSS-treated (25-200μg·mL-1) groups were much better than that of non-PSS-treated group, especially in 100μg·mL-1 PSS final concentration group. The growth of PMVEC in 400μg·mL-1 PSS final concentration group was inhibited. PSS can strengthen the growth of LPS-induced PMVEC.3. Compared with C group, the adhesion between PMVEC and PMN was increased significantly in L group (P<0.01). But compared with L group, PSS could reduce the adhesion (P< 0.05).4. Compared with those in C group, protein expressions of ICAM-1 and TNF-αin L group significantly increased (P<0.01). Compared with those in L group, PSS could significantly inhibit their expressions (P<0.01).5. The activity of p38MAPK and NF-κB in L group was significantly higher than those in group C (P<0.01). But compared with those in L group, the activity of p38MAPK and NF-κB in LP group decreased significantly (P<0.05).6. L group had significantly higher the mRNA and protein expression levels of PAI-1 and t-PA than C group (P<0.01), and PAI-1/t-PA ratio increased in the L group, In comparison with L group, the protein expression levels of PAI-1 and the mRNA expression levels of PAI-1 and t-PA in LP group markedly decreased (P<0.05), and PAI-1/t-PA ratio decreased in the LP group.7. Compared with those in C group, W/D ratio, protein expression of TNF-αand ICAM-1 in lung tissue, concentration of PAI-1 and t-PA, PAI-1/t-PA ratio, pathomorphological score of lung tissue in L group increased significantly (P<0.01), and LPS increased the nuclear translocation after NF-κB activation observed by optical microscop (P<0.01). There was no significant difference between C group and P group in above indexes (P>0.05). However, compared with L group, the above indexes in LP group improved significantly except concentration of t-PA (P<0.01), and PSS could significantly reduce the nuclear translocation after NF-κB activation. In coagulation index, compared with those in C group, APTT and TT were lengthened in P group (P<0.01), but PT, APTT and TT were all shortened (P<0.01). PT, APTT and TT in LP group were all lengthened than those in L group (P<0.01-0.05).Conclusion1. In a certain range of dosage, PSS could promote the proliferation of normal cultured PMVEC. LPS can injury PMVEC; PSS can improve LPS-induced PMVEC injury.2. PSS can have the role of inhibition of adhesion between PMN and LPS-induced PMVEC.3. PSS can reduce the protein expression of TNF-αand ICAM-1.4. The inhibition of PSS on p38MAPK and NF-κB activation seems to be the cause of attenuated ICAM-1 and TNF-αoverexpression induced by LPS.5. PSS can attenuate the inhibition of fibrinolysis system induced by LPS by decreasing the mRNA and protein expression of PAI-1.6. PSS has a protective effect on LPS-induced acute lung injury in mice which might be mediated through NF-κB pathway, and which is related with anticoagulation and activing fibrinolysis system.
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