| Objective: Inflammatory lung disease (ILD) is caused by a variety ofintrapulmonary and extrapulmonary factors such as infection, poisoning andimmunodeficiency. Lipopolysaccharide (LPS) is one of the most importantfactors in ILD. The alveolar macrophage (AM) is the first defensive line of thelung and plays an important role in initiation and maintenance of inflammation.LPS activates the receptors of AM, sequently induces the intracellularsignaling cascade and therefore produces a large number of proinflammatorycytokines, such as tumor necrosis factor (TNF)-α, but also of antiinflammatorycytokines, such as interleukin (IL)-10. P38mitogen-activated protein kinase(p38MAPK) is an important kinase involved in intracellular signaltransduction and inflammatory response, and also signal transducers andactivators of transcription-3(STAT3) has been shown to play roles in theinflammatory signaling cascades triggered by LPS. SB203580, p38MAPKspecific inhibitor, can inhibit acute lung injury (ALI) induced by LPS. Weassumed that there was an association between p38MAPK and STAT3signaling pathway. In the study, we observed the influences of SB203580onTNF-α and IL-10production and the activation of STAT3in mouse alveolarmacrophage cell line (MH-S) stimulated by LPS, explored the relationshipbetween p38MAPK and STAT3and this may provide new ideas for clinicaltreatment of ILD.Methods:(1) IL-10production in MH-S cell measured by ELISA. Thecell concentration was adjusted to5×106/ml. Cells were placed in24-wellplates overnight and were stimulated randomly with different agents asfollows:①Control group: with equal volume of RPMI1640of serum-freemedium;②LPS group: LPS stimulation at final concentration of100ng/ml;③LPS+SB5group:5μΜ SB203580was pretreated20mins before100 ng/ml LPS stimulation;④LPS+SB10group:10μΜ SB203580was pretreated20mins before100ng/ml LPS stimulation;⑤LPS+SB15group:15μΜSB203580was pretreated20mins before100ng/ml LPS stimulation. All ofthe supernatant was collected after LPS stimulation for90min,2h,4h,6h,12hand then measured by ELISA.(2) Immunocytochemical determination ofSTAT3and p-STAT3expression. The cell concentration was adjusted to5×106/ml and the cells were stimulated randomly with different agents asfollows:①Control group: with equal volume of RPMI1640of serum-freemedium;②LPS15min group: LPS stimulation at final concentration of100ng/ml for15min;③LPS15min+SB10group:10μΜ SB203580was pretreated20mins before100ng/ml LPS stimulation for15min.The data were expressed as means±SEM. The differences betweengroups were analyzed by one-way analysis of variance (ANOVA). Ifsignificant, the data were analyzed by Student-Newman-Keuls (SNK-q) test.P<0.05was statistically significant.Results:(1) The IL-10levels increased after LPS stimulation, started toincrease at90min, reached peak level at6h, decreased at12h. It wassignificantly different between stimulated group and control group (P<0.05);the levels of IL-10in SB203580(5μΜ,10μΜ and15μΜ) intervention groupdecreased, which were significantly different with LPS group (P<0.05), but itwas not in a concentration dependent manner.(2)①STAT3expressionchanges: cytoplasmic was stained yellow in each group. There was no statistalsignificance between each group (P＞0.05).②p-STAT3expression changes:There was almost no stain in control group. In LPS15min group, the nucleusstained yellow, while LPS15min+SB10group nucleus stained lighter.p-STAT3expression of the two experimental groups inceased significantlycomparing with the control group (P <0.05); Compared with LPS15mingroup, p-STAT3expression decreased significantly in LPS15min+SB10group (P <0.05). While compared with control group, p-STAT3expression ofLPS15min+SB10group increased significantly. Conclusions:1The level of IL-10increased in LPS-stimulated MH-Scell supernatant; the level of IL-10decreased after SB203580intervention,while it was still higher than control group. However the inhibitory effect ofSB203580did not show concentration dependence. It suggested that higherconcentration of SB203580may have no significant impact on the secretion ofthe anti-inflammatory cytokine IL-10.2Non-activated STAT3existed in thecytoplasm. In LPS-stimulated MH-S cells, STAT3was activated into STAT3phosphorylative and entered into the nucleus. After the SB203580intervention,the nuclei stained lighter. It indicated that SB203580may reduce the STAT3phosphorylation expression through p38MAPK and there may be a relationbetween p38MAPK and STAT3signal pathway3. These results suggestedthat SB203580may inhibite STAT3activation through p38MAPK, andtherefore inhibited the secretion of IL-10. |
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