| Objective: A variety of pathogenic factors (such as trauma, infection, ischemia-reperfusion, burns, etc.) can lead to acute lung injury (ALI), which appears as progressing acute respiratory failure, severe ALI is defined as acute respiratory distress syndrome (ARDS), the pathogenesis has not yet entirely clear. Lipopolysaccharide (LPS), a cell wall component unique to Gram-negative bacteria, can induce severe pneumonia, which led to ARDS. ALI is rapid onset, rapid development, yet no specific treatment. In recent years, despite the adoption of new ventilation technology and other therapeutic measures such as glucocorticoid, mortality rate of ARDS is still as high as 40% -60%. So how to prevent the development of ALI aroused great attention. Research shows that the outcome of ALI is thought to depend, in part, on the balance between proinflammatory cytokines and anti-inflammatory cytokines.Since more types of inflammatory cytokines produced, less signal transduction pathway, we study the pathogenesis of ALI from the signal transduction pathway.Mitogen-activated protein kinase (MAPK) is the focal point of different signal pathway. In macrophages, MAP kinases are crucial regulators of the production of proinflammatory cytokines in response to bacterial infection. Signal transduction and transcription-activating factor (STAT) is a kind of deoxyribonucleic acid binding protein, which is the key regulator of cytokines. The study of LPS and cytokines revealed that the intervention of LPS signal transduction in cells is an important way controlling the inflammatory cascade. A series of research shows that MAPKs play an important role in the LPS signal transduction. It is a potential target in the control of inflammatory response. The p-STAT3 expression and the level of TNF-αin LPS stimulated MH-S cell lines were observed, with or without SB203580 intervention. We explore the mechanism of LPS-induced ALI from the signal transduction pathways, which may supply new theoretical basis for the prevention and treatment of ALI.Methods:1. TNF-αlevels of cell supernatant were measured by ELISA. MH-S cells were grown in medium and the cell concentration was adjusted to 5×10~6/ml. Cells were cultured overnight in twenty-four orifice, the next day stimulated with different agents, and divided into 5 groups randomly:①Control group: with equal volume of RPMI1640;②LPS group: LPS final concentration of 100 ng/ml, the supernatant was collected after LPS stimulation for 90 min, 2h, 4 h, 6 h, 12 h.③SB5 + LPS group: 5μΜSB203580 was added 20 mins before 100 ng/ml LPS stimulation. after LPS stimulation for 90 min, 2h, 4 h, 6 h, 12h, the supernatant was collected;④SB10 + LPS group:10μΜSB203580 was added 20 mins before 100 ng/ml LPS stimulation. after LPS stimulation for 90 min, 2h, 4 h, 6 h, 12 h , the supernatant was collected;⑤SB15 + LPS group 15μΜSB203580 was added 20 mins before 100 ng/ml LPS stimulation. after LPS stimulation for 90 min, 2h, 4 h, 6 h, 12 h , the supernatant was collected . Supernatant was preserved at -80℃, TNF-αlevels of cell supernatant were measured by ELISA2. p-STAT3 expression was examined by Western blot The cells were stimulated by LPS and/or SB203580 and divided into 5 groups randomly:①Control group: with equal volume of RPMI1640;②LPS15min group: LPS final concentration of 100 ng/ml, 15 minutes stimulation;③LPS30min group: LPS final concentration of 100 ng/ml and stimulation 30 minutes;④SB10 + LPS15min group:10μΜSB203580 was added 20 mins before 100 ng/ml LPS stimulation for 15 minutes;⑤SB10 + LPS30min group: final concentration of SB203580 was 10μΜ, the final concentration of LPS was 100ng/ml, SB intervention 20 minutes before LPS stimulation for 30 min. Cells were collected after centrifugation (1500 rpm, 4℃, 10 min), abandon the supernatant, -80℃for the preservation of total protein extracts for Western-blot3. The cells crawl in six orifice and stimulated by LPS and SB203580, p-STAT3 was examined by Immunocytochemical in MH-S. The groups were the same as described in the Western-blot method Data are expressed as means±SEM. The group differences were analyzed by one-way analysis of variance (ANOVA) using SPSS13.0 software. If significant, the data were futher analyzed by Student-Newman-Keuls(SNK-q) test and P<0.05 was considered statistically significantResults:(1) The change of TNF-αlevels in MH-S cell supernatant. The TNF-αlevels increased after LPS-stimulation, start to increase at 90 min , reached peak levels at 4 h and 6 h, decreased at 12 h. The data were analyzed by one-way analysis of variance (ANOVA). There were significant diference between stimulated group and control group (P<0.05); the levels of TNF-αdecreased in SB203580 (10μΜand 15μΜ) intervention group,which have significant deferences comparing to LPS group(P<0.05), The levels of TNF-αdecreased as the concentration of SB203580 increased.The levels of TNF-αdecreased in SB203580(5μΜ) intervention group, but there was no significant deference comparing to LPS group (P>0.05)(2) Western blots showed phosphorylation of STAT3 protein levels in LPS15min and LPS30min group were higher than that of the control group and SB group, p-STAT3 expression levels of LPS30min group were higher than that of LPS15min group.(3) Immunocytochemical results show almost no stain in control Group. Cell began stained in LPS15min group and strengthened in LPS30min group, while after intervention by SB203580 cell stained smeared out. Groups were analyzed by one-way analysis of variance (ANOVA). There were statistal significant between stimulated-group and control-group (P<0.05), LPS15min and SB10+LPS15min, LPS30min and SB10+LPS30min (P<0.05).Conclusions:1. In the MH-S cell supernatant, TNF-αincreased after LPS stimulation and decreased by SB203580.2. The p-STAT3 expression levels in LPS15min group and LPS30min group were stronger than that of the control group and SB203580 group, LPS30min group stronger than LPS15min group. The results suggest SB203580 can reduce the expression of p-STAT3 and the mechanism may be SB203580 inhibited STAT3 through p38MAPK.
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