| Objective: Glucocorticoids （GC） has long history as anti-inflammatorydrug in the therapy of inflammatory lung disease （ILD）. ILD is due toinflammation, immune factors cause numerous inflammtory cells of lung andbronchus infiltration, such as neutrophils and macrophage, the release of alarge inflammtory mediators produce falls to cascade, resulting the imbalancebetween proinflammatory mediators and anti-inflammatory mediators in thelung causes lung disease. Lipopolysaccharide （LPS）, through stimulatesvarious kinds of the inflammtory cells induce production of inflammatorymediator, thereby promote the development of ILD. The alveolar macrophage（AM） is the main effector cell of LPS, release the endogenous inflammationfactors, which led to cascade falls, is the basic reason of inflammationoccurrence and development. Activation of signal transducers and activatorsof transcription-3（STAT3） plays the key role on the regulation of ILD. Therelease of inflammatory cytokines can be reduced by inhibitingmitogen-activated protein kinase （MAPK） signaling pathway. In this study, theinfluences of LPS stimulated TNF-α and IL-10production in mouse alveolarmacrophage were observed, intervened by glucocorticoids and/or SB203580, aspecific inhibitor of p38MAPK. Analyzing whether there is synergetic effectbetween glucocorticoids and SB203580, further exploring signal transductioneffect of STAT, supply new theoretical basis about clinical application ofglucocorticoids and p38MAPK inhibitors for the treatment of ILD.Methods:1.The levels of TNF-α in supernatant of LPS stimulated cell lines weremeasured by ELISA MH-S cells lines were cultured in culture medium.Cell suspension wascollected and added to twenty-four well polystyrene tissue culture plates at1×106cells/ml of culture medium. After24h of growth，the cells wereallocated randomly:①Control group: with equal volume of RPMI1640ofserum-free medium;②LPS+DEX group: the cells were preincubated withDEX at a final concentration of1×10-6mol/L for2h, followed by addition ofLPS for2h,6h,12h;③DEX group: the cells were stimulated with DEXat a final concentration of1×10-6mol/L for2h,6h,12h;④DEX+SB203580+LPS group: the cells were preincubated with both DEX at afinal concentration of1×10-6mol/L for2h and SB203580at a finalconcentration of10μmol/L for20min, followed by addition of LPS for2h,6h,12h;⑤LPS+SB203580group: the cells were preincubated with SB203580at a final concentration of10μmol/L for20min, followed by addition of LPSfor2h,6h,12h;⑥LPS group: the cells were stimulated with LPS at a finalconcentration of100ng/ml for2h,6h,12h;The supernatant of six groups was collected after stimulation for themeasurement of TNF-α by ELISA.2. The levels of IL-10in supernatant of LPS stimulated cell lines weremeasured by ELISAMethods and groups were the same as determination of TNF-α.3. Phosphorylation of STAT3expression in MH-S cell was examined byImmunocytochemistry （ICC）.MH-S cells lines were cultured in culture medium.Cell suspension of thecollection was added to twenty-four well polystyrene tissue culture plates at1×106cells/ml of culture medium to crawl on the plates. After overnight ofgrowth, the cells were allocated randomly:①Control group: with equalvolume of RPMI1640of serum-free medium;②LPS+DEX group: the cellswere preincubated with DEX at a final concentration of1×10-6mol/L for2h,followed by addition of LPS stimulated for30min;③DEX group: the cellswere stimulated with DEX at a final concentration of1×10-6mol/L for30min;④DEX+SB203580+LPS group: the cells were preincubated with both DEX at a final concentration of1×10-6mol/L for2h and SB203580at a finalconcentration of10μ mol/L for20min, followed by addition of LPSstimulated for30min;⑤LPS+SB203580group: the cells were preincubatedwith SB203580at a final concentration of10μmol/L for20min, followed byaddition of LPS stimulated for30min;⑥LPS group: the cells were stimulatedwith LPS at a final concentration of100ng/ml for30min; Phosphorylation ofSTAT3expression was examined by Immunocytochemistry in MH-S cells.4. Phosphorylation of STAT3，STAT3expression in MH-S cell wereexamined by Western blot.Groups were the same as determination of ICC. Cell suspension of thecollection was added to six well polystyrene tissue culture plates.Afterstimulation, the cells of groups were collected for the examination ofphosphorylation of STAT3，STAT3expression in MH-S cell.Data are expressed as means±SEM. The group differences wereanalyzed by one-way analysis of variance （ANOVA） using SPSS13.0software.If significant, the data were futher analyzed by Student-Newman-Keuls（SNK-q） test and P<0.05was considered statistically significant.Results:1.The change of TNF-α levels in MH-S cell supernatant: TNF-α levelexhibited an increase at2h after LPS-stimulation, reached peak levels at6h,slight decrease at12h. This trend was not observed in the control group（P<0.05）. DEX treatment inhibited TNF-α in supernatant of LPS stimulatedcells. Cells treated with DEX produced significantly less TNF-α comparedwith LPS group at2h,6h,12h respectively（P<0.05）. SB203580treatmentinhibited TNF-α in supernatant of LPS stimulated cells. Cells treated withSB203580produced significantly less TNF-α compared with LPS group at2h,6h,12h respectively（P<0.05）. While cells treated with both DEX andSB203580further inhibited TNF-α in supernatant of LPS stimulated cells,which showed synergistic effect. Cells treated with both DEX and SB203580produced significantly less TNF-α compared with DEX+LPS group andSB+LPS group at2h,6h,12h respectively（P<0.05）. 2.The change of IL-10levels in MH-S cell supernatant: IL-10levelexhibited increase at2h after LPS-stimulation, reached peak levels at6h,slight decrease at12h. This trend was not observed in the control group（P<0.05）. DEX treatment inhibited IL-10in supernatant of LPS stimulatedcells. Cells treated with DEX produced significantly less IL-10comparedwith LPS group at2h,6h,12h respectively（P<0.05）. SB203580treatmentinhibited IL-10in supernatant of LPS stimulated cells. Cells treated withSB203580produced significantly less IL-10compared with LPS group at2h,6h,12h respectively（P<0.05）. While treated with both DEX and SB203580which play synergistic effect further inhibited IL-10in supernatant of LPSstimulated cells. Cells treated with both DEX and SB203580producedsignificantly less IL-10compared with DEX+LPS group and SB+LPS groupat2h,6h,12h respectively（P<0.05）.3. ICC results showed faint stain in control group. Stain of cells was thestrongest in LPS group.While after intervention by DEX, cell stained smearedout, after intervention by SB203580, cell stained smeared out also. Howeverafter intervention by both DEX and SB203580which play synergistic effect,cell stained further smeared out. After intervention by DEX, the non-LPS-stimulated cells showed faint stain. There were statistical significance onphosphorylation of STAT3expression between LPS group, LPS+Dex group,LPS+SB203580group, Dex+SB203580+LPS group and control group（P<0.05）.Compared with LPS group, the phosphorylation of STAT3expression decreased significantly in LPS+Dex group, LPS+SB203580group,Dex+SB203580+LPS group（P<0.05）. Compared with LPS+Dex group,LPS+SB203580group, the expression further decreased significantly inDex+SB203580+LPS group（P<0.05）.4. Results of phosphorylation of STAT3expression examined by Westernblot showed faint express in control group. Expression was the strongest inLPS group. While after intervention by DEX, the expression decreased, afterintervention by SB203580, the expression decreased also. However afterintervention by both DEX and SB203580which play synergy, the expression further decreased. After intervention by DEX, the non-LPS-stimulated cellsshowed faint express. There were statistical significance on phosphorylation ofSTAT3expression between LPS group, LPS+Dex group, LPS+SB203580group, Dex+SB203580+LPS group and control group （P<0.05）. Comparedwith LPS group, the phosphorylation of STAT3expression decreasedsignificantly in LPS+Dex group, LPS+SB203580group,Dex+SB203580+LPS group（P<0.05）. Compared with LPS+Dex group,LPS+SB203580group, the expression further decreased significantly inDex+SB203580+LPS group（P<0.05）.5. Results of STAT3expression examined by Western blot showed thereis no significant difference between the groups（P>0.05）.Conclusions:1. In the MH-S cell supernatant, the levels of TNF-α and IL-10increasedafter LPS stimulation, however the levels of TNF-α and IL-10decreased afterintervention by DEX or SB203580respectively, DEX and SB203580collaboratively inhibited secretion of TNF-α and IL-10. The results indicatedthat p38MAPK inhibitor SB203580may improve steroid resistance of ILD bysynergic effect with GC.2. Phosphorylation of STAT3expression increased in MH-S cell afterLPS stimulation, however the expression of phosphorylation of STAT3decreased after intervention by DEX and SB203580which play synergisticeffect. The results indicated that they inhibited the levels of TNF-α and IL-10through the phosphorylation of STAT3mediate.3. The above results indicated p38MAPK inhibitor SB203580mayimprove steroid resistance of ILD by synergistic effect with GC from the cellfactors and signaling pathways. |
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