| Objective:Inflammatory lung disease is characterized by numerous inflammtory cell infiltration , inflammtory mediator release and proinflammatory/anti-inflammatory system imbalance . Glucocorticoids (GC) has long history as anti-inflammatory drug in the therapy of inflammatory lung disease, but there is individual differences on therapeutic effect ,and signal transduction mechanism of GC is unknown in the therapy of inflammatory lung disease. The alveolar macrophage (AM) is the main inflammatory cell and plays important role in the development of inflammatory lung disease.Lipopolysaccharide (LPS), the main active ingredients of endotoxin produced by gram-negative bacteria, can stimulate the immunocytes and induce production of inflammatory mediator, thereby promote the development of inflammatory lung disease. Signal transducers and activators of transcription-3 (STAT3) can inhibit the production of inflammatory cytokines. To study the role of STAT3 on influence of dexamethasone to TNF-αand IL-10 secretion by mouse AM and supply theoretical basis for clinical application of GC in therapy of inflammatory lung disease,MH-S cell line was studied in the experiment and effect of DEX on TNF-αand IL-10 secretion induced by LPS was investigated.Methods:1. TNF-αlevels of cell supernatant were measured by ELISAMH-S cells lines were cultureed in cell medium and the concentration of cell was adjusted to 5×106/ml. Cells were stimulated with different agents, and divided into six groups randomly:①Control group: with equal volume of RPMI1640;②LPS group: LPS final concentration of 100 ng/ml stimulated for 2h, 6 h, 12 h;③DEX 10-6mol/l + LPS group: DEX final concentration of 10-6mol/l pretreatment 2h before 100 ng/ml LPS stimulation for 2h, 6 h, 12h ;④DEX 10-8mol/l + LPS group: DEX final concentration of 10-8mol/l pretreatment 2h before 100 ng/ml LPS stimulation for 2h, 6 h, 12h ;⑤DEX 10-6mol/l group: DEX final concentration of 1×10-6mol/l stimulated for 2h, 6 h, 12 h;⑥DEX 10-8mol/l group: DEX final concentration of 1×10-8mol/l DEX stimulated for 2h, 6 h, 12 h.The supernatant was collected after stimulation and TNF-αlevels of cell supernatant were measured by ELISA.2. IL-10 levels of cell supernatant were measured by ELISA .The method and groups were the same as described in the method of TNF-αmeasure.3. p-STAT3 expression in MH-S cell was examined by Immunocytochemistry.The cells crawled in culture plates and the concentration of cell was adjusted to 5×106/ml.The cells were divided into four groups randomly:①Control group: with equal volume of RPMI1640;②LPS group: LPS final concentration was 100 ng/ml and stimulated for 30 min, 2h, 4h;③Dex+LPS group: DEX pretreatment 2h before 100 ng/ml LPS stimulation for 30 min, 2h, 4h and DEX final concentration was 1×10-6mol/l;④DEX group: DEX final concentration was 1×10-6mol/l and stimulated for 30 min, 2h, 4h; p-STAT3 expression was examined by Immunocytochemistry in MH-S.Data were expressed as means±SEM. The differences between groups 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 significant.Results:1. Changes of TNF-αin cell supernatant: TNF-αlevel was(164.29±0.44)pg/ml in LPS2h group(,532.90±0.99)pg/ml in LPS6h group,(350.78±10.29)pg/ml LPS12h group. Compared with control group(30.81±3.81)pg/ml, levels of TNF-αincreased significantly in LPS groups(P<0.05). TNF-αlevel was(135.07±1.08)pg/ml in DEX10﹣6 mol/L +LPS2h group, (397.65±0.49)pg/ml in DEX10﹣6 mol/L +LPS6h group,(351.20±1.56)pg/ml in DEX10﹣6 mol/L +LPS12h group,(162.05±0.35)pg/ml in DEX10﹣8 mol/L +LPS2h group,(468.86±1.36)pg/ml in DEX10﹣8 mol/L +LPS6h group,(304.92±0.26)pg/ml in DEX10﹣8 mol/L +LPS12h group, which were higher significantly than control group(P<0.05); While compared with LPS6h group, levels of TNF-αdecreased significantly in DEX10﹣6 mol/L +LPS6h group and DEX10﹣8mol/L+LPS6h group(P<0.05), and the inhibitory effect of DEX was dose-dependent; Level of TNF-αwas lower in DEX10﹣6 mol/L +LPS2h group than LPS2h group(P<0.05),but without difference between DEX10﹣8 mol/L +LPS2h group and LPS2h group (P>0.05); Level of TNF-αwas lower in DEX10﹣8 mol/L +LPS12h group than LPS12h group (P<0.05), but without difference between DEX10﹣6 mol/L +LPS12h group and LPS12h group (P>0.05).2. Changes of IL-10 levels in cell supernatant: IL-10 level was(24.89±0.24)pg/ml in LPS2h group and without difference between LPS2h group and control group(23.21±0.35)pg/ml (P>0.05); IL-10 level was(33.32±0.30)pg/ml in LPS6h group,(44.93±0.99)pg/ml in LPS12h group,which were higher than control group(P<0.05). IL-10 level was(31.05±0.54)pg/ml in DEX10﹣6 mol/L +LPS2h,(50.00±0.33)pg/ml in DEX10﹣6 mol/L +LPS6h,(85.00±0.64)pg/ml in DEX10﹣6 mol/L +LPS12h,(27.74±0.46)pg/ml in DEX10﹣8 mol/L +LPS2h,(54.23±0.64)pg/ml in DEX10﹣8 mol/L +LPS6h,(78.05±0.30)pg/ml in DEX10﹣8 mol/L +LPS12h, which were higher significantly than control group and LPS group (P<0.05) ,and the promotion effect of DEX was dose-dependent. When compared with control group and LPS group, IL-10 levels increased significantly in DEX10﹣6 mol/L2h group(26.85±0.24)pg/ml, DEX10﹣6 mol/L6h group(50.64±0.08)pg/ml, DEX10﹣6 mol/L12h group(76.22±0.95)pg/ml, DEX10﹣8 mol/L6h group(45.93±0.76)pg/ml, DEX10﹣8 mol/L12h group(66.09±1.48)pg/ml (P<0.05) , but without change in DEX10﹣8 mol/L2h group(24.93±1.46)pg/ml (P>0.05). 3. Immunocytochemical results: Cell stain began in LPS30min group and optical density value was (0.17±0.001), reached peak in LPS2h group(0.22±0.007), began to weaken in LPS4h group(0.20±0.005), while cell stain strengthened futher after pretreatment by DEX and optical density value was (0.25±0.004) in DEX+LPS30min group, (0.34±0.011) in DEX+LPS2h group, (0.34±0.013) in DEX+LPS4h group.The data were analyzed by one-way analysis of variance (ANOVA), compareed with control group(0.12±0.013),the p-STAT3 expresstion increased significantly in experimental group (P<0.05); compareed with LPS group, the p-STAT3 expresstion increased significantly in DEX+LPS group(P<0.05).Conclusions:1. In the MH-S cell supernatant, the levels of TNF-αand IL-10 increased after LPS stimulation ,but the levels of TNF-αdecreased and IL-10 increased further after DEX intervention,which were dose- dependent.2. p-STAT3 expresstion increased in MH-S cell after LPS stimulation and increased further after DEX intervention, which indicated STAT3 was involved in the effect of DEX on inhibition of TNF-αand promotion of IL-10.
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