[The effect of lipopolysaccharide on gene expression of TLR4 and MD-2 in rat alveolar macrophage and its secretion of inflammation cytokines].
OBJECTIVE
To investigate the effect of lipopolysaccharide (LPS) on gene expression of Toll-Like receptor 4 (TLR4) and myeloid differentiation protein-2 (MD-2) in rat alveolar macrophage NR8383 cell and its secretion of inflammation cytokines with or without small interference RNA target MD-2 (MD2-siRNA) treatment.


METHODS
NR8383 cell was cultured with F-12K medium and stimulated with LPS (0.01 approximately 10 mg/L) for 2 h or stimulated with 1 microg/ml LPS for 2-24 h. MD2-siRNA oligo was transfected into NR8383 cell by Lipofectamine 2000. The expression of TLR4 mRNA and MD-2 mRNA in cell were detected by semi-quantitative revels transcription polymerase (RT-PCR). The contents of TNF-alpha, IL-6 and IL-1beta in the cell cultured supernatant were tested by ELISA. One way ANOVA was used for difference comparison during groups and Pearson correlation was used for correlation analysis.


RESULTS
(1) The mRNA expression of TLR4 and MD-2 in control NR8383 cell were 0.52+/-0.05 and 0.44+/-0.09, respectively. There was no obviously change after 0.01 mg/L LPS stimulation. The increase occurred in a dose dependent manner from 0.1 mg/L to 10 mg/L. The highest TLR4 and MD-2 mRNA expression were 0.72+/-0.06 and 0.65+/-0.10 (F=17.255 and 6.045, P<0.01). The changes of TNF-alpha, IL-6 and IL-1beta contents in cell cultured supernatant were similar with that of TLR4 and MD-2 gene expression. TNF-alpha, IL-6 and IL-1beta content were (25.8+/-3.4) ng/L, (62.4+/-4.7) ng/L and (31.6+/-1.7) ng/L in control group, while the corresponding contents were (58.9+/-5.3) ng/L, (96.5+/-3.9) ng/L and (55.4+/-5.4) ng/L after 10 mg/L LPS simulation (F=29.55, 54. 47 and 31.45, P<0. 01). (2) When stimulated with 1 microg/ml LPS, the mRNA expressions of TLR4 and MD-2 in NR8383 cell were increased from hour 2. The highest mRNA expressions of TLR4 and MD-2 occurred at hour 6, and then decreased slowly from hour 8. The mRNA expressions at hour 24 were still higher than those in cells without LPS stimulation (F=5.279 and 4.106, P<0.01). The changes of TNF-alpha, IL-6 and IL-1beta content in cell cultured supernatant were also similar with that of gene expression (F=10.64, 11.23 and 17.58, P<0. 01). The secretion peaks of TNF-alpha and IL-1beta occurred from hour 6 to hour 8, and the secretion peak of IL-6 occurred from hour 8 to hour12. (3) The mRNA expression of MD-2 was related with that of TLR4 positively (r=0.513, P<0.01). (4) The interfere efficiency of MD-2 siRNA was 67%. There was no obvious increase of TNF-alpha, Il-1beta and IL-6 in MD-2 siRNA treated group after LPS stimulation.


CONCLUSIONS
Higher dose LPS can up-regulate the gene expression of TLR4 and MD-2 in rat alveolar macrophage NR8383 cell with a long time and promote the secretion of TNF-alpha, IL-6 and IL-1beta. MD-2 siRNA can inhibit NR8383 cell secrete TNF-alpha, IL-6 and IL-1beta induced by LPS.
Ren
Wei-ying
WY
Department of Pulmonary Medicine, Research Institute of Respiratory Disease, Fudan University, Zhongshan Hospital, Shanghai 200032, China.
Zhu
Lei
L
Hua
Feng
F
Jin
Jian-jun
JJ
Cai
Ying-yun
YY
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D015870:Gene Expression / Q000187:drug effects*; D007249:Inflammation; D053583:Interleukin-1beta / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051218:Lymphocyte Antigen 96 / Q000235:genetics*; D016676:Macrophages, Alveolar / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger; D051381:Rats; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D053583:Interleukin-1beta; D015850:Interleukin-6; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D012333:RNA, Messenger; C495347:Tlr4 protein, rat; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha