Effect of Jianpi Huoxue decoction-containing serum on tumor necrosis factor-alpha secretion and gene expression of endotoxin receptors in RAW264.7 cells induced by lipopolysaccharide.
- Author:
Jing-hua PENG
1
;
Yi-yang HU
;
Qin FENG
;
Yang CHENG
;
Li-li XU
;
Shao-dong CHEN
;
Qing TAO
;
Feng-hua LI
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Cell Line; Culture Media; pharmacology; Drugs, Chinese Herbal; pharmacology; Gene Expression; drug effects; Lipopolysaccharide Receptors; genetics; Lipopolysaccharides; pharmacology; Macrophages; cytology; drug effects; metabolism; Male; RNA, Messenger; metabolism; Rats; Rats, Sprague-Dawley; Receptors, Immunologic; genetics; Serum; Signal Transduction; drug effects; Toll-Like Receptor 2; genetics; Toll-Like Receptor 4; genetics; Tumor Necrosis Factor-alpha; metabolism; secretion
- From: Chinese journal of integrative medicine 2009;15(3):198-203
- CountryChina
- Language:English
-
Abstract:
OBJECTIVETo evaluate the effect of Jianpi Huoxue decoction (JHD)-containing serum on tumor necrosis factor-alpha (TNF-alpha) secretion and endotoxin receptor gene expression in RAW264.7 cells induced by lipopolysaccharide (LPS).
METHODSThe cytotoxicity of blank-control serum and JHD-containing serum at different concentrations were evaluated through the lactate dehydrogenase (LDH) assay in RAW264.7 cells. RAW264.7 cells were divided into six groups: 5% blank-control serum group (C1, n=3), 5% blank-control serum plus LPS group (L1, n=4), 5% JHD-containing serum plus LPS group (J1, n=4), 10% blank-control serum group (C2, n=3), 10% blank-control serum plus LPS group (L2, n=4), and 10% JHD-containing serum plus LPS group (J2, n=4). After cultured with the corresponding serum for 1 h, cells in L1, L2, J1 and J2 were treated with LPS (0.1 microg/mL) for 12 h without rinse. The supernate, cells, protein and RNA were collected for assay. TNF-alpha in the culture supernate was assayed by the enzyme linked immunosorbent assay (ELISA). Protein expression of TNF-alpha in RAW cells was detected by Western-blot. TNF-alpha, Toll-like receptor 2 (TLR2), TLR4 and CD14 mRNA expression in RAW cells were detected by real-time RT-PCR.
RESULTSThe LDH assay supported that cultured for 24 h or less with the JHD-containing serum at the concentration of 10% or lower, RAW264.7 cells showed no cytotoxicity. After stimulation with LPS for 2 h, TNF-alpha in the culture supernate of the 5% blank-control serum plus LPS group (L1, P=0.03), 10% blank-control serum plus LPS group (L2, P=0.002) and in the cell layer (P=0.01) of these groups increased remarkably. After stimulation with LPS for 1 h, the mRNA expression of TNF-alpha (P=0.004), TLR (P=0.03), CD14 (P=0.004) was up-regulated obviously. In the 10% JHD-containing serum plus LPS group (J2), the protein expression of TNF-alpha in both supernate (P=0.04) and cell layer (P=0.04), gene expression of TNF-alpha (P=0.03), TLR4 (P=0.001), CD14 (P=0.001) were all inhibited. On the other hand, the TLR2 mRNA expression was not up-regulated after LPS stimulation in the 10% blank-control serum plus LPS group (L2).
CONCLUSIONJHD-containing serum inhibited the LPS-induced cytokines expression in RAW264.7 which was probably associated with its inhibitory effect on the mRNA expression of LPS receptors TLR and CD14.
