Effect of Jianpi Huoxue decoction (健脾活血方)-containing serum on tumor necrosis factor-α secretion and gene Expression of endotoxin receptors in RAW264.7 cells induced by lipopolysaccharide
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- Peng, J., Hu, Y., Feng, Q. et al. Chin. J. Integr. Med. (2009) 15: 198. doi:10.1007/s11655-009-9001-x
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To evaluate the effect of Jianpi Huoxue decoction (健脾活血方, JHD)-containing serum on tumor necrosis factor-α (TNF-α) secretion and endotoxin receptor gene expression in RAW264.7 cells induced by lipopolysaccharide (LPS).
The 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 μ g/mL) for 12 h without rinse. The supernate, cells, protein and RNA were collected for assay. TNF-α in the culture supernate was assayed by the enzyme linked immunosorbent assay (ELISA). Protein expression of TNF-α in RAW cells was detected by Western-blot. TNF-α, Toll-like receptor 2 (TLR2), TLR4 and CD14 mRNA expression in RAW cells were detected by real-time RT-PCR.
The 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-α 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-α (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-α in both supernate (P=0.04) and cell layer (P=0.04), gene expression of TNF-α (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).
JHD-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.