Effect of sophoridine and TLR4/MD-2 blocking agent on pathway of LPS-induced RAW264.7 macrophage TLR4-NF-κB-TNF-α

Abstract

To study the effect of sophoridine and TLR4/MD-2 blocking agent on pathway of LPS-induced RAW264. 7 macrophage TLR4-NF-kappaB-TNF-alpha in and its pharmacological mechanism of antiendotoxin. RAW264. 7 macrophages were cultured and divided into 5 groups, namely the blank control group, the LPS model group, the sophoridine + LPS group, the TLR4/MD-2 blocking agent + LPS group and the anti-TLR4/MD-2 + sophoridine + LPS group. Cells and cell culture fluids were collected at 120 min after the each group was processed. The expression of TLR4 protein was measured by western blot, the distribution and expression of NF-kappaB protein were measured by immunocytochemistry, and the expression of NF-kappaB and TNF-alpha mRNA were measured by western blot and reverse transcriptase polymerase chain reaction (RT-PCR). The content of TNF-alpha in the cell supernatant was detected by using radioimmunoassay. Compared with the LPS group, the expression of TLR4 protein, NF-kappaB mRNA, the rate of NF-kappaB entry the nucleus, TNF-alpha mRNA and TNF-alpha content in the cell supernatant were significantly decreased in the sophoridine + LPS group (P < 0.01). The rate of NF-kappaB entry the nucleus and TNF-alpha in the TLR4/MD-2 blocking agent + LPS group and the TLR4/MD-2 blocking agent + sophoridine + LPS group were notablly lower than that of the LPS model group (P < 0.01), close to that of the blank control group. However, there was no statistical significance between the two groups. TLR4/MD-2 may be one of sophoridine's targets. Sophordine's inhibitory effect on pathway activity of TLR4-NF-kappaB-TNF-alpha may be one of its antiendotoxin mechanisms.