The Effect Of Forsythia Suspense On Anti-endotoxin And LPS-TLR4 Signal-transducing Pathway

Objective: To observe the effects of AFS on the cell viability,the expression of TLR4 and TNF-αmRNA in macrophage RAW264.7 induced by LPS, and to observe the effects of aqueous of Forsythia suspense(AFS) on the expression of TLR4 in splenocytes and levels of ET in peripheral blood of rats 8h after burn, to explore the anti-endotoxin and its mechanisms .Methods: Cultured RAW264.7 cells have been randomly divided into six groups: control group: without any treatment; LPS group: adding LPS at final concentration of 10ng/ml for 16h; AFS1+LPS,AFS2+LPS,AFS3+LPS group: 30mins of AFS (50mg/ml,25mg/ml,12.5mg/ml in final concentration) preconditioning before adding LPS; Ploy B+LPS group: 30mins of Ploy B (10ug/ml in final concentration) preconditioning before adding LPS. The intracorporal experiment were divided into six groups: control group; burn group(8PBH);AFS1+LPS,AFS2+LPS,AFS3+LPS group; Ploy B+LPS group. The rats of AFS1 guoup,AFS2 group and AFS3 group of them were given AFS 5g/kg,2.5g/kg,1.25g/kg once a day by Po. pathway for seven days before burns, respectively. The cell viability of different groups were measured by MTT analysis; The expression of TLR4 and TNF-αmRNA in RAW264.7 cells and splenocytes were measured by RT-PCR, respectively , The expression of TLR4 protein in RAW264.7 cells and splenocytes were measured by western blot; The LPS concentration of plasma was detected by limulus lysate test.Results: 1. Effects of AFS on TLR4 signal-transducing pathway in RAW264.7 cells induced by LPS 1.1 MTT analysis: Compared with control group, the cell viability of LPS group significantly decreased (P<0.01); AFS1+LPS,AFS2+LPS,AFS3+LPS group significantly enhanced the cell viability(P<0.01), which was dose-dependent; there was no significant difference between AFS1+LPS group and Ploy B+LPS group. 1.2 TLR4 mRNA and protein expression: It was found that TLR4 mRNA and protein had a basic expression in RAW264.7 cells under normal condition; and it was increased significantly after LPS acted on RAW264.7 cells than that of control group at 16h; the expression of TLR4 in AFS1+LPS guoup, AFS2+LPS group and AFS3+LPS group decreased remarkably than that of LPS group, which was dose-dependent. 1.3 TNF-αmRNA expression TNF-αmRNA expression in RAW264.7 cells significantly increased in LPS group compared with the control group (p<0.01); AFS1,AFS2,AFS3 significantly decreased TNF-αmRNA expression (P<0.01), which was dose-dependent;there was no significant difference between AFS1+LPS group and Ploy B+LPS group. 2. Effects of AFS on the plasma level of ET and the expression of TLR4 in splenocytes of burnt rats Compared with control group, the expression of TLR4 and TNF-αmRNA in splenocytes were upregulated markedly (P<0. 01) and ET were significantly increased in the 8PBH (post burn hour-8) group. The protein expression of TLR4 in splenocytes was consistent with the mRNA expression. AFS1,AFS2,AFS3and Ploy B significantly attenuated these increases, and there was no significant difference between AFS1group and Ploy B group.Conclusions: AFS preconditioning can inhibit the up-regulation of TLR4 in macrophage induced by LPS, and can decrease levels of ET in peripheral blood of severely burnt rats, and can down-regulate the expression of TLR4 in severely burnt rats; It was verified that Forsythia suspensa had the effect of anti-endotoxin from the level of cells and entire animal probably by affecting LPS-TLR4 signal-transducing pathway.