DOI: 10.57760/sciencedb.j00217.02372
title: Study on the mechanism of inhibiting ragweed pollen and LPS-induced inflammation in RAW264.7 cells by Petroleum ether extract of Trametes suaveolens
title: 香栓菌石油醚提取物抑制豚草花粉和LPS诱导的RAW264.7细胞炎症机制研究

description: Objective To study the effect of the active fraction (BFT) in petroleum ether extract of the Trametes suaveolens on the inflammatory response induced by ragweed pollen extract (RWE) and lipopolysaccharide (LPS) in RAW264.7 macrophages and to investigate its mechanism of action.Methods Silica gel column chromatography was used to separate the active fractions in the petroleum ether extract of Trametes suaveolens, and GC-MS was used for analysis. CCK8 method and the FDA method to detect the cell vitality; DCFH-DA was used to detect the level of ROS in cells. The levels of TNF-α, IL-18, IL-1β, IL-6, NO and PGE2 in cell supernatant were detected by ELISA kit. Western blot to detect iNOS, COX-2, p-p65, p-IκBα, NLRP3, ASC, Caspase-1 protein expression levels in cells; The expressions of IL-6, TNF-α, IL-6, IL-18 and IL-1β in cells were detected by qRT-PCR. The expression of NLRP3 in cells was detected by immunofluorescence technique.Results The fraction of petroleum ether extract of Trametes suaveolens (BFT) obtained by silica gel column chromatography accounted for 56.81% of the total content. There were no toxic effects of BFT on RAW264.7 macrophages. Compared with the control group, co-stimulation with RWE (10 μg/mL) and LPS (100 ng/mL) significantly decreased cell viability (P&lt;0.01), but after pretreatment with BFT, cell viability was significantly increased (P&lt;0.01), and the levels of inflammatory factors and ROS were significantly decreased (P&lt;0.01) in RAW264.7 cells. Compared with the model group, BFT significantly reduced the phosphorylation levels of p65, IκBα, and the protein expression levels of iNOS, COX-2, NLRP3, Caspase-1 and ASC were significantly reduced in RAW264.7 macrophages (P&lt;0.01) The qRT-PCR results showed that BFT significantly down-regulated the expression of IL-6, TNF-α, IL-6, IL-18, and IL-1β in cells; Compared with the model group, NLRP3 fluorescence intensity was attenuated in RAW264.7 macrophages after treatment with BFT.Conclusion BFT inhibits RAW264.7 macrophage inflammation induced by RWE and LPS through the ROS/NF-κB/NLRP3 signalling pathway.
description: Objective To study the effect of the active fraction (BFT) in petroleum ether extract of the Trametes suaveolens on the inflammatory response induced by ragweed pollen extract (RWE) and lipopolysaccharide (LPS) in RAW264.7 macrophages and to investigate its mechanism of action.Methods Silica gel column chromatography was used to separate the active fractions in the petroleum ether extract of Trametes suaveolens, and GC-MS was used for analysis. CCK8 method and the FDA method to detect the cell vitality; DCFH-DA was used to detect the level of ROS in cells. The levels of TNF-α, IL-18, IL-1β, IL-6, NO and PGE2 in cell supernatant were detected by ELISA kit. Western blot to detect iNOS, COX-2, p-p65, p-IκBα, NLRP3, ASC, Caspase-1 protein expression levels in cells; The expressions of IL-6, TNF-α, IL-6, IL-18 and IL-1β in cells were detected by qRT-PCR. The expression of NLRP3 in cells was detected by immunofluorescence technique.Results The fraction of petroleum ether extract of Trametes suaveolens (BFT) obtained by silica gel column chromatography accounted for 56.81% of the total content. There were no toxic effects of BFT on RAW264.7 macrophages. Compared with the control group, co-stimulation with RWE (10 μg/mL) and LPS (100 ng/mL) significantly decreased cell viability (P&lt;0.01), but after pretreatment with BFT, cell viability was significantly increased (P&lt;0.01), and the levels of inflammatory factors and ROS were significantly decreased (P&lt;0.01) in RAW264.7 cells. Compared with the model group, BFT significantly reduced the phosphorylation levels of p65, IκBα, and the protein expression levels of iNOS, COX-2, NLRP3, Caspase-1 and ASC were significantly reduced in RAW264.7 macrophages (P&lt;0.01) The qRT-PCR results showed that BFT significantly down-regulated the expression of IL-6, TNF-α, IL-6, IL-18, and IL-1β in cells; Compared with the model group, NLRP3 fluorescence intensity was attenuated in RAW264.7 macrophages after treatment with BFT.Conclusion BFT inhibits RAW264.7 macrophage inflammation induced by RWE and LPS through the ROS/NF-κB/NLRP3 signalling pathway.