摘要
目的:研究香栓菌石油醚提取物中活性组分(BFT)对豚草花粉提取物(RWE)和脂多糖(LPS)诱导的RAW264.7巨噬细胞炎症反应的影响,并探究其作用机制。方法:采用硅胶柱层析法分离香栓菌石油醚提取物中的BFT,并采用GC-MS法进行分析。CCK-8法和FDA法检测细胞活力;DCFH-DA法检测细胞中ROS水平;ELISA检测细胞上清中炎症因子TNF-α、IL-18、IL-1β、IL-6、NO和PGE2水平;Western blot检测细胞中iNOS、COX-2、p-p65、p-IκBα、NLRP3、ASC、Caspase-1蛋白表达;qRT-PCR检测细胞中IL-6、TNF-α、IL-6、IL-18、IL-1β表达;免疫荧光技术检测细胞中NLRP3表达。结果:硅胶柱层析法得到的BFT中甾类成分占总含量的56.81%。BFT对RAW264.7巨噬细胞活性无显著影响。与对照组相比,RWE(10µg/mL)和LPS(100 ng/mL)共同刺激显著降低细胞活力(P<0.01),但BFT预处理后,RAW264.7细胞活力显著升高(P<0.01),炎症因子和ROS水平显著降低(P<0.01)。与模型组相比,BFT可显著降低RAW264.7巨噬细胞中p65、IκBα磷酸化水平,显著降低iNOS、COX-2、NLRP3、Caspase-1和ASC蛋白表达(P<0.01);qRT-PCR结果显示BFT可显著下调细胞中TNF-α、IL-6、IL-18、IL-1β表达;与模型组相比,BFT处理后,RAW264.7巨噬细胞中NLRP3荧光强度减弱。结论:BFT通过ROS/NF-κB/NLRP3信号通路抑制RWE和LPS共同诱导的RAW264.7巨噬细胞炎症。
Objective:To study effect of bioactive fraction from petroleum ether extract of Trametes suaveolens(BFT)on inflammatory response induced by ragweed pollen extract(RWE)and lipopolysaccharide(LPS)in RAW264.7 macrophages and to investigate its mechanism.Methods:Silica gel column chromatography was used to separate BFT in petroleum ether extract of Trametes suaveolens,and GC-MS was used for analysis.CCK-8 method and FDA method were used to detect cell vitality;DCFH-DA was used to detect level of ROS in cells.TNF-α,IL-18,IL-1β,IL-6,NO and PGE2 levels in cell supernatant were detected by ELISA.Western blot was used to detect iNOS,COX-2,p-p65,p-IκBα,NLRP3,ASC,Caspase-1 protein expressions in cells;expressions of IL-6,TNF-α,IL-6,IL-18 and IL-1βin cells were detected by qRT-PCR.NLRP3 expression in cells was detected by immunofluorescence.Results:Steroid components in BFT obtained by silica gel column chromatography accounted for 56.81%of total content.There were no toxic effects of BFT on RAW264.7 macrophages.Compared with control group,co-stimulation with RWE(10μg/mL)and LPS(100 ng/mL)significantly decreased cell viability(P<0.01),but after pretreatment with BFT,cell viability was significantly increased(P<0.01),levels of inflammatory factors and ROS were significantly decreased(P<0.01)in RAW264.7 cells.Compared with model group,BFT significantly reduced phosphorylation levels of p65,IκBα,and protein expressions of iNOS,COX-2,NLRP3,Caspase-1 and ASC were significantly reduced in RAW264.7 macrophages(P<0.01);qRT-PCR results showed that BFT significantly down-regulated expression of TNF-α,IL-6,IL-18 and IL-1βin cells;compared with 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 ROS/NF-κB/NLRP3 signalling pathway.
作者
赵晶晶
包海鹰
梁海龙
屠琦
ZHAO Jingjing;BAO Haiying;LIANG Hailong;TU Qi(College of Chinese Medicinal Materials,Jilin Agri-cultural University,Changchun 130118,China;Key Laboratory of Edible Fungi Resources and Utilization(North),Ministry of Agriculture and Rural Affairs,Jilin Agricultural University,Changchun 130118,China)
出处
《中国免疫学杂志》
北大核心
2026年第1期47-52,共6页
Chinese Journal of Immunology
基金
长江学者和创新团队发展计划项目(IRT1134)。
