核桃分心木提取物对脂多糖诱导RAW264.7细胞的抗炎作用

Anti-Inflammatory Effect of Diaphragma juglandis Fructus Extract on LPS-Induced RAW264.7 Cells

以核桃分心木为原料,探讨核桃分心木水提物(Diaphragma juglandis fructus water extract,DJF-W)和核桃分心木醇提物(Diaphragma juglandis fructus ethanol extract,DJF-E)对脂多糖诱导RAW264.7细胞的抗炎作用。利用超高效液相色谱串联质谱分析DJF成分,采用噻唑蓝比色法检测细胞活力,Griess检测细胞NO分泌水平,实时荧光定量PCR检测iNOS、COX-2、TNF-αmRNA表达量,免疫荧光检测iNOS和p38荧光强度,Westernblot法检测MAPK信号通路相关蛋白表达。结果显示,DJF中鉴定出132种物质,包括黄酮类40种、酚类24种、有机酸及其衍生物14种、苷类14种、苯丙素类9种、脂质类8种、萜类8种、其他15种;DJF-W浓度在25~1600μg/mL,DJF-E浓度在25~200μg/m L范围时对细胞活性无影响;经DJF-W和DJF-E干预后,与模型组相比DJF-W和DJF-E能抑制NO分泌水平,减少iNOS、COX-2、TNF-αmRNA表达量,降低iNOS和p38表达和MAPK信号通路相关蛋白表达;相同浓度下DJF-E抗炎效果优于DJF-W。综上,DJF-W和DJF-E可抑制脂多糖诱导RAW264.7细胞相关炎症因子分泌和表达以及MAPK信号通路来减轻炎症反应。

In this study,Diaphragma juglandis Fructus was used as raw material to investigate the antiinflammatory effects of Diaphragma juglandis fructus water extract(DJF-W)and Diaphragma juglandis fructus ethanol extract(DJF-E)on Lipopolysaccharide(LPS)induced RAW264.7 cells.Ultra-high performance liquid chromatography tandem mass spectrometry was employed to analyse the composition of DJF.Thiazolyl blue colorimetry(MTT)was utilised to detect cell viability.Griess was employed to determine the level of cellular NO secretion.Real-time fluorescence quantitative PCR was employed to the expression of mRNA for iNOS,COX-2,and TNF-α.Immunofluorescence was used to measure the fluorescence intensity of iNOS and p38.Western blot was employed to detect the expression of protein related to the MAPK signalling pathway.The results showed that 132 substances were identified in DJF,including 40 flavonoids,24 phenols,14 organic acids and their derivatives,14 glycosides,9phenylpropanoids,8 lipids,8 terpenoids,and 15 others;the concentration of DJF-W in the range of 25–1600μg/mL and the concentration of DJF-E in the range of 25–200μg/mL had no effect on cellular activity;after DJF-W and DJF-E interventions,DJF-W and DJF-E inhibited NO secretion level,reduced iNOS,COX-2,and TNF-αmRNA expression,and decreased iNOS and p38 expression and MAPK signalling pathway-related protein expression compared with the model group;the anti-inflammatory effect of DJF-E was better than that of DJF-W at the same concentration.In conclusion,DJF-W and DJF-E inhibited LPS-induced secretion and expression of RAW264.7 cell-associated inflammatory factors and the MAPK signalling pathway to attenuate the inflammatory response.