丹皮酚对脂多糖刺激的RAW264.7细胞炎症与自噬的影响

Effects of Paeonol on inflammation and autophagy of RAW264.7 cells stimulated by lipopolysaccharide

目的:以脂多糖(lipopolysaccharide,LPS)刺激的RAW264.7细胞,以自噬诱导剂雷帕霉素(rapamycin,Rap)促进自噬为主要模型,研究丹皮酚(paeonol,P)对RAW264.7自噬与免疫功能的影响。方法:使用LPS 1μg·ml-1刺激RAW264.7细胞6 h,丹皮酚200μmol/L处理12 h,Rap 100 ng·ml-1作用6 h,蛋白免疫印迹法(western blot,WB)检测自噬相关因子LC3B、Beclin、P62和炎症相关因子TN F-α、IL-1β的表达水平,激光共聚焦荧光共定位法(immunofluorescence,IF)检测LC3B、P62蛋白的荧光表达和定位;酶联免疫分析(enzyme-linked immunosorbent assay,ELISA)检测RAW264.7细胞TNF-α的分泌。结果:在脂多糖诱导RAW264.7炎症反应的细胞模型中,添加丹皮酚药物后LC3B、Beclin表达增多,P62表达减少,LC3B-l向LC3B-ll的转换效率增强;雷帕霉素抑制IL-1β、TNF-α的表达,与丹皮酚联用进一步抑制IL-1β、TNF-α的表达和分泌。结论:丹皮酚通过促进细胞自噬来抑制炎症反应,丹皮酚与雷帕霉素联用加强了对炎症的抑制作用。

Objective:We used LPS-stimulated RAW264.7 cells model to study the effect of Paeonol on the modulation of autophagy and immune reaction in the cells.Methods:RAW264.7 cells were stimulated with LPS at 11μg·mL-1 LPS for 6 hours,200μM Paeonol for 12 hours,and Rapamycin at 100 ng·mL-1 for 6 hours.Autophagy associated factors LC3B,Beclin and P62 and inflammatory factors TNF-αand IL-1βprotein levels were detected by WB.Expression and colocalization of LC3B and P62 were observed by Immunofluorescence.The secretion content of TNF-αin the culture medium was detected by ELISA.Results:In the inflammatory environment induced by LPS,RAW264.7 cells were preconditioned with Paeonol,the protein expression level of LC3B and Beclin increased and P62 decreased,the conversion efficiency of LC3B-1 to LC3B-ll increased;the Rapamycin down-regulated IL-1βand TNF-α;the combination of Rapamycin and Paeonol further reduced the expression and secretion of IL-1βand TNF-α.Conclusion:Paeonol inhibits the RAW264.7 cells which induced inflammatory response by LPS by promoting autophagy flux in the cells.The combination treatment of Paeonol and Rapamycin reduced the TNF-αsecretion level of RAW264.7 cells induced by LPS.