粗毛纤孔菌多糖提取工艺优化及体外抗炎作用

Extraction Optimization and In Vitro Anti-inflammatory Effect of Polysaccharide from Inonotus hispidus

通过单因素实验和响应面实验优化粗毛纤孔菌(Inonotus hispidus)多糖提取工艺,纯化获得均一多糖(INP);采用ELISA法测定NO和细胞因子IL-6、TNF-α含量,采用RT-PCR方法测定炎症相关基因表达量,采用蛋白免疫印迹方法检测p38MAPK/NF-κB、AKT信号通路相关蛋白表达量,研究INP(低、中、高剂量分别为125、250、500μg·mL^(-1))对脂多糖(LPS)诱导小鼠巨噬细胞RAW 264.7炎症的影响。结果表明:多糖提取工艺为料液比1︰18(g︰mL)、提取温度80.5℃、提取时间1.9 h,在此条件下多糖得率为(0.612±0.057)%。与模型组相比,INP中剂量组的NO、TNF-α含量显著降低,高剂量组非常显著降低;高剂量组的IL-6含量显著降低。INP中、高剂量组的IL-1β表达量均非常显著降低,高剂量组的TNF-α表达量非常显著降低,高剂量组的IL-6表达量显著降低。INP中、高剂量组的p-p38表达量分别非常、极显著降低,高剂量组的p-Jnk1/2表达量极显著降低,低、高剂量组的p-Erk1/2表达量均极显著降低,高剂量组的p-p65表达量非常显著降低,中、高剂量组的IκBα表达量均非常显著降低,低、中、高剂量组的p-AKT表达量均极显著降低。研究结果表明INP可减轻LPS诱导的细胞炎症损伤,将为粗毛纤孔菌多糖进一步开发利用提供参考。

A homogeneous polysaccharide named INP was extracted from Inonotus hispidus through optimizing the extraction conditions by single-factor and response surface experiments.Then INP was studied for its effects on lipopolysaccharide(LPS)-induced inflammation in mouse RAW 264.7 macrophages.Three doses of INP,low dose INP at 125μg·mL^(-1),medium dose INP at 250μg·mL^(-1),and high dose INP at 500μg·mL^(-1)were set up.Contents of NO,IL-6,TNF-αwere determined by ELISA.RT-PCR was used to determine gene expression levels of inflammatory factors,and Western blotting was used to determine protein expression in p38MAPK/NF-κB and AKT signaling pathways.The results showed that the optimized polysaccharide extraction conditions were material-liquid ratio 1:18(g:mL),extraction temperature 80.5℃,and extraction time 1.9 h.Under these conditions,the polysaccharide yield was(0.612±0.057)%.Both NO and TNF-αwere decreased in the medium dose(P<0.05)and high dose(P<0.01)INP groups compared with those in the model group.IL-6 content and IL-6 expression were decreased in the high dose INP group.Both medium and high INP doses significantly(P<0.01)down regulated the expression of TNF-αand IL-1β.Low dose INP decreased the expression of p-Erk1/2(P<0.001)and p-AKT(P<0.001).Medium dose INP decreased the expression of p-p38(P<0.01),IκBα(P<0.01),and p-AKT(P<0.001).High dose INP decreased the expression of p-p38(P<0.01),p-Jnk1/2(P<0.001),p-Erk1/2(P<0.001),p-p65(P<0.01),IκBα(P<0.01),and p-AKT(P<0.01).INP alleviated LPS-induced cell inflammatory damage,and thus provided a reference for development and utilization of polysaccharides from I.hispidus in the future.