芜菁多糖除蛋白及抑制RAW264.7巨噬细胞焦亡作用研究

Study on the deproteinization and inhibitory effects on the pyroptosis of RAW264.7 macrophages of Brassica rapa L. polysaccharide

中文关键词:  芜菁  多糖  细胞焦亡  除蛋白

英文关键词:Brassica rapa L.  polysaccharide  pyroptosis  deproteinization

中文摘要:

      目的 考察芜菁多糖两种方法除蛋白效果, 并探讨其对RAW264.7巨噬细胞焦亡的抑制作用。方法 采用AB-8大孔吸附树脂法、Savage法去除蛋白, 苯酚-硫酸法测定多糖含量, 采用脂多糖(lipopolysaccharide, LPS)及三磷酸腺苷(adenosine triphosphate, ATP)建立细胞焦亡模型, 细胞毒性检测试剂盒(cell counting kit-8, CCK8)检测芜菁多糖对RAW264.7细胞增殖率的影响, 免疫印迹实验(Western-Blot, WB)检测消皮素D (gasdermin D, GSDMD)相对表达量, 酶联免疫吸附测定法检测白细胞介素-18 (interleukin18, IL-18)、IL-1β、肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)、IL-6水平。结果 AB-8大孔吸附树脂除蛋白, 多糖保留率为85.9%, 蛋白去除率为91.0%; Savage法多糖保留率为77.6%, 蛋白去除率为72.8%, 精密度、稳定性、重复性和加样回收率的相对标准偏差(relative standard deviation, RSD)均小于2.0%。与模型组相比, 芜菁多糖各剂量能显著下调GSDMD的表达(P<0.05), 减少LPS/ATP诱导的RAW264.7细胞炎性因子IL-18、IL-1β、TNF-α、IL-6的释放(P<0.05)。结论 AB-8大孔吸附树脂法较Savage法, 蛋白去除率及多糖保留率更高; 芜菁多糖对LPS/ATP诱导的RAW264.7细胞焦亡具有抑制作用, 能在一定程度上减轻细胞损伤, 这可能与其抑制LPS/ATP诱导焦亡过程中的炎性反应和调控GSDMD蛋白的表达有关。

英文摘要:

      Objective To observe the deproteinizing effects of Brassica rapa L. polysaccharide by 2 kinds of methods, and explore the inhibitory effects on pyroptosis of RAW264.7 macrophages. Methods The protein was removed by AB-8 macroporous adsorption resin method and Savage method. The content of Brassica rapa L. polysaccharide was determined by phenol-sulfuric acid method. The pyroptosis model was established with lipopolysaccharide (LPS) and adenosine triphosphate (ATP), and the cell counting kit-8 (CCK8) was used to detect the effect of turnip polysaccharide on the proliferation rate of RAW264.7 cells. The influence of Brassica rapa L. polysaccharide on the expression of gasdermin D (GSDMD) was detected by Western-Blot (WB). The levels of interleukin-18 (IL-18)、IL-1β、tumor necrosis factor-α (TNF-α), and IL-6 were measured by enzyme-linked immunosorbent assay. Results AB-8 macroporous adsorption resin was used to remove protein, and the polysaccharide retention rate and protein removal rate were 85.9% and 91.0%, respectively, the polysaccharide retention rate and protein removal rate of Savage method were 77.6% and 72.8%, respectively. The relative standard deviation (RSDs) of precision, stability, repeatability and recovery were all less than 2.0%. Compared with the model group, each dose of Brassica rapa L. polysaccharide could significantly decrease the expression of GSDMD (P<0.05), and reduce the release of LPS/ATP-induced inflammatory factors IL-18、IL-1β、TNF-α, and IL-6 in RAW264.7 cells (P<0.05). Conclusion AB-8 macroporous adsorption resin method has higher protein removal rate and polysaccharide retention rate than Savage method. Brassica rapa L. polysaccharide can inhibit pyroptosis of RAW264.7 cells induced by LPS/ATP, and can alleviate cell damage to a certain extent, which may be related to the inhibition of inflammatory response during LPS/ATP-induced pyroptosis as well as the regulation of the expression of GSDMD protein.