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。
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.
作者
古丽米拉·卡德尔
阿吉然姆·阿布拉
任国瑞
海力茜·陶尔大洪
杨飞
GULIMILA Ka-De-Er;AJIRANMU A-Bu-La;REN Guo-Rui;HAILIQIAN Tao-Er-Da-Hong;YANG Fei(School of Pharmacy,Xinjiang Medical University,Urumqi 830011,China)
出处
《食品安全质量检测学报》
CAS
北大核心
2023年第2期183-190,共8页
Journal of Food Safety and Quality
基金
国家自然科学基金项目(81960765)。
关键词
芜菁
多糖
细胞焦亡
除蛋白
Brassica rapa L.
polysaccharide
pyroptosis
deproteinization
