siRNA干扰TLR4影响两种酵母多糖处理RAW264.7细胞NF-κB/MAPK信号通路的激活

扫码查看

原文链接

国家科技期刊平台
NETL
NSTL
万方数据

中文摘要：为了探究Toll样受体4(TLR4)在两种酵母多糖激活NF-κB/MAPK信号通路中发挥的作用,本研究利用CCK8法检测酵母β-葡聚糖和甘露聚糖的细胞毒性,并利用多种方法筛选效果最佳的TLR4 siRNA,然后利用ELISA、RT-qPCR方法检测相关炎症因子的分泌量及其mRNA表达量的变化,最后通过Western-blot检测NF-κB/MAPK信号通路关键蛋白磷酸化水平.结果显示,2.5～20 µg/mL β-葡聚糖及2.5～50 μg/mL甘露聚糖对RAW264.7细胞的活力没有影响;TLR4 siRNA-2可以高效敲低TLR4 mRNA和蛋白表达水平;两种酵母多糖处理敲低 TLR4 的细胞后,IL-6、IL-12、IL-1β、TNF-α、NO 分泌量及 IL-6、IL-12、IL-1β、TNF-α、iNOSmRNA表达水平显著下降,NF-κB/MAPK信号通路关键蛋白p-P65、p-P38、p-ERK1/2、p-JNK和p-IκB水平显著降低.结果表明,两种酵母多糖可以通过TLR4调控NF-κB/MAPK信号通路及相关细胞因子的分泌,最终确定TLR4是两种酵母多糖发挥免疫调节作用的重要受体.

外文标题：Knockdown of TLR4 by siRNA affects the activation of NF-κB/MAPK signaling pathway in RAW264.7 cells treated with two kinds of zymosan

外文摘要：In order to explore the role of Toll-like receptor 4(TLR4)in the activation of NF-κB/MAPK signaling pathway by two yeast polysaccharides.In this study,CCK8 method was used to detect the cytotoxicity of yeast β-glucan and mannan,and various methods were used to screen the best TLR4 siRNA.Then,ELISA and RT-qPCR were used to detect the secretion of related inflammatory factors and their mRNA expression.Finally,the phosphorylation level of key proteins in NF-κB/MAPK signaling pathway was detected by Western-blot.The results showed that 2.5-20 μg/mL β-glucan and 2.5-50 μg/mL mannan had no effect on the viability of RAW264.7 cells.TLR4 siRNA-2 can effectively knock down TLR4 mRNA and protein expression levels.The secretion of IL-6,IL-12,IL-1β,TNF-α,NO and the mRNA expression levels of IL-6,IL-12,IL-1β,TNF-α and iNOS were significantly decreased after TLR4 knockdown cells were treated with two yeast polysaccharides.The levels of key proteins p-P65,p-P38,p-ERK1/2,p-JNK and p-IκB in the NF-κB/MAPK signaling pathway were significantly decreased.The results showed that the two yeast polysaccharides could regulate the secretion of NF-κB/MAPK signaling pathway and related cytokines through TLR4,and finally determined that TLR4 was an important receptor for the immune regulation of the two yeast polysaccharides.

外文关键词：

yeast β-glucanyeast mannanTLR4RAW264.7 cellsNF-κB/MAPK signaling pathway

作者：

杜蕊、陈灰、张宇飞、李松建、刘世雄、李薛强、徐子萱、薛晨、刘大程

展开 >

作者单位：

内蒙古农业大学兽医学院,内蒙古呼和浩特 010018

农业农村部动物疾病临床诊疗技术重点实验室,内蒙古呼和浩特 010018

包头市固阳县畜牧业技术服务中心,内蒙古包头 014200

关键词：

酵母β-葡聚糖酵母甘露聚糖 Toll样受体4 RAW264.7细胞 NF-κB/MAPK信号通路

基金：

国家自然科学基金项目内蒙古自治区科技计划项目内蒙古自治区科技计划项目政府间国际科技创新合作项目高等教育改革发展项目-研究生科研创新资助项目

项目编号：

320607742020GG00362022YFDZ00512023YFE0100400S20231099Z

出版年：

2024

DOI：

10.16656/j.issn.1673-4696.2024.0118

中国兽医科学

中国农业科学院兰州兽医研究所

中国兽医科学

CSTPCD北大核心

影响因子：0.524

ISSN：1673-4696

年,卷(期)：2024.54(7)