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
Knockdown of TLR4 by siRNA affects the activation of NF-κB/MAPK signaling pathway in RAW264.7 cells treated with two kinds of zymosan
杜蕊 1陈灰 1张宇飞 1李松建 1刘世雄 1李薛强 1徐子萱 1薛晨 2刘大程1
作者信息
- 1. 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
- 2. 包头市固阳县畜牧业技术服务中心,内蒙古包头 014200
- 折叠
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.
Abstract
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.
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Key words
yeast β-glucan/yeast mannan/TLR4/RAW264.7 cells/NF-κB/MAPK signaling pathway引用本文复制引用
基金项目
国家自然科学基金项目(32060774)
内蒙古自治区科技计划项目(2020GG0036)
内蒙古自治区科技计划项目(2022YFDZ0051)
政府间国际科技创新合作项目(2023YFE0100400)
高等教育改革发展项目-研究生科研创新资助项目(S20231099Z)
出版年
2024
国家科技期刊平台
NSTL
万方数据