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

作　　者：杜蕊 陈灰 张宇飞[1,2] 李松建 刘世雄[1,2] 李薛强 徐子萱 薛晨 刘大程 DU Rui;CHEN Hui;ZHANG Yufei;LI Songjian;LIU Shixiong;LI Xueqiang;XU Zixuan;XUE Chen;LIU Dacheng(College of Veterinary Medicine,Inner Mongolia A gricultural University,Huhhot 010018,China;Key Laboratory of Clinical Diagnosis and Treatment Technology for Animal Diseases,Ministry of A griculture and Rural Development,Huhhot 010018,China;Animal Husbandry Technical Service Center of Guyang County,Baotou 014200,China)

机构地区：[1]内蒙古农业大学兽医学院,内蒙古呼和浩特010018 [2]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 [3]包头市固阳县畜牧业技术服务中心,内蒙古包头014200

出　　处：《中国兽医科学》2024年第7期974-981,共8页Chinese Veterinary Science

基　　金：国家自然科学基金项目(32060774);内蒙古自治区科技计划项目(2020GG0036;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)。

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m RNA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,2.5~20μg/m Lβ-葡聚糖及2.5~50μg/m L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 m RNA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、i NOSm RNA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。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 yeastβ-glucan and mannan,and various methods were used to screen the best TLR4 si RNA.Then,ELISA and RT-q PCR were used to detect the secretion of related inflammatory factors and their m RNA 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—20μg/m Lβ-glucan and 2.5—50μg/m L mannan had no effect on the viability of RAW264.7 cells.TLR4 si RNA-2 can effectively knock down TLR4 m RNA and protein expression levels.The secretion of IL-6,IL-12,IL-1β,TNF-α,NO and the m RNA expression levels of IL-6,IL-12,IL-1β,TNF-αand i NOS 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 

分 类 号：S852.33[农业科学—基础兽医学]