槐定碱通过自噬调控巨噬细胞抗内毒素炎症效应  

Sophoridine Regulates the Antiendotoxin Inflammatory Effects of Macrophages through Autophagy

作　　者：马小霞 聂志倩 杨静 锁俊涛 韩怀钦[1] 梁锦屏[1] MA Xiaoxia;NIE Zhiqian;YANG Jing;SUO Juntao;HAN Huaiqin;LIANG Jinping(School of Basic Medical Sciences,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学基础医学院,银川750004

出　　处：《宁夏医科大学学报》2024年第10期973-982,共10页Journal of Ningxia Medical University

基　　金：宁夏自然科学基金项目(2022AAC02028,2022AAC03213)。

摘　　要：目的探究槐定碱(SRI)通过自噬途径调控脂多糖(LPS)诱导的巨噬细胞炎症反应的作用机制。方法利用LPS处理巨噬细胞RAW264.7建立炎症反应模型。实验分为两部分,第1部分使用SRI、自噬抑制剂3-甲基腺嘌呤(3-MA)处理细胞,分为空白对照组、SRI组、LPS组、SRI+LPS组、3-MA+LPS组;第2部分运用Toll样受体4(TLR4)抑制剂瑞沙托维(TAK-242)处理细胞,分为TAK-242组、TAK-242+SRI组、TAK-242+LPS组、TAK-242+SRI+LPS组、TAK-242+3-MA+LPS组。利用ELISA检测细胞上清液中肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)分泌水平;RT-qPCR检测TNF-α、IL-6、白细胞介素-1β(IL-1β)mRNA水平表达;Western blot检测TLR4、NF-κB、自噬效应蛋白(Beclin-1)和自噬蛋白5(ATG5)表达水平;荧光显微镜观察细胞活性氧(ROS)表达及自噬蛋白LC3B表达。结果SRI、TAK-242、3-MA、TAK-242联合SRI或3-MA预处理LPS诱导的RAW264.7细胞,炎症因子TNF-α、IL-6、IL-1β表达均减小(P均<0.05);细胞内ROS表达减小(P<0.05);TLR4、磷酸化NF-κB蛋白表达均下调(P均<0.05);Beclin-1、ATG5表达均下调(P均<0.05);LC3B蛋白荧光强度减弱(P<0.05),且联合干预的效果优于单独使用抑制剂。结论SRI可能通过TLR4/NF-κB信号通路调节巨噬细胞自噬发挥抗炎和抗氧化应激作用,拮抗内毒素炎症反应。Objective To investigate how sophoridine(SRI)regulates the inflammatory response induced by lipopolysaccharide(LPS)in macrophages through the autophagy pathway.Methods RAW264.7 cells were treated with LPS to establish an inflammatory response model.The experiment was divided into two parts.In the first part of the experiment,cells were treated with SRI and autophagy inhibitor 3-methyladenine(3-MA).The experimental groups were blank control group,SRI group,LPS group,SRI+LPS group,3-MA+LPS group.In the second part of the experiment,cells were pretreated with toll-like receptor 4(TLR4)inhibitor resatorvid(TAK-242).The experiment consisted of five groups,TAK-242 group,TAK-242+SRI group,TAK-242+LPS group,TAK-242+SRI+LPS group,and TAK-242+3-MA+LPS group.The levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in cell supernatant were measured using ELISA.The mRNA expression levels of TNF-α,IL-6 and IL-1βwere determined using RT-qPCR.Protein expressionlevels of TLR4,NF-κB,Beclin-1 and ATG5 were analyzed by Western blot.Fluorescence microscopy was used to observe the expression of reactive oxygen species(ROS)and autophagy protein LC3B.Results Pretreatment of LPS-induced RAW264.7 cells with SRI or inhibitors such as TAK-242,3-MA or their combinations significantly reduced secretion levels of TNF-α,IL-6,IL-1βcompared to control groups(P all<0.05).Reduced ROS production was observed in treated cells(P<0.05).Moreover,the protein expression levels of TLR4 and phosphorylated NF-κB decreased(P all<0.05).Additionally,the expressionlevels of Beclin-1 and ATG5 proteins involved in autophagy regulation also decreased(P all<0.05).Decreased fluorescence intensity for LC3B protein indicated a decrease in cellular autophagic activity(P<0.05),with combined interventions showing better effects than single-inhibitor treatmen.Conclusion SRI exerted an anti-inflammatory and antioxidative effects by modulating macrophage autophagy pathway through TLR4/NF-κB axis,and antagonize the inflammatory response of endotoxin.

关 键 词：槐定碱 自噬 TOLL样受体4 

分 类 号：R967[医药卫生—药理学]