肉桂醛通过IRAK3/p38MAPK信号通路抑制巨噬细胞内结核分枝杆菌存活的实验研究

An experimental study on the inhibition of Mycobacterium tuberculosis survival within macrophages by cinnamaldehyde through the IRAK3/p38MAPK signaling pathway

摘要

目的 基于IRAK3/p38MAPK信号通路探讨肉桂醛（CA）对巨噬细胞内结核分枝杆菌（MTB）存活的影响。方法 通过细胞毒性试验（CCK-8）检测CA对巨噬细胞的毒性作用；利用透射电镜观察CA对巨噬细胞形态结构的影响；通过菌落形成单位（CFU）计数评估CA对巨噬细胞内MTB存活的影响；高效液相色谱技术（HPLC）检测巨噬细胞内CA浓度；转录组学测序（RNA-Seq）检测CA处理后MTB感染的巨噬细胞与模型组的差异表达基因；定量逆转录聚合酶链式反应（RT-qPCR）检测炎症通路TNF-α、IL-1β、IL-6、IRAK3和p38等基因的mRNA水平,酶联免疫吸附测定（ELISA）检测细胞培养上清中炎症因子TNF-α、IL-1β、IL-6的分泌水平,免疫印迹法（WB）检测炎症通路关键蛋白IRAK3、p38和磷酸化p38的表达水平。结果 CCK-8结果显示,30和60μmol/L CA对巨噬细胞无明显毒性作用,透射电镜观察到60μmol/L CA处理后的巨噬细胞形态结构无明显改变。CFU结果显示,CA处理组较模型组菌落数量明显减少,提示CA可抑制巨噬细胞内MTB的存活。HPLC检出巨噬细胞内CA浓度＜0.8μmol/L,提示CA进入细胞内直接作用于MTB不是其发挥抗菌作用的主要途径。RNA-Seq富集到CA处理后MTB感染的巨噬细胞与模型组炎症通路的差异表达基因。RT-qPCR结果显示,CA处理组较模型组炎症因子TNF-α、IL-1β、IL-6及炎症通路下游基因p38β表达显著降低,负反馈调节基因IRAK3表达显著升高。ELISA结果显示,CA处理组较模型组炎症因子TNF-α、IL-1β、IL-6分泌显著降低。WB结果显示,CA处理组较模型组IRAK3表达显著升高,磷酸化P38表达显著降低。结论 CA能够抑制巨噬细胞内MTB的存活,其机制可能与上调IRAK3表达、抑制p38MAPK磷酸化,进而降低MTB感染巨噬细胞的TNF-α、IL-1β、IL-6炎症因子分泌相关。

Abstract

Objective Based on the IRAK3/p38MAPK signaling pathway, this study explores the effect of cinnamaldehyde (CA) on the survival of Mycobacterium tuberculosis (MTB) within macrophages. Methods The cytotoxicity of CA on macrophages was detected by the CCK-8 assay; Transmission electron microscopy was employed to observe the impact of CA on the morphological structure of macrophages; The effect of CA on the survival of MTB within macrophages was evaluated by CFU counting; The concentration of CA was detected by HPLC; RNA-Seq was used to detect the gene expression differences; RT-qPCR was used to detect the mRNA levels of inflammatory factors such as TNF-α, IL-1β, IL-6, IRAK3 and p38; ELISA was used to detect the secretion levels of inflammatory factors TNF-α, IL-1β, and IL-6 in cell culture supernatants; WB was used to detect the expression levels of IRAK3, p38, and phosphorylated p38. Results The CCK-8 results indicated that 30 and 60μmol/L CA had no significant toxic effect on macrophages. Transmission electron microscopy revealed that the morphological structure of macrophages treated with 60μmol/L CA showed no significant change. CFU results showed that the number of colonies in the CA treatment group was significantly lower, suggesting that CA could inhibit the survival of MTB within macrophages. HPLC detected that the concentration of CA in macrophages was <0.8μmol/L, indicating that the direct action of CA entering the cells on MTB was not the main way for it to exert antibacterial effects. RNA-Seq identified the differentially expressed genes in the inflammatory pathways. RT-qPCR results showed that the expression of inflammatory factors TNF-α, IL-1β, IL-6 and downstream gene p38β of the inflammatory pathways was significantly lower in the CA treatment group compared to the model group, while the expression of negative feedback regulatory gene IRAK3 was significantly increased. ELISA results showed that the secretion of inflammatory factors TNF-α, IL-1β, and IL-6 in the CA treatment group was significantly lower. WB results showed that the expression of IRAK3 was significantly increased and the phosphorylated p38 expression was decreased in the CA treatment group. Conclusion CA can inhibit the survival of MTB within macrophages. The mechanism may be related to the upregulation of IRAK3 expression, inhibition of p38MAPK phosphorylation, and thereby reducing the secretion of TNF-α, IL-1β, and IL-6 inflammatory factors by MTB-infected macrophages.

关键词

肉桂醛 / 白细胞介素-1受体相关激酶3 / p38丝裂原活化蛋白激酶 / 巨噬细胞 / 结核分枝杆菌

Key words

cinnamaldehyde / interleukin-1 receptor-associated kinase 3 / p38 mitogen-activated protein kinase / macrophage / Mycobacterium tuberculosis

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基金资助

贵州省科技计划项目(黔科合基础-ZK(2023)一般581)

贵州省科技创新平台科研项目(黔科合平台CXPTXM(2025)017)