基于转录组测序探讨枸杞多糖调控巨噬细胞极化的机制

Molecular Mechanism of Lycium barbarum Polysaccharides Regulating Macrophage Polarization Based on Transcriptome Sequencing

摘要: 目的：利用转录组测序技术(RNA-seq)探讨预防性使用枸杞多糖是否影响巨噬细胞的极化状态并探讨其可能的作用机制。方法：用佛波酯(PMA)诱导THP-1细胞使其分化为巨噬细胞样细胞(PMA组)；在PMA处理细胞48 h后，利用LPS和IFN-γ将细胞诱导为M1型巨噬细胞(PLI组)；在PMA处理细胞48 h后，利用枸杞多糖处理细胞24 h，再利用LPS和IFN-γ将细胞诱导为M1型巨噬细胞(PLLI组)。对三组细胞进行转录组测序筛选差异表达基因，并通过Gene ontology (GO)、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes, KEGG)等数据库分析，筛选出差异表达基因富集的相关信号通路，最后用Western blot对分析结果进行验证。结果：经过比较分析发现，与PMA组细胞相比，PLI组细胞中有2760个基因表达上调，3290个基因表达下调；而PLLI组与PLI组相比，则显示262个基因表达上调，288个基因表达下调。值得注意的是，134个基因表现出动态的变化模式：在PLI组相对于PMA组表达升高，却在PLLI组相对于PLI组表达降低，这一特殊调控模式的基因包括ZBP1、HSPD1P6和CCL19等，其中，CCL19是NF-κB信号通路的关键趋化因子。进一步的Western blot实验证实，LBPs处理能够显著下调促炎型标志基因iNOS以及NF-κB信号通路关键蛋白和CCL19的表达水平，同时上调抑炎型巨噬细胞标志基因Arg-1和CD163的表达。结论：预防性使用枸杞多糖可抑制NF-κB信号通路，从而阻碍巨噬细胞向促炎性表型极化。

Abstract: Objective: RNA-seq technology was employed to investigate whether the preventive use of Lycium barbarum polysaccharides (LBPs) influences the polarization state of macrophages and to preliminarily explore its potential mechanism of action. Method: THP-1 cells were induced to differentiate into macrophage-like cells using phorbol myristate acetate (PMA) (PMA group). After 48 hours of PMA treatment, the cells were polarized into M1-type macrophages using LPS and IFN-γ (PLI group). After 48 hours of PMA treatment, the cells were treated with LBPs for 24 hours before being induced into M1-type macrophages with LPS and IFN-γ (PLLI group). Transcriptome sequencing was performed on the three groups of cells to identify differentially expressed genes (DEGs). These DEGs were subsequently analyzed using databases such as Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) to identify relevant signaling pathways. Finally, the results were validated using Western blot analysis. Results: Comparative analysis revealed that, compared to the PMA group, 2760 genes were upregulated and 3290 were downregulated in the PLI group. In the comparison between the PLLI group and the PLI group, 262 genes were upregulated and 288 were downregulated. Notably, 134 genes exhibited a dynamic expression pattern: their expression increased in the PLI group compared to the PMA group but decreased in the PLLI group compared to the PLI group. Genes with this regulatory pattern included ZBP1, HSPD1P6, and CCL19, among which CCL19 is a key chemokine in the NF-κB signaling pathway. Further Western blot analysis confirmed that LBPs treatment significantly downregulated the expression of the pro-inflammatory marker gene iNOS, as well as key proteins in the NF-κB signaling pathway and CCL19, while upregulating the expression of anti-inflammatory macrophage marker genes Arg-1 and CD163. Conclusion: The preventive use of Lycium barbarum polysaccharides can inhibit the NF-κB signaling pathway, thereby preventing macrophages from polarizing toward an inflammatory phenotype.