基于网络药理学与实验验证探讨升降散调控脓毒症免疫功能紊乱的作用机制

Mechanism of regulating immune dysfunction of sepsis based on network pharmacology and experimental verification

目的验证升降散在脓毒症中的作用并探讨其分子机制。方法通过TCMSP、Disgenet等数据库搜索药物活性成分作用靶点与疾病相关靶点,并将二者取交集。采用DAVID数据库对交集靶点进行GO、KEGG通路富集分析,将药物活性成分与关键通路的核心基因进行分子对接。采用盲肠结扎穿刺(cecal ligation puncture,CLP)构建脓毒症小鼠模型,收集小鼠脾脏组织与血清,采用流式细胞术检测脾脏中不同T细胞亚群百分比,采用ELISA检测血清中IL-6、IL-10水平。结果网络药理学分析搜索获得升降散活性成分25个,活性成分作用靶点238个,疾病相关靶点2797个,活性成分与疾病相关靶点交集基因有90个,潜在靶标为AKT、JUN、EGFR、MMP9等;GO功能富集分析,得到条目数1021条,其中生物过程942条,细胞组成23条,分子功能55条。KEGG通路分析发现交集基因主要富集在PD-1/PD-L1信号通路、HIF-1信号通路、TNF信号通路、炎症介质信号通路等,表明其治疗效果可能和这些通路有关。分子对接结果显示,槲皮素、山奈酚、大黄素等核心化合物与关键基因都能较好的结合。流式细胞术结果显示,CLP 7 d后小鼠脾脏CD4^(+)T细胞比例减少,CD4^(+)PD-1^(+)T细胞比例增加,IL-6释放减少,IL-10含量增加,小鼠呈免疫抑制状态。给予升降散灌胃治疗后小鼠脾脏CD4^(+)T细胞百分比增加,CD4^(+)PD-1^(+)T细胞数量减少,IL-6释放增强,IL-10含量降低,小鼠免疫抑制得以改善。结论升降散能增加脓毒症晚期促炎症细胞因子IL-6释放,提高CD4^(+)T/CD8^(+)T细胞比值,降低抗炎性细胞因子IL-10表达,以改善脓毒症晚期免疫抑制,提高小鼠生存率。

Aim To verify the role of Shengjiang Powder in sepsis and explore its molecular mechanism.Methods The targets of drug active ingredients and disease-related targets were searched by TCMSP,Disgenet and other databases,and the intersection of the two was selected.DAVID database was used to carry out enrichment analysis of GO and KEGG pathways for intersection targets,and molecular docking was performed between drug active ingredients and core genes of key pathways.Mouse model of sepsis was constructed by cecal ligation puncture(CLP).Spleen tissue and serum of mice were collected.The percentage of T cell subsets in spleen was detected by flow cytometry,and IL-6 and IL-10 levels in serum were detected by ELISA.Results A total of 25 active ingredients,238 targets of active ingredients,2797 disease-related targets,90 genes of intersection between active ingredients and disease-related targets,potential targets were AKT,JUN,EGFR,MMP9,etc.GO enrichment analysis showed 1021 items,including 942 biological processes,23 cell compositions and 55 molecular functions.KEGG pathway analysis found that the intersection genes were mainly enriched in THE PD-1/PD-L1 signaling pathway,HIF-1 signaling pathway,TNF signaling pathway and inflammatory mediators signaling pathway,indicating that the therapeutic effect may be related to these pathways.The molecular docking results showed that quercetin,kamanol,emodin and other core compounds could be well combined with key genes.Flow cytometry results showed that after seven days of CLP,the proportion of CD4^(+)T cells in spleen decreased,the proportion of CD4^(+)PD-1^(+)T cells increased,the release of IL-6 decreased,the content of IL-10 increased,and the mice were immunosup-pressed.The percentage of CD4^(+)T cells in spleen increased,the number of CD4^(+)PD-1^(+)T cells decreased,the release of IL-6 was enhanced,the content of IL-10 decreased,and the immunosuppression was improved.Conclusions It is proved that Shengjiang Powder can increase the release of pro-inflammatory cytokine IL-6,increase the ratio of CD4^(+)T/CD8^(+)T cells,and decrease the expression of anti-inflammatory cytokine IL-10 in the late stage of sepsis,so as to improve immune suppression in the late stage of sepsis and improve the survival rate of mice.