DataSheet1_Qingfei Jiedu decoction inhibits PD-L1 expression in lung adenocarcinoma based on network pharmacology analysis, molecular docking and experimental verification.ZIP

Objective: We aim at investigating the molecular mechanisms through which the Qingfei Jiedu decoction (QFJDD) regulates PD-L1 expression in lung adenocarcinoma (LUAD).
Methods: Bioactive compounds and targets of QFJDD were screened from TCMSP, BATMAN-TCM, and literature. Then, GeneCard, OMIM, PharmGKB, Therapeutic Target, and DrugBank databases were used to identify LUAD-related genes. The protein-protein interaction (PPI) network was constructed using overlapping targets of bioactive compounds in LUAD with the Cytoscape software and STRING database. The potential functions and pathways in which the hub genes were enriched by GO, KEGG, and DAVID pathway analyses. Molecular docking of bioactive compounds and key genes was executed via AutoDock Vina. Qualitative and quantitative analyses of QFJDD were performed using UPLC-Q-TOF-MS and UPLC. Expressions of key genes were determined by qRT-PCR, immunoreactivity score (IRS) of PD-L1 was assessed by immunohistochemistry (IHC), while the CD8+PD-1+T% derived from spleen tissues of Lewis lung cancer (LLC) bearing-mice was calculated using flow cytometry (FCM).
Results: A total of 53 bioactive compounds and 288 targets of QFJDD as well as 8151 LUAD associated genes were obtained. Further, six bioactive compounds, including quercetin, luteolin, kaempferol, wogonin, baicalein, and acacetin, and 22 hub genes were identified. The GO analysis showed that the hub genes were mainly enriched in DNA or RNA transcription. KEGG and DAVID pathway analyses revealed that 20 hub genes were primarily enriched in virus, cancer, immune, endocrine, and cardiovascular pathways. The EGFR, JUN, RELA, HIF1A, NFKBIA, AKT1, MAPK1, and MAPK14 hub genes were identified as key genes in PD-L1 expression and PD-1 checkpoint pathway. Moreover, ideal affinity and regions were identified between core compounds and key genes. Notably, QFJDD downregulated EGFR, JUN, RELA, HIF1A, NFKBIA, and CD274 expressions (p < 0.05), while it upregulated AKT1 and MAPK1 (p < 0.05) levels in A549 cells. The PD-L1 IRS of LLC tissue in the QFJDD high dose (Hd) group was lower than model group (p < 0.01). CD8+PD-1+T% was higher in the QFJDD Hd group than in normal and model groups (p < 0.05).
Conclusion: QFJDD downregulates PD-L1 expression and increases CD8+PD-1+T% via regulating HIF-1, EGFR, JUN and NFκB signaling pathways. Therefore, QFJDD is a potential treatment option for LUAD.

研究目的：本研究旨在探究清肺解毒方（QFJDD）调节肺腺癌（LUAD）中程序性死亡配体1（PD-L1）表达的分子机制。
研究方法：本研究从中药系统药理学数据库与分析平台（TCMSP）、BATMAN-TCM及相关文献中筛选清肺解毒方的活性成分及其作用靶点。随后通过基因卡（GeneCard）数据库、在线人类孟德尔遗传（OMIM）数据库、药物基因组学知识库（PharmGKB）、治疗靶点（Therapeutic Target）数据库及药物银行（DrugBank）数据库获取肺腺癌相关基因。利用STRING数据库与Cytoscape软件构建清肺解毒方活性成分与肺腺癌重叠靶点的蛋白质相互作用（PPI）网络。通过基因本体（GO）、京都基因与基因组百科全书（KEGG）及DAVID通路富集分析，明确核心靶点基因的潜在功能与富集通路。采用AutoDock Vina软件开展活性成分与核心基因的分子对接验证。使用超高效液相色谱-四极杆-飞行时间质谱（UPLC-Q-TOF-MS）与超高效液相色谱（UPLC）对清肺解毒方进行定性与定量分析。通过实时荧光定量聚合酶链反应（qRT-PCR）检测核心基因的表达水平，采用免疫组化（IHC）法评估PD-L1的免疫反应评分（IRS），并通过流式细胞术（FCM）计算荷Lewis肺癌（LLC）小鼠脾脏组织中CD8+PD-1+T细胞占比。
研究结果：本研究共筛选得到清肺解毒方的53种活性成分与288个作用靶点，以及8151个肺腺癌相关基因。进一步筛选得到槲皮素、木犀草素、山奈酚、汉黄芩素、黄芩素及芹菜素在内的6种活性成分，以及22个核心靶点基因。GO富集分析结果显示，核心靶点基因主要富集于DNA或RNA转录相关生物学过程。KEGG与DAVID通路富集分析表明，20个核心靶点基因主要富集于病毒感染、肿瘤、免疫、内分泌及心血管相关通路。其中EGFR、JUN、RELA、HIF1A、NFKBIA、AKT1、MAPK1及MAPK14核心基因被鉴定为PD-L1表达与PD-1检查点通路中的关键基因。此外，核心活性成分与关键基因间呈现出理想的结合亲和力与结合位点。值得注意的是，在A549细胞中，清肺解毒方能下调EGFR、JUN、RELA、HIF1A、NFKBIA及CD274的表达水平（p < 0.05），同时上调AKT1与MAPK1的表达水平（p < 0.05）。高剂量清肺解毒方组荷Lewis肺癌小鼠的肿瘤组织PD-L1免疫反应评分显著低于模型组（p < 0.01）。高剂量清肺解毒方组小鼠脾脏CD8+PD-1+T细胞占比显著高于正常对照组与模型组（p < 0.05）。
研究结论：清肺解毒方可通过调控HIF-1、EGFR、JUN及NFκB信号通路，下调PD-L1表达并升高CD8+PD-1+T细胞占比，因此其有望成为肺腺癌的潜在治疗方案。