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

Abstract

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 (H_d) group was lower than model group (p < 0.01). CD8⁺PD-1⁺T% was higher in the QFJDD H_d 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.

Keywords: CD8+PD-1+T; Qingfei Jiedu decoction; lung adenocarcinoma; molecular docking; network pharmacology; programmed cell death ligand-1.

FIGURE 1

The flowchart showing the molecular mechanisms by which QFJDD inhibits PD-L1 expression in LUAD.

FIGURE 2

Herb-Compound-Target Network.

FIGURE 3

(A) A Venn diagram of the intersecting LUAD-related genes constructed using DrugBank, GeneCard, OMIM, PharmGKB, and TTD databases. (B) The overlapping genes between QFJDD and LUAD. (C–E) The process of identifying twenty two hub genes. (F) The PPI network of twenty two hub genes targeted by QFJDD in LUAD. In the PPI diagram, one gene is represented by a circle, and the protein structure is displayed in the center of the circle.

FIGURE 4

(A,B) The GO and KEGG enrichment analyses of hub genes of QFJDD. (C) Network of twenty hub genes and thirty signaling pathways. The pink square represents hub gene, and the green triangle indicates signaling pathway. The square and triangle size reflects node degree. The higher the degree value, the bigger the node size.

FIGURE 5

(A) The KEGG analysis of PD-L1 expression and PD-1 checkpoint pathway in cancer (hsa05235 pathway). (B) Key genes of QFJDD acting on the hsa05235 pathway are labeled in red.

FIGURE 6

Molecular docking simulation analysis of representative receptor ligand pairs. (A) Quercetin act on EGFR. (B) Quercetin act on JUN. (C) Quercetin act on HIF1A. (D) Quercetin act on NFKBIA. (E) Quercetin act on RELA.

FIGURE 7

UPLC-Q-TOF-MS chromatogram of QFJDD in (A) POS and (B) NEG mode. POS, positive; NEG, negative.

FIGURE 8

In vitro experimental verification. The relative mRNA expression level of (A) JUN, (B) RELA, (C) HIF1A, (D) NFKBIA, (E) EGFR, (F) AKT1, (G) MAPK1, and (H) CD274 in A549 cells. N = 3. Data are shown as means ± SD. The significance of the results was assessed using the ANOVA. ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001 (vs. Control group); ^Δ P<0.05; ^ΔΔ P<0.01; ^ΔΔΔ P<0.001 (vs. QFJDD-containing serum group).

FIGURE 9

In vivo experimental validation. (A–E) The representative images (200×) of PD-L1 IHC staining of Lewis lung cancer tissues in each group. (F) Intergroup comparison of PD-L1 IRS in LLC tissues. N = 5. Data are means ± SD. Statistical significance was tested with Kruskal-Wallis nonparametric analysis of variance. ^** p < 0.01 (vs. H_d group). (G–M) Comparison of CD8⁺PD-1⁺T% between groups. N = 5. Data are presented as means ± SD. Significance was analyzed by the Kruskal-Wallis nonparametric analysis of variance. ^* p < 0.05 (vs. Normal group); ^Δ P<0.01 (vs. Model group).