Prediction of the Mechanism of Sodium Butyrate against Radiation-Induced Lung Injury in Non-Small Cell Lung Cancer Based on Network Pharmacology and Molecular Dynamic Simulations and Molecular Dynamic Simulations

Abstract

Background: Radiation-induced lung injury (RILI) is a severe side effect of radiotherapy for non-small cell lung cancer (NSCLC) ,and one of the major hindrances to improve the efficacy of radiotherapy. Previous studies have confirmed that sodium butyrate (NaB) has potential of anti-radiation toxicity. However, the mechanism of the protective effect of NaB against RILI has not yet been clarified. This study aimed to explore the underlying protective mechanisms of NaB against RILI in NSCLC through network pharmacology, molecular docking, molecular dynamic simulations and in vivo experiments.

Methods: The predictive target genes of NaB were obtained from the PharmMapper database and the literature review. The involved genes of RILI and NSCLC were predicted using OMIM and GeneCards database. The intersectional genes of drug and disease were identified using the Venny tool and uploaded to the Cytoscape software to identify 5 core target genes of NaB associated with RILI. The correlations between the 5 core target genes and EGFR, PD-L1, immune infiltrates, chemokines and chemokine receptors were analyzed using TIMER 2.0, TIMER and TISIDB databases. We constructed the mechanism maps of the 3 key signaling pathways using the KEGG database based on the results of GO and KEGG analyses from Metascape database. The 5 core target genes and drug were docked using the AutoDock Vina tool and visualized using PyMOL software. GROMACS software was used to perform 100 ns molecular dynamics simulation. Irradiation-induced lung injury model in mice were established to assess the therapeutic effects of NaB.

Results: A total of 51 intersectional genes involved in NaB against RILI in NSCLC were identified. The 5 core target genes were AKT1, TP53, NOTCH1, SIRT1, and PTEN. The expressions of the 5 core target genes were significantly associated with EGFR, PD-L1, immune infiltrates, chemokines and chemokine receptors, respectively. The results from GO analysis of the 51 intersectional genes revealed that the biological processes were focused on the regulation of smooth muscle cell proliferation, oxidative stress and cell death, while the three key KEGG pathways were enriched in PI3K-Akt signal pathway, p53 signal pathway, and FOXO signal pathway. The docking of NaB with the 5 core target genes showed affinity and stability, especially AKT1. In vivo experiments showed that NaB treatment significantly protected mice from RILI, with reduced lung histological damage. In addition, NaB treatment significantly inhibited the PI3K/Akt signaling pathway.

Conclusions: NaB may protect patients from RILI in NSCLC through multiple target genes including AKT1, TP53, NOTCH1, SIRT1 and PTEN, with multiple signaling pathways involving, including PI3K-Akt pathway, p53 pathway, and FOXO pathways. Our findings effectively provide a feasible theoretical basis to further elucidate the mechanism of NaB in the treatment of RILI.

Keywords: molecular docking; molecular dynamics simulation; network pharmacology; non-small cell lung cancer; radiation-induced lung injury; signaling pathway; sodium butyrate; target gene.

Figure 1

Flowchart of bioinformatic analysis.

Figure 2

2D structural information of sodium butyrate (A); Venn diagram of involved genes of sodium butyrate, RILI and NSCLC (B).

Figure 3

Protein-protein interaction (PPI) network of the 51 intersectional genes was analyzed by STRING database (A) and the hub targets network including the top 10 targets were constructed by cytoHubba plug-in (B). In the hub targets network, the larger the node and the redder the color represent the stronger the interaction degree.

Figure 4

Correlations between EGFR and the 5 core target genes in LUAD and LUSC were studied using TIMER 2.0 database. (A) AKT1-EGFR. (B) TP53-EGFR. (C) NOTCH1-EGFR. (D) SIRT1-EGFR. (E) PTEN- EGFR.

Figure 5

Correlations between PD-L1 (CD274) and the 5 core target genes were evaluated using TIMER 2.0 database. (A) TP53-PD-L1 in LUAD. (B) TP53-PD-L1 in LUSC. (C) NOTCH1-PD-L1 in LUAD. (D) PTEN-PD-L1 in LUAD.

Figure 6

Correlations between the 5 core target genes and immune infiltration level in LUAD were analyzed using TIMER database. (A) AKT1. (B) TP53. (C) NOTCH1. (D) SIRT1. (E) PTEN.

Figure 7

Correlations between the 5 core target genes and immune infiltration level in LUSC were analyzed using TIMER database. (A) AKT1, (B) TP53, (C) NOTCH1, (D) SIRT1, (E) PTEN.

Figure 8

Correlations between chemokines and the 5 core target genes were explored using TISIDB database. (A) AKT1-chemokines. (B) TP53-chemokines. (C) NOTCH1-chemokines. (D) SIRT1-chemokines. (E) PTEN-chemokines.

Figure 9

Correlations between chemokine receptors and the 5 core target genes were analyzed using TISIDB database. (A) AKT1-chemokines receptors. (B) TP53-chemokines receptors. (C) NOTCH1-chemokines receptors. (D) SIRT1- chemokines receptors. (E) PTEN-chemokines receptors.

Figure 10

GO analysis and KEGG analysis of the 51 intersectional genes were performed using Metascape database. (A) GO analysis of target genes. (B) KEGG pathway analysis of target genes.

Figure 11

Compound-targets-pathways map was constructed using Cytoscape 3.8.2 software. The compound (sodium butyrate) was green node, targets were blue and red nodes, and pathways were showed by yellow nodes, respectively. The red represents the 5 core target genes. The edges represent the interactions among them.

Figure 12

Key KEGG signaling pathways were visualized using the KEGG database. (A) PI3k-Akt signaling pathway. (B) p53 signaling pathway (C) FOXO signaling pathway. Red rectangles represent some of the target genes of the 51 intersectional genes. Green rectangles indicate unidentified proteins.

Figure 13

Stereogram and docking energy score of molecular docking using AutoDock Vina tool and PyMOL software. (A) AKT1-NaB. (B) TP53-NaB. (C) NOTCH1-NaB. (D) SIRT1-NaB. (E) PTEN-NaB. The yellow dotted line represents the interaction between NaB and the target protein. NaB, sodium butyrate.

Figure 14

100 ns molecular dynamic simulations of AKT1-NaB using the GROMACS software. (A) The 3D stereoscopic molecular map of AKT1 and NaB docking. (B) 2D display of water and hydrogen bond docking between AKT1 and NaB. (C) RMSD and (D) the RMSF of molecular dynamic simulations. NaB, sodium butyrate.

Figure 15

Sodium butyrate treatment attenuates radiation-induced lung injury. (A) Hematoxylin and eosin staining of lung tissues 1 week after radiation administration. Scale bar 100 μm. (B) The protein levels of p-PI3K, PI3K, p-AKT and AKT in lung tissue were detected by Western blot. (C, D) Quantitative analysis of (B). Results are representative of three independent experiments, and the differences between data were evaluated by one-way ANOVA, with p value less than 0.05 considered statistical significance. **p < 0.001 vs. Control; ^## p < 0.05 vs. IR.