UPLC-Q-TOF/MS, network pharmacology, molecular docking, and experimental validation to explore the mechanisms of toad clothing on rheumatoid arthritis

Abstract

Toad clothing (toad slough) detoxifies, reduces oedema and analgesia, is anti-inflammatory and anti-tumour, and regulates the immune system. Toad clothing's mechanism is unclear. The molecular mechanism of toad clothing in rheumatoid arthritis (RA) therapy will be investigated in this research. We used Ultra-Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UPLC-Q-TOF/MS), network pharmacology, molecular docking, and in vitro experiments to investigate the mechanism behind the anti-RA action of toad apparel. First, UPLC-Q-TOF/MS revealed 24 toad clothing ingredients. Network pharmacology predicted five key elements, six core targets, and the Phosphatidylinositol 3-Kinase/ Protein Kinase B (PI3K/AKT) signalling pathway. Then, molecular docking was used to validate the binding ability of core chemical constituents and core targets of toad clothing. The results showed that the primary constituent, resibufogenin, was bound tightly to six core targets and had a good affinity for key RA targets such as Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), Epidermal Growth Factor Receptor (EGFR), and Janus Kinase 2 (JAK2). Cellular tests demonstrated that resibufogenin dramatically lowered Tumour Necrosis Factor-alpha (TNF-α), Interleukin-6 (IL-6), and Interleukin-1 beta (IL-1β) levels and regulated the PI3K/AKT signalling pathway. This research showed toad clothing's anti-RA pharmacodynamic material basis and mechanism, offering a theoretical foundation for its creation, use, and clinical application.

Fig. 1

This study used a schematic diagram of network pharmacology and molecular docking, as well as experimental verification.

Fig. 2

Representative BPI diagram of toad clothing extract in different ionization modes. (A) Negative ion mode. (B) Positive ion mode. BPI, base peak ion.

Fig. 3

Mass Spectrum Fragmentation Pattern. (A) Mass Spectrum Fragmentation Pattern of constituent 1. (B) Mass Spectrum Fragmentation Pattern of constituent 6. (C) Mass Spectrum Fragmentation Pattern of constituent 7.

Fig. 4

Venn diagram of the targets of toad clothing in Rheumatoid Arthritis.

Fig. 5

PPI network of potential target on Rheumatoid Arthritis. (A) Constructing a protein interaction network of Rheumatoid Arthritis target genes induced by toad clothing; (B) Screening of core targets of toad clothing in treating Rheumatoid Arthritis. PPI, Protein–Protein Interaction.

Fig. 6

GO and KEGG pathway analysis. (A) The top 10 significance of enriched GO terms analysis of therapy target genes of toad clothing on Rheumatoid Arthritis (P < 0.05). (B) Top 20 significance of enriched KEGG pathways and analysis of PI3K-AKT signal path in KEGG database (P < 0.05). (C) The font marked in red represents the target in the PI3K-AKT signal pathway closely related to treating Rheumatoid Arthritis with toad clothing. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; PI3K-AKT, Phosphatidylinositol 3-Kinase/Protein Kinase B.

Fig. 7

The constituent‑target‑pathway-disease network of toad clothing. Diamond shapes are used to represent signaling pathways, hexagons are used to represent bioactive constituents, blue circular nodes indicate target proteins, green triangle nodes indicate toad clothing drug, red ellipse nodes represent Rheumatoid Arthritis diseases, and the black edge stated that there was a connection between constituents, targets, drug, pathway and disease.

Fig. 8

Molecular docking heat map analysis and results of the lowest binding energy in each target with the selected bioactive constituent of toad clothing. (A) Heat map analysis of molecular docking of core constituents to core targets;(B) resibufogenin, EGFR, −9.8 kcal/mol; arenobufagin, EGFR, −9.8 kcal/mol; cinobufagin, JAK2, −9.7 kcal/mol; resibufogenin, PIK3CA, −8.3 kcal/mol. EGFR, epidermal growth factor receptor; JAK2, Janus Kinase 2; PIK3CA, hosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha.

Fig. 9

Toxicity of Resibufogenin (RBG) on RAW264.7 cells. The chemical structure of RBG is drawn using ChemDraw software (A). RAW264.7 cells were treated with RBG (0.25–160 μM) in the absence (B)or presence (C) of LPS (1 μg/ml) for 24 h, then the cell viability was detected by CCK-8 assay. RAW264.7 cells were treated with RBG (10,20,40,80 μM) in the absence of LPS (1 μg/ml) for 24 h; then the supernatant was collected for the detection of NO with Griess reagent(D). Data are mean ± SD of a minimum of three independent experiments. ^#P < 0.05, ^##P < 0.01 versus Control; ^*P < 0.05 and ^**P < 0.01 versus LPS. RBG, resibufogenin; LPS, Lipopolysaccharide; CCK, Cell Counting Kit.

Fig. 10

Effect of RBG on RAW264.7 cells. (A-C) Effects of RBG on TNF-α, IL-6 and IL-1β secretion in RAW264.7 cells stimulated by LPS. ^#P < 0.05, ^##P < 0.01 versus Control; ^*P < 0.05 and ^**P < 0.01 versus LPS. RBG, resibufogenin; TNF-α, Tumour Necrosis Factor-alpha; IL-6, Interleukin-6; IL-1β, Interleukin-1 beta; LPS, Lipopolysaccharide.

Fig. 11

Effects of RBG on LPS-treated RAW264.7 cells. (A-C) the mRNA expression levels of PIK3CA, EGFR, and JAK2 in RAW264.7 cells stimulated by LPS. ^#P < 0.05, ^##P < 0.01 versus Control; ^*P < 0.05 and ^**P < 0.01 versus LPS. RBG, resibufogenin; LPS, Lipopolysaccharide; PIK3CA, hosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; EGFR, epidermal growth factor receptor; JAK2, Janus Kinase 2.

Fig. 12

The protein expression levels of p-PI3K and p-AKT in RAW264.7 cells treated by RBG. (A) The protein expression levels of p-PI3K and p-AKT were measured by western blot assay. (B, C) Quantitative analysis of protein expression levels of p-PI3K and p-AKT. ^#P < 0.05, ^##P < 0.01 versus Control; ^*P < 0.05 and ^**P < 0.01 versus LPS. All data were analyzed as mean ± SD (n = 3)—one-way ANOVA followed by the SNK method. p-PI3, Phosphorylated Phosphoinositide 3-kinase; p-AKT, phosphorylated Protein Kinase B; RBG, resibufogenin; LPS, Lipopolysaccharide.