Integrating network pharmacology and experimental verification to decipher the multitarget pharmacological mechanism of Cinnamomum zeylanicum essential oil in treating inflammation

Abstract

Inflammatory diseases contribute to more than 50 % of global deaths. Research suggests that network pharmacology can reveal the biological mechanisms underlying inflammatory diseases and drug effects at the molecular level. The aim of the study was to clarify the biological mechanism of Cinnamomum zeylanicum essential oil (CZEO) and predict molecular targets of CZEO against inflammation by employing network pharmacology and in vitro assays. First, the genes related to inflammation were identified from the Genecards and Online Mendelian Inheritance in Man (OMIM) databases. The CZEO targets were obtained from the SwissTargetPrediction and Similarity Ensemble Approach (SEA) database. A total of 1057 CZEO and 526 anti-inflammation targets were obtained. The core hub target of CZEO anti-inflammatory was obtained using the protein-protein interaction network. KEGG pathway analysis suggested CZEO to exert anti-inflammatory effect mainly through Tumor necrosis factor, Toll-like receptor and IL-17 signalling pathway. Molecular docking of active ingredients-core targets interactions was modelled using Pyrx software. Docking and simulation studies revealed benzyl benzoate to exhibit good binding affinity towards IL8 protein. MTT assay revealed CZEO to have non-cytotoxic effect on RAW 264.7 cells. CZEO also inhibited the production of NO, PGE₂, IL-6, IL-1β and TNF-α and promoted the activity of endogenous antioxidant enzymes in LPS-stimulated RAW 264.7 cells. Additionally, CZEO inhibited intracellular ROS generation, NF-kB nuclear translocation and modulated the expression of downstream genes involved in Toll-like receptor signalling pathway. The results deciphered the mechanism of CZEO in treating inflammation and provided a theoretical basis for its clinical application.

Keywords: Anti-inflammatory; Cinnamomum zeylanicum bark essential oil; Molecular docking; Network pharmacology; RAW 264.7 cells.

Graphical abstract

Fig. 1

Schematic flowchart of the proposed work.

Fig. 2

Total ion chromatogram of Cinnamomum zeylanicum bark essential oil.

Fig. 3

Compound target network and protein protein interaction (PPI) analysis. (A) Venn diagram representing 124 targets of CZEO against inflammation. (B) Compound-target network constructed using Cytoscape version 3.9.1. Pink diamonds represent hub compound with highest degree values and orange arrows represent targets. (C) Protein protein interaction network of 124 common targets resulted from STRING database containing 124 nodes and 1522 edges. (D) Topological screening of PPI network representing 6 hub genes.

Fig. 4

Pathway enrichment analysis of hub targets. (A) Gene Ontology enrichment analysis of top 10 biological processes, cellular components and molecular functions represented in red, green and blue bars respectively. (B) Bubble plot diagram of KEGG enrichment analysis of top 20 pathways where X-axis and Y-axis represent fold enrichment and names of pathways, respectively. The size and color of each bubble corresponds to number of enriched genes and p value.

Fig. 5

Toll-like receptor signaling pathway. Red rectangles represent the hub targets.

Fig. 6

Molecular docking analysis of core constituents of CZEO with hub targets. (A) Heat map showing binding affinities between hub targets with probable drug ligands. (B) Molecular docking simulation showing interaction between protein and ligand of highest binding affinity i.e. IL8 with benzyl benzoate.

Fig. 7

MD simulation of IL8 and benzyl benzoate complex (A) Protein-ligand RMSD of IL8-benzyl benzoate complex during 100 ns MD simulation (B) C-alpha root mean square fluctuation of IL8 during 100 ns MD simulation (C) Histogram of protein-ligand contacts resulted from MD simulation of IL8 with benzyl benzoate complex.

Fig. 8

Protective role of Cinnamomum zeylanicum essential oil (CZEO) on cell viability and morphological alteration in RAW 264.7 cells. (A) Cytotoxicity effect of CZEO on RAW 264.7 cells as measured by MTT assay (B) Cells were treated with various concentration of CZEO (12.5–100 μg/ml) for 24 h and the morphology was observed under microscope (scale bar 50 μm). Data are expressed as mean ± SD of three independent experiments.

Fig. 9

Effect of CZEO on LPS-induced nitric oxide (NO) and prostaglandin-E₂ (PGE₂) production in RAW 264.7 cells. Cells were treated with LPS (1 μg/ml) for 2 h followed by treatment with CZEO (12.5 and 100 μg/ml) for 24 h. Measurement of (A) NO and (B) PGE₂ levels. Data are expressed as mean ± SD (n = 3). Statistical significance was measured by using one way analysis of variance followed by Tukey test. ^#p<0.05 between untreated and LPS-treated group; *p < 0.05, **p < 0.01 between LPS and CZEO treated group.

Fig. 10

Effect of CZEO on LPS-induced pro-inflammatory cytokine expression in RAW 264.7 cells. Cells were treated with LPS (1 μg/ml) for 2 h followed by treatment with CZEO (12.5 and 100 μg/ml) for 24 h. Cell-free supernatants were collected to estimate the level of (A) TNF-α, (B) IL-1β and (C) IL6 cytokines. Data are expressed as mean ± SD (n = 3). Statistical significance was measured by using one way analysis of variance followed by Tukey test. ^#p < 0.05 between untreated and LPS-treated group; *p < 0.05 and **p < 0.01 between LPS and CZEO treated group.

Fig. 11

Effect of CZEO on the endogenous anti-oxidant enzymes in LPS stimulated RAW 264.7 cells. Cells were treated with LPS (1 μg/ml) for 2 h followed by treatment with CZEO (12.5 and 100 μg/ml) for 24 h. Antioxidant enzymes including (A) SOD, (B) CAT, (C) GSH, and (D) GPx levels were assessed from cell free supernatant. Data are expressed as mean ± SD (n = 3). Statistical significance was measured by using one way analysis of variance followed by Tukey test. ^#p < 0.05 between untreated and LPS-treated group; *p < 0.05 and **p < 0.01 between LPS and CZEO treated group.

Fig. 12

Change in intracellular ROS levels in LPS stimulated RAW 264.7 cells after treatment with CZEO as measured by flow cytometry. Cells were treated with LPS (1 μg/ml) for 2 h followed by treatment with CZEO (12.5 and 100 μg/ml) for 24 h. After incubation cells were incubated with DCFH-DA for 30 min. (A) Fluorescence intensity plots of DCF dye in untreated cells (black color) and cells exposed to LPS (red color) and then treated with 12.5 μg/ml of CZEO (green color) and 100 μg/ml of CZEO (blue color). The probe DCFH-DA was incubated in cells for 30 min. (B) Quantitative analysis represented as relative fluorescence intensity of each treated group. Data are expressed as mean ± SD (n = 3). Statistical significance was measured by using one way analysis of variance followed by Tukey test. ^#p < 0.05 between untreated and LPS-treated group; *p < 0.05 and **p < 0.01 between LPS and CZEO treated group.

Fig. 13

Analysis of change in mitochondrial membrane potential in LPS stimulated RAW 264.7 cells after treatment with CZEO as measured by flow cytometry using JC-1 assay. Cells were treated with LPS (1 μg/ml) for 2 h followed by treatment with CZEO (12.5 and 100 μg/ml) for 24 h. After incubation cells were stained with JC-1 dye for 30 min. Data are presented as dot plot of JC-1 red fluorescence representing healthy mitochondria (Y-axis) against JC-1 green fluorescence representing damaged mitochondria (X-axis). (A) Untreated group (B) LPS treated group (C) CZEO treated group (12.5 μg/ml) (D) CZEO treated group (100 μg/ml).

Fig. 14

Effect of CZEO on LPS induced nuclear translocation of NF-kB p65 in RAW 264.7 cells. Cells were treated with LPS (1 μg/ml) for 2 h followed by treatment with CZEO (12.5 and 100 μg/ml) for 24 h. (A) Representative confocal images of NF-kB p65 nuclear translocation in RAW 264.7 cells in different treated groups. RAW 264.7 cells are stained with DAPI (blue color) and immunolabelled for NFkB p65 (red color). Bar represents 50 μm (B) Quantitative analysis represented as relative mean fluorescence intensity of NF-kB p65 of each treated group. Data are expressed as mean ± SD (n = 3). Statistical significance was measured by using one way analysis of variance followed by Turkey test. ^#p < 0.01 between untreated and LPS treated group; **p < 0.01 between LPS and CZEO treated group.

Fig. 15

Effect of CZEO on expression level of downstream genes involved in Toll like receptor pathway. mRNA expression of (A) TLR4 (B) IL8 (C) IL6 (D) NFkB and (E) TNF genes identified by RT-qPCR. Cells were treated with LPS (1 μg/ml) for 2 h followed by treatment with CZEO (12.5 and 100 μg/ml) for 24 h. Data are expressed as mean ± SD (n = 3). Statistical significance was measured by using one way analysis of variance followed by Tukey test. ^#p < 0.05 between untreated and LPS treated group; ^nsp>0.05, *p < 0.05 and **p < 0.01 between LPS and CZEO treated group.