Activity and mechanism of naringin in the treatment of post-infectious cough

Abstract

Objective: To explore activity and mechanism of naringin in the treatment of PIC (Post-Infectious Cough) by virtue of network pharmacology and animal studies.

Methods: The targets associated with naringin were obtained from the SwissTargetPrediction and Super-PRED databases. Disease-related targets were collected from GeneCards and OMIM (Online Mendelian Inheritance in Man). Venny was utilized to identify the overlapping targets between naringin and the disease. PPI (Protein-Protein Interaction) networks for disease-related targets were constructed using STRING and Cytoscape 3.10.1. GO (Gene Ontology) functional annotation and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses were performed with Metascape. Molecular docking between key targets and naringin was conducted using AutoDock Vina. In the animal experiments, PIC models were established in guinea pigs via intranasal inoculation with A/PR/8 virus, and cough frequency was measured after citric acid-induced coughing. Morphological changes in lung tissue and airways were assessed using the HE (Hematoxylin-Eosin) staining method. The relative expression levels of IL-8 (Interleukin-8), IL-1β (Interleukin-1β), TNF-α (Tumor Necrosis Factor-Alpha), and NF-κB p65 (Nuclear Factor Kappa-B p65 Subunit) mRNA were analyzed by RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction). SOD (Superoxide Dismutase) activity in lung tissue was measured using a colorimetric assay.

Results: After screening, naringin may contribute to the treatment of post-infectious cough by targeting proteins expressed by core genes such as HSP90AA1 (HSP 90-Alpha), TLR4 (Toll-like Receptor 4), MTOR (Mechanistic Target of Rapamycin), HIF1A (Hypoxia-Inducible Factor Alpha), and NF-κB1 (Nuclear Factor Kappa-B Subunit 1). KEGG enrichment analysis revealed involvement in pathways including the HIF-1 (Hypoxia-Inducible Factor 1) signaling pathway and the PD-L1 (Programmed Death-Ligand 1) expression and PD-1 (Programmed Cell Death Protein 1) checkpoint pathway in cancer. Molecular docking results indicated that naringin exhibited strong binding affinity with HSP90AA1, TLR4, MTOR, HIF1A, NF-κB1, NOS3 (Nitric Oxide Synthase 3), and GRB2 (Growth Factor Receptor-Bound Protein 2). In animal experiments, compared to the normal group, guinea pigs in the model group exhibited a significantly higher number of coughs, pronounced lung tissue hyperplasia, and inflammatory cell infiltration. Additionally, the relative expression of IL-8, IL-1β, TNF-α, and NF-κB p65 mRNA was significantly increased, while lung tissue SOD activity was decreased. Treatment with naringin significantly reduced the number of coughs, attenuated pathological changes in lung tissue, lowered the lung index, decreased the relative expression of IL-8, IL-1β, TNF-α, and NF-κB p65 mRNA, and significantly increased SOD activity in lung tissue compared to the model group.

Conclusion: Naringin shows therapeutic potential to alleviate PIC symptoms in a guinea pig model through anti-inflammatory and antioxidant mechanisms.

Keywords: Anti-inflammatory; Antioxidant; Naringin; Network pharmacology; Post-infectious cough.

Fig. 1

A Chemical structure of naringin (PubChem CID: 442,428). B Venn diagram of active ingredient targets and disease targets of naringin. The middle part is the 45 intersecting targets

Fig. 2

“Disease-Component-Target” interaction network. Red arrows represent diseases, blue circles represent intersecting gene targets, and green hexagons represent naringin

Fig. 3

PPI network diagram. A The circle indicates the intersection target, the darker the color of the circle, the larger the Degree value of the target. B The circles represent different proteins, and the connecting lines between the circles indicate the interactions between the proteins

Fig. 4

GO functional enrichment analysis. The X-axis showed enriched gene ontology categories of the targets, and the Y-axis showed the enrichment scores of these terms

Fig. 5

KEGG pathway enrichment analysis. The X-axis represents the gene ratio (number of overlapping targets in a pathway divided by total genes in that pathway), the Y-axis displays enriched pathways. Dot size corresponds to the count of overlapping targets, and color intensity reflects the -log10 (p-value) significance level. Top 10 enriched pathways are shown

Fig. 6

Molecular docking of naringin to key target proteins. A Naringin with HSP90AA1. B Naringin with MTOR. C Naringin with NOS3. D Naringin with TLR4. E Naringin with HIF1A. F Naringin with NF-κB. G Naringin with GRB2

Fig. 7

The cough frequency in each group on the seventh day after viral infection (n = 5–6). Data were shown as mean ± SEM. ### p < 0.001, compared with the control group. * p < 0.05, ** p < 0.01, compared with A/PR/8 group

Fig. 8

A-C The mRNA levels of IL-8, TNF-α and NF-κB p65 in the lung tissues were determined by RT-qPCR (n = 6). Data were shown as mean ± SEM. # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the control group. * p < 0.05, ** p < 0.01, compared with A/PR/8 group

Fig. 9

A-F Histological observations of lung tissues for guinea pigs (n = 3). (Scale bar = 50 μm). G Histopathologic scoring of guinea pig lung tissue. Data were shown as mean ± SEM. ## p < 0.01, compared with the control group. * p < 0.05, ** p < 0.01, compared with A/PR/8 group

Fig. 10

The activity of SOD in lung tissue was measured by colorimetric assay (n = 5–6). Data were shown as mean ± SEM. *** p < 0.001, compared with A/PR/8 group