Therapeutic Effects of Naringin in Rheumatoid Arthritis: Network Pharmacology and Experimental Validation

Abstract

Rheumatoid arthritis is a chronic autoimmune disease characterized by persistent hyperplasia of the synovial membrane and progressive erosion of articular cartilage. Disequilibrium between the proliferation and death of RA fibroblast-like synoviocytes (RA-FLSs) is the critical factor in progression of RA. Naringin has been reported to exert anti-inflammatory and antioxidant effect in acute and chronic animal models of RA. However, the therapeutic effect and underlying mechanisms of naringin in human RA-FLS remain unclear. Based on network pharmacology, the corresponding targets of naringin were identified using SwissTargetPrediction database, STITCH database, and Comparative Toxicogenomics Database. Deferentially expressed genes (DEGs) in RA were obtained from the GEO database. The protein-protein interaction (PPI) networks of intersected targets were constructed using the STRING database and visualized using Cytoscape. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed, and the pathways directly related to pathogenesis of RA were integrated manually. Further, in vitro studies were carried out based on network pharmacology. 99 target genes were intersected between targets of naringin and DEGs. The PPI network and topological analysis indicated that IL-6, MAPK8, MMP-9, TNF, and MAPK1 shared the highest centrality among all. GO analysis and KEGG analysis indicated that target genes were mostly enriched in (hsa05200) pathways in cancer, (hsa05161) hepatitis B, (hsa04380) osteoclast differentiation, (hsa04151) PI3K-Akt signaling pathway, and (hsa05142) Chagas disease (American trypanosomiasis). In vitro studies revealed that naringin exposure was found to promote apoptosis of RA-FLS, increased the activation of caspase-3, and increased the ratio of Bax/Bcl-2 in a dose-dependent manner. Furthermore, treatment of naringin attenuated the production of inflammatory cytokines and matrix metalloproteinases (MMPs) in TNF-ɑ-induced RA-FLS. Moreover, treatment of naringin inhibited the phosphorylation of Akt and ERK in RA-FLS. Network pharmacology provides a predicative strategy to investigate the therapeutic effects and mechanisms of herbs and compounds. Naringin inhibits inflammation and MMPs production and promotes apoptosis in RA-FLS via PI3K/Akt and MAPK/ERK signaling pathways.

Keywords: MAPK/ERK signaling pathway; PI3k/Akt signaling pathway; naringin; network pharmacology; rheumatoid arthritis.

FIGURE 1

Technical strategy of the current study.

FIGURE 2

Target genes of naringin and DEGs in GSE55235. (A) Chemical structure of naringin; (B) DEGs in GSE55235 (upregulated genes were marked in red and downregulated genes were marked in green). (C) Venn diagram of naringin target genes and DEGs. (D) Clustered heat map of overlapped genes.

FIGURE 3

(A) Gene ontology (GO) enrichment analysis for key targets (top 10 were listed). (B) KEGG pathway enrichment analysis of key targets (top 25 were listed); the abscissa label represents Fold Enrichment of pathways.

FIGURE 4

Potential naringin pathway constructed in the present research. Abbreviations: TNF-α, tumor necrosis factor alpha; TNFR, tumor necrosis factor receptor; TRADD, tumor necrosis factor receptor type 1 associated death domain protein; FADD, Fas associated via death domain; PI3K, phosphatidylinositol 3-kinase; PDK1, pyruvate dehydrogenase kinase 1; AKT, protein kinase B; MDM2, murine double minute2; Bcl-2, B-cell lymphoma-2; Bax, BCL2 associated X; BAD, BCL2 associated agonist of cell death; BIM, BCL2-like 11; Bak, BCL2 antagonist/killer; BID, BH3 interacting domain death agonist; MAPK1, mitogen-activated protein kinase 1; NF-κB, nuclear factor kappa B. BH3, Bcl-2 homology domain; CytoC, cytochrome C; ROS, reactive oxygen species.

FIGURE 5

PPI network construction. (A) PPI network construction of key targets (subnetworks from MCODE analysis were arranged in circles). (B) (C) KEGG pathway analysis of genes in the subnetwork (top five were listed).

FIGURE 6

Topological analysis of key targets. (A) (B) (C) Top 10 genes with the highest BC, CC, and DC. (D) Venn diagram summarizing overlapped genes in three sections. Abbreviations: BC, betweenness centrality; CC, closeness centrality; degree centrality (DC).

FIGURE 7

Identification of RA-FLS and therapeutic effect of naringin. (A) Representative images of RA-FLS stained with 5% crystal violet (100×, bar scale = 100 μm and 200×, bar scale = 50 μm). (B) Representative images of immunofluorescence staining for Vimentin (200×, bar scale = 50 μm). (C) Cell viability was measured using CCK-8 assay. Naringin reduces cell viability of RA-FLS in a dose-dependent manner. *p < 0.05. Each experiment was repeated for three times in different individuals.

FIGURE 8

Naringin induces apoptosis of RA-FLS in a dose-dependent manner. (A) Representative flow cytometric plots and statistical graph. (B) Effect of naringin on apoptosis-related proteins expression via Western blot analysis and statistical graphs. *p < 0.05. Each experiment was repeated for three times in different individuals.

FIGURE 9

Naringin inhibits inflammatory cytokines, MMPs and p-Akt expression in RA-FLS. (A) Naringin inhibits inflammatory cytokines and MMPs expression in TNF-α induced RA-FLS. (B) Naringin dose dependently inhibits MMPs expression in RA-FLS. (C) Naringin inhibits p-ERK and p-Akt expression in RA-FLS. *p < 0.05 and **p < 0.01. Each experiment was repeated for three times in different individuals.