Berberine inhibits RA-FLS cell proliferation and adhesion by regulating RAS/MAPK/FOXO/HIF-1 signal pathway in the treatment of rheumatoid arthritis

Abstract

Aims: Rheumatoid arthritis (RA) is a common chronic immune disease. Berberine, as its main active ingredient, was also contained in a variety of medicinal plants such as Berberaceae, Buttercup, and Rutaceae, which are widely used in digestive system diseases in traditional Chinese medicine with anti-inflammatory and antibacterial effects. The aims of this article were to explore the therapeutic effect and mechanism of berberine on rheumatoid arthritis.

Methods: Cell Counting Kit-8 was used to evaluate the effect of berberine on the proliferation of RA fibroblast-like synoviocyte (RA-FLS) cells. The effect of berberine on matrix metalloproteinase (MMP)-1, MMP-3, receptor activator of nuclear factor kappa-Β ligand (RANKL), tumour necrosis factor alpha (TNF-α), and other factors was determined by enzyme-linked immunoassay (ELISA) kit. Transcriptome technology was used to screen related pathways and the potential targets after berberine treatment, which were verified by reverse transcription-polymerase chain reaction (RT-qPCR) and Western blot (WB) technology.

Results: Berberine inhibited proliferation and adhesion of RA-FLS cells, and significantly reduced the expression of MMP-1, MMP-3, RANKL, and TNF-α. Transcriptional results suggested that berberine intervention mainly regulated forkhead box O (FOXO) signal pathway, prolactin signal pathway, neurotrophic factor signal pathway, and hypoxia-inducible factor 1 (HIF-1) signal pathway.

Conclusion: The effect of berberine on RA was related to the regulation of RAS/mitogen-activated protein kinase/FOXO/HIF-1 signal pathway in RA-FLS cells.Cite this article: Bone Joint Res 2023;12(2):91-102.

Keywords: Berberine; Enzyme-linked immunosorbent assay; MMP-3; Rheumatoid arthritis; Traditional Chinese medicine; Western blot; arthritis; fibroblast-like synoviocytes; hypoxia; matrix metalloproteinases (MMPs); polymerase chain reaction; quantitative polymerase chain reaction; rheumatoid arthritis.

Fig. 1

The molecular structure of berberine.

Fig. 2

The effect of berberine on proliferation and adhesion of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs). a) The results of Cell Counting Kit-8. b) Cell adhesion test results. c) to f) Changes of matrix metalloproteinase (MMP)-1, MMP-3, receptor activator of nuclear factor kappa-Β ligand (RANKL), and tumour necrosis factor alpha (TNF-α) (*p = 0.05, **p = 0.010, ***p < 0.001, n = 3).

Fig. 3

a) Clustering heat map of differential transcripts. b) The principal component analysis (PCA). c) Volcanic map of berberine-regulated transcripts. d) Gene ontology (GO) bar chart of differential transcripts. e) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway bubble diagram. AMPK, AMP-activated protein kinase; FOXO, forkhead box O.

Fig. 4

Analysis of the key targets of berberine against rheumatoid arthritis. a) Berberine core upregulated transcriptional protein-protein interaction (PPI) diagram. b) Berberine core downregulated transcriptional PPI diagram.

Fig. 5

The pathways and molecular docking diagrams of berberine with the key targets. CDK2, cyclin-dependent kinase 2; EGFR, estimated glomerular filtration rate; ENO1, enolase 1; ERK, extracellular signal-regulated kinase; Grb2, growth factor receptor-bound protein 2; FOXO, forkhead box O; HIF-1, hypoxia-inducible factor 1; IKBKB, inhibitor of nuclear factor kappa-B kinase subunit β; IL6-R, interleukin-6 receptor; INS, insulin preproprotein; IRS, insulin receptor substrate; NFkB, nuclear factor kappa B; PLK, polo-like kinase-1; RAS, renin-angiotensin system; TAB1, TAK1-binding protein 1; TAK1, TGF-activated kinase 1; SOS, salt overly sensitive; STAT3, signal transducer and activator of transcription 3.

Fig. 6

The binding energies for the key targets docked into the berberine. a) AKT2-berberine (-9.1); b) AKT3 -berberine (-7.0); c) cyclin-dependent kinase 2 (CDK2)-berberine (-10.1); d) enolase 1 (ENO1)-berberine (-7.1); e) forkhead box 3 (FOXO3)-berberine (-8.9); f) FOXO4-berberine (-9.0); g) hypoxia-inducible factor 1 alpha (HIF-1α)-berberine (-6.0); h) HRAS-berberine (-7.3); i) inhibitor of nuclear factor kappa-B kinase subunit β (IKBKB)-berberine (-7.7); j) mitogen-activated protein kinase 1 (MAPK1)-berberine (-9.1); k) polo-like kinase-1 (PLK1)-berberine (-9.0); l) PLK4-berberine (-6.6); and m) signal transducer and activator of transcription 3 (STAT3)-berberine (-7.9).

Fig. 7

Effect of berberine on core transcripts of forkhead box O (FOXO) and hypoxia-inducible factor 1 (HIF-1) signalling pathway. a) to m) The effect of berberine on the transcriptional levels of mitogen-activated protein kinase 1 (MAPK1), signal transducer and activator of transcription 3 (STAT3), cyclin-dependent kinase 2 (CDK2), inhibitor of nuclear factor kappa-B kinase subunit β (IKBKB), FOXO3, polo-like kinase 1 (PLK1), HRAS, RAC serine/threonine protein kinase 2 (AKT2), AKT3, FOXO4, PLK4, enolase 1 (ENO1), and HIF-1 α after 24-hour administration (*p < 0.050, **p < 0.0, ***p < 0.001, ****p < 0.0001; n = 3). mRNA, messenger RNA.

Fig. 8

Effect of berberine on the expression of core proteins in forkhead box O (FOXO) and hypoxia-inducible factor 1 (HIF-1) signalling pathway. a) Western blot-related bands of related proteins. b) to g) Bar charts representing the relative quantitative results of HRAS, mitogen-activated protein kinase 1 (MAPK1), p-MAPK1, FOXO3, p-FOXO3, and HIF-1 α protein expression levels in turn (*p < 0.05, **p < 0.01, ***p < 0.001; n = 3). GADPH, glyceraldehyde 3-phosphate dehydrogenase.