Genistein inhibits angiogenesis developed during rheumatoid arthritis through the IL-6/JAK2/STAT3/VEGF signalling pathway

Abstract

Background: Angiogenesis plays an important role in the development of rheumatoid arthritis (RA), which increases the supply of nutrients, cytokines, and inflammatory cells to the synovial membrane. Genistein (GEN), a soy-derived isoflavone, has been validated that can effectively inhibit the angiogenesis of several tumours. We thus carried out a study in vitro to investigate the effect of GEN in vascular endothelial growth factor (VEGF) expression and angiogenesis induced by the inflammatory environment of RA.

Methods: MH7A cells were used to verify whether GEN can inhibit the expression of VEGF in MH7A cells under inflammatory conditions and demonstrate the mechanism. EA.hy926 ​cells were used to verify whether GEN can inhibit the migration and tube formation of vascular endothelial cells in inflammatory environment.

Results: GEN dose-dependently inhibited the expression and secretion of interleukin (IL)-6 and VEGF, as well as the nucleus translocation of Signal transducer and activator of transcription 3 (STAT3) in MH7A. Furthermore, GEN inhibited IL-6-induced vascular endothelial cell migration and tube formation in vitro.

Conclusion: GEN inhibits IL-6-induced VEGF expression and angiogenesis partially through the Janus kinase 2 (JAK2)/STAT3 pathway in RA, which has provided a novel insight into the antiangiogenic activity of GEN in RA.

The translational potential of this article: Our study provides scientific guidance for the clinical translational research of GEN in the RA treatment.

Keywords: Angiogenesis; Genistein; Rheumatoid arthritis.

Figure 1

Cytotoxicity of GEN on MH7A and EA.hy926 ​cells. Cells were treated with different concentrations of GEN for 24 and 48 ​h, and their viability was determined using Cell counting kit-8 (CCK-8) assay. (A) Cytotoxicity of GEN on EA.hy926 ​cells; (B) cytotoxicity of GEN on MH7A cells ​(means ​± ​SD, n ​= ​6, *P ​< ​0.05, **P ​< ​0.01). GEN = genistein; SD = standard deviation.

Figure 2

Protein and mRNA expression of cytokines in MH7A cells. MH7A cells were pretreated with GEN at different concentrations for 2 ​h ​and then stimulated with TNF-α (10 ​ng/mL) for 24 ​h; the protein content of IL-6 and VEGF in the MH7A cell culture supernatant was measured by ELISA, and the mRNA levels of IL-6 and VEGF in MH7A cells were analysed by qPCR. (A) Protein content of IL-6 in the MH7A cell culture supernatant; (B) protein content of VEGF in the MH7A cell culture supernatant; (C) mRNA expression of IL-6 in MH7A cells; (D) mRNA expression of VEGF in MH7A cells (means ​± ​SD, n ​= ​6, *P ​< ​0.05, **P ​< ​0.01). ELISA = enzyme-linked immunosorbent assay; GEN = genistein; IL-6 = interleukin-6; SD = standard deviation; TNF-α = tumour necrosis factor-α; VEGF = vascular endothelial growth factor.

Figure 3

Migration and tube formation of EA.hy926 ​cells. (A) Migration of EA.hy926 ​cells on membranes of the transwell; (B) wound healing of EA.hy926 ​cells; (C) tube formation of EA.hy926 ​cells on Matrigel; (D) the number of EA.hy926 ​cells on membranes of the transwell in each group; (E) % of wound healing in 24 ​hh; (F) branching points of the tube in a field; (G) master segment ​length of the tube in a filed ​(means ​± ​SD, n ​= ​8, *P ​< ​0.05, **P ​< ​0.01). GEN = genistein; IL-6 = interleukin-6; SD = standard deviation.

Figure 4

The number of EA.hy926 ​cells on membranes of the transwell when cocultured with MH7A. (A) Control; (B) EA.hy926 ​cells on membranes of the transwell when the medium in the lower chamber contains TNF-α; (C) EA.hy926 ​cells on membranes of the transwell when cocultured with MH7A cells; (D) EA.hy926 ​cells on membranes of the transwell when MH7A cells were treated with TNF-α; (E) EA.hy926 ​cells on membranes of the transwell when MH7A cells were treated with TNF-α and AG490 (25 ​ng/ml); (F) EA.hy926 ​cells on membranes of the transwell when MH7A cells were treated with TNF-α and GEN (25 ​μmol/L); (G) The number of EA.hy926 ​cells on membranes of the transwell in each group ​(means ​± ​SD, n ​= ​10, *P ​< ​0.05, **P ​< ​0.01). GEN = genistein; SD = standard deviation; TNF-α = tumour necrosis factor-α.

Figure 5

GEN inhibits IL-6–induced VEGF expression via JAK/STAT signalling pathway. (A) Protein level of VEGF in the MH7A cell culture supernatant; (B) mRNA expression of VEGF in MH7A cells; (C) mRNA expression of JAK2 in MH7A cells; (D) mRNA expression of STAT3 in MH7A cells; (E) Western blotting of STAT3 and p-STAT3; (F) immunofluorescence image of p-STAT3 in the nuclear of MH7A (40 ×); (G) immunofluorescence image of p-STAT3 in the nuclear of MH7A (63 ×) ​(*P ​< ​0.05, **P ​< ​0.01). GEN = genistein; IL-6 = interleukin-6; VEGF = vascular endothelial growth factor; DAPi=4',6-diamidino-2-phenylindole; GAPDH=Glyceraldehyde 3-phosphate dehydrogenase.