Coix seed oil alleviates synovial angiogenesis through suppressing HIF-1α/VEGF-A signaling pathways via SIRT1 in collagen-induced arthritis rats

Abstract

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by symmetric arthritis. Coix Seed Oil (CSO) has been shown to reduce inflammation in collagen induced arthritis (CIA) rats. However, the effect of CSO on synovial angiogenesis in RA is unknown. In this study, we aimed to explore whether CSO could inhibit RA synovial angiogenesis and elucidate the underlying mechanisms.

Methods: CIA rat models were established and subjected to different doses of CSO treatments for four weeks in vivo. Arthritis index, paw swelling, and weight were recorded to assess clinical symptoms. Hematoxylin and Eosin staining, Safarnin O fast green staining, Micro-CT, Immunohistochemical, and Immunofluorescence (IF) staining were performed to examined changes in synovial and joint tissues. The serum HIF-1α and VEGF-A levels were evaluated through enzyme-linked immunosorbent assay. Fibroblast-like synoviocytes (FLS) of rats was stimulated with tumor necrosis factor-α (TNF-α) for developing inflammatory model in vitro. Optimal concentrations of CSO and TNF-α for stimulation were measured through Cell Counting Kit-8 test. Wound healing and Transwell migration experiments were employed to determine FLS migratory ability. IF staining was performed to assess HIF-1α nuclear translocation in FLS. Protein levels of SIRT1, HIF-1α, VEGF-A, and CD31 were assessed through Western blot. The isolated aortic rings were induced with recombinant rat VEGF-A 165 (VEGF-A₁₆₅) to observe the CSO inhibitory impact on angiogenesis ex vivo.

Results: CSO attenuated the progression of arthritis in CIA rats, mitigated histopathological deterioration in synovial and joint tissues, significantly inhibited immature vessels labeled with CD31⁺/αSMA^-, and reduced the micro-vessels in VEGF-A₁₆₅ induced aortic rings. Moreover, it upregulated SIRT1 protein levels in CIA rats and TNF-α induced FLS, but decreased HIF-1α and VEGF-A protein levels. Furthermore, CSO inhibited the migration ability and HIF-1α nuclear translocation of TNF-α induced FLS. Finally, suppressing SIRT1 levels in TNF-α induced FLS enhanced their migration ability, HIF-1α nuclear translocation, and the protein levels of HIF-1α, VEGF-A, and CD31, whereas the inhibitory effect of CSO on TNF-α induced FLS was severely constrained.

Conclusions: This study indicates that CSO can alleviate synovial angiogenesis through suppressing HIF-1α/VEGF-A signaling pathways via SIRT1 in CIA rats.

Keywords: Angiogenesis; Coix seed oil; HIF-1α; Rheumatoid arthritis; SIRT1.

Fig. 1

CSO alleviated the clinical symptoms of CIA rats. A An illustration of the Experimental procedure. B Representative picture for ankle joints. C Arthritis index of rats. D Paw swelling of rats (mm). E Weight of rats (g). Datasets are shown as the mean ± SD. *P < 0.05, **P < 0.01 vs. CON group; ^#P < 0.05, ^##P < 0.01 vs. CIA group, n = 6

Fig. 2

CSO improved histopathological deteriorations in the joint and synovium of CIA rats. A H&E staining and Safranin O fast green staining of ankle joints. Blue arrows indicate inflammatory infiltration. Yellow arrows indicate cartilage erosion. B H&E staining of synovial tissues. Black arrows indicate vessel-like structures. C H&E score of joint. D Safranin O score of joint. E The number of vessel-like structures in synovial tissues. Datasets are shown as the mean ± SD. *P < 0.05, **P < 0.01 vs. CON group; ^#P < 0.05, ^##P < 0.01 vs. CIA group, n = 6

Fig. 3

CSO reduced joint destruction of CIA rats. A Representative micro-CT pictures of the ankle joints. White arrows indicate joint destruction. B Representative micro-CT scans of the calcaneus in three planes (Transverse, Coronal, and Sagittal). The red area designates the ROI in the calcaneus. C Representative 3D reconstruction pictures of the ROI. D BV/TV (%). E BMD (g/cm³). F Tb.N (1/mm). G Tb.Th (mm). H Tb.Sp (mm). I Tb.Pf (1/mm). Datasets are shown as the mean ± SD. *P < 0.05, **P < 0.01 vs. CON group; ^#P < 0.05, ^##P < 0.01 vs. CIA group, n = 6

Fig. 4

CSO reduced the serum levels of HIF-1α and VEGF-A in CIA rats. A Serum level of HIF-1α in rats. B Serum level of VEGF-A in rats. Datasets are shown as the mean ± SD. *P < 0.05, **P < 0.01 vs. CON group; ^#P < 0.05, ^##P < 0.01 vs. CIA group, n = 6

Fig. 5

CSO inhibited angiogenesis in synovial tissues of CIA rats. A IHC staining of CD31 and IF staining of CD31/αSMA. Black arrows indicate positive staining of CD31, White arrows indicate immature vessels labeled with CD31⁺/αSMA⁻. B Integral Optical Density (IOD) of CD31. C The number of CD31⁺/αSMA⁻ vessels. D The number of CD31⁺/αSMA⁺ vessels. E The number of Total vessels. Datasets are shown as the mean ± SD. *P < 0.05, **P < 0.01 vs. CON group; ^#P < 0.05, ^##P < 0.01 vs. CIA group, n = 3

Fig. 6

CSO regulated the expression levels of SIRT1, HIF-1α, VEGF-A, and CD31 in CIA rats. A Representative picture of Western blot. B SIRT1/GAPDH. C HIF-1α/GAPDH. D VEGF-A/GAPDH. E CD31/GAPDH. Datasets are shown as the mean ± SD. *P < 0.05, **P < 0.01 vs. CON group; ^#P < 0.05, ^##P < 0.01 vs. CIA group, n = 3

Fig. 7

Identification of primary FLS of rats. A Representative picture of FLS at different days and generations. B Representative picture of Vimentin-positive fluorescent staining. C Vimentin positive rate. Datasets are shown as the mean ± SD. *P < 0.05, **P < 0.01 vs. DAPI, n = 3

Fig. 8

Effects of different concentrations of CSO and TNF-α on cell proliferation and SIRT1 expression. A FLS Proliferation rate (%). B Representative picture of SIRT1 levels. C SIRT1/GAPDH. D Cell survival rate (%). Datasets are shown as the mean ± SD. *P < 0.05, **P < 0.01 vs. 0 ng/ml TNF-α group, n = 3

Fig. 9

CSO inhibited the migration ability of TNF-α induced FLS proliferation. A Representative image of wound-healing assay. B Representative image of Transwell migration assay. C FLS-covered area (%). D Relative number of migration cells. Datasets are shown as the mean ± SD. *P < 0.05, **P < 0.01 vs. NC group; ^#P < 0.05, ^##P < 0.01 vs. TNF-α group; n = 3. ^&P < 0.05, ^&&P < 0.01 vs. TNF-α + CSO group, n = 3

Fig. 10

CSO modulated SIRT1, HIF-1α, VEGF-A, and CD31 expression in TNF-α induced FLS. A Representative picture of Western blot. B SIRT1/GAPDH. C HIF-1α/GAPDH. D VEGF-A/GAPDH. E CD31/GAPDH. Datasets are shown as the mean ± SD. *P < 0.05, **P < 0.01 vs. NC group; ^#P < 0.05, ^##P < 0.01 vs. TNF-α group; ^&P < 0.05, ^&&P < 0.01 vs. TNF-α + CSO group, n = 3

Fig. 11

CSO inhibited TNF-α induced nuclear translocation of HIF-1α in TNF-α induced FLS. A Representative picture of HIF-1α translocation. B Nuclear HIF-1α rate. Datasets are shown as the mean ± SD. *P < 0.05, **P < 0.01 vs. NC group; ^#P < 0.05, ^##P < 0.01 vs. TNF-α group; ^&P < 0.05, ^&&P < 0.01 vs. TNF-α + CSO group, n = 3

Fig. 12

CSO inhibited the isolated aortic rings’ angiogenesis. A Representative picture of aortic rings angiogenesis. B Numbers of micro-vessels. C Micro-vessel length (pixels). Datasets are shown as the mean ± SD. *P < 0.05, **P < 0.01 vs. NC group; ^#P < 0.05, ^##P < 0.01 vs. VEGF-A₁₆₅ group, n = 3