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, 8 (10), e77513
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Anti-angiogenic Effect of Triptolide in Rheumatoid Arthritis by Targeting Angiogenic Cascade

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Anti-angiogenic Effect of Triptolide in Rheumatoid Arthritis by Targeting Angiogenic Cascade

Xiangying Kong et al. PLoS One.

Abstract

Rheumatoid arthritis (RA) is characterized by a pre-vascular seriously inflammatory phase, followed by a vascular phase with high increase in vessel growth. Since angiogenesis has been considered as an essential event in perpetuating inflammatory and immune responses, as well as supporting pannus growth and development of RA, inhibition of angiogenesis has been proposed as a novel therapeutic strategy for RA. Triptolide, a diterpenoid triepoxide from Tripterygium wilfordii Hook F, has been extensively used in treatment of RA patients. It also acts as a small molecule inhibitor of tumor angiogenesis in several cancer types. However, it is unclear whether triptolide possesses an anti-angiogenic effect in RA. To address this problem, we constructed collagen-induced arthritis (CIA) model using DA rats by the injection of bovine type II collagen. Then, CIA rats were treated with triptolide (11-45 µg/kg/day) starting on the day 1 after first immunization. The arthritis scores (P<0.05) and the arthritis incidence (P<0.05) of inflamed joints were both significantly decreased in triptolide-treated CIA rats compared to vehicle CIA rats. More interestingly, doses of 11~45 µg/kg triptolide could markedly reduce the capillaries, small, medium and large vessel density in synovial membrane tissues of inflamed joints (all P<0.05). Moreover, triptolide inhibited matrigel-induced cell adhesion of HFLS-RA and HUVEC. It also disrupted tube formation of HUVEC on matrigel and suppressed the VEGF-induced chemotactic migration of HFLS-RA and HUVEC, respectively. Furthermore, triptolide significantly reduced the expression of angiogenic activators including TNF-α, IL-17, VEGF, VEGFR, Ang-1, Ang-2 and Tie2, as well as suppressed the IL1-β-induced phosphorylated of ERK, p38 and JNK at protein levels. In conclusion, our data suggest for the first time that triptolide may possess anti-angiogenic effect in RA both in vivo and in vitro assay systems by downregulating the angiogenic activators and inhibiting the activation of mitogen-activated protein kinase downstream signal pathway.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Triptolide decreases severity of arthritis in collagen-induced arthritis (CIA) rats.
Rats were orally administered triptolide (Trip, 11, 22, and 45 µg/kg, respectively), or vehicle for 28 days from the day of first immunization. At the end of the experiment, the arthritis score and arthritis incidence were evaluated, and micro-CT scan was operated. (A) Doses of 11∼45 µg/kg triptolide significantly decreased the mean arthritis score in a dose-dependent manner compared with vehicle-treated rats; (B) Doses of 11∼45 µg/kg triptolide significantly decreased the arthritis incidence in a dose-dependent manner compared with vehicle-treated rats; (C) macroscopic evidence of arthritis such as erythema or swelling was markedly observed in untreated CIA rats, while doses of 45 µg/kg triptolide significantly attenuated arthritis severity in CIA rats; (D) the three-dimensionally reconstructed images of knee joints showed that dose of 45 µg/kg triptolide markedly reduced the extent of joint destruction compared with vehicle-treated rats. Data are represented as the mean ± SEM (n = 16). *P<0.05, **P<0.01, and ***P<0.001, comparison with the vehicle group.
Figure 2
Figure 2. Vessel density in synovial membrane tissues of inflamed joints in collagen-induced arthritis (CIA) rats.
(A), Platelet endothelial cell adhesion molecule 1 (CD31) immunohistochemical staining photomicrographs of synovial membrane tissues from knee joints of normal, CIA model and CIA rats treated with 45 µg/kg triptolide (Trip), respectively. (B), Hematoxylin and eosin staining photomicrographs of synovial membrane tissues from knee joints of normal, vehicle and 45 µg/kg triptolide-treated CIA rats, respectively. (C), Doses of 11∼45 µg/kg triptolide significantly decreased the capillaries, small, medium and large vessel density (CD31 immunohistochemistry) in synovial membrane tissues of inflamed joints in CIA rats. (D), Doses of 11∼45 µg/kg triptolide significantly decreased the vessel density (assessed on hematoxylin and eosin-stained paraffin sections) in synovial membrane tissues of knee joints in CIA rats. Data are represented as means ± SE (n = 16). ### P<0.001, comparison with the control group. *P<0.05, **P<0.01, and ***P<0.001, comparison with the vehicle group.
Figure 3
Figure 3. Inhibitory effect of triptolide on VEGF induced chemotatic migration of HFLS–RA (A) and HUVEC (B) using the Transwell culture insert.
Cells were placed in transwells and allowed to migrate for 4(VEGF) with or without triptolide [Control, Vehicle and triptolide-treated (Trip) groups]. Migrated HFLS–RA and HUVECs were respectively fixed, stained and counted in eight random fields (magnification x40). All experiments were done in triplicate. Mean ± SE was calculated from independent experiments. # P<0.05 and ## P<0.01, comparison with the control group. *P<0.05 and **P<0.01, comparison with the vehicle group.
Figure 4
Figure 4. Inhibitory effects of triptolide on cell adhesiveness of HFLS–RA (A) and HUVEC (B) using the adhesive assay, and on tube formation of HUVEC (C).
The cells were divided into six groups: negative control (cells were seeded in BSA coated 96-well plates), blank control (cells were seeded in FN coated 96-well plates), vehicle group (cells were seeded in FN coated 96-well plates with the presence of IL-1β), and three Trip groups (cells were seeded in FN coated 96-well plates with the presence of IL-1β and treated with 1, 10 and 50 ng/mL of triptolide, respectively). Compared with the vehicle group, triptolide at a concentration ranging from 1 to 50 ng/mL significantly suppressed the cell adhesiveness of HFLS–RA (P<0.01, A) and HUVEC (P<0.0, B) in a dose-dependent manner. (C) HUVEC were plated on the matrigel coated 96-well culture plates (Control), plated on the matrigel coated 96-well culture plates with the presence of VEGF (Vehicle), seeded in matrigel coated 96-well plates with the presence of VEGF and treated with 1, 10 and 50 ng/mL of triptolide, respectively(Trip 1, Trip 10 and Trip 50). Quantitation of the anti-angiogenic activities of triptolide on tube formation by counting the number of branch points, magnification x40. All experiments were done in triplicate. Mean ± SE was calculated from independent experiments. # P<0.05 and ## P<0.01, comparison with the control group. *P<0.05, **P<0.01, and ***P<0.001, comparison with the vehicle group.
Figure 5
Figure 5. Triptolide reduces the expression levels of tumor necrosis factor (TNF)-α (A), Interleukin (IL)-1β (B) and VEGF (C) in sera of CIA rats.
Rats were orally administered triptolide (Trip, 11, 22, and 45 µg/kg, respectively), or vehicle for 28 days from the day of first immunization. At the end of the experiment, sera were obtained from the rats and tested for TNF-α), IL-1β and VEGF by ELISA. All experiments were done in triplicate. ## P<0.01 and ### P<0.001, comparison with the control group. *P<0.05, **P<0.01, and ***P<0.001, comparison with the vehicle group.
Figure 6
Figure 6. Triptolide reduces the expression levels of tumor necrosis factor (TNF)-α (A), Interleukin (IL)-1β (B), VEGF (C), Angiopoietin (Ang)-1 (D), Ang-2 (E) in supernatants of IL-1β-stimulated HFLS–RA cells, VEGF receptor (VEGFR, F) and Tie2 (G) in HUVEC.
The cells were divided into five groups:Control–normal cultured cells; Vehicle–IL-1β induced cells; Trip groups–cells treated with various concentrations of triptolide (1, 10 and 50 ng/mL)] were detected by ELISA assay. All experiments were done in triplicate. Mean ± SE was calculated from independent experiments. # P<0.05, ## P<0.01, ### P<0.001, comparison with the control group. *P<0.05, **P<0.01, and ***P<0.001, comparison with the vehicle group.
Figure 7
Figure 7. Triptolide reduced IL-1β-activated p-ERK, p-p38 and p-JNK in HFLS–RA cells detected by western blot analysis.
Confluent HFLS-RA at passage 4–8 were incubated with various concentrations of triptolide (1, 10 and 50 ng/mL, respectively) for 24 h, followed by the addition of IL1-β (10 ng/mL). After 15 min incubation, cells were analyzed for detection of p-ERK, p-p38 and p-JNK by western blot analysis. All experiments were done in triplicate. Mean ± SE was calculated from independent experiments. ### P<0.001, comparison with the control group. *P<0.05, **P<0.01, and ***P<0.001, comparison with the vehicle group.

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Grant support

This study was supported by grants from the national drug research and development projects (2009ZX09502-019, 2009ZX09301-005-007), the project of China Academy of Chinese Medical Sciences (ZZ20090105), the Beijing Natural Science Foundation (7062051), and the National Natural Science Foundation of China (81274167, 30672647). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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