α-Mangostin Alleviated HIF-1α-Mediated Angiogenesis in Rats With Adjuvant-Induced Arthritis by Suppressing Aerobic Glycolysis

Abstract

A previously validated anti-rheumatic compound α-mangostin (MAN) shows significant metabolism regulatory effects. The current study aimed to clarify whether this property contributed to its inhibition on synovial angiogenesis. Male wistar rats with adjuvant-induced arthritis (AIA) were orally treated by MAN for 32 days. Afterwards, biochemical parameters and cytokines in plasma were determined by corresponding kits, and glycometabolism-related metabolites were further accurately quantified by LC-MS method. Anti-angiogenic effects of MAN were preliminarily assessed by joints based-immunohistochemical examination and matrigel plug assay. Obtained results were then validated by experiments in vitro. AIA-caused increase in circulating transforming growth factor beta, interleukin 6, hypoxia inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in blood and local HIF-1α/VEGF expression in joints was abrogated by MAN treatment, and pannus formation within matrigel plugs implanted in AIA rats was inhibited too. Scratch and transwell assays revealed the inhibitory effects of MAN on human umbilical vein endothelial cells (HUVECs) migration. Furthermore, MAN inhibited tubule formation capability of HUVECs and growth potential of rat arterial ring-derived endothelial cells in vitro. Meanwhile, MAN eased oxidative stress, and altered glucose metabolism in vivo. Glycolysis-related metabolites including glucose 6-phosphate, fructose 6-phosphate, 3-phosphoglyceric acid and phosphoenolpyruvic acid in AIA rats were decreased by MAN, while the impaired pyruvate-synthesizing capability of lactate dehydrogenase (LDH) was recovered. Consistently, MAN restored lipopolysaccharide-elicited changes on levels of glucose and LDH in HUVECs culture system, and exerted similar effects with LDH inhibitor stiripentol on glycometabolism and VEGF production as well as tubule formation capability of HUVECs. These evidences show that MAN treatment inhibited aerobic glycolysis in AIA rats, which consequently eased inflammation-related hypoxia, and hampered pathological neovascularization.

Keywords: glycometabolism; hypoxia inducible factor-1α (HIF-1α); metabolism reprogramming; oxidative stress MAN suppressed glycolysis-related angiogenesis; rheumatoid arthritis (RA).

FIGURE 1

MAN alleviated severity of AIA in rats after continuous oral treatment. (A), periodic changes of arthritis scores and body weight of AIA rats (n = 6); (B), CBC analysis of anticoagulation blood obtained upon the sacrifice of rats (n = 6); (C), morphological observation of paws by the end of experimental period. Statistical significances in image A: ^* p < 0.05 and ^** p < 0.01 compared with AIA model controls.

FIGURE 2

MAN inhibited pathological angiogenesis in AIA rats. (A), levels of angiogenic cytokines in plasma (n = 6); (B), levels of VEGF in different tissues of rats (n = 3); (C), local expression of HIF-1α and VEGF in cartilage of joints; (D), morphological observation of matrigel plugs implanted in different rats; (E), H&E staining-based histological examination of above matrigel plugs.

FIGURE 3

MAN inhibited angiogenesis potentials of endothelial cells in vitro. (A), the effects of MAN on HUVECs viability, assessed by MTT assay (n = 6); (B), MAN inhibited VEGF production in LPS-primed HUVECs in a concentration-dependent manner (n = 3); (C), MAN inhibited the migration of RA serum-cultured HUVECs in scratch assay; (D), the quantification result of assay C; (E), MAN decreased the counts of HUVECs infiltrated through transwell in the existence of VEGF; (F), the infiltrated HUVECs was dyed with crystal violet in the replicate of assay E; (G), MAN impaired the capacity of VEGF-primed HUVECs to form tubes in vitro; (H), suppressive effects of MAN on migration/growth of rat arterial ring-derived endothelial cells under both normal and AIA conditions. Statistical significances in image B: ^** p < 0.01 compared with LPS-primed cells.

FIGURE 4

MAN restored oxidative stress and glycometabolism alteration in AIA rats. (A), levels of NOX, MDA, GSH and SOD in plasma (n = 6), and all parameters in this and following assays were detected using rat blood samples collected at day 27; (B), levels of glucose, pyruvate, lactate and citric acid in plasma (n = 6); (C), catalytic activity of LDH in the rat plasma (assessed by the production of pyruvate ex vivo, n = 6).

FIGURE 5

MAN rebalanced glycolysis and aerobic oxidation in AIA rats. (A), levels of glycolysis-related differentiating metabolites among groups (n = 3), and all the metabolites shown in this and following images were detected by LC-MS in plasma obtained upon the sacrifice of rats; (B), levels of aerobic oxidation-related differentiating metabolites among groups (n = 3); (C), changes of metabolic flow occurred in AIA and MAN-treated AIA rats (dotted line indicated undetected compounds, colors of the frame indicated MAN-brought changes, colors of the text indicated AIA-related changes, blue and red colors indicated down-regulation and up-regulation respectively).

FIGURE 6

Supporting evidences for the regulation of MAN on glucose metabolism in AIA rats. (A), levels of glycometabolism metabolites indirectly related to glycolysis and aerobic oxidation of glucose detected in rat plasma (n = 3); (B), levels of high-energy phosphate compound in rat plasma (n = 3).

FIGURE 7

MAN affected glucose oxidation of HUVECs in vitro. (A), levels of glucose, LDH and SOD in culture medium from the cells receiving MAN treatments at various concentrations (n = 3); (B), levels of glucose, LDH and SOD in culture medium from the cells treated by LPS or in the combination of MAN (n = 3); (C), levels of angiogenic cytokines released by cells from assay B. Statistical significances in image A: ^** p < 0.01 compared with untreated cells.

FIGURE 8

Inhibition of MAN on inflammation-caused aerobic glycolysis contributed to its anti-angiogenesis properties. (A), levels of citric acid, lactate, pyruvate and HIF-1α in culture medium from LPS-primed HUVECs receiving PKM2 inhibitor CK or/plus MAN stimulus (n = 3); (B), levels of lactate, pyruvate and VEGF in culture medium as well as intracellular ATP of LPS-primed HUVECs receiving LDH inhibitor STI or/plus MAN stimulus (n = 3); (C), dynamic changes of intracellular ATP and extracellular glucose in HUVECs culture system in the presence of LPS or LPS + MAN (n = 3); D, STI and MAN exerted similar and synergistic effects on tube-forming capability of LPS-primed HUVECs in vitro. Statistical significance in image A and B: ^* p < 0.05 and ^** p < 0.01 compared with LPS-primed cells; ^# p < 0.05 and ^## p < 0.01 compared with cells receiving LPS + MAN treatment.