Andrographolide Ameliorates Abdominal Aortic Aneurysm Progression by Inhibiting Inflammatory Cell Infiltration through Downregulation of Cytokine and Integrin Expression

Abstract

Abdominal aortic aneurysm (AAA), characterized by exuberant inflammation and tissue deterioration, is a common aortic disease associated with a high mortality rate. There is currently no established pharmacological therapy to treat this progressive disease. Andrographolide (Andro), a major bioactive component of the herbaceous plant Andrographis paniculata, has been found to exhibit potent anti-inflammatory properties by inhibiting nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activity in several disease models. In this study, we investigated the ability of Andro to suppress inflammation associated with aneurysms, and whether it may be used to block the progression of AAA. Whereas diseased aortae continued to expand in the solvent-treated group, daily administration of Andro to mice with small aneurysms significantly attenuated aneurysm growth, as measured by the diminished expansion of aortic diameter (165.68 ± 15.85% vs. 90.62 ± 22.91%, P < 0.05). Immunohistochemistry analyses revealed that Andro decreased infiltration of monocytes/macrophages and T cells. Mechanistically, Andro inhibited arterial NF-κB activation and reduced the production of proinflammatory cytokines [CCL2, CXCL10, tumor necrosis factor α, and interferon-γ] in the treated aortae. Furthermore, Andro suppressed α4 integrin expression and attenuated the ability of monocytes/macrophages to adhere to activated endothelial cells. These results indicate that Andro suppresses progression of AAA, likely through inhibition of inflammatory cell infiltration via downregulation of NF-κB-mediated cytokine production and α4 integrin expression. Thus, Andro may offer a pharmacological therapy to slow disease progression in patients with small aneurysms.

Fig. 1.

Andro inhibits progression of small AAAs. (A) Schematic diagram of Andro treatment in mice. (B) Representative photos and aortic expansion of Andro- and solvent-treated mice, taken 14 days after surgery. E, elastase; IE, inactivated elastase. An AAA is defined as a percentage increase in aortic diameter that is equal to or greater than 100% (red dashed line). All values represent the mean ± S.E.M. (n = 3–9; *P < 0.05, one-way analysis of variance). (C) Representative images of arterial sections stained with H&E, Verhoeff–Van Gieson (VVG), and smooth muscle myosin heavy chain 11 (SM MHC 11; green) overlaid with 4′,6-diamidino-2-phenylindole (blue). Arteries were harvested 14 days after surgery. Scale bar = 200 μm.

Fig. 2.

Andro attenuates NF-κB activation in aortic tissues. (A and B) Representative images of arterial sections coimmunostained for activated p65 (green) and smooth muscle α-actin (smooth muscle α-actin; red) (A) or activated p65 (red) and MOMA-2 )green) (B) overlaid with 4′,6-diamidino-2-phenylindole (blue). Areas highlighted by white dashed boxes are shown at higher magnification on the right. Arteries were harvested 7 days after elastase perfusion. Scale bar = 50 μm. (C) Representative images of arterial sections immunohistochemically stained for activated p65. E, elastase; IE, inactivated elastase. Areas highlighted by black dashed boxes are shown at higher magnification at the bottom. Arteries were harvested 14 days after elastase perfusion. Scale bar = 100 μm.

Fig. 3.

Andro inhibits NF-κB activation and cytokine expression in TNFα-treated SMCs. (A and B) SMCs were pretreated with solvent (DMSO) or Andro (15 μM) for 1 hour before incubation with TNFα (10 ng/ml) for the indicated times. Whole-cell lysates were subjected to immunoblotting analysis with indicated antibodies. (C–I) SMCs were pretreated with solvent (DMSO) or Andro (15 μM) for 1 hour before incubation with TNFα (10 ng/ml) for 6 hours. mRNA expression of inflammatory chemokines and cytokines was analyzed by quantitative real-time PCR. All values represent the mean ± S.E.M. (n = 3–6; *P < 0.05, one-way analysis of variance (A–I). P-p65, phosphorylated p65.

Fig. 4.

Andro inhibits NF-κB activation and cytokine expression in LPS-treated monocytes/macrophages. (A and B) RAW264.7 cells were pretreated with solvent (DMSO) or Andro (15 μM) for 1 hour before incubation with LPS (100 ng/ml) for the indicated times. Whole-cell lysates were subjected to immunoblotting analysis with indicated antibodies. (C–I) RAW264.7 cells were pretreated with solvent (DMSO) or Andro (15 μM) for 1 hour before incubation with LPS (100 ng/ml) for 6 hours. mRNA expression of inflammatory chemokines and cytokines was analyzed by quantitative real-time PCR. All values represent the mean ± S.E.M. [n = 4–5; *P < 0.05, one-way analysis of variance (A–F and H) and two-tailed Student’s t test (G and I)]. P-p65, phosphorylated p65.

Fig. 5.

Andro reduces the adherence of monocytes/macrophages to ECs. (A) Fluorescently labeled RAW264.7 cells, pretreated with Andro (15 μM) or DMSO, were incubated with TNFα-treated (10 ng/ml) ECs. After washing vigorously, adhered RAW264.7 cells were imaged (upper panel) and quantified (lower panel). Data are expressed as the mean number of adhered cells/field ± S.E.M. (n = 4; *P < 0.05, one-way analysis of variance). (B–E) RAW264.7 cells were treated with or without Andro (15 μM) or solvent for the indicated time. mRNA expression of α4 (B), β1 (C), αL (D), and β2 (E) integrins was analyzed by quantitative real-time PCR. All values represent the mean ± S.E.M. (n = 4–6; *P < 0.05, one-way analysis of variance). N.S., not significant.

Fig. 6.

Andro inhibits inflammatory cytokine expression in elastase-perfused aortic tissues. mRNA expression of Ccl2 (A), Cxcl10 (B), Tnf (C), Ifng (D), Ccl5 (E), Ccl7 (F), and Cxcl16 (G) in aorta of solvent- or Andro-treated animals 14 days after surgery was analyzed by quantitative real-time PCR. All values represent the mean ± S.E.M. (n = 3–8; *P < 0.05, one-way analysis of variance). E, elastase; IE, inactivated elastase.

Fig. 7.

Andro decreases infiltration of inflammatory cells in elastase-perfused aortic tissues. (A) Representative images of arterial sections immunohistochemically stained for MOMA-2 (a monocyte and macrophage marker), CD3 (a T cell marker), and Ly6G (a neutrophil marker). Higher-magnification views of boxed areas are shown at the lower-left corner. E, elastase. Scale bar = 200μm. (B) Quantification of inflammatory cell infiltration in aortic tissue is expressed as positive cells/nuclei. All values represent the mean ± S.E.M. (n = 6–10; *P < 0.05, two-tailed Student’s t test).