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, 107 (21), 9795-800

Vinpocetine Inhibits NF-kappaB-dependent Inflammation via an IKK-dependent but PDE-independent Mechanism

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Vinpocetine Inhibits NF-kappaB-dependent Inflammation via an IKK-dependent but PDE-independent Mechanism

Kye-Im Jeon et al. Proc Natl Acad Sci U S A.

Abstract

Inflammation is a hallmark of many diseases, such as atherosclerosis, chronic obstructive pulmonary disease, arthritis, infectious diseases, and cancer. Although steroids and cyclooxygenase inhibitors are effective antiinflammatory therapeutical agents, they may cause serious side effects. Therefore, developing unique antiinflammatory agents without significant adverse effects is urgently needed. Vinpocetine, a derivative of the alkaloid vincamine, has long been used for cerebrovascular disorders and cognitive impairment. Its role in inhibiting inflammation, however, remains unexplored. Here, we show that vinpocetine acts as an antiinflammatory agent in vitro and in vivo. In particular, vinpocetine inhibits TNF-alpha-induced NF-kappaB activation and the subsequent induction of proinflammatory mediators in multiple cell types, including vascular smooth muscle cells, endothelial cells, macrophages, and epithelial cells. We also show that vinpocetine inhibits monocyte adhesion and chemotaxis, which are critical processes during inflammation. Moreover, vinpocetine potently inhibits TNF-alpha- or LPS-induced up-regulation of proinflammatory mediators, including TNF-alpha, IL-1beta, and macrophage inflammatory protein-2, and decreases interstitial infiltration of polymorphonuclear leukocytes in a mouse model of TNF-alpha- or LPS-induced lung inflammation. Interestingly, vinpocetine inhibits NF-kappaB-dependent inflammatory responses by directly targeting IKK, independent of its well-known inhibitory effects on phosphodiesterase and Ca(2+) regulation. These studies thus identify vinpocetine as a unique antiinflammatory agent that may be repositioned for the treatment of many inflammatory diseases.

Conflict of interest statement

Drs. Li, Yan, and Berk have formed a start-up company, with the hope of licensing the intellectual property rights from the University of Rochester and commercializing this technology.

Figures

Fig. 1.
Fig. 1.
Vinpocetine (Vinp) inhibits TNF-α–induced NF-κB–dependent promoter activity in a variety of cell types. Rat aortic VSMCs (A) or HUVECs (B), lung epithelial A549 cells (C), and macrophage RAW264.7 cells (D) transfected with NF-κB–luciferase reporter plasmid were pretreated with Vinp for 60 min before treatment with or without TNF-α (10 ng/mL) for 6 h in the continued presence or absence of various doses of Vinp, as indicated in A, or 50 μM Vinp, as indicated in B, C, and D. Cells were then lysed for luciferase assay. Data represent the mean ± SD of at least three independent experiments, and each experiment was performed in triplicate. *P < 0.05 vs. control; #P < 0.05 vs. TNF-α alone.
Fig. 2.
Fig. 2.
Vinpocetine (Vinp) inhibits TNF-α–induced expression of proinflammatory mediators in a variety of cell types. Rat aortic VSMCs (A), HUVECs (B), lung epithelial A549 cells (C), or macrophage RAW264.7 cells (D) were pretreated with 50 μM Vinp for 60 min before treatment with or without TNF-α (10 ng/mL) for 6 h in the continued presence or absence of Vinp (50 μM). Expression of TNF-α, IL-1β, IL-8, MCP-1, VCAM-1, ICAM-1, and MIP-1 at mRNA levels was measured by real-time quantitative RT-PCR. Data represent the mean ± SD of at least three independent experiments, and each experiment was performed in triplicate. *P < 0.05 vs. control; #P < 0.05 vs. TNF-α alone.
Fig. 3.
Fig. 3.
Vinpocetine (Vinp) inhibits monocyte adhesion of HUVECs and chemotactic activity of VSMCs. (A) Microscopic images showing U937 monocytes adhering to HUVECs as assessed by in vitro adhesion assay. HUVECs were pretreated with vehicle DMSO (0.5% final concentration) or 50 μM Vinp for 60 min before treatment with TNF-α (10 ng/mL) or vehicle for 6 h in the continued presence or absence of Vinp. U937 monocyte adhesion to TNF-α- or vehicle–stimulated HUVECs was analyzed. Con, control. (B) Quantitative monocyte adhesion to HUVECs. (C) Monocyte chemotaxis to VSMCs measured by transwell migration. Rat aortic VSMCs were treated with or without TNF-α (10 ng/mL) for 9 h in the presence or absence of various doses of Vinp (5–50 μM). VSMC-conditional medium was collected and used for monocyte chemotaxis assays in Boyden chambers. Data represent the mean ± SD of at least three independent experiments, and each experiment was performed in triplicate. P < 0.05 vs. control; #P < 0.05 vs. TNF-α alone.
Fig. 4.
Fig. 4.
Vinpocetine (Vinp) inhibits lung inflammatory response in vivo. (A) Vinp administered i.p. (at 2.5, 5, and 10 mg/kg of body weight) significantly inhibited induction of TNF-α, IL-1β, and MIP-2 mRNA in the lungs of mice by intratracheal administration of LPS (2 μg per mouse). Data represent the mean ± SD of at least three independent experiments. *P < 0.05 vs. untreated group; #P < 0.05 vs. LPS alone. (B) Histological analysis (H&E stain) showing that Vinp (10 mg/kg of body weight) inhibited PMN infiltration in BAL fluids from the lungs of mice treated with LPS. Arrows point to PMN. Con, control. (C) Histological analysis showing that Vinp (10 mg/kg of body weight) inhibited leukocyte infiltration in peribronchial and interstitial areas of the lung (H&E stain, magnification ×200).
Fig. 5.
Fig. 5.
Vinpocetine (Vinp) inhibits TNF-α–induced NF-κB activation by targeting IKK. (A) Effects of Vinp on TNF-α–induced IκBα phosphorylation and degradation. Rat aortic VSMCs were treated with TNF-α (10 ng/mL) for different time periods (0–30 min) as indicated, in the presence or absence of 50 μM Vinp. Western blotting analysis was carried out to evaluate the levels of phosphorylated IκBα, total IκBα, and β-actin. (B) Vinp inhibits TNF-α–induced IKK kinase activity in rat aortic VSMCs. VSMCs were treated with TNF-α (10 ng/mL) for 10 min in the presence of Vinp (50 μM) or vehicle. IKK kinase activity was analyzed by an immune complex kinase assay. Effects of Vinp on NF-κB activation induced by expressing CA-MEKK1 (C), CA-IKKα (D), CA-IKKβ (E), or WT p65 (F) in VSMCs are shown. Data represent the mean ± SD of at least three independent experiments. *P < 0.05 vs. vector control group; #P < 0.05 vs. either CA-MEKK1, CA-IKKα, CA-IKKβ, or WT p65 alone. (G) Effects of Vinp on LPS-induced IκBα phosphorylation and degradation in mouse lungs in vivo. The lung tissues of mice treated with or without Vinp and LPS were subjected to Western blotting analyses for the levels of phospho-IκBα and total IκBα. The relative phospho-IκBα (H) and total IκBα (I) levels were quantified by densitometry and normalized to β-actin. Data represent the mean ± SD of at least three animals. *P < 0.05 vs. vehicle control group; #P < 0.05 vs. LPS-alone group.
Fig. 6.
Fig. 6.
Vinpocetine (Vinp) directly inhibits IKK activity. IKKβ kinase activity was analyzed by in vitro kinase assay using recombinant IKKβ. Kinase assays were conducted using GST-IκBα and [γ-32P]ATP in the presence of various doses of Vinp for 10 min. (A) Representative autoradiogram showing IKK kinase activity (Upper) and Western blot analysis showing IKKβ levels (Lower). (B) Relative IKK activity was indicated. Intensities of the GST-IκBα bands in the autoradiogram were measured by densitometric scanning. Results were normalized to the control (Vinp = 0), which is arbitrarily set to 100%. Curve fitting was performed with GraphPad Prism. The IC50 values are defined as the concentrations of Vinp required to produce 50% inhibition. Data represent the mean ± SD from three independent experiments. *P < 0.05 vs. Vinp = 0. (C) Effects of Ca2+ and PDE-1 inhibition on TNF-α–induced IKK kinase activity, IκBα phosphorylation, and IκBα degradation. Rat aortic VSMCs were treated with TNF-α (10 ng/mL) for 10 min in the presence of either 50 μM Vinp, 30 μM nifedipine (Ca2+ channel blocker), 15 μM IC86340 (PDE-1 inhibitor), 2 mM EGTA (extracellular Ca2+ chelator), or 30 μM BAPTA/AM (intracellular Ca2+ chelator). A representative autoradiogram shows IKK kinase activity analyzed by an IKK immune complex kinase assay as described (panel 1). Western blotting analysis was carried out to evaluate the levels of phosphorylated IκBα (panel 2), total IκBα (panel 3), and β-actin (panel 4). Data represent at least three independent experiments.
Fig. 7.
Fig. 7.
Schematic diagram depicting how vinpocetine inhibits NF-κB–dependent inflammatory response in vitro and in vivo. As indicated, vinpocetine inhibits NF-κB–dependent inflammatory response by directly targeting IKK, independent of its well-known action on PDE-1, Na+, and Ca2+ regulation.

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