Vinpocetine inhibits NF-κB–dependent inflammation via an IKK-dependent but PDE-independent mechanism

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-α–induced NF-κB 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-α- or LPS-induced up-regulation of proinflammatory mediators, including TNF-α, IL-1β, and macrophage inflammatory protein-2, and decreases interstitial infiltration of polymorphonuclear leukocytes in a mouse model of TNF-α- or LPS-induced lung inflammation. Interestingly, vinpocetine inhibits NF-κB–dependent inflammatory responses by directly targeting IKK, independent of its well-known inhibitory effects on phosphodiesterase and Ca²⁺ regulation. These studies thus identify vinpocetine as a unique antiinflammatory agent that may be repositioned for the treatment of many inflammatory diseases.

Inflammation is a hallmark of many human diseases, including atherosclerosis (1), pulmonary inflammatory diseases (e.g., chronic obstructive pulmonary disease, asthma) (2), arthritis (3), infectious diseases, and cancer (4, 5). Steroids and cyclooxygenase inhibitors have long been used as the main therapeutical antiinflammatory agents, but they are frequently associated with significant detrimental effects in patients (4, 5). Thus, there is an urgent need for the development of unique antiinflammatory agents.

NF-κB is a key transcriptional factor involved in regulating expression of proinflammatory mediators, including cytokines, chemokines, and adhesion molecules, thereby playing a critical role in mediating inflammatory responses (6). NF-κB is a dimeric transcription factor consisting of homo- or heterodimers of Rel-related proteins (7). In the inactive state, NF-κB resides in the cytoplasm and forms a multiprotein complex with an inhibitory subunit, inhibitor of NF-κB (IκB). On activation by external stimuli, the inflammatory signal converges on and activates a set of IκB kinases known as the IκB kinase (IKK) complex. The activated IKK complex phosphorylates two conserved N-terminal serine residues of IκBα, leading to its ubiquitination and degradation by the proteasome. The liberated NF-κB then enters the nucleus, interacts with κB elements in the promoter region of a variety of inflammatory response genes, and activates their transcription (6). Thus, phosphorylation of IκBα appears to be the central point where diverse stimuli converge to regulate NF-κB. Two major IKKs, IKK-α (IKK-1) and IKK-β (IKK-2), have been identified and shown to be key components of the multiprotein IKK complex (8, 9). Both IKK-α and IKK-β are Ser/Thr kinases, and each of them directly phosphorylates IκB proteins (10). Several other molecules in the IKK complex have also been identified, such as signal-regulated ERK kinase kinase 1 (MEKK-1), NF-κB–inducing kinase, NF-κB essential modulator/IKKAP1/IKK-γ, and IKK complex-associated protein (11–14). These molecules have been shown to be essential for transmitting upstream signals to IKK-α and IKK-β by acting as a kinase, regulatory protein, or scaffold protein (11–14).

Vinpocetine is produced by slightly altering the vincamine molecule, an alkaloid extracted from the periwinkle plant, Vinca minor. Vinpocetine was originally discovered and marketed in 1978 under the trade name Cavinton (Hungary). Since then, vinpocetine has been widely used in many countries for the prevention of cerebrovascular disorders and cognitive impairment, including stroke, senile dementia, and memory disturbances (15). For instance, different types of vinpocetine-containing memory enhancers (Intelectol in Europe and Memolead in Japan) are currently used as dietary supplements worldwide. Vinpocetine is a cerebral vasodilator that improves brain blood flow (16). Vinpocetine has also been shown to act as a cerebral metabolic enhancer by enhancing oxygen and glucose uptake from blood and increasing neuronal ATP production (17). Vinpocetine appears to affect several cellular targets, such as Ca²⁺/calmodulin-stimulated cyclical nucleotide phosphodiesterase-1 (PDE-1) and voltage-dependent Na⁺ channels and Ca²⁺ channels (18). To date, there have been no reports of its significant side effects, toxicity, or contraindications at therapeutical doses (19).

PDEs, via regulation of cAMP and/or cGMP, have been known to regulate inflammatory responses. Because vinpocetine has been shown to inhibit PDE activity, we hypothesized that vinpocetine, a drug already available in the clinic, may act as an antiinflammatory agent. Here, we show that vinpocetine indeed inhibits NF-κB–dependent inflammatory responses in vitro and in vivo. Unexpectedly, the inhibitory effect of vinpocetine on NF-κB signaling is exerted via direct inhibition of IKK but not PDE. These studies thus identify vinpocetine as a unique antiinflammatory agent that may be repositioned as a therapeutical agent for the treatment of various human inflammatory diseases.

Results

Vinpocetine Inhibits NF-κB Activation in a Variety of Cell Types.

Because NF-κB plays a critical role in regulating inflammatory response, we investigated if vinpocetine acts as an antiinflammatory agent by inhibiting NF-κB. We first evaluated the effects of vinpocetine on NF-κB–dependent transcriptional activity by using a NF-κB–luciferase reporter plasmid transiently transfected in several cell types. As shown in Fig. 1A, vinpocetine potently inhibited TNF-α–induced NF-κB–dependent transcriptional activity in vascular smooth muscle cells (VSMCs) in a dose-dependent manner. Similar results were also observed in human umbilical vein endothelial cells (HUVECs) (Fig. 1B), human lung epithelial A549 cells (Fig. 1C), and the macrophage cell line RAW264.7 (Fig. 1D). Dose–response analysis further revealed that vinpocetine inhibits TNF-α–stimulated NF-κB–dependent transcriptional activity with an approximate IC₅₀ value of 25 μM (Fig. S1A). Cell viability analysis showed that vinpocetine did not have a significant effect on cell viability (Fig. S1B).

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.

Vinpocetine Inhibits TNF-α–Induced Proinflammatory Mediators in Several Cell Types.

We next determined if vinpocetine inhibits TNF-α–induced up-regulation of NF-κB–dependent proinflammatory mediators at the mRNA level by real-time quantitative RT-PCR. As shown in Fig. 2A and Fig. S2A, vinpocetine potently inhibited TNF-α–induced up-regulation of TNF-α, IL-1β, cytokine-induced neutrophil chemoattractant (CINC-1; rat form of IL-8), monocyte chemotactic protein-1 (MCP-1), and vascular cell adhesion molecule-1 (VCAM-1) transcripts in VSMCs. Similarly, vinpocetine inhibited TNF-α–induced up-regulation of TNF-α, IL-1β, IL-8, MCP-1, VCAM-1, and intercellular adhesion molecule-1 (ICAM-1) transcripts in HUVECs (Fig. 2B and Fig. S2B); up-regulation of TNF-α, IL-1β, and IL-8 transcripts in A549 cells (Fig. 2C); and up-regulation of TNF-α, IL-1β, and macrophage inflammatory protein-2 (MIP-2) transcripts in RAW264.7 (Fig. 2D).

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.

Vinpocetine Inhibits Monocyte Adhesion of HUVECs and Chemotactic Activity of VSMCs.

To evaluate the physiological consequences of the inhibitory effect of vinpocetine on induction of proinflammatory mediators further, we assessed two critical processes during inflammation, monocyte adhesion to HUVECs and chemotaxis to VSMCs, which are known to be dependent on adhesion molecules (e.g., ICAM-1, VCAM-1) and chemokines (e.g., MCP-1). As shown in Fig. 3 A and B, TNF-α–induced monocyte adhesion to HUVECs was significantly inhibited by vinpocetine. Monocyte chemotaxis to the conditional medium collected from VSMCs was measured by transwell migration using a Boyden chamber (Corning Life Science). As shown in Fig. 3C, monocyte chemotaxis induced by VSMCs treated with TNF-α was dose-dependently inhibited by vinpocetine.

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.

Vinpocetine Inhibits Lung Inflammatory Response in Vivo.

To confirm if vinpocetine also inhibits inflammatory responses in vivo, we evaluated the effects of vinpocetine on lung inflammation induced by intratracheal inoculation with LPS or TNF-α for 6 h, a well-established mouse model of lung inflammation (20). As shown in Fig. 4, i.p. administration of vinpocetine dose-dependently inhibited induction of TNF-α, IL-1β, and MIP-2 mRNA expression in the lungs of mice after intratracheal administration of LPS (Fig. 4A). Consistent with these results, vinpocetine also significantly inhibited polymorphonuclear neutrophil (PMN) infiltration in bronchoalveolar lavage (BAL) fluids (Fig. 4B) as well as in peribronchial and interstitial areas of mouse lungs treated with LPS (Fig. 4C). Similarly, vinpocetine also inhibited induction of these inflammatory mediators and PMN infiltration in the lungs of mice treated with TNF-α by intratracheal administration (Fig. S3). Taken together, these results strongly suggest that vinpocetine is a potent inhibitor of inflammatory response in vitro and in vivo.

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).

Vinpocetine Inhibits TNF-α–Induced NF-κB Activation by Targeting IKK.

Having identified vinpocetine as an inhibitor for NF-κB–dependent inflammation, we next determined the molecular target of vinpocetine. Because IKK-dependent phosphorylation and degradation of IκBα play very important roles in mediating TNF-α–induced activation of NF-κB and the subsequent up-regulation of NF-κB–dependent proinflammatory mediators, we first evaluated the effects of vinpocetine on IκB phosphorylation and IκB degradation induced by TNF-α in VSMCs. As shown in Fig. 5A, TNF-α induced phosphorylation and degradation of IκBα in a time-dependent manner, as measured by Western blot analysis using an antiphospho-Ser32 IκBα antibody and a nonphosphorylated IκB antibody. Pretreatment with vinpocetine markedly inhibited TNF-α–induced IκBα phosphorylation and degradation. These results suggest that vinpocetine inhibits TNF-α–induced NF-κB activation by preventing IκBα phosphorylation and degradation, thus acting at or upstream of IκBα.

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.

Because IKK represents the major upstream kinase for IκBα phosphorylation, we next determined whether vinpocetine inhibits IKK. IKK kinase activity was measured by in vitro kinase assay of the IKK immunocomplex, with GST-IκBα as a substrate in the presence of [γ-³²P]-ATP. As shown in Fig. 5B, IKK activity was markedly increased in the IKK immunocomplex from cells treated with TNF-α. Vinpocetine significantly inhibited TNF-α–induced IKK activity, thereby suggesting that vinpocetine inhibits TNF-α–induced NF-κB activation at the level (or upstream) of IKK. The dose–response analysis further revealed that the IC₅₀ value of vinpocetine on IKK inhibition in the cell is ≈26 μM (Fig. S4), similar to the IC₅₀ value for vinpocetine inhibition of NF-κB–dependent transcriptional activity (Fig. S1). In contrast, we did not observe significant inhibitory effects of vinpocetine on TNF-α–induced activation of MAP kinases ERK1/2, JNK, and p38, thereby demonstrating the specificity of vinpocetine in inhibiting IKK-NF-κB–dependent inflammation (Fig. S5).

Next, we determined whether vinpocetine inhibits TNF-α–induced NF-κB activation via inhibition of IKK or its major upstream kinase, MEKK1 (21). As shown in Fig. 5C, ectopic expression of constitutively active (CA) MEKK1 alone induced potent NF-κB–dependent luciferase activity. Pretreatment with vinpocetine significantly inhibited CA-MEKK1–induced NF-κB activation, indicating that vinpocetine acts at the level (or downstream) of MEKK1. Furthermore, vinpocetine inhibited NF-κB activation induced by ectopically expressed CA-IKKα (with serines 176 and 180 mutated to glutamic acid) and CA-IKKβ (with serines 177 and 181 mutated to glutamic acid) (Fig. 5 D and E) but not by expressed downstream molecule NF-κB p65 subunit (WT) (Fig. 5F). Collectively, these data suggest that vinpocetine inhibits TNF-α–induced NF-κB activation, likely by targeting IKK.

To determine whether vinpocetine functions as an inhibitor of IKK/NF-κB signaling in vivo, we measured phosphorylation and degradation of IκB, the immediate downstream substrate of IKK, in lung tissues from LPS-treated mice. As shown in Fig. 5, vinpocetine significantly inhibited LPS-induced IκB-phosphorylation (Fig. 5 G and H) and degradation (Fig. 5 G and I) in the lung. This result indicates that vinpocetine also inhibits IKK-NF-κB signaling in vivo.

Vinpocetine Directly Inhibits IKK Kinase Activity.

To determine whether vinpocetine directly targets IKK, we examined the effects of vinpocetine on IKK kinase activity by directly applying vinpocetine to recombinant IKKβ protein in a cell-free system. As shown in Fig. 6A, vinpocetine dose-dependently inhibited IKKβ kinase activity, as assessed by in vitro kinase assay using GST-IκBα as a substrate. The IC₅₀ value of vinpocetine on direct IKK inhibition in the cell-free system is ≈17.17 μM (Fig. 6B).

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.

Inhibitory Effect of Vinpocetine on IKK Kinase Activity Is Independent of Its Known Actions on PDE Inhibition and Ca²⁺ Regulation.

Vinpocetine is a well-known PDE-1 inhibitor (18, 22). Therefore, we determined whether the inhibitory effect of vinpocetine on NF-κB signaling is mediated via inhibition of PDE-1 by examining the effects of specific PDE-1 inhibitors on NF-κB–dependent transcriptional activity. IC86340, a highly selective PDE-1 inhibitor that inhibits VSMC growth (23), did not inhibit TNF-α–stimulated NF-κB–luciferase activity (Fig. S6A). Consistently, IC86340 had minimal inhibitory effects on TNF-α–induced IKK kinase activity, IκBα phosphorylation, and IκBα protein degradation (Fig. 6C). Because vinpocetine is also known to inhibit Ca²⁺ channels (24, 25) in neurons, we next determined whether the inhibitory effect of vinpocetine on TNF-α–induced IKK activity may involve altered intracellular Ca²⁺ homeostasis. The effects of nifedipine (Ca²⁺ channel blocker), EGTA (extracellular Ca²⁺ chelator), and 1,2-bis(o-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic Acid Tetra(acetoxymethyl) Ester (BAPTA/AM; an intracellular Ca²⁺ chelator) on TNF-α–induced NF-κB signaling were examined in VSMCs. As shown in Fig. 6C, none of these treatments exhibited any significant inhibitory effect on TNF-α–induced IKK kinase activity, IκB phosphorylation, and IκB degradation. It has also been reported that vinpocetine inhibits neuronal voltage-dependent Na⁺ channels. We found that the Na⁺ channel inhibitor tetrodotoxin did not inhibit TNF-α–stimulated NF-κB–dependent transcriptional activity in VSMCs (Fig. S6B). Finally, it has been recently reported that vinpocetine may inhibit PDE-4 activity in vitro (26). Because PDE-4 is a well-known antiinflammatory target (27), we analyzed the ability of vinpocetine to inhibit PDE-4 by PDE assay. As shown in Fig. S6, vinpocetine inhibited PDE-1A as expected (Fig. S6C) but did not effectively inhibit PDE-4D activity compared with the PDE-4 inhibitor rolipram (Fig. S6D), suggesting that the inhibitory effect of vinpocetine on NF-κB signaling is unlikely through PDE-4. Together, we conclude that vinpocetine inhibits TNF-α–induced IKK-dependent NF-κB activation independent of its known actions on PDE, Ca²⁺, and Na⁺ regulation, thereby revealing a previously undescribed action of vinpocetine on IKK-NF-κB signaling.

Discussion

Of particular interest in this study is the identification of vinpocetine as a unique antiinflammatory agent in vitro and in vivo. We show that vinpocetine inhibited TNF-α–induced NF-κB activation and the subsequent induction of proinflammatory mediators in several cell types. Moreover, we show that vinpocetine potently inhibited LPS- or TNF-α–induced inflammatory responses in the lungs of mice. Interestingly, vinpocetine inhibited NF-κB–dependent inflammatory responses by directly targeting IKK (Fig. 7). These findings may lead to the development of previously undescribed therapeutical repositioning strategies for the treatment of various human inflammatory diseases, including pulmonary inflammatory disease, arthritis, and atherosclerosis (3).

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.

Steroids and cyclooxygenase inhibitors have long been used as major therapeutical antiinflammatory agents. However, they often cause serious side effects in patients (4, 5). Thus, developing unique antiinflammatory agents is currently in high demand. Vinpocetine, a derivative of the alkaloid vincamine, has long been used in the clinic for treatment of cerebrovascular disorders and cognitive impairment (15). Vinpocetine is well known to enhance cerebral circulation and cognitive function and is currently used as a dietary supplement in many countries for preventative treatment of cerebrovascular disorders and related symptoms associated with aging. Vinpocetine dilates blood vessels and enhances circulation in the brain. This improves oxygen utilization and glucose uptake from blood, which activate cerebral metabolism and neuronal ATP bioenergy production (16, 17). In addition, vinpocetine elicits neuronal protective effects that increase the resistance of the brain to hypoxia and ischemic injury (28). In the present study, we show that vinpocetine is an antiinflammatory agent in vitro and in vivo. Thus, our finding, together with the fact that vinpocetine is purified from natural products and has already been used in the clinic for decades, identifies vinpocetine as a highly promising and attractive antiinflammatory candidate for the treatment of inflammatory diseases.

Of additional interest is that the inhibitory effect of vinpocetine on NF-κB–dependent transcription is independent of its known actions on PDE, Na⁺, and Ca²⁺ regulation. The first molecular target identified for vinpocetine was Ca²⁺/calmodulin-stimulated PDE (PDE-1). Vinpocetine inhibits PDE-1 and elevates vascular cGMP levels at IC₅₀ values around 15–30 μM (Table S1). The positive cerebral vasodilating effect of vinpocetine is at least partially attributable to its effect on PDE-1 inhibition (18). In addition, vinpocetine inhibits neuronal voltage-dependent Na⁺ channels and protects neurons against Na⁺ influx in a variety of cell types (Table S1). Moreover, vinpocetine has been shown to regulate Ca²⁺ signaling at relatively high concentrations in neurons through interacting with glutamate receptors or inhibiting voltage-gated Ca²⁺ channels (Table S1). These effects contribute, at least partially, to the neuroprotective effect of vinpocetine (18). In the present study, we found that vinpocetine directly inhibits IKKβ kinase activity in vitro with an IC₅₀ value of 17.17 μM (Fig. 6). Vinpocetine inhibits intracellular IKK kinase activation and NF-κB–dependent transcriptional activity with an IC₅₀ value of about 25 μM (Figs. S1 and S4). Interestingly, the specific PDE-1 inhibitor IC86340 exhibited no inhibitory effect on IKK activation, nor did decreasing intracellular Ca²⁺ concentration by BAPTA/AM, depleting extracellular Ca²⁺ by EGTA, or inhibiting voltage-gated Ca²⁺ channel by nifedipine (Fig. 6C and Fig. S6). Furthermore, the voltage-gated sodium channel antagonist tetrodotoxin did not alter NF-κB–dependent transcription (Fig. S6). Together, these data suggest that the inhibitory effect of vinpocetine on the NF-κB–dependent inflammatory response is likely to be independent of its known actions on PDE-1 as well as on Na⁺ and Ca²⁺ channels, thereby revealing a previously undescribed action of vinpocetine. It should be noted that the potency of the antiinflammatory effect of vinpocetine is comparable to that of its other well-known effects (Fig. 6, Figs. S1 and S4, and Table S1).

In conclusion, it is evident that vinpocetine acts as an antiinflammatory agent in vitro and in vivo. Vinpocetine inhibits NF-κB–dependent inflammatory responses by directly targeting IKK, independent of its well-known action on PDE-1 activity and Ca²⁺ regulation. Vinpocetine thus becomes an attractive therapeutical candidate for treating inflammatory diseases.

Materials and Methods

General Overview.

Information on reagents, cell culture and treatment, Western blot analysis, RNA isolation, real-time RT-PCR assay, dual-luciferase reporter assay, in vitro IKK kinase assay, monocyte adhesion/chemotaxis assay, and statistic analysis is provided in SI Materials and Methods.

Mouse Model of Lung Inflammation.

C57BL/6 mice at 8 weeks of age were used, as previously described (20). Under anesthesia, mice were intratracheally inoculated with LPS (Escherichia coli serotype 055:B5, 2 μg per mouse; Sigma) in 50 μL of PBS vehicle or TNF-α (500 ng per mouse) or with same volume of saline as control for 6 h. Vinpocetine (10 mg/kg of body weight) or an equal volume of vehicle control was administered via an i.p. route 2 h before the intratracheal inoculation of LPS or TNF-α. Lung tissues were collected and then stored at −80 °C for mRNA expression analysis. For histological analysis, dissected lung was inflated and fixed with 10% (vol/vol) buffered formaldehyde, embedded in paraffin, and sectioned at a thickness of 5 μm. Sections were then stained with H&E to visualize inflammatory responses and pathological changes in the lung. For PMN recruitment analysis, BAL was performed by cannulating the trachea with sterilized PBS, and cells from BAL were stained with Hemacolor (EM Science) after cytocentrifugation (Shandon Cytospin4; Thermo Electronic Co.). Three mice were used for each inoculation group. All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Rochester.