HMC05, Herbal Formula, Inhibits TNF-α-Induced Inflammatory Response in Human Umbilical Vein Endothelial Cells.

Vascular inflammation has been implicated in the progression of cardiovascular diseases such as atherosclerosis. In the present study, we found that HMC05, an extract from eight different herbal mixtures, dose-dependently inhibited tumor necrosis factor-α (TNF-α)-induced adhesion of monocytes to endothelial cells. Such inhibitory effect of HMC05 correlated with suppressed expression of monocyte chemoattractant protein-1, CC chemokine receptor 2, vascular cell adhesion molecule-1 and intercellular cell adhesion molecule-1. In addition, HMC05 significantly inhibited production of reactive oxygen species (ROS) and nuclear factor (NF)-κB activation by TNF-α. Those inhibitory effects of HMC05 (1-10 μg mL(-1)) on the TNF-α-induced inflammatory event was similar to those of berberine (1-10 μM), which is a major component of HMC05 and one of herbal compounds known to have vasorelaxing and lipid-lowering activities. However, berberine significantly reduced the viability of HUVECs in a time- and concentration-dependent manner. In contrast, HMC05 (1-10 μg ml(-1)) did not affect the cell viability for up to 48 h treatment. In conclusion, we propose that HMC05 may be a safe and potent herbal formula against vascular inflammation, and its action may be attributable to the inhibition of ROS- and NF-κB-dependent expression of adhesion molecules and chemokines.


Introduction
Vascular endothelium plays a central role in modulating the inflammatory response in a way that they attract inflammatory cells, activate the coagulant and complement systems and increase vascular permeability [1]. Activation of endothelial cells by pro-inflammatory mediators express increased level of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) and chemokines, which facilitate adhesion of monocytes to endothelial cells [2,3]. Such endothelial activation is one of the earliest and important processes in the pathophysiology of vascular diseases such as atherosclerosis and hypertension [4,5]. Production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) can be stimulated by various pathophysiological phenomena including modified low density lipoprotein (LDL) [6,7], hemodynamic stress [8,9] and hypertension [10].
HMC05 is a water extract of eight herbal drug mixtures originated and modified from a popular traditional herbal medicine, Banhabackchulchunmatang [11]. Previous studies on each of herbal drug components have revealed protective activities against atherosclerosis and inflammation through promotion of anti-inflammatory mediator production [12,13], and inhibition of peroxynitrite-induced oxidative damage [14]. HMC05 has showed an anti-atherosclerotic activity through inhibition of pro-inflammatory cytokine production in a recent study with standardized HMC05 based on berberine and hesperidine [15]. However, the effects of HMC05 on endothelial inflammatory response have not been clearly demonstrated. In addition, previously, we have shown that berberine, an alkaloid isolated from the traditional Chinese herbal medicine, huanglian (Coptis chinensis), exert a cytotoxic effect on endothelial cells, whereas a berberine-containing herbal formula, Zoagumhwan, does not possess cytotoxicity on the cells [16]. HMC05, which contains berberine as a major component, has not been clearly demonstrated for its cytotoxicity profile on endothelial cells. Therefore, in the present study, we investigated the effects of HMC05 on TNF-α-induced inflammatory response and viability of endothelial cells in comparison to the effect of berberine alone.

Measurement of Cell
Viability. The cell viability was assessed using the MTT staining method. The cells from 4-to 5-day-old cultures were seeded in 96-well plates at a density of 1 × 10 4 cells well −1 . The volume of the medium in the wells was 100 μl. In the control experiments, the cells were grown in the same media containing the drug-free vehicle. The cells were incubated with the drug for 48 h, and then with 10 μl of MTT (5 g l −1 ) for 4 h. Two hundred microliters of dimethyl sulfoxide were added to each culture and mixed by pipetting to dissolve the reduced MTT crystals. The relative cell viability was determined by measuring optical density in a microplate reader (Molecular Devices, Versa MAX Sunnyvale, CA, USA) at 540 nm.

Fluorescent Labeling of U937 Cells and Cell Adhesion
Assay. U937 human premonocytic cells were used to investigate leukocyte-endothelial cell interaction [17], and these cells are known to express CCR2, a receptor for MCP-1 [18]. U937 cells were labeled with 2 ,7 -bis(2-carboxyethyl)-5(6)-carboxyfluorescein acethoxymethyl ester (BCECF/AM, 10 μg ml −1 ) for 1 h at 37 • C. HUVECs cultured in 24-well plate were pretreated with HMC05 water extract or berberine for 30 min and then incubated with TNF-α for an additional 3 h. Then, HUVECs were co-incubated with BCECF/AMprelabeled U937 cells (1 × 10 6 cells well −1 ) for 30 min at 37 • C. Non-adhering U937 cells were removed, and the cells were washed twice with PBS. A set of cells was taken and imaged by inverted microscopy connected to digital camera (TMS; Nikon, Japan), and in other sets, cells were lysed in 0.1% Triton X-100 in 0.1 mol l −1 Tris. Fluorescence was measured by using a Fluostar optima microplate reader (BMG LABTECH GmbH, Germany) using excitation at 485 nm and emission at 520 nm.

Immunocytochemical Analysis for ICAM-1 and VCAM-1
Expression. The cellular localization of adhesion molecules, ICAM-1 and VCAM-1, were examined by an immunocytochemical method. HUVECs were cultured in sterile eightwell chambered slides (Nalge Nunc International, Naperville, IL, USA) with medium alone or medium containing HMC05 or vehicle for 1 h before an additional 3 h treatment of TNF-α (10 ng ml −1 ). The cells were then fixed with ice-cold methanol for 5 min at −20 • C. After air drying for 10 min, the slides were blocked with 3% bovine serum albumin (BSA) for 1 h and incubated with ICAM-1 or VCAM-1 monoclonal antibody (1 : 20 dilution of a 200 μg ml −1 solution) at 4 • C overnight in humidified chamber. The cells were washed three times with TBS-T (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% Tween 20) for 5 min and then incubated with Alexa Fluor 488 donkey anti-mouse IgG (1 : 200) in TBS with 3% BSA for 1 h at room temperature. After washing in TBS-T for 30 min, the cells were stained with 600 nM 4 ,6diamidino-2-phenylindole (DAPI) for 2 min. After several brief washings, the cells were mounted with Prolong Gold Antifade reagent and covered with a coverslip. Finally, the cells were observed using a Nikon microscope (TE-2000U) with appropriate excitation/emission filter pairs.

Measurement of Intracellular Reactive Oxygen Species.
Intracellular reactive oxygen species (ROS) generation was measured using 2 ,7 -dichlorofluorescein diacetate (DCF-DA; Sigma), a fluorescent dye. Confluent cells were subjected to serum starvation (0.5% FBS) for 24 h and then used for experiments. Starved cells were treated with HMC05 or berberine in the presence of TNF-α (10 ng ml −1 ) for 1 h. The cells were loaded with DCF-DA (5 μM) for 5 min at 37 • C, and then, imaged by inverted fluorescence microscopy (TE2000-U; Nikon, Japan), and analyzed with an image analysis system (ImageInside Ver 2.32).

Protein Extraction and Western Blot
Analysis. Cells were incubated with TNF-α in the presence or absence of HMC05. For the detection of NF-κB in HUVECs, nuclear protein extraction was carried out by using an NE-PER kit as per manufacturer's instructions (Pierce, Rockford, IL, USA). In case of CCR2 detection, total proteins were extracted from U937 cells using RIPA buffer containing protease inhibitor cocktail (Sigma). Protein content was measured with BCA protein assay reagent (Pierce). Equal amount of proteins (20 μg nuclear or 70 μg total proteins) were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk in TBS-T for 2 h, and incubated with specific antibodies in TBS containing 1% non-fat milk at 4 • C overnight. After washing three times with TBS-T, the membranes were hybridized with horseradish peroxidase-conjugated secondary antibody in skim-milk-TBS for 1 h at room temperature. The immunoreactive proteins were visualized using an ECL kit.

NF-κB Reporter Gene Dual-Luciferase
Assay. NF-κB transactivation was studied by using NF-κB luciferase reporter construct (firefly luciferase) in conjunction with 0.05 μg/ml of pRL-TK (renilla luciferase as a transfection control) using lipofectamine transfection reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. HUVECs (1 × 10 5 cells well −1 ) were incubated with transfection mixture at 37 • C for 5 h, mixed with the same volume of growth medium, and kept in an incubator at 37 • C overnight. These cells were then treated with HMC05 or BER in the presence of 10 ng ml −1 TNF-α. After 3 h, the cells were washed with PBS and then lysed by repeated freezing and thawing. Cells were then scraped gently and the lysates were centrifuged at 10 000 rpm for 5 min. Firefly and renilla luciferase activities were measured using Dual-Luciferase Reporter Assay Kit (Promega Corporation, Madison, WI, USA) on a Turner TD20/20 luminometer (Turner Biosystems, CA, USA).

Statistical
Analysis. The data are expressed as mean ± standard error of mean (SEM) of more than three independent experiments and were analyzed using one-way analysis of variance (ANOVA) and the Student-Newman-Keul's test for individual comparisons. P values of < .05 were considered statistically significant.

HMC05 Inhibits Monocyte Adhesion to Endothelial Cells
Induced by oxLDL and TNF-α. We first examined the effect of HMC05 on the oxidized LDL (oxLDL)-induced vascular inflammation by measuring the adhesion of inflammatory leukocytes to endothelial cells, a crucial step in inflammatory process. As shown in Figure 1(a), HMC05 inhibited oxLDLinduced adhesion of leukocytes to HUVECs. Since in a variety of vascular diseases including atherosclerosis, TNF-α level is increased in the lesion site and contributes to vascular inflammatory process [19,20], we then examined the effect of HMC05 on TNF-α-treated endothelial response. HUVECs stimulated with TNF-α (10 ng ml −1 ) for 3 h significantly increased adhesion of leukocytes to the endothelial cells compared with untreated (control) cells (Figures 1(b) and 1(c)). The TNF-α-induced adhesion was significantly inhibited by HMC05 (1 and 10 μg ml −1 ) in a concentration-dependent manner. The inhibitory activity of HMC05 (1-10 μg ml −1 ) on TNF-α-induced adhesion of monocytes to HUVECs was similar to those of berberine (1-10 μM), a major component of HMC05, or simvastatin (5 μM), a hypolipidemic and cardiovascular risk-reducing drug. promotes the adhesion of leukocytes, which is regarded as the molecular basis for the inflammatory response observed in various diseases [20][21][22]. Real-time reverse transcription polymerase chain reaction (RT-PCR) was carried out to examine the effects of HMC05 on the mRNA expression of MCP-1, one of the key factors to initiate inflammatory process in vascular inflammation, and adhesion molecules, VCAM-1 and ICAM-1. Treatmentof HUVECs with TNF-α increased MCP-1 (Figure 2(a)) and VCAM-1 (Figure 2 to the effect of berberine (10 μM), whereas suppression of MCP-1 and ICAM-1 expression by HMC05 was stronger than by berberine. To further assess the effect of HMC05 on protein expression of the adhesion molecules, immunocytochemistry were performed. As shown in Figures 3(a) and  3(b), VCAM-1 and ICAM-1 protein expression levels were high in TNF-α-stimulated HUVECs, which was suppressed by HMC05 and berberine treatment.

CCR2 Expression in TNF-α-treated Human Monocytic U937 Cells Was Inhibited by HMC05.
Since MCP-1 binds only to CCR2 [23], the expression of CCR2 plays an important role in monocyte recruitment and in many inflammatory states of vessels. The CCR2 mRNA expression was increased by the treatment with TNF-α for 3 h in U937 cells, but this effect was significantly inhibited by co-treatment with HMC05 (10 μg ml −1 ) (Figure 4(a)). The effect of berberine (10 μM) on the TNF-α-induced CCR2 expression was similar to that of HMC05. Similarly, we also noted the protein expression of CCR2 in U937 cells was increased by TNF-α in a western blot analysis, which upon HMC05 treatment, significantly downregulated in a concentration-dependent manner (Figure 4(b)).  analysis (b). The blot is a representative of three independent experiments, and the bar graphs represent relative density of CCR2/Actin. Data are expressed as the mean ± SEM of three independent experiments. * P < .01 compared with untreated control group. # P < .01 compared with TNF-α-treated group.

Geranylgeranyl Pyrophosphate Reverses the Inhibitory
TNF-α increases ROS production which is a major cause of vascular inflammation, we examined the effect of HMC05 on TNF-α-induced ROS increase by measuring DCF fluorescence using a microfluorometry. Co-treatment of the cells with HMC05 and TNF-α significantly inhibited the TNF- The intensity of DCF fluorescence detecting intracellular ROS was analyzed by using ImageInside program. Data are expressed as the mean ± SEM of three independent experiments. * P < .01 compared with untreated control group. # P < .01 compared with the TNF-αtreated group. $ P < .01 compared with TNF-α and HMC05-treated group.
α-induced ROS production ( Figure 5). Similarly, treatment with berberine also suppressed the TNF-α-induced ROS increase. It is well known that TNF-α-induced ROS increase in endothelial cells is associated with activation of NADPH oxidase through prenylation of Rac1 by geranylgeranyl pyrophosphate (GGPP) [24][25][26]. To assess the involvement of NADPH oxidase through Rac prenylation in TNFα-induced ROS production, we examined the effects of exogenous application of GGPP. The pretreatment with exogenously applied GGPP in combination with HMC05 completely reversed the inhibitory effects of HMC05 on TNF-α-induced ROS generation ( Figure 5).

Discussion
Pro-inflammatory cytokines are thought to be central players in the development of vascular inflammation, which leads to the development of cardiovascular complications [27,28]. TNF-α, a commonly found cytokine in atherosclerotic lesions, induces cell adhesion molecules, which contribute to the inflammatory process [20]. In addition, it has been highlighted that cytokine-activated endothelial cells secrete monocyte-specific chemoattractant molecules to recruit monocytes at the sites of vascular injury and inflammation [29].
The present study clearly showed that HMC05 significantly inhibited oxLDL-and TNF-α-induced monocyte adhesion onto endothelial cells in along with reduced expressions of MCP-1, VCAM-1 and ICAM-1 in HUVECs. In addition, HMC05 also blocked the TNF-α-increased expression of CCR2, the MCP-1 receptor, in monocytes. Since the MCP-1/CCR2 binding is the major regulator of monocyte recruitment and seems to play a primary role in many inflammatory states of vessels, our results suggest that HMC05 is an effective formula for vascular inflammation by suppressing both the expression of endothelial chemokine and monocyte chemokine receptors.
Oxidative stress is a characteristic feature of the vascular inflammatory response and in the pathogenesis of atherosclerosis [30]. The expression of adhesion molecules on the endothelial cell surface during primary inflammatory  Figure 8: Schematic diagram showing signaling pathway HMC05 action against TNF-α-induced vascular inflammation. HMC05 may protect vascular inflammation through suppression of ROS production and the expression of NF-κB-related adhesion molecules in endothelial cells, which in turn, prevents monocyte adhesion to endothelial cells. TNFR: tumor necrosis factor-α receptor. The solid arrow and broken arrow represent activation, and migration, respectively. response is dependent on the ROS-sensitive nuclear transcription factor NF-κB and AP-1 activation [2,[31][32][33]. Our results showing that HMC05 inhibited TNF-α-induced ROS and NF-κB translocation indicate that inhibitory effect of HMC05 on TNF-α-induced monocyte adhesion to endothelial cells is possibly mediated through suppression of ROS and subsequent NF-κB activation.
Most importantly, our results clearly showed the difference between HMC05 and berberine, a major component of HMC05. The inhibitory effects of HMC05 on the monocyteendothelial adhesion were similar to those of berberine, whereas HMC05 did not affect the cell viability in contrast to berberine. Although berberine is known to have lipidlowering effect, and vasorelaxing activities as well as ameliorating the cardiovascular diseases such as hypertension and atherosclerosis [34][35][36], recent study revealed that berberine caused prothrombotic effects on endothelial cells, indicating the limitation on therapeutic application of berberine as a monotherapy [37]. Furthermore, the cytotoxic property of berberine is the most important and frequently tested biological activity [38]. Similar to the previous reports, in the present study, berberine showed significant cytotoxicity in HUVECs. However, HMC05 did not show any cytotoxic effect on HUVECs, suggesting that berberine-containing HMC05 may be a safer remedy in the treatment of vascular inflammation than berberine alone. Although we had not determined the amount and activity of exact component, HMC05 contains high levels of phenolic compounds that have wide range of biological activities including antioxidant effects. Recently, the main components of HMC05 were screened and identified as one flavonoid, hesperidin and three alkaloids, coptisine, palmatine and berberine by simultaneous determination method [39]. For example, hesperidin, one of the major components of HMC05, is also reported to have many biological effects including antiinflammatory, antimicrobial, anticarcinogenic, antioxidant and radioprotective effects [40,41]. Based on those reports, the reduced cytotoxicity of HMC05 compared with berberine alone may possibly be due to the compensating action of flavonoid, hesperidin, against cytotoxic nature of berberine.
In conclusion, our results indicate that HMC05 may protect vascular inflammation through suppression of ROS production and regulation of the expression of NF-κBrelated adhesion molecules in endothelial cells (Figure 8).

Funding
Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (grant no B080031) and the Regional Technology Innovation Program of the Ministry of Commerce, Industry and Energy (MOCIE) (grant no RTI04-01-04).