Ursolic Acid and Derivatives Exhibit Anti-atherosclerotic Activity by Inhibiting the Expression of Cell Adhesion Molecules Induced by TNF-alpha

Abstract

Ursolic acid (UA) has been implicated as one of the major components of traditional medicinal plants. In this study, we investigated the effect and mechanisms of UA, by comparison with other triterpenes, Glycyrrhetic acid (GA), Oleanolic acid (OA), Uvaol (Uv) and alpha-amyrin on the vascular cell adhesion molecule-1 (VCAM-1) in Human Umbilical Vein Endothelial Cells (HUVECs). UA inhibited the induction of the VCAM-1 expression in a dose-dependent manner after stimulation with TNF-alpha, whereas GA and OA did not show any inhibitory effect. Furthermore, UA inhibited the adhesion of HL-60 to the TNF-alpha-stimulated HUVECs. The activity difference indicated that the ursane structures with a highly polar group at the 28-position are critical components of UA for regulating the VCAM-1 expression. These results suggested that the anti-inflammatory effect of the triterpenes might depend on their chemical structure.

Introduction

Atherosclerosis is known to be an inflammatory disease and is the leading cause of cardiovascular disease (CVD) mortality (Ross, 1999). The rupture of these atherosclerotic plaques is considered to be critical in the subsequent clinically overt ischemic events (Falk, 2006; Blake and Ridker, 2001). Immune cells, such as lymphocytes and monocytes, are key players in the development of the atherosclerotic lesion. In addition, the adhesion of circulating monocytes to the endothelium and their subsequent migration into the vascular wall are critical steps in both the vascular inflammatory responses and the atherosclerotic processes (Ikeda et al., 1998). The binding of monocytes to the vascular endothelium is mediated by the cross-linkage of the cell adhesion molecules (CAMs), such as the intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and the cell surface expression of these molecules is significantly increased at the atherosclerosis sites, showing the localized pro-inflammatory environment (Blankenberg et al., 2003). Thus, these cell contacts, such as recruitment of monocytes by adhesion to the vascular wall and endothelial cells, play a major role in amplifying the atherosclerosis events through the induction of cell adhesion molecules (Libby, 2002). Lotito et al. reported the structure-function relationships of dietary flavonoids with the TNF-α induced CAMs expression in human aortic endothelial cells. The 5′7-di-hydroxyl substitution of a flavonoid A-ring and the 2,3-double bond and 4-keto group of the C-ring appeared to be the main structural requirements for inhibition of the adhesion molecule expression activity (Lotito and Frei, 2006).

As a preliminary experiment of this study, we screened 48 medicinal plants and found that the extract of Salvia officinlis has a strong activity for inhibiting the expression of CAMs (ICAM-1, VCAM-1, and E-selectin), and identified that its active component is ursolic acid (UA; 3-β-hydroxy-urs 12-en-28 oic acid), (data not shown). UA, a pentacyclic triterpene compound, widely exists in natural plants, such as Rosmarinus officinalis and Ocimum basilicum, and they show several biological effects including anti-tumor (You et al., 2001), anti-microbial (Fontanay et al., 2008), and anti-inflammatory activities (Subbaramaiah et al., 2000). Zeller et al. reported UA interfeses with TNF-α-mediated expression of adhesion molecules downstream NF-κB activation (Zeller et al., 2012). UA inhibited NF-κB, NF-AT, and AP-1 in lymphocytes (Checker et al., 2012). Oleanolic acid (OA) derivative, one of the triterpene, inhibited NF-κB pathway in a p-IκBα and p-p65 (Gao et al., 2016). Moreover, Yan et al. reported that clematichinenoside, one of the triterpene saponin, suppressed cell adhesion expression through NADPH oxidase-dependent IKK/NF-κB pathways in TNF-α-induced HUVECs (Yan et al., 2015). Although, inhibitory effect of UA on the NF-κB signal pathway depends on experimental conditions (Yokomichi et al., 2011).

In this study, we investigated four naturally-occurring triterpenes similar to the UA structure; i.e., OA, Glycyrrhetic acid (GA), Uvaol (Uv), and α-Amyrin (α-A). Among those triterpenes, OA and GA have an oleanane structure while UA, Uv and α-A have ursane structures (Fig 1A). The Ursane structure has two methyl groups on the 19- and 20-positions of the E ring, while the oleanane structure has two methyl group on the 20-position of the E ring. These compounds are biologically active and similar in chemical structure (Hiramatsu et al., 2015; Zeller et al., 2012). In contrast to these reports, we focused the structure-function relationships of the triterpenoid, particularly other ursane type chemicals, at the inflammation site, such as CAMs.

Fig. 1.

Structure of ursane (a) and oleanane (b) backbone (A) and these compounds' derivatives used in this experiment (B)

We are investigated how and by what mechanisms ursane type structures attenuates the expression of the endothelial adhesion molecules. We confirmed that the attenuation of the adhesion molecules by them was strictly dependent on their molecular structure. Furthermore, we revealed whether UA lead to inhibition of the cell-cell interaction by reducing the expression of VCAM-1, the primary cause of atherosclerosis.

Materials and Methods

Materials    UA and Glycyrrhetic acid (GA) were purchested from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Oleanolic acid (OA) and Uvaol (Uv) were obtained from Sigma. α-Amyrin (α-A) and the primers of VCAM-1 were purcheased from Sigma-Aldrich. TNF-α was purchased from Peprotech.

Cell cultures    HUVECs were prepared by the method previously described (Mochizuki et al., 2010). The HL-60 cells were obtained from the American Type Culture Collection and were grown in RPMI-1640 medium (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10 % fetal bovine serum, 100 µg/mL penicillin, and 100 units/mL streptomycine in a 5 % CO₂-containing atmosphere. The confluent HUVECs were pre-incubated with 20 µmol/L of the triterpenes for 4 h prior to the incubation with 10 ng/mL TNF-α, then the cell surface expression of VCAM-1 was assessed by a Western blot analysis. The HUVECs cultured with the triterpenes, such as UA and TNF-α, did not cause any toxicity as determined by the MTT assay (data not shown).

Western blot analysis    Cells were washed twice with phosphatate-buffered saline, pH7.0, and lysed with RIPA buffer [50 mmol/L Tris-HCl (pH7.4), 150 mmol/L NaCl., 1 % NP-40, 0.5 % sodium deoxycholate, 0.1 % SDS]. After protein quantification, equal amounts of the protein (total protein, 40–60 µg) were boiled with Leammli sample butter for 5 minutes at 100 °C. The proteins in the cell homogenates were separated by 10 % SDS-PAGE and transferred to a PVDF membrane. The membrane was washed with TTBS [10 mmol/L Tris-HCl (pH7.6), 150 mmol/L NaCl, 0.05 % Tween-20], blocked with 1 % skim milk powder in TTBS for 1 h, and incubated with the appropriate primary antibody at a 1:1000 dilution in TTBS at 4 °C. After washing 3 times with TTBS, the blots were further incubated for 1 h at room temperature with the IgG antibody coupled to horseradish peroxidase in TTBS. The blots were then washed in TTBS before visualization. An ECL kit was used for the detection.

RNA Isolation and Reverse Transcription (RT)-PCR    The total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA). The RNA concentration was determined by measuring the absorbance at 260 nm. The RT reaction was performed with 5 µg of the totalRNA and an oligo (dT) primer using the First-stand cDNA synthesis kit. The PCR reactions were carried out using 0.5 µL of cDNA in 25 µL of 10 mmol/L Tris-HCl, pH 8.3, containing 50 mmol/L KCl, 0.1 %Triton X-100, 200 µmol/L dNTPs, 1 µmol/L concentration of each forward and reverse primer, and 2 units of rTaqDNA polymerase (Toyobo Co., Osaka, Japan). The primers were prepared by the method previously described (Mochizuki et al., 2010)

Preparation of cytoplasmic and unclear extracts    The stimulated HUVECs were harvested with NP-40 buffer [10 mmol/L Tris-HCl (pH7.4), 10 mmol/L NaCl, 3 mmol/L MgCl₂, 0.1 mmol/L EDTA, 0.5 %(v/v) NP-40] and left for 20 min on ice to lyse the cells. The cells were centrifuged at 1500 rpm for 5 min at 4 °C and separated into the supernatants and pellets. The pellets were washed with NP-40 buffer, then resuspended with RIPA buffer and centrifuged at 11,000 rpm for 5 min at 4 °C to harvest as the nuclear extracts. The supernatants were harvested as the cytoplasmic extracts.

Leukocytes-endothelial cells adhesion assay    The HUVECs were grown to confluence in wells of a 24-well culture plate, pretreated with UA at 15 µmol/L for 4 h, and after it was removed, stimulated with TNF-α for 5 h. The HL-60 was cultured in a 60-mm dish and treated by the same method as the HUVECs. This was followed by the addition of 10⁶ cells mL⁻¹ of the HL-60 to the HUVECs monolayer and incubated in a CO₂ incubator for 10 min. The plate was them placed on the shaker at 70 rpm for 10 min. The non-adherent HL-60 was removed by washing with PBS. The HUVECs and adherent HL-60 were fixed with 2 % paraformaldehyde overnight at 4 °C, washed with PBS, and measured by counting the number of both cells, and the adhesion rate of the HL-60 to the HUVECs was calculated by the following formula: number of adhered HL-60/number of HUVECs.

Statistical analysis    All analyses were preformed using IBM SPSS version 25 software (Inc., Chicago, IL, USA). Statical comparisons were evaluated with a one-way ANOVA followed by the Tukey test for multiple comparisons. Differences were considered significant at p < 0.01. Unless otherwise stated, all error bars represent the SD.

Results

Structure-dependent Effect of Triterpenes on the Expression of Endothelial Cell Adhesion Molecules    To investigate the structural requirement of triterpenes on the VCAM-1 expression inhibitory activity, we compared the various structurally-related triterpenes, i.e., Ursolic acid (UA), Glycyrrhetic acid (GA), Oleanolic acid (OA), Uvaol (Uv) and α-amyrin (α-A) (Fig. 1B). Figure 1 shows the chemical structures of the triterpenes. They have major variations in the nature of the E ring. The expression of VCAM-1 was relatively weak in the TNF-α -untreated cells (negative control), and significantly enhanced in the TNF-α -stimulated cells (positive control).

We first compared UA, OA and GA. OA and GA have the oleanane structure, while UA has the ursane structure (Fig. 1A), and OA is structurally identical to UA except for the position of the methyl group in the E-ring (Fig. 1B). We confirmed that UA significantly inhibited the TNF-α-induced VCAM-1 expression (Fig. 2A), whereas OA and GA did not show any inhibition. This result suggests that the basic structure required for the inhibition of the CAM expression is the ursane structure, not the oleanane structure. We next focused on the ursane-type triterpenes, UA, Uv and α-A, and evaluated their inhibitory activity. We found that UA and Uv showed a significant effect on attenuating the expression of VCAM-1, while α-A showed no inhibitory effect (Fig. 2B). In the E ring on their structures, UA has a carboxy group, Uv has a hydroxymethyl group, and α-A has a methyl group. UA and Uv lowered VCAM-1 expression by 91 % and 68 %, respectively, as compared to the TNF-α induced group. UA exhibited a slightly stronger activity than Uv. It is suggested that the 28-position of the ursane structure needs to be oxidized to a polar functional group, such as the carboxy group or hydroxymethyl group, for the VCAM-1 inhibition activity.

Fig. 2.

Inhibitory effect of triterpenes on expression of VCAM-1 in cultured HUVECs treated with TNF-α.

The HUVECs were treated with 20 µmol/L triterpenes for 4 h, and stimulated with TNF-α at 10 ng/mL for 5 h.

A western blot analysis was carried out as described in the Materials and Methods. (A) is the comparion among the popular tritepenes, (B) is the ursan-structured ones.

Effect of UA Concentration on Endothelial Adhesion Molecule Expression    We analyzed the gene and protein expression levels of VCAM-1 by western blotting. The cultured HUVECs with UA and TNF-α did not cause any toxicity, as determined by the MTT assay (data not shown). The expression of VCAM-1 was relatively weak in the TNF-α -untreated cells (negative control), and significantly enhanced in the TNF-α -stimulated cells (positive control). We confirmed that UA inhibited it in time-dependent manner, particularly 4 h (Fig. 3A). The mRNA expression showed completely inhibited with addition of 20 µmol/L UA (Fig. 3B). With cell-based ELISA, we also revealed that UA exhibited suppressive activities for the expression of the other CAMs. The mRNA level of GAPDH did not change, suggesting that the stability of mRNA was not affected by UA. These results suggest that UA inhibits the CAMs expression at both the transcriptional and protein expression levels.

Fig. 3.

Activity of UA against the protein expression of VCAM-1. Suppressive effect of UA on the NF-κB activation by TNF-α.

(A) Western blot assays showed that UA has an inhibitory activity against the expression of VCAM-1 in a time-course manner. HUVECs were pre-treated with 20 µmol/L UA for 0, 0.25, 0.5, 1, 2, 4h and stimulated with TNF-α (10 ng/mL) for 5 h.

The protein extracts were prepared and VCAM-1 analyzed. (B) A RT-PCR analysis showed the ability of inhibit VCAM-1 mRNA in a dose-dependent manner. HUVECs were incubated with UA (0-20 µmol/L) for 4 h before exposure to 10 ng/mL TNF-α for 5 h. The total RNA of the cells was then isolated and analyzed by RT-PCR, as described in “Materials and Methods”. (C) Effect of UA on TNF-α-induced activation of NF-κB. HUVECs were pre-treated with 0-20 µmol/L UA for 4 h and stimulated with TNF-α (10 ng/mL) for 1 h. The cytosol extracts were prepared and the IκB degradation analyzed. Nuclear extracts were prepared and analyzed for nuclear translocation of the NF-κB activation.

UA Blocked Degradation of IκB and Induction of NF-κB Activation by TNF-α    The expression of CAMs is one of the inflammatory responses activated by TNF-α, and Cytokine-dependent CAM induction is regulated at the gene level by the activity of the transcription factors, such as nuclear factor-κB (NF-κB) (Hayden and Ghosh, 2008). Before NF-κB was activated, its regulatory protein, IκB, was phosphorylated and ubiquitinated, then decomposed in the proteasome system. Therefore, we examined the effect of UA on the degradation of IκB and the activation of NF-κB by the TNF-α stimuli. The HUVECs were pre-incubated with different concentrations of UA for 4 h, then treated with TNF-α for 1 h. The cytosolic extracts were prepared and tested for the IκB degradation and the nuclear extracts tested for the NF-κB activation. As shown in Fig. 3C, UA tended to inhibit the NF-κB activation in the nuclear extracts in a dose-dependent manner, parallel to the TNF-α -mediated IκB degradation in the cytosolic extracts. These results suggest that UA suppressed the NF-κB activation by inhibiting the degradation of IκB.

UA Inhibited the Leukocytes-Endothelial Cells Interactions    Leukocyte adhesion to endothelial cells is crucial in vascular inflammation and this interaction can be induced by chemotactic cytokines. This phenomenon leads to forming the primary lesions of atherosclerosis. Therefore, we examined whether UA could inhibit the adhesion of HL-60 to the TNF-α-stimulated HUVECs. We found that stimulation with TNF-α produced an approximately 10-fold increase in the adhesion of HL-60 to the HUVEC monolayer when compared to the untreated group. Pre-treatment of the HUVECs with UA showed a statistically significant reduction of the adherent HL-60 cells to the HUVECs (28.3 % of the TNF-α-treated group, P < 0.01) (Fig. 4). This result suggests that UA inhibits the leukocyte-endothelial cell interaction by suppression of the CAMs expression.

Fig. 4.

Ursolic acid suppressed the adhesion of HUVEC and HL-60 stimulated by TNF-α.

(A) Representative image of the reduction of TNF-α-stimulated adhesion of HL-60 to HUVEC monolayer after pre-incubation with the indicated doses of UA for 4 h. (B) HUVEC were pre-incubated with the indicated doses of UA for 4 h and stimulated with TNF-α (10 ng/mL) for 5 h. HL-60 was added to HUVEC monolayer and these cells were shaken at 70 rpm for 10 min. The adhered cells were measured as described in the Materials and methods. Data are from n=5 experiments. *P < 0.01 compared to TNF-α alone.

Discussion

Atherosclerosis is a major cause of death in Japan, and approximately 1out of 3 dies of arteriosclerotic disease such as cardiovascular and cerebrovascular diseases. The formation of atherosclerotic lesions involves blood leukocyte recruitment to the arterial intima, engulfment of lipids derived from ox-LDL, and transformation into macrophage foam cells (Libby, 2002). Endothelial cells are also activated in this process and express CAMs and chemokines that attribute to atherosclerosis by regulating different steps of the leukocyte recruitment process (Blankenberg et al., 2003).

Endothelial cells express a variety of CAMs to regulate the adhesion of leukocytes to endothelial cells and also to regulate the adhesion of the endothelial cells to the extracellular matrix (ECM). It is known that only mononuclear cells (lymphocytes, monocytes, eosinophils) express receptors for VCAM-1, while all leukocytes express other receptors for ICAM-1 and E-selectin, but not VCAM-1 (Gille et al., 1999; Chen et al., 2003; Chen et al., 2002). Therefore, it can be said that among the CAMs, VCAM-1 is mainly involved in vascular diseases by controlling the adherence of the mononuclear cells to the endothelium (Elices et al., 1990).

UA is a multifunctional triterpene with biological activities such as anti-inflammatory (Subbaramaiah et al., 2000; Liu, 1995), anti-microbial (Fontanay et al., 2008; Ngouela et al., 2005), and anti-proliferative activities (Uto et al., 2013). Triterpenes have many types of structures, their structures are related to various properties and activities, and many have an anti-inflammatory activity. We focused on the anti-atherosclerotic activities of UA, the activities of UA, structure-dependence of its activities, and the relation to the inflammatory activities. Among the 5 triterpenes, we selected as the control UA that showed the most effective ability for suppressing VCAM-1 against TNF-α stimuli. We confirmed that UA and Uv, which have a ursane-type structure with methyl groups on the 19- and 20-positions of the E ring (Fig. 1B), were able to inhibit the VCAM-1 expression (Fig. 2B), in contrast to OA and GA, which have oleanoane-type structures with methyl groups on the 20-positions of the E ring (Fig. 1B). Hiramatsu et al. reported that UA inhibited the intracellar transport of ICAM-1 to the cell surface in human lung carcinoma A549 cells. They also suggested that the position of a methyl group at the E ring (UA) may be critical for the selectivity to inhibit the intracellular trafficking of glycoproteins (Hiramatsu et al., 2015). In contrast to these reports, we compared three ursane-type structure triterpenes (UA, Uv, and α-A). UA and Uv, both oxidized at the 28-position, inhibited the VCAM-1 expression, and UA showed a slightly stronger inhibition than Uv. On the other hand, α-A, which has a carbon group at the 28-position, that is not oxidized, did not show any inhibitory activity on the VCAM-1 expression. The 28-position's functional group, which is the highly polar carboxy group or hydroxymethyl group, is considered to be very important.

Uto et al. previously identified that the C₂-C₃ trans-dihydroxyl group at the A ring and the C₁₉-C₂₀ trans-dimethyl group at the E ring are important for suppressing cell proliferation in the leukemia cell lines. (Uto et al., 2013) In our study, we also confirmed that the high concentration of UA, OA and betulinic acid, which all have a carboxy group at the 28-position, suppressed cell growth in the HUVECs by the MTT assay (data not shown). Thus, the carboxy group at the 28-position is also another important region for the suppression of cell growth. While there is no significant difference in the structure and activity between UA and Uv, since UA contains a higher proportion than Uv in the medical plants, we further investigated the activity of UA.

Zeller et al. reported that UA, which pretreated for 30 min before TNF-α was added, reduced VCAM-1 expressions by Real-time PCR and Western blotting in dose-dependent manners (Zeller I et al., 2012). These results suggested that UA inhibits the CAMs expression at both the transcriptional and protein expression levels (Zeller I et al., 2012). Therefore, we confirmed that UA inhibited VCAM-1 protein expression level in time-dependent manners (pretreated time) (Fig. 3A). It is likely to incorporate UA into endothelial cells surface in time-dependent. RT-PCR analysis also showed the activity of the VCAM-1 mRNA expression level in a dose-dependent manner (Fig. 3B). The mRNA expression showed similar trends by ICAM-1 (Yokomichi et al., 2011) and E-selectin (Takada et al., 2010) expressions, UA inhibited cell adhesion expression by transcriptional level. In addition, UA inhibited the degradation of IκB in the cytosolic extracts and the translocation of NF-κB in the nuclear extracts (Fig 3C). According to previous reports, NF-κB signaling pathway differed in cell-type or experimental conditions (Yokomichi et al., 2011). However, Shishodia et al. reported that UA inhibited I κ B degradation, I κ B phosphorylation , p65 phosphorylation in Jurkat cells (Shishodia et al., 2003). And then, Yokomichi et al. showed that UA slightly inhibited IκBα degradation in A549 cells (Yokomichi et al., 2011). Moreover, Takada et al. suggested that UA and OA inhibited TNF-α-induced NF-κB/E-selectin activation in HUVEC (Takada et al., 2010). In this report, we showed that UA inhibited the NF-κB signal pathway in HUVEC, as their previous reports. But, the degradation of IκB was slightly blurry image. So, we have to investigate upstream signal transduction of NF-κB activation such as IκBα phosphorylation and IKK. IKK is upstream of translation of cell adhesion, and IKK activation regulars IκBα. IKK activation inhibited by UA in Jurkat cells and inhibition of it might be related in some way to upstream kinase (Shishodia et al., 2003). It is not clear that UA inhibit IKK activation in HUVECs. We need to confirm IKK activation using HUVECs.

UA directly binds to a promoter region related to the expression of the CAMs on DNA, and works on the activation of the transcriptional factors.

These results indicated that UA allows inhibition of the CAMs by repressing the degradation of IκB. UA has previously been reported to have an enzyme activity inhibition such as P450 (Kim et al., 2004), leukocyte elastase (Ying et al., 1991), lipoxygenase and cyclooxygenase (Najid et al., 1992), and protein tyrosine phosphatase-1B (Zhang et al., 2006). Furthermore, we showed that the number of adherent HL-60s to the HUVECs was significantly decreased by the UA treatment using the flow condition which created the artificial blood flow (Fig. 4.).

Overall, we demonstrated that UA, the major component in the leaf extract of Salvia officinalis, prevents the cytokine-induced VCAM-1 expression. The ursane structures with the highly polar group at the 28-position of UA are critical for the inhibition of the VCAM-1 expression in time-dependent, which regulates the TNF-α-induced the degradation of IκB and the translocation of NF-κB. Moreover, we also revealed that the inhibition of the CAMs by UA affects the leukocyte-endothelial cell adhesion. We concluded that UA is a promising functional factor, which is substrate from food, such as plants, with an anti-atherosclerotic activity.

Abbreviations

α-A

α-amyrin

GA

Glycyrrhetic acid

OA

oleanolic acid

UA

ursolic acid

Uv

uvaol

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