Introduction

According to recent statistics from World Health Organization (WHO), it was shown that the mortality rate for people suffering from cardiovascular disease is higher than any other diseases [1]. Atherosclerosis is the principal cause for cardiovascular diseases such as myocardial infarction and stroke. It is characterized by accumulation of lipids and cholesterol within the arterial walls [2]. Blood monocytes that are recruited to the vessel wall are the precursors of the lipid-laden macrophages that form fatty streaks—one of the earliest manifestations of atherosclerosis. In the last decade there have been a great deal of research on anti-inflammatory therapies being developed for clinical intervention of atherosclerosis. Maslinic acid (Pubchem CID: 73659) is a natural pentacyclic triterpenoid found in medicinal plants. The anti-inflammatory effect of maslinic acid has been confirmed in a number of studies, demonstrating that it reduces susceptibility of plasma and hepatocyte membranes to lipid peroxidation [3] and inhibits production of nitric oxide, tumor necrosis factor alpha, and cyclooxygenase-2 [4, 5]. In a study conducted by Moneriz et al. (2011), secreted phospholipase A2 (sPLA2) was discovered as one of the novel putative targets of maslinic acid [6].

Mammalian sPLA2 enzymes are classified based on their structural features into one conventional group consisting of Group I, II, V, X and otoconin-90, and two other atypical groups which include Group III and XII [7]. Individual sPLA2 function as an enzyme targeting membrane phospholipids, including cellular membranes, non-cellular lipids such as surfactant and lipoproteins, foreign phospholipids in bacterial membranes and dietary phospholipids, or serving as a ligand for membrane-bound and soluble receptors [8]. So far, only human Group IIA (hGIIA)-sPLA2 subtype is found in human sera in different physiological and pathological conditions [9, 10]. The levels of hGIIA-sPLA2 in the circulatory system or inflammatory exudates corresponds to the degree of severity in the inflammatory diseases and tissue injuries [11, 12].

hGIIA-sPLA2 was also detected in the vascular smooth muscle cell of the media and the intima layers as well as in the macrophage-rich region of atherosclerotic plaques. It has been reported that hGIIA-sPLA2 induce monocyte differentiation and migration that leads to inflammatory reaction in atherosclerotic lesion [13]. Another research described that hGIIA-sPLA2 plays a role in modifying the lipoproteins and release lipid mediators in the arterial walls [14]. The modification of lipoproteins also leads to the expression of chemokines as a result of inflammatory reaction which then induces the recruitment of monocyte towards the intima layer where differentiation of monocyte occurs [15].

Given that sPLA2 are an emerging class of mediators for inflammation and it possess the capacity to hydrolyze lipoproteins and release lipid mediator from cellular membranes, this study sought to investigate the interaction between maslinic acid and hGIIA-sPLA2. The interaction was examined by monitoring changes in the enzyme’s relative intrinsic fluorescence and its inhibitory effect on the enzyme hydrolytic activity. The anti-inflammatory effect of maslinic acid targeting hGIIA-sPLA2 was performed in THP-1 promonocytic cells by determining its effect in suppressing hGIIA-sPLA2-induced monocyte-macrophage differentiation and migration.

Materials and Methods

Maslinic acid was purchased from Cayman Chemicals; Fatty-acid free BSA was from Sigma. hGIIA-sPLA2 protein was expressed, purified, and quantified as described [16]. The fluorescent substrate for PLA2 assay, 1-hexadecanoyl-2-(10- pyrenedecanoyl)-sn-glycero-3-phosphoglycerol, ammonium salt (β-py-C10-PG) was from Molecular Probes (Eugene).

Cell Culture

THP-1 cells were cultured at 37 °C under a 5 % CO2 atmosphere in RPMI 1640 (GIBCO) with 100 U/mL penicillin/streptomycin, 2 mM glutamine, 5 % fetal bovine serum and maintained at 5 × 105 cells/mL.

Fluorescence Spectroscopy

The relative intrinsic fluorescence intensity of hGIIA-sPLA2 enzyme with and without maslinic acid was determined by fluorescence spectroscopy as described previously [17]. The hGIIA-sPLA2 enzyme was dissolved in Phosphate Buffered Saline (PBS) buffer to a final concentration of 100 μM. Maslinic acid solution (1 mM) was prepared in ethanol. A series of samples containing different amounts of maslinic acid and a constant amount of hGIIA-sPLA2 were prepared in a 20:80 % ethanol: buffer mixture. The final concentrations of maslinic acid were 3.13, 6.25, 12.5, 25, 50, 100 μM, while the hGIIA-sPLA2 protein concentration was fixed at 100 μM. Each dilution comprising 100 µL was transferred to a black, flat-bottom 96-well microplate (Corning). Fluorescence was measured at 25 °C on a BMG FLUOstar OPTIMA Microplate Reader with Ex = 280 nm and Em = 340 nm.

In Vitro Phospholipase Assay

PLA2 activity was evaluated as previously described [18] with β-py-C10-PG used as a substrate. To each well of a 96-well microtiter plate, 97 µL of solution A (27 µM bovine serum albumin, 50 mM KCl, 1 mM CaCl2, 50 mM Tris– HCl, pH 8.0) and 3 µL of maslinic acid or 3 µL of DMSO only for negative control reactions were added. Solution B was delivered in 100-µL portions to the wells and they have the same composition as Solution A and hGIIA-sPLA2 enzyme (200 ng). Blank contain an additional 100-µL portion of Solution A minus the enzymes. The assay was initiated by adding 100 µL of Solution C [4.2 µM of β-py-C10-PG (molecular probes) vesicles in assay buffer] with a repeating micropipette to all wells. The fluorescence (excitation = 342 nm, emission = 395 nm) was read with the BMG FLUOstar OPTIMA Microplate Reader. The increase in fluorescence was continuously recorded for 1 min, and the enzyme activity was calculated based on the initial slopes of fluorescence versus time. Enzyme activity was normalized to the vehicle-treated controls.

Molecular Docking

To understand the mechanism of action, inhibition of hGIIA-sPLA2 by maslinic acid was studied in silico using computational methods to support the in vitro results. Maslinic acid was docked into the active site of hGIIA-sPLA2 to determine their interaction. The crystal structure of hGIIA-sPLA2 (PDB code: 3U8D) was retrieved from protein data bank (www.rcsb.org). Protein structure is prepared and energy-minimized. Molecular docking is performed using CDOCKER program in Discovery Studio suite 4.0.

THP-1 Cell Treatment

THP-1 cells (2 × 105 cells/mL) were seeded into a 24-well plate. The cells were either untreated (negative control), treated with 1 µg/mL of hGIIA-sPLA2 only (positive control) or treated with hGIIA-sPLA2 in the presence of increasing concentrations of maslinic acid (5, 10, 20, and 50 µM). The cells were incubated in 5 % CO2 incubator. After 24 h, the cell suspension were transferred to a microcentrifuge tube and spun at 14000 rpm for 10 min. The supernatant collected was kept at −20 °C for the migration assay. The 24-well plate was then rinsed four times with PBS and the remaining adherent cells were used for adhesion assay.

Adhesion Assay

The adherent cells were fixed with methanol for 15 min at room temperature. The fixed cells were then stained with Giemsa stain for 30 min and washed with PBS to remove excess stain. The plates were air dried and examined under the inverted microscope. The adhesion score was measured by counting the number of adhered THP-1 cells per six different microscopic fields under high power field.

Migration Assay

The migration assay was performed in a six-well plate using tissue culture polycarbonate filter inserts (8 um pore, Corning, Costar, Cambridge, MA, USA). THP-1 cells (0.5 × 106 cells/mL) were resuspended in 1 mL of serum-free medium and loaded into the upper chamber of an insert. The lower chamber contained medium collected from treated cells (as indicated in THP-1 cell treatment). The cells were then incubated for 24 h in the 5 % CO2 incubator. After the incubation period, the remaining cells in the upper chambers of the filter inserts were removed and washed with PBS. The migrated cells on the lower side of the filter insert were then fixed with 3.7 % of formaldehyde in PBS and permeabilized with methanol before Giemsa staining. The stained cells were counted in 6 different microscopic fields under a high power field.

Statistical Analysis

Statistical analyses were conducted using SPSS (version 22). Results are expressed as means ± SD for the number of independent experiments indicated. Statistical analysis involved the use of an ANOVA test, followed by a Tukey’s test where several experimental groups were compared to the control group. A p < 0.05 was considered statistically significant.

Results

Intrinsic Fluorescence Interaction of Maslinic Acid with hGIIA-sPLA2 Enzyme

The change in intrinsic fluorescence reflects conformational change in protein due to substrate or ligand interaction. Thus it is expected that fluorescence measurement of hGIIA-sPLA2 together with maslinic acid would provide information regarding the formation of enzyme-inhibitor complex. The results showed that maslinic acid enhanced intrinsic fluorescence of hGIIA-sPLA2 (Fig. 1). Enhancement of relative fluorescence was observed with increasing concentration of maslinic acid (3.13–100 µM). The results indicated that maslinic acid may directly interact with hGIIA-sPLA2 enzyme.

Fig. 1
figure 1

Effect of maslinic acid on hGIIA-sPLA2 intrinsic fluorescence. A series of samples containing different amounts of maslinic acid and a constant amount of hGIIA-sPLA2 were prepared. The final concentrations of maslinic acid were 3.13, 6.25, 12.5, 25, 50 and 100 μM, while hGIIA-sPLA2 protein concentration was fixed at 100 μM. Fluorescence was measured at 25 °C on a BMG FLUOstar OPTIMA Microplate Reader with Ex 280 nm and Em 340 nm. Values are expressed as means ± SD of three independent experiments. Asterisks represent p < 0.05 compared to reactions containing hGIIA-sPLA2 and ethanol only

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Effect of Maslinic Acid on hGIIA-sPLA2 Enzyme Activity

One important feature of the hGIIA-sPLA2 enzyme is in hydrolyzing the sn-2 position of glycerophospholipid, producing arachidonic acid which is a precursor for eicosanoid components such as prostaglandins and leukotrienes. This enzyme has also been shown to hydrolyze phosphatidylcholine in lipoproteins to generate lysophosphatidylcholine and free fatty acids, thereby contributing to the accumulation of these lipids products at the atherosclerotic lesion site. It is hypothesized that reduction of the eicosanoids production may suppress inflammation. Maslinic acid inhibited hGIIA-sPLA2 enzyme activity (as shown in Fig. 2) in a concentration-dependent manner. More than 60 % of inhibition (p < 0.05) was achieved at 100 μM concentration. To elucidate the kinetics of inhibition, hGIIA-sPLA2 enzyme activity was determined in the presence of increasing substrate concentrations. Lineweaver–Burk double reciprocal plot (Supplementary Figure 1) showed that the V max for hGIIA-sPLA2 enzyme activity in the presence of maslinic acid was less than uninhibited enzyme and that both conditions (with maslinic acid and without inhibitor) have same K m, indicating non-competitive inhibition.

Fig. 2
figure 2

Effect of Maslinic acid on hGIIA-sPLA2 enzymatic activity. The enzyme hGIIA-sPLA2 was incubated with Py-PG-containing phospholipid vesicles (4.2 μM) in the presence of increasing concentrations of maslinic acid. Enzyme activity was determined according to the procedure described in the “Materials and Methods” section. Enzyme activity was normalized to negative control reactions containing hGIIA-sPLA2 and β-py-C10-PG substrate only, and expressed as the mean percentage inhibition ± SD of three independent experiments. Asterisks represents p < 0.05 compared to negative controls containing hGIIA-sPLA2 and β-py-C10-PG substrate only

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Molecular Interaction Between Maslinic Acid and hGIIA-sPLA2 Enzyme

The crystal structure of hGIIA-sPLA2 (3U8D) enzyme was obtained from PDB. The binding region of hGIIA-sPLA2 consists of hydrophobic site with Leu2, Phe5, Ile9, Ala17, Ala18, Tyr21, Cys28, Cys44, and Phe98 while the catalytic site consists of hydrophilic residues His47 and Asp48 [19]. His47 and Asp48 are important hydrogen bond acceptors and calcium binding site [20]. The presence of His47 and Asp48 residue on the active site facilitates the hydrolysis mechanism by abstracting a proton from the water molecule then followed by nucleophilic attack on the sn-2 bond [21]. The docking interaction (Fig. 3) shows that maslinic acid binds to His47 and Asp48 residues via hydrogen bonding, suggesting that it inhibits the access of the catalytic calcium ion required for the enzymatic reaction. In addition, maslinic acid also formed hydrophobic interactions with hGIIA-sPLA2 enzyme at Leu2, Phe5, Ala18, Cys44, which are the interfacial binding sites of the enzyme. The interfacial binding site of sPLA2 enzyme represents the point of contact between the enzyme and the phospholipid bilayer substrate [22]. This indicates that apart from blocking the calcium binding site, maslinic acid may also interfere with enzyme binding to the membrane phospholipid, thereby inhibiting the enzymatic activity of hGIIA-sPLA2.

Fig. 3
figure 3

Model of hGIIA-sPLA2 (PDB id 3U8D) with maslinic acid. Maslinic acid binds to His47 and Asp48 residues via hydrogen bonding, and also formed hydrophobic interactions with Leu2, Phe5, Ala18 and Cys44

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Effect of Maslinic Acid on hGIIA-sPLA2-Induced THP-1 Cell Differentiation

To further elucidate the role of maslinic acid in regulating hGIIA-sPLA2-mediated inflammatory processes, an adhesion assay was performed in hGIIA-sPLA2-induced THP-1 cells. THP-1 is a human pro-monocytic cell line and it has been used as a model system for monocyte-macrophage differentiation. According to previous studies, promonocytic THP-1 cells are able to differentiate when stimulated with inflammatory activators. THP-1 cells in its differentiated state exhibit phenotypic modification such as adhering to culture plate surfaces as well as changes in its morphology [23]. Based on the result obtained in Fig. 4, hGIIA-sPLA2 significantly induces THP-1 differentiation as indicated from the increased number of adherent cells. Treatment with maslinic acid significantly reduces the number of adherent cells at 10, 20 and 50 µM concentrations (p < 0.05), indicating that maslinic acid has the capacity to suppress hGIIA-sPLA2-induced THP-1 cell differentiation. Considering that maslinic acid has been reported to inhibit cell proliferation and apoptosis through a mitochondrial-mediated pathway [24], a set of negative control cell treatments with maslinic acid alone was performed. Our results demonstrated that maslinic acid did not significantly affect the number of adherent cells (Supplementary Figure 2). The basal level of differentiated cells (adhered) in the absence of hGIIA-sPLA2stimulation was relatively low and that maslinic acid treatment (5, 10, 20 and 50 uM) has no significant impact on the cellular process.

Fig. 4
figure 4

Effect of maslinic acid on hGIIA-sPLA2-induced THP-1 cell adhesion. THP-1 cells were untreated, treated with 1 µg/mL hGIIA-sPLA2, or treated with 1 µg/mL hGIIA-sPLA2 in the presence of various concentrations of maslinic acid (5, 10, 20 and 50 µM). The cells were incubated at 37 °C for 24 h, and the number of adherent cells was determined as indicated in the “Materials and Methods” section. Each bar represents the mean number of adherent cells of three independent experiments ± SD; n = 3. #Signifies p < 0.05 compared to untreated cells and Asterisks represent p < 0.05 compared to cells treated with hGIIA-sPLA2 only

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Effect of Maslinic Acid on hGIIA-sPLA2-Induced THP-1 Cell Migration

To determine the migratory capacity of the cells in response to hGIIA-sPLA2, THP-1 cells were placed in the upper chambers of Transwell inserts. The lower chambers contained supernatants from non-stimulated cells, 24 h hGIIA-sPLA2-stimulated cells, or 24 h hGIIA-sPLA2-stimulated cells in the presence of various concentrations of maslinic acid. THP-1 cells showed migration in response to supernatants from hGIIA-sPLA2-stimulated cells. Based on the results obtained, maslinic acid significantly reduced the number of cells migrated in response to hGIIA-sPLA2 treatment at the concentration of 10, 20 and 50 µM (Fig. 5) (p < 0.05). The inhibitory effect was specific to hGIIA-sPLA2 as cells treated with maslinic acid alone did not induce significant cell migration (Supplementary Figure 3).

Fig. 5
figure 5

Effect of maslinic acid on hGIIA-sPLA2-induced THP-1 cell migration. THP-1 cells were untreated, treated with 1 µg/mL hGIIA-sPLA2, or treated with 1 µg/mL hGIIA-sPLA2 in the presence of various concentrations of maslinic acid (5, 10, 20 and 50 µM). The cells were incubated at 37 °C for 24 h, and the numbers of cells migrated were determined as indicated in “Materials and Methods”. Each bar represents mean number of migrated cells of three independent experiments ± SD; n = 3. #Signifies p < 0.05 compared to untreated cells and *represents p < 0.05 compared to cells treated with hGIIA-sPLA2 only

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Discussion

Considering its mechanism of action in catalyzing hydrolysis of membrane phospholipids which contributes to eicosanoids production, it is hypothesized that sPLA2 inhibitors may act as potential anti-inflammatory agents. Maslinic acid has been widely accepted as a natural compound with anti-inflammatory effects. Recent studies have elucidated its molecular mechanism and potential binding targets. Nevertheless, it is still unknown how maslinic acid regulates the inflammatory signaling pathways which contribute to the inhibition of iNOS/COX-2 activity and eicosanoid release. Given that sPLA2s are an emerging class of mediators for inflammation and it possess the capacity to hydrolyze lipoproteins and release lipid mediator from cellular membranes, this study sought to investigate the interaction between maslinic acid and hGIIA-sPLA2.

The results of this study demonstrated that maslinic acid enhances the intrinsic fluorescence of purified hGIIA-sPLA2 enzyme, thus suggesting maslinic acid’s direct interaction with the enzyme to form an enzyme-maslinic acid complex. In addition, maslinic acid inhibits hGIIA-sPLA2 enzyme activity and the mode of inhibition is non-competitive. The inhibitory activity of maslinic acid observed is in agreement with other studies reporting the inhibition of ursolic acid, an ursane-type pentacyclic triterpene, on sPLA2 enzymes isolated from snake venom and human inflammatory exudates [25]. The same study also showed that ursolic acid-mediated inhibition of hGIIA-sPLA2 enzyme is non-competitive and irreversible. The molecular interaction between maslinic acid and hGIIA-sPLA2 was further evaluated. We showed that maslinic acid forms a hydrogen bond with His47 and Asp48 amino acid residue. Both His47 and Asp48 are important hydrogen bond acceptors and calcium binding site which facilitates the hydrolysis mechanism by abstracting a proton from the water molecule then followed by nucleophilic attack on the sn-2 bond [20, 21]. In addition, maslinic acid also binds to Leu2, Phe5, Ala18, and Cys44 hydrophobic site. Specifically, Leu2 and Ala18 are interfacial residues of the hGIIA-sPLA2 enzyme. According to Winget et al., the interface is a flat surface which constitutes the active site of the PLA2 enzyme where it “sits” on the membrane phospholipid before executing its enzymatic activity [22]. We suggest that maslinic acid blocks the access of catalytic calcium ion required for enzymatic reaction and inhibits the binding of hGIIA-sPLA2 enzyme to the membrane phospholipid, thereby inhibiting the enzymatic activity of hGIIA-sPLA2.

Previous studies have established the immuno-modulatory function of hGIIA-sPLA2 in mediating monocyte recruitment and differentiation [13]. Immune cells recruited to the atherosclerotic lesions amplify the inflammatory response in that area and contributes to plaque instability. Our results show that, upon treatment, maslinic acid significantly reduced hGIIA-sPLA2-induced THP-1 cell differentiation and migration. The effect observed is specific to hGIIA-sPLA2 as cells treated with maslinic acid alone did not significantly affect the number of adherent and migrated cells. The basal level of differentiated and migrated cells in the absence of hGIIA-sPLA2 stimulation was relatively low and that maslinic acid treatment has no significant impact on the cellular process. Nevertheless, the molecular mechanism for the anti-inflammatory actions of maslinic acid in inhibiting hGIIA-sPLA2-induced cell differentiation and migration remains poorly understood. The hGIIA-sPLA2 enzyme is known to hydrolyze glyceroacylphospholipids from lipoproteins and cellular membranes, producing NEFA and lysophospholipids. The NEFA and lysophospholipids in turn induce monocyte recruitment to the lesional endothelial site. We hypothesized that maslinic acid binds and inhibits hGIIA-sPLA2 enzymatic activity, thereby reduces the release of NEFA and lysophospholipids which stimulates monocyte migration and differentiation.

Some findings have demonstrated that hGIIA-sPLA2-induced cell migration was mediated by protein kinase C (PKC) pathway [26]. Our previous study showed that maslinic acid suppresses PKC ßI, δ, and ζ activation and constitutive NF-κB activation in Raji B lymphoma cells [27]. Whether maslinic acid-mediated suppression of PKC/NF-κB activation is dependent on inhibition of hGIIA-sPLA2 in THP-1 monocytes requires further investigation. Recent studies have also found that compounds that inhibit the interaction between hGIIA-sPLA2 and integrin suppressed hGIIA-sPLA2-induced U937 human monocytic leukaemia cells adhesion and migration [28]. Subsequent research is also needed to establish the possibility of maslinic acid interfering with the hGIIA-sPLA2 activity in a manner independent of their lipolytic enzymatic activity.

Conclusion

Our findings provide insightful clues into the molecular interaction of maslinic acid and hGIIA-sPLA2. The results suggest that maslinic acid directly interacts with hGIIA-sPLA2 and inhibit their enzyme activity by binding to the calcium binding and phospholipid interfacial site which blocks the enzyme catalytic reaction. Maslinic acid also inhibited hGIIA-sPLA2-induced THP-1 cell differentiation and migration. There are possibilities that maslinic acid targets hGIIA-sPLA2 enzymatic activity itself and or acts as an inhibitor that blocks the direct binding of hGIIA-sPLA2 to integrins. Thus, it is suggested that further study needs to be conducted in regards to the mechanism involved.