Moringa oleifera Lam. leaf extract and its bio-active compound attenuate TNF-α-induced transendothelial migration of monocytes

doi:10.21203/rs.3.rs-2062774/v1

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Moringa oleifera Lam. leaf extract and its bio-active compound attenuate TNF-α-induced transendothelial migration of monocytes

https://doi.org/10.21203/rs.3.rs-2062774/v1

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Endothelial cells maintain blood fluidity, control regional blood flow and contribute to regulation of blood pressure, inflammation and blood clotting. The purpose of this study was to investigate into the effects of a bioactive compound from moringa leaf on endothelial cell production of inflammatory mediators and on monocyte adhesion and transmigration. EA.hy926 cells were pre-treated with crude Moringa oleifera Lam. leaf extract, the constituent fraction 6 of this extract and the bioactive component 3-HBI followed by stimulation with TNF-α. Endothelial cells incubated with the crude extract, fraction 6 or 3-hydroxy-β-ionone (3-HBI) and stimulated with TNF-α showed significantly lower production of pro-inflammatory mediators including IL-6, IL-8, and MCP-1 compared to untreated but TNF-α stimulated cells. The extract and active compound inhibited phosphorylation of IκB-α and reduced expression of the NF-κB p65, suppressing its nuclear translocation. They also reduced the expression of key adhesion molecules (ICAM-1, VCAM-1 and PECAM-1). Moreover, they decreased adhesion of THP-1 monocytes to EA.hy926 cells and monocyte transmigration through an endothelial cell monolayer. According to these findings, 3-HBI and crude moringa leaf extract have the possibility of preventing the development and progression of vascular diseases like atherosclerosis and neuroinflammation by inhibiting the release of pro-inflammatory cytokines, chemokines, and adhesion molecules.

Moringa oleifera Lam.

3-hydroxy-β-ionone

Endothelial cell

Monocyte

Anti-inflammation

Endothelial cells (ECs) form the monolayer which lines the lumen of blood and lymphatic vessels creating a semi-permeable barrier between the vasculature and the surrounding tissues. The endothelium is not only a barrier between blood and tissues but also an endocrine organ ¹. ECs maintain blood fluidity and control regional blood flow. They are important in regulating vessel wall permeability, platelet adhesion and aggregation, and leukocyte activation ².

Inflammation is normally a protective mechanism that is part of the first line of body host defense against microbial infection and response to tissue injury. Infection and injury stimulate resident cells of the innate immune system, such as mast cells, macrophages and dendritic cells, which leads to the production of cytokines and other proinflammatory mediators including interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α that activate nearby ECs ³. ECs also produce these pro-inflammatory mediators and the change in inflammatory state results in changes in thrombosis, vasomotor tone, and barrier function ⁴.

Pro-inflammatory mediators also stimulate ECs to express adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), and chemoattractants. Chemoattractants act to attract leukocytes including neutrophils and monocytes to the ECs while the adhesion molecules mediate the capture, rolling, arrest and crawling of leukocytes on the luminal EC surface ⁵. Leukocytes can subsequently migrate directly through the basement membrane and smooth muscle layer into the intima of the vessel wall. Several EC adhesion molecules and receptors participate in this transit across the endothelial barrier, including junctional adhesion molecules (JAMs) and platelet endothelial cell adhesion molecule 1 (PECAM-1) ^5,6. These actions induce leukocyte delivery to the inflammatory site ².

If acute inflammatory reactions fail to eliminate the triggering agent, or if there is some interference with the healing process, an acute inflammatory response may progress to the chronic stage. Although chronic inflammation progresses silently, it is considered a major contributor to several diseases including cardiovascular disease (CVD), neuroinflammation, diabetes, rheumatoid arthritis, and cancer ^7,8. CVD is the most common, with increasing prevalence worldwide and a high burden of morbidity and mortality. The number of deaths from CVD globally increased from 12.1 million in 1990, reaching 18.6 million in 2019 ⁹. Atherosclerosis is the primary cause of CVD which includes coronary artery disease, stroke and peripheral vascular disease ¹⁰. Atherosclerosis is a pathologic process affecting arterial walls. It is characterized by an accumulation of lipid droplets and immune cells such as macrophages, T cells, dendritic cells, and mast cells and a fibrous cap with smooth muscle cells and collagen in subendothelial regions ^11,12. Plaque growth can cause narrowing of the arterial lumen (stenosis) which can contribute to ischemia in the surrounding tissue. Atherosclerotic plaque rupture is the most common trigger of acute thrombosis of coronary arteries that causes myocardial infarction ^13,14.

Vascular ECs are involved in the initiation and progression of atherosclerosis and other inflammatory processes through controlling adhesion and transendothelial migration of circulating leukocytes ⁵. Decreasing the inflammatory responses of ECs and lowering the transmigration of leukocytes may be potential targets to inhibit the atherosclerotic process. Recently, Baker et al reported that eicosapentaenoic acid and docosahexaenoic acid from fatty fish can reduce the production of inflammatory cytokines and chemokines and the surface expression of ICAM-1 by EA.hy926 ECs ¹⁵. Another study showed that andrographolide effectively inhibits TNF-α-induced ICAM-1 expression and monocyte adhesion in EA.hy926 cells through suppression of the IKK/NF-κB signaling pathway ¹⁶.

Moringa oleifera Lam. (MO) is a tree that grows widely in many tropical and subtropical countries particularly in Thailand. MO is a source of traditional medicines and extracts and compounds with pharmacological properties ¹⁷. For example, MO leaves have been reported to be anti-inflammatory by inhibiting lipopolysaccharide (LPS)-induced production of nitric oxide and pro-inflammatory cytokines by RAW264.7 cells ¹⁸. In a previous study, we found that MO leaf extract and its bioactive component 3-hydroxy-β-ionone (3-HBI) inhibit inflammation through regulation of the NF-κB pathway ¹⁹. Furthermore, MO leaf extract lowers arterial blood pressure through inducing relaxation of small resistance arteries mainly via activating the eNOS-NO-sGC pathway ²⁰. Despite these findings, the anti-inflammatory effect of MO leaf extract and its bioconstituents on ECs has been underexplored. Therefore, this study aimed to investigate the effect of MO leaf extract and its bioactive compound 3-HBI on cultured EA.hy926 ECs following stimulation with TNF-α.

Cytotoxicity of moringa leaf extract and its bioactive compound on EA.hy926 cells

Viability of EA.hy926 cells was measured using the MTT assay. Five percent (IC5) and half maximal (IC50) inhibitory concentrations were calculated by dose–response relationships/sigmoidal curve fitting analysis (Fig. 1). The IC5 for crude MO extract, fraction 6, and 3-HBI on EA.hy926 cells was 65.1 µg/mL, 57.3 µg/mL, and 32.95 µg/mL, respectively (Fig. 1a). IC5 of MO crude extract, fraction 6, and 3-HBI, followed by 24 hours TNF-α (1 ng/ mL) exposure was 105.49 µg/mL, 147.88 µg/mL, and 73.4 µg/mL (Fig. 1b). Cell viability after treatment with crude MO extract, fraction 6, and 3-HBI with or without stimulation with TNF-α was always more than 80%. Cell exposure to TNF-α at 1 ng/mL for 24 h had no effect on cell viability (Fig. 1c).

Effects of moringa leaf extract and its bioactive compound on cytokine and chemokine production by EA.hy926 cells

Pro-inflammatory mediators were measured by Bio-Plex multiplex immunoassay system. TNF-α stimulation of EA.hy926 cells for 24 hours at 1 ng/mL led to markedly increased concentrations of all inflammatory mediators compared to unstimulated control cells cultured in DMEM. The concentrations of IL-6, IL-8, and MCP-1 were significantly lower in the medium of cells incubated with MO extract, fraction 6, or 3-HBI when compared with the TNF-α stimulated control (Fig. 2a-c). These results demonstrate that crude MO leaf extract and its constituent 3-HBI have anti-inflammatory potential.

Effect of moringa leaf extract and its bioactive compound on ICAM-1 (CD54) and PECAM-1 (CD31) expression on EA.hy926 cells

EA.hy926 cells were treated with crude moringa leaf extract, fraction 6, or 3-HBI for 24 hours followed by incubation with TNF-α (1 ng/mL) for 6 hours and then analyzed by flow cytometry. TNF-α significantly up-regulated cell surface ICAM-1 (85.47% positive cells) and PECAM-1 expression (93.54% positive cells) on EA.hy926 cells. The cells were 63.88%, 58.09%, and 68.96% ICAM-1 positive while the percentages of PECAM-1 positive cells were 84.69%, 83.08%, and 89.81% after culture with crude MO extract, fraction 6 and 3-HBI, respectively. Moringa extract, fraction 6 and 3-HBI treatment significantly decreased both ICAM-1 and PECAM-1 positive cells compared to TNF-α stimulated control (Fig. 3a-d). Moreover, crude MO leaf extract, fraction 6, and 3-HBI treatment significantly decreased the level of PECAM-1 and ICAM-1 expression on positive cells (MFI) compared to TNF-α stimulation (Fig. 3e-f). However, 3-HBI treatment did not affect the level of PECAM-1 expression (MFI).

Effects of crude moringa leaf extract and its bioactive compound on THP-1 adhesion to EA.hy926 cells

To understand how crude MO leaf extract and its bioactive compound influence the interaction between monocytes and endothelial cells, we analyzed whether crude extract and 3-HBI could affect the adhesion of monocytic THP-1 cells to an endothelial cell monolayer in our experimental conditions. Figure 4b demonstrates that TNF-α induced endothelial cell stimulation dramatically increasing the adherence of calcein-labeled THP-1 cells to EA.hy926 cells when compared to unstimulated cells (p ≤ 0.001). Pre-treatment of the TNF-α-activated EA.hy926 cells with crude extract, fraction 6 or 3-HBI significantly decreased calcein-labeled THP-1 adhesion, compared to TNF-α stimulated cells alone (p ≤ 0.001). Figure 4a shows images of fluorescence-labelled THP-1 monocytes bound to EA.hy926 cells with and without crude extract and 3-HBI exposure.

Effects of crude moringa leaf extract and its bioactive compound on THP-1 monocyte transmigration through an endothelial cell monolayer

In order to further confirm the effects of crude MO leaf extract and 3-HBI on monocyte migration, we analyzed the transmigration of monocytic THP-1 cells through an endothelial cell monolayer using a transwell transmigration assay. Endothelial cells were added into an insert and cultured until they formed a monolayer. Cells were pre-treated with MO leaf extract, fraction 6, or 3-HBI and stimulated with TNF-α (Fig. 5a). Then, THP-1 monocytes were labeled with fluorescent substrate (calcein AM) and placed into the endothelial cell monolayer upper chamber (Fig. 5b). TNF-α stimulation of the endothelial cells significantly increased the migration of THP-1 monocytes compared to unstimulated controls (p < 0.001). Pre-treatment with crude MO extract, fraction 6, or 3-HBI followed by TNF-α stimulation significantly decreased THP-1 monocyte transmigration through the endothelial monolayer (Fig. 5c).

Effects of crude moringa leaf extract and its bioactive compound on ICAM-1, VCAM-1, pNF-ᴋB P65 and pIkB-α protein expression in EA.hy926 cells

We next determined whether the TNF-α-induced NF-κB activation and downstream signaling pathway were inhibited by MO leaf extract and 3-HBI. TNF-α induced both NF-κB P65 and IκB phosphorylation, and the activation of NF-κB P65 and IκB-α was significantly attenuated by crude MO leaf extract, fraction 6, and 3-HBI pre-treatment of EA.hy926 cells (Fig. 6a, b). Moringa leaf extract and its compound significantly decreased ICAM-1 and VCAM-1 expression when compared to stimulation of TNF-α alone (p < 0.001) (Fig. 6b). These results suggest that MO extract and 3-HBI might reduce the expression adhesion molecules (ICAM-1 and VCAM-1) in EA.hy926 cells via suppression of NF-κB pathway.

An important arterial condition known as atherosclerosis is characterized by chronic inflammation that develops as a result of the recruitment of leukocytes, particularly monocytes, into the subendothelial region, followed by the production of several cytokines and the transformation of monocytes into foam cells ²¹. ECs play a critical role in the initiation and progression of atherosclerosis through leukocyte recruitment, expression of adhesion molecules and promoting transmigration of leukocytes into the intimal layer ¹⁴. Previously, Soe et al. showed that resveratrol blocks LPS-stimulated monocyte binding to ECs by inhibiting ICAM-1 expression ²². Another study found that MO leaf extract lowers arterial blood pressure through inducing relaxation of small resistance arteries mainly via activating the eNOS-NO-sGC pathway ²⁰. Our previous research highlights both anti-inflammatory and anti-cancer potential of MO leaf extract and its bioactive component 3-HBI ^19,23. The current study examines the activity of MO leaf extract, fraction 6 of this extract, and 3-HBI in TNF-α activated ECs. We found that MO leaf extract and 3-HBI significantly decrease production of cytokines and chemokines including IL-6, IL-8, and MCP-1 (Fig. 2), and decrease cell surface expression of adhesion molecules (ICAM-1, VCAM-1, and PECAM-1). This finding confirms the ability of MO leaf extract, fraction 6, and 3-HBI to exert anti-inflammatory activity on ECs by suppression of cytokine and chemokine secretion and adhesion molecule expression. Baker et al. described that plant-derived fatty acids, gamma-linolenic acid (GLA), pinolenic acid (PLA), dihomo-gamma-linolenic acid (DGLA), and eicosatrienoic acid (ETA) decrease production of ICAM-1, MCP-1, RANTES, and IL-6 by EA.hy926 cells ²⁴. Another study from Bork et al. demonstrated that a low ratio of linoleic acid (LA) to alpha-linoleic acid (ALA) had anti-inflammatory effects by lowering the secretion of VEGF, RANTES, ICAM-1, MCP-1 and IL-6 by TNF-α stimulated EA.hy926 cells ²⁵.

IL-6 is a cytokine with effects on regulation of the immune response and vascular inflammatory response via upregulation of adhesion molecules on the endothelium ²⁶. MCP-1 and IL-8 are chemoattractants facilitating the recruitment and attachment of circulating leukocytes to the sub-endothelial layer ²⁷. Hence, we further investigate the activity of crude MO leaf extract and 3-HBI on monocyte and endothelial cell interaction and monocyte transmigration. The results showed that adhesion of calcein-labeled THP-1 cells to EA.hy926 cells was significantly increased with TNF-α stimulation of ECs. Pre-treatment of TNFα-activated Ea.hy926 cells with crude extract, fraction 6 or 3-HBI significantly decreased monocytic THP-1 cell adhesion to EA.hy926 cell monolayers, compared to TNF-α stimulated cells alone (p ≤ 0.001). In addition, we also analyzed the transmigration of THP-1 monocytes through an EC monolayer by using a transwell transmigration assay. As demonstrated in Fig. 5, incubation with crude MO extract, fraction 6, or 3-HBI followed by TNF-α stimulation significantly decreased monocytic THP-1 cells transmigration through the EC monolayer. Before being able to transmigrate through the endothelial monolayer, monocytes have to roll over the monolayer and then firmly adhere to it. Activated ECs express adhesion molecules that interact with ligands on monocytes. This interaction allows monocytes to roll on the endothelium and transmigrate ²⁸. Our results highlight that MO leaf extract blocks monocyte adhesion and transendothelial migration by reducing inflammatory cytokine production and suppressing the expression of adhesion molecules on ECs. These effects of MO leaf extract and its contained compound 3-HBI could influence several pathological states, which involve monocyte-mediated inflammation, including atherosclerosis and neuroinflammation. Monocytes contribute critically to the initiation, progression, and thrombus formation stages of atherosclerosis and myocardial infarction; hence our findings are of significance. Monocytes are cells of the immune system that are central to the inflammatory response seen in hypercholesterolemia-associated trafficking to inflamed arteries, mainly dependent on chemokines, extravasation, subendothelial accumulation, and differentiation into macrophages. Eventually, these cells transform into pathogenic cholesterol-laden foam cells characteristic of atherosclerotic plaques ^21,28,29.

There are some studies reporting a role for TNF-α-induced NF-kB activation driving the expression of cytokines, chemokines and adhesion molecules (ICAM-1, VCAM-1 and PECAM-1) in ECs ^30,31. NF-κB is the major transcription factor involved in the regulation of inflammatory and immune genes linked to endothelial dysfunction and atherosclerosis ^32,33. Studies have shown the beneficial effect of Astragalus polysaccharides in suppressing ICAM-1 and VCAM-1 expression in TNF-α-treated human umbilical vein endothelial cells (HUVECs) ³⁴ and chrysin attenuates VCAM-1 expression and monocyte adhesion in LPS-stimulated brain endothelial cells by preventing NF-κB signaling ³⁵. Our current study shows that ICAM-1 and VCAM-1 expression increased in response to TNF-α as did both NF-κBP65 and IκB phosphorylation, and that this upregulation of ICAM-1, VCAM-1 and NF-κB signaling was significantly attenuated by crude MO leaf extract, fraction 6, and 3-HBI pre-treatment in EA.hy926 cells. Thus, these results indicate that MO leaf extract and 3-HBI might reduce the expression adhesion molecules (ICAM-1 and VCAM-1) in EA.hy926 cells and monocyte transendothelial migration via suppression of the NF-κB pathway. Our present findings are consistent with a previous study showing that resveratrol, a natural compound in grapes and red wine, suppressed TNF-α-induced monocyte adhesion genes and proteins in EA.hy926 cells ³⁶. Similarly, andrographolide down-regulates TNF-α-induced ICAM-1 expression at least in part via attenuation of activation of NF-κB in EA.hy926 cells ¹⁶.

The main message from this research is the ability of crude MO leaf extract and the bioactive compound 3-HBI to reduce several aspects of the inflammatory response in cultured ECs. If this effect occurred in vivo, it may at least partly prevent the initiation and progression of atherosclerosis by suppression of pro-inflammatory cytokine and chemokine secretion and adhesion molecule expression. Further we identify that the effects of the extract and 3-HBI are at least partially through suppression of the IKB-α/NF-κB signaling pathway. The activity and the mechanism of action of MO leaf extract and 3-HBI should be further explored in other cell systems and in relevant animal models.

In summary, the results of this study demonstrate that the production of cytokines (IL-6) and chemokines (IL-8 and MCP-1) and the upregulation of adhesion molecules (ICAM-1, VCAM-1, and PECAM-1) in ECs were triggered by TNF-α. Furthermore, adhesion of monocytic THP-1 cells to EA.hy926 cells and transendothelial migration of THP-1 monocyte were shown to significantly increase with TNF-α stimulation of endothelial cells. All these consequences of TNF-α stimulation can be reversed by pre-treatment with MO leaf extract (Fig. 7), which appears to be at least partially through suppression of the IKB-α/NF-κB signaling pathway. We suggest a possible link between the ability of MO leaf extract and 3-HBI, the bioactive compound in the extract, to protect against vascular endothelial inflammation and its down-regulating effect on the NF-κB signaling pathway. Our findings highlight MO leaf extract and 3-HBI as potential novel agents that can provide protection against inflammation and inflammatory diseases, especially where the endothelium is involved.

Preparation of MO leaf extract and its bioactive component

Growing of Moringa oleifera Lam. in the Thai province of Uthai Thani followed all applicable guidelines of Thai agricultural standard TAS 9001 − 2013 (good agricultural practices for food crop), and the leaf collection method and experimental use were certified as such by the Asia medical and agricultural laboratory and research center (AMARC 9001 − 2556). Plant dried powder (Lot. No.5534) were obtained from Khaolaor Laboratories Co., Ltd. (Samut Prakan, Thailand) as shown in supplementary material, Figure S1. The method for plant extraction were in accordance with all the relevant guidelines³⁷. Briefly, moringa leaf were macerated with EtOAc at room temperature. Extract was filtered through Whatman No.1 filter paper before rotary evaporation at 40°C. As described in our previous research¹⁹, fractions were separated from the crude EtOAc extract using flash column chromatography (FCC) (Merck, Darmstadt, Germany); gradient elution was performed using a solvent system with gradually increasing polarity including hexane, hexane-EtOAc and EtOAc-methanol (MeOH) and received 15 fractions (Fr.1–Fr.15). All fractions were subjected to screening for the inhibition of pro-inflammatory cytokine production. The fraction no. 6 was strongest activity then subjected for secondary fractionation by FCC. There were 17 sub-fractions (Fr.6.1–Fr.6.17), with only sub-fraction no.6.17 showing active anti-inflammation. Then it was partitioned by silica gel column chromatography, the result showed only one active anti-inflammation fraction (no.6.17.2) (supplementary material, Figure S2). Therefore, it was subjected to compound identification by LC-ESI-QTOF-MS/MS. MO extract and fractions were stored in vials at -20°C and protected from light until use.

By comparing the active compounds with MS and MS/MS data to available databases, a preliminary identification was made. An active substance, 3-hydroxy-β-ionone (3-HBI), was discovered in moringa sub-fractions. It was purchased for the experiments described here from Santa Cruz Biotechnology Inc. in Dallas, Texas, USA.

Cell lines and culture conditions

EA.hy926 ECs (ATCC® CRL926) were obtained from American Type Culture Collection (ATCC). Cells were cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic (Thermo Fisher Scientific, Inc., New York, NY, USA), and 1% HAT (100 µM hypoxanthine, 0.4 µM aminopterin and 16 µM thymidine) (Sigma-Aldrich, Missouri, MO, USA). THP-1 cells were maintained in RPMI 1640 Complete Medium supplemented with 10% FBS and 1% antibiotic-antimycotic (Thermo Fisher Scientific, NY, USA). All cells were incubated at 37°C with 5% CO₂, with medium renewal every 2–3 days.

Cellular cytotoxicity test

The MTT assay was used to determine the cytotoxicity of MO leaf extract and active compound. EA.hy926 cells were seeded in a 96-well plate at a density of 1 x 10⁴ cells/well and treated with various concentrations of extract, fraction 6 and active compound (3-HBI) and further exposed to complete medium with or without TNF-α (1 ng/ mL final concentration) for 24 hours. The supernatant was removed and 50 µl of MTT (0.5 mg/mL) (Invitrogen, CA, USA) in serum free medium was added and plates were incubated at 37°C for 3 hours. MTT reagent was removed, and formazan crystals were dissolved in 100 µl of DMSO. The absorbance was measured at 590 nm by a microplate reader (PerkinElmer, Inc., MA, USA). This method followed a previous protocol ³⁸. Five percent inhibitory concentration (IC5) and half-maximal inhibitory concentration (IC50) of crude EtOAc extract and 3-HBI were calculated by concentration response relationships/sigmoidal curve fitting analysis.

Measurement of pro-inflammatory mediator concentrations

EA.hy926 cells were seeded in 24 well plates and incubated for 24 hours. Cells were pre-treated with MO leaf extract (75 µg/ mL), fraction 6 (75 µg/ mL), or 3-HBI (50 µg/ mL) for 24 hours followed by stimulation with TNF-α (1 ng/ mL) for 24 hours. The concentrations of pro-inflammatory cytokines and chemokines including interleukin 6 (IL-6), interleukin 8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) were evaluated by using a Bio-Plex multiplex immunoassay system (Bio-Rad Laboratories Inc. United States). Reagents and standards were prepared in accordance with the instructions from the manufacturer. Plates were read using a Bio-Plex 200 multiplex reader (Bio-Rad Laboratories Inc. United States).

Determination of cell surface ICAM-1 (CD54) and PECAM-1 (CD31) expression

Cell surface markers were analyzed by immunostaining with PE-conjugated anti-CD54 and FITC-conjugated anti-CD31 (BD Biosciences, San Jose, CA, USA). EA.hy926 cells were seeded in 12 well plates and incubated at 37°C for 24 hours. Cells were exposed to MO leaf extract (75 µg/mL), fraction 6 (75 µg/mL), or 3-HBI (50 µg/mL) for 24 hours followed by stimulation with TNF-α (1 ng/ mL) for 6 hours. Cells from each condition were harvested and washed with PBS. Cells were suspended in FACS buffer solution and directly stained with fluorescence-conjugated antibodies to CD54 or CD31. The reaction was incubated at 4°C for 1 hour in the dark and the cells were then washed twice with PBS. Cell pellets were resuspended in staining buffer and cells analyzed using a FC 500 Flow Cytometer and CytoFLEX S Flow Cytometer (Beckman Coulter, Inc., Indianapolis, IN, USA). FITC-conjugated mouse IgG1, κ isotype control and PE-conjugated mouse IgG1, κ isotype control (BioLegend, Inc., San Diego, CA, USA) were used as a negative control.

Monocyte adhesion to endothelial cells

Adhesion of monocytic THP-1 cells to EA.hy926 cells was investigated by using the Vybrant Cell Adhesion Assay Kit (Thermo Fisher Scientific). EA.hy926 cells were incubated in 96 well plates for 24 hours. Cells were pre-treated with MO leaf extract (75 µg/ mL), fraction 6 (75 µg/ mL), or 3-HBI (50 µg/ mL) for 24 hours followed by incubation with TNF-α (1 ng/ mL) for 6 hours. THP-1 monocytes were prepared as a cell suspension at 3 x 10⁵ cells/mL. Calcein was added to THP-1 cell suspensions in serum free medium and the cells incubated for 30 minutes at 37°C. THP-1 monocytes were washed three times with serum free medium. Calcein-labeled THP-1 cells were co-cultured with treated EA.hy926 cells for 1 hour at 37°C. Non-adherent THP-1cells were removed by gentle washing, 100 µL PBS were added to each well and fluorescence was detected using a fluorescence plate reader at 480 nm/520 nm. THP-1 monocyte adhesion was measured as a percentage of control (TNF-α stimulated cells by without treatment with extract, sub-fraction of bioactive compound). Images of fluorescence-labeled THP-1monocytes bound to EA.hy926 cells were taken with a Nikon H600l Microscope.

Monocyte transmigration assay

THP-1 migration through EC monolayers was investigated by using a leukocyte transendothelial migration assay. EA.hy926 cells (5 x 10⁴) in 400 µL medium were added to each insert in a 24-well plate containing 500 µL of culture medium. Cells were cultured for 72 hours until the endothelial cells formed a monolayer. Cells were pre-treated with MO leaf extract, fraction 6, or 3-HBI and stimulation with TNF-α as described above. The culture medium was carefully removed and the insert transferred to a new well containing 500 µL of DMEM including Formyl-Methionine-Leucine-Phenylalanine (FMLP; 20 nM). Fluorescence-labeled THP-1 cell suspension was prepared and added to each insert and cultured for 12 hours at 37°C. The medium below the insert with the migrated cells was transferred to a 96-well plate suitable for fluorescence measurement on a fluorescence plate reader at 480 nm/520 nm.

SDS-PAGE and Western blot analysis

Total proteins of the EA.hy926 cells from each condition were extracted into ice-cold RIPA lysis buffer (Bio Basic Inc., NY, USA) in the presence of Halt Protease/Phosphatase Inhibitor Cocktails (Thermo Fisher Scientific, NY, USA), and centrifuged at 12,000 rpm for 15 minutes at 4°C. Quantification of total protein concentration was performed using the Bradford Coomassie-binding colorimetric method. The protein extract was mixed with an equal volume of 4 x Laemmli loading buffer and heated at 95°C for 5 minutes to denature the proteins. Samples were loaded into wells of 10 or 12% SDS-polyacrylamide gels alongside a blultra prestained protein ladder (6.5–270 kDa) which were subjected to electrophoresis (PAGE). Gels were run at 100 V for 10 minutes to get the proteins lined up in the gel followed by increasing the voltage to 150 V for 1–1.5 hours. Then proteins were transferred from the gels to a polyvinylidene fluoride membrane (Bio-Rad Laboratories Inc., Hercules, CA, USA). The membrane was blocked for 2 hours at room temperature with blocking buffer containing 5% bovine serum albumin (Capricorn Scientific GmbH, Germany) in Tris-buffered saline with Tween 20 (TBST) buffer. The membrane was then blotted using primary antibodies specific to β-actin, phospho-NFkB p65 (Ser536) (Thermo Fisher Scientific), phospho-IkB-α (Ser32) (Cell signaling Technology, Massachusetts, USA), ICAM-1, and VCAM-1 (Abcam Inc., MA, USA) on a rotator at 4°C for overnight. The membrane was then washed with TBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (H + L) secondary antibody (Thermo Fisher Scientific, NY, USA) for one hour at room temperature. The membrane was observed by soaking in chemiluminescence substrate for 5 minutes and placed in a ChemiDoc XRS + Imaging System (Bio-Rad Laboratories Inc., Hercules, CA, USA). The chemiluminescence signal of the blotted membrane was detected by Image Studio Lite software (LI-COR Corporate, Lincoln, NE, USA).

Statistical analysis

All experimental conditions were triplicated to provide reliable results. One-way ANOVA and Bonferroni multiple comparisons test were used for data analysis which was conducted using GraphPad Prism software. Confidential interval of 95% (p = 0.05) was used in all statistical analysis.

Data availability

All data generated or analysed during this study are included in this published article “Bioactive compounds in Moringa oleifera Lam. leaves inhibit the pro-inflammatory mediators in lipopolysaccharide-induced human monocyte-derived macrophages” (doi: 10.3390/molecules25010191).

Acknowledgements

This study was supported by Thailand Science Research and Innovation, and Naresuan University.

Author contributions

This research was contributed by authors “conceptualization, K.U. and Y.T.; methodology, T.L.; formal analysis, T.L.; investigation, T.L.; resources, P.P.; data curation, K.U., and Y.T.; writing—original draft preparation, T.L.; writing—review and editing, T.L., P.C. and K.U.; visualization, K.U. and T.L.; supervision, K.U.; funding acquisition, K.U., P.P. and Y.T. All authors have read and agreed to the published version of the manuscript.

Competing interests

The authors declare no competing interests.

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Moringa oleifera Lam. leaf extract and its bio-active compound attenuate TNF-α-induced transendothelial migration of monocytes

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Abstract

Figures

Introduction

Results

Cytotoxicity of moringa leaf extract and its bioactive compound on EA.hy926 cells

Discussion

Materials And Methods

Preparation of MO leaf extract and its bioactive component

Cell lines and culture conditions

Cellular cytotoxicity test

Measurement of pro-inflammatory mediator concentrations

Determination of cell surface ICAM-1 (CD54) and PECAM-1 (CD31) expression

Monocyte adhesion to endothelial cells

Monocyte transmigration assay

SDS-PAGE and Western blot analysis

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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