International Journal of Nutrition, Pharmacology, Neurological Diseases

ORIGINAL ARTICLE
Year
: 2022  |  Volume : 12  |  Issue : 3  |  Page : 112--119

The Combination of Decaffeinated Coffee (Coffea canephora) and Green Tea (Camellia sinensis) Reduced PSGL-1 Glycosylation by GALNT2 in Ox-LDL-induced RAW 264.7


Rohman Mohammad Saifur1, Sishartami Lintang Widya2, Widodo Nashi3, Rachmawati Ermin4, Lukitasari Mifetika5,  
1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Brawijaya, Malang, East Java, Indonesia; Brawijaya Cardiovascular Research Centre, Universitas Brawijaya, Malang, East Java, Indonesia
2 Master Program in Biomedical Sciences, Faculty of Medicine, Universitas Brawijaya; Department of Public Health, Faculty of Sport Science, Universitas Negeri Malang, Malang, East Java, Indonesia
3 Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya, Malang, East Java, Indonesia
4 Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, UIN Maulana Malik Ibrahim Malang, Malang, East Java, Indonesia
5 Brawijaya Cardiovascular Research Centre, Universitas Brawijaya, Malang, East Java; School of Nursing, Faculty of Health Sciences, Universitas Brawijaya, Malang, East Java, Indonesia

Correspondence Address:
Sishartami Lintang Widya
Faculty of Sport Science, Semarang Street Number 5, Universitas Negeri Malang, Malang City, East Java 65145, Indonesia
Indonesia

Abstract

Context: Coffee and green tea extract combination is expected to reduce macrophage migration. Aims: We investigated the effects of combination of coffee and green tea extracts on GALNT2 gene expression and PSGL-1 protein expression in Ox-LDL-induced RAW 264.7 cells. Materials and methods: RAW 264.7 cells were treated with a combination of coffee and green tea extracts with three different doses for 24 hours (coffee 80 μg/mL + green tea 80 μg/mL; coffee 160 μg/mL + green tea 160 μg/mL; coffee 320 μg/mL + green tea 320 μg/mL), respectively. Then, cells stimulated with 8 μg/mL Ox-LDL for 20 hours. GALNT2 mRNA expression was analyzed by reverse transcription polymerase chain reaction and western blot for PSGL-1 protein expression. Results: GALNT2 gene expression and PSGL-1 protein expression decreased significantly (P < 0.05) with treatment by combination of coffee and green tea extracts in dose-dependent manner. Conclusion: Coffee and green tea extract combination administration significantly reduced GALNT2 gene expression and PSGL-1 protein expression in Ox-LDL-induced RAW 264.7 cells.



How to cite this article:
Mohammad Saifur R, Lintang Widya S, Nashi W, Ermin R, Mifetika L. The Combination of Decaffeinated Coffee (Coffea canephora) and Green Tea (Camellia sinensis) Reduced PSGL-1 Glycosylation by GALNT2 in Ox-LDL-induced RAW 264.7.Int J Nutr Pharmacol Neurol Dis 2022;12:112-119


How to cite this URL:
Mohammad Saifur R, Lintang Widya S, Nashi W, Ermin R, Mifetika L. The Combination of Decaffeinated Coffee (Coffea canephora) and Green Tea (Camellia sinensis) Reduced PSGL-1 Glycosylation by GALNT2 in Ox-LDL-induced RAW 264.7. Int J Nutr Pharmacol Neurol Dis [serial online] 2022 [cited 2022 Dec 5 ];12:112-119
Available from: https://www.ijnpnd.com/text.asp?2022/12/3/112/357250


Full Text



Key Messages: This study showed that the administration of a combination of decaffeinated coffee and green tea extracts could reduce GALNT2 gene expression and PSGL-1 protein expression thus possibly inhibit the macrophages migration to the subendothelium in atherosclerotic conditions.

 Introduction



Cardiovascular diseases (CVDs) remain a major cause of death globally.[1] CVD can refer to a class of diseases that involves the heart or blood vessels, such as coronary heart disease (CHD).[2] In 2019, CHD causes 9.14 million deaths in the world.[1] The standard therapy for CHD is percutaneous coronary intervention with stent implantation to prevent restenosis. However, narrowing of the arterial diameter after stent placement or in-stent restenosis (ISR) can still occur at 10% patients after drug-eluting stent (DES) insertion.[3] ISR was also found to be associated with delayed neoatherosclerosis, which is generally associated with a poor survival prognosis in patients.[4] Neoatherosclerosis is the acceleration of atherosclerosis caused by various factors, such as chronic inflammation caused by endothelial dysfunction.[5],[6] Infiltration of macrophages plays a key role to the development of neoatherosclerosis. In the subendothelium, macrophages ingest oxidized lipoproteins and then macrophages will apoptosis which can promote the development of necrotic core.[6] In addition, macrophages death can release lipid that contribute to free cholesterol pooling, thereby forming a necrotic core.[7]

Macrophages have the GALNT2 gene encoding the polypeptide N-acetylgalactosaminyl-transferase 2 (ppGalNAcT2) which functions to add N-acetyl galactosamine to the hydroxyl group at the serine/threonine residue that occurs in the first step of O-linked oligosaccharide biosynthesis.[8],[9] In a study conducted in Taiwan, it was found that the GALNT2 gene is associated with the incidence of ISR in DES.[10] Macrophages express P-selectin glycoprotein ligand 1 (PSGL-1) that is a posttranslational modified (O-linked glycosylated) protein by ppGalNacTs encoded by the GALNTs gene, which regulates macrophages migration by binding to P-selectins on the endothelium.[11] The presence of an early stage of glycosylation by ppGalNacTs will trigger a further series of posttranslational modifications. Thus, GALNT2 and PSGL-1 could be therapeutic targets to reduce macrophages migration.

Coffee and tea are the most common consumed beverages around the world. Recently, coffee and tea consumption has been associated with CVD, but there are conflicting findings.[12] Some studies showed that coffee intake related to the risk of myocardial infarction.[13],[14],[15] But the other studies revealed that long-term coffee consumption is not associated with an increased risk of CHD and premature death from CVD in healthy individuals.[16],[17] One of the main compounds in coffee is caffeine that can increase acute blood pressure.[18] Tea also contains caffeine, although the amount is less than coffee.[19] Epidemiologic evidence suggests an association between consumption of green tea and a decreased risk of CVD.[20],[21] A study conducted in Japan suggests a moderate intake of coffee and green tea was associated with a lower risk of mortality from CVD.[22] In a previous study, it was shown that the administration of a combination of green tea and green coffee extracts showed a better effect on improving fasting blood glucose, triglyceride levels, and blood pressure in metabolic syndrome rat models.[23]

In this study, we used decaffeinated coffee and green tea extracts and combination doses. Many studies had conducted using coffee or green tea on macrophages. However, there has been no study about GALNT2 and PSGL-1 with coffee and green tea treatment. This study aimed to investigate the effect of decaffeinated coffee and green tea extracts on the migration of macrophages that play a role in the inflammatory process in atherosclerosis. This study was conducted in RAW 264.7 cells. This study suggested that combination of decaffeinated coffee and green tea could reduce the expression of GALNT2 gene and PSGL-1 protein in RAW 264.7 cells.

 Materials and Methods



This research was conducted in the Central Laboratory of Life Sciences and Molecular Biology Laboratory, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya, Malang, Indonesia.

Preparation of coffee and green tea extracts

The extract preparation of coffee and green tea are referred in previous studies.[24] Coffee was extracted from Robusta green coffee beans obtained from the Dampit, district of Malang, Indonesia. Green coffee beans roasted at 180°C for 3 minutes until first crack. The infusion method was used to extract coffee, by boiling mineral water for 10 minutes at 90°C, then it was filtered using fine filter paper. After that, the extract proceeds to the decaffeination process using activated carbon for 8 hours at 60°C.[25] Green tea leaves were obtained from Ciwidey, Bandung. Dried leaf rolls were used which were pale green in color and smelled like tea leaves. The dried tea leaves were decaffeinated by boiling with mineral water for 5 minutes at 50°C, then filtered using Whatman paper no. 1.[26] The filtered liquid was processed with infuse method for 29 minutes at 90°C. The decaffeination process of coffee and green tea has been optimized based on previous study.[24] Coffee and tea extracts used for culture treatment were prepared by dissolving in phosphate-buffered saline and then filtered using a 0.22-m membrane filter.

Ethics

This research has obtained ethical recommendation Number: 191/EC/KEPK-S2/11/2020 published by the Health Research Ethics Committee, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia.

Cell culture

The RAW 264.7 mouse macrophage cell line was obtained from Elabscience (Houston, TX, USA). RAW 264.7 cells were maintained in Dulbecco modified eagle medium (DMEM high glucose, Gibco 11965092) supplemented with 10% fetal bovine serum (Gibco 16000036), 100 IU/mL penicillin and 100 μg/mL streptomycin (P/S, Gibco 15140122) at 37°C in 5% CO2 atmosphere. Cells were randomly divided into five groups: N: cells + medium; C+: cells + Ox-LDL 8 μg/mL; P1: cells + Ox-LDL 8 μg/mL + coffee extract 80 μg/mL + green tea extract 80 μg/mL; P2: cells + Ox-LDL 8 μg/mL + coffee extract 160 μg/mL + green tea extract 160 μg/mL; P3: cells + Ox-LDL 8 μg/mL + coffee extract 320 μg/mL + green tea extract 320 μg/mL.

Cell viability assay

The effect of coffee and green tea treatment on cell viability was measured by the WST-1 assay, purchased from Roche Diagnostics (Mannheim, Germany). On this assay, the living cells had the ability to reduce the tetrazolium salt to a soluble formazan product which results in a color change. RAW 264.7 cells were cultured in a 96-well plate with initial cell density 5 × 105 cells/well in triplicate for 24 hours and treated with combination extract of coffee and green tea at different concentration ranging from 0 to 640/640 μg/mL for 24 hours. After that, WST-1 reagent was added to each well and blank samples followed by incubation at 37°C in 5% CO2 atmosphere for 1 hour. Cell viability was determined by the formazan dye formed by live cells and the absorbance measured using a microplate reader BioTek ELx808 and Gen5 reader software at the wavelength of 450 nm. In control cells, the optical density of formazan was considered as 100% of viable cells.

Loewe additivity model measurement

Synergy Finder 2.0: visual analytics of multidrug combination synergies was used to determine the synergism of coffee and green tea extract. The combination response (7 × 7) matrix represented percentages of viability cells, with control cells without treatment set to 100%. Concentrations of coffee and green tea extracts ranged from 0 to 320 μg/mL were used individually as well as in a 7 × 7 concentration matrix. The synergy of combination coffee and green tea was measured using the Loewe additivity model.[27] The Loewe additivity model calculates the desired response as if the two drugs were the same. Thus, slices through the response surface for each selected effect should show a linear relationship between the doses of the two drugs.

Reverse transcription polymerase chain reaction (RT-PCR) analysis

RAW 264.7 cells were cultured (2 × 105 cells/well) in a 12-well plate overnight. Cells were treated with a combination of coffee and tea with three different doses for 24 hours (coffee 80 μg/mL + green tea 80 μg/mL; coffee 160 μg/mL + green tea 160 μg/mL; coffee 320 μg/mL + green tea 320 μg/mL), respectively. Then, cells stimulated with 8 μg/mL ox-LDL for 20 hours. Ox-LDL was purchased from Invitrogen by Thermo Fisher Scientific (Life Technologies Corporation, Eugene, OR, USA). Negative control cells were not treated and stimulated. Total RNA was extracted using PRImeZOL (Canvax, Córdoba, Spain) and used for cDNA synthesis (Promega Co, Madison, WI, USA) following the manufacturer’s instruction. The PCR primers used in this study were listed: GALNT2 forward 5′-CATTTCTACTACGCAGCGGT-3′, GALNT2 reverse 5′-TCCTTTGTCAAGGCCCATTC-3′; β-actin forward 5′-TGAGAGGGAAATCGTGCGTGACAT-3′, β-actin reverse 5′-ACCGCTCATTGCCGATAGTGATGA-3′. PCR was setup under the following sequence: denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds followed by final extension for 7 minutes at the end of 29 cycles. PCR products were carried out on 1.5% (w/v) agarose gels and photographed using ImageQuart LAS 500. The band intensities were quantified by ImageJ software (version 1.50i for Windows; NIH, Bethesda, MD, USA) and were expressed relative to β-actin band.

Western blot analysis

RAW 264.7 cells were cultured in 100 mm dish (1 × 107 cells/dish) for each group and incubated for 24 hours at 37°C. The next day, cells were treated with combination coffee 80 μg/mL + green tea 80 μg/mL; coffee 160 μg/mL + green tea 160 μg/mL; coffee 320 μg/mL + green tea 320 μg/mL for 24 hours then stimulated with Ox-LDL for 20 hours. Cells were harvested and centrifugated. After removal of supernatants, the pelleted cells were lysed and protein was extracted by adding RIPA buffer (Cell Signaling, Beverly, MA, USA). Equal amounts of protein (20 μL) were separated by electrophoresis on 12% sodium dodecyl sulfate‑polyacrylamide gels and transferred to a polyvinylidene fluoride membrane using The Bio-Rad Trans-Blot Semi-Dry (Bio-Rad Laboratories, Inc., California, USA). The membrane was blocked for 1 hour with 5% skimmed milk in Tris-buffered saline (Tris-HCl pH 7.5) contained 0.05% Tween-20 to block nonspecific binding. The membranes were then incubated with primary antibody against PSGL-1 (MyBiosource, San Diego, CA, USA) for overnight at 4°C and washed three times for 10 minutes each with TBS-T. TBS-T is a mixture of tris-buffered saline (TBS) (a buffer solution) and Polysorbate 20 (a polysorbate-type nonionic surfactant). Polysorbate 20 is also known as Tween 20, a commercial brand name. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Invitrogen by Thermo Fisher Scientific, Life Technologies Corporation) for 1 hour at room temperature and washed with TBS-T three times. Then, the bands were visualized with an enhanced chemiluminescence kit western blotting detection reagents (Thermo Scientific, Rockford, IL, USA). The protein bands were measured by ImageJ software.

Statistical analysis

The data are presented as mean values ± standard deviations (SD) of the results of at least three replicates and analyzed using the SPSS version 25.0 software (SPSS Inc., Chicago, IL, USA). The mean difference between groups was measured using one-way analysis of variance followed by post hoc test with P < 0.05 was considered statistically significant.

 Results



Effect of combination of decaffeinated coffee and green tea extracts on viability of RAW 264.7 macrophages

Viability test was carried out to determine the toxicity and the concentration range for experiments. Treatment of RAW 264.7 cells with combination coffee and green tea extracts enhanced the viability of cells with no cytotoxic effect [Figure 1]. [Figure 1] shows that the cell viability was above 100% for all combination doses. At combination doses less than 40/40 μg/mL, the cell viability was above 160%. At combination doses between 40/40 and 320/320 μg/mL, the cell viability was stable at 120% to 140%. The lowest cell viability was found at combination dose of 640/640 μg/mL with the range of 100% to 120%. From previous study, there is no cytotoxic effect of coffee single extract and green tea single extract. Moreover, combination of coffee and green tea could inhibit foam cell formation at dose 160/160 and 320/320 μg/mL.[28] Therefore, this experiment used three different combination doses (80/80, 160/160, and 320/320 μg/mL) of coffee and green tea extracts [Table 1].{Figure 1}{Table 1}

Synergistic effects of decaffeinated coffee and green tea extracts

Loewe additivity model was used to measure the synergistic effects of coffee and green tea extracts through Synergy Finder 2.0. [Figure 2] shows red, white, and green color. As the red color intensity gets higher, the higher the synergistic score obtained. The Loewe score above 10 showed that coffee and green tea extracts have synergistic effects. At doses above 80/80 μg/mL, the higher red color intensity indicated Loewe energy scores more than 10. A Loewe synergy score of 13.52 was observed in combination-treated cells which supported the synergistic effects of coffee and green tea extracts.{Figure 2}

Effects of combination of decaffeinated coffee and green tea extracts on mRNA level GALNT2

The analyses showed that mRNA levels GALNT2 were increased (P = 0.000) by treatment of Ox-LDL compared to the control group [Figure 3]. The results revealed that mRNA levels GALNT2 were significantly lower (P = 0.000) in a doses-dependent manner by treatment with combination of coffee and green tea extracts compared to the C(+) group. Cells treated with combination of coffee and green tea extracts at the dose of 320/320 had significantly lower GALNT2 mRNA expression compared to 80/80 group (P = 0.017) and C(+) group (P = 0.000). Moreover, no significant difference in GALNT2 mRNA expression observed between 80/80 and 160/160 group or 160/160 and 320/320 group [Table 2].{Figure 3}{Table 2}

Effects of combination of decaffeinated coffee and green tea extracts on PSGL-1 protein expression

The results showed that PSGL-1 protein expression in RAW 264.7 cells was increased (P = 0.000) after Ox-LDL administration compared to the control group [Figure 4]. The analysis revealed that PSGL-1 protein expression was significantly lower (P = 0.000) by combination of coffee and green tea extracts in a dose-dependent manner compared to the C(+) group. In the group treated with dose 320/320 μg/mL, there was a significantly lower (P = 0.049) of PSGL-1 protein expression compared to the group treated with dose 80/80 μg/mL. Cells that treated with dose 160/160 μg/mL had no significant difference of PSGL-1 protein expression compared to groups treated with dose 80/80 μg/mL (P = 0.427) and 320/320 μg/mL (P = 0.189) [Table 3].{Figure 4}{Table 3}

 Discussion



This study demonstrated the effects of combination of coffee and green tea extracts on RAW 264.7 cells. Coffee contains many polyphenols, especially chlorogenic acid (CGA) which is known to have antioxidant abilities.[29] A study using rat cardiomyocytes showed that CGA was not cytotoxic and could stabilized cardiomyocyte membranes.[30] Green tea is rich in polyphenols (30% dry weight), including flavanols, flavandiols, flavonoids, and phenolic acids. Most green tea polyphenols are flavonols, which are better known as catechins. Catechins mostly consist of epigallocatechin gallate (EGCG), epigallocatechin, epicatechin gallate, and epicatechin.[31]

RAW 264.7 cells were induced with Ox-LDL as observed in the early stages of atherosclerosis. Oxidized LDL displaying characteristics different from native LDL that make them proatherogenic and Ox-LDL increase adherence of monocytes to the endothelium.[32],[33],[34] In this study, PSGL-1 protein was increased in Ox-LDL-induced cells. A study was reported that PSGL-1 plays pivotal roles in the inflammatory process of atherogenesis.[35] Furthermore, this study revealed that PSGL-1 protein expression decreased significantly with administration of combination of coffee and green tea extracts. Previous study showed that administration of coffee extract with a concentration of 500 μg/mL could downregulate the CXCL13 gene and reduced tumor necrosis factor-α, interleukin-6, and MCP-1 (Monocyte chemoattractant protein-1) in LPS(Lipopolysaccharides)-induced THP-1 cell line is a suitable in vitro cell model to study modulation of monocyte and macrophage functions.[36],[37] Moreover, a study by Hong et al. demonstrated that EGCG inhibits MCP-1 expression by blocking p38 MAPK induced by PMA (phorbol 12-myristate 13-acetate), AP-1 (The Activator Protein-1), and nuclear factor-κB in endothelial cells, thereby reducing migration of monocytes.[38] The other studies revealed that EGCG prevents endothelial adhesion molecule expression, reduces monocyte adhesion, and inhibits neutrophil migration through cultured endothelial monolayers.[39],[40],[41],[42]

Recruitment of monocytes/macrophages to the vascular wall is a key feature in the pathogenesis of atherosclerotic lesions.[34] Phillips et al. showed that the PSGL-1 blocking significantly limits neointima formation and macrophage accumulation after carotid denudation injury at 28 days in the atherosclerosis-prone apoE-/- mouse on a Western diet.[43] A study using PSGL-1–deficient mice demonstrated that early neutrophil migration after chemically induced peritonitis is impaired in PSGL-1–deficient mice. These experiments using PSGL-1–deficient mice have established that PSGL-1 is a critical P-selectin ligand early in inflammation.[44] PSGL-1 deficiency could inhibit the adhesion of endothelial cells and leukocytes through cytokines, and reduced the atherosclerosis in apoE-/- mice.[45] It suggested that decreased in PSGL-1 could inhibited leukocytes migration and inhibit development of atherosclerosis.

In the context of leukocyte recruitment, protein glycosylation plays an important role in selectin–selectin ligands mediating leukocyte capture and scrolling. PSGL-1 or CD162 is a highly glycosylated homodimeric sialomucin, which binds to P-, E-, and L-selectin under both static and dynamic conditions.[46] PSGL-1 is a 2-O-glycan core protein. The initial stage of O-glycan biosynthesis is the addition of galactosamine (GalNAc) to serine or threonine residues in proteins, catalyzed by polypeptide GalNAc transferase (ppGalNAcT).[11] Polypeptide GalNAc-transferases (ppGalNAc T2), an enzyme that initiates mucin-type O-glycans, are well known to be produced in monocytes/macrophages.[8] Studies have shown that O-glycans and GALNTs genes play critical roles in a variety of biologic functions and human disease development. GALNT2 gene expression was found to be upregulated by Ox-LDL in RAW 264.7 cells in this study. This study showed that treatment with combination of coffee and green tea extracts significantly improved GALNT2 gene expression in dose-dependent manner.

GALNT2 is the gene that encodes UDP-N-acetyl-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyl transferase 2 also known as ppGalNacT2. ppGalNacT2 is a member of the GalNac-transferase family, enzymes that transfer N-acetyl galactosamine to the hydroxyl group of serine/threonine in the first step of O-oligosaccharide biosynthesis.[9] Moreover, single-nucleotide polymorphisms of GALNT2 have been associated with coronary artery disease.[47] So far, GALNT2 studies revealed its effects on lipid profiles and cancer cell proliferation.[48],[49],[50],[51],[52],[53] However, this study explored GALNT2 in RAW 264.7 macrophages cells. GALNT2 expected to play a role for early step of PSGL-1 glycosylation. This study demonstrated that the increase of GALNT2 gene expression had similar pattern with PSGL-1 protein expression. These results indicated possible role of GALNT2 on PSGL-1 glycosylation for monocytes/macrophages migration.

The results of this study are expected that combination of coffee and green tea extracts can reduce the migration of macrophages/monocytes in atherosclerotic conditions. The limitation of this study was the exact mechanism of coffee and green tea extracts on GALNT2 gene expression and PSGL-1 protein expression still unclear. This study did not examine direct pathway of GALNT2 for PSGL-1 glycosylation and did not analyze macrophage migration ability. Further studies are needed on the PSGL-1 glycosylation pathway by GALNT2 and the effect of combination of coffee and green tea extracts on macrophage migration.

 Conclusion



In conclusion, this study revealed the effects of combination of coffee and green tea extracts on GALNT2 gene expression and PSGL-1 protein expression in Ox-LDL-induced RAW 264.7 cells. This study can be used as a reference for further research on the benefits of combination of coffee and green tea extracts both in vitro and in vivo.

Financial support and sponsorship

This research was support by Ministry of Research, Technology, and Higher Education of the Republic of Indonesia.

Conflicts of interest

There are no conflicts of interest.

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