Decaffeinated coffee and green tea extract inhibit foam cell atherosclerosis by lowering inflammation and improving cholesterol influx/efflux balance through upregulation of PPARγ and miR-155

Coffee and green tea extraction

The raw material used in this study was Robusta green coffee beans obtained from the the Dampit area, Malang Regency, Indonesia. The sample used was in the form of powdered green coffee beans that had been roasted at 180° C for 3 minutes (until the first crack of the bean). Three green tea leaves were retrieved from the highest part of trees in the Ciwidey area in Bandung, for the tea extract preparation. The selection of dried green tea was based on final appearance: a rolls shape, a pale green colour, and a sweet scent.

Coffee decaffeination was carried out following the Fischer method.²⁴ The green tea decaffeination process was performed using the blanching method by Liang, 2007,²⁵ whereas green tea and coffee extraction followed the process performed by Vuong et al., 2011, with a water-to-tea/coffee ratio of 20:1 ml/g.²⁶ The optimized boiling time for coffee and tea extraction, boiling water time for tea decaffeination, and addition of agent adsorption of caffeine in coffee decaffeination were decided using response surface methodology (RSM) Box-Behnken design (BBD) in Design Expert 7.1.5 (StatEase) software.

Briefly, the light-roasted ground coffee beans were boiled in mineral water for 10 minutes at 90°C, followed by filtration with fine filter paper. The filtrate then was subjected to the decaffeination process, using activated charcoal for 8 hours at 60° C.²⁷ The tea dried leaves were boiled in mineral water for 5 minutes at 50°C, then filtered using Whatman paper No 1.^25,28 The filtrate was infused in 90°C water for 30 minutes. Stock solutions were freshly prepared by dissolving the coffee and tea extract (64 mg/ml) and filtering the solution through a 0.22 μm-pore size membrane filter.

Raw 264.7 cell culture

Macrophages from the RAW 264.7 mouse cell line were obtained from ATCC (CVCL_0493). The passage number 10-20 was used in this study. Raw 264.7 cells were cultured in complete medium containing Dulbecco’s modified Eagle medium (DMEM) high glucose, Gibco 11965092), supplemented with 10% fetal bovine serum (FBS, Gibco 16000036) and 1% penicillin/streptomycin (P/S, Gibco15140122). The cells were incubated in a humidifier incubator at 37° with 5% CO₂. To induce macrophage type 2 (M2) differentiation and ultimately establish a foam cell model, Raw 264.7 cells were cultured with recombinant mouse M-CSF 50 ng/ml (BioLegend 576406) for 24 hours, then stimulated with 50 μg/ml oxLDL (Thermofisher L34357) for another 24 hours. DCGTE was administered at 2 different doses (160/160 and 320/320 μg/ml). The cells were divided into 4 groups, K, K+, P1, P2 as follows: complete medium + 50 ng/ml M-CSF (K), complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL (K+); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 160/160 μg/ml DCGTE (P1); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 320/320 μg/ml DCGTE (P2).

Oil red O staining

Briefly, the cells were cultured at 15,000 cells/well in 24-well plates placed with a coverslip until reaching 60-70% confluency. The medium was discarded and cells were rinsed twice with PBS, then fixed in 10% paraformaldehyde for 10 minutes. The cells were washed with phosphate buffer saline (PBS) twice (for 3 minutes) again, and were incubated with 60% isopropanol for 30 minutes to facilitate the staining of neutral lipids. The cells were stained with filtered Oil Red O (ORO) solution (Sigma-Aldrich, MAK194) at 37°C for 30 minutes in darkness. The cells were washed with 60% isopropanol for 5 seconds, followed by 5 repeated washing steps with PBS. Positively stained (red cytoplasm) cells were macrophage-derived foam cells, which were observed via light microscope (Olympus Bx51), photographed with Optilab Advance Plus MTN016, and analysed using Optilab Viewer v3.0 software.²⁹

Lipid absorbance quantification

100% 2-propanol was added to the ORO-stained plates (500 μl per well on the 24-well plates). The plates were incubated for 40 minutes at room temperature on an orbital shaker in darkness. 200 μl eluates were transferred to a clear 96-well microtiter plate (polystyrene). As a control, 3 microtiter plate wells were filled with 200 μl of 2-propanol. Absorption was measured in duplicates at 492 nm.³⁰

CD36 and ABCA1 expression

The cells were pipetted into a 24-well plate with a 15,000 cells/well initial cell density. Cultured cells were stained by plating the cells on glass coverslips. After treatment, the cells were washed 3 times with PBS and fixed with 4% paraformaldehyde for 30 minutes at room temperature. The cells were washed twice with PBS (three minutes), blocked with 2% bovine serum albumin (BSA) for 1 hour at room temperature, and incubated with anti-CD36 monoclonal antibody 1:150 (Santa Cruz, Cat. No. sc3709) and Rabbit antiphospo-ABCA1 antibody 1:200 (Bioss, Cat. No. bs-12956R) at 4°C overnight. The cells were washed 3 times with PBS and incubated with anti-rabbit IgG antibody rhodamine conjugated (Rockland Cat. No. 6111002-0500, RRID: AB_11181881) and Fab Goat anti-mouse IgG antibody FITC (Rockland Cat. No. 710-1202; RRID: AB_218843) for 1.5 hours in light-shielded conditions. Nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI; BioLegend Cat. No. 422801 ID = BLG2181) for five minutes in the dark. After thre washes with PBS, coverslips were mounted with glycerol. The cells were visualized using an Olympus IX71 Fluorescent Microscope at 40× magnification, and then photographed with Olympus Cell Sens software version 3.2. The pixel intensities in the cell cytoplasm that reflecting the expression levels were quantified using Image J (Fiji) software and presented as percentages of CD36 or ABCA1 expression, then compared statistically.

Tumor Necrosis Factor (TNF) α and Interleukin (IL)10 expression

RAW 264.7 cells were seeded in 24-well plates at 1.5·104 cells/well to get confluency 80-100% in 5^th day. Media were collected and centrifuged at 2500 rpm for 7 min. TNFα and IL10 content of the media was then determined by a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) using the Mouse TNFα ELISA Kit (BT Lab E0117Mo) and mouse IL10 ELISA kit (BT Lab E002Mo) according to the manufacturer’s instructions in ELISA reader BioTek ELx808 using Gen5 reader software.

miR-155 expression

This procedure was followed in the following steps: isolation of total RNA, cDNA synthesis, and Real-Time PCR. Total RNA from cells was extracted by using the miRVana (Ambion Thermofisher AM1560) reagent kit according to the manufacturer’s protocol.³¹ Reverse transcription polymerase chain reaction (RT-PCR) was conducted in a single process using an RT-PCR kit from Qiagen (miRCURY LNA RT kit 33430): incubation for 60 minutes at 42°C, followed by incubation for 5 minutes at 95°C to heat-inactivate the reverse transcriptases, and cooling at 4°C.

The RT-qPCR was run using a PCR kit (Qiagen miRCURY LNA SyBR Green PCR kit 33435) in a, Bio-Rad CFX. The primer sequences for miR-155 amplification were: F 5′ TGC GGT TAA TGC TAA TTG TGA TA 3′; R 5′ GTG CAG GGT CCG AGG T 3′. Normalisation of miR-155 expression was done using U6 as a housekeeping gene. Each PCR amplification was performed under the following conditions: PCR initial heat activation for two minutes at 95°C, denaturation at 95°C for 10 minutes, combined annealing/extension at 56°C for 60 seconds through 40 cycles.

Purification of cellular nuclear extracts

The cells were grown to 80% confluency on a 100 mm plate dish culture. After the cells were washed with PBS twice, they were scraped into a 15 ml conical tube. The cells were centrifuged for 5 minutes at 1000 rpm. The nuclear protein isolation was done using a nuclear extraction kit from ABCAM ab113474. The pellet was resuspended in 100 μl 1X pre-extraction buffer, supplemented with dithiothreitol (DTT) and protease inhibitor cocktail (PIC) per 10⁶ cells, then incubated on ice for 10 minutes. The pellet was vortexed vigorously for 10 seconds and centrifuged for 1 minute at 12,000 rpm. The nuclear pellet was added with two volumes of extraction buffer containing DTT and PIC at about 10 μl per 10⁶ cells, then were incubated on ice for 15 minutes and vortexed (five seconds) every 3 minutes. The last step was centrifugation of the suspension for 10 minutes at 14.000 rpm at 4°C. The supernatant contained protein nuclear extract.

PPARγ expression and activity

Primers were designed according to the sequences in GenBank as follows: β-actin: F: 5′ TGA GAG GGA AAT CGT GCG TGA CAT 3′, R: 5′ ACC GCT CAT TGC CGA TAG TGA TGA 3′; PPARγ: F: 5′AAC TGC AGG GTG AAA CTC TGG GAG ATT CTC C-3′, R: 5′ GGA TTC AGC AAC CAT TGG GTC AGC TCT3′. Standard 25 μL-solution polymerase chain reaction (PCR) with 2 μL of cDNA after reverse transcriptase reaction was performed, with the following parameters: initial heat activation at 95°C for two seconds, denaturation process at 95°C for 40 seconds (three cycles), annealing for 40 seconds at 57°C (three cycles), 55°C (three cycles), 52°C (29 cycles), extension for 45 seconds at 72°C with TaKaRa Ex Taq Hot Start Version (TaKaRa, Japan), in a MJ Research PTC-200 Peltier Thermal Cycler. The PCR reaction product (10 μL) was run through 1,5% agarose gel by electrophoresis. Densitometric quantification of the band intensities was conducted using Image J (Fiji) software.

PPARγ activity was analysed using the PPARγ Transcription Factor Assay Kit (ab133101, Abcam, MA, USA) according to the manufacturer's protocol. Briefly, the nuclear protein extracts were collected as described earlier and added to a 96-well plate containing a specific double stranded DNA (dsDNA) sequence, including the peroxisome proliferator response element (PPRE) at the bottom of the wells. Then, briefly, the primary antibody was added to each well, followed by goat anti-rabbit HRP, and developing and stop solutions supplied in the kit, respectively. PPARγ activity was detected using a BioTek ELx808 ELISA reader, connected with a Gen5 3.0 software at OD 450 nm.

Statistical analysis

Data were expressed as mean ± standard error (SE). All parameters were measured in at least three independent experiments (triplicate). One-way analyses of variance (ANOVA) were performed. The significant statistical difference threshold was defined for p < 0.05.

The decaffeinated coffee and green tea extract inhibit foam cell formation

First, we investigated the effects of the decaffeinated coffee and tea extract (DCGTE) on Raw 264.7 macrophages treated with 50 ng/ml M-CSF and 50 μg/ml ox-LDL. The assessment of foam cells characteristics was performed using ORO staining, followed by measuring the absorbance to quantify the lipid content in the cell. Raw 264.7 cells exposed to DCGTE at a concentration of 160/160 and 320/320 μg/ml elicited an augmentation in foam cell numbers and lipid accumulation at 24 hours compared to the positive control (50 μg/ml oxLDL) (p = 0.000**, Figure 1).

Figure 1. Effect of DCGTE on foam cell number and lipid content in oxLDL-stimulated Raw 264.7 cells.

The foam cell was characterized by red color in cytoplasmic part observed with microscope Olympus BX51 200× magnification. ∗ p < 0.05, relative to C, ∧ p < 0.05 relative to C(+), # p < 0.05 relative to P1.

Culture groups: complete medium + 50 ng/ml M-CSF (K); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL (K+); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 160/160 μg/ml DCGTE (P1); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 320/320 μg/ml DCGTE (P2).

The decaffeinated coffee and green tea extract reduced CD36 and increased ABCA1 expression in ox-LDL stimulated macrophages

Foam cells could be formed due to a dysregulation of the lipid metabolism, which was determined by the continuous uptake of ox-LDL and failure in lipid efflux from macrophages.^1,2,6 The endocytosis of ox-LDL is mainly regulated by CD36,^8,9 whereas ABCA1 plays a significant role in cholesterol efflux.^32,33 Thus, this study aimed to investigate the effect of the extract in the cells examined using the immunofluorescence technique. Figure 2 depicts the CD36 and ABCA1 expression after being incubated with the extract for 24 hours in M-CSF and ox-LDL-stimulated Raw 264.7. One-way ANOVA tests showed significant differences in CD36 (p = 0.000**) and ABCA1 (p = 0.000**) expression in DCGTE-exposed samples compared to positive control (K+). Therefore, we concluded that the administration of decaffeinated coffee and green tea extract could suppress CD36 and induce ABCA1 expression in Raw 264.7 cells.

Figure 2. Effects of DCGTE on CD36 and ABCA1 expression in oxLDL-stimulated macrophages.

The blue colour indicated nuclear staining, green for CD36 with FITC, red for ABCA1 with rhodamin fluorochrome. The expression was observed using a fluorescent microscope 400× magnification and quantify with Image J as percentage/200 cells. ∗ p < 0.05, relative to C, ∧ p < 0.05 relative to C(+), # p < 0.05 relative to P1.

Culture groups: complete medium + 50 ng/ml M-CSF (K); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL (K+); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 160/160 μg/ml DCGTE (P1); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 320/320 μg/ml DCGTE (P2).

The decaffeinated coffee and green tea extract attenuated TNFα and enhanced IL10 secretion in ox-LDL-stimulated macrophages

The inflammation process mediates the increase in CD36 expression¹¹and degradation of ABCA1.^34,35 Hence, this current study also tried to investigate the effect of the extract in inhibiting the inflammation process, by examining TNFα and IL10 production. Figure 3 shows a reduced TNFα concentration in the group administered with 160/160 μg/ml and 320/320 μg/ml extracts compared to control (p = 0.012; p = 0.000). Consistently with the result from inflammatory cytokine production, the data shows that IL10, an anti-inflammatory cytokine, was higher in groups treated with the 160/160 μg/ml and 320/320 μg/ml extracts compared to control (p = 0.002; p = 0.000). Therefore, we concluded that administration of DCGTE at concentrations of 160/160 and 320/320 μg/ml gradually inhibited TNFα and stimulated IL10 production in Raw 264.7 cells.

Figure 3. DCGTE effect on inflammation process.

On the 4th day after seeding, the cell medium was taken and analyzed for cytokine protein content by ELISA. ∗ p < 0.05, relative to K, ∧ p < 0.05 relative to K(+), # p < 0.05 relative to P1.

Culture groups: complete medium + 50 ng/ml M-CSF (K); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL (K+); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 160/160 μg/ml DCGTE (P1); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 320/320 μg/ml DCGTE (P2).

The decaffeinated coffee and green tea extract increased mRNA and PPARγ activity in ox-LDL-stimulated macrophages

The signalling pathway that mediates DCGTE is also explored in this present study. PPARγ is an antiatherogenic transcription factor that contributes to the suppression of foam cell formation.³⁶ Figure 4 demonstrates that both the expression and activity of PPARγ, after cells were administered with DCGTE at 160/160 and 320/320 μg/ml were increased compared to the K+ group (p = 0.000** and p = 0.001**). Additionally, PPARγ expression and activity showed their lowest level in the K (+) group.

Figure 4. DCGTE increased PPARγ expression and activity.

The mRNA expressions of PPARγ were analyzed using PCR. The reactions were loaded onto an agarose gel, resolved by size via electrophoresis, and visualized with ethidium bromide. The bands were quantified using Image J and normalized with βactin. PPARγ activitiy was analysed using ELISA. ∗ p < 0.05, relative to K, ∧ p < 0.05 relative to K(+), # p < 0.05 relative to P1.

Culture groups: complete medium + 50 ng/ml M-CSF (K); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL (K+); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 160/160 μg/ml DCGTE (P1); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 320/320 μg/ml DCGTE (P2).

The decaffeinated coffee and green tea extract reduced the miR-155 expression in ox-LDL-stimulated Raw 264.7

Recent reports showed the importance of miRNA in post-transcriptional gene expression regulation.^37–39 miR-55 plays a significant role in inflammation. In addition, miR-155 has been proven to target CD36, SOCS1, and Vav3 based on in silico results.³⁴ Regarding this function, this study explored the effect of DCGTE on miR-155 expression, and results can be seen in Figure 5. The results demonstrated a significant increase in relative miR-155 expression in cells treated with 160/160 and 320/320 μg/ml DCGTE compared to positive control (K+) (p = 0.000**). Surprisingly, the data showed the expression of miR-155 higher in positive control (K+) (p = 0.000**) compared to the control group.

Figure 5. DCGTE increased the miR-155 expression.

On the 4th day after seeding, the RNA of the cells was extracted, converted into cDNA and followed by qPCR. The house keeping gene was U6. The expressions were quantified using Livax Method 2 ^-(∆∆Ct) n. ∗ p < 0.05, relative to K, ∧ p < 0.05 relative to K(+), # p < 0.05 relative to P1.

Culture groups: complete medium + 50 ng/ml M-CSF (K); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL (K+); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 160/160 μg/ml DCGTE (P1); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 320/320 μg/ml DCGTE (P2).

Underlying data

Figshare: the supplementary data.xlsx, https://doi.org/10.6084/m9.figshare.16664983.v9.⁶⁶

This project contains the following underlying data:

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).