Ⅰ.INTRODUCTION

RAW 264.7 cells are macrophages derived from mouse, and they can primarily phagocytose foreign molecules^1-3). The coffee molecules, mostly chlorogenic acid, may function as foreign molecules, if they are introduced directly to RAW 264.7 cells. Therefore, in the present study the immediate reaction of RAW 264.7 cells to the coffee extract was assessed through different protein expressions for cellular signal transduction. Instead of the purified coffee molecules, the present study used the dialyzed coffee extract purified by cellulose bag (Spectrum laboratories, Inc, CA, USA) to separate small coffee molecules less than 6-8 kDa from the ordinary crude coffee extract.

The dialyzed coffee molecules are expected to be freely absorbed through murine gastrointestinal tract in vivo, therefore, it is presumed that the dialyzed coffee molecules may be appropriate for the application in vitro cell culture experiment. The drinking coffee has been known to enhance the immunity as well as cardiovascular stimulation^4-6). However, for the cell biological investigation of dialyzed coffee molecules, the present study utilized the murine macrophage cell line, RAW 264.7, to perform different biological analyses including histological observation and protein expression assays.

Ⅱ.MATERIALS and METHODS

Dialyzed coffee extract production

The coffee bean (Coffea arabica L.) used for this study was grown and harvested in Nepal, 2015, roasted in Chuncheon, Korea, and followed by hot water extraction using grounded coffee bean. In order to exclude the macromolecules which are not easily absorbed through human intestinal mucosa, the crude coffee extract solution was dialyzed using cellulose bag (Spectra/Por, Spectrum, CA, USA) which can filtrate small molecules less than 6-8 kDa (Fig. 1).

The dialysis of coffee extract was performed in a tank filled with double distilled water, and its molecular diffusion was accelerated by stirring a magnetic bar in 4° C refrigerator. The dialyzed coffee extract was also gel-filtrated with high performance liquid chromatography (HPLC) machine (Hewlett Packard 1050, USA) using Superose column (Superose^TM 12, 10/300 GL, Sweden) at 0.5 mL/min running buffer (20mM KH₂PO₄, 20mM K₂HPO₄).

The concentration of dialyzed coffee extract was determined by UV absorption method using the following mathematical formula⁷⁾.

Molecular concentration (mg/mL) = 1 .55A 280 - 0 .76A 260

As the dialyzed coffee extract was measured 0.445 at A₂₈₀ and 0.372 at A₂₆₀, the concentration of the present dialyzed coffee extract was calculated into 4.07mg/mL.

The chromatography clearly disclosed three peaks (Fig. 2), therefore, the three peaks were fractionated using fraction collector (Gilson FC204, USA), and followed by thin layer chromatography. The thin layer chromatography (Sigma, USA) was performed with running buffer solution (ethyl acetate:ethanol:water = 8:2:1) in order to compare the three fractions with the other coffee molecules, i.e., chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, 4-coffeoylquinic acid and 5-coffeoylquinic acid.

RAW cell culture treated with dialyzed coffee extract

RAW 264.7 cells (ATCC, USA), murine immortalized macrophage cell line, were cultured in Dulbecco’s modified Eagle’s medium (WelGene Inc. Korea) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (WelGene Inc. Korea), 100 unit/mL penicillin, 100μg/mL streptomycin, 250ng/mL amphotericin B (WelGene Inc. Korea), at 5% CO₂, 37.5°C. The cells were tested for mycoplasma on a regular basis to ensure that only mycoplasma-free cell lines were studied in the assays.

During the active growth of RAW 264.7 cells, the experimental groups were treated with 20μg/mL and 40μ g/mL of dialyzed coffee extract for 24 hours separately, while the control group was treated with normal saline. The concentration of 20μg/mL dialyzed coffee extract was estimated to the similar level of 5 cups of coffee drinking a day, which was usually recommended as the maximum limit a day. Therefore, in this study the concentration of 20μ g/mL dialyzed coffee extract was simply called as the low dose of coffee extract, and the concentration of 40μg/mL as the high dose of coffee extract.

After the cell culture the RAW 264.7 cells were fixed with 10% neutral formalin, stained with hematoxylin and eosin, and observed under light microscope.

Their microscopic images were captured by a digital camera (DP-70^®, Olympus Co., Japan), and the representative 20 images in high magnification (x400) were used for cell counting through image analyzing program of IMT i-solution (ver 21.1, Vancouver, Canada).

Immunoprecipitation HPLC analysis for the protein extract obtained from RAW 264.7 cell culture

After 12 hours culture of RAW 264.7 cell treated with dialyzed coffee extract at 20μg/mL and 40μg/mL concentration separately, the RAW 264.7 cells were harvested with protein lysis buffer (0.3% SDS, 50 mM Tris-HCl pH 8.0, 0.3% β-mercaptoethanol, 1 mM PMSF, 1 mM EDTA) containing protein inhibitor cocktail (Sigma, USA). And then the protein extracts were kept in -70°C deep freezer to prevent further protein degradation.8)Fig. 3

Each 100μg of protein extract was applied to immunoprecipitation procedures using protein A/G agarose column (Amicogen, Korea). The protein A/G agarose columns were separately pre-incubated with 1μg of 25 different antisera, including β-actin, Ki-67, NFkB, TNFα, and LC3 (SantaCruz Biotech. USA). Briefly, the protein samples were mixed with 5 mL binding buffer (150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM PMSF and 0.5% NP-40), and incubated in the protein A/G agarose columns at 10°C for 1 hour. The columns were placed on the rotating stirrer during the incubation time. After washing each column with sufficient amount of PBS solution (pH 7.3, 137 mM NaCl, 2.7 mM KCl, 43 mM Na₂HPO₄-7H₂O and 1.4 mM KH₂PO₄), the target protein was eluted with 250 μL IgG elution buffer (Pierce, USA). The immunoprecipitated proteins were analyzed by HPLC (1100 series, Agilent, USA) using a reverse phase column (YMC-Pack SIL, Japan), running with 0.15M NaCl, 20% acetonitrile solution at 0.4 mL/min for 30 min, and analyzed by UV spectroscope at 280 nm. Both control and experimental groups were simultaneously performed for the IP-HPLC. In the results of IP-HPLC, the sample protein peak areas (mAU*s) obtained from HPLC analysis were eliminated by the antibody peak area (mAU*s) in the negative control. And the data were mathematically reduced into the square root value as the unit level for molecular concentration, and subsequently compared with each other between the experimental and control groups. All the square root values of protein peak areas were plotted into a graph depending on the characteristic protein groups.⁹⁾

Ⅲ.RESULTS

Observation of RAW 264.7 cells

RAW 264.7 cells treated with 20μg/mL and 40μg/mL of dialyzed coffee extract were cultured on petri dish for 24 hours separately, stained with hematoxylin and eosin, and subsequently observed under light microscope. The RAW 264.7 cells grew confluently on the surface of petri dish, and there appeared rare feature of cellular necrosis (Fig. 4). However, when the cell number was counted in the high magnification view (x400), the low dose of coffee extract (20μg/mL) increased the RAW 264.7 cell number (1919.8 ± 321.9), while the high dose of coffee extract (40μg/mL) decreased the RAW 264.7 cell number (1137.9 ± 22.1) compared to the control (1420.2 ± 215) (Fig. 5).

Thin layer chromatography of dialyzed coffee extract in the comparison with essential coffee elements

The three peak fractions (A, B, and C) obtained from the gel filtration chromatography (Fig. 2) were performed for thin layer chromatography, and it turned out that the three fractions were not identical to the coffee elements including chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, 4-coffeoylquinic acid and 5-coffeoylquinic acid. The three fractions were almost smeared into the area of lower molecular weight compared to those essential coffee elements. Therefore, it is presumed that the dialyzed coffee extract may be composed of small molecular elements which have not been characterized yet (Fig. 3).

IP-HPLC analysis for RAW 264.7 cell culture treated with dialyzed coffee extract

The protein extracts obtained from the RAW 264.7 cell culture treated with dialyzed coffee extract, 20μg/mL and 40μg/mL separately, were analyzed through IP-HPLC analysis. The low dose of dialyzed coffee extract enhanced the expression of β-actin (106.6 ± 7.0%) and Ki-67 (111.7 ± 17.8%), but reduced the expression of NFkB (84.3 ± 18.2%), TNFα (92.2 ± 10.1%), and LC3 (71.1 ± 11.0%). Whereas the high dose of dialyzed coffee extract reduced the expression of β-actin (90.3 ± 9.0%), Ki-67 (77.5 ± 13.3%), but enhanced the expression of TNFα (109.0 ± 4.3%). The expressions of NFkB and LC3 were slightly recovered by the treatment of the high dose of dialyzed coffee extract compared to the control up to 99.6 ± 12.4% and 91.8 ± 8.0%, respectively (Fig. 6)

Ⅳ.DISCUSSIONS

RAW 264.7 cells are derived from murine macrophages, which were immortalized with molecular genetic methods in order to utilize in vitro culture system^10-12). The dialyzed coffee extract may be primarily engulfed by RAW 264.7 cells in culture system. However, as the RAW 264.7 cells are antigen presenting cells, the endocytosed coffee elements may induce molecular signaling for inflammation in their cytoplasms. In the present study the histological observation of RAW 264.7 cells revealed no remarkable cytotoxic effect of dialyzed coffee extract in the low and high dose treatments of dialyzed coffee extract. The dialyzed coffee extract contains almost small-sized coffee elements, which are able to be absorbed freely into gastrointestinal mucosa epithelium¹³⁾. The absorbed coffee elements may directly induce the cellular signaling in macrophages located at the submucosal tissue. This common phenomenon occurring in the gastrointestinal mucosa epithelium may indicate that the dialyzed coffee extract is also applicable to in vitro cell culture experiment instead of the purified coffee elements obtained through multiple biochemical procedures.

RAW 264.7 cells, as macrophages, are reactive to antigenic materials depending on their antigenic potentials. This preliminary study used the low and high dose of dialyzed coffee extract, 20μg/mL and 40μg/mL, respectively, to know the biological effect of dialyzed coffee extract on RAW 264.7 cells. The low dose of dialyzed coffee extract dramatically induced the proliferation of RAW 264.7 cells, while the high dose of dialyzed coffee extract reduced those effects. Because the level of 20-40μg/mL coffee extract was still great amount for cell culture experiment as shown in the previous studies done by other authors^14,15), it must be true that the treatment of the high dose of dialyzed coffee (about 10 cups of coffee) may affect some cytotoxic damage in the cells. Therefore, from the present study it was supposed that the concentration of dialyzed coffee extract less than 20μg/mL level be investigated more in the following study.

The protein extract obtained from RAW 264.7 cell culture treated with dialyzed coffee extract were also analyzed through IP-HPLC. Resultantly, the low dose of dialyzed coffee extract enhanced the growth of RAW 264.7 cells with the increased expression of β-actin and Ki-67, and also induced the anti-inflammatory effect by decreasing the expression of NFkB, TNFα, and LC3 compared to the high dose of dialyzed coffee extract . Although this study was a preliminary experiment, the fact that the dialyzed coffee extract played a role for the modulatory effect of cellular proliferation^16,17) and inflammation^4,18,19) might provide a new vision to explore the biological effect of coffee extract, which is the most familiar beverage to modern human beings²⁰⁾.

Regarding the anti-inflammatory effect of coffee extract, the low dose of dialyzed coffee extract decreased the protein expressions of NFkB, TNFα, and LC3, while the high dose of dialyzed coffee extract slightly recovered those expressions up to the level of the control (Fig. 6). These findings were almost identical to the previous reports done by other authors^{1,4,18,21,22)}. Particularly, it was known that chlorogenic acid of coffee extract can effectively suppress chronic ventricular remodeling after myocardial ischemia because it is critically involved in the suppression of macrophage infiltration.²³⁾, and that 3-methyl-1,2- cyclopentanedione (3-MCP) in coffee extracts is proposed to be an effective anti-inflammatory agent through the modulation of age-related NFkB signaling cascade and its pro-inflammatory genes²⁴⁾.

Although the present study was performed as a preliminary pilot study to detect any cytotoxic effect of dialyzed coffee extract depending on its concentration compared to the essential coffee elements, which were widely reported in the literature^15,25-28), the dialyzed coffee extract showed conspicuous anti-inflammatory effect in the dose of 20μg/mL but not in the dose of 40μg/mL. These findings may directly indicate the dose-dependent biological effect of dialyzed coffee extract, and provide a guide line for the following study to perform with the lower concentration of dialyzed coffee extract less than 20μg/mL level.

In summary, this study was performed to develop an in vitro cell culture method to utilize the dialyzed coffee extract. The low dose of dialyzed coffee extract produced almost no harmful effect on RAW 264.7 cell culture for 12 hours, but it produced stimulatory effect on RAW 264.7 cells by increasing the cell number and enhancing the protein expression of β-actin, Ki-67. Therefore, it was thought that the low dose of dialyzed coffee extract was applicable to cell culture experiment without difficult purification procedures of coffee elements. In addition, as the contrast cellular effect between the low and high dose of coffee extract was found in this study, it was also presumed that the low dose of coffee extract may play an important role in the inflammatory reaction of murine macrophages compared to the high dose of coffee extract. However, the additional investigation should be followed to explore more biological effect of dialyzed coffee extract with precise molecular and biochemical methods.