피부 각질세포에 대한 비파엽 에탄올 추출물의 PGE₂ 조절 작용과 상처치료 효과

1. Plant materials and extraction

Eriobotryae folium was collected from Wando, Jeonnam, Korea in June 2013. It was authenticated by Professor BS Pyo of the department of Oriental Medicine Materials, Dongshin University and the voucher specimen (No. DSUBIC-13-02) was preserved in the herbarium of the Biotechnology Industrialization Center of Dongshin University. Plant material was shade dried at room temperature, chopped followed by pulverization. Two hundred grams of powdered plant material were extracted by reflux with 94.0% ethanol for 3 h. The extract was evaporated to dryness by using a rotary vacuum evaporator at 50℃ to get crude extract (EFEE, 38 g).

2. Instruments and reagent

PGE₂, NAD⁺, NADH, gluthathione-sepharose 4B, dithiothreitol (DTT), sodium dodecyl sulfate (SDS), EDTA, reduced glutathione, and rest of essential chemicals and reagents were purchased from Sigma (St. Louis, MO, USA). The GST gene fusion pGEX-2T expression vector was purchased from Pharmacia Crop. (Piscataway, New Jersey, USA). The cDNA of human 15-PGDH was cloned from a human placenta cDNA library, as described previously (Ensor et al., 1990). The UV spectra were obtained using a UV-VIS spectrophotometer (Shimadzu, Kyoto, Japan). PGE₂ enzyme immunoassay kit was purchased from Thermo Scientific (Rockford, IL, USA). Real-time PCR was performed with the Light Cycler 2.0 Instrument (Roche, Mannheim, Germany).

3. Expression and purification of 15-PGDH

15-PGDH cDNA plasmid containing BamHI and EcoRI sites of the pGEX-2T expression vector was used to transform Escherichia coli BL-21 lysS. The cells were grown in 500mL medium containing 50㎍/mL ampicillin at 37℃ and 220 rpm until the OD₆₀₀ reached 0.6. Isopropyl B-D-thiogalactoside (1 M stock solution) of 500 μL was added and the cells were allowed to grow for 12 h at 25℃. Then the cells were harvested by centrifugation at 4000 × g for 30 min at 4℃. The cell pellets were resuspended in 20 mL cold cell Lysis buffer (1 × PBS buffer pH 7.4 containing 1 mM EDTA and 0.1 mM DTT) and sonicated (4 × 10 s at 4℃). The disrupted cells were centrifuged at 4000 × g for 20 min at 4℃. The supernatant was applied slowly to a gluththione-sepharose 4B column, which was equilibrated at 4℃ with a lysis buffer. The column was washed with lysis buffer until OD₂₈₀ reached below 0.005. The 15-PGDH was eluted from the glutathione-sepharose 4B column by incubation at room temperature for 5 min with the elution buffer (50 mM Tris-HCl pH 8.0 containing 10mM reduced glutathione, 1mM EDTA and 0.1mM DTT). The concentration of enzyme was determined and the purity of the 15-PGDH was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

4. 15-PGDH assay

Assay for the activity of the 15-PGDH inhibitors was performed using a fluorescence spectrophotometer by measuring the formation of NADH at 468㎚ following excitation at 340㎚. Tris-HCl buffer (50 mM, pH 7.5) containing 0.1 mM DTT, 0.25 mM NAD⁺, purified enzyme (10㎍), 21 μM PGE₂ and various concentrations of EFEE (total 2 mL) were added to each cell. Each concentration was assayed in triplicate. The absorbance at 340㎚ after incubation with EFEE was determined from a standard curve of various concentrations of NADH.

5. Cell culture and cell viability assay

HaCaT cells, a human keratinocyte cell line, were cultured in Dulbecco’s modified Eagle’s media (DMEM). The cultured media were supplemented with 10% heat inactivated fetal bovine serum (Sigma, St. Louis, MO, USA) and 100㎍/mL penicillin, in 5% CO₂ at 37℃. Cell viability was determined by the MTT assay. HaCaT cells (1 × 10⁴) were seeded in 96 well plates per 90 μL of DMEM medium. After the overnight of incubation, EFEE was treated for 72 h followed by 4 h of incubation with 10 μL of MTT (5㎎/mL stock solution). Then medium was removed and followed by addition of 150 μL of DMSO to dissolve formazan. Absorbance was measured at 540㎚ using an microplate reader (Perkin-Elmer, Waltham, MA, USA).

6. Determination of PGE₂ release

HaCaT cells was seeded (5 × 10⁵ cells/well) on to 6-well culture plates in DMEM medium containing fetal bovine serum and antibiotic for overnight in 5% CO₂ incubator at 37℃. EFEE of different concentrations were treated and media was collected after 12 h of sample treatment. PGE₂ levels were determined by PGE₂ enzyme immunoassay kits according to manufacturer’s protocol.

7. Quantitative real-time PCR

Total cellular RNA was isolated from HaCaT cells using TRI reagent (RNAiso Plus, Takara Bio Inc., Shiga, Japan) according to manufacturer’s protocol. cDNA for each RNA sample was synthesized in 20 μL reactions using the SuperScript First Strand synthesis system for reverse transcription-PCR (Invitrogen, Carlsbad, CA, USA) following manufacturer’s protocol. PCR reaction contained 4 μL of 1 : 5 diluted cDNA, 4 mM MgCl₂ 10 pmole of each primer and 4 μL of Fast Starter Mix buffer (dNTPs, SYBR Green dye and Tag polymerase). Primers used for real-time PCR were as follows: Human PGT forward, 5’- GGATGCTGTTTGGAGGAATCCTCA-3’ and reverse, 5’- GCAC GATCCTGTCTTTGCTGAA-3’; MRP4 forward, 5’- AACCTCTAACC GACATTCCTG-3’ and reverse, 5’-TCAA CATATTACAGCCACCATC-3’; COX-1 forward 5’-CCTCAT GTTTGCCTTCTTTGC-3’ and reverse 5’-GGCGGGTACATT TCTCCATC-3’; COX-2 forward, 5’-GATCTACCCTCCTCA A-3’ and reverse 5’-GAACAACTGCTCATCAC-3’ and β-actin forward 5’-GACTATGACTTAGTTGCGTTA-3’ and reverse 5’- GTTGAAC TCTCTACATACTTCCG-3’.

8. Wound healing effect by in vitro scratch assay

For the in vitro scratch assay (Hintermann et al., 2001; Koivisto et al., 2006), HaCaT cells were seeded onto six well plates in a density of 5 × 10⁵ cells/well, and grown until they reached about 80% confluence. Then the media was changed with serum free DMEM containing mitomycin (10㎍/mL) and incubated for 2 h to prevent wound proliferation followed by extensive washing with PBS. A scratch was made using a sterile 200 μL pipette tip and cells were washed. TGF-β1 (300 pg/mL) as a positive control and EFEE as a 15-PGDH inhibitor were added to the medium. Pictures were taken exactly at the same position before and after the incubation to document the wound healing process. Experiments were repeated twice and delegate pictures are shown. Scratches were picture under the microscope (× 100) immediately after scratch making and once more after 48 h incubation at 37℃, 5% CO₂ incubator.

9. Statistical analysis

The results are expressed as the mean ± SD. Data between groups were analyzed by a Student’s unpaired two-tailed t-test and p-values less than 0.05 were considered significant.

1. Determination of extracellular and intracellular PGE₂ levels

PGE₂ has been known as an important mediator of healing of gastric ulcer, dermal wound and so on (Chatterjee et al., 2012; Yamamoto et al., 2012; Choi et al., 2013; Li et al., 2011). It is reported that 15-PGDH is inhibited by a variety of pharmacological agents including NSAIDs such as indomethacin, anti-paltelet aggregatory drugs such as panaxynol (Moustafa et al., 2013; Fujimoto et al., 1998), anti-allergic drugs such as flavonoid baicalein (Iijima et al., 1980), and so on. As shown in Table 2, EFEE increased 18.33% of extracellular PGE₂ level and 39.68% of intracellular PGE₂ level compared to control group in HaCaT cells.

Table 2.

Intracellular and extracellular PGE2 levels after the treatment of ethanol extract of Eriobotryae folium (EFEE) in HaCaT cells.

2. 15-PGDH inhibitory activity and cytotoxicity

15-PGDH is the key metabolic senzyme of PGE₂ (Choi et al., 2013). Therefore, inhibition of 15-PGDH is supposed to facilitate previous biological effects by increasing PGE₂. Inhibitors of 15-PGDH will be valuable for the disease therapy requiring PGE₂ elevation (Moustafa et al., 2013). For this reason, we have searched EFEE for potential 15- PGDH inhibitory activity. The effective dose for 50% inhibition (ED₅₀) was used for the expression of 15-PGDH inhibitory activity. We found that EFEE contained 15- PGDH inhibitor ED₅₀ value, 168.4㎍/mL (Table 1). On the other hand, cytotoxicity is very important for pharmaceutical application. The cytotoxicity of EFEE was determined by MTT assay. IC₅₀ (concentration for 50% of cell survival) of EFEE in HaCaT cells appeared was 250.0㎍/mL.

Table 1.

15-PGDH inhibitory activity and cytotoxicity of ethanol extract of Eriobotryae folium (EFEE) in HaCaT cells.

3. COX-1, COX-2, MRP4 and PGT expression

COX pathway increased PGE₂ level by inducing the synthesis of PGE₂ from arachidonic acid in biological system. In this connection, expression of COX-1, COX-2, MRP4 and PGT is essential for PGE₂ synthesis and transportation. PGE₂ can cross through the membrane by simple diffusion or via a prostaglandin efflux transporter, such as MRP4. After acting via its EPR, pericellular PGE₂ is cleared via re-uptake by PGT and then rapidly metabolized by cytosolic 15-PGDH (Lee et al., 2012). Thus, PGE₂ levels would be functions of expression levels of these genes. Therefore, we treated HaCaT cells with EFEE and checked the regulation of COX-1, COX-2, MRP4 and PGT. Real-time PCR assay showed that EFEE increased expression of COX-1 and COX-2 (Fig. 1). On the other hand, expression of MRP4 and PGT decreased by EFEE treatment. In Table 2 and Fig. 1, intracellular and extracellular PGE₂ level in HaCaT cells elevated by the increasing of COX-1 and COX-2 expression, and the decreasing of MRP4 and PGT expression. In addition, EFEE contained 15-PGDH inhibitory effect (Table 1). Interestingly, it has been reported that Eriobotryae folium extract suppressed LPS-induced nitric oxide and PGE₂ production through the inhibition of inducible nitric oxide synthase and COX-2 expression in lipopolysaccharidestimulated RAW264 cells (Uto et al., 2010). Therefore, it is estimated that EFEE has different functions working in different cells such as murine macrophages, keratinocytes.

Fig. 1.

Effect of ethanol extract of Eriobotryae folium (EFEE) on mRNA expression of COX-1, COX-2 (A) and MRP4, PGT (B).HaCaT cells were treated with EFEE (168.4㎍/mL) for 12 h. Values are mean ± SD (n = 4). *p < 0.05 and **p < 0.001 compared to the control group.

4. Wound healing effect by in vitro scratch assay

For the wound healing study, in vitro scratch assay was performed to compare the result of samples. EFEE facilitated wound healing as compared to the negative control. Moreover, the wound healing effect of EFEE (168.4㎍/mL) was comparable to that of TGF-β1 (300 pg/ mL) as a positive control. TGF-β1 is one of endogenous growth factors, including EGF, FGF and PDGF, that are released from some T cells at the around site and presumed to be a necessary part of wound healing (Liu et al., 2006; Strutz et al., 2001). By measuring the distance of scratch, the recovery % of samples compared with negative control was calculated. EFEE treatment group showed 228% recovery, while TGF-β1 treatment group showed 241% recovery of scratched wounds (Fig. 2).

Fig. 2.

Pictures (A) of wound healing effect (B) in HaCaT cells by in vitro scratch assay.EFEE; ethanol extract of Eriobotryae folium. HaCaT cells were treated with EFEE (168.4 ㎍/mL) and TGF-β1 (300 pg/mL) as a positive control. The pictures were taken at 0 h and 48 h after treatment of samples. Values are mean ± SD (n = 3). **p < 0.001 compared to the control group.

Many studies have reported that PGE₂ is an important mediator of dermal wound healing (Kolodsick et al., 2003; Savla et al., 2001). Likewise, there is a close connection between wound healing effect of EFEE and PGE₂ upregulation by controlled expression of MRP4, PGT, COX-1 and COX-2. Moreover, 15-PGDH inhibition effect of EFEE facilitated PGE₂ up-regulation. These results indicate that EFEE could be applicable to therapeutic materials for the treatment of PGE₂ level dependent diseases such as dermal wound, gastric ulcer and so on.