Effects of epigallocatechin-3-gallate (EGCG) on a scleroderma model of fibrosis

Animals

Skin fibrosis was induced in 6–8-week-old, weighing about 20–22 g, pathogen-free female Balb/c mice (DEU, Department of Experimental Animals) by local injection of bleomycin for 21 days [19], [23], [25]. The animals were kept under standard animal laboratory conditions: temperature of 20–22°C, humidity of 55–60%, photoperiod of 12:12 h light: dark cycle. They had free access to standard laboratory feed and water. All methods including the euthanasia procedure were conducted in accordance with Guide for Care and Use of Laboratory Animals, Institute for Laboratory Animal Research, National Institute of Health.

Experimental design

The experimental design was performed according to Yamamato et al. [19], [23], [25]. Bleomycin was used for scleroderma model (Onko Koçsel, Turkey). EGCG (Sigma-Aldrich, USA) and bleomycin solutions were prepared at 1 μg/1 μL concentration before the experiment. A total of 32 female Balb/c mice were used and randomly divided into 4 groups of 8 animals each, as follows:

1. Control group (n:8) was given subcutaneous (sc) saline (SF) once a day, intraperitoneal (ip) SF twice a week,

2. BLM group (n:8) was given 100 μL of BLM sc once a day, 100 μL of SF ip twice a week,

3. BLM+EGCG group (n:8) was given 100 μL of BLM sc once a day, 100 μL of EGCG ip twice a week,

4. EGCG group (n:8) was given 100 μL of SF sc once a day, 100 μL of ECCG ip twice a week.

After 21 days, the animals were sacrificed under ether anesthesia in sterile condition. The injected skin was removed and processed for analysis. Skin tissue samples were separated for immunohistochemistry, histochemistry, western blotting analyses and qPCR analysis.

Immunohistochemical and histological analyses

The injected sections of skin were fixed in 10% formalin and embedded in paraffin. Four micrometer (μm) sections were stained with hematoxylin and eosin for the determination of dermal thickness. For the analysis of connective tissue, Masson’s trichrome staining was performed. Dermal thicknesses of the injected sections were analyzed with an Nikon Eclipse C1 microscope (Nikon, Japan) at 200-fold magnification and digitalized by Nikon DS-Fi2 camera. Dermal sections were measured (as μm) on 10 areas. In each series of experiments, the dermal thicknesses were evaluated statistically.

α-Smooth muscle actin (α-SMA) (Thermo, USA) was used for immunohistochemical analyses. Four micrometer sections were cut, baked at 60°C for 30 min, and cooled. Sections were deparaffinized and rehydrated through graded alcohols to water. Antigen retrieval was required and it was done as follows: Slides were placed in Tris-buffered saline with 0.1% Tween-20 and loaded onto a Ventana Autostainer (Roche, USA). Blocking was done and the primary antibody (Thermo Fisher Scientific, USA) was applied for 30 min at room temperature. A horseradish peroxidase (HRP) derived secondary antibody (Thermo Fisher Scientific, USA) was applied. Slides were washed with wash buffer, incubated with liquid diaminobenzidine tetrahydrochloride (DAB) substrate (Agilent Technologies, Dako, USA) for 7 min, and rinsed with water. They were counterstained for 2 min with hematoxylin (Agilent Technologies, Dako, USA), blued with wash buffer, rinsed with water, dehydrated, cleared, and coverslipped. α-SMA positive cells were counted on five areas. Images were visualized with a Nikon Eclipse C1 microscope (Nikon, Japan) at 200-fold magnification and digitalized by Nikon DS-Fi2 camera. In each series of experiments, α-SMA positive cells were counted and evaluated statistically.

Preparation of tissue extracts for western blotting and quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analysis

Skin tissues were prepared for the western blotting and qPCR analysis. Each sample was homogenated with the trizol reagent (Roche, USA) using TissueLyser II (Qiagene, USA). The homogenate was incubated for 5min at room temperature. A 1:5 volume of chloroform was added, and the tube was vortexed and subjected to centrifugation at 12,000g for 15min. The aqueous phase was isolated, and the total RNA was precipitated by cold absolute ethanol. After centrifugation and washing, the total RNA was finally eluted in 20μL of RNase, DNase free water. The quantity was characterized using a UV spectrophotometer (NanoDrop 8000, Thermo Scientific, USA). RNA was isolated from the homogenates. The isolated RNA had a 260/280 ratio of 1.9–2.1.

For the isolation of total protein the organic phase was obtained at the bottom of the tube as described above (12,000 g centrifigation/15 min) Isopropanol in 1% glycerol (Sigma Aldrich, USA) was added to bottom of the tube. This protein precipitate was washed three times with 0.3 M guanidine hydrochloride in 95% ethanol. In addition, the protein precipitate was dried and solubilized in 1% sodium dodecyl sulfate (SDS). To ensure maximum dissolution, the protein precipitate was incubated at 50°C with intermittent vortexing in 1% SDS. This produced a fairly good amount of protein. Total protein extracts were transferred to fresh tubes and stored at −80°C.

Detection of MMP-1, MMP-8, MMP-13 and pSMAD 2/3 by western blotting

The protein concentration was quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Pierce, USA). A total 25 μg of protein was separated by SDS–polyacrylamide gel electrophoresis and transferred to a 0.45 μm pore size, Polyvinylidene difluoride (PVDF) membrane (Millipore, USA) which was then blocked for 1 h at room temperature using 5% skim milk/Tris buffered saline-Tween (TBST). The blots (For each MMP type, four blots-representing four experimental groups) were incubated overnight with a 1:1000 dilution of appropriate primary antibody in 5% skim milk/TBST. The following antibodies were used: Rabbit MMP-1, MMP-8, MMP-13 (ThermoFisher Scientific, USA) and total Smad 2/3, P-Smad 2/3 (Santa Cruz Biotechnology, USA). After the primary incubation, membranes were washed with TBST, the blots were incubated with the appropriate horseradish peroxidase–conjugated mouse anti-rabbit secondary antibody (Santa Cruz Biotechnology, USA).

The following steps involved the processing of beta actin in a similar way, to be further used for comparison with the intensity of the MMP bands:

After visualizing the bands, MMP-1, MMP-8 and MMP-13 membranes were stripped, [using a solution (Thermo, USA) to dissociate the appropriate antibody] and the remaining protein band was re-incubated for β-actin using a monoclonal antibody (Santa Cruz Biotechnology, USA).

For the analysis of Smad and p-Smad: p-Smad membrane (following visualation for p-Smad) was stripped [using a solution (Thermo, USA) to dissociate the appropriate antibody] and re-incubated for Smad 2/3 with the Smad 2/3 antibody.

All protein bands were visualized using enhanced chemiluminescence (ECL) (Millipore, USA) and were quantitated using Image-J, NIH Image densitometry software. Relative expressions of proteins were calculated using the ratios: MMP-1/β-actin, MMP-8/β-actin, MMP-13/β-actin and P-Smad 2-3/Smad 2-3 (Figure 6). All experiments were performed in triplicate and the western blotting images were presented as the representative of three images.

Detection of TGF-β1 mRNA levels by quantitative reverse transcriptase– polymerase chain reaction (RT-PCR)

First-strand cDNA was synthesized from 1μg total RNA in 20μL by reverse transcription using high capacity cDNA kit (Applied Biosystems, CA, USA) according to the manufacturer’s instructions. Reverse transcription reaction consisted of 2μL Oligo-dT (50μM), 2μL of 10× reverse transcriptase buffer, 0.8 μL of deoxynucleoside triphosphate (25 mM), 1 μL of RNase inhibitor (40 U/μL), 1 μL of MultiScribe Reverse Transcriptase (50 U/μL), and RNase free dH₂O, up to a final volume of 20 μL. The cDNA was then stored at −20°C for the gene expression study.

Real time quantitative PCR was performed to detect the gene expression of TGF-β1 in skin tissue using SYBR master mix (ThermoFisher Scientific, USA) at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s and the reaction was performed on Roche Lightcycler (Roche, USA). GAPDH, mouse sequence 5′-AGGTCGGTGTGAACGGATTTG-3′ and reverse 5′-TGTAGACCATGTAGTTGAGGTCA-3′, was used as an internal control. The primers that were used are TGF-β1 mouse sequence Forward 5′-GCCTGAGTGGCTGTCTTTTGA-3′ and Reverse 5′-CTGTATTCCGTCTCCTTGGTTCA-3′.

A relative quantification was performed using the 2^ΔΔCt method [26], [27]. The experiments were performed in triplicate and were repeated twice.

Statistical analysis

Data were presented as means±SD and for comparisons between the groups, ANOVA Sidak analysis of variance and for dual comparisons Mann-Whitney U-test were used. Statistical evaluations were performed using the SPSS package program, version 20.0 (IBM, USA). A p-value of <0.05 was considered to be statistically significant.

Investigation of the effects of EGCG on the expression of antifibrotic molecules using histochemical and immunohistochemical staining

H&E and Masson Trichrome staining were performed and the histological examination of the skin tissues from the mice is shown in Figures 1 and 2. The dermal thicknesses in the bleomycin+EGCG injected mice was significantly lower than in the bleomycin injected mice respectively (p<0.05, Figures 1 and 2). Alpha smooth muscle actin immunohistochemical staining score was lower in the BLM+EGCG injected mice than bleomycin injected mice (p<0.05, Figures 3 and 4). EGCG shows inhibitory effect on fibrosis.

Figure 1:

Masson-Trichrome staining.

Evidence of fibrosis (green color) was more prominent in the BLM group than BLM+EGCG group. Original magnification 20×. (A) Control group, (B) BLM group, (C) BLM+EGCG group, (D) EGCG group.

Figure 2:

Staining score for Masson trichrome.

Results are presented as mean+SEM. *p<0.05.

Figure 3:

α-SMA staining of dermal tissue sections.

Alpha smooth muscle actin (α-SMA) is a marker for activated fibroblasts. The number of α-smooth muscle actin-positive fibroblast cells (black arrows) was higher in the BLM group than BLM+EGCG group. Original magnification 20×.

Figure 4:

Staining score for alpha smooth muscle actin.

Results are presented as mean+SEM. *p<0.05.

Effects of EGCG on MMP-1, MMP-8, MMP-13 and p-Smad2/3 protein expressions in skin tissues

As mentioned above, all experiments were performed in triplicate. For this reason, all images obtained from three independent western blotting experiments (MMP-1, MMP-8, MMP-13, p-smad 2/3) were evaluated statistically following the calculation of MMP(s)/β-Actin band area ratios obtained from densitometric measurements. The western blotting analysis showed that EGCG increased the MMP-1, MMP-8 levels in the bleomycin induced mice. Also, p-SMAD 2-3/Total SMAD 2-3 protein level ratio was decreased (p<0.05, Figures 5 and 6) in the bleomycin+EGCG group. MMP-13 was not found to be significantly different in comparison with the BLM controls (p>0.05, Figures 5 and 6).

Figure 5:

Effects of EGCG treatment on MMPs and Smad pathway proteins in scleroderma.

Western blottings were performed to measure protein levels of MMP1, MMP8, MMP13, SMAD 2/3 and pSMAD 2/3, as described under materials and methods. (1) Control group, (2) BLM group, (3) BLM+EGCG group, (4) EGCG group.

Figure 6:

Effects of EGCG treatment on MMPs and Smad pathway proteins in scleroderma.

Quantitative representation of protein expression in all groups were obtained by densitometric analysis. (1) Control group, (2) BLM group, (3) BLM+EGCG group, (4) EGCG group. *p<0.05.

Effect of EGCG on TGF-β1 mRNA levels in SSc tissues

As expected in a succesful bleomycin induced scleroderma model, the levels of TGF-β mRNAs in the BLM group was significantly increased (p<0.01) compared with the control group. TGF-β mRNA levels in BLM+EGCG group was found to be lower (p<0.01) in comparison with the BLM group (Figure 7).

Figure 7:

EGCG decreased TGF-β1 mRNA.

mRNA expression of TGF-β1 was analyzed by quantitative real-time qPCR. Results are presented as mean+SEM. *p<0.01.