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Ellagic acid exerts anti-fibrotic effects on hypertrophic scar fibroblasts via inhibition of TGF-β1/Smad2/3 pathway

Ellagic acid exerts anti-fibrotic effects on hypertrophic scar fibroblasts via inhibition of... Hypertrophic scar (HS) is a kind of serious pathological scar with no currently effective treatment. HS fibroblasts (HSFs) are the main effector cells for HS formation. Ellagic acid (EA) exerts regulatory effects in some diseases, but its role in HS remains unclear. This study aimed to evaluate the effect of EA on the fibrotic phenotypes of HSFs and to further investigate the downstream signaling mechanism. The cell counting kit-8 (CCK-8) assay was used to perform cytotoxicity and proliferation assays. HSFs migration was assessed using wound healing and transwell assays. HSFs contraction was measured by a collagen lattice contraction assay and detection of α-smooth muscle actin (α-SMA) expression. The levels of mRNA and protein were determined by qPCR and western blotting, respectively. The results showed that EA inhibited the proliferation, migration, and contraction of HSFs and collagen expression in HSFs in a dose-dependent manner. Furthermore, EA not only suppressed the Smad2/3 pathway but also reversed TGF-β1- induced activation of the Smad2/3 pathway and up-regulation of the fibrotic cellular phenotypes in HSFs. These findings demonstrate that EA exerts anti-fibrotic effects on HSFs by blocking the TGF-β1/Smad2/3 pathway, which indicates that EA is a potential therapeutic candidate for treatment of HS. Keywords: Collagen, Contraction, Ellagic acid, Hypertrophic scar fibroblasts, Migration, Proliferation, Smad pathway, TGF-β1 Introduction the pathological mechanism of HS formation is mainly Hypertrophic scar (HS) is a severe skin fibrotic disease identified by aberrant deposition of extracellular matrix that often occurs after burn injury. HS is generally raised, (ECM), especially collagen I and III, and overactive red, itchy, or painful, and can even leads to movement hypertrophic scar fibroblasts (HSFs) are the effector cells dysfunction. These adverse symptoms result in not only contributing to the progression of HS [3]. It is meaning- physical but also psychological injury to the patients ful to explore an approach that could effectively attenuate [1]. Statistics show that up to 70% of burn patients suf- the fibrotic cellular phenotypes of HSFs, including prolif - fer from HS [2]. In recent years, a few treatments have eration, migration, contraction, and collagen expression. been developed; however, there is currently no effective Ellagic acid (EA) (molecular formula C H O , Fig. 1A) 14 6 8 therapy for HS. Previous studies have demonstrated that is a natural plant polyphenolic compound that is abun- dant in various fruits and nuts. Previous studies have mostly focused on the antioxidant, antihepatotoxic, and antitumor effects of EA [4–6]. Additionally, a few studies *Correspondence: mengxljdyy@jlu.edu.cn; yuja@jlu.edu.cn Xianjun Liu and Xinxin Gao contributed equally to this work have indicated that EA exerts anti-fibrotic effects in liver, Department of Burns Surgery, The First Hospital of Jilin University, No. pancreatic, and cardiac fibrosis [7–9]. However, the effect 1409 Ximinzhu Street, Changchun 130000, China of EA on HS formation remains unclear. Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Liu et al. Appl Biol Chem (2021) 64:67 Page 2 of 10 Fig. 1 EA inhibited HSFs proliferation in a dose-dependent manner. A The structural formula of EA. B Cytotoxicity assay result for HDFs, as detected using CCK-8 assay (n = 3). HSFs proliferation in different experimental groups, as detected using CCK-8 assay at the indicated timepoints (n = 5). C Over trends in growth and D inter-group comparisons are shown. Data are presented as mean ± SD; #P > 0.05, *P < 0.05 and **P < 0.01 The Smad2/3 signaling pathway is a canonical fibro - Affinity), anti-Smad2 (1:5000; cat. no. ab40855; Abcam), sis-associated signal transduction pathway, activation anti-Smad2 (phospho S255) (1:5000; cat. no. ab188334; of which regulates a spectrum of pathological cellular Abcam), anti-Smad3 (1:5000; cat. no. ab40854; Abcam), behaviors of HSFs [3]. TGF-β1, a well-known pro-fibrotic anti-Smad3 (phospho S423 + S425) (1:2000; cat. no. growth factor, is a significant upstream stimulator of the ab52903; Abcam), and anti-GAPDH (1:5000, cat. no. Smad2/3 pathway. Previous studies have found that TGF- 10494-1-AP; Proteintech, IL, USA). Secondary antibod- β1 promotes the proliferation, migration, and contraction ies (1:5000; goat anti-mouse cat. no. SA00001-1; goat of HSFs and collagen synthesis in HSFs by activating the anti-rabbit cat. no. SA00001-2; Proteintech) were used Smad2/3 pathway [10]. Proper regulation of the TGF- for western blotting. EA (cat. no. B21073; Yuanye Bio- β1/Smad2/3 pathway in HSFs is considered a promising tech, Shanghai, China) was dissolved in 1 M NaOH. The strategy for hindering HS formation. final concentration of NaOH was ≤ 0.1% (v/v) and did not In this study, we evaluated the effects of EA on the contribute to toxicity. TGF-β1 (cat. no. bs-2266P; Bioss, proliferation, migration, and contraction of HSFs and Beijing, China) was diluted with sterile double-distilled collagen expression in HSFs. Importantly, we fur- water. ther investigated the association between the TGF-β1/ Smad2/3 pathway and EA-induced regulation of HSFs. Cells isolation and culture Normal human dermal fibroblasts (HDFs) (cat. no. Materials and methods #2320) were purchased from Sciencell, and three cell Antibodies and drugs lines were obtained for subsequent cytotoxicity assay by The primary antibodies used for western blotting in parallel culture. HSFs were primarily cultured from the this study were as follows: anti-α-smooth muscle actin scar tissues of five patients (3 men and 2 women; aged (α-SMA) (1:1000; cat. no. ab32575; Abcam, Cambridge, 10–50  years) who suffered HS and had a cicatrectomy UK), anti-collagen I (1:1000; cat. no. AF7001; Affinity, in the Department of Burns Surgery of the First Hospi- OH, USA), anti-collagen III (1:1000; cat. no. AF0136; tal of Jilin University between January and April 2018. Liu  et al. Appl Biol Chem (2021) 64:67 Page 3 of 10 Protocols were approved by the Ethics Committee of EA or TGF-β1 according to experimental requirements. the First Hospital of Jilin University (no. 2017-088) and At the time points 0, 24, and 48 h after scratching, images were conducted according to the principles expressed in were acquired at three random fields of view using an the Declaration of Helsinki. Written informed consent inverted microscope (magnification 40 × ; Olympus was obtained from all donors before surgery. Briefly, the Corporation, Tokyo, Japan). Wound area was measured epidermis and subcutaneous adipose tissue of HS tis- using ImageJ software (version 1.51w). Migration ability sue masses were shaped. Next, the tissues were cut into of HSFs was evaluated using the data shown as migration approximately 1 mm sections under sterile conditions. rate (%) = (initial wound area – wound area at 24 or 48 h) After washing, sections were placed in a cell culture dish / initial wound area × 100. and incubated in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and Transwell assay double antibiotics (penicillin, 100 U/mL; streptomycin, A transwell assay was used to assess the migration of 0.1  mg/mL) at 37  °C with 5% CO . All cell culture rea- HSFs. Briefly, 2 × 10 HSFs were seeded in the upper gents were supplied by Gibco (Thermo Fisher Scientific, chambers of transwell plates (6-well, 8  μm; Millicell, MA, USA). When fibroblasts reached approximately MA, USA) incubated in serum-free DMEM, and the 90% confluence, they were trypsinized and passaged. lower chambers were supplied with DMEM contain- Cells from passages 2–4 were used in the subsequent ing 10% FBS, with EA or TGF-β1 according to experi- experiments. mental requirements. After 24  h, non-migrated HSFs on the upper face of the polycarbonate membrane were Cytotoxicity and proliferation assays removed with a cotton swab, and migrated HSFs were A cell counting kit 8 (CCK-8) assay was used for cyto- fixed with methanol and stained with Giemsa dye solu - toxicity and proliferation assays. Viable HDFs or HSFs tion (cat. no. R20649; Yuanye Biotech, Shanghai, China). counts were indirectly detected by measuring the opti- Images of each well were captured at three random fields cal density (OD) values. Cytotoxicity assay was per- of view using an OLYMPUS inverted microscope. The formed as described below. Briefly, HDFs were seeded cells were counted using ImageJ software. at 2 × 10 cells per well in a 96-well culture plate, with three replicates per sample, excluding the use of external Collagen lattice contraction assay rows and columns, to avoid edge effects. After complete A collagen lattice contraction assay was used to assess cell attachment, HDFs were processed according to the the contraction ability of HSFs. HSFs were mixed with experimental requirements. After treatment for 48  h, 10 rat tail tendon collagen (cat. no. C8062; Solarbio, Bei- µL of CCK-8 (APExBIO, Houston, USA) solution was jing, China). The final collagen concentration of the mix - added to each well. After incubation for 2 h at 37 °C, the ture was 1  mg/mL, with a cell density of 2.5 × 10 cells/ absorbance was measured at 450 nm wavelength using a mL. The mixture was added to a 24-well cell culture plate microplate reader (Thermo Fisher Scientific, MA, USA). (200 μL/well) and incubated at 37 °C for 1 h. After coag- The cytotoxicity assay results were expressed as cell sur - ulation, 500 μL serum-free DMEM, with EA or TGF-β1 vival rates. Cell survival rate (%) = (OD value of treated according to experimental requirements, was added onto group – OD value of blank)/ (OD value of control group the gel. A picture of each sample was captured at 48  h. - OD value of blank) × 100. As for the proliferation assay, The area of the gels calculated using ImageJ software. The HSFs were seeded at 5 × 10 cells per well in a 96-well contraction rate was presented as a measure of HSFs con- culture plate, and the OD values were tested at time traction: contraction rate (%) = (well area – gel area)/well points 0, 24, 48, and 72 h after treatment. In addition to area × 100. these, the method of OD value detection was the same as the cytotoxicity assay. The proliferation assay results were Real‑time quantitative PCR (qPCR) expressed as OD values. RNA was isolated, and qPCR was carried out. Briefly, total RNA was isolated from the samples using TRI- Wound healing assay zol reagent (Invitrogen, USA). cDNA was synthe- A wound healing assay was used to assess the migration sized using the TransScript All-in-One First-Strand of HSFs. In brief, HSFs were cultured in a 6-well culture cDNA Synthesis SuperMix (TransGen Biotech, Beijing, 4 2 plate at a density of 1 × 10 cells/cm with DMEM con- China). FastStart Universal SYBR Green Master (ROX) taining 10% FBS until the cell confluence reached 100%. (Roche Diagnostics, Mannheim, Germany) was used A scratch wound was made in the middle of each well for qPCR with a Stratagene Mx3005P instrument (Agi- using a 1-mL pipette tip. After washing with PBS, the cul- lent Technologies, USA). The reaction conditions were ture medium was changed to serum-free DMEM, with as follows: initial denaturation at 95  °C for 10  min; 40 Liu et al. Appl Biol Chem (2021) 64:67 Page 4 of 10 cycles of denaturation at 95 °C for 30 s and annealing at GraphPad Prism (version 7.00) statistical package. A 60 °C for 1 min; dissociation at 95 °C for 1 min, anneal- value of P < 0.05 indicated a statistically significant ing at 55  °C for 30  s, and final extension at 95  °C for difference. 30  s. The primers used in qPCR are listed in Table  1. The expression levels of target genes were normalized Results and discussion −C to that of GAPDH, and the 2 method was used to EA inhibited HSFs proliferation in a dose‑dependent calculate the relative mRNA levels [11]. Each sample manner was run in triplicate. First, we performed cytotoxicity assay for EA to HDFs by a CCK-8 assay. As shown in Fig. 1B, there was no sig- nificant difference in cell survival rate between each EA- Western blotting treated group (concentration ≤ 150  μM) and the control Harvested fibroblasts were processed with RIPA lysis group. This result suggested a concentration range, at buffer (CST, USA) supplemented with phenylmethyl - which EA is harmless to normal HDFs. Then, to evalu - sulphonyl fluoride (Thermo Fisher Scientific, MA, ate the effect of EA on HSFs proliferation, a CCK-8 assay USA), protease inhibitor cocktail (TransGen Biotech, was performed. As shown in Fig.  1C and D, HSFs pro- Beijing, China), and phosphatase inhibitor cocktail liferation in the EA-treated groups was attenuated as (TransGen Biotech). Protein concentration was meas- compared with that of the control group. There was no ured using a bicinchoninic acid (BCA) protein assay significant difference in cell viability between the 10  μM kit (Beyotime Institute of Biotechnology, Nantong, group and the control group at 24 and 48  h. The data China). Samples were separated using SDS-PAGE gels, demonstrated a significant decrease in HSFs proliferation and subsequently electro-transferred to PVDF mem- in the 10 μM group as compared with that of the control branes (Millipore, MA, USA) for immunoblotting group at 72  h; HSFs proliferation in the 50 and 100  μM analysis. After blocking with 5% bovine serum albumin groups was significantly lower than that of the control (Sigma-Aldrich, MO, USA) in TBST (0.5% Tween-20) group at all time points. Additionally, HSFs proliferation for 1  h at room temperature, the PVDF membranes in the 50 and 100 μM groups was significantly lower than were incubated with primary antibodies at 4  °C over- that of the 10  μM group at all time points. HSFs prolif- night. Following incubation with the appropriate horse- eration in the 100 μM group was significantly lower than radish peroxidase-conjugated secondary antibodies for that of the 50 μM group at all time points except for 24 h. 1  h at room temperature, proteins were detected with All these data indicate that EA inhibited HSFs prolifera- the EasySee western blotting kit (TransGen Biotech) tion in a dose-dependent manner. using a myECL imager (Thermo Fisher Scientific), and the band intensities were quantified by calculation of EA inhibited HSFs migration in a dose‑dependent manner gray values using ImageJ software. Data were collected The effect of EA on HSFs migration was assessed using from three independent experiments. wound healing and transwell assays. As shown in Fig. 2A and 2B, at 24 and 48 h after scratching, the migration rate of HSFs significantly decreased with increasing EA dose. Statistical analysis In addition, the results of the transwell assay (Fig. 2C and The quantified data are presented as the mean ± stand- 2D) showed that the migrated HSFs were significantly ard deviation (SD). The differences between the two reduced with increasing EA dose, consistent with the groups were analyzed by Student’s t-test using the results of wound healing assay. These data demonstrate that EA inhibited HSFs migration in a dose-dependent manner. Table 1 Sequences of primers for qPCR EA inhibited HSFs contraction in a dose‑dependent Forward (5’ → 3’) Reverse (5’ → 3’) manner To evaluate the effect of EA on HSFs contraction, a col - ACTA2GAC AAT GGC TCT GGG CTC TGT GCT TCG TCA CCC ACG TA TGTAA lagen lattice contraction assay was performed, and the COL1A1ACC AGT CTC CAT GTT GCA CGT GAC CTC AAG ATG TGC CACT expression of α-SMA, which is correlated with the con- GAAGA tractive ability of HSFs, was detected using qPCR and COL1A2GAG GGC AAC AGC AGG TTC TCA GCA CCA CCG ATG TCC A western blotting. As shown in Fig. 3A and B, with increas- ACTTA ing EA dose, the contraction rate significantly decreased. COL3A1CCA CGG AAA CAC TGG TGG ACGCC AGC TGC ACA TCA AGG AC As for the detection of α-SMA, encoded by ACTA2 gene, GAPDHGCA CCG TCA AGC TGA GAA CTGG TGA AGA CGC CAG TGG A the data of qPCR showed that the mRNA level of ACTA2 Liu  et al. Appl Biol Chem (2021) 64:67 Page 5 of 10 Fig. 2 EA inhibited HSFs migration in a dose-dependent manner. HSFs proliferation in different experimental groups, as detected using a wound healing assay at the indicated timepoints (n = 5). A Representative pictures (scale bar = 200 μm), and B quantitative analysis of migration rate (%) and inter-group comparison are shown. HSFs proliferation in different experimental groups, as detected using a transwell assay after 24 h of treatment (n = 5). C Representative pictures (scale bar = 100 μm) and D quantitative analysis of migrated cell numbers are shown. Data are presented as mean ± SD; *P < 0.05 and **P < 0.01 decreased with an increase in EA dose (Fig. 3C). Western EA inhibited TGF‑β1/Smad2/3 signaling pathway in HSFs blotting showed that the change in α-SMA protein level Based on the above findings, we assessed the effect of was in line with that of its mRNA level. All these meas- EA on HS-associated signaling pathway in HSFs. The ures indicated that EA inhibited HSFs contraction in a protein levels of total (t)-Smad2, p-Smad2, t-Smad3, dose-dependent manner. and p-Smad3 were detected using western blotting, and the ratios of p-Smad2/t-Smad2 and p-Smad3/t-Smad3 EA inhibited the expression of collagen I and III in HSFs were calculated to evaluate the Smad2/3 pathway. As in a dose‑dependent manner shown in Fig. 5A and B, the ratios of p-Smad2/t-Smad2 To evaluate the effect of EA on collagen synthesis in and p-Smad3/t-Smad3 significantly decreased in the HSFs, qPCR and western blotting were used to detect the EA-treated group (100 μM), and significantly increased mRNA and protein levels of collagen I and III, respec- in the TGF-β1-treated group (10  ng/mL) as compared tively. As shown in Fig. 4A, the mRNA levels of COL1A1, with those in the control group. Furthermore, EA COL1A2, and COL3A1 were significantly reduced with treatment significantly reversed TGF-β1-induced up- increasing EA dose, in addition to COL1A2 expression regulated phosphorylation of Smad2 and 3. These data in the 10 μM group. The change in protein levels of col - indicated that EA inhibited the TGF-β1/Smad2/3 sign- lagen I and III was consistent with the change in mRNA aling pathway in HSFs. levels (Fig. 4B). These results demonstrate that EA inhib - ited the expression of collagen I and III in HSFs in a dose- dependent manner. Liu et al. Appl Biol Chem (2021) 64:67 Page 6 of 10 Fig. 3 EA inhibited HSFs contraction in a dose-dependent manner. HSFs contraction in different experimental groups, as detected using a collagen lattice contraction assay after 48 h of treatment (n = 5). A Representative pictures, and B quantitative analysis of contraction rate (%) and inter-group comparison are shown. C Relative mRNA level of ACTA2 in different experimental groups, as detected using qPCR (n = 5). D Protein levels of α-SMA and GAPDH in different experimental groups, as detected using western blotting, data were collected from three independent experiments. Data are presented as mean ± SD; *P < 0.05 and **P < 0.01 Fig. 4 EA inhibited the expression of collagen I and III in HSFs in a dose-dependent manner. A The relative mRNA levels of COL1A1, COL1A2, and COL3A1 in different experimental groups, as detected using qPCR (n = 5). B The protein levels of collagen I, collagen III, and GAPDH in different experimental groups, as detected using western blotting, data were collected from three independent experiments. Data are presented as the mean ± SD; #P > 0.05, *P < 0.05 and **P < 0.01 EA inhibited TGF‑β1‑induced up‑regulation of HSFs transwell assay (Fig. 6C), contraction as assessed by colla- proliferation, migration, and contraction, and collagen gen lattice contraction assay (Fig. 6D) and α-SMA detec- synthesis in HSFs tion (Fig. 6E and F), and collagen synthesis as assessed by To further investigate the association between EA- collagen I and III detection (Fig. 6E and F). These results induced inhibition of fibrotic phenotypes and TGF-β1/ demonstrate that EA exerted inhibitory effects on TGF- Smad2/3 signaling pathway in HSFs, we measured the β1-induced fibrotic phenotypes of HSFs. effect of EA (100 μM) on TGF-β1-induced up-regulation of HSFs proliferation, migration, and contraction, and Discussion collagen synthesis in HSFs. The results showed that EA HS, which often occurs after burn injury, is gradu- significantly reversed TGF-β1-induced up-regulation of ally becoming a challenge for clinical workers [1]. proliferation as assessed by CCK-8 assay (Fig. 6A), migra- Although a few therapies, such as laser therapy, com- tion as assessed by wound healing assay (Fig.  6B) and pression garments, and topical drug application, have Liu  et al. Appl Biol Chem (2021) 64:67 Page 7 of 10 Fig. 5 EA inhibited TGF-β1/Smad2/3 signaling pathway in HSFs. A Protein levels of t-Smad2, p-Smad2, t-Smad3, p-Smad3, and GAPDH in different experimental groups, as detected using western blotting, data were collected from three independent experiments. B Quantitative analysis of proteins expression and inter-group comparison are shown. Data are presented as mean ± SD; #P > 0.05, *P < 0.05 and **P < 0.01 been developed, there is not yet a satisfactory treatment These findings illustrate the similar effect of EA on the for HS [12]. In recent years, pre-clinical studies have same phenotypes of different diseases cells. indicated that some natural plant extracts exhibit anti- Collagen, particularly collagen I and III, the most abun- fibrotic properties in  vitro and/or in  vivo [13–15]. Nat - dant components of ECM deposition in HS, is consid- ural extracts are often well-tolerated. Our work, for the ered to be associated with the specific manifestations of first time, has demonstrated that EA attenuated multiple HS that is hard and raised above the skin [20]. A previ- fibrotic phenotypes of HSFs in vitro. ous study by Reanmongkol et al. found that EA has a role The over-proliferation of HSFs is considered to be asso - in wound heling by a rat dermal wound model, while it ciated with HS formation. In studies on the role of EA is not effective on collagen accumulation [21]. In our in fibrotic diseases, to date, EA was reported to exert a study, EA reduced the expression of collagen I and III in hindering effect on the growth of rat PDGF-treated pan - HSFs (Fig.  4), similar to a recent study conducted by Lin creatic stellate cells [16] and rat cardiac fibroblasts [9]. et  al. which demonstrated that EA inhibited the expres- Similarly, our work showed that EA significantly inhib - sion of collagen I and III in rat cardiac fibroblasts[9]. This ited HSFs proliferation (Fig. 1). Moreover, various studies indicates that EA possesses therapeutic potential against focusing on the role of EA in multiple cancer types have fibrosis, which makes it worthy of further research. also reported its inhibitory effect on cell proliferation[6]. The Smad2/3 pathway is widely recognized as a major Studies on enhanced migration, a pathological cellular signaling pathway that leads to HS formation [3, 22]. behavior of HSFs, are gradually increasing. In addition to Activation of the Smad2/3 pathway causes multiple the inhibitory action of EA on the migration of a few can- fibrotic phenotypes of HSFs. TGF-β1 induces the phos - cer cell lines [6], Masamune et al. found that EA attenu- phorylation of Smad2 and 3 by binding to cell surface ated PDGF-induced migration of pancreatic stellate cells TGF-β receptors. Phosphorylated Smad2 and 3 translo- [16], and Lin et  al. found similar inhibitory effect of EA cate to the nucleus with cooperation of Smad4, and these on rat cardiac fibroblasts [9]. Consistent with these stud - molecules, in the form of a transcriptional complex, ies, our work found that EA suppressed HSFs migration regulate downstream target genes expression [23]. Many (Fig. 2). studies have validated that blocking the TGF-β1/Smad2/3 The contraction of human dermal fibroblasts plays an pathway can effectively suppress the pathological pheno - important role in wound healing, but excessive contrac- types of HSFs [24]. Our study demonstrated that EA sup- tion of HSFs contributes significantly to the inelasticity pressed HSFs proliferation, migration, and contraction, and contracture of HS[17]. Some previous studies have and expression of collagen I and III in HSFs by inhibiting reported that EA could reduce the ventricular myocytes the TGF-β1/Smad2/3 signaling pathway (Fig.  7, created of mice and rats in  vitro [18, 19]. Similarly, our results with BioRender.com). However, the upstream mecha- indicated that EA inhibited HSFs contraction (Fig.  3). nism driving EA-induced inhibition of this pathway still Liu et al. Appl Biol Chem (2021) 64:67 Page 8 of 10 Fig. 6 EA inhibited TGF-β1-induced up-regulation of HSFs proliferation, migration, and contraction, and collagen synthesis in HSFs. A HSFs proliferation detected using CCK-8 assay; HSFs migration detected using B wound healing assay (scale bar = 200 μm) and C transwell assay (scale bar = 100 μm); D HSFs contraction detected using collagen lattice contraction assay; E relative mRNA levels of ATCA2, COL1A1, COL1A2, and COL3A1 detected using qPCR; F protein levels of α-SMA, collagen I, collagen III, and GAPDH detected using western blotting; and the corresponding quantitative analysis and inter-group comparison are shown. Data are presented as mean ± SD; *P < 0.05 and **P < 0.01 needs further investigation. In addition to mechanism These findings present EA as a promising novel drug study, further in vivo experiment is needed. A rabbit ear candidate for treatment of HS, and provide theoreti- scar model should be a good candidate, which is widely cal support for further in  vivo experiments and clini- used in the studies on HS [25]. cal application in the future. Moreover, this research In conclusion, this study demonstrates that EA extends our understanding of the biological activity of exerts inhibitory effects on HSFs proliferation, migra - EA. tion, and contraction, and collagen synthesis in HSFs by inhibiting the TGF-β1/Smad2/3 signaling pathway. Liu  et al. Appl Biol Chem (2021) 64:67 Page 9 of 10 Fig. 7 Schematic illustration of EA regulatory effects in HSFs Acknowledgements Engineering Normal University, No. 3050 Kaixuan Street, Changchun 130000, This study was supported by the PhD Research Project of Jilin Engineer- China. ing Normal University (no. BSKJ201923), the Fund Project of the Finance Department of Jilin Province (no. JLSWSRCZX2020-002), the Fund Project of Received: 23 June 2021 Accepted: 31 August 2021 the Development and Reform Commission of Jilin Province (no. 2020C049), the Fund Project of Jilin Provincial Science and Technology Department (no. 20200404166YY ), and the grants from the First Hospital of Jilin University (no. JDYYGH2019006 and no. BQEGCZX2019004). We thank Bethune Institute of Epigenetic Medicine of the First Hospital of References Jilin University for providing experimental conditions for part of cell experi- 1. Finnerty C, Jeschke M, Branski L, Barret J, Dziewulski P, Herndon D (2016) ments in this study. Hypertrophic scarring: the greatest unmet challenge after burn injury. Lancet (London, England) 388(10052):1427–1436 Authors’ contributions 2. Bombaro K, Engrav L, Carrougher G, Wiechman S, Faucher L, Costa B et al JY and XM designed the concept of the study, reviewed and revised the (2003) What is the prevalence of hypertrophic scarring following burns? manuscript. 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Ellagic acid exerts anti-fibrotic effects on hypertrophic scar fibroblasts via inhibition of TGF-β1/Smad2/3 pathway

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Abstract

Hypertrophic scar (HS) is a kind of serious pathological scar with no currently effective treatment. HS fibroblasts (HSFs) are the main effector cells for HS formation. Ellagic acid (EA) exerts regulatory effects in some diseases, but its role in HS remains unclear. This study aimed to evaluate the effect of EA on the fibrotic phenotypes of HSFs and to further investigate the downstream signaling mechanism. The cell counting kit-8 (CCK-8) assay was used to perform cytotoxicity and proliferation assays. HSFs migration was assessed using wound healing and transwell assays. HSFs contraction was measured by a collagen lattice contraction assay and detection of α-smooth muscle actin (α-SMA) expression. The levels of mRNA and protein were determined by qPCR and western blotting, respectively. The results showed that EA inhibited the proliferation, migration, and contraction of HSFs and collagen expression in HSFs in a dose-dependent manner. Furthermore, EA not only suppressed the Smad2/3 pathway but also reversed TGF-β1- induced activation of the Smad2/3 pathway and up-regulation of the fibrotic cellular phenotypes in HSFs. These findings demonstrate that EA exerts anti-fibrotic effects on HSFs by blocking the TGF-β1/Smad2/3 pathway, which indicates that EA is a potential therapeutic candidate for treatment of HS. Keywords: Collagen, Contraction, Ellagic acid, Hypertrophic scar fibroblasts, Migration, Proliferation, Smad pathway, TGF-β1 Introduction the pathological mechanism of HS formation is mainly Hypertrophic scar (HS) is a severe skin fibrotic disease identified by aberrant deposition of extracellular matrix that often occurs after burn injury. HS is generally raised, (ECM), especially collagen I and III, and overactive red, itchy, or painful, and can even leads to movement hypertrophic scar fibroblasts (HSFs) are the effector cells dysfunction. These adverse symptoms result in not only contributing to the progression of HS [3]. It is meaning- physical but also psychological injury to the patients ful to explore an approach that could effectively attenuate [1]. Statistics show that up to 70% of burn patients suf- the fibrotic cellular phenotypes of HSFs, including prolif - fer from HS [2]. In recent years, a few treatments have eration, migration, contraction, and collagen expression. been developed; however, there is currently no effective Ellagic acid (EA) (molecular formula C H O , Fig. 1A) 14 6 8 therapy for HS. Previous studies have demonstrated that is a natural plant polyphenolic compound that is abun- dant in various fruits and nuts. Previous studies have mostly focused on the antioxidant, antihepatotoxic, and antitumor effects of EA [4–6]. Additionally, a few studies *Correspondence: mengxljdyy@jlu.edu.cn; yuja@jlu.edu.cn Xianjun Liu and Xinxin Gao contributed equally to this work have indicated that EA exerts anti-fibrotic effects in liver, Department of Burns Surgery, The First Hospital of Jilin University, No. pancreatic, and cardiac fibrosis [7–9]. However, the effect 1409 Ximinzhu Street, Changchun 130000, China of EA on HS formation remains unclear. Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Liu et al. Appl Biol Chem (2021) 64:67 Page 2 of 10 Fig. 1 EA inhibited HSFs proliferation in a dose-dependent manner. A The structural formula of EA. B Cytotoxicity assay result for HDFs, as detected using CCK-8 assay (n = 3). HSFs proliferation in different experimental groups, as detected using CCK-8 assay at the indicated timepoints (n = 5). C Over trends in growth and D inter-group comparisons are shown. Data are presented as mean ± SD; #P > 0.05, *P < 0.05 and **P < 0.01 The Smad2/3 signaling pathway is a canonical fibro - Affinity), anti-Smad2 (1:5000; cat. no. ab40855; Abcam), sis-associated signal transduction pathway, activation anti-Smad2 (phospho S255) (1:5000; cat. no. ab188334; of which regulates a spectrum of pathological cellular Abcam), anti-Smad3 (1:5000; cat. no. ab40854; Abcam), behaviors of HSFs [3]. TGF-β1, a well-known pro-fibrotic anti-Smad3 (phospho S423 + S425) (1:2000; cat. no. growth factor, is a significant upstream stimulator of the ab52903; Abcam), and anti-GAPDH (1:5000, cat. no. Smad2/3 pathway. Previous studies have found that TGF- 10494-1-AP; Proteintech, IL, USA). Secondary antibod- β1 promotes the proliferation, migration, and contraction ies (1:5000; goat anti-mouse cat. no. SA00001-1; goat of HSFs and collagen synthesis in HSFs by activating the anti-rabbit cat. no. SA00001-2; Proteintech) were used Smad2/3 pathway [10]. Proper regulation of the TGF- for western blotting. EA (cat. no. B21073; Yuanye Bio- β1/Smad2/3 pathway in HSFs is considered a promising tech, Shanghai, China) was dissolved in 1 M NaOH. The strategy for hindering HS formation. final concentration of NaOH was ≤ 0.1% (v/v) and did not In this study, we evaluated the effects of EA on the contribute to toxicity. TGF-β1 (cat. no. bs-2266P; Bioss, proliferation, migration, and contraction of HSFs and Beijing, China) was diluted with sterile double-distilled collagen expression in HSFs. Importantly, we fur- water. ther investigated the association between the TGF-β1/ Smad2/3 pathway and EA-induced regulation of HSFs. Cells isolation and culture Normal human dermal fibroblasts (HDFs) (cat. no. Materials and methods #2320) were purchased from Sciencell, and three cell Antibodies and drugs lines were obtained for subsequent cytotoxicity assay by The primary antibodies used for western blotting in parallel culture. HSFs were primarily cultured from the this study were as follows: anti-α-smooth muscle actin scar tissues of five patients (3 men and 2 women; aged (α-SMA) (1:1000; cat. no. ab32575; Abcam, Cambridge, 10–50  years) who suffered HS and had a cicatrectomy UK), anti-collagen I (1:1000; cat. no. AF7001; Affinity, in the Department of Burns Surgery of the First Hospi- OH, USA), anti-collagen III (1:1000; cat. no. AF0136; tal of Jilin University between January and April 2018. Liu  et al. Appl Biol Chem (2021) 64:67 Page 3 of 10 Protocols were approved by the Ethics Committee of EA or TGF-β1 according to experimental requirements. the First Hospital of Jilin University (no. 2017-088) and At the time points 0, 24, and 48 h after scratching, images were conducted according to the principles expressed in were acquired at three random fields of view using an the Declaration of Helsinki. Written informed consent inverted microscope (magnification 40 × ; Olympus was obtained from all donors before surgery. Briefly, the Corporation, Tokyo, Japan). Wound area was measured epidermis and subcutaneous adipose tissue of HS tis- using ImageJ software (version 1.51w). Migration ability sue masses were shaped. Next, the tissues were cut into of HSFs was evaluated using the data shown as migration approximately 1 mm sections under sterile conditions. rate (%) = (initial wound area – wound area at 24 or 48 h) After washing, sections were placed in a cell culture dish / initial wound area × 100. and incubated in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and Transwell assay double antibiotics (penicillin, 100 U/mL; streptomycin, A transwell assay was used to assess the migration of 0.1  mg/mL) at 37  °C with 5% CO . All cell culture rea- HSFs. Briefly, 2 × 10 HSFs were seeded in the upper gents were supplied by Gibco (Thermo Fisher Scientific, chambers of transwell plates (6-well, 8  μm; Millicell, MA, USA). When fibroblasts reached approximately MA, USA) incubated in serum-free DMEM, and the 90% confluence, they were trypsinized and passaged. lower chambers were supplied with DMEM contain- Cells from passages 2–4 were used in the subsequent ing 10% FBS, with EA or TGF-β1 according to experi- experiments. mental requirements. After 24  h, non-migrated HSFs on the upper face of the polycarbonate membrane were Cytotoxicity and proliferation assays removed with a cotton swab, and migrated HSFs were A cell counting kit 8 (CCK-8) assay was used for cyto- fixed with methanol and stained with Giemsa dye solu - toxicity and proliferation assays. Viable HDFs or HSFs tion (cat. no. R20649; Yuanye Biotech, Shanghai, China). counts were indirectly detected by measuring the opti- Images of each well were captured at three random fields cal density (OD) values. Cytotoxicity assay was per- of view using an OLYMPUS inverted microscope. The formed as described below. Briefly, HDFs were seeded cells were counted using ImageJ software. at 2 × 10 cells per well in a 96-well culture plate, with three replicates per sample, excluding the use of external Collagen lattice contraction assay rows and columns, to avoid edge effects. After complete A collagen lattice contraction assay was used to assess cell attachment, HDFs were processed according to the the contraction ability of HSFs. HSFs were mixed with experimental requirements. After treatment for 48  h, 10 rat tail tendon collagen (cat. no. C8062; Solarbio, Bei- µL of CCK-8 (APExBIO, Houston, USA) solution was jing, China). The final collagen concentration of the mix - added to each well. After incubation for 2 h at 37 °C, the ture was 1  mg/mL, with a cell density of 2.5 × 10 cells/ absorbance was measured at 450 nm wavelength using a mL. The mixture was added to a 24-well cell culture plate microplate reader (Thermo Fisher Scientific, MA, USA). (200 μL/well) and incubated at 37 °C for 1 h. After coag- The cytotoxicity assay results were expressed as cell sur - ulation, 500 μL serum-free DMEM, with EA or TGF-β1 vival rates. Cell survival rate (%) = (OD value of treated according to experimental requirements, was added onto group – OD value of blank)/ (OD value of control group the gel. A picture of each sample was captured at 48  h. - OD value of blank) × 100. As for the proliferation assay, The area of the gels calculated using ImageJ software. The HSFs were seeded at 5 × 10 cells per well in a 96-well contraction rate was presented as a measure of HSFs con- culture plate, and the OD values were tested at time traction: contraction rate (%) = (well area – gel area)/well points 0, 24, 48, and 72 h after treatment. In addition to area × 100. these, the method of OD value detection was the same as the cytotoxicity assay. The proliferation assay results were Real‑time quantitative PCR (qPCR) expressed as OD values. RNA was isolated, and qPCR was carried out. Briefly, total RNA was isolated from the samples using TRI- Wound healing assay zol reagent (Invitrogen, USA). cDNA was synthe- A wound healing assay was used to assess the migration sized using the TransScript All-in-One First-Strand of HSFs. In brief, HSFs were cultured in a 6-well culture cDNA Synthesis SuperMix (TransGen Biotech, Beijing, 4 2 plate at a density of 1 × 10 cells/cm with DMEM con- China). FastStart Universal SYBR Green Master (ROX) taining 10% FBS until the cell confluence reached 100%. (Roche Diagnostics, Mannheim, Germany) was used A scratch wound was made in the middle of each well for qPCR with a Stratagene Mx3005P instrument (Agi- using a 1-mL pipette tip. After washing with PBS, the cul- lent Technologies, USA). The reaction conditions were ture medium was changed to serum-free DMEM, with as follows: initial denaturation at 95  °C for 10  min; 40 Liu et al. Appl Biol Chem (2021) 64:67 Page 4 of 10 cycles of denaturation at 95 °C for 30 s and annealing at GraphPad Prism (version 7.00) statistical package. A 60 °C for 1 min; dissociation at 95 °C for 1 min, anneal- value of P < 0.05 indicated a statistically significant ing at 55  °C for 30  s, and final extension at 95  °C for difference. 30  s. The primers used in qPCR are listed in Table  1. The expression levels of target genes were normalized Results and discussion −C to that of GAPDH, and the 2 method was used to EA inhibited HSFs proliferation in a dose‑dependent calculate the relative mRNA levels [11]. Each sample manner was run in triplicate. First, we performed cytotoxicity assay for EA to HDFs by a CCK-8 assay. As shown in Fig. 1B, there was no sig- nificant difference in cell survival rate between each EA- Western blotting treated group (concentration ≤ 150  μM) and the control Harvested fibroblasts were processed with RIPA lysis group. This result suggested a concentration range, at buffer (CST, USA) supplemented with phenylmethyl - which EA is harmless to normal HDFs. Then, to evalu - sulphonyl fluoride (Thermo Fisher Scientific, MA, ate the effect of EA on HSFs proliferation, a CCK-8 assay USA), protease inhibitor cocktail (TransGen Biotech, was performed. As shown in Fig.  1C and D, HSFs pro- Beijing, China), and phosphatase inhibitor cocktail liferation in the EA-treated groups was attenuated as (TransGen Biotech). Protein concentration was meas- compared with that of the control group. There was no ured using a bicinchoninic acid (BCA) protein assay significant difference in cell viability between the 10  μM kit (Beyotime Institute of Biotechnology, Nantong, group and the control group at 24 and 48  h. The data China). Samples were separated using SDS-PAGE gels, demonstrated a significant decrease in HSFs proliferation and subsequently electro-transferred to PVDF mem- in the 10 μM group as compared with that of the control branes (Millipore, MA, USA) for immunoblotting group at 72  h; HSFs proliferation in the 50 and 100  μM analysis. After blocking with 5% bovine serum albumin groups was significantly lower than that of the control (Sigma-Aldrich, MO, USA) in TBST (0.5% Tween-20) group at all time points. Additionally, HSFs proliferation for 1  h at room temperature, the PVDF membranes in the 50 and 100 μM groups was significantly lower than were incubated with primary antibodies at 4  °C over- that of the 10  μM group at all time points. HSFs prolif- night. Following incubation with the appropriate horse- eration in the 100 μM group was significantly lower than radish peroxidase-conjugated secondary antibodies for that of the 50 μM group at all time points except for 24 h. 1  h at room temperature, proteins were detected with All these data indicate that EA inhibited HSFs prolifera- the EasySee western blotting kit (TransGen Biotech) tion in a dose-dependent manner. using a myECL imager (Thermo Fisher Scientific), and the band intensities were quantified by calculation of EA inhibited HSFs migration in a dose‑dependent manner gray values using ImageJ software. Data were collected The effect of EA on HSFs migration was assessed using from three independent experiments. wound healing and transwell assays. As shown in Fig. 2A and 2B, at 24 and 48 h after scratching, the migration rate of HSFs significantly decreased with increasing EA dose. Statistical analysis In addition, the results of the transwell assay (Fig. 2C and The quantified data are presented as the mean ± stand- 2D) showed that the migrated HSFs were significantly ard deviation (SD). The differences between the two reduced with increasing EA dose, consistent with the groups were analyzed by Student’s t-test using the results of wound healing assay. These data demonstrate that EA inhibited HSFs migration in a dose-dependent manner. Table 1 Sequences of primers for qPCR EA inhibited HSFs contraction in a dose‑dependent Forward (5’ → 3’) Reverse (5’ → 3’) manner To evaluate the effect of EA on HSFs contraction, a col - ACTA2GAC AAT GGC TCT GGG CTC TGT GCT TCG TCA CCC ACG TA TGTAA lagen lattice contraction assay was performed, and the COL1A1ACC AGT CTC CAT GTT GCA CGT GAC CTC AAG ATG TGC CACT expression of α-SMA, which is correlated with the con- GAAGA tractive ability of HSFs, was detected using qPCR and COL1A2GAG GGC AAC AGC AGG TTC TCA GCA CCA CCG ATG TCC A western blotting. As shown in Fig. 3A and B, with increas- ACTTA ing EA dose, the contraction rate significantly decreased. COL3A1CCA CGG AAA CAC TGG TGG ACGCC AGC TGC ACA TCA AGG AC As for the detection of α-SMA, encoded by ACTA2 gene, GAPDHGCA CCG TCA AGC TGA GAA CTGG TGA AGA CGC CAG TGG A the data of qPCR showed that the mRNA level of ACTA2 Liu  et al. Appl Biol Chem (2021) 64:67 Page 5 of 10 Fig. 2 EA inhibited HSFs migration in a dose-dependent manner. HSFs proliferation in different experimental groups, as detected using a wound healing assay at the indicated timepoints (n = 5). A Representative pictures (scale bar = 200 μm), and B quantitative analysis of migration rate (%) and inter-group comparison are shown. HSFs proliferation in different experimental groups, as detected using a transwell assay after 24 h of treatment (n = 5). C Representative pictures (scale bar = 100 μm) and D quantitative analysis of migrated cell numbers are shown. Data are presented as mean ± SD; *P < 0.05 and **P < 0.01 decreased with an increase in EA dose (Fig. 3C). Western EA inhibited TGF‑β1/Smad2/3 signaling pathway in HSFs blotting showed that the change in α-SMA protein level Based on the above findings, we assessed the effect of was in line with that of its mRNA level. All these meas- EA on HS-associated signaling pathway in HSFs. The ures indicated that EA inhibited HSFs contraction in a protein levels of total (t)-Smad2, p-Smad2, t-Smad3, dose-dependent manner. and p-Smad3 were detected using western blotting, and the ratios of p-Smad2/t-Smad2 and p-Smad3/t-Smad3 EA inhibited the expression of collagen I and III in HSFs were calculated to evaluate the Smad2/3 pathway. As in a dose‑dependent manner shown in Fig. 5A and B, the ratios of p-Smad2/t-Smad2 To evaluate the effect of EA on collagen synthesis in and p-Smad3/t-Smad3 significantly decreased in the HSFs, qPCR and western blotting were used to detect the EA-treated group (100 μM), and significantly increased mRNA and protein levels of collagen I and III, respec- in the TGF-β1-treated group (10  ng/mL) as compared tively. As shown in Fig. 4A, the mRNA levels of COL1A1, with those in the control group. Furthermore, EA COL1A2, and COL3A1 were significantly reduced with treatment significantly reversed TGF-β1-induced up- increasing EA dose, in addition to COL1A2 expression regulated phosphorylation of Smad2 and 3. These data in the 10 μM group. The change in protein levels of col - indicated that EA inhibited the TGF-β1/Smad2/3 sign- lagen I and III was consistent with the change in mRNA aling pathway in HSFs. levels (Fig. 4B). These results demonstrate that EA inhib - ited the expression of collagen I and III in HSFs in a dose- dependent manner. Liu et al. Appl Biol Chem (2021) 64:67 Page 6 of 10 Fig. 3 EA inhibited HSFs contraction in a dose-dependent manner. HSFs contraction in different experimental groups, as detected using a collagen lattice contraction assay after 48 h of treatment (n = 5). A Representative pictures, and B quantitative analysis of contraction rate (%) and inter-group comparison are shown. C Relative mRNA level of ACTA2 in different experimental groups, as detected using qPCR (n = 5). D Protein levels of α-SMA and GAPDH in different experimental groups, as detected using western blotting, data were collected from three independent experiments. Data are presented as mean ± SD; *P < 0.05 and **P < 0.01 Fig. 4 EA inhibited the expression of collagen I and III in HSFs in a dose-dependent manner. A The relative mRNA levels of COL1A1, COL1A2, and COL3A1 in different experimental groups, as detected using qPCR (n = 5). B The protein levels of collagen I, collagen III, and GAPDH in different experimental groups, as detected using western blotting, data were collected from three independent experiments. Data are presented as the mean ± SD; #P > 0.05, *P < 0.05 and **P < 0.01 EA inhibited TGF‑β1‑induced up‑regulation of HSFs transwell assay (Fig. 6C), contraction as assessed by colla- proliferation, migration, and contraction, and collagen gen lattice contraction assay (Fig. 6D) and α-SMA detec- synthesis in HSFs tion (Fig. 6E and F), and collagen synthesis as assessed by To further investigate the association between EA- collagen I and III detection (Fig. 6E and F). These results induced inhibition of fibrotic phenotypes and TGF-β1/ demonstrate that EA exerted inhibitory effects on TGF- Smad2/3 signaling pathway in HSFs, we measured the β1-induced fibrotic phenotypes of HSFs. effect of EA (100 μM) on TGF-β1-induced up-regulation of HSFs proliferation, migration, and contraction, and Discussion collagen synthesis in HSFs. The results showed that EA HS, which often occurs after burn injury, is gradu- significantly reversed TGF-β1-induced up-regulation of ally becoming a challenge for clinical workers [1]. proliferation as assessed by CCK-8 assay (Fig. 6A), migra- Although a few therapies, such as laser therapy, com- tion as assessed by wound healing assay (Fig.  6B) and pression garments, and topical drug application, have Liu  et al. Appl Biol Chem (2021) 64:67 Page 7 of 10 Fig. 5 EA inhibited TGF-β1/Smad2/3 signaling pathway in HSFs. A Protein levels of t-Smad2, p-Smad2, t-Smad3, p-Smad3, and GAPDH in different experimental groups, as detected using western blotting, data were collected from three independent experiments. B Quantitative analysis of proteins expression and inter-group comparison are shown. Data are presented as mean ± SD; #P > 0.05, *P < 0.05 and **P < 0.01 been developed, there is not yet a satisfactory treatment These findings illustrate the similar effect of EA on the for HS [12]. In recent years, pre-clinical studies have same phenotypes of different diseases cells. indicated that some natural plant extracts exhibit anti- Collagen, particularly collagen I and III, the most abun- fibrotic properties in  vitro and/or in  vivo [13–15]. Nat - dant components of ECM deposition in HS, is consid- ural extracts are often well-tolerated. Our work, for the ered to be associated with the specific manifestations of first time, has demonstrated that EA attenuated multiple HS that is hard and raised above the skin [20]. A previ- fibrotic phenotypes of HSFs in vitro. ous study by Reanmongkol et al. found that EA has a role The over-proliferation of HSFs is considered to be asso - in wound heling by a rat dermal wound model, while it ciated with HS formation. In studies on the role of EA is not effective on collagen accumulation [21]. In our in fibrotic diseases, to date, EA was reported to exert a study, EA reduced the expression of collagen I and III in hindering effect on the growth of rat PDGF-treated pan - HSFs (Fig.  4), similar to a recent study conducted by Lin creatic stellate cells [16] and rat cardiac fibroblasts [9]. et  al. which demonstrated that EA inhibited the expres- Similarly, our work showed that EA significantly inhib - sion of collagen I and III in rat cardiac fibroblasts[9]. This ited HSFs proliferation (Fig. 1). Moreover, various studies indicates that EA possesses therapeutic potential against focusing on the role of EA in multiple cancer types have fibrosis, which makes it worthy of further research. also reported its inhibitory effect on cell proliferation[6]. The Smad2/3 pathway is widely recognized as a major Studies on enhanced migration, a pathological cellular signaling pathway that leads to HS formation [3, 22]. behavior of HSFs, are gradually increasing. In addition to Activation of the Smad2/3 pathway causes multiple the inhibitory action of EA on the migration of a few can- fibrotic phenotypes of HSFs. TGF-β1 induces the phos - cer cell lines [6], Masamune et al. found that EA attenu- phorylation of Smad2 and 3 by binding to cell surface ated PDGF-induced migration of pancreatic stellate cells TGF-β receptors. Phosphorylated Smad2 and 3 translo- [16], and Lin et  al. found similar inhibitory effect of EA cate to the nucleus with cooperation of Smad4, and these on rat cardiac fibroblasts [9]. Consistent with these stud - molecules, in the form of a transcriptional complex, ies, our work found that EA suppressed HSFs migration regulate downstream target genes expression [23]. Many (Fig. 2). studies have validated that blocking the TGF-β1/Smad2/3 The contraction of human dermal fibroblasts plays an pathway can effectively suppress the pathological pheno - important role in wound healing, but excessive contrac- types of HSFs [24]. Our study demonstrated that EA sup- tion of HSFs contributes significantly to the inelasticity pressed HSFs proliferation, migration, and contraction, and contracture of HS[17]. Some previous studies have and expression of collagen I and III in HSFs by inhibiting reported that EA could reduce the ventricular myocytes the TGF-β1/Smad2/3 signaling pathway (Fig.  7, created of mice and rats in  vitro [18, 19]. Similarly, our results with BioRender.com). However, the upstream mecha- indicated that EA inhibited HSFs contraction (Fig.  3). nism driving EA-induced inhibition of this pathway still Liu et al. Appl Biol Chem (2021) 64:67 Page 8 of 10 Fig. 6 EA inhibited TGF-β1-induced up-regulation of HSFs proliferation, migration, and contraction, and collagen synthesis in HSFs. A HSFs proliferation detected using CCK-8 assay; HSFs migration detected using B wound healing assay (scale bar = 200 μm) and C transwell assay (scale bar = 100 μm); D HSFs contraction detected using collagen lattice contraction assay; E relative mRNA levels of ATCA2, COL1A1, COL1A2, and COL3A1 detected using qPCR; F protein levels of α-SMA, collagen I, collagen III, and GAPDH detected using western blotting; and the corresponding quantitative analysis and inter-group comparison are shown. Data are presented as mean ± SD; *P < 0.05 and **P < 0.01 needs further investigation. In addition to mechanism These findings present EA as a promising novel drug study, further in vivo experiment is needed. A rabbit ear candidate for treatment of HS, and provide theoreti- scar model should be a good candidate, which is widely cal support for further in  vivo experiments and clini- used in the studies on HS [25]. cal application in the future. Moreover, this research In conclusion, this study demonstrates that EA extends our understanding of the biological activity of exerts inhibitory effects on HSFs proliferation, migra - EA. tion, and contraction, and collagen synthesis in HSFs by inhibiting the TGF-β1/Smad2/3 signaling pathway. Liu  et al. Appl Biol Chem (2021) 64:67 Page 9 of 10 Fig. 7 Schematic illustration of EA regulatory effects in HSFs Acknowledgements Engineering Normal University, No. 3050 Kaixuan Street, Changchun 130000, This study was supported by the PhD Research Project of Jilin Engineer- China. ing Normal University (no. BSKJ201923), the Fund Project of the Finance Department of Jilin Province (no. 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Journal

Applied Biological ChemistrySpringer Journals

Published: Dec 1, 2021

Keywords: Collagen; Contraction; Ellagic acid; Hypertrophic scar fibroblasts; Migration; Proliferation; Smad pathway; TGF-β1

References