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Abstract. – OBJECTIVE: Keloids are a skin disorder where the skin goes beyond the origi-nal border of the wound or trauma, resulting in functional and cosmetic deformities, displea-sure, itching, pain, psychological stress, and patient dissatisfaction. This study aimed to ex-plore the therapeutic effect of interleukin-10 (IL-10) on the proliferation of keloid fibroblasts.

PATIENTS AND METHODS: Keloid fibroblasts were isolated, primarily cultured, and treated with IL-10 at different concentrations. Normal skin fibroblasts were used as normal control. Immunofluorescent staining was performed to identify the establishment of keloid, as well as normal skin fibroblast. Cell Counting Kit-8 (CCK-8) was carried out to monitor the prolif-erative variation, while Western blot was con-ducted to detect the expression variation of key members involved in the TGF-β/Smad sig-naling pathway.

RESULTS: Identified by the IF staining of Vi-mentin, a classical biomarker of fibroblast, both primary culture of keloid and normal skin fibro-blasts have been established. Compared with control, the proliferation of Keloid fibroblasts was shown to be significantly suppressed on treatment with IL-10 in a time and dose-de-pendent manner. Expression of P-Smad2/3 and Smad4 were increasingly down-regulated, whereas Smad-7 was up-regulated with the in-creasing concentration of IL-10. By contrast, the variation of Smad 2/3 expressions was hardly in-fluenced. Furthermore, the Collagen Type I and Collagen Type II were found to be markedly de-creased after treatment with IL-10.

CONCLUSIONS: IL-10 was shown to be able to significantly inhibit the proliferation of keloid fibroblasts, which was explicitly and strongly suggestive of its potential therapeutic effect in the management of keloid.

Key Words:Keloid, Fibroblast, IL-10, Proliferation, TGF-β/Smad.

Introduction

Keloids are a skin disorder where the skin goes beyond the borders of the original wound, although keloids of unrecognized origin also occur1-3. Con-ventional wisdom holds that keloids have been seen as the result of aberrant wound healing involving excessive fibroblast participation that is marked by hyalinized collagen bundles, even if this characterization has been questioned4,5. Keloid scars are often regarded as cosmetically unattractive and frustrating problems that follow injuries and can result in functional and cosmetic deformities, displeasure, itching, pain, psycho-logical stress, and patient dissatisfaction. Also, they can possibly affect joint movement, signifi-cantly reducing the quality of life6-8. Therefore, various methods have been used, ranging from surgical to non-surgical approaches, in an attempt to improve keloid scars. Nevertheless, it remains difficult to identify a universal treatment that can produce optimal results for all types of scars due to the range of factors influencing the efficacy of the treatment, including both demographic and anatomical position of the lesion. Indeed, there is still a need to explore the new therapeutic method for the treatment of keloid.

Although many attempts have been made to understand the pathophysiology and the molec-ular abnormalities9,10, the pathogenesis of keloid is yet to be determined. Recently, several evi-

European Review for Medical and Pharmacological Sciences 2019; 23: 9085-9092

C.-K. SHI1, Y.-P. ZHAO2, P. GE2, G.-B. HUANG3

1Dermatological Department, Qilu Children’s Hospital of Shandong University, Jinan, Shandong, PR. China2Burn and Plastic Surgery, The People’s Hospital of Zhangqiu Area, Zhangqiu Area, Jinan, Shandong, PR. China3Burn and Plastic Surgery, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong, PR. China

Corresponding Author: Guobao Huang, MD; e-mail: djl198166@163.com

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dence11,12 revealed that inflammatory mediators play an important role in the keloid microenvi-ronment and may be crucial for keloid fibroblast abnormalities, thus strongly suggesting that in-flammatory factors might hold potential as an anti-scarring therapeutic. Actually, this hypoth-esis has been replicated as earlier as in the year 2000 by Liechty et al13 using a murine model of wound healing. In their study, the absence of IL-10 in fetal mouse skin was shown to lead to scar formation, which was indicative of the potential of IL-10 that may be anti-wound. Unfortunately, the follow-up reports surrounding IL-10 in keloid or scar have been hardly emerged. Enlightened by the aforementioned work, we are determined to continue spinning around the anti-scarring potential of the IL-10 in vitro cell culture system in keloid.

Patients and Methods

Collection of Keloid TissuesThe current investigation was approved by the

Medical Ethics Committee of the Qilu Children’s Hospital of Shandong University, and written informed consent was obtained from each patient before undergoing surgical excision. Six patients, who have not received any previous treatment for keloid scar before surgical excision, were included in the research. Among them, three males and three females, were age-ranged from 23 to 42 years. The location of keloid scar lesions was on the back and arm. The diagnosis of the keloid scar was histopathologically confirmed by the hematoxylin and eosin (HE) staining of skin tissues. The normal skin tissue in the peripheral margin of hypertrophic scar lesion was used as normal control.

Primary Culture of Fibroblasts andTreatment with IL-10

Keloid scar and normal skin tissue were cut into pieces with the size of 1×1 mm, while the epidermis and dermis were isolated by scissors. Then, these pieces were placed in culture dish with a diameter of 10 cm (Corning, Corning, NY, USA). They were subsequently added with 5 ml culture medium containing Dulbecco’s Modi-fied Eagle’s Medium (DMEM; Hyclone, South Logan, UT, USA) with 100 U/mL penicillin and 0.1 mg/mL streptomycin, and 10% fetal bovine serum (FBS; Hyclone, South Logan, UT, USA) at 37°C in a humidified air incubator containing

5% CO2. The medium was changed with 5 ml of culture medium every 3 days. Keloid fibroblasts and normal skin fibroblasts can reach 100% con-fluence for 14 days. Next, they were respectively subcultured (1:3) into 10 cm culture dish. Investi-gations were performed with early passage cells (4th through 6th passage), which were divided into five groups: (A) Control group: without treatment with IL-10; (B, C, D) Experimental group: fibro-blasts were treated with 5, 10, and 20 ng/mL of IL-10; (E) Positive group: treated with 0.2 mg/mL of 5-FU. Once keloid fibroblasts were added with IL-10, the morphological changes of the keloid fibroblasts were observed under an inverted mi-croscope and photos were taken for 48 h.

Cell Proliferation AssayCell proliferation assays were performed using

Cell Counting Kit-8 (CCK-8) to monitor growth. The Keloid fibroblasts (KF) and normal skin fi-broblasts (NF) (1×105) were seeded, respectively, into 96-well plates with 100 mL of growth medi-um per well and allowed to attach and grow over-night. The medium was then replaced with 100 uL of growth medium containing 0,5, 10, and 20 ng/mL of IL-10, and 0.2 mg/mL 5-Fu was taken as positive group. After incubation for 48h, 10 mL of solutions of CCK-8 was added and continued to incubate at 37°C for 3h. The optical density (OD) of plates was read at 520 nm in a plate read-er continuously from the 1st to the 11th day. The experiment was performed independently at least three times.

Immunofluorescence (IF) Staining The cells grown on coverslips were fixed with

4% formaldehyde for 30 min at 4°C, and washed three times with phosphate-buffered saline (PBS); followed by Triton ×100 (0.5%) for 0.5 h at room temperature, and washed three times with PBS; blocked with goat serum (10 mg/mL) for 30 min at 37°C. Cells were then probed with primary antibody Smad7 (sc-365846, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Smad4 (sc-73040, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), P-Smad2/3 (sc-133098, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in Tris-Buffered Saline con-taining 0.5% Tween-20 (TBST) overnight at 4°C. The cells were washed three times with TBST, and incubated with the appropriate secondary antibody (Dylight®-conjugated anti-rabbit-594, anti-rabbit-550 or anti-mouse-488) at 1:1000 for 2 h in a black box at room temperature after using

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the DIPA for 5 min. Images of labeled cells were acquired using a confocal laser scanning micro-scope (Leica, Wetzlar, Germany).

Western BlotThe cells were harvested and washed three

times with ice-cold PBS, followed by lysed with radio immunoprecipitation (RIPA) assay. Cell lysates (50 ug of protein) were subjected to run with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a 10-12% gel, and then transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated with prima-ry antibody at 4°C overnight. After washing for 3 times, with each for 5 min, the membrane was then incubated with alkaline phosphatase (AP)-conju-gated goat anti-rabbit/mouse secondary antibody (diluted at 1:800) at room temperature for 1 h. The membrane blot was visualized with West-ern-Breeze kit (Invitrogen, Carlsbad, CA, USA). The densities of the bands were normalized with those of glyceraldehyde-3-phosphate dehydroge-nase (GAPDH). Primary antibodies were TGF-β1 (#5154, 1:1000; Cell Signaling Technology, Dan-vers, MA,USA), Smad2/3, P-Smad2/3, Smad4, Smad7, Collagen Type I (sc-376350,1:500, San-ta Cruz Biotechnology, Santa Cruz, CA, USA), Collagen Type III (sc-514601,1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and GAP-DH (sc-47724,1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Statistical AnalysisData were processed using SPSS 17.0 software

(SPSS, Inc., Chicago, IL, USA). The data are expressed as mean ± standard error of the mean (SEM), independent sample t-test was applied for paired-comparisons, while one-way ANOVA (Bonferroni’s analysis) was performed for multi-ple comparisons between the groups. The statis-tical significance was set at p<0.05.

Results

Morphological Variation of Keloid Fibroblasts Treated with IL-10

To investigate the effect of IL-10 on keloid, the primary culture of both keloid fibroblasts and nor-mal skin fibroblasts, used as normal control, have been established in vitro in our study (Figure 1A). Morphologically, a remarkably visible difference can be present between keloid fibroblasts and normal skin fibroblasts under the microscope (Figure 1A). The normal skin fibroblasts gradu-ally took on a long spindle, flat star, part of the triangular, 2-3 protruding from the cytoplasmic length from different passages; while, compared with normal fibroblasts, the keloid fibroblasts did not stretch the round cell; many cells arranged in clusters, swirling, or weave pattern. Before conducting the functional analysis of IL-10, both keloid fibroblasts and normal skin fibroblasts were subjected to identification using IF staining

Figure 1. Morphological variation and identification of Keloid fibroblasts and normal skin fibroblasts primarily cultured. A, Establishment of primary culture of both keloid fibroblasts (KF) and normal skin fibroblasts (NF). Clearly, KF growth was more radiated, swirling, or weave pattern compared with the NF on the 3rd passage; B, Identification of the fibroblasts using IF staining (Dylight-488) with Vimentin (sc-80975, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Expectedly, Vimentin was seen mainly in the cytoplasm of Keloid fibroblast and normal skin fibroblast (scale bar=50 μm). The magnification of passage 1st and 3rd was 100 fold and 200 fold, respectively. The magnification of IF staining (Figure 1B) was 200 fold.

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with Vimentin, a classically well-accepted differ-entiating biomarker for fibroblasts. Results from the IF staining showed that Vimentin was, as expected, expressed mainly in the cytoplasm of fibroblasts, explicitly suggesting that the primary culture of keloid and normal skin fibroblasts have been successfully established (Figure 1B).

IL-10 Significantly Suppresses the Proliferation of Keloid Fibroblasts

Both keloid fibroblasts and normal skin fibro-blasts were treated with a gradient concentration of IL-10 for 48 h, respectively. As shown in Figure 2A, in terms of morphological variation, keloid fibroblasts took on a long spindle-shaped character

with larger cell bodies; cytoplasm of fibroblast was rich and showed multiple angle shape which grew by two or three synaptic lengths after treatment with IL-10 for 48 h. While, no significant morpho-logical variation of normal skin fibroblast can be observed (Figure 2B). Moreover, the number of keloid fibroblasts treated with the different con-centrations of IL-10 was increasingly decreased compared with control (Figure 2A). By contrast, the number of normal skin fibroblasts seems to be hardly influenced (Figure 2B). Notably, morpho-logical changes of keloid fibroblasts appear to be dose-dependent. At 20 ng/mL, the spindle-shaped keloid fibroblasts were remarkably shorter and smaller, intercellular gaps were wider, and the

Figure 2. IL-10 can suppress the growth of Keloid fibroblasts. A, Gradual loss of fibroblastic characteristics of Keloid fibroblasts after treatment with different concentration of IL-10 for 48 h. B, By contrast, the morphology and growth of normal skin fibroblasts (NF) were hardly influenced after treatment with different concentration of IL-10 for 48 h. The magnification fold was ×200. C, Proliferation of Keloid fibroblast was faster than that of NF from 3rd day to 8th day. From 2nd day, all proliferation of Keloid fibroblast groups treated with different concentrations of IL-10 ng/mL began to be suppressed (p<0.05). There was also a significant difference between 5-FU group and experimental groups treated with different concentration of IL-10 (p<0.05). D, No significant effect of IL-10 on the normal skin fibroblasts (NF) was observed, irrespective of different concentrations of IL-10 ranging from 0 ng/mL, 5 ng/mL, 10 ng/mL to 20 ng/mL) (p>0.05).

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number of cells gradually decreased with the in-creasing of IL-10, compared with the control. The data obtained evidently indicate that IL-10 could markedly suppress the growth of keloid fibroblast, as opposed to normal skin fibroblasts. To further confirm, the CCK-8 method was adopted to quan-titatively monitor the growth variation. Results of CCK-8 exhibited that in terms of proliferative capacity, the keloid fibroblasts ability was remark-ably much more than that of normal fibroblasts. Additionally, the proliferation of keloid fibroblasts was increasingly suppressed with the increasing gradient concentration of IL-10 (5 ng/mL, 10 ng/mL, and 20 ng/mL) relative to control (Figure 2C). The group where keloid fibroblasts were treated with 5-FU (0.2 mg/mL) was used as positive con-trol. It was exhibited that IL-10 has no effect on the proliferation of normal fibroblasts (Figure 2D). Results demonstrate that IL-10 could significant-ly inhibit the proliferation of keloid fibroblasts, whereas having no significant effect on the growth of normal fibroblasts.

Expression Variation of TGF-β/Smad Signaling Pathway after Treatment with IL-10 in Keloid Fibroblasts

Having observed that IL-10 can suppress the proliferation of keloid fibroblasts, as opposed to

normal skin fibroblast, we sought to investigate the mechanism by which IL-10 works in the keloid fibroblasts. It has been reported that the TGFβ/Smad signaling pathway was actively in-volved in the formation of keloid scar. Therefore, we hypothesized that IL-10 could influence the TGFβ/Smad signal pathway. To test this hypoth-esis, the translocation, as well as the variation of p-Smad2/3, Smad4, and Smad7 were detected using the IF staining. Results of the IF staining showed that, compared with control, the expres-sion of Smad7 was increased with the treatment of IL-10. While, the p-Smad2/3 and Smad 4 sup-posed to be in the nucleus, they were observed to translocate from the nucleus to the cytoplasm. Additionally, the expression of p-Smad2/3 and Smad 4 were found to be down-regulated after the treatment of IL-10 (Figure 3A). To further confirm the results obtained using the IF stain-ing, Western blot was employed to evaluate the translocation, as well as the expression variation of p-Smad2/3, Smad4, and Smad7 in keloid fibro-blasts in the presence and absence of IL-10 treat-ment. As expected, the variation of p-Smad2/3, Smad4, and Smad7 expression was highly consis-tent with what we observed using the IF staining method (Figure 3B). Furthermore, both Collagen type I and Collagen type III expression in keloid

Figure 3. Variation of TGF-β/Smad signal pathway in keloid fibroblasts treated with IL-10. A, Smad 7 was significantly up-regulated whereas p-Smad 2/3 and Smad 4 was pronouncedly down-regulated after treatment with IL-10, as exemplified by IF staining. The expression of Smad7 (primary antibody 1:500; Dylight-649 1:1000 ), sub-localized in the cytoplasm of fibroblasts, was gradually strengthened with the increasing concentration of IL-10; Smad4 (primary antibody 1:500; Dylight-549 1:1000) and P-Smad2/3 (primary antibody 1:500; Dylight-488 1:1000) translocated from nucleus to cytoplasm, and the expression of Smad 4 and p-Smad1/3 were gradually weakened with the increasing concentration of IL-10 (scale bar=50 μm). B, Variation of TGF-β1, Smad7, Smad2/3, p-Smad 2/3, and Smad 4 expressions were evaluated by Western blotting. Meanwhile, expression of Collagen type I and Collagen type III was evaluated using Western blot.

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fibroblasts were detected using Western blot in the absence and presence of treatment with IL-10. It was exhibited that Collagen type I and Colla-gen type III were inhibited after treatment with IL-10, relative to control group (Figure 3B).

Discussion

IL-10 was found to be capable of markedly suppressing the growth of fibroblasts derived from keloid in a dose-dependent manner. Also, p-Smad 2/3 and Smad 4 were translocated from cytoplasm to the nuclear compartment in keloid fibroblasts treated with IL-10. In contrast, Smad 7 was translocated from the nuclear compart-ment to the cytoplasm. Smad 7 expression was gradually up-regulated, whereas TGF-β1 was de-creased. In addition, expression of Collagen I and III was remarkably reduced after the treatment with IL-10 in keloid fibroblasts, suggesting that it is by inhibiting the TGF-β1/Smad signal pathway that IL-10 suppresses the expression of Collagen of keloid fibroblasts, thereby preventing the for-mation of keloids.

Despite IL-10 had been demonstrated to be able to suppress the formation of keloid scars using the animal model13, little is known regard-ing the underlying mechanism by which IL-10 operates in the inhibition of keloid scar. The following studies related to IL-10 in keloid were, therefore, extended from in vivo animal model to in vitro cell culture system. The original report investigating IL-10 operated in would healing using the in vitro cell culture system came from human skin fibroblasts14. This work reported that IL-10 itself had no effect on the growth of fibro-blast; however, it was capable of reducing TNF-a induced fibroblast proliferation, which was sug-gestive of the inhibitory role of IL-10 in the remodeling of the ECM during wound healing. Later, this notion was supported by another inde-pendent mechanistic report15 showing that IL-10 apparently inhibited fibrosis by activating AKT and STAT3 phosphorylation. In recent studies16,17 with regard to the potential suppressing role of IL-10 in fibrosis, IL-10 was observed to be able to suppress the fibrosis by inhibiting the TGF-β/Smad signaling pathway. Indeed, we demon-strated that IL-10 had the potential to suppress fibroblasts proliferation and reduce extracellular collagen deposition by regulating the TGF-β/Smad signaling pathway. Enlightened by these phenomena previously reported, we were trying

to evaluate the effect exerted by IL-10 on the proliferation and differentiation of fibroblasts de-rived from keloid scars and normal skin tissues, hypothesizing that IL-10 might suppress the pro-liferation of fibroblasts derived from Keloid. Our results displayed that IL-10 was significantly able to prevent the proliferation and differentiation of fibroblasts derived from keloid scars by suppress-ing the TGF-β/Smad signaling pathway, which was in line with the previous findings made by Xu et al16. Moreover, the Collagen type I and type III, two main components of the extracellular matrix, were remarkably reduced after treatment with IL-10 in a dose-dependent manner, further strengthening the notion that IL-10 can inhibit Collagen I and III expression via suppressing the TGF-β/Smad signaling pathway.

Keloid is a complex and multifactorial fibrot-ic abnormality associated with excessive fibro-blast proliferation and collagen synthesis18,19. We discovered the inhibitory effects of IL-10 on keloid fibroblasts. Our results showed that IL-10 prevented keloid fibroblasts from Collagen I, Collagen III expression, and keloid fibroblasts proliferation in a time and dose-dependent man-ner. The reduction in collagen expression caused by IL-10 could be explained by the phenomenon of the suppression of the TGF-β/Smad signaling by IL-1016. Meanwhile, we did not observe the similar effects exerted by IL-10 over normal skin fibroblasts, which is indicative of its selective role of IL-10 that could only have a remarkable effect on keloid fibroblasts, as opposed to normal fibroblasts. The underlying mechanism remains unknown and needs to be further investigated. It should be noted that IL-10 itself can direct-ly suppress the growth of keloid fibroblasts, as opposed to normal skin fibroblasts, which was slightly different from the report of Moroguchi et al14, claiming that IL-10 itself had no effect on fi-broblast proliferation, but rather reduced the TN-Fa-induced fibroblast proliferation. The possible reasons leading to the discrepancy between our current study and the earlier one by Moroguchi et al14 may be that a different working concentra-tion of IL-10 was taken, and different cell lines were employed. In our research, IL-10, whose concentration was at 40 ng/mL was displayed to be significantly growth-inhibiting in keloid fibroblasts. While, in the study by Moroguchi et al14, the concentration of IL-10 at 10 ng/mL was taken to treat the normal human skin fibroblasts in their experiment. Besides, they employed the primary culture of normal human skin fibroblasts

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as an experiment subject, which was exactly set up as normal control in our own report. Actually, as stated above, we have not even observed the growth-inhibiting role of IL-10 on the normal human skin fibroblasts.

It should be noted that, in our study, we focused only on the effect exerted by IL-10 over-prolifer-ation of keloid fibroblasts. Other aspects, such as migration and invasion have not been evaluated. Besides, we have paid attention when interpret-ing our data. There are other interleukins, other than IL-10, for instance of IL-6 and IL-820, which have also been found able to directly suppress the growth of fibroblasts. Although we first showed that IL-10 was able to suppress the growth of ke-loid fibroblasts and Collagen synthesis by inhibit-ing the TGF-β/Smad signaling pathway, there are still two limitations in our study. First, our results require a further acknowledgment using animal model in vivo; second, since the TGF-β/Smad signaling pathway was Smad-dependent and-in-dependent, whether or not IL-10 could affect other signaling pathways remains to be further investigated.

Conclusions

IL-10 inhibited the proliferation of keloid fibro-blasts derived from keloid scars and collagen synthesis, and that IL-10 could negatively modu-late the TGF-β/Smad signaling pathway, thereby preventing keloid fibroblasts from proliferation and collagen production. These data strengthens the notion that IL-10 could have the potential translational significance to cure the keloid scar.

Conflict of InterestThe Authors declare that they have no conflict of interests.

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