Keloid Fibroblast Cells Research Articles

Single-cell RNA sequencing analysis reveals the role of TXNDC5 in keloid formation.

Thioredoxin domain-containing protein 5 (TXNDC5) is associated with fibrosis in a variety of organs, but its mechanism of action in keloid is unclear. In this study, we aimed to investigate the mechanism of TXNDC5 in keloid. Single-cell RNA sequencing data of keloid and normal scar samples obtained from public databases were normalized and clustered using the Seurat package. Pathway enrich analysis was conducted using biological process enrichment analysis and Gene Set Enrichment Analysis (GSEA). In addition, TXNDC5 expression and its effects on migration and invasion of keloid fibroblasts (KFs) were validated based on cell function experiments. A total of five cell types were obtained. The KF clusters were further clustered into two fibroblast subtypes (Fibroblast cells 1 and Fibroblast cells 2). Biological process enrichment analysis showed that transforming growth factor beta (TGF-β) signaling pathway was enriched in the two fibroblast subtypes. GSEA analysis demonstrated that genes in TGF-β signaling pathway were mainly enriched in Fibroblast cells 1, and that genes involved in cell proliferation, migration, and the TGF-β signaling pathway were all high-expressed in fibroblast cells 1. TXNDC5 was positively correlated with fibroblast proliferation, migration and TGF-β signaling pathway, and AUCell score. The cellular experiment confirmed that the messenger RNA and protein levels of TXNDC5 and TGF-β1 were high-expressed in KFs cells (P<0.001), and that knockdown of TXNDC5 downregulated TGF-β1 expression and inhibited migration and invasion of KFs (P<0.0001). Our study indicated that TGF-β signaling pathway was enriched in fibroblast cells, and TXNDC5 was positively correlated with proliferation, migration, and TGF-β signaling pathway. Cellular experiment demonstrated that knocking down TXNDC5 downregulated TGF-β1 expression, and suppressed migration and invasion of KFs. The current discoveries provided a new therapeutic strategy for the treatment of keloid.

Genistein inhibited endocytosis and fibrogenesis in keloid via CTGF signaling pathways

BackgroundThis study aimed to evaluate soy isoflavones' effect and potential use—specifically genistein—in treating human keloid fibroblast cells (KFs) and in a keloid tissue culture model.MethodsTo investigate the effects of genistein on keloid, a wound-healing assay was performed to detect cell migration. Flow cytometry was used to measure apoptosis. Western blotting and immunofluorescence staining were performed to detect the expression of target proteins. KF tissues were isolated, cultured, and divided into the control, silenced connective tissue growth factor (CTGF) proteins, and shNC (negative control) groups.ResultsGenistein suppressed cell proliferation and migration, triggering the cell cycle at the G2/M phase and increasing the expression of p53 dose-dependent in keloids. Genistein inhibited the expression of COL1A1, FN, and CTGF mRNA and protein. Knockdown CTGF reduced the migrated ability in KFs. Genistein also abated TGF-β1-induced keloid fibrosis through the endocytosis model. Separated and cultured the keloid patient’s tissues decreased the cell migration ability by genistein treatment and was time-dose dependent.ConclusionsThis study indicated that genistein-induced p53 undergoes cell cycle arrest via the CTGF pathway-inhibited keloid cultured cells, and genistein suppressed the primary keloid cell migration, suggesting that our research provides a new strategy for developing drugs for treating keloids.

Network Pharmacology, Molecular Docking Analysis and Experiment Validations on Molecular Targets and Mechanisms of the Dispel-Scar Ointment in Scar Treatment.

Dispel-Scar Ointment is used in Traditional Chinese Medicine to treat scarred tissue and increasing evidence has shown that DSO has potent therapeutic; however, its exact mechanism remains unexplored. This study explored the molecular mechanisms of action of DSO in scarring using network pharmacology, molecular docking, and experimental validation. Public databases were used to predict the bioactive ingredients and putative targets of DSO against scars. A compounds-targets network was constructed using the Cytoscape software. Enrichment analysis was performed using ClueGo and FunRich to specify the biological functions and associated pathways of hub targets. Molecular docking was used to verify the correlation between the major active components and hub targets, visualised using PyMol 2.3. Experimental validations were conducted to elucidate the influence of DSO on keloid fibroblast cells using the CCK-8, wound-scratch, cell reactive oxygen species, and western blot assays. Results：Network pharmacological analysis of DSO for scar treatment identified 146 ingredients and 1078 gene targets. Major targets included, prostaglandin-endoperoxide synthase 2 matrix metallopeptidases, and nitric oxide synthase 2. ClueGo analysis revealed 29 pathways (p&amp;amp;amp;lt;0.05) and FunRich 345 pathways (p&amp;amp;amp;lt;0.05), mainly toll-like receptor, TGF-β, interleukin-4/13, glypican, and tumour necrosis factor-related apoptosis-inducing ligand pathways. Molecular docking showed MMP2-flavoxanthin, MMP9-luteolin and MMP-9-kaempferol bound best to DSO. DSO could inhibit the proliferation and migration of scar fibroblasts and promote their apoptosis in a concentration-dependent manner. DSO also decreased TGF-β1, -βR2, pSMAD2, pSMAD3, SMAD4, CoL1a1, and MMP2 expression. Network pharmacology, molecular docking, and experimental validation showed DSO&amp;amp;#039;s potential in treating scars. It may inhibit scars via the TGF-β1/SMADs/MMPs signalling pathway, providing a basis for DSO&amp;amp;#039;s scar treatment application.

Targeting the Epigenome Reduces Keloid Fibroblast Cell Proliferation, Migration, and Invasion

Keloids are pathological fibroproliferative scars resulting from abnormal collagen deposition within and beyond the margins of the initial cutaneous insult. Keloids negatively impact quality of life functionally and cosmetically, with current treatment modalities unsatisfactory. Recent studies indicate that epigenetic dysregulation is central to the development and progression of keloids. Here we evaluate the functional significance of epigenetic targeting strategies in vitro using patient-derived keloid fibroblasts treated with small molecule inhibitors of HDACs, LSD1, CoREST and p300, as potential therapies for keloids. We find that both the dual-acting CoREST inhibitor, corin, and the HDAC inhibitor, entinostat, reduce fibroblast proliferation more than the LSD1 inhibitor, GSK-LSD1; additionally, corin was the most effective inhibitor of migration and invasion across keloid fibroblasts. RNA-seq analysis of keloid fibroblasts treated with corin demonstrates coordinate upregulation of many genes including key mediators of cell adhesion such as claudins. Corin also downregulates gene sets involved in cell cycle progression, including reduced expression of cyclins A1 and B2 compared to DMSO. These results highlight a significant role for epigenetic regulation of pathologic mediators of keloidal scarring and suggest that inhibitors of the epigenetic CoREST repressor complex may prove beneficial in the prevention and/or treatment of keloidal scarring in patients.

BMP1 Promotes Keloid by Inducing Fibroblast Inflammation and Fibrogenesis.

Keloid is a typical fibrotic and inflammatory skin disease with unclear mechanisms and few therapeutic targets. In this study, we found that BMP1 was significantly increased in a collagen high-expressing subtype of fibroblast by reanalyzing a public single-cell RNA-sequence data set of keloid. The number of BMP1-positive fibroblast cells was increased in keloid fibrotic loci. Increased levels of BMP1 were further validated in the skin tissues and fibroblasts from keloid patients. Additionally, a positive correlation between BMP1 and the Keloid Area and Severity Index was found in keloid patients. In vitro analysis revealed collagen production, the phosphorylation levels of p65, and the IL-1β secretion decreased in BMP1 interfered keloid fibroblasts. Besides, the knockdown of BMP1 inhibited the growth and migration of keloid fibroblast cells. Mechanistically, BMP1 inhibition downregulated the noncanonical TGF-β pathways, including p-p38 and p-ERK1/2 signaling. Furthermore, we found the delivery of BMP1 siRNAs could significantly alleviate keloid in human keloid-bearing nude mice. Collectively, our results indicated that BMP1 exhibited various pathogenic effects on keloids as promoting cell proliferation, migration, inflammation, and ECM deposition of fibroblast cells by regulating the noncanonical TGF-β/p38 MAPK, and TGF-β/ERK pathways. BMP1-lowing strategies may appear as a potential new therapeutic target for keloid.

Effects of Green Tea Leaf Extract on Viability, Apoptosis, and Expression of Interleukin-6 in Keloid Fibroblasts

Green tea leaf (Camellia sinensis) extract contains polyphenols, anti-inflammatory and anti-carcinogenic agents that may effectively to as keloid therapy. This study aims to examine the effect of green tea leaf extract on viability, apoptosis, and interleukin-6 expression in keloid fibroblasts. Keloid fibroblast cultures were divided into groups that were cultivated with green tea extract at dosages of 100, 200, 400, and 800 μg/mL, negative control (10% Fetal Bovine Serum/FBS), and positive control (100 μM dexamethasone). This study used post-test-only control group design. The highest mean IL-6 expression was in the GTE 200 (275,942.7 pg/mL), while the lowest was in the GTE 800 ( -2,890.6 pg/mL). The viability and apoptosis was analized by using one-way ANOVA, continued with Least Significant Difference (LSD) with a significance of p 0.05 in the GTE 800 treatment group compared to the entire intervention group. Kruskal-Wallis followed by a Mann-Whitney was carried out for interleukin-6 (IL-6) with a significance of p 0.05 in the extract therapy group at dosages of 200, 400, 800 μg/ml and 100 μM dexamethasone. In conclusion, green tea leaf extract at 800 μg/ml could reduce viability interleukin-6 expression and increase apoptotic caspase-3 expression in keloid fibroblast cells.

Structure-based screening of inhibitor platelet-derived growth factor from ethanol extract of Uncaria gambir (Hunter) Roxb. as an antifibrotic in keloid fibroblast cells

Molecular Docking and Simulation studies of Farnesyl Trasnferase with the potential inhibitor TheflavinR. Balajee, M. S. Dhana Rajan

Therapeutic candidates for keloid scars identified by qualitative review of scratch assay research for wound healing.

The scratch assay is an in vitro technique used to analyze cell migration, proliferation, and cell-to-cell interaction. In the assay, cells are grown to confluence and then 'scratched' with a sterile instrument. For the cells in the leading edge, the resulting polarity induces migration and proliferation in attempt to 'heal' the modeled wound. Keloid scars are known to have an accelerated wound closure phenotype in the scratch assay, representing an overactivation of wound healing. We performed a qualitative review of the recent literature searching for inhibitors of scratch assay activity that were already available in topical formulations under the hypothesis that such compounds may offer therapeutic potential in keloid treatment. Although several shortcomings in the scratch assay literature were identified, caffeine and allicin successfully inhibited the scratch assay closure and inflammatory abnormalities in the commercially available keloid fibroblast cell line. Caffeine and allicin also impacted ATP production in keloid cells, most notably with inhibition of non-mitochondrial oxygen consumption. The traditional Chinese medicine, shikonin, was also successful in inhibiting scratch closure but displayed less dramatic impacts on metabolism. Together, our results partially summarize the strengths and limitations of current scratch assay literature and suggest clinical assessment of the therapeutic potential for these identified compounds against keloid scars may be warranted.

Analysis of HOX gene expression and the effects of HOXA9 overexpression in fibroblasts derived from keloid lesions and normal skin.

Keloids are fibroproliferative lesions resulting from an abnormal wound healing process due to pathological mechanisms that remain incompletely understood. Keloids tend to occur more frequently in anterior versus posterior body regions (e.g., ears, face, upper torso); this has been attributed to higher skin tension in those areas, although this has not yet been conclusively proven. Previous studies reported reduced expression of multiple homeobox (HOX) genes in keloid versus normal fibroblasts, suggesting a role for HOX genes in keloid pathology. However, HOX genes are differentially expressed along the anterior-posterior axis. Hypothetically, differential HOX expression may be due to differences in body sites, as matched donor sites are often unavailable for keloids and normal skin. To better understand the basis for differential HOX gene expression in cells from keloids compared with normal skin, we compared HOXA7, HOXA9, HOXC8 and HOXC11 expression in keloid and normal skin-derived fibroblasts from various body sites. When keloid (N= 20) and normal (N= 12) fibroblast cell strains were evaluated, expression of HOXA7, HOXA9 and HOXC8 was significantly lower in keloid versus normal fibroblasts. However, HOX gene expression was lower in fibroblasts from more anterior versus posterior body sites. When keloid and normal cells from similar body sites were compared, differential HOX expression was not observed. To investigate the phenotypic relevance of HOX expression, HOXA9 was overexpressed in keloid and normal fibroblasts. HOXA9 overexpression did not affect proliferation but significantly reduced fibroblast migration and altered gene expression. The results suggest that differential HOX expression may be due to differences in positional identity between keloid and normal fibroblasts. However, HOX genes can potentially regulate fibroblast phenotype, suggesting that differential HOX gene expression may play a role in keloid development in anterior body sites.

Antifibrotic effects of Hypocrellin A combined with LED red light irradiation on keloid fibroblasts by counteracting the TGF-β/Smad/autophagy/apoptosis signalling pathway.

Keloids are characterized by abnormal proliferation of fibroblasts and continuous deposition of extracellular matrix (ECM) components. In the field of dermopathy, photodynamic therapy (PDT) with visible light has been increasingly investigated. The natural photosensitizer Hypocrellin A (HA) was shown to have excellent light induced anticancer, antimicrobial and antiviral activities. In this experiment, we investigated the impacts of HA united light-emitting diode (LED) red light irradiation on human keloid fibroblast cells (KFs). Our results showed that HA combined with red light irradiation treatment (HA-R-PDT) decreased KF viability, reduced KF collagen production and ECM accumulation, inhibited cell proliferation, suppressed cell invasion and induced cell apoptosis. Moreover, our observations demonstrated that the TGF-β/Smad signalling pathway and autophagy were restrained by HA-R-PDT. TGF-β1 could promote autophagy in KFs through both the Smad and ERK pathways, while inhibition of autophagy altered the TGF-β1 levels through negative feedback. Therefore, HA-R-PDT suppressed cell hyperproliferation, collagen synthesis and ECM accumulation of KFs by regulating the TGF-β1-ERK-autophagy-apoptosis signalling pathway. HA-R-PDT deserves systematic investigation as a potential therapeutic strategy for keloids, and autophagy might be a promising candidate in the treatment of KFs.

Activating transcription factor 3 (ATF3) regulates cell growth, apoptosis, invasion and collagen synthesis in keloid fibroblast through transforming growth factor beta (TGF-beta)/SMAD signaling pathway

ABSTRACT The successful treatment of keloids is a great challenge in the plastic surgery field. Activating transcription factor 3 (ATF3) is discovered as an adaptive responsive gene, which plays a critical role in fibroblast activation. This study aimed to investigate the expression and biological role of ATF3 in the pathogenesis of keloids. ATF3 expression in normal skins and keloids was evaluated by real-time PCR, western blot and immunohistochemistry. Effects of ATF3 on cell growth, apoptosis, invasion and collagen production were evaluated in keloid fibroblast cells overexpressing or downregulating ATF3. ATF3 expression was significantly elevated in keloid tissues when compared with that of normal skins and parakeloidal skin tissues. Moreover, ATF3 promoted cell proliferation and collagen production in keloid fibroblast cells. Conversely, transfection with siRNA targeting ATF3 led to decreased cell viability and collagen synthesis via inhibiting transforming growth factor-β1 (TGF-β1) and fibroblast growth factor 2/8 (FGF2/8) production in keloid fibroblasts. ATF3 could reduce the apoptosis rate of keloid fibroblast cells. Molecularly, we found that ATF3 promoted BCL2 level and inhibit the expression of BCL2 associated agonist of cell death (Bad), Caspase3 and Caspase9 in keloid fibroblast cells. ATF3 also enhanced the invasive potential via upregulating the expression of Matrix Metalloproteinases (MMP) family members (MMP1, MMP2, MMP9 and MMP13). ATF3 could induce activation of TGF-β/Smad signaling pathway in fibroblasts. Collectively, ATF3 could promote growth and invasion, and inhibit apoptosis via TGF-β/Smad pathway in keloid fibroblast cells, suggesting that ATF3 might be considered as a novel therapeutic target for the management of keloid.

The Effect of Roselle (Hibiscus sabdariffa L.) Flowers Extract on the Apoptosis of Fibroblast Proliferation in Keloids

BACKGROUND: Keloid is a benign fibroproliferative dermal disorder as a result of dysregulation wound healing process in susceptible individuals. The pathogenesis is not clearly known yet, but upregulation of transforming growth factor-β1 (TGF-β1) was found to have a significant role in inducing hyperproliferation of fibroblast cells. Roselle (Hibiscus sabdariffa L.) flower extract has been found to have high content of polyphenols. Some studies have shown inhibition effect of H. sabdariffa polyphenols extract on TGF-β1, and as result affects fibroblast proliferation. Therefore, roselle flower extract might have a significant role in the prevention of keloid formation.&#x0D; AIM: The objective of the study was to determine the effect of roselle flower extract on fibroblast cells proliferation in human keloid.&#x0D; METHODS: An experimental controlled trial of 10 different concentrations (1.96 μg/ml, 3.91 μg/ml, 7.81 μg/ml, 15.63 μg/ml, 31.25 μg/ml, 62.50 μg/ml, 125 μg/ml, 250 μg/ml, 500 μg/ml, and 1000 μg/ml) of roselle (H. sabdariffa L.) flower extract was done on cultured fibroblast cells originated from human keloid biopsied tissue. Tunnel assay was done to evaluate the apoptosis rate of the cultured fibroblast cells on each concentration. Determination of TGF-β1 titer of the cultured human keloid fibroblast cells in and cytotoxicity assay of the extract on cultured normal human fibroblast cells in each concentration were done with enzyme-linked immunosorbent assay method. All the assays were done in triple repetition. Statistical analysis using linear regression test was done to determine the association between the concentration of roselle flower extract with apoptosis rate and TGF-β1 titer. One-way ANOVA was used to analyze the results of cytotoxicity assay.&#x0D; RESULTS: Apoptosis rate of the cultured fibroblast cells was found to be increased dose dependently with roselle flower extract concentration (r2 = 0.797; p &lt; 0.05). TGF-β1 titer was inversely related with the extract concentration (r2 = 0.501; p &lt; 0.05). Cytotoxicity assay revealed that no differences in absorbance value and viability cells were found in each concentration.&#x0D; CONCLUSION: Roselle (H. sabdariffa L.) flower extract was found to induce apoptosis of the cultured fibroblast cells and reduction of TGF-β1 titer in dose-dependent pattern, without cytotoxicity effect against human fibroblast cells.

TSG-6 Inhibits the Growth of Keloid Fibroblasts Via Mediating the TGF-β1/Smad Signaling Pathway

Background The cytokine TNF-α-stimulated gene-6 (TSG-6) had been verified to have a certain inhibitory effect on the inflammation. During wound healing, fibroblasts increasingly proliferated and deposited collagen fibers, leading to the formation of pathological scars. We sought to elucidate the mechanism by which the TGF-β1/Smad pathway was mediated by TSG-6 in human keloid fibroblasts. Materials and methods Human keloid fibroblast cells were isolated from keloid tissue by enzyme digestion and identified by immunocytochemistry. Lentiviral vectors pLVX-puro-TSG-6 and pLVX-shRNA1-TSG-6 were constructed which were then transfected into human keloid fibroblasts. The mRNA and protein levels of TSG-6 were detected respectively by RT-PCR and western blot assay. The intracellular localization of TGF-β1-induced proteins and phosphorylated (p)-Smad2/3 in keloid fibroblasts were investigated using an immunofluorescence assay. Plasminogen activator inhibitor-1 (PAI-1) transcriptional activity was detected by RT-PCR. Results TSG-6 could effectively interfere the TGF-β1/Smad signal transduction pathway in keloid fibroblasts rather than in normal fibroblasts. The phosphorylation levels of Smad2/3 were notably reduced by TSG-6 treatment. TSG-6 blocked the complex formation of Smad2/3/4, and their nuclear translocation. However, it upregulated Smad7 expression, presenting dose dependence. PAI-1 was also suppressed by TSG-6 treatment. Conclusions TSG-6 inhibits proliferation by inducing apoptosis in keloid fibroblasts, which may be associated with TGF-β1/Smad pathway.

The effect of 5\u03b1-oleandrin on keloid fibroblast activities

BackgroundKeloids develop due to hyperactivity of keloid fibroblast (KF) in proliferation, migration, and collagen deposition along with low rates of collagen degradation. These are a result of the Wnt/β catenin signaling pathways under stimulation of TGF-β. 5α-oleandrin can suppress Wnt-targeted genes of osteosarcoma cells. We aimed to evaluate the anti-fibrotic effects of 5α-oleandrin on KF activities.MethodsWe collected the core of keloid materials from six patients who underwent keloid debulking surgery. Passage 4 of KF cells were then treated with mitomycin-C, 5α-oleandrin, and dilution medium as the negative control. To determine the effective dose of 5α-oleandrin, we diluted 5α-oleandrin into various concentrations. The incubation periods were 24 h, 48 h, and 72 h. The anti-proliferation and anti-fibrotic properties were measured using standard assay.ResultsBoth the mitomycin-C and 5α-oleandrin treated groups indicated decrease in proliferation index (86.16 ± 4.20% and 73.76 ± 4.94%, respectively), collagen deposition index (90.26 ± 1.72% and 71.35 ± 4.26%, respectively), and migration capacity (33.51 ± 1.50% and 28.57 ± 1.58%, respectively). These were significant changes (p ≤ 0.05) compared to the non-treated group. Antifibrotic activities of 5α-oleandrin in cellular proliferation and collagen deposition were better than mitomycin-C.ConclusionsThe 5α-oleandrin has good antifibrotic effect in keloid fibroblast activities.

STAT3 signalling pathway is implicated in keloid pathogenesis by preliminary transcriptome and open chromatin analyses.

Keloids are wounding-induced fibroproliferative human tumor-like skin scars of complex genetic makeup and poorly defined pathogenesis. To reveal dynamic epigenetic and transcriptome changes of keloid fibroblasts, we performed RNA-seq and ATAC-seq analysis on an early passage keloid fibroblast cell strain and its paired normal control fibroblasts. This keloid strain produced keloid-like scars in a plasma clot-based skin equivalent humanized keloid animal model. RNA-seq analysis reveals gene ontology terms including hepatic fibrosis, Wnt-β-catenin, TGF-β, regulation of epithelial-mesenchymal transition (EMT), STAT3 and adherens junction. ATAC-seq analysis suggests STAT3 signalling is the most significantly enriched gene ontology term in keloid fibroblasts, followed by Wnt signalling (Wnt5) and regulation of the EMT pathway. Immunohistochemistry confirms that STAT3 (Tyr705 phospho-STAT3) is activated and β-catenin is up-regulated in the dermis of keloid clinical specimens and keloid skin equivalent implants from the humanized mouse model. A non-linear dose-response of cucurbitacin I, a selective JAK2/STAT3 inhibitor, in collagen type I expression of keloid-derived plasma clot-based skin equivalents implicates a likely role of STAT3 signalling in keloid pathogenesis. This work also demonstrates the utility of the recently established humanized keloid mouse model in exploring the mechanism of keloid formation.

Downregulation of miR‑637 promotes proliferation and metastasis by targeting Smad3 in keloids.

Keloids are a type of abnormal scar tissue. MicroRNAs (miRNAs) exhibit a pivotal role in the regulation of cell proliferation and metastasis of keloids. miRNA microarray revealed that miR-637 was one of the most frequently altered miRNAs in keloids. Furthermore, upregulation of miR-637 inhibited cell proliferation and metastasis by targeting mothers against decapentaplegic homolog (Smad)3, one of the important proteins that affects the formation of keloids. Further studies demonstrated that miR-637 regulated the proliferation and metastasis of human keloid fibroblast (HKF) cells by mediating the Smad3 signaling pathway. Overall, the present findings suggest that miR-637 may be a promising therapeutic target in keloids.

Antifibrotic Effect of Ethanolic Extract of Nerium indicum Mill. Standardized 5α-Oleandrin on Keloid Fibroblasts Cells

Keloid is a skin lesion caused by an abnormal proliferation of fibroblasts and the deposition of collagen in the wound healing process. Therapy for keloids is relatively limited and mostly has side effects. Recent studies show that 5α-oleandrin from N. indicum Mill. (N. indicum) has anti-keloid effect by inhibiting (proliferation, migration activity and TGF-β1 expression) keloid fibroblast. Further studies with N. indicum extract standardized 5α-oleandrin should be conducted to develop it as an anti-keloid agent by performing at proliferative effects, collagen accumulation, and keloid fibroblast migration. The antifibrotic activity of N. indicum extract on the proliferation of keloid fibroblasts was measured by MTT assay, whereas collagen deposits were observed by Sirius Red method. Observation of keloid fibroblast migration by scratch assay is according to Liang et al. (2009). Ethanol extract of N. indicum standardized 5α-oleandrin can inhibit the proliferation of keloid fibroblas with IC50 0.458 μg/mL, also inhibited collagen deposits with IC50 0.055 μg/mL at incubation for 72 hours. In incubation 48 hours after treatment with ethanol extract of 1.0 μg/mL occurred inhibition of migration significantly compared with control.

Efficacy and possible mechanisms of Botulinum Toxin type A on hypertrophic scarring.

Clinical observations indicate that Botulinum toxin type A (BTXA) can inhibit the growth and improve the eventual appearance of hypertrophic scarring. However, the molecular mechanism remains unclear. We used human keloid fibroblasts to investigate the molecular mechanism of BTXA on hypertrophic scarring. Different concentrations of BTXA (0.01, 0.1, 1, and 10U/L) were used to treat keloid fibroblasts. Changes in cellular morphology, viability, proliferation, cell cycle, and apoptosis were observed by immunofluorescence, MTT assay, and flow cytometry. In addition, real-time qPCR and Western blotting were used to explore the potential molecular mechanisms. Keloid fibroblast viability decreased with increasing BTXA dose. After BTXA treatment, the volume of keloid fibroblasts cells increased, but the nucleus of cells shrunk. Long thin dendrites were formed as the concentration of BTXA increased. Furthermore, the proliferation and S phase of keloid fibroblasts were inhibited by BTXA. Matrix metalloproteinase (, MMP)-1 and -2 RNA and protein showed high expression, but TGF-β1 and MMP-9 showed low expression than the control. Botulinum toxin type A may promote the healing of scars by inhibiting the proliferation of keloid fibroblasts and regulating the expression of TGF-β1, which could affect the expression of MMP-1 and MMP-2. This study provides theoretical support for the clinical application of BTXA to control hypertrophic scarring.

The mechanisms of β-catenin on keloid fibroblast cells proliferation and apoptosis.

To investigate the role of β-catenin siRNA on proliferation and apoptosis of keloid fibroblast cell. Real-time polymerase chain reaction (RT-PCR) and Western blot were performed to monitor the mRNA and protein expression levels of β-catenin in pathological scar tissue and adjacent normal tissue. Human keloid fibroblast cells (KFB) were isolated from the keloid's tissue by enzyme digestion assay and identified by immunocytochemistry assay. Keloid fibroblast cell lines in vitro were transfected with 3 pairs of specific β-catenin small interfering RNA (siRNA); RT-PCR and Western blot were performed to identify the best siRNA. The proliferation and apoptosis of KFB transfected with β-catenin were estimated by MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and flow cytometry (FCM). In addition, the expression levels of Bcl-2, p53, and active-caspase-3 were detected by Western blot. The RT-PCR and Western blot assay results showed that the expression levels of β-catenin mRNA and protein in pathological scar tissue were significantly higher than those in adjacent normal tissue (p<0.05). KFB were successfully separated from human pathological scar tissue, and immunofluorescence staining results showed that cells were spindle and positively stained with vimentin. The β-catenin siRNA2 remarkably inhibited the expression of β-catenin at mRNA and proteins levels in the human keloid fibroblasts. Compared with the control group, cell proliferation was decreased, and apoptotic rate was increased in β-catenin siRNA2 group. Knockdown of β-catenin significantly decreased the proliferation and increased apoptosis of KFB, which could inhibit the formation of pathological scar.

Three-dimensional poly lactic-co-glycolic acid scaffold containing autologous platelet-rich plasma supports keloid fibroblast growth and contributes to keloid formation in a nude mouse model

BackgroundThere is a lack of proper animal models to study keloid formation. AimTo create three-dimensional poly lactic-co-glycolic acid (PLGA) scaffolds containing autologous platelet-rich plasma (PRP) as an in vitro culture environment for keloid fibroblasts (KL), and to study their implantation into nude mice to mimic the process of keloid formation. MethodsNormal fibroblasts (FB) and KL cells were isolated from surgical specimens and transduced with lentivirus loaded with green fluorescent protein (GFP) and luciferase genes. The FB and KL cells were three-dimensionally cultured for 14–18days in PLGA scaffolds containing PRP. Ten mice were implanted with KL cells in their left forelimbs(KL), and FB-scaffolds (FB+PLGA) in their right forelimbs. An additional ten mice were implanted with PLGA scaffolds without cells (PLGA) in their left forelimbs, and KL-scaffolds (KL+PLGA) in their right forelimbs. Graft volume and collagen content were analyzed 120days after the implantation. Resultsin vivo luminescence cell imaging showed that the FB cells proliferated in the PLGA scaffolds within 60days after implantation, and reached a plateau afterwards until 120days after implantation. The KL cells continuously proliferated in the PLGA scaffolds until 120days after implantation. The KL+PLGA group showed higher graft volumes than the FB+PLGA group 120days after the implantation (median volume, 166.95 vs. 63.34mm3); however, the difference is not statistically significant (P=0.743), due to a large variation of the graft volume within each group. Furthermore, Sirius red staining revealed increased collagen I deposition, and immunohistochemistry showed large-scale accumulation of α-smooth muscle actin (α-SMA), collagen I, and collagen III in the KL+PLGA grafts. ConclusionThe three-dimensional PLGA scaffold containing PRP supports keloid fibroblast growth and contributes to keloid formation in a nude mouse model.