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Botulinum Toxin Type a Prevents the Phenotypic Transformation of Fibroblasts Induced by TGF‑Β1 Via the PTEN/PI3K/Akt Signaling Pathway

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Botulinum Toxin Type a Prevents the Phenotypic Transformation of Fibroblasts Induced by TGF‑Β1 Via the PTEN/PI3K/Akt Signaling Pathway INTERNATIONAL JOURNAL OF MOleCular meDICine 44: 661-671, 2019 Botulinum toxin type A prevents the phenotypic transformation of fibroblasts induced by TGF‑β1 via the PTEN/PI3K/Akt signaling pathway XUE ZHANG, DONG LAN, SHUHUA NING, HONGXIA JIA and SISI YU Department of Dermatology and Plastic Surgery, Beijing Chaoyang Hospital, Jingxi Campus, Capital Medical University, Beijing 100043, P.R. China Received August 21, 2018; Accepted May 24, 2019 DOI: 10.3892/ijmm.2019.4226 Abstract. Hypertrophic scar (HS) is a common type of On the whole, the findings of this study indicate that BTXA dermatosis. Botulinum toxin type A (BTXA) can exert an may inhibit fibroblast phenotypic transformation by regulating anti-HS effect; however, the regulatory mechanisms under- PTEN methylation and the phosphorylation of related path- lying this effect remain unclear. Thus, the aim of this study ways. The findings of this study can provide a theoretical basis was to examine the effects of BTXA on phosphatase and for HS treatment. tensin homolog deleted on chromosome ten (PTEN) expres- sion and the fibroblast phenotypic transformation induced by Introduction transforming growth factor (TGF)-β1, which is an important regulatory factor involved in the process of HS. For this Skin scars could be divided into several categories, for example, purpose, fibroblasts were treated with various concentra- hypertrophic scar (HS), keloid scar and atrophic scar (or sunken tions of BTXA and then treated with 10 ng/ml of TGF-β1 scar). Among these, HS, a benign hyperproliferative growth with gradient concentrations of BTXA. The proliferation of dermal collagen, originates from unbalanced fibroblast and apoptosis of fibroblasts were measured by cell counting cellular dynamics that result from an elevated proliferation kit‑8 assay (CCK‑8) and flow cytometry, respectively. PTEN and a reduced apoptosis of fibroblasts (1). HS is an aberrant methylation was analyzed by methylation‑specific PCR (MSP) healing response, secondary to traumatic injuries, empyrosis and DNA methyltransferase (DNMT) activity was determined and surgical trauma (2). The formation of HS has been demon- using a corresponding kit. RT‑qPCR and western blot analysis strated to be relevant to a proliferative stage of wound healing, were performed to detect the transcription and translation during which dermal tissue hyperplasia and the over-deposition levels. The results revealed that BTXA suppressed the prolif- of extracellular matrix (ECM) proteins derived from fibroblasts eration and increased the apoptosis of fibroblasts treated with can cause durative fibrosis and inflammation (3). TGF-β1 in a dose‑dependent manner. BTXA in combination Fibroblast‑to‑myofibroblast transformation is a vital event with TGF-β1 suppressed the expression of molecules related during wound healing and hypertrophic scar formation (4). to the extracellular matrix (ECM), epithelial-mesenchymal Fibroblasts arise at the wound site at the ultimate inflammatory transition (EMT) and apoptosis. BTXA reduced the PTEN stage and the initial proliferative phase of healing wound (5). methylation level and downregulated the expression levels Myofibroblasts, which are differentiated from fibroblasts, are of methylation‑associated genes. BTXA also inhibited the primitively produced in the local derma and hypodermal tissues phosphorylation of phosphoinositide 3-kinase (PI3K) and Akt. around the injured skin in HS (6) in addition to other origins, for instance, bone marrow‑derived fibrocytes (7), tissue‑specific stem cells (8), pericytes and vascular smooth muscle cells (9) and tubular epithelial cells with epithelial‑mesenchymal transi- tion (EMT) (10). Myofibroblasts in granulation tissues can lead Correspondence to: Dr Xue Zhang, Department of Dermatology to HS formation; however, the roles of these myofibroblasts in and Plastic Surgery, Beijing Chaoyang Hospital, Jingxi Campus, the pathogenesis of HS remain unclear. Capital Medical University, 5 Jingyuan Road, Shijingshan, Transforming growth factor-β (TGF-β) may serve as a Beijing 100043, P.R. China E-mail: [email protected] therapeutic target for HS and may be an important regulatory factor in the process of HS (4). TGF-β1/Smad2 signaling can Key words: botulinum toxin type A, transforming growth factor‑β1, promote collagen synthesis and enhance the proliferation of hypertrophic scar, phosphatase and tensin homolog deleted on human HS fibroblasts (11). A previous study revealed that chromosome ten, phosphoinositide 3‑kinase/Akt, fibroblast, methylation TGF-β can exert differential temporal effects on HS forma- tion (12). Moreover, TGF-β1 is able to induce Smad2 nuclear translocation, finally causing the transcription of target genes, including collagens I and III (13). Moreover, the silencing of 662 ZHANG et al: BTXA PREVENTS THE FIBROBLAST PHENOTYPIC SWITCH Smad2 can prevent type I and III collagen overproduction in 1:100; BosterBio) for 1 h at room temperature. The fibroblasts HS fibroblasts (14). Recently, multiple agents, such as baica- were then incubated with goat anti‑rabbit IgG H&L secondary lein (15), loureirin B (16), tetrandrine (17) and botulinum toxin antibodies (1:5,000; ab6721; Abcam) for 1 h. The nuclei were type A (BTXA) (1), have been reported to exert anti‑HS effects stained with Hoechst 33258 (Beyotime) for 20 min at 37˚C by inhibiting TGF‑β1 signaling. in the dark and the stained fibroblasts were observed under a Botulinum toxin is an effective neurotoxin generated fluorescence microscope (Nikon). from Clostridium botulinum and it has been shown to have potential for use in the treatment of wounds following trauma, Fibroblast treatment and viability assay. Fibroblasts (2x105) burns or surgery (18). BTXA is available for clinical use in a were maintained in 96-well plates for 24 h, and fresh media number of countries as its safe and has effective properties in were then added with various concentrations of BTXA the treatment of hypertrophic scarring (20). Rasaii reported (0, 0.125, 0.25, 0.5, 1 and 2 UI/ml; Lanzhou Biochemical Co.). that triamcinolone in combination with BTXA was ineffec- Cell counting kit-8 (CCK-8; Beyotime) solution (20 µl), with the tive for keloid scars (21), while BTXA was demonstrated to original concentration in the kit, was incubated with the fibro- attenuate HS growth and improve the symptoms of HS in blasts for an additional 2 h at 37˚C when the fibroblasts were clinical practice (22). Currently, an increasing number of incubated for 12, 24 and 48 h, respectively. Thereafter, other studies have demonstrated that BTXA can alleviate fibrosis fresh fibroblasts were incubated with TGF‑β1 (10 ng/ml) (4) by regulating fibroblast behaviors in HS and capsular contrac- for 3 h, and the fibroblasts were subsequently treated with ture. For instance, BTXA can attenuate HS and capsular low, medium and high concentrations of BTXA (0.25, 0.5 contracture by suppressing the phenotypic transformation and 1 UI/ml, respectively). Subsequently, 20 µl CCK‑8 were of fibroblasts to myofibroblasts, according to in vitro and incubated with the fibroblasts for an additional 2 h at 37˚C, in vivo experiments (1,19). BTXA can markedly reduce the for the determination of fibroblast viability. The absorbance of collagen deposition in HS (22-24). Furthermore, BTXA has 450 nm was determined using a multimode detector (BioTek been demonstrated to inhibit fibroblast proliferation and to Instruments). reduce the level of α-smooth muscle actin (α-SMA) (25,26). Furthermore, BTXA can also regulate the expression levels Apoptosis analysis. Apoptosis was detected according to of matrix metalloproteinase (MMP)-2 MMP-9, and collagen the protocol of the Annexin V-fluorescein isothiocyanate types I and III, in addition to inhibiting capsule formation (27). (FITC)/propidium iodide (PI) apoptosis detection kit (KeyGEN). HS fibroblasts have been reported to possess malignant The results were analyzed using a FACSCalibur flow cytom- characteristics, including hyperproliferation, anti-apoptosis eter (BD Biosciences). The cells were then identified using and atypical differentiation (28-31). Tansdifferentiation, Annexin V-FITC/PI double fluorescence staining as follows: proliferation and apoptosis have been demonstrated to be Unlabeled, viable cells; PI‑stained cells, necrotic cells; regulated by the phosphatidylinositol 3‑kinase (PI3K)/Akt Annexin V‑FITC‑bounded cells, early apoptotic cells; and signaling pathway in HS fibroblasts (32‑34). Moreover, Akt double‑labeled cells, late apoptotic cells. is also involved in cell motility and ECM deposition (35,36). Phosphatase and tensin homologue deleted on chromosome Immunofluorescence assay. Fibroblasts were fixed with 4% ten (PTEN) protein has reported as a tumor suppressor and it paraformaldehyde for 20 min and washed with PBS. After can negatively regulate Akt signaling to inhibit the prolifera- being blocked in bovine serum albumin (BSA), the fibroblasts tion of HS fibroblasts (37). Inactive PTEN expression has been were incubated first with anti‑α‑SMA antibody (A03744, 1:200; observed in several fibrotic diseases (38‑40). Therefore, HS BosterBio) overnight at 4˚C and then with Cy3‑conjugated goat may be relevant to the antagonistic association between PTEN anti‑rabbit secondary antibodies (BA1032, 1:500; Beyotime) and Akt. In the present study, our aim was to explore the for 0.5 h. Subsequently, the fibroblasts were stained with potential mechanisms involved in PTEN//PI3K/Akt signaling 4',6-diamidino-2-phenylindole (DAPI; Beyotime)
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