Hypertrophic Scar Tissue Research Articles

Botulinum toxin type a intralesional monotherapy for treating human hypertrophic scar in a dose-dependent manner: In an animal model

The effect of Botulinum toxin type A (BTX-A) in treating or preventing a hypertrophic scar (HS) had been reported in clinical studies. However, the dose-effect relationship remains unclear. To study the dose-effect relationship of BTX-A intralesional monotherapy treating human HS. Six HS tissues were collected from six patients. Each tissue was segmented into 24 specimens and split into four groups: negative control (group A), 0.5U BTX-A (group B), 1U BTX-A (group C), and 2U BTX-A (group D). Six nude mice, each was prepared by implanting four specimens (one from each group) into the back for a total of 24 specimens. The process mentioned above were repeated six times. A re-entry operation was performed to obtain the specimens after 8 weeks. The weight of HS, the expression of decorin and TGF-β1, the proliferation, and migration ability of hypertrophic scar fibroblasts (HSFBs) were compared among groups. The weight of HS, the expression of decorin and TGF-β1, the proliferation, and migration ability of HSFBs showed significant differences in groups C and D as compared to group A; there has been no statistical significance in group B. BTX-A showed significant therapeutic efficacy when compared with the negative control group in a dose-dependent manner. BTX-A can reduce the weight of HS, upregulate the expression of decorin, downregulate the expression of TGF-β1, and inhibit HSFBs proliferation and migration ability. This study indicates that BTX-A intralesional monotherapy treating HS should reach a threshold dose to achieve an effective treatment, and a high dose of BTX-A is more effective than a low dose.

Expression and effect of microRNA-627 in human hypertrophic scar

Objective: To investigate the expression and effect of microRNA-627 (miR-627) in human hypertrophic scar. Methods: The experimental research method was used. From October 2019 to January 2020, hypertrophic scar tissue from 6 patients with hypertrophic scar (2 males and 4 females, aged (34±11) years) and the remaining normal skin tissue from 6 trauma patients (3 males and 3 females, aged (35±13) years) after flap transplantation were collected. The above-mentioned 12 patients were admitted to the General Hospital of Northern Theater Command and met the inclusion criteria. The mRNA expression of miR-627 was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The 3rd to 5th passages of fibroblasts (Fbs) were isolated from hypertrophic scar tissue and cultured for subsequent experiments after identification. Fbs from hypertrophic scar were divided into miR-627 negative control group, miR-627 mimic group, and miR-627 inhibitor group. The corresponding sequences were transfected respectively. At 0 (immediately), 12, 24, 36, and 48 h after transfection, the cell viability was detected by thiazolyl blue method; at 24 h after transfection, the apoptosis was detected by flow cytometry; at 24 h after transfection, the protein expression levels of insulin-like growth factor Ⅰ (IGF-Ⅰ), type Ⅰ collagen, and α smooth muscle actin (α-SMA) were detected by Western blotting. Two batches of Fbs from hypertrophic scar were used, one batch was divided into IGF-Ⅰ wild type+miR-627 negative control group and IGF-Ⅰ wild type+miR-627 mimic group, and the other batch was divided into IGF-Ⅰ mutant+miR-627 negative control group and IGF-Ⅰ mutant+miR-627 mimic group. The corresponding sequences were transfected respectively. At 48 h after transfection, the expressions of luciferase and renal luciferase were detected by luciferase reporter gene detection kit, and the ratio of the two was calculated to reflect the activity of IGF-Ⅰ. Fbs from hypertrophic scar were divided into miR-627 negative control group, miR-627 mimic alone group, and miR-627 mimic+IGF-Ⅰ group, and were transfected with the corresponding sequences respectively. At 24 h after transfection, the protein expression levels of IGF-Ⅰ, type Ⅰ collagen, and α-SMA were detected by Western blotting. The number of samples in cell experiment was 3. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, independent sample t test, and chi-square test. Results: The expression of miR-627 mRNA in hypertrophic scar tissue was 0.47±0.06, which was significantly lower than 1.12±0.23 in normal skin tissue (t=15.090, P<0.01). At 12, 24, 36, and 48 hours after transfection, the cell viability of miR-627 mimic group was significantly lower than that of miR-627 negative control group (t=9.918, 34.370, 13.580, 61.550, P<0.05 or P<0.01); the cell viability of miR-627 inhibitor group was significantly higher than that of miR-627 negative control group (t=4.722, 8.616, 13.330, 14.000, P<0.05 or P<0.01). At 24 h after transfection, compared with the apoptosis rate (8.42±0.47)% in miR-627 negative control group, (10.89±0.35)% in miR-627 mimic group was significantly higher (t=7.301, P<0.01), and (5.00±0.22)% in miR-627 inhibitor group was significantly lower (t=11.510, P<0.01). At 24 h after transfection, compared with the cell protein expressions of IGF-Ⅰ, type Ⅰ collagen, and α-SMA in miR-627 negative control group, those in miR-627 mimic group were significantly lower (t=25.470, 5.282, 7.415, P<0.01), and those in miR-627 inhibitor group were significantly higher (t=15.930, 8.857, 9.763, P<0.01). At 48 h after transfection, the luciferase/renal luciferase ratio of IGF-Ⅰ of cells in IGF-Ⅰ wild type+miR-627 mimic group was 0.463±0.061, which was significantly lower than 0.999±0.011 in IGF-Ⅰ wild type+miR-627 negative control group (t=16.852, P<0.01); the luciferase/renal luciferase ratio of IGF-Ⅰ of cells in IGF-Ⅰ mutant+miR-627 mimic group was 0.934±0.021, which was similar to 0.930±0.023 in IGF-Ⅰ mutant+miR-627 negative control group (t=1.959, P>0.05). At 24 h after transfection, the protein expressions of IGF-Ⅰ, type Ⅰ collagen, and α-SMA of cells in miR-627 mimic alone group were 1.623±0.070, 1.363±0.042, and 1.617±0.025, which were significantly lower than 2.723±0.045, 2.147±0.067, and 2.533±0.055 in miR-627 negative control group (t=22.831, 7.280, 26.220, P<0.01); the protein expressions of IGF-Ⅰ, type Ⅰ collagen, and α-SMA of cells in mimic+IGF-Ⅰ group were 2.477±0.102, 1.760±0.046, and 2.387±0.049, which were significantly higher than those of miR-627 mimic alone group (t=3.830, 8.286, 3.436, P<0.05 or P<0.01). Conclusions: miR-627 expression in human hypertrophic scars is down-regulated; miR-627 can inhibit the proliferation and promote the apoptosis of Fbs in human hypertrophic scar by targeted inhibition of IGF-Ⅰ expression.

LncRNA NORAD regulates scar hypertrophy via miRNA-26a mediating the regulation of TGFβR1/2.

Transforming growth factor-β (TGF-β) pathway presents dysregulation in pathological scarring and mediates hypertrophic scar (HS) formation. The study aims to analyze the potential mechanism of long non-coding RNA NORAD (LncRNA NORAD) and microRNA (miR-26a) regulation of the TGF-β pathway in hypertrophic scar fibroblasts (HSFs). Hypertrophic scar tissues were collected and assayed for LncRNA NORAD, miR-26a, transforming growth factor β receptor I (TGF-βR1) and TGF-βR2, with enzyme-linked immunosorbent assay (ELISA) or qualitative polymerase chain reaction (qPCR). LncRNA NORAD interfering plasmids were transfected into HSFs and induced with TGF-β1. Cell Counting Kit-8 (CCK-8) assays were performed to assess HSF proliferation, and flow cytometry to analyze apoptosis and the cell cycle. TGF-βR1, TGF-βR2, Smad2, and p-Smad2 levels were detected using western blot (WB). The related proteins (p21, cyclin D1 and cyclin-dependent kinase 4 (CDK4)) regulating the cell cycle, and apoptosis-related proteins (caspase-3 and Bcl-2) were also detected using WB. The binding sites of miRNA-26a and LncRNA NORAD, TGF-βR2, or UBE3A were predicted using Starbase and confirmed with dual luciferase reporter assay. RNA immunoprecipitation (RIP) was utilized to explore the interplay of miR-26a with its target genes. LncRNA NORAD is decreased, miR-26a is increased and TGF-β receptors show abnormal expression in scar tissue. LncRNA NORAD knockdown inhibits proliferation of HSF cells induced by TGF-β1 treatment. In addition, cell apoptotic levels are markedly increased and cell numbers in G0/G1 phase are increased. Moreover, the TGF-β/Smad pathway is regulated by decreasing endogenous LncRNA NORAD levels, possibly by affecting the relative levels of TGF-βR1. p21 is notably upregulated, while cyclin D1 and CDK4 are downregulated. Apoptosis-related proteins are significantly affected. LncRNA NORAD may act as a sponge, binding miR-26a and changing its expression. Finally, RIP shows that miR-26a targets the 3'UTRs of TGF-βR2 and UBE3A. LncRNA NORAD regulates HSF proliferation via miR-26a mediating the regulation of TGF-βR2/R1. LncRNA NORAD/miR-26a could be a potential target for treating HS.

LncRNA MIR503HG promotes hypertrophic scar progression via miR-143-3p-mediated Smad3 expression.

Hypertrophic scars (HSs) form due to unchecked proliferation of fibrous tissue after an injury to the skin. Recently, lncRNA MIR503HG was shown to be involved in HS. However, the mechanism by which MIR503HG affects the formation and progression of HS still needs further study. qRT-PCR was applied to examine the levels of MIR503HG and miR-143-3p in HS tissues and human hypertrophic scar fibroblasts (hHSFs). The relationships of MIR503HG, miR-143-3p and Smad3 were explored with a dual-luciferase reporter assay. Cell proliferation, apoptosis, and invasion were measured by CCK-8 assay, flow cytometry and transwell assay, respectively. The protein level of Smad3 was tested via Western blotting. MIR503HG was upregulated and miR-143-3p was downregulated in HS versus normal skin tissues. The knockdown of MIR503HG and the overexpression of miR-143-3p suppressed the proliferation and invasion of hHSF, and promoted cell apoptosis. MIR503HG bound to miR-143-3p while miR-143-3p directly targeted Smad3 to inhibit its expression. Suppression of miR-143-3p and overexpression of Smad3, respectively, reversed these effects of knockdown of MIR503HG and overexpression of miR-143-3p on hHSFs. Our research supports a model in which the MIR503HG/miR-143-3p/Smad3 axis serves as a critical regulator of HS, highlighting a promising therapeutic option for HS.

MicroRNA-181a promotes the proliferation of hypertrophic scar fibroblasts and inhibits their apoptosis via targeting phosphatase and tensin homolog.

This study aimed to compare the expression of microRNA (miR)-181a and phosphatase and tensin homolog (PTEN) in hypertrophic scar tissue and cells and to explore the effects of miR-181a and PTEN on the proliferation and apoptosis of the human scar fibroblast cell line HSFb. HSFb cells were transfected with miR-negative control (miR-NC), miR-181a mimics, miR-181a inhibitor, pcDNA3.1-PTEN (pc-PTEN), or small interfence-PTEN (si-PTEN) plasmid using a Lipofectamine 2000 transfection kit. The effects of miR-181a and PTEN on the proliferation and apoptosis of HSFb were determined using a Cell Counting Kit (CCK)-8 experiment and flow cytometry, respectively. The effects of miR-181a and PTEN on the expression of apoptosis-related proteins in HSFb, including type I collagen (Col-1) and type III collagen (Col-3), were measured by western blot. Finally, the relationship between miR-181a and PTEN was explored by the dual-luciferase reporter gene experiment. The miR-181a in hypertrophic scar tissues and HSFb were significantly up-regulated compared to embryo skin fibroblast (ESF-1) cells and normal tissues (P<0.05), whereas the opposite results were seen for PTEN expression (P<0.05). Inhibiting miR-181a or upregulating the expression of PTEN significantly suppressed the proliferation of HSFb (P<0.05) and induced their apoptosis (P<0.05). Western blot revealed that inhibiting and upregulating miR-181a and PTEN, respectively, decreased the expression of the B-cell lymphoma-2 (Bcl-2), Col-1, and Col-3 proteins in HSFb, but significantly up-regulated the expression of Bcl-2-associated X protein (Bax), cleaved caspase-3 (c-caspase-3), and cleaved caspase-9 (c-caspase-9) (P<0.05). The dual-luciferase reporter gene experiment results confirmed PTEN to be the downstream target gene of miR-181a. Simultaneous upregulation of miR-181a and PTEN expression had no significant effect on the proliferation and apoptosis of HSFb. miR-181a promotes the up-regulation of Col-1 and Col-3, and regulates the proliferation and apoptosis of HSFb by targeting PTEN, thereby enhancing the formation of hypertrophic scarring (HS). Therefore, miR-181a and PTEN may be potential therapeutic targets for the treatment of HS.

MiR-124-3p targeting of TGF-β1 inhibits the proliferation of hypertrophic scar fibroblasts.

microRNAs are involved in a variety of physiological and pathophysiological processes, but their role in the pathogenesis of hypertrophic scars (HS) is not fully understood. Transforming growth factor β1 (TGF-β1) plays an important role in the genesis and development of HS. In this study, we hypothesized that a post-translational miRNA mechanism regulates the expression of TGF-β1 in HS fibroblasts (HSFBs) and participates in the development of HS. Predictions from EBCORI, PicTar and miRBase databases showed that miR-124-3p can target and regulate the expression of TGF-β1. We collected HS tissue and corresponding normal tissue from 25 patients with HS who had been operated on for the first time. The expression level of miR-124-3p in HS tissue was significantly lower than in normal tissue, while the expression level of TGF-β1 mRNA was significantly higher than in normal tissue (p < 0.05), showing a negative correlation between them. Results from a luciferase reporter assay showed that miR-124-3p targets the 3'-UTR of TGF-β1 and inhibits its expression. After miR-124-3p mimics were transfected into HSFBs, the expression of TGF-β1, α-smooth muscle actin (α-SMA), collagen I, survivin, and Bcl-2 were reduced and the expression of Bax was increased, with significant decreases in DNA synthesis, proliferation and survival. However, after a miR-124-3p inhibitor was transfected into HSFBs, these effects were reversed as the expression of TGF-β1, α-SMA, collagen I, survivin, and Bcl-2 increased, expression of Bax decreased, and DNA synthesis, proliferation and survival cells increased significantly. miR-124-3p can inhibit the proliferation of HSFBs by targeting TGF-β1, and miR-124-3p may thus be a potential therapeutic target in HS.

Proliferation of hypertrophic scar fibroblasts inhibited by microRNA-627 targeting IGF-Ⅰin hypertrophic scar

Objective: To investigate the mechanism of microRNA-627(miR-627) inhibiting the proliferation of hypertrophic scar fibroblasts (Fbs) by targeting IGF-I. Methods: The experimental method was used. From October 2019 to January 2020, hypertrophic scar tissues from 6 patients with hypertrophic scar (2 males and 4 females, aged (34±11) years) and the remaining normal skin tissues from 6 patients with trauma (3 males and 3 females, aged (35±13) years) after skin flap transplantation were collected. the above-mentioned 12 patients were admitted to the General Hospital of Northern Theater Command and met inclusion criteria. The mRNA expression of miR-627 was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The 3rd to 5th generations of Fbs were cultured from hypertrophic scar tissue for subsequent experiments. Fbs from hypertrophic scar were divided into miR-627 control group, miR-627 mimic group and miR-627 inhibitor group. The corresponding sequences were transfected respectively. At 0 (immediate), 12, 24, 36 and 48 h after transfection, the cell viability was detected by thiazolyl blue reagent; at 24 h after transfection, the apoptosis was detected by Annexin V-fluorescein-5-isothiocyanate/propidium iodide kit; at 24 h after transfection, the expression levels of IGF-Ⅰ, collagen I and a-SMA were detected by Western blot. The hypertrophic scar Fbs were divided into IGF-Ⅰ wild type + miR-627 control group, IGF- wild type + miR-627 mimics group, IGF-Ⅰ mutant + miR-627 control group. At 48 hours after transfection, the expression of luciferase and renal luciferase were detected by luciferase reporter gene detection kit, and the ratio of the two was calculated to reflect the activity of IGF-Ⅰ. Fbs from hypertrophic scar were divided into miR-627 control group, miR-627 mimic group and miR-627 mimic + IGF-I group, and were transfected with corresponding sequences respectively. At 24 h after transfection, the expression levels of IGF-Ⅰ, type I collagen and a-SMA were detected by Western blot. The number of samples in cell experiment was 3. Analysis of variance, one-way analysis of variance, t test and chi-square test were used to statistic the data. Results: The expression of miR-627 mRNA in hypertrophic scar tissue was 0.47±0.06, which was significantly lower than that in normal tissue 1.12±0.23 (t=15.090, P<0.01). At 12, 24, 36 and 48 hours after transfection, the cell viability of miR-627 mimic group was significantly lower than that of miR-627 control group (t=9.918, 34.370, 13.580, 61.550, P<0.05 or P<0.01); the cell viability of miR-627 inhibitor group was significantly higher than that of miR-627 control group (t=4.722, 8.616, 13.330, 14.000, P<0.05 or P<0.01). At 24 h after transfection, the apoptosis rate of miR-627 mimic group was (10.89±0.35)% significantly higher than that of miR-627 control group (8.42±0.47)% (t=7.301, P<0.01), and that of miR-627 inhibitor group was (5.00±0.22)% significantly lower significantly (t=11.510, P<0.01). At 24 h after transfection, compared with miR-627 control group, miR-627 mimics could significantly down regulate the expression of IGF-Ⅰ, type I collagen and a-SMA (t=25.470, 5.282, 7.415, P<0.05); miR-627 inhibitor could up regulate the expression of IGF-Ⅰ, type I collagen and a-SMA (t=15.930, 8.857, 9.763, P<0.05). At 48 h after transfection, the luciferase/renal luciferase ratio of IGF-Ⅰ in IGF-Ⅰ wild type + miR-627 mimic group was 0.463±0.061, which was significantly lower than that of IGF-Ⅰ wild type + miR-627 control group 0.999±0.011 (t=16.852, P<0.01), The luciferase/renal luciferase ratio of IGF-mutant + miR-627 mimic group was 0.934±0.021, which was similar to that of IGF-Ⅰ mutant+miR-627 control group 0.930±0.023 (t=1.959, P>0.05). After 24 hours of transfection, the protein expressions of IGF-Ⅰ, collagen I and a-SMA in miR-627 mimic group were 1.623±0.070, 1.363±0.042 and 1.617±0.025, which were significantly lower than those in miR-627 control group 2.723±0.045, 2.147±0.067 and 2.533±0.055 (t=22.831, 7.280 and 26.220, P<0.05); The protein expression of miR-627 mimic+IGF-Ⅰ group was 2.477±0.102, 1.760±0.046, 2.387±0.049, which was significantly higher than that of miR-627 mimic group (t=3.83, 8.286, 3.436, P<0.05). Conclusion: miR-627 can inhibit the proliferation of Fbs in hypertrophic scar by targeting IGF-Ⅰ.

Effects and mechanism of eleutheroside E on the growth of human hypertrophic scar fibroblasts

Objective: To investigate the effects and mechanism of Eleutheroside E on the growth of human hypertrophic scar fibroblasts (Fbs). Methods: The experimental research method was used. The hypertrophic scar tissue was collected from 6 patients with hypertrophic scar [1 male and 5 females, aged 20 to 51 (37±8) years] admitted to the Department of Burns and Plastic Surgery of General Hospital of Northern Theater Command, from October 2018 to March 2019. The third to seventh generations of human hypertrophic scar Fbs were cultured for the follow-up experiments. Cells were divided into normal saline group, 100 μmol/L Eleutheroside E group, 200 μmol/L Eleutheroside E group, and 400 μmol/L Eleutheroside E group, and 10 μL normal saline, Eleutheroside E at the final molarity of 100, 200, and 400 μmol/L were added to cells in the corresponding groups. Cells were divided into small interfering RNA (siRNA)-negative control alone group, siRNA-thrombospondin 1 (THBS1) alone group, siRNA-negative control+400 μmol/L Eleutheroside E group, and siRNA-THBS1+400 μmol/L Eleutheroside E group. Cells in siRNA-negative control alone group and siRNA-negative control+400 μmol/L Eleutheroside E group were transfected with siRNA-negative control, cells in siRNA-THBS1 alone group and siRNA-THBS1+400 μmol/L Eleutheroside E group were transfected with siRNA-THBS1. At 24 h after transfection, cells in siRNA-negative control alone group and siRNA-THBS1 alone group were added with 10 μL normal saline, cells in siRNA-negative control+400 μmol/L Eleutheroside E group and siRNA-THBS1+400 μmol/L Eleutheroside E group were added with Eleutheroside E at the final molarity of 400 μmol/L, respectively. At 0 (immediately), 12, 24, 36 and 48 h after treatment, the cell proliferation activity was detected by thiazolyl blue assay. Cells were divided into normal saline group, 200 μmol/L Eleutheroside E group, and 400 μmol/L Eleutheroside E group, siRNA-negative control alone group, siRNA-THBS1 alone group, siRNA-negative control+400 μmol/L Eleutheroside E group and siRNA-THBS1+400 μmol/L Eleutheroside E group. The corresponding treatments were the same as before. At 24 h after treatment, the apoptosis was observed by Hoechst 33258 staining. Cells were divided into normal saline group, 100 μmol/L Eleutheroside E group, 200 μmol/L Eleutheroside E group, and 400 μmol/L Eleutheroside E group, siRNA-negative control alone group, siRNA-THBS1 alone group, siRNA-negative control+400 μmol/L Eleutheroside E group, and siRNA-THBS1+400 μmol/L Eleutheroside E group. The corresponding treatments were the same as before. At 24 h after treatment, the protein expression level of THBS1 of cells was detected by Western blotting. The number of sample in each group at each time point was all 3. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, independent sample t test, and Bonferroni correction. Results: At 0 h after treatment, the absorbance values of cells in normal saline group, 100 μmol/L Eleutheroside E group, 200 μmol/L Eleutheroside E group, and 400 μmol/L Eleutheroside E group were similar (P>0.05). At 12, 24, 36, and 48 h after treatment, the cell absorbance values of cells in 100 μmol/L Eleutheroside E group, 200 μmol/L Eleutheroside E group, and 400 μmol/L Eleutheroside E group were significantly lower than those of normal saline group (t=7.64, 28.94, 13.69, 5.87, 6.96, 22.83, 14.75, 11.52, 21.09, 20.15, 29.52, 23.12, P 0.05). At 12, 24, 36 and 48 h after treatment, the cell absorbance values of cells in siRNA-THBS1 alone group and siRNA-negative control+400 μmol/L Eleutheroside E group were significantly lower than those in siRNA-negative control alone group (t=7.14, 44.87, 20.67, 40.98, 9.26, 11.08, 15.33, 20.56, P 0.05). Compared with that in normal saline group, the number of apoptotic cells in 200 μmol/L Eleutheroside E group and 400 μmol/L Eleutheroside E group were increased at 24 h after treatment. At 24 h after treatment, compared with that in siRNA-negative control alone group, the numbers of apoptotic cells in siRNA-THBS1 alone group and siRNA-negative control+400 μmol/L Eleutheroside E group were increased, while the number of apoptotic cells in siRNA-THBS1 alone group, siRNA-negative control+400 μmol/L Eleutheroside E group, and siRNA-THBS1+400 μmol/L Eleutheroside E group were similar. At 24 h after treatment, the protein expression levels of THBS1 of cells in 100 μmol/L Eleutheroside E group, 200 μmol/L Eleutheroside E group, and 400 μmol/L Eleutheroside E group (0.87±0.12, 0.38±0.07, 0.20±0.09) were significantly lower than 1.83±0.17 in normal saline group (t=16.61, 16.17, 17.29, P 0.05). Conclusions: Eleutheroside E can inhibit the growth of human hypertrophic scar Fbs by down regulating the expression of THBS1.

LncRNA TRHDE-AS1 inhibit the scar fibroblasts proliferation via miR-181a-5p/PTEN axis

Hypertrophic scar (HS), a fibroproliferative disorder caused by abnormal wound healing after skin injury, which is characterized by excessive deposition of extracellular matrix and invasive growth of fibroblasts. Recent studies have shown that some non-coding RNA implicated the formation of HS, but the mechanism remains unclear. In this study, we found that lncRNA TRHDE-AS1 was downregulated in HS tissues and HSFs, and the level of lncRNA TRHDE-AS1 negatively correlated with the level of miR-181a-5p in HS tissue and HSFs. Overexpressed lncRNA TRHDE-AS1 significantly suppressed miR-181a-5p level, while promoted HSFs apoptosis and inhibited HSFs proliferation. Further study shown that PTEN was a direct target of miR-181a-5p, and lncRNA TRHDE-AS1 served as a molecular sponge for miR-181a-5p to regulate the expression of PTEN. Overexpression of PTEN could eliminate lncRNA TRHDE-AS1-mediated proliferation suppression of HSFs. In conclusion, our study suggested that lncRNA TRHDE-AS1/miR-181a-5p/PTEN axis plays an important role in promoting hypertrophic scar formation, which may be effectively used as a therapeutic target for hypertrophic scar treatment.

Mechanical stretch promotes hypertrophic scar formation through mechanically activated cation channel Piezo1

Hypertrophic scar (HS) formation is a skin fibroproliferative disease that occurs following a cutaneous injury, leading to functional and cosmetic impairment. To date, few therapeutic treatments exhibit satisfactory outcomes. The mechanical force has been shown to be a key regulator of HS formation, but the underlying mechanism is not completely understood. The Piezo1 channel has been identified as a novel mechanically activated cation channel (MAC) and is reportedly capable of regulating force-mediated cellular biological behaviors. However, the mechanotransduction role of Piezo1 in HS formation has not been investigated. In this work, we found that Piezo1 was overexpressed in myofibroblasts of human and rat HS tissues. In vitro, cyclic mechanical stretch (CMS) increased Piezo1 expression and Piezo1-mediated calcium influx in human dermal fibroblasts (HDFs). In addition, Piezo1 activity promoted HDFs proliferation, motility, and differentiation in response to CMS. More importantly, intradermal injection of GsMTx4, a Piezo1-blocking peptide, protected rats from stretch-induced HS formation. Together, Piezo1 was shown to participate in HS formation and could be a novel target for the development of promising therapies for HS formation.

Retrospective Evaluation of Progenitor Biological Bandage Use: A Complementary and Safe Therapeutic Management Option for Prevention of Hypertrophic Scarring in Pediatric Burn Care.

Progenitor Biological Bandages (PBB) have been continuously applied clinically in the Lausanne Burn Center for over two decades. Vast translational experience and hindsight have been gathered, specifically for cutaneous healing promotion of donor-site grafts and second-degree pediatric burns. PBBs constitute combined Advanced Therapy Medicinal Products, containing viable cultured allogeneic fetal dermal progenitor fibroblasts. Such constructs may partly favor repair and regeneration of functional cutaneous tissues by releasing cytokines and growth factors, potentially negating the need for subsequent skin grafting, while reducing the formation of hypertrophic scar tissues. This retrospective case-control study (2010–2018) of pediatric second-degree burn patients comprehensively compared two initial wound treatment options (i.e., PBBs versus Aquacel® Ag, applied during ten to twelve days post-trauma). Results confirmed clinical safety of PBBs with regard to morbidity, mortality, and overall complications. No difference was detected between groups for length of hospitalization or initial relative burn surface decreasing rates. Nevertheless, a trend was observed in younger patients treated with PBBs, requiring fewer corrective interventions or subsequent skin grafting. Importantly, significant improvements were observed in the PBB group regarding hypertrophic scarring (i.e., reduced number of scar complications and related corrective interventions). Such results establish evidence of clinical benefits yielded by the Swiss fetal progenitor cell transplantation program and favor further implementation of specific cell therapies in highly specialized regenerative medicine.

Functional Transdermal Nanoethosomes Enhance Photodynamic Therapy of Hypertrophic Scars via Self-Generating Oxygen.

Photodynamic therapy (PDT) is a new therapeutic strategy for hypertrophic scars (HSs), and nanoethosomes (ES) have attracted considerable attention as an efficient transdermal delivery system for PDT of HSs (HS-PDT). However, the delivery of photosensitizers and the hypoxic microenvironment of HSs limit HS-PDT efficacy. Consequently, functional transdermal ES (A/A-ES) that are loaded with the photosensitizer, 5-aminolevulinic acid (ALA), and immobilized nanoenzyme Au nanoclusters (ANCs) within the ES surface have been developed that exhibit superior co-delivery characteristics and produce catalase that enhances HS-PDT efficacy. The unique structure of A/A-ES enables them to co-deliver ALA and ANCs into the HS tissue and to efficiently decompose the endogenous hydrogen peroxide in the HS to generate oxygen. The findings from in vitro and in vivo experiments demonstrated that A/A-ES efficiently co-delivered ALA and ANCs into the HS tissue and that they improved the hypoxic microenvironment of the HS. Systematic assessments reveal that A/A-ES enhance HS-PDT efficacy and that they are highly effective at improving the morphology and promoting HS fibroblast apoptosis and the rearrangement of collagen. These works give rise to an effective treatment option for HSs that integrates the transdermal co-delivery of ALA and nanoenzymes, thereby enabling them to exert their respective beneficial effects, and they highlight the enhancement of HS-PDT efficacy via self-generating oxygen.

Inhibitory Effect of the LY2109761 on the Development of Human Keloid Fibroblasts.

Keloids are scars characterized by abnormal proliferation of fibroblasts and overproduction of extracellular matrix components including collagen. We previously showed that LY2109761, a transforming growth factor- (TGF-) β receptor inhibitor, suppressed the secretion of matrix components and slowed the proliferation of fibroblasts derived from human hypertrophic scar tissue. However, the exact mechanism underlying this effect remains unclear. Here, we replicated the above results in keloid-derived fibroblasts and show that LY2109761 promoted apoptosis, decreased the phosphorylation of Smad2 and Smad3, and suppressed TGF-β1. These results suggest that the development and pathogenesis of keloids are positively regulated by the Smad2/3 signaling pathway and the upregulation of TGF-β1 receptors. LY2109761 and other inhibitors of these processes may therefore serve as therapeutic targets to limit excessive scarring after injury.

Expression and effect of microRNA-205 in hypertrophic scar

Objective: To investigate the expression and effect of microRNA-205 (miR-205) in hypertrophic scar. Methods: The experimental research method were applied. From October 2019 to January 2020, hypertrophic scar tissue from 6 patients with hypertrophic scar [1 male and 5 females, aged (36±7) years], and remaining normal skin tissue from 6 trauma patients [2 males and 4 females, aged (38±9) years] after flap transplantation operation were collected. The above-mentioned 12 patients were admitted to the General Hospital of Northern Theater Command and met the inclusion criteria. Real time fluorescent quantitative polymerase chain reaction was used to detect the mRNA expressions of miR-205 and thrombospondin-1 (TSP-1). The hypertrophic scar tissue was taken to culture the 3rd to 5th passage of fibroblasts (Fbs) for the follow-up experiments. Fbs of hypertrophic scar was divided into TSP-1+miR-205 control group, TSP-1+miR-205 mimic group, TSP-1 mutant+miR-205 control group, TSP-1 mutant +miR-205 mimic group, which were transfected with the corresponding sequences. At 48 h after transfection, the expressions of luciferase and renal luciferase were detected by luciferase reporter gene detection kit, and the luciferase/renal luciferase ratio was calculated to indicate the activity of TSP-1. Two batches of hypertrophic scar Fbs were collected and divided into miR-205 control group, miR-205 mimic group, and miR-205 inhibitor group and miR-205 control group, miR-205 mimic group, and miR-205 mimic+TSP-1 group, respectively, which were transfected with the corresponding sequences. At 0 (immediately), 12, 24, 36, and 48 h after transfection, the cell viability was detected by microplate reader. Two batches of hypertrophic scar Fbs were collected, grouped, and treated as the cell viability detecting experiment. At 24 h after transfection, Hoechst 33258 staining was performed to observe the nuclear shrinkage, so as to reflect the apoptosis of Fbs. The number of samples in cell experiment was 3. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, and t test. Results: The mRNA expression of miR-205 in hypertrophic scar tissue was 0.54±0.05, which was significantly lower than 1.26±0.07 in normal skin tissue (t=8.213, P<0.01). The expression of TSP-1 mRNA in hypertrophic scar tissue was 1.46±0.07, which was significantly higher than 0.68±0.11 in normal skin tissue (t=6.031, P<0.01). At 48 h after transfection, the luciferase/renal luciferase ratio reflecting the TSP-1 activity of cells in TSP-1+miR-205 mimic group was 0.532±0.028, which was significantly lower than 0.998±0.012 in TSP-1+miR-205 control group (t=26.500, P<0.01), and the luciferase/renal luciferase ratio of cells in TSP-1 mutant+miR-205 mimic group was 0.963±0.012, which was close to 0.976±0.010 in TSP-1 mutant+miR-205 control group (t=0.816, P>0.05). At 12, 24, 36, and 48 h after transfection, the cell viability in miR-205 mimic group was significantly lower than that in miR-205 control group (t=6.169, 12.670, 27.130, 12.670, P<0.05 or P<0.01). At 0, 12, 24, 36, and 48 h after transfection, the cell viability in miR-205 inhibitor group was significantly higher than that in miR-205 control group (t=6.169, 7.221, 7.787, 7.835, 13.030, P<0.05 or P<0.01). At 12, 24, 36, and 48 h after transfection, the cell viability in miR-205 mimic group was significantly lower than that in miR-205 control group and miR-205 mimic+TSP-1 group (t=8.118, 26.970, 39.550, 42.490, 14.570, 12.240, 36.830, 45.220, P<0.05 or P<0.01). At 24 h after transfection, compared with miR-205 control group, the cell apoptosis in miR-205 mimic group was increased, and the cell apoptosis in miR-205 inhibitor group was decreased. At 24 h after transfection, compared with miR-205 mimic group, the cell apoptosis in miR-205 control group miR-205 mimic+TSP-1 group were decreased. Conclusions: miR-205 can inhibit the proliferation and promote the apoptosis of Fbs in hypertrophic scar by inhibiting the expression of TSP-1, which has the potential to be the therapeutic target for hypertrophic scar.

Expression and effect of microRNA-205 in human hypertrophic scar

Objective: To investigate the expression and effect of microRNA-205 (miR-205) in human hypertrophic scar. Methods: The experimental research method was applied. From October 2019 to January 2020, hypertrophic scar tissue from 6 patients with hypertrophic scar (1 male and 5 females, aged (36±7) years) and remaining normal skin tissue from 6 trauma patients (2 males and 4 females, aged (38±9) years) after flap transplantation operation were collected. The above-mentioned 12 patients were admitted to the General Hospital of Northern Theater Command and met the inclusion criteria. Real-time fluorescent quantitative reverse transcription polymerase chain reaction was used to detect the mRNA expressions of miR-205 and thrombospondin-1 (TSP-1). The hypertrophic scar tissue was taken to culture the 3rd to 5th passage of fibroblasts (Fbs) for the follow-up experiments. Two batches of hypertrophic scar Fbs were divided into TSP-1+ miR-205 control group, TSP-1+ miR-205 mimic group, and TSP-1 mutant+ miR-205 control group, TSP-1 mutant+ miR-205 mimic group, which were transfected with the corresponding sequences. At 48 h after transfection, the expressions of luciferase and renal luciferase were detected by luciferase reporter gene detection kit, and the luciferase/renal luciferase ratio was calculated to indicate the activity of TSP-1. Two batches of hypertrophic scar Fbs were collected and divided into miR-205 control group, miR-205 mimic group, and miR-205 inhibitor group and miR-205 control group, miR-205 mimic group, and miR-205 mimic+ TSP-1 group, respectively, which were transfected with the corresponding sequences. At 0 (immediately), 12, 24, 36, and 48 h after transfection, the cell viability was detected by microplate reader. Two batches of hypertrophic scar Fbs were grouped and treated as described in the cell viability detecting experiment. At 24 h after transfection, Hoechst 33258 staining was performed to observe the nuclear shrinkage, so as to reflect the apoptosis of Fbs. The number of samples in cell experiment was three. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, t test, and chi-square test. Results: The mRNA expression of miR-205 in hypertrophic scar tissue was 0.54±0.05, which was significantly lower than 1.26±0.07 in normal skin tissue (t=8.213, P<0.01). The expression of TSP-1 mRNA in hypertrophic scar tissue was 1.46±0.07, which was significantly higher than 0.68±0.11 in normal skin tissue (t=6.031, P<0.01). At 48 h after transfection, the luciferase/renal luciferase ratio reflecting the TSP-1 activity of cells in TSP-1+ miR-205 mimic group was 0.532±0.028, which was significantly lower than 0.998±0.012 in TSP-1+ miR-205 control group (t=26.500, P<0.01), and the luciferase/renal luciferase ratio of cells in TSP-1 mutant+ miR-205 mimic group was 0.963±0.012, which was close to 0.976±0.010 in TSP-1 mutant+ miR-205 control group (t=0.816, P>0.05). At 12, 24, 36, and 48 h after transfection, the cell viability in miR-205 mimic group was significantly lower than that in miR-205 control group (t=6.169, 12.670, 27.130, 12.670, P<0.05 or P<0.01). At 0, 12, 24, 36, and 48 h after transfection, the cell viability in miR-205 inhibitor group was significantly higher than that in miR-205 control group (t=6.169, 7.221, 7.787, 7.835, 13.030, P<0.05 or P<0.01). At 12, 24, 36, and 48 h after transfection, the cell viability in miR-205 mimic group was significantly lower than that in miR-205 control group and miR-205 mimic+ TSP-1 group (t=8.118, 26.970, 39.550, 42.490, 14.570, 12.240, 36.830, 45.220, P<0.05 or P<0.01). At 24 h after transfection, compared with miR-205 control group, the cell apoptosis in miR-205 mimic group was increased, and the cell apoptosis in miR-205 inhibitor group was decreased. At 24 h after transfection, compared with miR-205 mimic group, the cell apoptosis in miR-205 control group and miR-205 mimic+ TSP-1 group were decreased. Conclusions: miR-205 can inhibit the proliferation and promote the apoptosis of Fbs in human hypertrophic scar by inhibiting the expression of TSP-1, which has the potential to be a therapeutic target for hypertrophic scar.

ABT-263 Reduces Hypertrophic Scars by Targeting Apoptosis of Myofibroblasts.

Background: Inhibiting proliferation and inducing apoptosis of myofibroblasts is becoming one of the promising and effective ways to treat hypertrophic scar. ABT-263, as an orally bioavailable BCL-2 family inhibitor, has showed great antitumor characteristics by targeting tumor cell apoptosis. The objective of this study was to explore whether ABT-263 could target apoptosis of overactivated myofibroblasts in hypertrophic scar. Methods: In vivo, we used ABT-263 to treat scars in a rabbit ear scar model. Photographs and ultrasound examination were taken weekly, and scars were harvested on day 42 for further Masson trichrome staining. In vitro, the expression levels of BCL-2 family members, including prosurvival proteins, activators, and effectors, were detected systematically in hypertrophic scar tissues and adjacent normal skin tissues, as well as in human hypertrophic scar fibroblasts (HSFs) and human normal dermal fibroblasts (HFBs). The roles of ABT-263 in apoptosis and proliferation of HSFs and HFBs were determined by annexin V/PI assay, CCK-8 kit, and cell cycle analysis. Mitochondrial membrane potential was evaluated by JC-1 staining and the expression of type I/III collagen and α-SMA was measured by PCR, western blotting, and immunofluorescence staining. Furthermore, immunoprecipitation was performed to explore the potential mechanism. Results: In vivo, ABT-263 could significantly improve the scar appearance and collagen arrangement, decrease scar elevation index (SEI), and induce cell apoptosis. In vitro, the expression levels of BCL-2, BCL-XL, and BIM were significantly higher in scar tissues and HSFs than those in normal skin tissues and HFBs. ABT-263 selectively induced HSFs apoptosis by releasing BIM from binding with prosurvival proteins. Moreover, ABT-263 inhibited HSFs proliferation and reduced the expression of α-SMA and type I/III collagen in a concentration- and time- dependent manner. Conclusion: HSFs showed increased mitochondrial priming with higher level of proapoptotic activator BIM and were primed to death. ABT-263 showed great therapeutic ability in the treatment of hypertrophic scar by targeting HSFs.

A six-herb Chinese medicine composition ointment as a promising candidate for treatment of hypertrophic scars

ObjectiveTo study the anti-hypertrophic scar effect of the six-herb Chinese medicine composition (SCMC) ointment on the rabbit ear hypertrophic scar models. MethodsThe optimal formulation of SCMC ointment matrix was screened by the orthogonal designs and a series of evaluation tests. The SCMC ointment was prepared through emulsifying method. The rabbit ear hypertrophic scar models were established and used to investigate the anti-hypertrophic scar effect of SCMC ointment. ResultsOur results demonstrated that all the quality control indications of the SCMC ointment met the requirements. Anti-hypertrophic scar activity results showed that all the rabbit ear scar tissues appeared different degrees of shrink and fading, and took an unobvious but palpable shift from hard to soft texture with the low, middle and high concentration SCMC ointments treatments in vivo. Additionally, on 21st day the scar area and thickness in different concentrations of SCMC ointment groups were significantly reduced than control group, in a concentration-dependent manner. The immunohistochemical results also indicated that the SCMC ointment had good anti-hypertrophic scar properties and could inhibit hypertrophic scar formation. ConclusionThe SCMC ointment could improve the blood circulation condition of hypertrophic scar tissues. Our research has demonstrated the Chinese medicine composition ointment with good anti-hypertrophic scar properties that could be used to treat hypertrophic scars. Meanwhile, it provides a theoretical basis for further clinical application.

Effect of THBS1 on the Biological Function of Hypertrophic Scar Fibroblasts.

Hypertrophic scarring is a skin collagen disease that can occur following skin damage and is unlikely to heal or subside naturally. Since surgical treatment often worsens scarring, it is important to investigate the pathogenesis and prevention of hypertrophic scarring. Thrombospondin-1 (THBS1) is a matrix glycoprotein that can affect fibrosis by activating TGF-β1, which plays a key role in wound repair and tissue regeneration; therefore, we investigated the effects of THBS1 on the biological function of hypertrophic scar fibroblasts. THBS1 expression was measured in hypertrophic scars and adjacent tissues as well as normal fibroblasts, normal scar fibroblasts, and hypertrophic scar fibroblasts. In addition, THBS1 was overexpressed or silenced in hypertrophic scar fibroblasts to determine the effects of THBS1 on cell proliferation, apoptosis, and migration, as well as TGF-β1 expression. Finally, the role of THBS1 in hypertrophic scarring was confirmed in vivo using a mouse model. We found that THBS1 expression was increased in hypertrophic scar tissues and fibroblasts and promoted the growth and migration of hypertrophic scar fibroblasts as well as TGF-β1 expression. Interestingly, we found that si-THBS1 inhibited the occurrence and development of bleomycin-induced hypertrophic scars in vivo and downregulated TGF-β1 expression. Together, our findings suggest that THBS1 is abnormally expressed in hypertrophic scars and can induce the growth of hypertrophic scar fibroblasts by regulating TGF-β1. Consequently, THBS1 could be an ideal target for treating hypertrophic scarring.

MiR-3613-3p inhibits hypertrophic scar formation by down-regulating arginine and glutamate-rich 1.

Hypertrophic scar (HS) is a severe skin disorder characterized by excessive extracellular matrix production and abnormal function of fibroblasts. Recent studies have demonstrated that microRNAs (miRNAs) play critical roles in HS formation. This study aims to investigate the role of miR-3613-3p in the formation of HS. The mRNA and miRNA levels were measured by quantitative RT-PCR analysis. The protein levels were examined by Western blot assay. Cell proliferation was determined by Cell Counting Kit-8 assay. The Caspase-3 and Caspase-9 activities were measured using flow cytometry assay. Dual-luciferase activity reporter assay and mRNA-miRNA pulldown assay were conducted to validate the target of miR-3613-3p. miR-3613-3p was downregulated, while arginine and glutamate-rich 1 (ARGLU1) was upregulated in HS fibroblasts (HSFs) and tissues. Overexpression of miR-3613-3p or knockdown of ARGLU1 markedly inhibited the expression of extracellular matrix (ECM) production-associated proteins and promoted Caspase-3 and Caspase-9 activations in HSFs. ARGLU1 was further identified as a direct target of miR-3613-3p. Restoration of ARGLU1 abrogated the suppressive effect of miR-3613-3p on cell proliferation and ECM protein expression of HSFs. Our results demonstrated that miR-3613-3p inhibited HS formation via targeting ARGLU1, which may provide potential therapeutic targets for the management of HS.

Tanshinone IIA down-regulated p-Smad3 signaling to inhibit TGF-β1-mediated fibroblast proliferation via lncRNA-HSRL/SNX9.

Tanshinone IIA (TSIIA), an active component of Salvia miltiorrhiza (Danshen), is reported to inhibit cell proliferation in hypertrophic scars (HS). In our previous study, we observed that lncRNA human-specific regulatory loci (HSRL) was up-regulated in HS tissues. However, whether TSIIA serves as an effective treatment for HS through affecting HSRL is still unexplored. TGF-β1-stimulated fibroblast were used as the in vitro HS model. The effects of TSIIA on cell proliferation were evaluated using CCK-8, Edu staining and colony formation assays. By performing loss-of-function and rescue experiments, we explored the role of HSRL and Sorting nexin 9 (SNX9) in TGF-β1-stimulated fibroblast. Employing RNA-protein pull-down assay and Co-immunoprecipitation, we further investigated the mechanisms through which TSIIA attenuated TGF-β1-stimulated fibroblast. Our data demonstrated that TSIIA could effectively attenuate TGF-β1-mediated fibroblast proliferation in a dose-dependent manner. Meanwhile, TSIIA could down-regulate the expression of α-SMA, VEGFA, Collagen 1, HSRL, SNX9 and p-Smad2/3 in TGF-β1-stimulated HSF. In addition, we found that SNX9 overexpression reversed the effects of HSRL knockdown on TGF-β1-stimulated HSF. Furthermore, we confirmed that TSIIA treatment weakens the interaction between p-Smad3 and SNX9 in HS models. Tanshinone IIA down-regulated p-Smad3 signaling to inhibit TGF-β1-mediated fibroblast proliferation via lncRNA-HSRL/SNX9.