The effects of Shikonin on the hypertrophic scar of rabbit ears via the TLR4/NF-κB signaling pathway.

As a traditional Chinese medicine, Zihuang Shengji Ointment has obvious effects on promoting postoperative wound healing and reducing scar formation in clinical application. Shikonin is the major phytochemical in Zihuang Shengji Ointment. As a kind of naphquinone compound with anti-tumor, anti-viral, anti-inflammatory, anti-bacterial and other biological activities extracted from Lithospermum erythrorhizon, shikonin exerts an important role in many diseases. Shikonin has impacts on the development of hypertrophic scars (HS), however, these effects are yet mostly unknown. As a result, we created the Newland white rabbit ear HS model, administered shikonin to it, and then assessed scar hypertrophy using HE and VG staining. The degree of scarring is assessed by HI, NA, as well as AA. The expression levels of collagen I, collagen III, as well as α-SMA as well as fibroblast proliferation, are also measured using real-time PCR, immunohistochemistry, and western blot. TUNEL tests are used to assess fibroblast apoptosis. In our work, HE staining and VG staining showed that the shikonin-treated group had normal bundles of collagen fibers and regular fibroblasts. Shikonin suppresses the production of HS, according to histopathological features, HI, NA, and AA measures. Shikonin also causes fibroblast apoptosis and lowers the production of α-SMA, collagen I, as well as collagen III in the HS rat. Notably, we discover that NF-κB activation and TLR4 activity are inhibited by shikonin. Overall, the results show that the signaling pathway of TLR4/NF-κB is modulated by shikonin's inhibitory effect on scar formation, which represses the levels of collagen I, collagen III, α-SMA, as well as fibroblasts.

Mechanosensitive Aspects of Cell Biology in Manual Scar Therapy for Deep Dermal Defects.

Deep dermal defects can result from burns, necrotizing fasciitis and severe soft tissue trauma. Physiological scar restriction during wound healing becomes increasingly relevant in proportion to the affected area. This massively restricts the general mobility of patients. External mechanical influences (activity or immobilization in everyday life) can lead to the formation of marked scar strands and adhesions. Overloading results in a renewed inflammatory reaction and thus in further restriction. Appropriate mechanical stimuli can have a positive influence on the scar tissue. "Use determines function," and even minimal external forces are sufficient to cause functional alignment (mechanotransduction). The first and second remarkable increases in connective tissue resistance (R1 and R2) seem to be relevant clinical indications of adequate dosage in the proliferation and remodulation phase, making it possible to counteract potential overdosage in deep dermal defects. The current state of research does not allow a direct transfer to the clinical treatment of large scars. However, the continuous clinical implementation of study results with regard to the mechanosensitivity of isolated fibroblasts, and the constant adaptation of manual techniques, has nevertheless created an evidence-base for manual scar therapy. The manual dosages are adapted to tissue physiology and to respective wound healing phases. Clinical observations show improved mobility of the affected regions and fewer relapses into the inflammatory phase due to mechanical overload.

Effect of UVA1 on hypertrophic scarring in the rabbit ear model.

Hypertrophic scars (HTSs) are common and cause functional and psychological morbidity. UVA1 (340-400 nm) phototherapy has been previously shown to be effective in the treatment of localized scleroderma, systemic sclerosis, and POEMS syndrome with minimal side effects, all of which are presented as collagen fibrils hyperplasia that is common with scarring in skin histology. In the present study, we aimed to investigate the impact of UVA1 on the protein expression of TGF-ß signal pathway and myofibroblasts in a rabbit model of cutaneous scarring. Full-thickness skin wounds (2 cm × 5 cm in diameter) were made in New Zealand white rabbits to establish the hypertrophic scarring model. New Zealand white rabbits were divided into two treatment groups (n=30 wounds per group with an equal number of controls): medium-dose of UVA1 phototherapy group: 60 J/cm2; high-dose of UVA1 phototherapy group: 110 J/cm2. Left ears were used for treatment and the right ones were used for control. Treatment was administered five times weekly for 6 weeks. Treated and untreated control wounds were harvested at various time points and examined by histologic examination, immunohistochemical assessment, and ultrastructural evaluation. The results showed that UVA1 phototherapy caused a significant reduction in dermal thickness by histological features, whereas the scar index was descended significantly in both medium- and high-dose UVA1 groups compared with the control group. Examination of immunohistochemistry also revealed a marked suppression of tissue growth factor-ß (TGF-ß) (both medium- and high-dose), α smooth muscle actin (α-SMA) (only high-dose), and tissue inhibitor of metalloproteinase 1 (TIMP-1) (only high-dose), and apparent increase in matrix metalloproteinases (MMP-1) (both medium- and high-dose) compared with the control. The ultrastructural evaluation showed the collagen fibers' diameter had shrunk, and that fibroblastic cytoplasm was not affluent and in a quiescent stage. These findings of the present study suggested that administration of UVA1 irradiation is effective to improve the experimental HTS model and raises a possibility of the therapeutic approach of UVA1 in the scar. Although not directly examined in the present study, MMP inhibition is hypothesized to be responsible for this effect. However, early UVA1 treatment could not prevent the formation of scar model.

Tetrandrine induces microRNA differential expression in human hypertrophic scar fibroblasts in vitro.

MicroRNAs (miRNAs) have recently been shown to play a role in normal wound healing process. miRNAs may be linked to pathologic wound healing and closely related to the formation of hypertrophic scars. This study aimed to explore the effects of tetrandrine on the miRNA expression profile in human hypertrophic scar fibroblasts (HSFs) in vitro. HSFs were randomly divided into two groups: the tetrandrine treatment group and the control group. The experimental and control groups were collected and analyzed by miRNA array after a 48-h culture. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to confirm the array results. The targets of differentially expressed miRNA were functionally annotated using bioinformatic approaches. miRNA microarray analysis identified 193 differentially expressed miRNAs and the expression of 186 miRNAs in the experimental group decreased while that of 7 miRNAs increased compared to the control group. The most significantly downregulated miRNA was hsa-miR-1246, and hsa-miR-27b had the highest expression level. Significant differentially expressed miRNAs were predicted to be related to several important signaling pathways related to scar wound healing. The differential miRNA expression identified in this study provides the experimental basis for further understanding the anti-fibrosis effect of tetrandrine.

Opuntia Extract Reduces Scar Formation in Rabbit Ear Model: A Randomized Controlled Study.

The purpose of this article is to investigate the effect of Opuntia stricta H (Cactaceae) extract on suppression of hypertrophic scar on ventral surface wounds of rabbit ears. Full thickness skin defection was established in a rabbit ear to simulate hypertrophic scar. Opuntia extract was sprayed on the wounds in the experimental group, and normal saline was used in the control group. After the wounds healed with scar formation, the hypertrophic scar tissue was harvested on days 22, 39, and 54 for histological analysis. The expression of type I and type III collagen and matrix metalloproteinase-1 (MMP-1) were evaluated by immunohistochemistry and real-time quantitative polymerase chain reaction. The results indicated that the scar of the control group is more prominent compared with the opuntia extract group. The expression of type I collagen in the opuntia extract group was lower than the control group, while type III collagen in opuntia extract group gradually increased and exceeded control group. The expression of MMP-1 decreased in the opuntia extract group, while the control group increased over time, but the amount of MMP-1 was much higher than that in the control group on day 22. In conclusion, opuntia extract reduces hypertrophic scar formation by means of type I collagen inhibition, and increasing type III collagen and MMP-1.T he novel application of opuntia extract may lead to innovative and effective antiscarring therapies.

Evaluation of the effect of fractional CO2 laser on histopathological picture and TGF-ß1 expression in hypertrophic scar.

BACKGROUND AND AIMS: Hypertrophic scar is a form of abnormal wound healing process in which tissue repair regulating mechanism is disrupted. Transforming growth factor ß1 has a particular importance in the fibrotic scarring response. Treatment of hypertrophic scar included many chemical, physical, and surgical options. Fractional CO2 laser devices have gained acceptance as a way for managing hypertrophic scar. Aims of this study are: (a) to determine the clinical and histopathological effects of fractional CO2 laser on hypertrophic scar, (b) to evaluate the expression pattern of transforming growth factor-ß1 (TGF-ß1) as an important fibrogenic factor before and 6 months after fractional CO2 laser treatment. PATIENTS AND METHODS: Forty patients of hypertrophic scar were selected, each patient was treated by four sessions with 1 month apart with fractional CO2 laser. Vancouver Scar Scale (VSS) was used to assess the patients before and after laser treatment. Skin biopsy was taken from eight cases before and 3 months after four fractional CO2 laser sessions and four normal skin control biopsies. All were assessed by hematoxylin-eosin (H&E), Masson's trichrome, Van Gieson and immunohistochemical (IHC) staining with TGF-ß1. The epidermal thickness was assessed before and after treatment by image analyzing system software. RESULTS: There was statistically significant difference in VSS before and after fractional CO2 laser (P > 0.001). The epidermal thickness showed significant increase after laser treatment (P > 0.001), and there was also thinning in stratum corneum and replacement of the irregular collagen bands with organized new collagen fibrils as demonstrated by H&E and the other special stains. The study also showed significant decrease in TGF-ß1 expression after laser therapy (P = 0.008). CONCLUSION: Fractional CO2 laser could be considered as a good way for hypertrophic scar management. It normalizes dermal collagen as imaged by histopathological picture and the change in TGF-ß1 expression.

Compound Astragalus and Salvia miltiorrhiza extract suppresses rabbits' hypertrophic scar by modulating the TGF-ß/Smad signal.

BACKGROUND: Hypertrophic scar is a fibro-proliferative disease. Our previous studies demonstrate that compound Astragalus and Salvia miltiorrhiza extract (CASE) inhibits proliferation and invasion in keloid fibroblasts. OBJECTIVE: To investigate the effects of CASE on hypertrophic scar. METHODS: Rabbits were divided into the control, model and three dosage groups of CASE (0.94, 1.88, 3.76%). An animal model of hypertrophic scar was established and treated with CASE ointment or ointment base. The histopathological detection by hematoxylin & eosin and Masson's trichrome staining and protein expression of scars by Western blot were performed. RESULTS: The hydroxyproline content was decreased under CASE treatment. Transforming growth factor beta 1 (TGF-ß1) protein expression increased in the model group while it decreased under CASE treatment. The elevated expression of Smad4 protein was decreased under CASE treatment. Additionally, CASE promoted Smad7 protein expression. CONCLUSION: CASE could inhibit formation of hypertrophic scar by modulating TGF-ß/Smad signal and may be useful for the treatment of hyperplastic scars.

A high throughput screen identifies Nefopam as targeting cell proliferation in ß-catenin driven neoplastic and reactive fibroproliferative disorders.

Fibroproliferative disorders include neoplastic and reactive processes (e.g. desmoid tumor and hypertrophic scars). They are characterized by activation of ß-catenin signaling, and effective pharmacologic approaches are lacking. Here we undertook a high throughput screen using human desmoid tumor cell cultures to identify agents that would inhibit cell viability in tumor cells but not normal fibroblasts. Agents were then tested in additional cell cultures for an effect on cell proliferation, apoptosis, and ß-catenin protein level. Ultimately they were tested in Apc1638N mice, which develop desmoid tumors, as well as in wild type mice subjected to full thickness skin wounds. The screen identified Neofopam, as an agent that inhibited cell numbers to 42% of baseline in cell cultures from ß-catenin driven fibroproliferative disorders. Nefopam decreased cell proliferation and ß-catenin protein level to 50% of baseline in these same cell cultures. The half maximal effective concentration in-vitro was 0.5 uM and there was a plateau in the effect after 48 hours of treatment. Nefopam caused a 45% decline in tumor number, 33% decline in tumor volume, and a 40% decline in scar size when tested in mice. There was also a 50% decline in ß-catenin level in-vivo. Nefopam targets ß-catenin protein level in mesenchymal cells in-vitro and in-vivo, and may be an effective therapy for neoplastic and reactive processes driven by ß-catenin mediated signaling.

Oleanolic acid inhibits hypertrophic scarring in the rabbit ear model.

BACKGROUND: Hypertrophic scarring, a common proliferative disorder of dermal fibroblasts, results from an overproduction of collagen and excessive deposition of extracellular matrix. Although treatment with surgical excision or steroid hormones can modify the symptoms, numerous treatment-related complications have been described. AIM: To investigate the effects of oleanolic acid (OA), a naturally occurring triterpenoid, on hypertrophic scarring in a rabbit ear model. METHODS: A rabbit ear model of hypertrophic scarring was used, with wounds produced with a biopsy punch. Oleanolic acid (2.5%, 5% and 10%) was applied once daily to the scars for 22 days. On postoperative day 28, the scars were excised, and the tissue used for histological examination and assays of the levels of collagens I and III, matrix metalloproteinase (MMP)-1 and transforming growth factor (TGF)-ß(1). The scar elevation index (SEI) was also determined. RESULTS: Treatment with different concentrations of oleanolic acid (OA) for 22 days significantly inhibited hypertrophic scarring in rabbit ear tissue. Levels of TGF-ß(1), collagen I and collagen III were significantly decreased and levels of MMP-1 significantly increased in the scar tissue. SEI was also significantly reduced. Histological findings showed significant amelioration of the scar tissue. CONCLUSIONS: OA suppresses hypertrophic scarring in the rabbit ear model and may be an effective cure for human hypertrophic scarring.

[Effects of radix astragali on expression of transforming growth factor ß 1 and Smad 3 signal pathway in hypertrophic scar of rabbit].

OBJECTIVE: To observe the effects of radix astragali on expression of TGF-ß1 and Smad 3 signal pathway in hypertrophic scar of rabbits, and to analyze its therapeutic effect and mechanism on hypertrophic scar. METHODS: Twenty healthy adult Japanese big ear rabbits were inflicted with 4 full-thickness skin defects on ventral side of each ear, which formed scar later. Rabbits were divided into 5 groups: 1.00, 0.50, 0.25 g/mL radix astragali treatment groups [injected with radix astragali on post injury day (PID) 21, 25, 32, and 36 respectively], physiological saline group (PS, injected with 0.2 mL physiological saline in the same volume at the same time points as above groups), and blank control group (BC, without treatment) according to the random number table, with 32 scars in each group. Another 4 rabbits were enrolled as normal control group (NC). Structural changes of hypertrophic scar was observed with HE and Masson staining. Thickness and hardness of hypertrophic scar on PID 32 and 43 were respectively examined by chromoscope ultrasonic diagnostic equipment and hardness tester. Protein and mRNA expression of TGF-ß1 and Smad 3 in hypertrophic scar was respectively detected with RT-PCR and immunohistochemical analysis. Data were processed with t test and one-way analysis of variance. RESULTS: Compared with that in PS and BC groups, dermis of hypertrophic scar became thinner in radix astragali treatment groups on PID 32, 43, with fibroblasts and collagenous fibers arranged regularly on PID 43. Thickness and hardness of hypertrophic scar, levels of mRNA and protein of TGF-ß1 and Smad 3 decreased along with the increase in radix astragali concentration. Compared with those in PS group, levels of mRNA of TGF-ß1 and Smad 3 in 1.00 g/mL radix astragali treatment group on PID 32 decreased 26.1% and 28.2%. Protein levels of TGF-ß1 and Smad 3 in 1.00 g/mL radix astragali treatment group were 3.15 ± 0.80 and 4.72 ± 1.06, which were obviously lower than those in PS group (6.06 ± 0.85, 8.04 ± 0.63, with F value respectively 27.230 and 33.525, P < 0.05 or P < 0.01). There was significant statistical difference in all measurement indices except for mRNA of TGF-ß1 and Smad 3 among radix astragali treatment groups on PID 32 and 43 [with t values respectively 3.593-4.814 (thickness), 4.051-5.811 (hardness), 2.976-5.986 (TGF-ß1 protein), and 2.742-4.630 (Smad 3 protein), P < 0.05 or P < 0.01]. CONCLUSIONS: Radix astragali injection inhibits fibroblast proliferation in hypertrophic scars through down-regulating mRNA expression and protein synthesis of TGF-ß1 and Smad 3, thus inhibits hypertrophic scars formation. Its inhibition effect is drug concentration and duration dependent. The drug may be considered as a potential agent to prevent hypertrophic scar.

[Effect of curcumin on growth and function of fibroblast in human hyperplastic scar].

OBJECTIVE: To seek an effective drug for treatment of human hyperplastic scar through studying the effects of curcumin on fibroblast growth and collagen synthesis. METHODS: Fibroblasts derived from scar tissue and from normal epidermal tissue were isolated and cultured separately with tissue-block method, their morphology were observed under invert phase contrast microscope, their growth curve was drawn respectively to determine the speed of growth. Then, fibroblasts from scar were stimulated with curcumin in different concentrations (0, 12.5, 25, 50 and 100 micromol/L) for detecting the inhibitory effect of curcumin on growth of fibroblasts using MTT methods and that on activity of procollagen alpha-1 gene transcription in fibroblast was detected by RT-PCR. RESULTS: The cell growth curve showed that double-multiplying time was 5 days in fibroblasts from scar and 4 days in those from normal dermis, showing significant difference between them (P < 0.05). MTT showed that curcumin in 12.5 micromol/L showed a cell proliferation enhancing trend, and its absorbance value was significantly higher than that in the normal group, but the effect turned to inhibition when concentration increased to over 25-100 micromol/L, and became significant inhibition at concentration of 50 and 100 micromol/L. Besides, curcumin also showed markedly inhibition on collagen type I synthesis in fibroblasts (P < 0.01). CONCLUSION: High concentration curcumin can inhibit effectively the fibroblast proliferation and collagen I synthesis in hyperplastic scar, therefore, may has therapeutic effect on the disease in human being.

[Effects of citrus reticulata blanco extract on fibroblasts from human hypertrophic scar in vitro].

OBJECTIVE: To investigate the effects of citrus reticulata blanco extract on the proliferation and collagen metabolism of fibroblasts from human hypertrophic scar. METHODS: Human hypertrophic scar fibroblasts from two burn patients obtained from plastic surgery were cultured in vitro and divided into experimental group (n = 12, with basic culture medium and 2.5, 5.0, 10.0,25.0 mg/L citrus reticulata blanco extract, respectively, 3 bottles for each concentration of citrus reticulata blanco extract ), control group 1 (n = 3, with basic culture medium) , and control group 2 ( n = 3, with basic culture medium and 5% ethyl alcohol). The cell proliferation in each group was observed with MTT method, then the inhibition rate was calculated. Apoptosis and its index ( AI) in each group were determined after TUNEL staining . The changes in the content of ICTP and PINP in each group were observed by radioimmunity. RESULTS: The inhibition rate in the experimental group with the citrus reticulata blanco extract in concentration of 2. 5, 5.0, 10.0, 25. 0 microg/ ml were (7. 100+/-0.038)% , (8. 100+/- 0. 048)% , (10. 900+/-0. 055)%, (15.900+/-0. 097) %, respectively, which were significantly higher than those in other two groups ( P <0.05 ). The Al (69. 7% , 71.7%, 86.4% , 95.2% ), ICTP [(17.2+/-0.6), (18.3+/-0.6), (19.8+/-0.5), (23.2+/-0.6) microg/L] and PINP [ (101.7+/-1.4) , (107. 8+/-1. 1) , (111.6+/-1.2) , (124. 6+/-1.3) microg/L] in experimental group with the citrus reticulata blanco extract in concentration of 2.5, 5.0, 10.0 , 25.0 mg/L were also obviously higher than other two control groups( P <0.05) ,but these indices in control 1 group were similar to those in control 2 group( P >0. 05). CONCLUSION: The citrus reticulata blanco extract might be beneficial for the management of hypertrophic scar through inhibition of the proliferation of fibroblasts in hypertrophic scar, by promoting apoptosis and collagen degradation.

[Blocking action of tetrandrine on the scar-collagen matrix contraction stimulated by transforming growth factor beta].

OBJECTIVE: To investigate the effect of Tetrandrine on the scar-collagen matrix contraction stimulated by transforming growth factor beta (TGF-beta). METHODS: The scar-derived fibroblasts were suspended in a solution of collagen matrix, and TGF-beta, they were associated to form a scar model in vitro. The influence of tetrandrine on the contraction indexes was observed. RESULTS: Tetrandrine plays a blocking action in the scar-collagen matrix contraction stimulated by TGF-beta, which was in a dose-dependent manner. CONCLUSION: This function of tetrandrine could be responsible for the anti-scar mechanism.