Calpain inhibition ameliorates scald burn-induced acute lung injury in rats.

BACKGROUND: The molecular pattern of severe burn-induced acute lung injury, characterized by cell structure damage and leukocyte infiltration, remains unknown. This study aimed to determine whether calpain, a protease involved in both processes, mediates severe burn-induced acute lung injury. METHODS: Rats received full-thickness scald burns covering 30% of the total body surface area, followed by instant fluid resuscitation. MDL28170 (Tocris Bioscience), an inhibitor of calpain, was given intravenously 1 h before or after the scald burn. The histological score, wet/dry weight ratio, and caspase-3 activity were examined to evaluate the degree of lung damage. Calpain activity and its source were detected by an assay kit and immunofluorescence staining. The proteolysis of membrane skeleton proteins α-fodrin and ankyrin-B, which are substrates of calpain, was measured by Western blot. RESULTS: Time-course studies showed that tissue damage reached a peak between 1 and 6 h post-scald burn and gradually diminished at 24 h. More importantly, calpain activity reached peak levels at 1 h and was maintained until 24 h, paralleled by lung damage to some extent. Western blot showed that the levels of the proteolyzed forms of α-fodrin and ankyrin-B correlated well with the degree of damage. MDL28170 at a dose of 3 mg/kg b. w. given 1 h before burn injury not only antagonized the increase in calpain activity but also ameliorated scald burn-induced lung injury, including the degradation of α-fodrin and ankyrin-B. Immunofluorescence images revealed calpain 1 and CD45 double-positive cells in the lung tissue of rats exposed to scald burn injury, suggesting that leukocytes were a dominant source of calpain. Furthermore, this change was blocked by MDL28170. Finally, MDL28170 given at 1 h post-scald burn injury significantly ameliorated the wet/dry weight ratio compared with burn injury alone. CONCLUSIONS: Calpain, a product of infiltrating leukocytes, is a mediator of scald burn-induced acute lung injury that involves enhancement of inflammation and proteolysis of membrane skeleton proteins. Its late effects warrant further study.

MicroRNA-138 Aggravates Inflammatory Responses of Macrophages by Targeting SIRT1 and Regulating the NF-κB and AKT Pathways.

BACKGROUND/AIMS: With increased understanding of sepsis, mortality is decreasing. However, there is still a lack of effective therapeutic strategy. The inflammatory response of macrophages is critical during sepsis. METHODS: Macrophages were stimulated with LPS. Western blotting and qRT-PCR were used to detect inflammatory responses. Then, the inhibitor of microRNA-138 was transfected and Western blotting, qRT-PCR, H&E staining and ELISA were used to verify the role of microRNA-138 in inflammation. Then target gene prediction databases were used to predict the potential target of microRNA-138. Both animal and cell models under LPS challenges were established to verify the regulation of SIRT1 and microRNA-138 during inflammation. RESULTS: The present study showed that microRNA-138 was increased in macrophages stimulated with LPS. Additionally, the NF-κB and AKT pathways were both activated. The pre-treatment of microRNA-138 inhibitor decreased inflammatory factors, downregulated the NF-κB pathway, activated the AKT pathway and protected against organ damage in mice challenged with LPS. SIRT1 was demonstrated as a potential target of microRNA-138In macrophages stimulated with LPS, the inhibition effect of microRNA-138 inhibitor on inflammation was lost by SIRT1 siRNA pre-treatment. In the animal model, the protective effect of microRNA-138 antagomir disappeared in SIRT1 knockout mice. CONCLUSION: We demonstrated that miR-138 participated in the inflammatory process by inhibiting SIRT1 and activating the NF-κB pathway.

Melatonin prevents acute kidney injury in severely burned rats via the activation of SIRT1.

Acute kidney injury (AKI) is a common complication after severe burns. Melatonin has been reported to protect against multiple organ injuries by increasing the expression of SIRT1, a silent information regulator that regulates stress responses, inflammation, cellular senescence and apoptosis. This study aimed to investigate the protective effects of melatonin on renal tissues of burned rats and the role of SIRT1 involving the effects. Rat severely burned model was established, with or without the administration of melatonin and SIRT1 inhibitor. The renal function and histological manifestations were determined to evaluate the severity of kidney injury. The levels of acetylated-p53 (Ac-p53), acetylated-p65 (Ac-p65), NF-κB, acetylated-forkhead box O1 (Ac-FoxO1), Bcl-2 and Bax were analyzed to study the underlying mechanisms. Our results suggested that severe burns could induce acute kidney injury, which could be partially reversed by melatonin. Melatonin attenuated oxidative stress, inflammation and apoptosis accompanied by the increased expression of SIRT1. The protective effects of melatonin were abrogated by the inhibition of SIRT1. In conclusion, we demonstrate that melatonin improves severe burn-induced AKI via the activation of SIRT1 signaling.

Identification of sirtuin 1 as a promising therapeutic target for hypertrophic scars.

BACKGROUND AND PURPOSE: Sirtuin1 (SIRT1), the founding member of mammalian class III histone deacetylases, is reported to be a drug target involved in fibrotic diseases. However, whether it is an effective drug target in hypertrophic scar treatment is still not known. EXPERIMENTAL APPROACH: In the present study, we observed that SIRT1 localized to both the epidermis and the dermis of skin tissues by immunohistochemistry. After knock-down of SIRT1 by shRNA or up-regulating SIRT1 by resveratrol, the expression of α-SMA, Col1 and Col3 in fibroblasts were detected by western blots. A mouse excision wound healing model was used to observe the changes in collagen fibre associated with the different expression levels of SIRT1. KEY RESULTS: SIRT1 expression was inhibited in hypertrophic scar tissue. The down-regulation of SIRT1 resulted in an increased expression of α-SMA, Col1 and Col3 in hypertrophic scar-derived fibroblasts. In contrast, the up-regulation of SIRT1 not only inhibited the expression of α-SMA, Col1 and Col3 in hypertrophic scar-derived fibroblasts but also blocked the activation of TGFß1-induced normal skin-derived fibroblasts. In the mouse model of wound healing, the deletion of SIRT1 resulted in denser collagen fibres and a more disordered structure, whereas resveratrol treatment led to a more organized and thinner collagen fibre, which was similar to that observed during normal wound healing. CONCLUSIONS AND IMPLICATIONS: The results revealed that SIRT1 negatively regulates TGFß1-induced fibroblast activation and inhibits excessive scar formation and is, therefore, a promising drug target for hypertrophic scar formation.

Acute downregulation of miR-155 at wound sites leads to a reduced fibrosis through attenuating inflammatory response.

Fibrosis, tightly associated with wound healing, is a significant symptomatic clinical problem. Inflammatory response was reported to be one of the reasons. MiR-155 is relatively related with the development and requirement of inflammatory cells, so we thought reduce the expression of miR-155 in wound sites could improve the quality of healing through reduce inflammatory response. To test this hypothesis, locally antagonizing miR-155 by directly injecting antagomir to wound edge was used to reduce the expression of miR-155. We found wounds treated with miR-155 antagomir had an obvious defect in immune cells requirements, pro-inflammatory factors IL-1ß and TNF-α reduced while anti-inflammatory factor IL-10 increased. With treatment of miR-155 antagomir, the expression of α-smooth muscle actin (α-SMA), Col1 and Col3 at wound sites all reduced both from mRNA levels and protein expressions. Wounds injected with antagomir resulted in the structure improvement of collagen, the collagen fibers were more regularly arranged. Meanwhile the rate of healing did not change significantly. These results provide direct evidences that miR-155 play an important role in the pathogenesis of fibrosis and show that miR-155 antagomir has the potential therapy in prevention and reduction of skin fibrosis.

MicroRNA-21 regulates hTERT via PTEN in hypertrophic scar fibroblasts.

BACKGROUND: As an important oncogenic miRNA, microRNA-21 (miR-21) is associated with various malignant diseases. However, the precise biological function of miR-21 and its molecular mechanism in hypertrophic scar fibroblast cells has not been fully elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative Real-Time PCR (qRT-PCR) analysis revealed significant upregulation of miR-21 in hypertrophic scar fibroblast cells compared with that in normal skin fibroblast cells. The effects of miR-21 were then assessed in MTT and apoptosis assays through in vitro transfection with a miR-21 mimic or inhibitor. Next, PTEN (phosphatase and tensin homologue deleted on chromosome ten) was identified as a target gene of miR-21 in hypertrophic scar fibroblast cells. Furthermore, Western-blot and qRT-PCR analyses revealed that miR-21 increased the expression of human telomerase reverse transcriptase (hTERT) via the PTEN/PI3K/AKT pathway. Introduction of PTEN cDNA led to a remarkable depletion of hTERT and PI3K/AKT at the protein level as well as inhibition of miR-21-induced proliferation. In addition, Western-blot and qRT-PCR analyses confirmed that hTERT was the downstream target of PTEN. Finally, miR-21 and PTEN RNA expression levels in hypertrophic scar tissue samples were examined. Immunohistochemistry assays revealed an inverse correlation between PTEN and hTERT levels in high miR-21 RNA expressing-hypertrophic scar tissues. CONCLUSIONS/SIGNIFICANCE: These data indicate that miR-21 regulates hTERT expression via the PTEN/PI3K/AKT signaling pathway by directly targeting PTEN, therefore controlling hypertrophic scar fibroblast cell growth. MiR-21 may be a potential novel molecular target for the treatment of hypertrophic scarring.

[Influence of negative pressure wound therapy on the angiogenesis of wounds in diabetic rats].

OBJECTIVE: To observe the influence of negative pressure wound therapy on the angiogenesis of wounds in diabetic rats. METHODS: Diabetes model was reproduced by intraperitoneal injection of 20 g/L streptozotocin in the dosage of 65 mg/kg in 40 SD rats. Two weeks later, rats were divided into control group (C) and negative pressure group (NP) according to the random number table, with 20 rats in each group. A piece of full-thickness skin in the center of the back of each rat in the size of 2 cm×2 cm was excised to produce a wound. Immediately after injury, wounds in group C were given conventional dressing change; wounds in group NP were treated with continuous negative pressure (-16.0 kPa) therapy for four hours a day, which lasted for seven days. (1) Blood glucose and body weight of rats in two groups were respectively measured by glucose meter and electronic scale before treatment, and 1 and 2 week (s) after. (2) Wound blood flow was detected by laser Doppler perfusion imager before treatment and on post treatment day (PTD) 1, 3, 7, with 5 rats at each time point. (3) On PTD 3 and 7, respectively, five rats from each group were sacrificed. The wound tissue was excised and divided into two parts. The angiogenesis in the left part tissue was observed with immunohistochemical staining. The microvessel density was calculated. (4) The full-thickness skin excised before treatment and the right part tissue freeze on PTD 3 and 7 were collected. On PTD 1 and 14, wound tissue was excised in the above-mentioned method. The mRNA levels of the vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 1 (Fit-1), angiopoietin 1 (Ang-1), Ang-2, and tyrosine kinase receptor 2 (Tie-2) were determined with real-time fluorescence quantification PCR. Data were processed with two-way analysis of variance or LSD-t test. RESULTS: (1) No significant difference was observed between two groups in blood glucose level and body weight as a whole or at each time point (with F values respectively 0.667, 0.176, t values from 0.311 to 0.707, P values all above 0.05). (2) The difference in the overall wound blood flow of rats between two groups was significant (F = 24.66, P < 0.05). On PTD 1, 3, 7, values of wound blood flow of rats in group NP were (179 ± 24), (219 ± 12), (192 ± 30) perfusion unit, significantly higher than those of rats in group C[(127 ± 16), (179 ± 8), (144 ± 17) perfusion unit, with t values respectively 3.71, 5.57, 2.77, P < 0.05 or P < 0.01]. (3) The difference in the overall microvessel density in the wound of rats between two groups was significant (F = 33.25, P < 0.05). On PTD 3, the microvessel density in the wound of rats in group NP was (80 ± 12) per 100-time visual field, which was significantly higher than that of group C[(38 ± 4) per 100-time visual field, t = 9.257, P < 0.05]. On PTD 7, the microvessel density in the wound of rats in two groups were close (t = 1.159, P > 0.05), but the vessels in group NP were regularly arranged with spacious lumen, while the vessels in group C were disorderly arranged with narrow lumen. (4) On PTD 1, 3, mRNA expression levels of VEGF, Fit-1, and Ang-1 in group NP were obviously higher than those in group C (with t values from 1.28 to 11.60, P values all below 0.01). On PTD 7, the mRNA expression level of Ang-1 (27.59 ± 3.55) in group NP was obviously higher than that in group C (19.87 ± 1.86, t = 7.23, P < 0.001), while the mRNA level of its antagonist Ang-2 (5.79 ± 0.61) in group NP was obviously lower than that in group C (17.62 ± 0.85, t = 19.88, P < 0.001). On PTD 3, 7, 14, mRNA levels of Tie-2 in group NP were obviously lower than those in group C (with t values from 8.92 to 15.60, P values all below 0.01). CONCLUSIONS: Negative pressure wound therapy may promote wound angiogenesis by enhancing the expression of Ang-1 and lowering the expression of Ang-2 in diabetic rats.

Construction of the tissue engineering seed cell (HaCaT-EGF) and analysis of its biological characteristics.

OBJECTIVE: To construct the tissue engineering seed cell (HaCaT cell line) with stable expression of the human epidermal growth factor (EGF), and analyze the changes of its biological characteristics. METHODS: PCDNA3.1-EGF eukaryotic expression vector was transferred into HaCaT cell, and G418 was utilized to select the HaCaT-EGF cell line. Using an inverted microscope, PCR, ELISA method to detect the changes of the cell morphology, the expression of the EGF gene and protein, and the mRNA expression levels of apoptosis related molecule Caspase-3, the cell cycle related protein cyclin D1. RESULTS: The mRNA expression levels of the obtained HaCaT-EGF cell were more than 100 times higher than the level of ordinary HaCaT cell. The colony of the HaCaT-EGF cells was more focused and tight compared to the empty vector transfected HaCaT cells and normal HaCaT cells. The expression levels of apoptotic factor Caspase-3 and cyclin D1 in HaCaT-EGF cell were significantly higher than those in the empty vector HaCaT- pcDNA3.1 cell, and the differences were statistically significant (P<0.01), but there was no significant difference compared to the normal HaCaT cells (P>0.05). CONCLUSIONS: HaCaT-EGF cell can continuously secrete EGF, and the biological characteristic is stable. It can be used for tissue engineering experiment and is an ideal seed cell for constructing tissue engineered skin.

[Effects of adipose-derived stem cells on renal injury in burn mice with sepsis].

OBJECTIVE: To investigate the effect of adipose-derived stem cells (ADSC) on renal injury in mice with burn injury and sepsis and its underlying mechanism. METHODS: (1) Adipose tissue was collected from both inguinal regions of 5 C57BL/6J mice to isolate, culture and purify ADSC through enzyme digestion, density gradient centrifugation, and adherence method. Cells of the third passage were used in the experiment. The morphologic change in cells was observed and the growth curve of cells was determined. The expression of cell surface antigen phenotype was analyzed by flow cytometry, and the cells were identified by adipogenic and osteogenic differentiation. (2) Another 37 C57BL/6J mice were divided into normal control group (n = 5), saline group (n = 16), and group ADSC (n = 16) according to the random number table. The mice in saline group and group ADSC were injected with Pseudomonas aeruginosa after being subjected to 15% TBSA full-thickness burn on the back to reproduce septic burn model. Then the mice were injected with saline and ADSC through tail vein respectively. At post burn hour (PBH) 12, 24, 48, and 72, the pathological change in kidney tissue was observed, the levels of blood urea nitrogen and serum creatinine were determined, and the levels of TNF-α, IL-12, IL-10, and cyclooxygenase-2 (COX2) mRNA were determined with real-time fluorescence quantitative PCR in both groups. Above-mentioned indexes were also examined in the normal control group (without burn). Data were processed with multifactor analysis of variance and LSD- t test. RESULTS: (1) Cells in the third passage were orderly arranged with the shape similar to fibroblasts. The percentages of CD90(+), CD105(+), CD34(-), and CD45(-) cells were all above 90%. The cells could differentiate into osteoblasts and adipocytes. The cells were identified to be ADSC. (2) From PBH 12 to PBH 72, the neutrophil infiltration gradually increased, and the structure of kidney tubules and glomeruli were deranged in saline group. The pathological change in kidney tissue in group ADSC was less serious than that of normal control group at each time point. From PBH 12 to PBH 72, the levels of blood urea nitrogen and serum creatinine in saline group were significantly higher than those of normal control group and group ADSC (P values all below 0.01). Compared with those of the normal control group, the levels of TNF-α and IL-12 mRNA were higher in group ADSC and saline group at PBH 24 (P values all below 0.05). At PBH 24, the level of TNF-α mRNA in group ADSC (1.58 ± 0.19) was lower than that of saline group (3.36 ± 0.30, P < 0.05). At PBH 24, the levels of IL-10 and COX2 mRNA in group ADSC (2.89 ± 0.47, 4.90 ± 0.59) were higher than those in normal control group (1.00 ± 0.15, 1.00 ± 0.27) and saline group (1.32 ± 0.38, 1.57 ± 0.38, P values all below 0.05). CONCLUSIONS: ADSC can decrease the levels of blood urea nitrogen and serum creatinine, promote the production of anti-inflammatory cytokines IL-10 and COX2, and reduce the release of the pro-inflammatory cytokines TNF-α and IL-12 to offer protective effects against renal injury in burn mice with sepsis.

[Reproduction of a mouse model of deep partial-thickness scald and determination of hypoxia in the wound].

OBJECTIVE: To reproduce a stable mouse model of deep partial-thickness scald and to determine the hypoxia status in the wound. METHODS: (1) A homemade scald-producing apparatus with constant steam (92 °C) emission was used to reproduce scald injury on the back (2 cm in diameter) in 80 male BALB/c mice for different duration (2, 4, 6, and 8 s), with 20 mice for each scald duration. The nozzle was aligned perpendicularly to the back of mice, 2 cm above the skin surface. The gross condition of wound was observed with naked eyes immediately after injury. Skin samples of 5 mice with different burn duration were harvested 0, 12, 24, and 48 h after scald for histopathological observation with hematoxylin and eosin staining, to screen the scalding time and time for biopsy of scalded skin to determine proper scalding time for the experiment. (2) Model of deep partial-thickness scald was reproduced with the desired scalding time as shown in the preliminary experiment in another 5 BALB/c mice. The hypoxia status in subcutaneous tissue was observed with immunohistochemical staining 72 h after scald. Another 20 BALB/c mice were divided into normal control group (n = 5, without scald) and deep partial-thickness scald group (n = 15, scalded for a suitable duration as determined in the preliminary experiment) according to the random number table. The subcutaneous oxygen content in wound center, the margin of the wound, and the normal skin adjacent to the wound was detected with laser Doppler transcutaneous oxygen tension 72 h after scald, with 5 mice in each region. Data were processed with one-way analysis of variance. RESULTS: (1) The wound of mice with different scald durations was pale, clean, and no exudate was observed right after injury. (2) The burn depth developed gradually along with the scalding time and sample harvesting time, and it became stable 24 h after scalding. A deep partial-thickness injury was observed in the dermis of mice scalded for 4 s and harvested 24 h after scald, and it was shown that the external hair sheath was still present, and it was determined to be a deep partial-thickness scald. (3) Dense staining of pimonidazole (hypoxia) was found in deep partial-thickness scald wound 72 h after scald, especially in the marginal zones of the wounds. The partial oxygen pressure in the wound center, wound margin, and normal skin around the wound was respectively (36.2 ± 3.2), (37.0 ± 1.4), (37.4 ± 2.7) mm Hg (1 mm Hg = 0.133 kPa), showing no statistically significant difference among them (F = 74.705, P > 0.05), but they were significantly lower than that of the control group [(53.1 ± 2.4) mm Hg, with F values respectively 82.377, 91.375, 100.531, P values all below 0.05]. CONCLUSIONS: Deep partial-thickness scald model can be reproduced in (20.0 ± 1.0) g male BALB/c mice by scalding with 92 °C hot steam for 4 s, and the depth of wound becomes stable 24 h after scalding. Hypoxia can be found in the scalded wounds, especially in the marginal zones of the wounds.

Protection against TGF-ß1-induced fibrosis effects of IL-10 on dermal fibroblasts and its potential therapeutics for the reduction of skin scarring.

Scarring, tightly associated with fibrosis, is a significant symptomatic clinical problem. Interleukin 10 (IL-10) has been identified as a candidate scar-improving therapy based on preclinical studies. However, the molecular mechanism of IL-10 in scar improvement is still uncertain. In this study, human dermal fibroblasts stimulated with TGF-ß1 were treated with IL-10 to analyze the mRNA and some of proteins' expression levels of type I collagen (Col1), type III collagen (Col3), alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-1 (MMP1), MMP2, MMP8 and tissue inhibitor of metalloproteinase 1 (TIMP1), TIMP2 by real-time PCR and Western blot, to observe α-SMA-positive fibroblasts by immunocytochemistry. The contracture and improvement of fibroblast-populated collagen lattice (FPCL) and a murine model of wound healing were used to evaluate the scar-improving effects by histological staining. The results showed that IL-10 can significantly down-regulate the mRNA and protein expression levels of Col1, Col3, α-SMA, and up-regulate the mRNA expression levels of MMP1 and MMP8, and decrease α-SMA-positive fibroblasts. FPCL analysis showed that the IL-10 (20 ng/ml) can significantly inhibit the contracture, improve the architecture of FPCL. Wounds injected with IL-10 demonstrated that the appearance of scar was improved, the wound margin of scarring was narrow, and the deposition of collagens (Col1 and Col3) in regenerated tissue was relieved. These results provide direct evidences that IL-10 has the inhibitory effects on the excessive deposition of extracellular matrix components and fibroblast-to-myofibroblast transition, and show that IL-10 has the potential therapy in prevention and reduction of skin scarring.

[Effects of Wnt/beta-catenin signaling on the phenotype change of human dermal fibroblasts and its mechanism].

OBJECTIVE: To study the role of Wnt/beta-catenin signaling in the phenotype change of normal skin fibroblasts (NFb) into myofibroblasts and the underlying mechanism. METHODS: NFb were isolated by collagenase digestion and cultured. (1) Experiment one. NFb were divided into four groups according to the random number table. Cells in control group were cultured with serum-free DMEM nutrient solution (briefly called nutrient solution). Cells in TGF-beta1 group were cultured with nutrient solution containing 10 ng/mL recombinant human TGF-beta1 (the same concentration for following experiments). Cells in Wnt3a group were cultured with nutrient solution containing 150 ng/mL Wnt3a (the same concentration for following experiments). Cells in TGF-beta1 + Wnt3a group were cultured with nutrient solution containing TGF-beta1 and Wnt3a. The mRNA and protein expression levels of beta-catenin and alpha-smooth muscle actin (alpha-SMA) were determined by real-time fluorescent quantitative PCR and Western blotting at post culture hour (PCH) 48. (2) Experiment two. NFb were divided into four groups according to the random number table. Cells in control group and TGF-beta1 group were treated as those in the corresponding groups in experiment one. Cells in SB415286 (glycogen synthase kinase-3beta inhibitor) group were cultured with nutrient solution containing 10 micromol/L SB415286 (the same concentration for following experiments). Cells in TGF-beta1 + SB415286 group were cultured with nutrient solution containing TGF-beta1 and SB415286. The mRNA and protein expression levels of alpha-SMA were determined by real-time fluorescent quantitative PCR and Western blotting, and the alpha-SMA-positive myofibroblasts were detected by immunofluorescence cytochemical staining at PCH 48. The experiments were all repeated for three times. Data were processed with analysis of variance and LSD- t test. RESULTS: (1) Experiment one. There was no statistically significant difference among four groups in beta-catenin mRNA level (F = 0.302, P = 0.823). There were statistically significant differences among four groups in beta-catenin protein level (F = 16.713, P = 0.001). The protein level of beta-catenin was higher in TGF-beta1 group (0.73 +/- 0.12) and Wnt3a group (0.82 +/- 0.17) than in control group (0.34 +/- 0.11, with t values respectively 3.028, 3.727, P < 0.05 or P < 0.01). The protein level of beta-catenin in TGF-beta1 + Wnt3a group (1.23 +/- 0.21) was higher than that of the other three groups (with t values respectively 6.911, 3.883, 3.184, P values all below 0.01). There were statistically significant differences among four groups in alpha-SMA mRNA level (F = 31.830, P = 0.001). Compared with that of control group, the expression level of alpha-SMA mRNA was up-regulated in TGF-beta1 group and down-regulated in Wnt3a group (with t values respectively 6.759, 2.535, P < 0.05 or P < 0.01). The expression level of alpha-SMA mRNA in TGF-beta1 + Wnt3a group was lower than that of TGF-beta1 group (t = 4.532, P < 0.01). The protein levels of alpha-SMA in control, TGF-beta1, Wnt3a, and TGF-beta1 + Wnt3a groups were respectively 0.83 +/- 0.17, 1.43 +/- 0.20, 0.53 +/- 0.12, and 0.89 +/- 0.14 (F = 16.597, P = 0.001). Compared with that of control group, the protein level of alpha-SMA was up-regulated in TGF-beta1 group and down-regulated in Wnt3a group (with t values respectively 4.582, 2.291, P < 0.05 or P < 0.01). The protein level of alpha-SMA in TGF-beta1 + Wnt3a group was lower than that of TGF-beta1 group (t = 4.123, P < 0.01). (2) Experiment two. There were statistically significant differences among four groups in alpha-SMA mRNA level (F = 34.101, P = 0.001). The alpha-SMA mRNA level in SB415286 group was lower than that of control group (t = 2.511, P < 0.05). The alpha-SMA mRNA level in TGF-beta1 + SB415286 group was lower than that of TGF-beta1 group (t = 3.587, P < 0.01). There were statistically significant differences among four groups in alpha-SMA protein level (F = 11.381, P = 0.003). The alpha-SMA protein level was lower in SB415286 group than in control group (t = 2.364, P < 0.05). The alpha-SMA protein level was down-regulated in SB415286 +TGF-beta1 group as compared with that of TGF-beta1 group (t = 2.556, P < 0.05). There were few alpha-SMA-positive fibroblasts in control group. Compared with that of control group, the expression of alpha-SMA was significantly increased in TGF-beta1 group (t =11.198, P < 0.01), and the expression of alpha-SMA was down-regulated in SB415286 group. Meanwhile, the expression of alpha-SMA in TGF-beta1 + SB415286 group were significantly lower than that of TGF-beta1 group (t = 5.902, P < 0.01). CONCLUSIONS: The Wnt/beta-catenin signaling might be involved in the fibroblasts-myofibroblasts transition, and it negatively regulate the TGF-beta1 -mediated profibrotic effects.

[Effects of myrrh extract on proliferation and collagen mRNA expression of human fibroblasts in vitro].

OBJECTIVE: To observe the effects of myrrh extract on biological characteristics of human dermal fibroblasts (Fb), and to explore its possible mechanisms in promoting wound healing. METHODS: Normal Fb was isolated from human foreskin tissue and cultured in vitro. The third to fifth passages of Fb were used in the experiment. (1) Fb were planted onto 96-well plate and divided into control group, and 1 × 10(-4), 1 × 10(-3), 1 × 10(-2), 1 × 10(-1), 1, 10, 1 × 10(2) g/L myrrh water extract groups and myrrh ethanol extract groups according to the random number table. Fb in control group were cultured with DMEM medium containing 0.25% calf serum (briefly called low-concentration serum medium), and those in various concentrations of myrrh water extract and myrrh ethanol extract groups respectively with low-concentration serum medium containing corresponding concentration of 2 kinds of myrrh extract. After being cultured for 48 h, cell morphology was observed with inverted-phase contrast microscope, and Fb proliferation activity (denoted as absorbance value) was determined with MTT method. (2) Fb were respectively planted into flasks and dishes and divided into two groups according to the random number table. Fb in control group were cultured with low-concentration serum medium, and that in 1 g/L myrrh water extract group with low-concentration serum medium containing 1 g/L myrrh water extract. After being cultured for 72 h, Fb cell cycle and the type I and III collagen mRNA expression were respectively determined by flow cytometry and real-time fluorescent quantitative PCR. Data were processed with LSD-t test. RESULTS: (1) Fb in all groups grew in long-spindle shape, but the cell fusion was much obvious in 1 g/L myrrh water extract group than in control group. Fb absorbance value in 1 × 10(-3), 1 × 10(-2), 1 × 10(-1), 1, 10 g/L myrrh water extract groups was respectively 0.378 ± 0.032, 0.402 ± 0.007, 0.390 ± 0.038, 0.453 ± 0.036, 0.390 ± 0.037, all higher than that in control group (0.332 ± 0.044, with t value respectively 2.24, 2.93, 2.69, 5.73, 2.71, P values all below 0.05). Compared with that in control group, Fb absorbance value in 1 × 10(-4) g/L myrrh water extract group was not statistically different (0.312 ± 0.048, t = 2.84, P > 0.05), while that in 1 × 10(2) g/L myrrh water extract group was significantly lower (0.154 ± 0.009, t = 7.17, P < 0.05). Fb absorbance values in 1 × 10(-3), 1 × 10(-1), 1, 10, 1 × 10(2) g/L myrrh ethanol extract groups were significantly lower than that in control group (with t values from 2.30 to 24.79, P values all below 0.05). (2) Compared with those in control group [(82.2 ± 7.9)% and (13.3 ± 2.3)%, (4.5 ± 0.8)%], the percentage of cells in G0/G1 phase in 1 g/L myrrh water extract group was obviously decreased [(74.3 ± 6.3)%, t = 6.77, P < 0.05], while those in S and G2/M phases increased [(16.6 ± 3.4)%, (9.1 ± 1.6)%, with t value respectively 7.53, 6.34, P values below 0.05]. Compared with those in control group (1.00 ± 0.05, 1.00 ± 0.06), the mRNA level of collagen III in 1 g/L myrrh water extract group was significantly up-regulated (1.38 ± 0.12, t = 3.81, P < 0.01), while that of collagen I was not statistically different (0.89 ± 0.08, t = 1.17, P > 0.05). CONCLUSIONS: Myrrh water extract can notably promote the proliferation of Fb, accelerate the cell cycle of Fb, and up-regulate the mRNA expression of type III collagen in Fb, which may be related to its mechanisms in promoting wound healing.

[Effects of Angelica dahurica extract on biological behavior of dermal fibroblasts].

OBJECTIVE: To observe the effects of Angelica dahurica extracts on the biological characteristics of human dermal fibroblasts in vitro and to preliminary explore its possible therapeutic mechanism for wound healing. METHODS: The optimal concentration of Angelica dahurica extracts was identified by analysing of proliferation activity of human normal fibroblasts (Fb) that treated with different concentration of Angelica dahurica extracts through thiazole blue (MTT) colorimetric assay. Cell cycle, collagen I and collagen III mRNA levels of the optimal Angelica dahurica extracts treated Fb were detected by flow cytometry (FCM) and real-time PCR techniques. RESULTS: At concentrations of 5 × 10(-4) to 5 × 10(-2) g/L, the Angelica dahurica extracts significantly enhanced the proliferation of Fb. The most significant concentration was 5 × 10(-3) g/L (t = 5.79, P < 0.01), at which an increased percentage of G1 to S and S to G2 phase cells (t = 11.2, 5.69, 2.44, P < 0.05) as well as an increased level of collagen I (1.61 ± 0.26 vs. 1.00 ± 0.16) and collagen III mRNA (3.36 ± 0.40 vs. 1.00 ± 0.14) were obtained compared to the control group (t = 6.69, 7.64, P < 0.01). CONCLUSIONS: Angelica dahurica extracts can notably promote the proliferation of Fb and accelerating the cell cycle of Fb as well as up-regulating the expression of collagen I and collagen III, which may enhance the process of wound healing.

A potential skin substitute constructed with hEGF gene modified HaCaT cells for treatment of burn wounds in a rat model.

This study aimed to investigate the feasibility of using an immortal keratinocyte cell line, HaCaT cells, to effectively deliver epidermal growth factor (EGF) in a skin substitute to treat burn wounds. The skin equivalent was constructed with human EGF (hEGF) gene modified HaCaT cells obtained through stable gene transfection; these were applied to full thickness burn wounds in a rat model. The results showed that the hEGF gene modified HaCaT cells produced more than 390ng/l of bioactive hEGF in the culture supernatant. K19 and integrin-ß1 as keratinocyte differentiation markers were elevated in the hEGF gene modified HaCaT cells which were shown to be non-tumorigenic. The skin equivalent constructed with hEGF gene modified HaCaT cells demonstrated improved epidermal morphogenesis with a thick and compact epidermis. Wound healing was accelerated noticeably when applied with this skin substitute seeded with hEGF gene modified HaCaT cells in vivo. The results suggest that HaCaT cells modified with hEGF gene might be promising seed cells for construction of genetically modified skin substitute which can effectively secrete hEGF to accelerate wound repair and regeneration.

Reduced expression of microtubule-associated protein 1 light chain 3 in hypertrophic scars.

Autophagy is a tightly regulated physiological process essential for cellular maintenance, differentiation, development, and homeostasis. Aberration of this process associates with the pathogeneses of several diseases in mammals. Hypertrophic scar (HS) is characterized by an abundance of collagenous tissue with hypercellularity. However, the molecular mechanism in HS formation is poorly understood. We compared the autophagic capacity in HS and its normal skin (NS) counterparts and explored the molecular mechanism of autophagy during the formation of HS. Microtubule-associated protein 1 light chain 3 (LC3) proteins in HS and NS were detected by immunohistochemistry, Western blot and quantitative real-time PCR (qPCR). The data showed that LC3 positive staining in HS was less intensive relative to NS group (p < 0.05). Three forms of LC3, with molecular weights of about 19 kDa (proLC3), 18 kDa (LC3-I) and 16 kDa (LC3-II), respectively, expressed in NS by Western blot. In contrast, only proLC3 expressed while both LC3-I and LC3-II were significantly downregulated in HS. The protein level of beclin 1 in HS was significantly lower compared with NS (p < 0.05). LC3 and beclin 1 mRNA levels in HS were significantly lower than that in NS (p < 0.05). These results suggest that the generation of LC3-I and LC3-II are interrupted in HS, and that the resultant decrease of autophagic capacity may associate with the pathogenesis of HS.

[Efficacy of vacuum sealing drainage in mice infected with Pseudomonas aeruginosa and its mechanism].

OBJECTIVE: To observe the effect of vacuum sealing drainage (VSD) on the proliferation of Pseudomonas aeruginosa (PA) in infected wound, and to explore its possible mechanism. METHODS: Full-thickness skin wounds each with area of 1 cm x 1 cm were produced on the back of 40 C57 BL/6 mice, and then they were contaminated with wild type PA strains PAO1 marked with target gene of bacterial luciferase luxCDABE (PAO1-lux), they were dressed for 24 hours to reproduce PA infection model. Then mice were divided into experiment [E, with treatment of VSD (pressure value at -16.625 kPa)] and control (C, with treatment of conventional dressing change) groups according to the random number table, with 20 mice in each group. The fluorescence intensity of PAO1-lux and blood flow in wound was respectively measured by in vivo optical imaging system and laser Doppler perfusion imager before treatment and at post treatment hour (PTH) 24. The expression levels of IL-1beta and vascular endothelial growth factor (VEGF) mRNA in wound edge were determined by real-time fluorescence quantitative RT-PCR before treatment and at PTH 24. The specimens of wound edge tissue were collected for observation of pathological change at PTH 24. Data were processed with t test. RESULTS: There were no obvious difference in fluorescence intensity of PAO1-lux and blood flow in wound between E and C groups before treatment (with t value respectively 0.03, 0.50, P values all above 0.05). The fluorescence intensity of PAOl-lux and blood flow in wound in E group at PTH 24 [(2.69 +/- 0.75) photons x s(-1) x cm(-2) x sr(-1) and (96 +/- 9) PU] was respectively lower and higher than that inC group [(5.18 +/- 0.96) photons x s(-1) cm x (-2) x sr(-1) and (70 +/- 11) PU, with t value respectively 3.54, 3.13, P values all below 0.05]. The expression levels of IL-1beta and VEGF mRNA in both groups before treatment were similar (with t value respectively 0.19, 0.07, P values all above 0.05). The expression levels of IL-1beta and VEGF mRNA in E group at PTH 24 was respectively 4.72 +/- 0.37, 2.68 +/- 0.39, all markedly higher than those in C group (2.24 +/- 0.50, 1.22 +/- 0.13, with t value respectively 6.90, 6.12, P values all equal to 0.00). The number of inflammatory cell infiltrating the wound edge in E group at PTH 24 was increased by nearly 77% as compared with that in C group. CONCLUSIONS: Compared with conventional dressing change, VSD can reduce the amount of Pseudomonas aeruginosa in full-thickness skin defect wound at the early stage, it may be related with an increase in blood flow and number of inflammatory cells in wound tissue, promoting expression of IL-1beta and VEGF mRNA.

[Different expression of protein in the supernatant of heat injured keratinocytes].

OBJECTIVE: To compare the difference of protein expression in the supernatant of heat injured keratinocytes (KC) and normal KC. METHODS: A model of heat injured KC was produced in vitro. The supernatant of normal KC and heat injured KC was collected after culture for 12 hours, and was ultrafiltered and lyophilized to get the protein. The protein sample was separated by immobilized pH gradient based two dimensional gel electrophoresis (2-DE). The gel was stained and the different expression of protein was analyzed using ImageMaster 2D analysis software. RESULTS: (1) Average protein spots were 1,898 +/- 113, 1,877 +/- 97 in the supernatant of normal and heat injured KC and 1,118 protein spots could be used for statistical analysis. (2) Statistical result showed that 26 protein spots were significantly different between the two groups. 16 protein spots were higher in the supernatant of normal KC and then 10 protein spots were lower in the normal group. (3) 16 protein spots, which included 10 kinds of proteins, were identified successfully as different spots. Lower expression proteins were alpha-enolase, actin cytoplasmic 2, peroxiredoxin-4, phosphoglycerate mutase 1, G protein-regulated inducer of neurite outgrowth l in the supernatant of heat injured KC. Higher expression proteins in heat KC were purine nucleoside phosphorylase, tumor necrosis factor ligand superfamily member 10, proteasome subunit alpha type-7, UDP-glucose 6-dehydrogenase in the supernatant of heat injured KC. CONCLUSIONS: The result indicated that there are some significant different expression proteins in the supernatant of normal KC and heat injured KC. These findings provide new data for screening major molecules of tissue repair and finding the mechanism of wound repair.

Smad interacting protein 1 as a regulator of skin fibrosis in pathological scars.

Keloids and hypertrophic scars are significant symptomatic clinical problems characterized by the excessive and abnormal deposition of collagen-based extracellular matrix (ECM) components. However, the molecular basis of keloid and hypertrophic scar formation has not been fully elucidated. Here, we demonstrated that down-regulation of the transcription factor Smad interacting protein 1 (SIP1) could be relevant to keloid and hypertrophic scar formation. The results of the present study show that the level of SIP1 mRNA is significantly decreased in pathological scar tissues and in normal skin and pathological scar fibroblasts treated with transforming growth factor ß1 (TGF-ß1). In contrast, the expression of SIP1 mRNA is not decreased in normotrophic scar samples. The SIP1 mRNA level inversely correlates with the mRNA level of type I collagen (COL1A2) and directly correlates with the mRNA level of matrix metalloproteinase-1 (MMP1). Overexpression of SIP1 in keloid and hypertrophic scar fibroblasts represses TGF-ß1-stimulated COL1A2 expression and induces MMP1 expression. Alternatively, knockdown of SIP1 in normal skin fibroblasts enhance TGF-ß1-induced COL1A2 levels. These findings suggest that SIP1 could be a regulator of skin fibrosis, and depletion of SIP1 in pathological scar tissues could result in an up-regulation of collagen and down-regulation of matrix metalloproteinase, leading to an abnormal accumulation of ECM along with fibrosis and pathological scar formation.

[Effect of heat injured keratinocytes supernatant on biological behavior of fibroblasts].

OBJECTIVE: To observe the effect of the supernatant of heat injured keratinocytes (KC) on biological behavior of the dermal fibroblasts (Fb). METHODS: Human dermal Fb were isolated and cultured. A model of heat injured KC (HaCaT) was reproduced in vitro. Supernatant of normal KC and the supernatant of KC culture 12 hours after heat injury were collected and diluted with non-serum DMEM in 1:1 volume ratio to make normal KC conditioned medium (NKCM) and heat injury KC conditioned medium (HKCM) respectively. Fb was respectively treated with non-serum DMEM and 2 kinds of conditioned medium. (1) The proliferation of Fb was detected with MTT method at post culture hour (PCH) 12, 24, 36, 48. (2) The apoptosis of Fb was determined by flow cytometry at PCH 12 (Fb were heat injured in advance; Fb without heat treatment was used as control). (3) At PCH 24, expression of a-SMA in Fb cytoplasm was determined with immunofluorescence method; expression of a-SMA mRNA in Fb was determined with real-time quantified PCR. Data were processed with one-way analysis of variance, and pairwise comparison among groups with LSD-t test. RESULTS: (1) The proliferation of Fb: the absorbance value of Fb cultured with HKCM at PCH 12, 24, 36, 48 was respectively higher than that of Fb cultured with non-serum DMEM (with t value respectively 1.89, 2.35, 2.02, 1.94, and P values all below 0.01). There were significant statistical differences between the absorbance values of Fb cultured with HKCM and those of Fb cultured with NKCM at PCH 12, 24, and 48 (at PCH 12, t = 1.83, P < 0.01; at PCH 24, t = 2.91, P < 0.05; at PCH 48, t = 1.83, P < 0.05). (2) Apoptosis of Fb cultured with HKCM was diminished as compared with that of Fb cultured with NKCM and of Fb without treatment (t = 3.31, P < 0.05; t = 1.47, P < 0.01). (3) The expression of alpha-SMA (red fluorescence) in Fb cultured with non-serum DMEM or NKCM was less as seen under fluorescence scope, and it was obviously increased in Fb cultured with HKCM. (4) The relative expression amount of alpha-SMA mRNA in Fb cultured with HKCM was 1.32 +/- 0.06, which was higher than that both in Fb cultured with NKCM (1.14 +/- 0.07, t = 2.51, P < 0.05) and in Fb cultured with non-serum DMEM (1.00 +/- 0.09, t = 1.77, P < 0.05). CONCLUSIONS: The supernatant of KC 12 hours after heat injury can obviously promote the proliferation of Fb, inhibit its apoptosis and accelerate transdifferentiation of Fb to myofibroblasts.