Burning can cause all sorts of tissue damage depending on burn severity. Burns can be classified as first-, second-, and third-degree burns according to the involvement of skin and deeper tissues. Burns are so much different from other injuries that a separate medical superspeciality has been designated to treat them [
All male and female Wistar rats weighing 200–250 g were purchased from Shanghai SLAC Laboratory Animals Co., Ltd. (China). The 98 rats were randomly selected and divided into 7 groups: model group, vehicle group, administration groups (low-dose, middle-dose, and high-dose group), positive group, and normal group. They were housed in clear plastic cages with solid floors and hard wood chip bedding and placed in a temperature- and humidity-controlled environment. The experiment was conducted in the central lab of Affiliated Dongnan Hospital of Xiamen University in accordance with laboratory animal standards in China (GB14925-95) and the University Guidelines of the Ethics Committee for Animal Care and Use in Research. The experiment was performed according to the 3R principle of animals use and blinded treatment.
The herb (200 g) was cut into small pieces and extracted with purified water (400 mL, 1.5 h reflux, 100°C, ×2). The water solution was concentrated under a reduced pressure after filtration in a Buchner funnel, enriching the total flavones by macroporous resin. The extract content of rhamnocitrin-3-O-
The extract (60 g, crude drug 15 g/g) was resuspended in distilled water with continuous stirring. Then chitosan (60 g) was added to the stirring mixture till it became swollen completely. Next, borneol (10 g, dissolved into 20 mL glycerol) was added. Subsequently, sodium benzoate (1.2 g) was cast over the gel. Finally, volume was made with distilled water and was stirred continuously till a uniform high-dose gel weighing 1000 g formed. The gel (pH 6.0–7.0) had translucent appearance, good spreadability, uniform particle size distribution, and moderate viscosity (5.73 Pa·s). And the extracts in the middle-dose and low-dose gels were 50% and 25% of high-dose, respectively. The vehicle control gel was prepared without the extract. Model group were deep second-degree burn rats without administration, and normal group were rats without modeling and without administration. Vehicle (10 g/kg), low-dose, middle-dose, high-dose (12.5, 25 and 50 g/kg), and positive (silver sulfadiazine, 10 g/kg) group were painted for administration twice a day for 28 days to monitor adverse drug reactions and other accident situations. The doses and dose interval were used according to the preliminary laboratory experiment.
The model was built according to the scalded rat model from Fu and Wang [
The wound tissue sections were fixed in 4% formaldehyde. Then they were penetrated with paraffin and paraffin-embedded according to conventional methods. HE staining was conducted on days 7, 14, and 21 after burn injury. For this study 4
The rats were anesthetized by the vapor of ethyl alcohol after burn injury on days 1, 4, and 7, to take 2 mL blood at the inner canthus with glass capillary tube. Blood was allowed to coagulate for 15 min at room temperature. Then 200
Statistical analysis was performed using SPSS software version 17. All experimental parameters were expressed as the mean ± the standard error mean (SEM). Statistical comparisons were made using one-way analysis of variance (ANOVA) followed by LSD’s post hoc test.
The gels could serve as wound dressings to prepare an optimum wound bed without secretion. In OFG-treated groups, oedema and infiltration of inflammatory cells apparently decreased in burnt areas with good hyperplasia and incrustation (7 d). On day 14, new hair follicle and sebaceous glands were observed with almost complete epithelization and decrustation, in contrast with the incomplete epithelization in model and vehicle groups. On day 21, the administration rats showed a complete healing process contrasting the poor situation in model groups (Figure
Therapeutic effect of OFBGC on rats with II° burn (HE staining, ×100). The measurements were carried out on day 7, day 14, and day 21. Red areas showed incrustation and purple particles were inflammatory cells. (a) model group, (b) vehicle control, (c) low-dose group, (d) middle-dose group, (e) high-dose group, (f) positive control, and (g) normal control. 1: 7 d, 2: 14 d, and 3: 21 d.
Incrustation is the solid covering or layer that is formed from necrotic tissue. Decrustation is the scab being removed from skin surface with complete wound closure [
Healing time of wound in II° burn rats.
Treatment | Incrustation time (days) | Decrustation time (days) |
---|---|---|
Model | 8.29 ± 0.57 |
22.25 ± 0.56 |
Vehicle | 3.16 ± 0.42 |
16.91 ± 0.46 |
Low-dose | 2.31 ± 0.32 |
13.77 ± 0.39 |
Middle-dose | 2.16 ± 0.34 |
12.18 ± 0.58 |
High-dose | 1.88 ± 0.35 |
9.38 ± 0.51 |
Positive | 4.14 ± 0.45 |
13.96 ± 0.60 |
The productions of p38 and IL-1
Expression of p38 and IL-1
Treatment | p38 | IL-1 | ||||
---|---|---|---|---|---|---|
1 d | 4 d | 7 d | 1 d | 4 d | 7 d | |
Model | 79.89 ± 2.95# | 79.98 ± 4.37# | 80.92 ± 3.57# | 26.31 ± 1.66# | 27.68 ± 2.08# | 26.84 ± 1.66# |
Vehicle | 79.25 ± 3.58# | 76.42 ± 4.17 | 77.29 ± 4.13 | 25.23 ± 1.99 | 26.34 ± 1.43 | 25.83 ± 1.58 |
Low-dose | 76.21 ± 4.41 | 76.06 ± 4.72 | 75.70 ± 4.20 |
24.70 ± 1.96 |
25.67 ± 1.65 |
25.40 ± 1.19 |
Middle-dose | 75.32 ± 2.88 |
72.04 ± 4.95 |
72.34 ± 4.23 |
23.77 ± 1.66 |
25.60 ± 1.98 |
24.82 ± 1.53 |
High-dose | 72.40 ± 3.86 |
72.38 ± 4.81 |
72.29 ± 4.22 |
23.67 ± 1.87 |
24.70 ± 1.25 |
24.46 ± 1.28 |
Positive | 74.32 ± 5.12 |
73.16 ± 3.84 |
73.26 ± 3.26 |
23.36 ± 1.50 |
23.35 ± 1.11 |
23.50 ± 1.29 |
Normal | 74.40 ± 4.91 |
74.39 ± 4.62 |
74.31 ± 4.31 |
24.13 ± 1.22 |
25.12 ± 1.49 |
24.85 ± 1.31 |
Expression of EGF and VEGF in II° burns rat serum.
Treatment | EGF | VEGF |
---|---|---|
Model | 305.81 ± 15.52 | 100.29 ± 7.42 |
Vehicle | 310.60 ± 13.84 | 99.14 ± 7.07 |
Low-dose | 314.39 ± 10.18# | 110.07 ± 1.35# |
Middle-dose | 321.27 ± 7.20 |
104.40 ± 3.53 |
High-dose | 307.98 ± 10.03 | 101.98 ± 4.38 |
Positive | 318.17 ± 8.07# | 102.89 ± 4.19 |
Normal | 299.49 ± 16.28 | 101.47 ± 3.42 |
On day 7, the model, vehicle, and normal control groups showed no obvious increase in EGF and VEGF in burnt tissues compared with the normal group. But there were significant expressions of EGF in low-dose and middle-dose rats. The administration rats also showed obvious increases in VEGF (Figure
EGF and VEGF expression of immunohistochemical (×400) at day 7. The fuscescent ones, respectively, represented EGF and VEGF protein at different doses. 1: EGF and 2: VEGF; (a) model group, (b) vehicle control, (c) low-dose group, (d) middle-dose group, (e) high-dose group, (f) positive control, and (g) normal control.
Normal rats were found to be of no obvious expression of CD34 because no capillary formed. There were increases in CD34 in OFG-treated rats on day 7, especially in middle-dose group, and the expression in middle-dose rats was observed in subepidermic layer on day 14 and day 21. However, the model group showed a delayed increase in CD34 from day 7 to day 21 (Figure
CD34 expression of immunohistochemical (×400). The staining CD34 protein (fuscescent ones) could represent blood vessel. The capillary was new generated in the reparative process and the coarse vessels were existing. (a) Model group, (b) vehicle control, (c) low-dose group, (d) middle-dose group, (e) high-dose group, (f) positive control, and (g) normal control. 1: 7 d, 2: 14 d, and 3: 21 d.
In our study, the major constituent rhamnocitrin-3-O-
Extract of
Although the wound healing in vehicle-treated rats was not significant when compared with OFG-treated rats, it did show improved results in comparison with no treatment rats. The probable reason was that chitosan could prevent the loss of body fluid, prevent exudate buildup, protect the wounds from external contamination, have sufficient bactericidal activity to inhibit infection, and prepare an optimum wound bed for tissue repairing [
EGF binding to its receptor (EGFR) triggers rapid human skin fibroblast and keratinocyte locomotion. Additionally, this process plays a critical role in cell migration and human cutaneous wounds [
Similar studies on VEGF were carried out. OFG treatment can accelerate VEGF expression in a dose-dependent manner in healing tissue, but weak expression in the low-dose group was observed in serum on day 7. VEGF could promote angiogenesis by promoting the formation of endothelial cells and inducing newly formed blood vessels in wound healing [
Different from positive control, OFG-treated groups took less healing time for wound repair and showed more effective epithelialization by enhancing expression of EGF and in II° burn rats (Table
OFG can not only remarkably intensify incrustation and decrustation processes but also relieve the inflammatory reaction with deep second-degree burn. Based on these findings, the potential mechanisms are possibly related to the increasing synthesis and releasing of EGF and CD34 in wound healing, decreasing expression of p38 and IL-1
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors declare that they have no conflicts of interest.
This work was supported by the Medical Technology Innovation Topics from Nanjing Military Area Command of PLA of China (no. 14MS085 and no. MS098).