Resveratrol was isolated, as previously described by Park et al (19). Aloe emodin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle's medium and RPMI-1640 medium were purchased from Sigma-Aldrich and used as a culture media. A 100X antibiotic and antimycotic solution containing 10,000 U/ml penicillin, 10 mg/ml streptomycin and 25 µg/ml amphotericin B in 0.9% NaCl, was purchased from Sigma-Aldrich. Multi-well plates (6-, 12- or 48-well) were purchased from Corning Incorporated (Corning, NY, USA). Human and murine vascular endothelial growth factor (VEGF), murine IL-1β and murine monocyte chemoattractant protein-1 (MCP-1) ELISA kits were obtained from Cusabio Biotech Co., Ltd. (Wuhan, China). An ImmunoCruz™ staining kit for the detection of murine VEGF was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rat monoclonal anti-mouse MCP 1 (clone ECE.2; cat. no. CLYPAR-100-MCP1) (CCL2/MCP1 antibody was raised against synthetic peptide corresponding to residues 102–130 of mouse MCP-1 specific to mouse CCR2), rat monoclonal anti-mouse macrophage (clone BM8; cat. no. T-2028) (BM8 monoclonal antibody reacts with mouse F4/80 antigen specific to mouse and rat) and rabbit polyclonal anti-myeloperoxidase antibodies (cat. no. P05164) (rabbit polyclonal to myeloperoxidase raised from human granulocytes reacts specific to mouse, rat, human, pig and monkey) were purchased from Sanbio BV (Uden, Netherlands), BMA Biomedicals (Augst, Switzerland), and Thermo Fisher Scientific, Inc. (Waltham, MA, USA), respectively.

The THP-1 human acute monocytic leukemia and HaCaT human keratinocyte cell lines (American Type Culture Collection, Rockville, MD, USA) were and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (Sigma-Aldrich), penicillin (100 U/ml), streptomycin (100 µg/ml) and amphotericin (0.25 µg/ml). The plates were incubated overnight for 12 or 72 h.

Male BALB/c mice (5-week-old, n=30) were obtained from ShanDong University (Jinan China) and housed one per cage (to prevent attacks on wounds) for 1 week in a temperature-controlled room at 25±1°C with 60% relative humidity and a 12-h light-dark cycle, and provided with access to a standard laboratory diet and water ad libitum prior to experimentation. The ethical guidelines of the Animal Center of Yantai Yuhuangding Hospital, Yantai, Shandong Province, China. The mice were treated according to the ethical guidelines of the Animal Center of Yantai Yuhuangding Hospital (Yantai, China), and the Animal Studies Committee of the Shan Dong University approved all experimental procedures.

Following treatment with different concentrations of aloe emodin (1, 100 and 500 ng/ml) for 24 h, the cells were washed with phosphate-buffered saline (PBS; pH 7.2) and incubated in fresh medium for 4 days. The number of surviving cells was then indirectly determined using an MTT cytotoxicity assay (Sigma-Aldrich), according to the manufacturer's instructions. The absorbance was measured using a Multiskan EX ELISA plate reader (MTX Lab Systems, Inc., Vienna, VA, USA) at a wavelength of 570 nm, with a reference wavelength of 650 nm (20,21).

To examine the effects of aloe emodin and isolated resveratrol on the burn wound healing process, burn wounds were created on the backs of male BALB/c mice following anesthetization with an intraperitoneal injection of pentobarbital (50 µg/g; Hubei Nosk Chemical Co., Ltd., Hubei, China). Briefly, the hair on the backs of the mice was removed using a hair remover (Xiantao Topmed Nonwoven Protective Products, Co. Ltd., Hubei, China) under anaesthesia with pentobarbital, and the back was subsequently wiped with distilled water warmed to 37°C, followed by 70% ethanol. The back skin was subjected with a 100°C custom-made soldering iron tip (Shenzhen Kingdom Technology Co., Ltd. Guangdong, China) for 10 sec. A full-thickness excision of the burn skin wound was made in the dorsal skin by lifting a fold of skin at the midline and punching through two layers of skin using a sterile disposable biopsy punch (diameter 8 mm) on the same day. The mice were housed separately and provided with free access to a standard laboratory diet and water following the completion of the surgical procedure. Subsequently, the indicated quantities (100 mg ointment/mouse) of resveratrol [2×10⁻⁴ and 5×10⁻⁴% (w/w)] and aloe-emodin [1×10⁻⁸, 1×10⁻¹⁰ and 1×10⁻¹²% (w/w)] were layered on the burn wound surface for 19 consecutive days and covered with Opsite Flexigrid film dressing (Smith & Nephew, London, UK) to prevent infection or licking of the ointment samples. Clear Vaseline alone was applied to the control burn wound mice. On day 20, the mice were sacrificed by cervical dislocation and the skin of the wound area was removed. In the experiment of the effects of resveratrol on burn wound healing, a vehicle control group, which received treatment with clear Vaseline alone, and two treatment groups, treated with ointment containing 2×10⁻⁴ or 5×10⁻⁴% (w/w) aloe emodin (n=3 per group) were compared. In the experiments examining the effects of resveratrol, the number of animals in each treatment group were as follows: A vehicle control group treated with clear Vaseline alone (n=11); a 1×10⁻⁸% (w/w) aloe emodin-treated group (n=11); a 1×10⁻¹⁰ (w/w) aloe emodin-treated group (n=7); and a 1×10⁻¹²% (w/w) aloe emodin-treated group (n=7). The above topical applications were performed under anaesthesia with pentobarbital.

The backs of the mice were subjected to burn wounds using the same method as described above. A polyethylene filter pellet (~6 mm diameter; 2 mm thickness; Hebei Yuyin Trade Co., Ltd., Hebei, China) containing the above-mentioned quantities of aloe emodin and resveratrol was applied to the burn wound surface and covered with Opsite Flexigrid film dressing to prevent dislodging of the filter pellet. The filter pellets were removed after 1, 3, 5 and 7 days and replaced with fresh filter pellets. The control mice were treated with filter pellets containing 0.9% (isotonic) NaCl solution alone at the same time-points. On day 9, the mice were sacrificed and the filter pellets were removed prior to the addition of 200 µl PBS (pH 7.0) to the filter pellets. The solution was mixed for 10 min using a Vortex (vortex mixer-V8, 200–3,000 rpm/min; Seoulin Bioscience, Seoul, Korea). Following removal of the filter pellet, the mixture was centrifuged at 1,000 × g for 10 min at 4°C. The obtained cell pellets were resuspended in PBS.

The total number of leukocytes, including polymorpho-nuclear leukocytes and macrophages, were measured using a Vi-CELL XR Coulter cell counter (Beckman Coulter Inc., Brea, CA, USA), and the ratio of polymorphonuclear leukocytes to macrophages was determined using Giemsa-stained smear samples. Briefly, the blood samples were placed on clean slides, air dried, fixed in methanol and stained with Giemsa (Sigma- Aldrich). They were then observed under ×100 magnification.

For this assessment, seven mice were used for each treatment group, which consisted of a vehicle control group treated with 0.9% NaCl solution alone, and 1, 100, and 500 ng aloe emodin and resveratrol-treated groups. These experiments were also performed under anaesthesia with pentobarbital. Briefly, 100 mg tissue was rinsed with 1X PBS, homogenized in 1 ml of 1X PBS and stored overnight at −20°C. Two freeze-thaw cycles were then performed to break the cell membranes, and the homogenates were centrifuged for 5 min at 5,000 × g and 2–8°C. The supernatant was removed and assayed immediately. The expression levels of MCP-1, IL-1β and VEGF in the filter pellets were measured using mouse MCP-1, IL-1β and VEGF ELISA kits (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's instructions. The readings were taken at 450 nm using an ELISA reader (Thermo Fisher Scientific, Inc.).

Wound closure rate was assessed by tracing the wound 1, 2, 4, 8, 16 and 20 days post-wounding using transparency paper and a permanent marker. The time to wound closure was defined as the time-point at which the wound bed was completely re-epithelialised with new tissue. The wound area was measured by tracing the wound margin, and its surface area was calculated using Image J software (National Institutes of Health, Bethesda, MA, USA). The wound area was visually measured using an L×400 microscope (Labomed, Fremont, CA, USA) at ×10 magnification. The wound healing rate was calculated using the following formula: (Surface area of the original wound − surface area of the remaining wound)/surface area of the original wound × 100.

On days 4 and 10 of experimentation, seven mice were sacrificed from each group were sacrificed and the wounds were excised, together with the surrounding skin. The tissue samples were fixed in 10% neutral-buffered formalin (Shijiazhuang Xinlongwei Chemical Co., Ltd., Hebei, China) for at least 24 h, progressively dehydrated in solutions containing an increasing percentage of ethanol (70, 80, 95 and 100%), cleared in Histoclear (AS ONE Corp., Tokyo, Japan), embedded in paraffin (Raylabel Instrument Co., Ltd., Shanghai, China) under vacuum, sectioned (5 µm), de-paraffinised and stained with hematoxylin and eosin (Beijing Tidybio Science & Technology Co., Ltd., Beijing, China). The stained sections were examined for collagen, inflammatory cells and blood vessel markers of healing.

The data are expressed as the mean ± standard error of the mean. The results were analysed using one-way analysis of variance and Dunnett's multiple comparison test. Statistical analyses were performed using SPSS software version 16.0 (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The molecular structures of aloe emodin and resveratrol were similar to those reported previously (Fig. 1A and B). Treatment with either aloe emodin or resveratrol did not result in cytotoxicity in either the HaCaT cells or THP-1 macrophages at concentrations of 1, 100 and 500 ng/ml (Fig. 2). Furthermore, aloe emodin and resveratrol had no effect on the proliferation of the HaCaT cells or THP-1 macrophages.

Wound healing rates were measured on different days following wounding. In all groups, wound healing rates increased with increasing duration. Significant increases in wound-healing activity were observed in the mice treated with aloe emodin and resveratrol, compared with those that received control treatments. The effects of the treatments on the wound-healing activity levels in mice with excision wounds are presented in Tables I and II. In this model, the aloe emodin-treated mice exhibited a significant increase in wound healing rate (P<0.05), and a decreased time to epithelialization, compared with the control mice.

The wound injury area created by an iron bar heated at 100°C reached a maximum on days 6–8, and wound repair was subsequently observed. The burn wound area in the groups treated with the topical application of resveratrol at doses of 2×10⁻⁴ and 5×10⁻⁴% (w/w) decreased on days 8 and 19, compared with those in the vehicle-treated control mice (Fig. 3A–C). Previous studies have investigated the effect of natural products isolated from medicinal plants on skin regeneration in burn wound healing (22). The present study also demonstrated that the burn wound areas in mice treated with aloe emodin and resveratrol at doses of 1×10⁻⁸ and 1×10⁻¹²% were significantly (P<0.05) reduced after 6–18 days, compared with those of the control group, however, no significant difference was observed between the non-control treatment groups (Fig. 4A and B).

The levels of IL 1β, MCP-1 and VEGF in the exudates of vehicle-treated burn wounded control mice were analysed. The levels of IL-1β increased in a time-dependent manner over the 7 days following wound formation. It is well-known that the production of IL-1β by macrophages is stimulated by lipopolysaccharide. Aloe emodin (500 ng) significantly increased the level of IL-1β on days 1, 3, 5 and 9, compared with the level observed in the resveratrol (100 ng) and control groups (P<0.05) (Figs. 5A and B). At a dose of 1 ng, aloe emodin also increased the level of IL-1β on day 6. The migration of polymorphonuclear leukocytes was reduced by aloe emodin and resveratrol (1, 100 and 500 ng/ml) 1 or 3 days after treatment of the burn. The level of IL-1β production in the exudates of the burn wound area of treated mice increased time dependently 7 days after the tissue was wounded. The MCP-1 level in the exudates of the wound area of control mice reached a maximum level 1 day after burn treatment, and declined 3 days after burn treatment (Figs. 5C and D). The VEGF level in the exudates of the wound area of control mice increased until day 7. The application of asiaticoside at a dose of 100 ng/filter pellet increased the VEGF level on days 1 and 5 compared with that in control mice, and at a dose of 1 ng/filter pellet, it increased the VEGF level on day 9 (Figs. 5E and F).

H&E-stained sections of granulation tissue samples collected on days 4 and 10 following wound formation were examined for cellular infiltration, epithelial regeneration and matrix organization. Granulation tissue was collected on days 4 and 10 for H&E staining Histopathological analysis of the wounds on day 4 indicated increased cellular infiltration in the treated groups, compared with the control group, with no epidermal regeneration observed. After 10 days, the wounds exhibited steady and progressive wound healing in the control group. The eschar had separated, leaving space for the epidermis to grow to complete re-epithelisation. A reasonable level of collagen and numerous inflammatory cells were observed in the corium. The regeneration, stratification and polarity of the epithelial cells were markedly higher in the aloe emodin-treated burn wound, compared with the control. Fig. 6A and B shows the appearance of the mouse burn wound skin tissues at day 10 in the aloe emodin- and resveratrol-treated groups.