Compounds AE (molecular weight, 959.12 g/mol; molecular formula, C₄₈H₇₈O₁₉) and MA (molecular weight, 975.12 g/mol; molecular formula, C₄₈H₇₈O₂₀) were obtained from Sigma-Aldrich (Shanghai, China). RPMI-1640 medium and Dulbecco's modified Eagle's medium, were acquired from Sigma-Aldrich China, Inc., (Shanghai, China). Supplementary agents, including antibiotic/anti-mycotic cocktails containing 10 mg/ml streptomycin, 10,000 U/ml penicillin as well as 25 µg/ml amphotericin B dissolved in 0.9% NaCl solution, were also procured from Sigma-Aldrich. The 6-, 12- and 48-well plates used in the present study were obtained from Corning, Inc. (Corning, NY, USA).

Male Sprague-Dawley (SD) rats (n=150; weight, 250–300 g) were acquired from the National Laboratory Animal Center, Hospital of Taishan Medical College (Taishan, China). The rats were housed independently in cages at 25±1°C, with access to food pellets and water ad libitum. A period of 7 days was allowed for the acclimatization of the rats. The in vivo work is reported in accordance with the Animal Research: Reporting of In Vivo Experiments guidelines (31). The present study was approved by the Ethics Committee of Taishan Medical College.

Administration of AE and MA and the production of burn wounds was performed as previously described (32,33). Prior to the production of the lesion, the SD rats were anesthetized by intraperitoneal injection with sodium pentobarbital (60 mg/kg body; Sigma-Aldrich). By using lower portions of scapula, the back regions of the animals were then shaved and depilated. Subsequently, a burn wound of partial thickness was created by placing a 3.5×4.6-cm hot plate (75°C) on the targeted location for 10 sec, and ~10% of the entire body surface region was targeted for this purpose. Upon wound induction, 500 µl test compound (MA or AE) was applied topically to the wound once daily. Using millimeter-scale graph paper, the wound injury was examined every 2 days, on days 2, 8, 10 and 14. Upon reaching the end of the assessment period, the SD rats were sacrificed by overdose with sodium pentobarbital (intraperitoneal injection; 100 mg/kg body weight). For the purpose of histological examination, the tissues of healed wounds with dimensions 0.5×0.5 cm were obtained from individual rats based upon their respective groups. For the control group, a topical application of Vaseline (Sigma-Aldrich) alone was performed.

Upon burn injury induction, the wounds were assessed grossly on days 2, 8, 10 and 14. The following parameters were assessed: Swelling of the wound surface and wound bed, the presence of exudates and coloration and consistency of the tissues surrounding the wounds.

After the induction of burn injuries, color images of lesions were obtained on days 2, 8, 10 and 14 post-treatment using a digital camera. Using a permanent marker pen and millimeter-scale transparent graph paper, the wound dimensions were tentatively measured and accordingly sketched on the trace paper. With the data collected, the wound healing properties were numerated by utilizing the following formula:

Degree of wound healing (%) = 1-[wound area on the corresponding day (cm²)/wound area on day zero (cm²)] × 100

Tissue specimens with dimensions of 0.5×0.5 cm were isolated from the burnt area at the midriff sections. The tissues were then kept in freshly prepared, neutral buffered solution with 10% formaldehyde for a period of 24 h. Next, staining using hematoxylin and eosin were performed on the sections and further analyzed under a light microscope (Nikon 516609; Nikon Corporation, Tokyo, Japan) at objective magnifications of ×20 and ×40.

The human acute monocytic leukemia cell line (THP-1) was procured from American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI-1640 basal medium with supplementations of 10% fetal bovine serum (Thermo Fisher Scientific, Inc.), penicillin (100 U/cc), streptomycin (100 µg/ml) and amphotericin (0.25 µg/ml).

For the quantification of cell proliferation and viability, an MTT assay was performed (Sigma-Aldrich) according to the manufacturer's protocol. In the assay, typically only viable cells reduce MTT into insoluble purple formazan and the intensity of purple color directly correlates to the number of viable cells. The THP-1 cells were cultured on 96-well microtiter plate with seeding density of 5×10³ cells/well containing 100 µl RPMI media for a period of 48 h. The media was changed after 48 h and replaced with basal media supplemented with various concentrations of MA and AE: 1,000, 500, 250, 125, 62.5, 31.2, 15.6 and 7.8 ppm. The cells were then incubated in a humidified chamber with an atmosphere of 5% CO₂ at 37°C. Media was replaced as required. Evaluation of viable cells was initiated by the addition of 100 µl MTT solution into each well and incubating at 37°C in the dark for 4 h. Crystals formed during the incubation period were then dissolved using 100 µl of MTT organic solvent (prepared in 0.1 N HCl dissolved in anhydrous isopropanol). Subsequently, optical density was measured by means of an enzyme-linked immunosorbent assay (ELISA) reader, and was quantified at 570 nm with background subtraction at 690 nm (34,35). This was performed on days 1, 4 and 7 post-treatment.

Seeding density of THP-1 cells were fixed at 2×10⁵ cells/per well in 100-mm culture plates and subsequently maintained for a period of 48 h. Upon reaching 80% confluency, the cells were exposed to 10 pg/ml, 10 ng/ml or 100 ng/ml AE or MA for ≤24 h. The levels of VEGF and MCP-1 were detected respectively using ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer's protocol.

Data are presented as the mean ± standard error of mean. Statistical analyses were performed using SPSS software, version 21 (IBM SPSS, Armonk, NY, USA). The differential expressions among experimental groups were assessed using one-way analysis of variance, followed by Fisher's least significant difference test. P<0.05 was considered to indicate a statistically significant difference.

Compounds AE and MA (Fig. 1) were applied to treat burn wounds established in a rat model of burn wound healing. Following day 2, it was noted that wounds in the untreated, Vaseline-control group were swollen and bruised. Conversely, a modest level of swelling were observed in all AE- and MA-treated groups. The majority of injuries in the AE and MA groups began to heal and formed fibrous tissue. Notably, on day 8 the wounds in the untreated control, AE and MA groups were dark red in color, displayed wound size decrement at the injury site and remained unchanged in terms of physical magnitude. Wound contraction was also observed in all treatment groups compared with the control groups (data not shown).

The lesions in the AE- and MA-treated groups showed marked hair growth, and scabs were noted to be covering their respective lesion openings. Importantly, all wounds in the control, AE- and MA-treated groups exhibited a moderate level of exudation with no hair growth and scabs on top of lesion openings on day 10. Furthermore, the incidence of a dry surface, augmented hair development and progressive contraction of wound were noticeably present in all cases, in particular in the MA- and AE-treated groups. On the final day of the study (day 14), all burn wounds in the AE and MA groups, as well as the untreated group, exhibited marked levels of exudation and scab loss. In addition, increased hair formation was observed at the lesions in the AE- and MA-treated groups, which indicated that healing was taking place. Overall, the lesions in the AE- and MA-treated groups were significantly reduced in size after day 10 (Table I).

On day 14, the control group displayed distinct fibrinoid necrosis in the subepidermal area, as demonstrated by the presence of collagen and fibrin, as well as evidence of degenerative changes. Furthermore, due to only minor skin outgrowth, re-epithelialization was not achieved and exudates were shown to cover the wound surface. Fibrinoid necrosis in selected areas of the subepidermis were noted, particularly in the wounds of the AE and MA groups. Furthermore, edema was detected in interstitial zone near the dermal region of the AE and MA groups. Re-epithelialization was incomplete; however, the lesions in the AE and MA groups appeared to be more severe compared with the control group (Fig. 2).

The proliferation of THP-1 cells was measured following exposure to different concentrations of AE and MA at days 1, 4 and 7 post-treatment. Generally, it was observed that the cell viability increased in accordance with culture duration in all culture circumstances, except in cultures supplemented with 1,000 ppm AE and MA (Fig. 3). These results suggest that AE and MA at concentrations of ≤500 ppm have no severe effects on the viability and proliferative ability of THP-1 cells in vitro. An anti-proliferative effect on THP-1 cells was only observed at the higher concentration of 1,000 ppm AE or MA (Fig. 3).

As shown in Fig. 4, the levels of MCP-1 were significantly increased in THP-1 cells following treatment with 100 ng/ml AE for 6 and 12 h (P<0.05), as compared with the Vaseline-treated cells. After 12 h, MCP-1 expression became undetectable due to cell death leading to an insufficient number of cells. Furthermore, the levels of VEGF were significantly increased in THP-1 cells following treatment with 100 ng/ml MA for 6, 12, 24, 36 and 48 h (P<0.05; Fig. 4). However, the effect of MA and AE on VEGF production was not significant at day 7 post-treatment (Table II).