[Effects and mechanisms of cerium-myricetin nanosystem on wound healing in rats with full-thickness burns].

Abstract

Objective: To investigate the effects and mechanisms of cerium-myricetin nanosystem (Ce-MYR) on wound healing in rats with full-thickness burns. Methods: This study was an experimental study with a grouped design and repeated measurement design. Ce-MYR was prepared by coordination self-assembly and characterized. Mouse RAW264.7 cells were divided into control group cultured normally, hydrogen peroxide group treated with hydrogen peroxide, and low Ce-MYR group, medium Ce-MYR group, and high Ce-MYR group which were first exposed to hydrogen peroxide and subsequently treated with Ce-MYR at final mass concentrations of 5, 10, and 20 μg/mL, respectively. After 24 h of culture, intracellular reactive oxygen species (ROS) levels were detected using the fluorescent probe method. Mouse bone marrow-derived macrophages (BMDMs) were divided into control group cultured normally, lipopolysaccharide (LPS) group treated with LPS, and Ce-MYR group treated with LPS combined with Ce-MYR. After 24 h of culture, the percentages of CD86- and CD206-positive areas in cells were calculated after immunofluorescence staining. Additional mouse BMDMs were divided into the LPS group and the Ce-MYR group, as described above. After 24 h of culture, the protein expressions of heme oxygenase-1 (HO-1), arginase-1 (Arg-1), inducible nitric oxide synthase (iNOS), and NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) in cells were detected by Western blotting. The sample size in all the above experiments was 4. Ten 6-week-old male Sprague-Dawley rats were used to establish four full-thickness burn wounds on the back of each rat, which were divided into phosphate-buffered saline (PBS) group and Ce-MYR group according to the random number table method, with 5 rats in each group. PBS or Ce-MYR was subcutaneously injected at the wound margin at post-injury days 0 (immediately), 3, and 6, respectively. The percentages of residual wound area were calculated at post-injury days 3, 7, 14, and 21. At post-injury day 21, the wound tissue of rats was collected and the number of CD31-positive blood vessels was counted after immunohistochemical staining, the percentages of CD86- and CD206-positive areas were calculated after immunofluorescence staining, and the levels of interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay. Results: After 24 h of culture, the ROS level in RAW264.7 cells in hydrogen peroxide group was significantly higher than that in control group (P<0.05); the ROS level in RAW264.7 cells in medium Ce-MYR group was significantly lower than that in low Ce-MYR group (P<0.05) and significantly higher than that in high Ce-MYR group (P<0.05). After 24 h of culture, the percentage of CD86-positive area in BMDMs in Ce-MYR group was significantly lower than that in LPS group (P<0.05) and significantly higher than that in control group (P<0.05); the percentage of CD206-positive area in BMDMs in Ce-MYR group was significantly higher than that in control group and LPS group (with P values both <0.05). After 24 h of culture, compared with those in LPS group, the protein expressions of HO-1 and Arg-1 in BMDMs in Ce-MYR group were significantly increased (P<0.05), whereas the protein expressions of iNOS and NLRP3 were significantly decreased (P<0.05). At post-injury days 3, 7, 14, and 21, the percentages of residual wound area in rats in Ce-MYR group were (87.1±2.4)%, (65.1±2.2)%, (16.6±1.9)%, and (0.5±0.4)%, respectively, which were significantly lower than (101.5±3.5)%, (79.9±3.2)%, (36.2±3.9)%, and (14.5±1.9)% in PBS group (with t values of 7.604, 8.478, 10.193, and 16.166, respectively, P<0.05). At post-injury day 21, compared with those in PBS group, the number of CD31-positive blood vessels in wound tissue of rats in Ce-MYR group was significantly increased (t=3.395, P<0.05), the percentage of CD86-positive area was significantly decreased (t=4.474, P<0.05), the percentage of CD206-positive area was significantly increased (t=4.713, P<0.05), and the levels of IL-1β, IL-6, and TNF-α were significantly decreased (with t values of 3.999, 5.040, and 6.023, respectively, P<0.05). Conclusions: Ce-MYR may promote wound healing by reducing ROS levels, enhancing HO-1-related antioxidant responses in macrophages, thereby facilitating macrophage polarization from the M1 phenotype toward the M2 phenotype and improving oxidative stress and inflammatory imbalance in rats with full-thickness burn wounds.