Effect of small molecular weight soybean protein-derived peptide supplementation on attenuating burn injury-induced inflammation and accelerating wound healing in a rat model

Abstract

The populations most afflicted by burn injuries have limited abilities to support the significant specialized requirements and costs for acute and long-term burn injury care. This article describes the results of optimizing the use of readily absorbed small molecular weight soybean protein enzymolysis-derived peptide to attenuate rat burn injury-induced inflammation and accelerate wound healing. A major full-thickness 30% total body surface area burn-injury rat model was utilized and the systemic white blood cell (WBC) counts, the relative level of stimulation index of respiratory burst, and the inflammatory markers procalcitonin (PCT), tumor necrosis factor-α (TNF-α), chemokine (C-C motif) ligand 3 (CCL-3), chemokine (C-C motif) ligand 11 (CCL-11) and interleukin-10 (IL-10) were assessed. The burn injury-induced neutrophil and macrophage immune cell infiltration of the cutaneous tissues was detected by immunohistochemical analysis of the protein markers myeloperoxidase (MPO) and cluster of differentiation 68 (CD-68). The local induction of the burn injury-induced toll-like receptor 4/nuclear factor kappa-light-chain-enhancer of activated B (TLR4/NF-κB) signaling pathway in the effected cutaneous tissues was determined by the quantification of the protein expression of TLR4 and phosphorylated NF-κB/p65 using Western blots. In addition, burn wound size and healing rate were assessed biweekly for 8 weeks by imaging and measuring the burn wound surface area, and the angiogenesis protein marker of cluster of differentiation 31 (CD-31) expression in cutaneous tissues was also detected by immunohistochemical analysis. The results showed that nutrient supplementation with optimized readily absorbed small molecular weight soybean protein-derived peptide resulted in a dramatic anti-inflammatory effect as evidenced by the significant increase in the burn injury-induced systemic white blood cell counts and their relative level of stimulation index of respiratory burst, reduction in the burn injury-induced activation of NF-κB transcriptional signaling pathways, significant reduction in the local burn injury-induced cutaneous infiltration of neutrophils and macrophages at all measured time points, reduction in wound size and improved rate of burn injury wound healing with increased CD-31 protein expression. These results indicated that dietary supplementation with small molecular weight soybean-derived peptides could be used as an adjunct therapy in burn injury management to reduce inflammation and improve overall patient outcomes.

Fig. 1. Soybean protein-derived peptides supplementation regulates burn-induced leukocytosis. (A) WBC counts of sham experimental group rats administered either PBS or soybean protein-derived peptides (sham + PBS and sham + peptides, respectively) and 30% TBSA burn-injured rats administered either PBS or soybean protein-derived peptides (burn + PBS and burn + peptides, respectively), were determined 3, 7 and 14 days post-burn injury. (B) Induction of PMA-induced respiratory bursts in sham + PBS, sham + peptides, burn + PBS and burn + peptides experimental groups were also determined at 3, 7 and 14 days post-burn injury using flow cytometry detection of oxidation to fluorescent positive cells as compared to baseline fluorescence intensity in the same samples not treated with PMA. The unstimulated fluorescence was represented by the red histogram, while the PMA-stimulated oxidative burst in neutrophils was represented by the blue histogram as positive neutrophil counts. (C) The stimulation index (mean fluorescence intensity after PMA stimulation/mean fluorescence intensity of control) was measured. The star symbol (*) indicates a significant difference between sham experimental group administered PBS and the ‡ symbol represents a significant difference between burn-injured experimental group administered PBS (ANOVA, p < 0.05).

Fig. 2. Soybean protein-derived peptides supplementation produced anti-inflammatory systemic burn injury-induced cytokine profile. Induction of systemic inflammatory markers were measured in sham experimental group rats administered either PBS or soybean protein-derived peptides (sham + PBS and sham + peptides, respectively) and 30% TBSA burn-injured rats administered either PBS or soybean protein-derived peptides (burn + PBS and burn + peptides, respectively) at 3, 7 and 14 days post-burn injury. (A) Serum PCT levels were determined for each rat in each treatment group at the specified time-points using standard ELISA assay measurements. Inflammatory plasma protein cytokine levels were determined for each rat in each treatment group at the specified time-points using standard chemokine/cytokine Luminex™ assay measurements including (B) pro-inflammatory factor TNF-α, (C) chemokine CCL-3 (also known as MIP-1α), (D) chemokine CCL-11 (also known as eotaxin) and (E) anti-inflammatory cytokine IL-10, were shown. The star symbol (*) indicates a significant difference between sham experimental group administered PBS and the ‡ symbol represents a significant difference between burn-injured experimental group administered PBS (ANOVA, p < 0.05).

Fig. 3. Soybean protein-derived peptides supplementation inhibited local burn injury-induced inflammatory immune cell infiltration. Induction of local inflammatory immune cell infiltration of neutrophils and macrophages at the site of burn injury was evaluated in cutaneous fixed tissue section slices by immunohistochemical analysis using the (A) neutrophil cellular marker MPO and the (B) macrophage cellular marker CD-68 in sham experimental group rats administered either PBS or soybean protein-derived peptides (sham + PBS and sham + peptides, respectively) and 30% TBSA burn-injured rats administered either PBS or soybean protein-derived peptides (burn + PBS and burn + peptides, respectively) 3 days post-burn injury. And the enlargement scale of each image was 400 times. The quantitative analysis of MPO (C) and CD-68 (D) in different groups was measured by Image Pro Plus software.

Fig. 4. Soybean protein-derived peptides supplementation reduced burn injury induction of TLR-4/NF-κB inflammatory signaling pathway. Induction of the TLR-4/NF-κB inflammatory signaling pathway at the site of burn injury was evaluated in total protein extract of cutaneous tissue by western blot analysis following protein concentration determinations. (A) Equal total protein concentrations of each sample were loaded for gel-electrophoresis and protein expression levels were detected using TLR-4, β-actin, phospho-NF-κB p65/RELA and total NF-κB p65/RELA specific antibodies followed by appropriate secondary antibody via chemiluminescent evaluation in sham experimental group rats administered either PBS or soybean protein-derived peptides (sham + PBS and sham + peptides, respectively) and 30% TBSA burn-injured rats administered either PBS or soybean protein-derived peptides (burn + PBS and burn + peptides, respectively) 3 days post-burn injury. (B) Image J software densitometry measurements were used to quantify the western blot analysis protein expression and analyzed using Wilcoxon signed-rank test and relative protein expression levels were calculated normalizing TRL-4 expression to that of β-actin and phospho-NF-κB p65/RELA to that of total NF-κB p65/RELA. The star symbol (*) indicates a significant difference between sham experimental group administered PBS and the ‡ symbol represents a significant difference between burn-injured experimental group administered PBS (ANOVA, p < 0.05).

Fig. 5. Soybean protein-derived peptides supplementation promotes burn injury wound healing. (A) Injury area and wounds resulting from 30% TBSA burn injury were photographically captured in rats administered either PBS or soybean protein-derived peptides (burn + PBS and burn + peptides, respectively) immediately before burn trauma induction (week 0), 2, 4, 6 and 8 weeks post-burn injury. Quantitative scalar measurements of the burn wound area were measured (cm²) using Image-Pro Plus 5.1 software. (B) Relative burn wound extent (% wound area of total burn injured-area) was calculated as the absolute wound area (cm²) at either 4, 6 or 8 weeks post-injury/total approximated initial burn-injured area (cm²) × 100; (C) relative burn wound healing (% healed wound area of total wound) was calculated as (initial 4 week wound area (cm²) − wound area at a either 6 or 8 weeks post-injury (cm²)/initial wound area (cm²)) × 100; (D) exponential wound healing model wound closure predictions were projected. (E & F) CD-31, the marker of angiogenesis, was used to reveal the angiogenesis of burn wound (E) and quantitative analysis was done (F) at indicated groups. The star symbol (*) indicates a significant difference between burn injured rats administered PBS versus soybean protein-derived peptide (Student's t-test; p < 0.05).