Wound healing requires a prolonged period of time in diabetic rats. The results indicated that the time required to heal wounds was significantly decreased in diabetic rats supplemented with WP compared with diabetic rats treated with vehicle.
Discussion and conclusions
Wound healing begins immediately after injury and proceeds by well-organized integration and interaction between different types of cells and tissues. Wound healing is characterized by centripetal movement of the wound edge toward the center of the wound, producing wound closure. The process of wound healing can be divided into five phases – the cellular phase (granulation), narrowing of the wound area (wound contraction), collagen deposition (collagenation), epithelial covering (epithelialization) and scar remodeling (cicatrization). These phases are overlapping, and any agent that accelerates the process promotes wound healing [33, 34]. Diabetes mellitus delays wound healing by affecting these processes [35, 36].
Examination of the wounds in this study demonstrated that although diabetes mellitus slows wound healing, daily WP administration after wounding hastens wound closure in both diabetic and non-diabetic rats. These results are consistent with those of Ebaid et al. , who revealed that WP supplementation improved both the healing and closure of diabetic wounds.
The enhancement of wound healing in WP-treated diabetic rats was concomitant with a potential improvement in the glycemic state and alleviation of the lowered serum insulin levels. Furthermore, non-diabetic rats that were orally administered WP daily for 4 or 8 days exhibited an enormous increase in serum insulin levels consistent with enhanced wound closure, although serum fasting glucose levels were not significantly affected. These results are in concordance with previous publications [37, 38] indicating that WP has insulinotropic effects in non-diabetic and diabetic conditions. As reported in previous studies, WP contains many amino acids at varying concentrations . Most of these amino acids are likely to act as insulin secretagogues after WP supplementation. It is well known that amino acids stimulate insulin release from pancreatic β-cells both alone and in combination [39–41]. The mechanism by which amino acids induce insulin release from β-cells is complex, and several metabolic pathways are activated, depending on the type of amino acid [38, 40]. In addition to the insulinotropic action of amino acids, WP produced an increase in the plasma glucose-dependent insulinotropic polypeptide after ingestion . In addition to the stimulatory effects of whey protein on insulin secretion, the present study revealed that the administration of whey protein to normal and diabetic wounded rats enhanced the proliferation and multiplication of β-cells which seemed to be more granulated in the treated animals. Thus, this also provides evidence that support the hypothesis that whey protein increased insulin levels in both normal and diabetic rats.
The direct positive effect of insulin on the enhancement of wound healing has been unclear in previous publications. However, many studies [42–44] found that insulin treatment by topical application or by injection accelerated wound healing. By contrast, Weringer et al.  revealed that there was no detectable difference in the duration of the healing response in either insulin-treated or non-treated diabetic mice.
Hyperlipidemia with or without diabetes mellitus may impair wound healing [46, 47]. The diabetic rats in the present study exhibited a profound elevation in total cholesterol, triglycerides, LDL-cholesterol and vLDL-cholesterol levels. The cardiovascular risk indices, represented by the ratios of total cholesterol levels and LDL-cholesterol to HDL-cholesterol levels, were also remarkably increased. These changes may cause deterioration of the process of healing. Sen et al.  reported that re-epithelialization was reduced in hypercholesterolemic mice. Jang et al.  observed that hypercholesterolemia is associated with angiogenic impairment, which in turn delayed wound healing. WP treatment of wounded diabetic rats produced marked amelioration of the tested serum lipids and the calculated cardiovascular risk indices, which may be secondary to the improvement of serum insulin level. Based on our results and previous publications, it can be concluded that the alleviation of serum lipids and cardiovascular risk indices may be involved, at least in part, in the enhanced wound healing observed in diabetic rats treated with WP.
In our previous study , the early changes in inflammatory cytokines (IL-1β, TNF-α and IL-6) during the acute phase of the inflammatory response and the effect of WP were investigated at 6 and 24 h after wounding. The increase in these inflammatory cytokines as a result of neutrophil infiltration during the early phase after wounding is particularly essential for debridement and the clearing of infection by the absorption of wound exudates [6, 48]. Neutrophils, which are the first cells to arrive at the wound, eliminate microorganisms and then undergo apoptosis. Afterward, neutrophils are rapidly and efficiently consumed by macrophages in a process that does not lead to further inflammation . In the present study, we assessed the effect of WP on these inflammatory cytokines as well as on the anti-inflammatory cytokine IL-6, but during later periods, at 2, 4 and 8 days after wounding in normal and diabetic rats.
The serum TNF-α level was moderately increased in non-diabetic rats as a result of wounding, but it was strongly elevated in the wounded diabetic rats 4 days after wounding. The serum concentration of IL-1β was significantly increased in wounded non-diabetic rats on day 2 and 8 but was not significantly affected in wounded diabetic rats compared with the corresponding uninjured control. The serum IL-6 level was significantly increased in wounded non-diabetic rats, but it was not significantly affected in the wounded diabetic group. However, levels of the anti-inflammatory cytokine IL-10 in serum were significantly decreased in wounded non-diabetic rats but were not significantly affected in wounded diabetic rats. From these contrary results, it can be concluded that the deterioration of both inflammatory and anti-inflammatory cytokines in diabetic rats may play a crucial role in the delay of wound healing in diabetes mellitus.
WP treatment of wounded animals in this study did not significantly affect serum TNF-α, IL-6 and IL-10 levels after 2 days. When the treatment period was extended to 4 days, the levels of the inflammatory cytokines TNF-α, IL-1β and IL-6 decreased. The decrease in IL-1β and IL-6 levels continued as the treatment progressed for 8 days in both wounded non-diabetic and diabetic rats treated with WP, whereas the TNF-α concentration was unaffected in treated wounded diabetic rats after 8 days. The concentration of the anti-inflammatory cytokine IL-10 in serum was significantly increased after treatment with WP for 4 days, but it was not significantly altered after 8 days of treatment in both wounded non-diabetic and diabetic rats. Thus, it can be suggested that WP initially and acutely evokes the pro-inflammatory and inflammatory response at 6 and 24 hours  and then hastens the switch from inflammatory to anti-inflammatory responses during the process of wound healing. These findings agree with Peranteau et al. , who reported that overexpression of IL-10, an anti-inflammatory cytokine, decreases the inflammatory response to injury and creates an environment conducive to regenerative wound healing. Therefore, WP improved wound healing in diabetic mice because it significantly decreased the elevated pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and increased IL-10 in the plasma and wound tissues. Based on these previously described ideas, it can be concluded that WP limited prolonged inflammation and modulated the immune response during the progress of wound healing in both normal and diabetic animals.
An increase in free radicals and diminished antioxidant activity may worsen the situation and account for the delay in wound healing in diabetic patients. In the present study, liver lipid peroxidation, which is an indicator of oxidative stress, was markedly increased in wounded non-diabetic and diabetic rats compared with the corresponding uninjured animals. This result is in accordance with Rosenbaum et al. , who suggested that oxidative stress, regardless of its source, induces cellular dysfunction in endothelial and smooth muscle cells and reduces angiogenesis and the healing process.
In the present study, the treatment of wounded diabetic rats with WP profoundly decreased the elevated lipid peroxidation and improved the antioxidant defense system by increasing hepatic glutathione levels and the activity of glutathione-S-transferase, glutathione peroxidase and SOD. These results agree with those of Ebaid et al. , who discovered an increase in GSH level and a decrease in reactive oxygen species (ROS) and hydroperoxide in the wounded tissue of diabetic rats after treatment with WP. This suggests that WP may improve wound healing, at least in part, by the modulation of oxidative stress and the antioxidant defense system.
In conclusion, WP hastens the wound healing process in diabetic rats by limiting prolonged inflammation, improving hyperglycemic and hyperlipidemic states, increasing insulin level, and amelioration of oxidative stress and the antioxidant defense system. However, we continue to perform further studies to assess the effect of WP on the phases of wound healing, including granulation, collagenation, epithelialization and cicatrization at the cellular and molecular levels.