The present study demonstrates that topical administration of Delonix regia galactomannan in solution enhances the healing of excisional cutaneous wounds in mice, accompanied by the following events: 1) reduction of edema, hyperemia, nociception and inflammatory cell infiltrate; 2) increased number of fibroblasts/myofibroblasts; 3) increased deposition of type I collagen; 4) modulation of oxidative stress markers and cytokines expression.
Healing wounds occurs as overlapping phases including homeostasis, inflammation, proliferation and remodeling [1], but an imbalance during the inflammatory phase may cause either prolonged healing or excessive scar formation [25, 26]. Phlogistic signs of edema and hyperemia are found early after the induction, and are decurrent from pro-inflammatory cytokines released by infiltrating leukocytes, mainly neutrophils and macrophages, in the wound bed which also produce reactive species in order to prevent bacterial infection [26], being such signs reduced by GM-DR. Additionally, D. regia galactomannan inhibited both IL-1β and IL-6 expression, and TNF-α immunostaining also seemed to be diminished, although without reaching statistical significance. In view of this inhibition, it is suggested that GM-DR would be responsible for reducing leukocyte infiltrate and inflammatory signs of inflammation by the modulation of pro-inflammatory cytokines such as IL-1β and IL-6.
The present results were similar to previous obtained with different polysaccharides, such as those extracted from Caesalpinia ferrea barks in the same murine model [21], but are in apparent contradiction with those obtained from mice wounds after topical administration of a galactomannan extracted from Caesalpinia pulcherrima, which promoted IL-1β and IL-6 release [18]. It is known that proteins dragged during the galactomannan extraction may induce local inflammantion [10]. The extractive procedure of C. pulcherrima is similar to ours, except for an alkaline hydrolysis performed before the final precipitation. Such step was added to our procedure in order to remove remaining proteins without rupture of glycosidic bonding. Although we have used a different approach to determine % protein, its detection limit is similar to their colorimetric procedure (0.5%), as inferred from the original report of this technique [27]. Even considering the structural differences between theses galactomannans, as molar mass and distribution profile of side galactosyl groups, it is not to be excluded that low levels of remaining proteins would actually favored inflammation evocated by C. pulcherrima galactomannan on excisional wounds.
In our study, the reduction in the mechanical threshold applied on the wound edges was reversed by D. regia galactomannan as early as 6 hours after treatment. The inhibition of pro-inflammatory cytokines such as IL-1β and IL-6 and, consequently, the reduction of migration of these inflammatory cells may justify the antinociceptive effect of GM-DR. Our group had already demonstrated the antinociceptive effect of the polysaccharide extract of Caesalpinia ferrea stem barks topically applied to cutaneous wounds in mice probably by the reduced expression of IL-1β [21], and that polysaccharides extracted from Ximenia americana barks reduced peripheral hypernociception induced by carragennan in mice [28].
Crust detachment and scar formation were anticipated in the animals treated with D. regia galactomannan. GM-DR promoted healing by second intention and retraction of the cutaneous wound, being verified by the reduction of the area and increase of the wound index in the proliferative phase. It is documented that the wound contraction, a feature of the proliferative phase of the physiological healing process, involves the participation of differentiated myofibroblasts [29]. Myofibroblasts are characterized by the expression of a particular integrin smooth muscle alpha actin (α-SMA), which determines its contractility, and the increased synthesis of MEC proteins, such as collagen types I and III [30]. The treatment with GM-DR evoked the expression of α-SMA. Thus, it is suggested that this galactomannan may act in the proliferative phase inducing the differentiation of fibroblasts into myofibroblasts. In agreement with our study, the polysaccharides extracted from the leaves of Plantago australis at 500 and 1000 mg/kg increased the wound healing index seven days after the excision, as well the proliferation of keratinocytes in a horizontal migration model, modulating the levels of TNF-α [31]. In addition, galactomannans extracted from Cydonia oblonga increased tissue elasticity and the healing rate at higher concentrations (10–20%) [32] and that from C. pulcherrima increased the wound healing rate in the later stage of the healing process, at the day 10 [18]. The galactomannans of Cassia grandis seeds also increased the retraction of the wound and reduced the infiltration of inflammatory cells from the 3rd day of treatment [6].
The most important feature of the proliferative phase is the formation of granulation tissue (due to the granular appearance generated by the newly formed capillaries) proliferation and migration of fibroblasts to the lesion (fibroplasia) [33] under stimulation of TGF-β, fibroblasts differentiate in myofibroblasts [29], contributing to wound retraction. GM-DR increased the number of fibroblasts/myofibroblasts, which was accompanied by greater expression of α-SMA from the 7th to the 14th day, reinforcing the regulator mechanism of this growth factor, in order to accelerate the formation and maturation of the granulation tissue in the proliferative phase and an effective and coordinated tissue repair.
The profuse degradation of extracellular matrix (MEC) and type III collagen, along with formation of mature type I collagen are critical in this phase, which lasts some months and years until the formation of a paucicellular scar [30, 34]. Although total collagens had not been altered by GM-DR, the deposition was increased for type I collagen, but it was decreased for type III, which indicates an effect in the proliferative phase. GM-DR may also accelerate the production of granulation tissue, as this is correlated with the maturation of the newly formed tissue, as well as with the increase in the healing rate observed since the 7th day of treatment.
However, if the inflammatory infiltrate persists, it will hamper the proliferation of fibroblasts and vascular neoformation, contributing to the deficiency in the formation of granulation tissue [25]. Thus, the inhibitory effect of GM-DR on the inflammatory leukocytes infiltrate was accompanied by proliferation and migration of fibroblasts. According Mimosa tenuiflora polysaccharide promoted in vitro fibroblast stimulation [35] and C. pulcherrima galactomannan increased fibroplasia associated with collagen deposition at the 14th day post-ulceration [18].
Oxidative stress markers is often a secondary physiological event associated to inflammation, as evidenced cell stress under persistence dense infiltrate of inflammatory cells. In contrast, the inhibition of oxidative stress by superoxide dismutase, catalase or reduced glutathione prevents cell damage [25]. GM-DR reduced the levels of MDA at day 2, as well increased that of reduced glutathione at days 2 and 5, while reduced the polymorphonuclear infiltrate. Similar effects on polymorphonuclears and MDA were obtained C. ferrea polysaccharides treatment [21] or with the aqueous extract of Ocimum sanctum, which reduced MDA concomitant to increased activity of superoxide dismutase and catalase, and increased levels of reduced glutathione [36].
Although GM-DR had stimulated fibroplasia and collagen deposition, there was no increase in the number of blood vessels. It is known that neoangiogenesis is important to supply oxygen and nutrient demand of cells throughout the healing process. However studies have already shown that mild angiogenesis, but forming structured vascular network, favors the formation of adequate scar [37]. In contrast to that observed in our study, it was demonstrated that oral administration of an ethanolic extract of polysaccharides from the root of Sanguisorba officinalis L. in mice resulted in the acceleration of angiogenesis, via VEGF production [38], and skin wounds treated with C. pulcherrima galactomannan had a more pronounced neoangiogenesis when compared to controls [18]. Further studies involving specific markers of newly formed vessels, such as CD-31 and 34, may be useful to elucidate that discrepancy.
Delonix regia galactomannan (GM-DR) promotes tissue repair in mice excisional cutaneous wounds acting as anti-inflammatory via inhibition of cytokine pro-inflammatory (IL-1β, IL-6) and healing by stimulation fibroplasia and collagenesis via increase of TGF-β.