A skin wound can be defined as a disruption or break of the skin barrier. Acute wounds are normally resolved in a timely manner, whilst chronic ones present with slow healing phases. Depending on the lesion extension, a wound can be classified as clean/simple (when there is minimal loss of tissue and healing occurs within 48 h after lesion) or complicated (when there is the loss of a large tissue area and a slow healing process is present). Healing [1
] involves vascular (vasoconstriction followed by vasodilation) and inflammatory responses; the latter are characterized by plasma extravazation and leukocyte influx to the site of injury. Then, healing progresses into a proliferation phase in which connective tissue and novel vessels (granulation tissue) are formed; during this phase, the wound contracts and closes, forming a scar. Finally, during the maturation phase, the blood flow reduces and the scar is remodelled, becoming stronger by collagen deposition.
Skin wounds, especially those of a chronic or complicated nature, represent a major cause of morbidity and mortality, particularly in lower extremities. Wounds can become infected by bacteria, especially in patients in intensive care units and patients with different morbidities, including diabetes and poor skin perfusion [1
]. Infection may result in a biofilm-containing non-growing bacteria encased in a mucoid coat that stimulates inflammation [1
]. In this context, the inflammation-induced vascular leakage provides nutrients to the bacteria. Therefore, both the bacteria and the increased host vascular permeability contribute to delaying wound healing, resulting in a chronic wound phenotype [1
]. Indeed, re-epithelialisation of the wound cannot occur until biofilm-induced inflammation is removed.
Although the management of wounds costs billions of dollars yearly, there is no universally effective method for their treatment [1
]. Current clinical interventions include surgical debridement of lesions, complex dressings including alginate, foams, and silver, and hyperbaric oxygen [1
]. Of importance, systemic antibiotics and topical antimicrobials may be administered when bacterial infection is present; however, they are of limited efficacy as, in this scenario, bacteria are not reproducing and the biofilm limits exposure to antimicrobials.
is a biofilm-forming bacterium frequently detected in skin wounds, especially in deeper regions of the wound beds [4
]. Of note, wounds infected by this microorganism are characterized by larger areas of lesion and a delayed healing process [6
]. This, associated with the fact that P. aeruginosa
presents both intrinsic and acquired antibiotic resistance [7
], makes the clinical management of wounds infected by this pathogen a great challenge. Therefore, there is a great unmet need for inexpensive agents that can disrupt P. aeruginosa
biofilm and, at the same time, promote wound healing.
In this context, plant-derived compounds have potential as both antimicrobial and healing agents. Cinnamaldehyde, the major compound of the essential oil from Cinnamomum
sp. stem barks, is well known for its ability to increase skin blood flow and for its antimicrobial properties against different bacteria including P. aeruginosa
. These properties have been shown in different studies [9
]; however, there are few reports of its healing effects [17
A study by Takasao et al. [17
] showed that the in vitro incubation of cinnamaldehyde with human skin fibroblasts induces collagen synthesis. More recently, this compound was found to stimulate human endothelial cell proliferation in vitro [18
]. The same study demonstrated that the systemic administration of cinnamaldehyde in rats accelerates the healing of cutaneous wounds by inducing angiogenesis in the wounded area; however, the topical effects of this compound have not yet been addressed.
Herein, we investigated the in vitro antimicrobial actions of cinnamaldehyde against P. aeruginosa strains. The in vivo healing potential of the topical application of cinnamaldehyde on skin excision wounds infected or not with P. aeruginosa, as well as the mechanisms involved in this response, were also investigated in mice.
Skin wound healing can be impaired or delayed by the colonization of the wounds by microorganisms such as P. aeruginosa
, commonly resistant to the available antibiotic therapy [8
]. In this context, an ideal therapy for infected wounds should not only inhibit the pathogen, but also present healing activity.
Herein, cinnamaldehyde was antimicrobial against P. aeruginosa
strains, including those with a multidrug resistance phenotype; and it also attenuated bacterial virulence. These findings are supported by recent evidence on that this compound at MIC/2, disrupts pre-formed biofilms of P. aeruginosa
through inhibition of intracellular signalling processes involved in the control of biofilm formation by this pathogen [15
]. Similarly, a cinnamaldehyde-enriched oil exhibited anti-biofilm activity equivalent to that found in our study [20
]. Of note, anti-biofilm strategies have been considered interesting novel therapeutic approaches to prevent or disrupt biofilms in persistent infections by P. aeruginosa
Biofilm formation is an important mechanism of bacterial colonization of skin wounds [24
]. Therefore, we next explored the effects of the topical application of cinnamaldehyde on P. aeruginosa
-infected skin wounds in mice. Daily topical application of cinnamaldehyde reduced the load of P. aeruginosa
in skin wounds and also promoted faster healing of these wounds. Overall, cinnamaldehyde did not affect the area of the wounds not infected by P. aeruginosa
, although a healing response was observed one day earlier in these mice in comparison with vehicle controls. In addition, this compound did not affect the number of other bacteria colonizing these lesions. These results suggest that cinnamaldehyde healing effects may dependent on the pathogen present in the wounds, as different mechanisms may be involved in the host responses to such infections. Of note, the systemic administration of cinnamaldehyde accelerated skin wound healing rats [18
], suggesting that the route of administration of this compound may also interfere with healing.
TRPA1 is a well-documented target of cinnamaldehyde as an agonist [25
]. It is a member of the transient receptor potential (TRP) family expressed on neuronal and non-neuronal cells and it has been pointed as a key mediator of skin perfusion and also as a sensor of bacterial infection [9
]. Interestingly, TRPA1 activation in vitro induced the mRNA expression of genes involved in the control of keratinocyte proliferation and differentiation [29
]. Our data demonstrated that the repeated systemic administration of HC-030031 prevents cinnamaldehyde-induced healing in mice infected with P. aeruginosa
. Of note, the studies on TRPA1 as a bacterial sensor are few and have mainly concentrated on E. coli
]; thus, we present herein the first evidence that TRPA1 mediates host–P. aeruginosa
interactions in vivo.
Interestingly, mice administered with HC-030031 that were not infected with this pathogen presented larger lesions in comparison with the control group; this suggests that the endogenous activation of TRPA1 is important to healing, even in the absence of P. aeruginosa
. Few reports have assessed the role of TRPA1 in wound healing. Yang and collaborators [30
] indicated that burn patients with broader skin lesion areas express increased levels of TRPA1. It was also shown that the loss of TRPA1 signalling reduces inflammation and improves corneal healing in mice with chemical burns [31
]. Another study by Hayashi et al. [32
] suggested that TRPA1 activation inhibits the repair of the stomach epithelial wounds. All this evidence and the data gathered herein allow us to conclude that TRPA1’s role in wound healing may depend on the tissue type and stimuli.
An analysis of our model demonstrates that, at by 4 post-infection, P. aeruginosa
-infected wounds are characterized by an inflammatory milieu in comparison with non-infected controls. This response included the upregulation of IL-6 and 17, IL-17, VEGF and NO, all known to play a role in wound healing [19
]. It may seem counterintuitive that VEGF is upregulated in chronic wounds, but this has been observed in other chronic wounds, such as aphthous ulcers [33
]. The effect of VEGF in healing may be context-specific [34
]. In chronic inflammation, VEGF may have a preferential effect on vascular leak over revascularization. Therefore, angiogenesis inhibition might assist re-epithelialisation [33
Herein, it was found that cinnamaldehyde reduces the production of key inflammatory mediators (IL-17, VEGF and NO) in the wound beds of P. aeruginosa
-infected mice. Cinnamaldehyde anti-inflammatory effects are not novel, and different studies have demonstrated its ability to reduce NO and pro-inflammatory cytokine generation upon LPS stimuli [26
]. However, its modulatory role on VEGF expression is unclear. Contrary to the data presented herein, a recent study suggested that the systemic treatment with cinnamaldehyde induces skin wound healing in diabetic mice by increasing VEGF levels [18
]. It is possible that cinnamaldehyde effects on VEGF release during infection depend on the stimuli (infection versus diabetes) and treatment schemes (intraperitoneal versus topical administration). Of note, cinnamaldehyde presented no significant effects on the bacterial numbers or the levels of inflammatory mediators in skin lesions t infected with P. aeruginosa
The contribution of TRPA1 activation to cinnamaldehyde inhibitory actions in inflammatory mediator release in wound beds infected with P. aeruginosa
was also assessed in mice systemically treated with HC-030031. VEGF and IL-17 levels in the skin wounds of mice infected with P. aeruginosa
and treated with cinnamaldehyde, were partially attenuated by the systemic administration of the TRPA1 antagonist HC-030031; a drug that did not affect the number of bacteria in cinnamaldehyde-applied lesions. Interestingly, TRPA1 antagonism effects on NO release were similar to that of cinnamaldehyde. P. aeruginosa
-derived LPS was recently shown to activate TRPA1 in vitro, although in a smaller extent than E. coli
]. These evidences suggest that cinnamaldehyde and HC-030031 effects on NO release may be due to the ability of cinnamaldehyde and HC-030031 to compete with P. aeruginosa
LPS for a binding site on TRPA1. The partial recovery of IL-17 and VEGF production following TRPA1 antagonism in animals topically applied with cinnamaldehyde, indicates a complex scenario in terms of activation sites, which remains to be further elucidated. Also, considering the TRPA1 expression on different cells involved in skin wound healing such as neurones, keratinocytes and immune cells [38
], it is not yet known in which cells these molecules (LPS, cinnamaldehyde and HC-030031) are preferentially binding to in the wound beds infected by P. aeruginosa
in order to delay or promote skin healing. Further in vitro and in vivo studies are necessary to determine the specific actions of cinnamaldehyde as well as the role of TRPA1 on each cell type involved in skin healing (fibroblast, keratinocyte and endothelial cell culture) under infected and non-infected conditions. In this context, further histological and immunohistochemical analysis would be also valuable.
Overall, our data demonstrate that the repeated topical application of cinnamaldehyde promotes faster healing of skin wounds infected by P. aeruginosa
by decreasing bacterial colonization and attenuating the production of key inflammatory mediators of tissue regeneration such as IL-17, VEGF and NO (Figure 6
). The results also indicate that this anti-inflammatory effect is partially mediated by TRPA1 activation, although the cells involved in this process remain to be determined. Modification of both bacterial and host factors will likely be required for successful wound healing, and agents that disrupt P. aeruginosa
virulence without causing resistance might be especially valuable [39
]. We suggest that topical formulations (gel, nanoemulsion or aerosol formulations) containing sub-inhibitory concentrations of cinnamaldehyde may be a useful tool to treating skin infections induced by P. aeruginosa