Topical Application of Cinnamaldehyde Promotes Faster Healing of Skin Wounds Infected with Pseudomonas aeruginosa

Abstract

Wound healing can be delayed following colonization and infection with the common bacterium Pseudomonas aeruginosa. While multiple therapies are used for their treatment, these are ineffective, expensive, and labour-intensive. Thus, there is an enormous unmet need for the treatment of infected wounds. Cinnamaldehyde, the major component of cinnamon oil, is well known for its antimicrobial properties. Herein, we investigated the effects of sub-inhibitory concentrations of cinnamaldehyde in the virulence of P. aeruginosa. We also assessed its healing potential in P. aeruginosa-infected mouse skin wounds and the mechanisms involved in this response. Sub-inhibitory concentrations of cinnamaldehyde reduced P. aeruginosa metabolic rate and its ability to form biofilm and to cause haemolysis. Daily topical application of cinnamaldehyde on P. aeruginosa-infected skin wounds reduced tissue bacterial load and promoted faster healing. Lower interleukin-17 (IL-17), vascular endothelial growth factor (VEGF) and nitric oxide levels were detected in cinnamaldehyde-treated wound samples. Blockage of transient receptor potential ankyrin 1, the pharmacological target of cinnamaldehyde, abrogated its healing activity and partially reversed the inhibitory actions of this compound on VEGF and IL-17 generation. We suggest that topical application of sub-inhibitory concentrations of cinnamaldehyde may represent an interesting approach to improve the healing of P. aeruginosa-infected skin wounds.

Keywords: Pseudomonas aeruginosa; cinnamaldehyde; skin wound; wound healing.

Figure 1

Effects of sub-inhibitory concentrations of cinnamaldehyde on P. aeruginosa. P. aeruginosa ATCC 27853 (a) viability (Δ nm) and (b) metabolic rate (as percentage (%) of reduction of resazurin to resorufin). (c) Biofilm formation and (e) haemolysis induced by P. aeruginosa ATCC 27853. Effect of sub-inhibitory concentrations of cinnamaldehyde on erythrocytes in the absence of bacteria (d). Cinnamaldehyde was tested at MIC/2, MIC/4 and MIC/8. Vehicle (2% DMSO in PBS)-treated bacteria were used as controls. * p < 0.05, differs from the vehicle-treated group. n = 3.

Figure 2

Effects of topical cinnamaldehyde or systemic TRPA1 antagonism on skin wound bacterial population. Total bacteria (a), Gram-negative bacteria (b) and P. aeruginosa (c) population in skin wound samples of infected and non-infected mice on day 4 post-skin excision. Total bacteria (d), Gram-negative bacteria (e) and P. aeruginosa (f) population in skin wound samples of infected and non-infected mice on day 7 post-skin excision. Animals received either sterile saline or P. aeruginosa ATCC 27853 following skin excision. Cinnamaldehyde (0.5 mg/mL; 30 µL, n = 8) or 2% DMSO in sterile saline (30 µL, n = 8) were topically applied once a day for seven days. The TRPA1 antagonist HC-030031 (30 mg/kg, i.p., n = 8) or vehicle (8% DMSO in saline, i.p., n = 8) were administered to animals receiving or not cinnamaldehyde. * p < 0.05, differs from vehicle-treated mice. ND: not detected.

Figure 3

Effects of topical cinnamaldehyde or systemic TRPA1 antagonism on wound healing. Time course of (a) wound area and (b) healing in non-infected mice. Time course of (c) wound area and (d) healing in P. aeruginosa-infected mice. (e) Area under de curve (AUC) of wound area over a seven-day time-course. Animals received either sterile saline or P. aeruginosa ATCC 27853 following skin excision. Cinnamaldehyde (0.5 mg/mL; 30 µL, n = 6) or 2% DMSO in sterile saline (30 µL, n = 6) were topically applied once a day for sevn days. The TRPA1 antagonist HC-030031 (30 mg/kg, i.p., n = 6) or vehicle (8% DMSO in saline, i.p., n = 6) were administered to animals receiving or not cinnamaldehyde. * p < 0.05, differs from vehicle-treated non-infected mice; ^# p < 0.05, differs from vehicle-treated P. aeruginosa infected mice; ^@ p < 0.05, differs from cinnamaldehyde-treated P. aeruginosa infected mice.

Figure 4

Effects of topical cinnamaldehyde or systemic TRPA1 antagonism on wound severity. Wound severity was macroscopically evaluated. Representative photographs (a) and severity scores (b) of the wounds on days 4 and 7 post-skin excision. Animals received either sterile saline or P. aeruginosa ATCC 27853 following skin excision. Cinnamaldehyde (0.5 mg/mL; 30 µL, n = 6) or 2% DMSO in sterile saline (30 µL, n = 6) were topically applied once a day for over seven days. The TRPA1 antagonist HC-030031 (30 mg/kg, i.p., n = 6) or vehicle (8% DMSO in saline, i.p., n = 6) were administered to animals receiving cinnamaldehyde or not. * p < 0.05, differs from vehicle-treated non-infected mice; ^# p < 0.05, differs from vehicle-treated P. aeruginosa infected mice; ^@ p < 0.05, differs from cinnamaldehyde-treated P. aeruginosa infected mice.

Figure 5

Inflammatory mediator release in cinnamaldehyde-treated mouse skin wounds. The levels of (a) IL-6, (b) IL-17, (c) VEGF and (d) NO in skin wounds of animals infected or not with P. aeruginosa ATCC 27853. Cinnamaldehyde (0.5 mg/mL; 30 µL, n = 6) or 2% DMSO in sterile saline (30 µL, n = 6) were topically applied once a day for four days. The TRPA1 antagonist HC-030031 (30 mg/kg, i.p., n = 6) or vehicle (8% DMSO in saline, i.p., n = 6) were administered to animals receiving or not cinnamaldehyde. * p < 0.05, differs from vehicle-treated non-infected mice; ^# p < 0.05, differs from vehicle-treated P. aeruginosa infected mice; ^@ p < 0.05, differs from cinnamaldehyde-treated P. aeruginosa-infected mice. ND: not detected.

Figure 6

Summary of cinnamaldehyde effects in the wound healing of mice infected with P. aeruginosa. Mice infected with P. aeruginosa have delayed healing, associated with increased production of inflammatory mediators in the wound beds in response to this bacterial infection. The topical application of cinnamaldehyde reduces the bacterial load in the wound tissue and promotes wound healing, as denoted by reduction of the wounded area. This effect is associated with diminished levels of IL-17, VEGF and NO in the wound beds. The systemic TRPA1 antagonism by HC-030031 prevents cinnamaldehyde’s pro-healing action by increasing IL-17 and NO levels.