Anti-bacterial and anti-inflammatory properties of Vernonia arborea accelerate the healing of infected wounds in adult Zebrafish

Abstract

Background: Management of wounds and healing under impaired conditions are the major challenges faced globally by healthcare workers. Phytocompounds which are anti-microbial and capable of modulating inflammation contribute to overall wound healing and regain of the lost structure and function especially in wounds impaired with polymicrobial infection.

Methods: An acute cutaneous impaired wound model using adult zebrafish was validated to simulate mammalian wound pathophysiology. This model was used to evaluate phytofractions of Vernonia arborea in the present study, for reduction of infection; myeloperoxidase (MPO) as a marker of infection; neutrophil infiltration and resolution; kinetics of inflammatory cytokines; and wound repair kinetics (viz., nitrite levels and iNoS expression; reepithelisation).

Results: Four fractions which were active in-vitro against five selected wound microbes were shown to reduce ex-vivo microbial bioburden upto 96% in the infected wound tissue. The reduction in CFU correlated with the neutrophil kinetics and MPO enzyme levels in the treated, wound infected zebrafish. Expression of pro-inflammatory cytokines (IL-6 and TNF-α) was downregulated while upregulating anti-inflammatory cytokine (IL-10), and nitric oxide signalling with fourfold increase in iNOS expression. The adult zebrafish wound model could well serve as a standard tool for assessing phytoextracts such as V. arborea for wound healing with anti-microbial properties.

Keywords: Inducible nitric oxide synthase; Interleukins; Myeloperoxidase; Neutrophil; Wound infection; Zebrafish.

Fig. 1

Time kill kinetics of V. arborea Fraction 10 against all five test strains with the respective MIC and AUC of log CFU shown below. UC, untreated control; PC, positive control (ampicillin at inhibitory concentration); n = 3; values are mean ± SEM. ** p < 0.05, *** p < 0.01 compared to the untreated control (one-way ANOVA followed by Dunnett’s post hoc test)

Fig. 2

Bacterial populations in the zebrafish infected wound tissue at 6, 24 hpi and 3 dpi. Treatment with bioactive fractions (0.5%) showed a reduction in CFU comparable to the positive control. The untreated control showed a constant increase in CFU with time. Values are mean [n = 20] ± SD, p < 0.05, p < 0.01 compared to positive or untreated control, respectively

Fig. 3

H&E stained sections of the zebrafish infected wound tissue showing better resolution of neutrophil population [n = 20] 3 dpi after treatment with V. arborea F10 (0.5%) in comparison with the positive control

Fig. 4

Neutrophil population at the zebrafish infected wound site recorded on 0, 1 and 3 dpi. The bioactive fraction treated wounds exhibited high infiltration on 1 dpi and good resolution on 3 dpi. V. arborea F10 at 0.5% treatment showed maximum resolution of neutrophil population in comparison to the control. Values are mean [n = 20] ± SD, *p < 0.05, **p < 0.01 compared to positive or untreated control, respectively

Fig. 5

Myeloperoxidase enzyme activity measured across the zebrafish control and treatment groups on 1, 3 and 5 dpi. Values are mean [n = 20] ± SD, *p < 0.05, **p < 0.01 compared to positive or untreated control, respectively

Fig. 6

Correlation of three parameters in zebrafish infected wound tissue upon treatment of with V. arborea F10 (0.5%). Log₁₀ CFU (for E. coli for example), neutrophil population and myeloperoxidase enzyme activity on 1 and 3 dpi and reepithelialisation time for the PC, UC and F10 groups are indicated. PC, positive control, UC, untreated control. All parameters were found to increase with the UC

Fig. 7

Fold change in expression of inflammatory cytokines. The change across the control and treated zebrafish (0.5%) groups (V. arborea F10, F26, F28, F30 fractions) over time, days post infection (dpi) showed decrease of pro-inflammatory cytokines- IL-6 and TNF-α upon treatment. Increased expression of anti-inflammatory cytokine IL-10 from day1 to day 3 in the treatment groups was noted. Values are mean [n = 20] ±SD, *p < 0.05, **p < 0.01 compared to positive or untreated control, respectively. UC, untreated control; PC, positive control; VC, vehicle control

Fig. 8

Nitrite level in zebrafish infected wound tissue on 3, 5 and 7 dpi. V. arborea bioactive fractions show optimal increase in nitrite concentration compared to controls. Values are mean [n = 20] ±SD, *p < 0.05, **p < 0.01 compared to positive or untreated control, respectively

Fig. 9

Fold change in expression of iNOS in zebrafish control and treatment (0.5% V. arborea fractions) groups over time, days post infection (dpi). Values are mean [n = 20]± SD, *p < 0.05, **p < 0.01 compared to positive or untreated control, respectively. UC, untreated control; PC, positive control; VC, vehicle control

Fig. 10

Wound closure time observed across the zebrafish wound models with infection, treated with V. arborea. Values are mean [n = 20] ±SD, *p < 0.05, **p < 0.01 compared to positive or untreated control, respectively

Fig. 11

Healing model of Infected Wounds using V. arborea extracts in Adult Zebrafish –Kinetics using visual, CFU reduction, histopathology, cytology, biochemical and immune-molecular markers at different phases