Molecular Action of Hydroxytyrosol in Wound Healing: An In Vitro Evidence-Based Review

Hydroxytyrosol (HT) is an essential molecule isolated from the phenolic fraction of olive (Olea europaea). HT has been implicated for its health-stimulating effect mainly due to its antioxidative capacity. The current review summarises and discusses the available evidence, related to HT activities in wound healing enhancement. The literature search of related articles published within the year 2010 to 2020 was conducted using Medline via Ebscohost, Scopus, and Google Scholar databases. Studies were limited to in vitro research regarding the role of HT in wound closure, including anti-inflammation, antimicrobial, antioxidative, and its direct effect to the cells involved in wound healing. The literature search revealed 7136 potentially relevant records were obtained from the database search. Through the screening process, 13 relevant in vitro studies investigating the role of HT in wound repair were included. The included studies reported a proangiogenic, antioxidative, antiaging, anti-inflammatory and antimicrobial effect of HT. The current in vitro evidence-based review highlights the cellular and molecular action of HT in influencing positive outcomes toward wound healing. Based on this evidence, HT is a highly recommended bioactive compound to be used as a pharmaceutical product for wound care applications.


Wound Healing
A wound can be defined as a pathological condition involving the devastation of normal anatomic tissue microstructure and function. It is the consequence of chemical, thermal or physical injury leading to an inflammatory response from the penetration of foreign materials and organisms into the damaged tissue [1,2].
A 'wound' can be classified into acute and chronic based on the wound closure duration. Acute wounds like burns, traumatic injuries and non-infected surgically created wounds, take approximately 8-12 weeks to be repaired. On the contrary, chronic wounds may need more than 12 weeks to be closed, such as diabetic foot ulcers, venous leg ulcers and pressure ulcers [3][4][5].
The reduction of wound area is a dynamic biological process, triggered by ruptured tissue, with overlapping multicellular phenomenon involving numerous elements, such as platelets, A paramount reason for having the inflammatory phase is to clear foreign materials, including bacteria from the wound environment [8]. Chronic infections can also lead to the prolongation of the inflammatory state. Hence, an antimicrobial agent is the utmost importance in ensuring the progression of the normal wound healing process [16].
In chronic wounds, the oxygen concentration in tissue is low, due to low blood flow and high oxygen consumption [17]. The lack of oxygen or hypoxic condition is well-related to the increment of bacterial colonisation. including Staphylococcus species, Streptococcus species, Pseudomonas aeruginosa and Escherichia coli, which are dominant microorganisms found in wound site [18][19][20]. Besides, oxidative stress environment in non-healing wounds promotes extracellular matrix (ECM) destruction that supplies nutrition for the microbes [21]. Hence, an antioxidant could have a beneficial effect on alleviating bacterial infection, which in turn, allows the normal progression of wound healing.
Effectiveness of an antimicrobial agent is indicated by two values that can be obtained through bacterial culture experiments. They are minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) [22]. The MIC is defined as the minimal concentration of antibiotic that prevents a clear suspension of 10 5 colony-forming units (CFUs) of bacteria/mL from becoming turbid after overnight incubation. While MBC demonstrates the lowest level of antimicrobial agent needed that results in microbial death. The MBC is identified by determining the lowest concentration of antibacterial agent that reduces the viability of the initial bacterial inoculum by ≥99.9%.

Antioxidant Activity
Considering the oxidative stress reported in chronic wounds, an antioxidant agent should be beneficial in the progression of wound healing. Oxidative stress is the result of an imbalance between high levels of reactive oxygen species (ROS) formation and low level of antioxidative activity [23]. ROS is a short-lived and highly reactive molecule, in free radical form, secreted by neutrophils and macrophages immediately after an injury [24]. It has the ability to terminate bacterial survival, induce tissue destruction and inhibit cellular functions, such as cell proliferation and cell migration [25].
In addition, ROS production can be regulated through nuclear factor erythroid-2-related factor 2 (NRF-2) and heme-oxygenase 1 (HO-1) activation. NRF-2 is a helix-loop-helix basic leucine zipper transcription factor and a master regulator of cell responses to oxidative stress [26]. Activation of NRF-2 led to the production of HO-1, the rate-limiting enzyme that converted heme into carbon monoxide (CO) and biliverdin [27]. HO-1 significantly elevated in chronic wounds as protection against oxidative stress. Taken together, ROS, NRF-2 and HO-1 are good biomarkers to examine the antioxidant property and the progression of wound healing.

Cellular Events
Cell proliferation and migration are critical cellular events in the proliferative phase of wound healing. Dermal fibroblasts, epithelial cells and endothelial cells are the three main cellular components in the regeneration of the human skin. Upon clearance of inflammatory cells, fibroblast migrates to the wound site and immediately degrade the loose provisional fibrin matrix left by the inflammatory cells [11]. As the proliferative phase progresses, fibroblasts proliferate and migrate to form the granulation tissue covered with a newly synthesised matrix. This is followed by the re-epithelialisation of the wound by the epithelial cells, migrating over the new matrix laid out by the fibroblasts [6,28,29]. Formation of a new blood vessel by the endothelial cells occur concurrently throughout all the proliferative phase [30]. Therefore, the proliferation and migration of dermal fibroblasts, epithelial cells and endothelial cells are suitable to be used as parameters for wound healing evaluation.

Hydroxytyrosol
Among all phenolic constituents of olives, hydroxytyrosol (HT) is currently being an attractive molecule to be utilised in the pharmaceutical, medical, nutraceutical, cosmeceutical and food industry. HT or 4-(2-hydroxyethyl)-1,2-benzenediol is a polar secondary plant metabolite, obtained from olives, derived from oleuropein through enzymatic hydrolysis. It has a typical catechol structure with chemical formula C 8 H 10 O 3 and molecular weight of 154.16 g/mol [31].

Research Articles Search Strategy
A systematic review of the literature was conducted to identify relevant studies reporting the role of HT in wound healing. The Medline via Ebscohost (Ipswich, MA, USA), Scopus (Amsterdam, The Netherlands) and Google Scholar (Mountain View, CA, USA) databases were used to identify the relevant articles established between 2010 and 2020. The search strategy involved a combination of the following keywords: hydroxytyrosol* AND wound* OR acute* OR chronic*.

Selection of Research Articles
The results were limited only to the relevant articles published in the English language, due to limited resources for translation services. The research articles which primarily investigated the role of HT in wound care were included. Review articles, news, letter, editorials or case studies were excluded from the review. Research articles that were not in vitro experiments that investigated HT activity in promoting wound healing were removed.

Inclusion and Exclusion Criteria
For this evidence-based review, only in vitro studies that investigated the role of HT in wound healing were included. In vitro investigations included in this review must evaluate the role of HT in cellular events (cell proliferation, cell migration and cell viability) that are well-related to the enhancement of wound healing. Moreover, in vitro research examining the antioxidant, anti-inflammatory and antimicrobial property of HT, which can accelerate wound closure were also included. Studies that measure the changes in reactive oxygen species (ROS), nuclear factor erythroid-2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), inflammatory cytokines (TNF-α, IL-1β, Il-6 and IL-8), nuclear factor kappa B (NF-κB), matrix metalloproteinases (MMPs) and transforming growth factor-beta (TGF-β), whenever available, were included. For this evidence-based review, all in vivo experiments investigated the role of HT in wound healing are excluded.

Data Extraction and Management
Articles were screened in three phases prior to their inclusion and exclusion criteria in this systematic review. The first phase; the titles and abstracts were screened to ensure inclusion and exclusion criteria adhered to. Second phase; abstracts of the remaining articles were screened, and abstracts that did not meet the inclusion criteria were excluded. In the final phase, the remaining articles were read thoroughly by four independent reviewers to exclude any articles that did not meet the inclusion criteria followed by data extraction. The following data were recorded from the studies: [1] The types of study; [2] aim of study; [3] subject or sample; [4] methodology or parameters; [5] results; and [6] conclusion.

Literature Search
The extensive literature search successfully identified 7136 potentially relevant records. Initial screening of records resulted in the removal of 2681 records that were not the original article, not published in English language and a duplication. The articles were then screened based on the title for any inclusion criteria that resulted in the removal of 4425 articles. From the remaining 30 articles, 16 articles were removed after screening the abstracts for inclusion and exclusion criteria. Reviewers then read the full text of the remaining 14 papers, of which 1 article was excluded because they did not fulfil the inclusion criteria. Thirteen articles were finalised for data extraction. A flow chart of the article's retrieval process was shown in Figure 1.
In terms of the experimental model, endothelial cells were used to evaluate the effect of HT on the angiogenesis process that is crucial in the proliferative phase of wound healing. Five studies utilised human umbilical vascular endothelial cell (HUVEC) in their studies, while two studies by Zrelli et al. (2015Zrelli et al. ( , 2011 [36,37] utilised porcine endothelial cells. For the antioxidative studies, one study each utilised human keratinocyte and monocyte, while another one study had both human keratinocyte and dermal fibroblast. HT antiaging effect was investigated in dermal fibroblast. Regarding antimicrobial effect, HT was tested against Staphylococcus aureus and Staphylococcus epidermidis by Ghalandari et al.    All of the test compounds, whether alone or in combination, inhibits VEGFR-2 phosphorylation.

3.
Only HT inhibits PLCγ1, while HT, melatonin and serotonin shared enhancement effect on Akt and eNOS.

2.
Calabriso et al. 2018 [33] To investigate the HT effects on endothelial dysfunction under inflammatory conditions.
HT attenuates PMA-induced elevation of mitochondrial and cytosolic ROS level. 6.
HT suppresses PMA-induced elevation of SOD activity and MDA level. 7.
HT restores the PMA-induced depolarisation of the mitochondrial membrane potential. 8.
HT restores the PMA-induced reduction of ATP synthase activity. 9.
HT restores the PMA-induced downregulation of ATP5β. 10. HT restores the PMA-induced downregulation of PGC-1α and NRF-1, as well as the upregulation of TFAM. ROS production Glutathione (GSH) level.

4.
Gene and protein expression of HO-1.
1. HUVEC cell viability was not affected up to 100 µM concentrations of HT and its metabolite. 2.
HT and all metabolites suppress TNF-α induced upregulation of CCL2 and PTGS2.

1.
HT enhances PEMF-induced HUVEC proliferation with 30 µM concentration resulted in the highest cell proliferation.

2.
HT enhances PEMF-induced HUVEC cell migration compared with the control and PEMF treatment only.

3.
Combination of HT and PEMF treatment elevated the mRNA and protein levels of Akt, mTOR and TGF-β1, but not p53 when compared to the control.
Cell migration.
HT increased VECs migration rate.

6.
Zrelli et al. 2011 [37] To study the effect of HT on proliferation and protection against oxidative stress-induced damage in VECs. Inhibition of Akt, p38, and ERK1/2 impedes HT-induced elevation of nuclear NRF-2 protein level.
Inhibition of HO-1 impedes HT-induced cell proliferation, migration and protection against H 2 O 2 .
DNA damage.
HT reduced LED-BL-induced DNA damage in both HEKs and HDFs. 4.
HT reverses LED-BL-induced increase in MMP-1, MMP-12 and PCNA, as well as the reduction in collagen I gene and protein levels in both HEKs and HDFs. Cell cytotoxicity. 5.

Jeon and Choi 2018 [41]
To evaluate the anti-inflammatory and antiaging effects of HT via UVA-induced aging model in HDFs.

2.
Ratio of aged cells.

1.
Cell viability was not affected up to 30 µM of HT.

1.
HT-pAc at 0.80 ± 0.02 mmol was required to achieve half the maximal antioxidative effect.

2.
MIC value of HT-pAc for S. epidermidis is 2.5 mg/mL.

Role of HT in Angiogenesis
Formation of new vasculature network within the injured tissue is crucial in supporting its restoration back to its functional state. Thus, the angiogenic property of hydroxytyrosol has become a subject of interest to 6 out of the 13 studies included [32][33][34][35][36][37]. Contradicting results were reported whereby three studies reported antiangiogenic properties with HT, while the remaining three studies reported proangiogenic properties of HT.
Chronologically, the proangiogenic properties of HT were reported first before its antiangiogenic properties. Zrelli et al. (2011) first reported the enhancement of cell proliferation and migration in their porcine vascular endothelial cells. HT also demonstrated the antioxidative effect by reducing the formation of ROS. At the molecular level, they found that the effect exerted by HT can be attributed to the upregulation of NRF-2 and its subsequent effector, the HO-1 [37]. In the follow-up study by the same group, they succeeded in elucidating the involvement of PI3K/Akt and ERK1/2 signalling pathways in the proangiogenic effect of HT on porcine vascular endothelial cells [36].
Various forms of electrical stimulation, including direct current, combined electromagnetic fields and pulse electromagnetic field (PEMF), have been demonstrated to positively influence the process of angiogenesis [45]. Correspondingly, Cheng et al. (2017) investigated the effect of combining HT and PEMF on HUVEC proliferation and migration. Indeed, PEMF alone demonstrated enhancement of HUVEC proliferation and migration. HT addition enhances these effects on HUVEC [35].
Endothelial dysfunction and impaired angiogenesis during diabetes have been attributed as one of the contributors to a chronic non-healing wound in diabetic patients [46] [32]. In all three studies, HT exerts a regenerative effect on the HUVEC via suppression of oxidative stress, inflammation, cell adhesion and cell migration.

Role of HT in Oxidative Stress
Oxidative stress in wound microenvironment is the imbalance condition of reactive oxygen species (ROS) concentration and the ability of biological systems to rebuild the resulting damage [47]. Thus, the antioxidant property is essential to improve wound healing.  [38][39][40].
The earliest study focusing on the antioxidative effect of HT in the context of wound healing is by Guo et al. (2010) [40]. By utilizing the epidermal keratinocyte cell line, HaCat cells, they investigated the effect of HT on alleviating oxidative stress caused by UVB light. As expected, HT suppresses the oxidative stress caused by UVB light on HaCat cells indicated by the significant decrease in the detection of oxidised dichloro-dihydro-fluorescein diacetate (DCFH-DA) and 8-hydroxy-2 -deoxyguanosine (8-OHdG) within the cell.
In another study, Avola et al. (2018) utilised both keratinocytes and dermal fibroblasts to investigate the antioxidative effect of HT caused by light-emitting-diode-generated blue light (LED-BL) irradiation [38]. As anticipated, HT suppresses the oxidative stress caused by LED-BL on both keratinocytes and dermal fibroblasts indicated by the significant decrease in the detection of oxidised DCFH-DA and 8-OhdG within the cell.
HT antioxidative effect had also been investigated in the inflammatory model of lipopolysaccharide (LPS)-stimulated human THP-1 monocytes. In this study, Meschini et al. (2018) investigated enzyme-treated olive vegetation waste (OVW) instead of synthesised HT [39]. They were able to demonstrate a significantly higher yield of HT in enzyme-treated OVW when measured with gas chromatography mass spectrometry (GC-MS) compared to the non-treated OVW. When evaluated for antioxidative effect, all OVW were able to reduce the 2,2-diphenyl picrylhydrazyl (DPPH) activity. However, the enzyme-treated OVW shown significantly greater antioxidative effect compared to the non-enzyme-treated OVW. This suggested that a higher content of HT resulted in a greater antioxidative effect.

Antiaging Properties of HT
Skin aging can influence wound healing capabilities by causing decreased proliferative responses, delayed angiogenesis, delayed remodelling, and slower re-epithelialisation [48]. In dermal fibroblast, declined motility, proliferative capacity and chemotactic response can be observed with aged-related cellular defects [49]. Consequently, Jeon and Choi (2018) utilised dermal fibroblast to evaluate the antiaging effect of HT following photoaging induced by UVA light [41]. By measuring the senescence-associated β-galactosidase (SA-β-gal) in the dermal fibroblast, they found that HT treatment significantly reduces the number of cells presented with SA-β-gal, suggesting the antiaging effect of HT.

Antimicrobial Properties of HT
In terms of antimicrobial activity, three studies revealed that HT possesses the ability to inhibit bacterial growth. Studies conducted by Ghalandari et al. (2018) unravelled antimicrobial activity against S. aureus and S. epidermidis [42].  [43]. They included eight g-negative bacteria, namely, Erwinia carotovora, Klebsiella pneumonia, Pseudomonas aeruginosa, Yersinia enterocolitica, Salmonella thypimurium, Aeromonas hydrophila, Shigella sonnei, Escherichia coli and 4 g-positive bacteria, namely, Pediococcus acidilactici, Kocuria rhizophila, Listeria monocytogenes, and S. aureus. Generally, HT exhibited MIC of 1000 µg/mL against most bacteria except 400 µg/mL MIC against S. aureus and E. coli. They furthered their investigation using four different strains of E. coli and concluded that 200 and 400 of HT µg/mL significantly reduced bacterial growth rate compared to the control, while 1000 µg/mL dose resulted in complete inhibition of growth.
Finally, Crisante et al. (2015), demonstrated that their HT containing polyacrylate was able to reduce the growth of S. epidermidis with inhibition zone 10 mm at 10 mg/mL and MIC value 2.5 mg/mL [44].

Role of HT in Inflammation
Excessive levels of inflammatory cytokines, such as TNF-α, IL-6, IL-8 and IL-1β, have been implicated in delaying wound healing [50]. Therefore, anti-inflammatory agent is required to alleviate the inflammatory state and allow progression of wound healing. Inflammatory cytokines were evaluated in eight studies, complementary to the angiogenesis, antioxidative, and antiaging parameters.
TGF-β1 has been implicated in promoting angiogenesis among its other functions. Thus, Cheng et al. (2017) decided to investigate the TGF-β1 expression and its underlying molecular mechanism [35]. In their PEMF induced HUVEC, significant upregulation of TGF-β1 compared to the untreated HUVEC was observed. This upregulation was amplified with the treatment of HT. This regulation of TGF-β1 by PEMF and HT was found to be mediated by the Akt and mTOR signalling pathways.
During inflammation, the nuclear factor κβ (NF-κβ) initiate the transcription of many cytokines gene [14]. Therefore, the inhibition of NF-κβ has become a target in controlling inflammatory cytokines. HT has been shown to suppress the upregulation of NF-κB and p53 in HaCat cells. Lopez et al. (2017) went further to investigate the effect of HT on the regulators of NF-κβ, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) and inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) [34]. In their HUVEC, HT suppresses the TNF-α induced activity of IκBα and IKKβ. This will, in turn, shut down the NF-κβ signalling pathway and cease the production of inflammatory cytokine as evidence by the downregulation of the chemokine (CAC) motif ligand 2 (CCL2), prostaglandin-endoperoxidase synthase 2 (PTGS2).
Tumour necrosis factor α (TNF-α) is the pro-inflammatory cytokines that are responsible for a diverse range of signalling events during inflammation, leading to necrosis or apoptosis of target cells [51]. Exacerbation of TNF-α has been implicated in delayed wound healing. Hence, suppressing its production can serve as beneficial to wound healing progression. Calabriso et al. (2018) managed to show the TNF-α suppression by HT in their PMA-induced HUVEC [33]. The TNF-α suppressing effect of HT was also observed in LPS-induced THP-1 monocyte.
Suppression of interleukins (Ils) by HT was also reported in the studies included. In terms of IL-1β, HT has been shown to suppress its upregulation in PMA-induced HUVEC, TNF-α-induced THP-1 monocyte, and UVA-induced dermal fibroblast. Other Ils, such as IL-6 and IL-8, were also investigated. HT displays suppression of IL-6 in TNF-α-induced THP-1 monocyte and UVA-induced dermal fibroblast. IL-8 was found to be suppresses in UVA-induced dermal fibroblast.

Discussion
In this in-vitro evidence-based review, we summarised findings from 13 experiments evaluating the role of hydroxytyrosol (HT) in wound healing. Among factors that can impede wound repair are oxidative stress, abnormal angiogenesis, persistent inflammation and microbial colonisation. The evidence supported HT's high potential as a bioactive phenolic molecule isolated from olives to prevent the abovementioned factors, due to its powerful antioxidant, angiogenesis modulation, anti-inflammatory and antibacterial activities.
Furthermore, the studies included employs a variety of cellular model that are involved in different phases of wound healing. This enables the understanding of HT role throughout the process of wound healing. Starting at the inflammatory phases, HT exerts inhibitory effect that may allow progression of wound healing into the proliferation phase. At the proliferation phase, HT displays positive effects on the granulation tissue formation, angiogenesis and re-epithelialisation process.
Regarding antioxidative effect of HT, the three studies included were limited to the measurement of ROS production and suppression. Oxidative stress can also be triggered by reduced activity of the endogenous antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GRx), and glutathione-S-transferase (GST) [24]. Assessment of the effect of HT on these enzymes can provide a wholesome picture of the role of HT in modulating oxidative stress in injured tissue.
Aging has long been associated with oxidative stress [52]. Although SA-β-gal in dermal fibroblasts indicates cellular aging, it can be interpreted too as the accumulation of oxidative stress. Similar to Avola et al. (2018) and Guo et al. (2010), Jeon and Choi (2018) also challenged their cells with irradiation of a specific spectrum of light [38,40,41]. It can be hypothesised that, if Jeon and Choi (2018) evaluated ROS, they would also find the antioxidative effect of HT [41]. Alternatively, if Avola et al. (2018) and Guo et al. (2010) measured antiaging parameters, they would also find the antiaging properties of HT [38,40].
Within the context of inflammation, an assortment of inflammatory mediators used by the studies included enabled the mapping of HT effect on the signalling network of inflammation. Figure 2 illustrates the effect of HT on different cytokines. This can serve as a guide for the therapeutic use of HT in modulating inflammation. Moreover, results from different studies, employing different cell types, were similar according to the cytokines. This suggests a consistent effect of HT across different cells involved in wound healing. However, these data have to be considered with caution as the number of published studies is not large enough to draw a consensus.

Conclusions
This in vitro evidence-based review provided data that support the potential of HT as a bioactive compound to enhance wound reduction, due to its antioxidant, angiogenesis modulation, antiinflammatory and antibacterial activities. The information in this review could potentially be applied to various current technology, including tissue engineering, drug delivery system and cell-free treatment that benefit different unresolved situation, such as chronic wound healing, severe ulcer, burn, postsurgical wound, and others.