Mechanisms of antioxidants in the treatment of vitiligo in vivo : a systematic review

Vitiligo is characterized by skin discoloration in different body regions


Introduction
Vitiligo is a chronic hypopigmenting pathology that does not cause systemic damage. Its main feature is the loss of melanocytes resulting in white macules on the skin without signs of inflammation or scales. Its prevalence is 0.5 to 1% of the world population (Gowda, et al., 2020;Rashighi & Harris, 2017;Seneschal, et al., 2021). Despite not being a potentially fatal disease, it deserves attention because it can be associated with psychological issues such as low self-esteem, insecurity, anxiety, and depression (Bergqvist & Ezzedine, 2020;Ezzedine, et al., 2015;Meneghin, 2021;Salzes, et al., 2015).
The skin is the largest organ in the body and accommodates the cells responsible for producing and storing melanina fundamental pigment in photoprotection (Cordero & Casadevall, 1997;Nichols & Katiyar, 2010). Melanocytes constitute 1% of the skin cells, and one of its organelles is the melanosome, which is responsible for synthesizing melanin. Each melanocyte is interconnected with 30 to 40 keratinocytes through dendrites, so melanosomes containing melanin are transported from melanocytes to keratinocytes and stored (Rzepka, et al., 2016;Videira, et al., 2013).
The purpose of this review was to search and describe the effects of the antioxidant compounds in the vitiligo treatment in vivo, aiming to gather the available data on the possible action mechanisms of in vivo antioxidants for vitiligo treatment.

Protocol and registration
This systematic review was prepared following the recommendations of PRISMA 2020 (Preferred Reporting Items for Systematic Reviews and Meta-Analysis). The review protocol was registered in the PROSPERO (International Prospective Systematic Reviews Registry) under registration number 300518.
The methodology employed in this systematic review followed the recommendations of De Luca Canto (2020).

Eligibility criteria
The research question this review aimed to answer was "What are the mechanisms of action of the antioxidants described in biological assays for the treatment of vitiligo?" The PICOS acronymous was used to development of the research question and to elaborate the search strategy (De Canto Luca, 2020).

Inclusion criteria
Articles that evaluated the induction of melanogenesis (melanin production, melanocyte viability, tyrosinase gene expression, tyrosinase activity) and activation of mechanisms such as regulation of MITF transcriptional activity by MAPK pathways (ERK, JNK, and p28) and regulation of TYR, TRP-1, and TRP-2 expression, as result of in vivo treatment with antioxidants.

Exclusion Criteria
Studies were excluded for the following reasons: studies that used compounds to treat vitiligo that did not have antioxidant activity, in vivo studies in other animal models than murine or zebrafish, in vitro studies only, human studies, ex vivo studies only, in silico studies only and studies without a control group.

Information Sources and Search Strategy
The search was performed in PubMed, Web of Science, Embase, ScienceDirect, and LILACS databases and grey literature by Open Grey and Google Scholar. An individual search strategy was elaborated for each database, comprising terms and MESH terms related to vitiligo, melanogenesis, antioxidants, and possible signaling pathways evolved in melanogenesis and antioxidant activity. The individual search used in each database is detailed in Supplemental Table 1. The searches included all articles up to October 16, 2021, with no restrictions on the initial publication date or language. In addition, a manual search was carried out in the reference list of selected articles in search of possible relevant studies.

Selection of studies
The articles were selected from the databases and exported to the Rayyan tool in the first step. Duplicate articles were excluded, and the titles and abstracts of the remaining articles were blindly examined by Paloma de Jesus (PJ) and Manuel Mera (MM). Based on the inclusion and exclusion criteria, the articles were selected. In the second stage, a complete reading Research, Society andDevelopment, v. 11, n. 16, e277111638060, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i16.38060 4 of the articles was performed using the same criteria to confirm the eligibility or not of the articles selected initially. At this stage, a discussion with the third author, Maria de Fátima (MF), was necessary for a mutual agreement.

Data collection and analysis process
Information collected included: authors, year, country, intervention, study design, sample size, sex, age, weight, treatment dose, trials, mechanisms, and main findings. The first author (PJ) collected the main information from the selected articles and structured the summary table. The second (MM) and third (MF) reviewers verified the information and the veracity of the data, and the divergences were discussed until reaching a consensus.
Due to the absence of quantitative data in some of the reported results, only qualitative analysis was performed. The measures of the effect of the antioxidants in the primary or secondary outcomes were summarized by dichotomous categorical variables (yes/no) (Brasil, 2012).

Risk of bias and quality assessment
To assess the risk of bias in the studies included in this review, the RoB tools from SYRCLE and CAMARADES were adopted (Hooijmans, et al., 2014;Macleod, et al., 2004). The first and second reviewers rated the 10-item articles as Yes, low risk of bias; No, high risk of bias; or Unclear, moderate risk of bias. Items evaluated included: peer review, temperature control, random allocation to treatment or control, blind induction of vitiligo, blinded outcome evaluation, use of an anesthetic without significant intrinsic protective activity to vitiligo, animal model characteristics, sample, compliance with welfare regulations, and declaration of potential conflict of interest. Differences were resolved with the third author.

Summary measures
For this SR, the primary outcome evaluated in the studies was the effect of antioxidants in the treatment of vitiligo, evaluated through the restoration of redox balance; regulation of MITF, TYR, TRP-1, TRP-2, tyrosine kinase receptor (kit), and dopachrome tautomerase (dct) expression; regulation of iNOS expression; regulation of NRF2 expression; regulation of the amount of melanin-containing cells and regulation of melanin synthesis. The secondary outcome was the induction of melanogenesis, melanocyte viability, and expression of genes related to melanogenesis (TYR, TRP-1, TRP-2, MAPK, p38 MAPK, PKA, NRF2, ERK, MITF, and kit). The format of data extraction was dichotomous (yes/no).

Results
The initial search identified 390 articles from the seven databases. After removing the duplicate articles, 245 articles remained for screening. Of these, 229 were excluded after reading the titles and abstracts, resulting in 16 articles being thoroughly evaluated. After a comprehensive analysis, 9 studies were excluded, and 7 were included in the qualitative synthesis. No additional articles were identified in the reference lists of selected studies. A detailed flowchart with the process of identification, exclusion, and inclusion of studies is represented in Figure 1.

Included
Flowchart adapted from: PRISMA Statement 2020: an up-to-date guide to publishing systematic reviews. Page, et al., (2021).

Study characteristics
The selected articles were published between 2014 and 2021, three from 2021 (Abuduaini, et al., 2021;Lai, et al., 2021;Zhai, et al., 2021). The language of all articles was English, and the country of publication of all articles was China.
It was a condition for inclusion in the systematic review that the articles had an in vivo study with zebrafish or mice. A constant feature in the selected studies is that, except for Huo, et al., 2017, they all brought images showing visible results of the effects in mice and zebrafish. It was possible to observe an increase in fur coloration in mice and the head and back in zebrafish embryos. Among the selected articles, all that mentioned the study pattern did so in a randomized manner, and all the mouse studies cited sample size, sex, age, intervention doses, and respective weight margins, except Zhong, et al., 2019, which did not report the weight of the animals. The zebrafish studies did not mention a study pattern. The descriptive characteristics of each article were compiled in Table 1. Research, Society and Development, v. 11, n. 16, e277111638060, 2022 (CC BY 4.  This study demonstrated that RY3-c has potent antioxidant activity, can repair cell damage induced by excessive oxidative stress, and act on melanogenesis via the MAPK pathway.
RY3-a, CY3-a-1 -CY3a-15, RY3-b, and RY3c, RY3-c stood out with higher melanogenic activity among the components studied. This component has potent antioxidant activity and was able to repair cell damage induced by excessive oxidative stress. In addition, RY3-c acted mainly in the p38-MAPK and ERK1/2 pathways and played an essential role in melanin synthesis. Research, Society and Development, v. 11, n. 16, e277111638060, 2022 (CC BY 4.

Melanogenesis and TYR activity in zebrafish:
Amount not mentioned

Risk of bias
The result of the risk of bias analysis using CAMARADES tool is summarized in Table 2. Criteria related to peerreviewed publication (item 1), compliance with animal welfare declaration regulations (item 9), and a potential conflict of interest were scored as 'yes' in all articles, showing a low risk of bias. References to blind induction of vitiligo (item 4), blind evaluation of the result (item 5), and use of anesthesia (item 6) were scored as "unclear item", not being possible to clearly define the quality of the information. Two studies scored 'no' because they lacked data about temperature control (item 2).
Three others also scored "no" for not fully describing the characteristics of animal models (item 7), and two for not reporting the random allocation to treatment or control (item 3). The missing information in all selected articles was about the sample size calculation (item 8). Consequently, there was a high risk of bias in these parameters.  (Macleod, et al., 2004).
CAMARADES (Collaborative Approach to Meta-analysis and Review of Experimental Studies in Animals) is a research group founded in 2004 whose focus is to minimize translational flaws in preclinical research. The Rob tool is an adaptation of the Cochrane RoB tool and aspects of bias are employed in animal intervention studies. The tools consist of a set of 10 questions related to selection bias, performance bias, detection bias, attrition bias, reporting bias, and other biases. For each of the questions, it is possible to answer "Yes", when the requested criterion is described, "No", when the study does not meet the requested criterion, and "Unclear" when the information is not present or was only partially answered.
The seven selected articles showed an uncertain risk of bias when evaluated with Rob from SYRCLE or CAMARADES tools. The studies did not describe all information about allocation of the animals, randomization, and blinding of studies. These characteristics are fundamental for assessing the quality of studies.

Discussion
Vitiligo is a pathology that affects both sexes without distinction, it is not potentially fatal, but due to its main physical characteristic (white macules on the skin), it may be related to psychological comorbidities (Ding, et al., 2021;Salzes C., et al., 2015). Current treatments for vitiligo may include pharmacological, surgical, cosmetic, and phototherapy interventions, but these interventions are still limited, and there is no satisfactory cure (Daniel & Wittal, 2015;Guerra, et al., 2010;Karagaiah, et al., 2020).
Melanin, the natural skin pigment responsible for photoprotection, is synthesized by the complex cascade of melanogenesis. The lack of skin color in the individual with vitiligo is due to the death of melanocytes or the blockage of the melanogenesis pathway. A master regulator of this pathway, MITF, is responsible for upregulating the enzymes tyrosinase (TYR), tyrosine-related protein-1 (TYRP-1), and tyrosine-related protein-2 (TIRP-2) (Hwang, et al., 2019;Niu, et al., 2018;Niu & Aisa, 2017). The tyrosinase enzyme deserves to be highlighted for being multifunctional and crucial in the synthesis of melanin by participating in several steps and limiting the process (Niu & Aisa, 2017). This enzyme is responsible for catalyzing the conversion of L-tyrosine to levodopa (L-DOPA) and L-DOPA to dopaquinone. Dopaquinone is then converted, via proteins TRP-1 and TRP-2, into eumelanin. On the other hand, pheomelanin depends on the conversion of dopaquinone through cysteine or glutathione (Rzepka, et al., 2016;Serre, et al., 2018).
The epidermis of a patient with vitiligo has an oxidative disorder that is not restricted to the injured regions. In this situation, excess H2O2 leads to cell apoptosis, which may be the initial triggering event for vitiligo (Glassman, 2011;Schallreuter, et al., 1999). Therefore, a potential mechanism for preventing the onset of pathologies related to oxidative stress is using substances with antioxidant properties (Zhang, et al., 2022).
Thus, this systematic review evaluated the in vivo assays effects of different antioxidants on melanogenesis by the following parameters: restoration of redox balance, upregulation of MITF, TYR, TRP-1, TRP-2, kit, and dct expression; upregulation of NRF2; upregulation of MAPK; upregulation of the amount of melanin-containing cells, direct upregulation in melanin synthesis, downregulation of iNOS expression, decreasing of malondialdehyde (MDA) content, and decreasing in the levels of cholinesterase (CHE)/acetylcholinesterase (Ache), as shown in Table 3. The seven studies reported that at least one of the antioxidants investigated in each article noticeably induces melanogenesis. Table 3 -Relationship between the mechanisms and the respective antioxidants tested.

Mechanism Compound evaluated
Upregulation of TYR expression As observed in Table 3, curcumin tablets, followed by butin and galangin, showed effect on the higher number of melanogenesis parameters evaluated.
The use of antioxidants as a therapeutic strategy has been increasingly studied (Jung, et al., 2018). In this systematic review, 12 investigated compounds were from natural origin (CWT,pip,chr,kaem,sco,2',3,4,4'-tetrahydrochalcone -RY3-a, butin, galangin, caffeic acid. luteolin and VitD3) and the CAP jet is the only exception. Malondialdehyde (MDA), a lipid peroxidation product, is an important biomarker in evaluating oxidative stress and was one of the parameters evaluated in the studies. This compound is often found at high levels in pathologies related to oxidative stress (Arican & Kurutas, 2008;Yildirim, et al., 2004). Another possible biochemical measurement to assess oxidative stress is the mensuration of monoamine oxidase (MAO) and cholinesterase (CHE). MAO is a monoamine-degrading enzyme present in animals, and its increase favors toxic levels of H2O2, which, despite not being a highly reactive molecule, can give rise to oxidative stress (Pizzinat, et al., 1999;Tipton, 2018). CHE is an essential enzyme in the body's redox balance because if it is high, it can reduce melanin synthesis and favor the onset of vitiligo (Schallreuter, et al., 2004;Schallreuter & Elwary, 2007). CWT, butin, and galangin can promote Research, Society andDevelopment, v. 11, n. 16, e277111638060, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i16.38060 13 a decrease in the content of MDA and levels of CHE in the blood and skin of animals. Abuduaini, et al., 2021, also showed a decrease in MAO by CWT. These results suggest that redox balance restoration stands out among the possible mechanisms by which CWT, butin, and galangin act in treating vitiligo. Another compound capable of restoring the redox balance recorded in articles included in this review was RY3-c. The tests performed showed that it was able to decrease the ROS content in a dosedependent manner.
Under normal conditions, NRF2 is located in the cytosol in a complex with Keap-1, but at the signal of oxidative stress, NRF2 is released and moves to the cell nucleus. In the nucleus, it combines with antioxidant response elements, and antioxidant enzymes are activated (Guo & Zhang, 2021). Among the reactive nitrogen species, nitric oxide is the main substance in tissue, and inducible nitric oxide synthase is largely responsible for stimulating cells to produce NO (Mansourpour, et al., 2019;Xia, et al., 2019). Thus, if a compound can perform the upregulation of NRF2 and downregulation of nitric oxide synthetase, it contributes to the restoration of redox balance, as CAP (Zhai, et al., 2021).
The MAP family is composed of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), which, after phosphorylation, can activate MITF, and thus the cascade common to all pathways for melanin synthesis is triggered (Wu, et al., 2011). MAPK can be indirectly activated by the alpha-MSH hormone, which binds to the melanocortin 1 receptor (MC1R), and by stem cell factor (SCF), which binds to the c-kit receptor on the surface of the melanocyte (Gonzalez & Montminy, 1989). RY3-c acted mainly in the p38-MAPK and ERK1/2 pathways and played an important role in melanin synthesis (Pillaiyar, et al., 2017).
The MITF and TYR genes are already well defined as melanogenic genes (Niu & Aisa, 2017). In addition to them, during the early color development of zebrafish, the dct and kit genes promote the differentiation of melanocyte stem cells into melanocytes (Rawls & Johnson, 2000). Starting from this point, the effects of the best combination of butin: caffeic acid: luteolin (1:4:10) on the expression of these melanogenic genes were investigated. The investigation concluded that the expression of tyr, kit, and dct in the eyes and on the back of zebrafish larvae increased, whereas the expression of MITF was only slightly increased on the back of the fish (Lai, et al., 2021). Thus, the increase in gene expression of tyr, kit, and dct is responsible for the increase in melanin production in zebrafish larvae treated with the combination of these compounds.

Conclusion
The studies show that the mechanisms of the antioxidants evaluated in these works, within in vivo models, included, mainly, the increase of tyrosinase activity by increase in the synthesis of TYR and TRP-1 proteins, the positive regulation of the number of epidermal cells, the restoration of redox balance, MITF activation, decrease in malondialdehyde content and decrease in cholinesterase levels and activity. The results suggest that compounds that have not yet been clinically tested should advance in this direction, as the in vivo results are promising. Furthermore, it was observed the total or partial absence of some information about the methodology used on the reported in vivo studies, evidenced by the evaluation of the risk of bias, showing the importance of a criterious description of the methodology of the studies to strength the confidence in the results reported.