|Year : 2021 | Volume
| Issue : 1 | Page : 22-25
Role of autophagy in nonsegmental vitiligo Naguid and Rashed
Rehab M Naguib MD 1, Laila A Rashed2
1 Department of Dermatology and Venerology, Faculty of Medicine, Beni-Suef University, Beni-Suef, Egypt
2 Department of Biochemistry, Faculty of Medicine, Cairo University, Cairo, Egypt
|Date of Submission||16-Feb-2020|
|Date of Acceptance||20-Jul-2020|
|Date of Web Publication||23-Dec-2020|
Rehab M Naguib
Department of Dermatology, Beni-Suef, 19 Port Saed Street 62511
Source of Support: None, Conflict of Interest: None
Background Vitiligo is a skin and less common hair disease characterized by decline in melanocyte function and depigmentation, with a prevalence of 0.5–1% in most populations. Autophagy is the degradation of components of the cytoplasm within lysosomes. This is distinct from endocytosis-mediated lysosomal degradation of extracellular and plasma membrane proteins.
Aim The aim was to detect biochemical parameter light chain 3 (LC3) to monitor autophagy in vitiligo skin of patients as compared with normal control persons to evaluate the role of autophagy in the vitiligo pathogenesis.
Materials and methods This case–control study included 60 patients with vitiligo and 60 age-matched and sex-matched healthy controls. Herein, 4 mm punch skin biopsy was taken from every patient (vitiligo lesion) and control and kept in lysis solution for the stability of the studied parameters and was kept frozen at −80°C till analysis of autophagy protein LC3 by qRT PCR.
Results The level of LC3 in lesional skin of vitiligo was significantly lower as compared with normal control persons.
Keywords: autophagy, light chain 3, vitiligo
|How to cite this article:|
Naguib RM, Rashed LA. Role of autophagy in nonsegmental vitiligo Naguid and Rashed. Egypt J Dermatol Venerol 2021;41:22-5
| Introduction|| |
Vitiligo is a skin and less common hair disease characterized by decline in the function of melanocytes (MCs) and depigmentation, with a prevalence of 0.5–1% in most populations .
Studies pointed to the role of genetic susceptibility to vitiligo . The theories have been shown to combine biochemical, immunological, and environmental events, in a genetic milieu .
Autophagy is a process that is present in all cells at low levels under normal conditions, but many stimuli like hypoxia or starvation may lead to its upregulation. Cytoplasmic components are broken down into basic components and returned to be reused in the cytosol .
Autophagy is mediated by an organelle called autophagosome. As autophagosomes engulf a part of cytoplasm, the autophagy is generally thought to be a nonselective degradation system .
The microtubule-associated protein 1 light chain 3 (MAP1LC3 or LC3) is a small protein of 18 kDa and 125 amino acids involved in the autophagosome formation that belongs to the MAP1LC3 family . LC3 had been thought to be included in regulation of assembly and disassembly of microtubules .
The purpose of this study was to evaluate the role of autophagy in the pathogenesis of vitiligo through the evaluation of LC3 expression in tissue biopsies from the normal and vitiliginous skin.
| Materials and methods|| |
This case–control study included 60 patients with nonsegmental active vitiligo ([Figure 1],[Figure 2],[Figure 3],[Figure 4]) and 60 age-matched and sex-matched healthy controls. The aim of our study was explained to each patient, the study was approved by the local research Ethics Committee of Faculty of Medicine, Beni–Suef University. The patients and controls were of Fitzpatrick skin type IV recruited from individuals attending the outpatient clinic of Beni-Suef University Hospital in the period from February 1, 2018 to June 31, 2018.
Exclusion criteria included use of any drugs that could affect the outcome of our study, such as topical or systemic vitiligo treatment. Subjects with other autoimmune diseases and subjects receiving phototherapy the last 6 months were excluded from the study.
Patient information was collected by a dermatologist, comprising age, sex, type of vitiligo, and affected body surface area according to the rule of nines .
Herein, 4 mm punch skin biopsy was taken from every patient (vitiligo lesions: 22 biopsies from back, 16 from abdomen, 12 from upper limb, and 10 from lower limb) and control, and it was kept in lysis solution for the stability of the studied parameters and had been kept frozen at −80°C till analysis of autophagy protein LC3 by qRT PCR for the estimation of the levels of LC3.
LC3 detection in tissue using qRT PCR
Total RNA has been isolated using Qiagen tissue extraction kit (Qiagen, Beckman, Housten, Texas, USA) according to instructions of manufacture and then 30 mg of the human tissue sample was excised and placed directly into a suitably sized vessel for disruption and homogenization. The tissue was disrupted and lysed in lysis Buffer RLT, and the lysate was homogenized by tissue homogenizer for 40 s. The lysate was centrifuged for 3 min at full speed, and the supernatant was carefully removed and transferred into a suitable microcentrifuge tube. One volume (350 μl) of 70% ethanol was then added to a cleared lysate. Overall, 700 μl of the sample was transferred to a RNeasy spin column placed in a suitable 2 ml collection tube and centrifuged for 15 s at greater than or equal to 8000 rpm. Then, 500 μl Buffer RPE was then added to RNeasy spin column, and centrifuged for 15 s at greater than or equal to 8000 rpm to wash the spin column membrane. RNeasy spin column had been then placed in a new suitable 1.5 ml collection tube. Then, 30–50 μl of RNase-free water was added directly to the spin column membrane and then centrifuged for 1 min at greater than or equal to 8000 rpm to elute RNA. The eluate was then transferred to a suitable new Eppendorf tube and then stored at −80°C for further use. Purity (A260/A280 ratio) and concentration of RNA had been obtained by the spectrophotometry (dual-wavelength Beckman, Spectrophotometer; Beckman, USA).
Total RNA (0.5–2 µg) was used for cDNA conversion by using the high-capacity cDNA reverse transcription kit (Fermentas, Housten,Texas, USA). A volume of 1 µl of the random primers had been added to 10 µl of RNA that had been denatured for 5 min at 65°C in a thermal cycler. RNA primer mixture had been then cooled to 4°C. cDNA master mix had been prepared according to kit instructions and was then added (for each sample). Last mixture was then incubated in programmed thermal cycler for one hour at 42°C followed by the inactivation of the enzymes at 95°C for 10 min, finally cooled at 4°C. RNA had been changed to cDNA. The converted cDNA was then stored at −20°C.
Real-time qPCR using SYBR Green I
Real-time qPCR amplification and analysis had been performed using Applied Biosystem with software version 3.1 (StepOne Plus; Elabscience Biotechnology Company Inc., Housten, Texas, USA). Then qPCR assay with primer sets had been optimized at annealing temperature.
Data were coded and entered using Statistical Package for the Social Sciences version 24 (IBM Corporation, New Orchad Road, Armonk, New York, USA). Data were summarized using mean and SD in the quantitative data and using frequency (count) and relative frequency (percentage) for the categorical data. Comparisons between the quantitative variables were then done using nonparametric Kruskal–Wallis and Mann–Whitney tests. Correlations between the quantitative variables were done using the Spearman correlation coefficient. P values less than 0.05 were considered as statistically significant.
| Results|| |
The sex ratio and age were not substantially different for each variable among patients with vitiligo (32 women and 28 men; mean±SD age 33.73±8.6 years) and healthy controls (40 women and 20 men; mean±SD age of 31.13±6.4 years). Clinical data of participants are presented in [Table 1].
|Table 1 Dermographic data and clinical characteristics of the patients and controls|
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The tissue LC3 expression:
The level of LC3 in lesional skin of vitiligo was significantly lower as compared with normal control persons (P<0.001), where the mean±SD was 0.37±0.3 and 1.03±0.1 in the cases and controls, respectively ([Table 2]).
We found no relation between LC3 expression and patient age, sex, disease duration family history, and affected body surface area, with P value more than 0.05 in all.
| Discussion|| |
The exact pathogenesis of vitiligo is unknown but researchers suggested that an interplay of many factors such as genetic, neural, oxidant–antioxidant, biochemical, minerals, and autoimmune process might induce vitiligo .
Vitiligo MCs are more sensitive to accumulate reactive oxygen species owing to the intrinsic antioxidant defects . This imbalance between pro-oxidants and antioxidant state can disrupt the homeostasis of MCs, causing accumulation of oxidized and damaged proteins or organelles leading to destruction of MCs .
Autophagy is a cellular self-digestion process in which damaged intracellular proteins or organelles are sequestered and transported to be degraded in the lysosomes for maintenance of cell homeostasis . It was discovered that autophagic activity is needed to prevent the oxidative damage and to achieve full proliferative capacity of normal MCs .
Human skin is exposed to many environmental insults such as ultraviolet (UV) light, which cause oxidative damage to the macromolecules. In response to the oxidative stress, cells activate the Nrf2 antioxidant response, and the epidermal keratinocytes activate autophagy in response to UVA and UV-oxidized phospholipid .
Genetic elimination of autophagy leads to accumulation of protein aggregates in stressed cells and increases apoptosis and intracellular reactive oxygen species levels .
LC3 is a classical protein marker for autophagy. Its cellular localization in the autophagosomes has been associated with closure, hemifusion, or transport of the autophagosome during its maturation. LC3 has been used to monitor the number of autophagosomes and autophagic activity .
All the aforementioned reasons prompted us to investigate role of autophagy in vitiligo, and to do so, we estimated LC3 level in the tissue.
Our results showed that the level of LC3 in lesional skin of vitiligo was significantly lower as compared with the normal control persons (P<0.001).
Consistent with our results, He et al.  examined autophagy levels of the primary normal human MC and they found microtubule-associated protein 1 light chain 3 (LC3), the most commonly used biomarker of the autophagosome formation, was significantly increased in normal MCs following H2O2 treatment.
Among the limitations of this study is likely that there are many gaps in the knowledge base, which need to be filled, and therefore, we could not find many published research studies on the detected biochemical parameter LC3 to monitor autophagy in vitiligo skin. Even in the study by He et al. , they included only one vitiligo MC cell line (PIG3V) and considering that genetic backgrounds vary in patients with vitiligo; the cell line that they used cannot completely represent features of the vitiligo MCs. So, their findings and also ours need to be confirmed in other MC lines extracted from the patients with vitiligo in the future studies based on these results.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]