1450 Biol. Pharm. Bull. 42, 1450–1455 (2019) Vol. 42, No. 9 Communication to the Editor

The Therapeutic Effects of Baicalin on (IL)-10 significantly higher than vitiligo-affected and per- ilesional skin.9) These studies confirmed the participation of Vitiligo Mice CD8 + T cells and cytokines in the pathological mechanisms a b a of vitiligo. Baicalin is a main active ingredient derived from Yiping Zhu, Liangrui Zhong, Jianzhong Peng, EORGI c ,a the radices of Scutellaria baicalensis G , which is a com- Qiang Yuan, and Aie Xu* monly used herbal medicine. It is a traditional Chinese herbal a Department of Dermatology, Zhejiang Chinese Medical medicine with several pharmacological effects, such as anti- University, Third People’s Hospital of Hangzhou, Hangzhou oxidation, anti-tumor, and anti-inflammation, and so on.10–12) Institute of Dermatology and Venerology; Hangzhou 310053, In the present study, we exploited our animal model for of China: b Department of Dermatology, Zhejiang Chinese Medical vitiligo to evaluate the potential use of Baicalins cytotherapy c University; Hangzhou 310053, China: and Department of the to induce recovery of from vitiligo lesions and inhibit the oc- College of Pharmacy, Zhejiang Chinese Medical University; currence and development of vitiligo. Hangzhou 310053, China. Received April 12, 2019; accepted June 5, 2019; advance MATERIALS AND METHODS publication released online June 19, 2019

Vitiligo is a commom disease of skin. Its pathogenesis is Mice All animal procedures were performed according complex, resulting in the incapacity to find a targeted cure. to the Guide for the Care and Use of Laboratory Animals Baicalin, which is isolated from Scutellariae radix, has been published by the U.S. National Institutes of Health. Female known to exhibit a number of pharmacological effects on C57BL/6 mic (4–5 weeks old, 10–15 g) were obtained from autoimmune diseases. In this study, we explored the effects of SLRC Laboratory Animal (Shanghai, China). The mice were Baicalin on the recovery of vitiligo stimulated by monophe- kept at room temperature (20–25°C) and humidity (40–50%) nylketone in mice. We observed that Baicalin slowed down in microisolator cages with sufficient supply of food and the progression of depigmentation, decreased the incidence water. of depigmentation, and reduced the area of depigmentation. Vitiligo Model and Baicalin Transplantation Female Moreover, reflectance confocal microscopy (RCM) shown SPF C57BL/6 mice at 4 weeks of age, were divided into three that Baicalin increased the epidermal melanocytes in depig- mented skin. Baicalin decreased CD8 T cell infiltration groups randomly, groups I–III contained 15 mice in each in mice skin, and decreased the expression of CXCL10 and group and 50 mg of oil-in-water (O/W) emulsion base were CXCR3. Baicalin significantly decreased the levels of serum used respectively every day (I). In groups II and III, mice cytokine (interleukin [IL]-6, tumor necrosis factor [TNF]-α, were treated with 40% monobenzone cream (4-benzyloxyphe- interferon-γ [IFN-γ], and IL-13). Collectively, these data sug- nol, Sigma, U.S.A.) cream were prepared by the Hangzhou gest that Baicalin play an important role in the occurrence Third people’s Hospital Medical Centre pharmacy for use in and development of vitiligo. animal experiments, shaved 2 × 2 cm on the abdomen. Spatu- Key words Baicalin; monobenzone; vitiligo las were used to massage the creams completely. The mice were intraperitoneally injected with 2 mg of Baicalin (Sigma-

Aldrich) in 100 µL of 5% NaHCO3 aqueous solution every day in group III. INTRODUCTION Depigmentation Evaluation In treatment groups, the de- pigmentation degree was objectively quantified by observers. Vitiligo is a skin disorder that does not cause fatality but We examined each exposed location and estimated the depig- results in significant psychosocial consequences. It is char- mentation degree as a percentage of the anatomic site. Points acterized by the progressive depigmentation of skin sections. were awarded as follows: no evidence of depigmentation was The cause of this disease remains to be determined; how- defined as 0 point; >5% was defined as 1 point; >5–25% ever, various factors have been suggested to be involved in was defined as 2 points; >25–50% was defined as 3 points; its pathogenesis. Recent work has suggested that a specific >50–75% was defined as 4 points; and 75–100% was defined cytotoxic T-cell (CD8 + T cell) reaction is responsible for the as 5 points. The sum of the scores was the depigmentation destruction of melanocytes.1) Moreover, melanocyte-specific score. The average individual score of the mice in that group cytotoxic CD8 + T cells have been detected in the peripheral represents the vitiligo score of each experimental group. blood and skin lesions,2,3) and the quantity of melanocyte- Reflectance Confocal Microscopy (RCM) RCM (Viva- specific cytotoxic CD8 + T cells correlates with disease se- scope 1500) was performed for medical imaging applications verity.4–6) CXCL10 could combine with its specific receptor in vivo. Vivascope 1500 contained a diode laser at 830 nm CXCR3 to activate T cells and exhibit the function for me- wavelength and power <35 mW at the tissue level. It also has diating immune responses. CXCL10 and CXCR3 were over- a × 30 objective lens with 0.9 numerical aperture. To reduce expressed in various autoimmune diseases, and they involved motion artifacts, an adhesive ring allowed the xation of the in T cells homing into the diseased tissues to aggravate the optic to the tissue. The immersion medium between adhesive degree of tissue damage.7,8) Recent observations support that plastic window and skin was distilled water. and ultrasound cytokines acted as a vital factor in the nosogenesis of vitiligo. gel was filled between plastic window and lens owing to their Compared to healthy control skin, the expression of tumor refractive indexes close to the epidermis (1.34). Compared necrosis factor (TNF)-α, interferon-γ (IFN-γ), and interleukin with the surrounding skin structures (1.4), elanin has a high

* To whom correspondence should be addressed. e-mail: [email protected] © 2019 The Pharmaceutical Society of Japan Vol. 42, No. 9 (2019) Biol. Pharm. Bull. 1451 reflectance index (1.7) to produce a strong backscatter that vi- sualize the melanocytes and pigmented keratinocytes clearly. It could further observe the cellular structures of the speci- mens in vivo owing to the images with a spatial resolution of 0.5–1.0 mm in the lateral and 3–5 mm in the axial dimen- sion.13,14) Histological Examination In order to detect the tissues from the perilesional skin of the vitiligo lesions and healthy skin by using microscopic. Tissue samples were taken out and formalin-fixed. Then embedded in paraffin, sectioned, and stained with hematoxylin and eosin were conducted according to standard methods. Immunofluorescence for Confocal Laser-Scanning Mi- croscopy The perilesional skin of the vitiligo lesions and healthy skin were formalin-fixed and embedded in paraffin. Then the parafn-embedded section (4-µm) were deparaf- finized using xylene and ethanol, washed thrice for 2 min with distilled water and soaked for 5 min with phosphate buffered saline (PBS). Furthermore, antigen retrieval was performed in a microwave oven for 30 min and washed twice for 2 min using distilled water, After that, we washed antigen retrieval with PBS twice for 2 min, and blocked for 30 min with serum. After washing with PBS, the sections were incubated in the rabbit anti-CD8 monoclonal antibody (ZSGB-BIO, China) overnight at 4°C. After that the sections were washed with PBS thrice for 5 min and incubated with fluorescein isothio- cyanate-conjugated goat anti-mouse antibody (ZSGB-BIO) for 2 h and washed with PBS thrice for 5 min. At last, we mounted the sections under a no. 0 coverslip and using a glycerol-based mounting medium to cover sections. Confocal laser scanning microscop were carried out to capture the images of immuno- fluorescence. Cytokine Analysis Enzyme-linked immunosorbent as- says (ELISA) were performed to detected the cytokines levels in serum in recipient mice, including IL-6, TNF-α, IFN-γ, and IL-13 using commercial kits (MultiSciences Biotech Co., Ltd., China). RNA Isolation and Real-Time RT-PCR Analysis SV total RNA purification kit (Promega, Shanghai, China) was carried out to extract total RNA from the perilesional skin of thevitiligo lesions from all groups. QuantiTect Reverse Transcription Kit (Qiagen, Germany) was used to reverse Fig. 1. The Occurrence and Development of Vitiligo Were Delayed by transcript reaction. Then Real time PCR was carried out Baicalin in Mice using QuantiFast SYBR Green PCR Kit (Qiagen). β-Actin was (a–c) After grafting, representative images of the depigmentation at Days 40 performed as endogenous control. The results were analyzed were shown compared among the three groups. The C57BL/6 mice that treated with monobenzone cream developed depigmentation on the monobenzone exposed using the comparative Ct method, and expressed as percentage site (red circle) and nonexposed site (red arrow). (d) The depigmentation suppressed of control, with the control as 1. decreasingly in the mice occurred after treating with Baicalin. (e) Baicalin retarded the occurrence of vitiligo. (f) The depigmentation degree also alleviated due to Statistical Analysis All values are presented as Baicalin. # p < 0.05 vs. control group. * p < 0.05 vs. model group. (Color figure can means ± S.D. The differences between mean values of nor- be accessed in the online version.) mally distributed data were estimated by one-way ANOVA (Dunnett’s t-test) and two-tailed Student’s t-test. * p < 0.05 indicated significant differences were noted by ns. white patches (depigmentation of non-exposed sites) were visible on the trunk, ears, and/or tail of the majority of mice RESULTS on the 23rd day after monobenzone treatment (Figs. 1a–c). The incidence in the model group was 73.33%, much higher Intragastric Administration of Baicalin Promoted Re- than that of group III, which was 26.67% (Fig. 1d). There was mission, and Slowed Down the Occurrence and Progres- a remarkably difference between groups III and II. At non- sion of Vitiligo On the 15th day, in 11 of 15 mice in the exposed sites, we observed that the occurrence time and the model group, in the drug-exposed area we observed small incidence of depigmentation patches were delayed, and the white patches first and then gradually expanded, whereas no depigmentation area score in group III was decreased signifi- patches were discovered in group III. We found that distant cantly (Figs. 1e, f), indicating that Baicalin may prevent the 1452 Biol. Pharm. Bull. Vol. 42, No. 9 (2019) occurrence and growth of vitiligo. CD8 + T cells were observed in healthy skin (group I) (Fig. Intragastric Administration of Baicalin Promoted Mela- 3d). nocyte Formation in Vitiligo Lesions We further examined Baicalin Could Reduce Expression of Chemokine melanocyte content in mice using in vivo RCM. The dermal CXCL10 and Its Receptor CXCR3 As demonstrated in papillary containing melanocytes and pigmented keratinocytes Fig. 4, high expression of CXCL10 and CXCR3 were obtained appeared as bright refractive granules under RCM in the un- in the group II. Contrast to the model group (group II), obvi- treated mice (Fig. 2a). The dermal papillary of the skin with ously reduced levels of CXCL10 and CXCR3 was observed in depigmentation indicated lower number of bright granules in the drug-treating group (group III). The level of CXCL10 and group II, suggesting the decrease of melanocytes and pigment- CXCR3 was lower in the mice of the control group (group I). ed keratinocytes (Fig. 2b). In group III mice, the skin con- Whereas Baicalin could inhibit the increase of CXCL10 and taining melanocytes and pigmented keratinocytes, exhibited CXCR3. bright refractive granules and dendrites under RCM (Fig. 2c). Intragastric Administration of Baicalin Inhibited In- Intragastric Administration of Baicalin Inhibited Leu- flammatory Cytokines Cytokines (IL-6, TNF-α, IFN-γ, and komonocytes and CD8+ T Cells in Vitiligo Lesions Histo- IL-13), analyzed from serum samples after the application of chemical assay suggested that the leukomonocyte infiltration monobenzone cream, were significantly increased in mono- was increased in the perilesional skin of distant depigmenta- benzone challenged animals when compared with those of the tion in group II (Fig. 3b). Only a small amount of leukomono- untreated group. This reached a maximum at the 25th day, cyte infiltration was observed in in group I and III (Figs. 3a, when depigmentation on non-exposed sites became evident, c). and declined thereafter (Fig. 5). Baicalin treatment efficiently The infiltrating lymphocytes included a substantial amount prevented the monobenzone-induced increase in IL-6, TNF-α, of CD8+ T cells in group II were measured by immunofluores- IFN-γ, and IL-13 when compared with cytokine levels in cence staining with an anti-CD8 antibody(Fig. 3e). CD8 + T group II (Fig. 5). cells were significantly reduced in group III (Fig. 3f), and few DISCUSSION

Vitiligo is a common skin disorder characterized by pro- gressive depigmentation of the skin due to selective loss of melanocytes, yet few successful therapies are currently avail- able. Therefore, it is essential to find a safe and effective natu- ral medicines. Baicalin, a small-molecule monomer, whichis derived from the dried root of Scutellaria, exhibits anti-inflammatory, anti- Fig. 2. (a) Respectively Image of the Healthy Skin at the Level of the oxidant, anti-apoptotic, and antibacterial properties. Baicalin Dermoepidermal Junctionin Were Captured by in Vivo RCM (Group I); or berberine loaded ultradeformable vesicles, which were able (b) Bright Refractive Granules Were Observed in Model Group Show as to incorporate high amounts of Baicalin and berberine, and Melanocytes and Pigmented Keratinocytes (Red Arrows); (c) In Group III Mice, the Skin Consisting of Melanocytes and Pigmented Keratino- promote their skin permeation. It showed remarkable antioxi- cytes, Exhibited as Bright Refractive Granules and Dentrites under RCM dant and photoprotective capabilities, presumably correlated (Color figure can be accessed in the online version.) with the stimulation of melanin production and tyrosinase

Fig. 3. Hematoxylin and Eosin Staining Were Performed to Detected the Presence of Leukomonocyte, and Immunofluorescence Staining for CD8+ T Cells in Depigmentation Skin Sections (a) Few leukomonocytes were observed in the healthy skin. (group I) (b) The results demonstrated that the leukomonocyte levels were remarkably increased in the perilesional skin of the progressive vitiligo lesions. (c) Compared with group II, the leukomonocytes were significantly decreased in group III. (d) In the healthy skin, we found only few CD8+ T cells in group I. (e) In group II, the infiltrated lymphocytes contained a substantial amount of CD8+ T cells. (f) Compared with group II, the CD8+ T cells were significantly decreased in group III. (Color figure can be accessed in the online version.) Vol. 42, No. 9 (2019) Biol. Pharm. Bull. 1453

activity.15) Based on its anti-inflammatory activity, Baicalin is widely used to treat various inflammatory diseases including atopic dermatitis and asthma,16) It was reported that Baicalin could decrease expression of transforming growth factor-β1, IL-13 and vascular endothelial growth factor.17) Besides, a study on a mouse model of vitiligo induced by monobenzone has reported previously.18) In this study, tyrosinase expression is dramatically decreased in the mice Fig. 4. Real-Time PCR Were Carrried Out to Validate Differentially treated with monobenzone, which is similar to that in vitiligo Expressed Genes patients’ lesional skins. In healthy people who apply mono- The expression of CXCL10 and CXCR3 was lower in the mice of the control benzone to acquire a lightened skin tone, this process can group (group I) and the drug-treating group (group III). High expression of # induce depigmentation that is clinically and histologically CXCL10 and CXCR3 was observed in the group II. p < 0.05 vs. control group. 19) * p < 0.05 vs. model group. (Color figure can be accessed in the online version.) indistinguishable from vitiligo. We demonstrated that de- pigmentation of exposed and found non-exposed sites on the trunk, ears, and/or tail of mice after treating with prolonged, indicating a systemic immune reaction to autologous melano- cytes. Therefore, we used this model to detect Baicalin effect on the vitiligo. Baicalin could efficiently alleviate vitiligo se- verity in mice induced by monobenzone, through prolonging the time to depigmentation, reducing the ratio of the mice that developed depigmentation distantly, and alleviated the degree of depigmentation and redepigmentation. Based on these re- sults, Baicalin could be used as a prophylactic, mitigating, and therapeutic approach to vitiligo. It has been reported that histological analysis of perilesional margins surrounding depigmented skin displays infiltration of activated T cells and other lymphocytes.20) Further, It had been demonstrated that the infiltration of active CD8 + T cells takes place in the vitiligo perilesional margins.21) Furthermore, those CD8 + T cells had significantly higher activation levels and higher cytotoxicity to autologous melanocytes than their counterparts from peripheral blood samples.21) These stud- ies have suggested that cytotoxic CD8 + T cells play a major role in the pathogenic mechanism of vitiligo. In this study, we found that monobenzone-induced depigmentation in mice also induced a loss of melanocytes in the lesional skin and CD8 + T infiltration in the perilesional area, which was simi- lar to lesions of human vitiligo. Baicalin showed significant effects on monobenzone-stimulated histopathological changes in skin by reducing inflammatory infiltration, especially CD8 + T cell infiltration. The results reflect the idea that Ba- icalin could attenuate vitiligo severity by inhibiting CD8 + T cell proliferation and inducing CD8 + T cell apoptosis. It indi- cates that Baicalin could be a potential candidate for interfer- ence in depigmentation. C-X-C motif chemokine ligands CXCL9, CXCL10 and CXCL11, and its receptor CXCR3 are linked to the Th1 pat- tern. It has been suggested as one of the most relevant che- mokine axes, which promote T cell migration in different au- toimmune and inflammatory processes.22) It has been proved that CXCL9, CXCL10 and CXCR3 are elevated in vitiligo patients with an increased expression in active phase.23,24) The keratinocytes were reported to produce CXCL9 and CXCL10, which binds to its specific receptor CXCR3, could be able to attract CD8+cells.25) In further, we explored the expres- sion of CXCL10 and CXCR3. In consistent, highly increased Fig. 5. Effects of Baicalin on Concentrations of IL-6, TNF-α, IFN-γ, expression of CXCL10 and CXCR3 was found in the mice and IL-13 in Serum of Vitiligo Mice treated with monobenzone. Contrast to the model group, Bai- On days 15, 25, and 50, we collected the serum and the inflammatory cytokines calin could obviously reduce the expression of CXCL10 and IL-6 (a), TNF-α (b), IFN-γ(c), and IL-13 (d) were detected, which were significantly suppressed by Baicalin. # p < 0.05 vs. control group. * p < 0.05 vs. model group. CXCR3. (Color figure can be accessed in the online version.) More and more evidences shown that cytokines function as 1454 Biol. Pharm. Bull. Vol. 42, No. 9 (2019) a vital factor in the pathogenesis of autoimmunity and play a predominantly directed against MelanA/MART1. J. Invest. Derma- role in depigmentation.26,27) The study by Swope et al.28) indi- tol., 116, 891–897 (2001). cated that cytokines including IL-1α, TNF-α, and IL-6 could 6) Mandelcorn-Monson RL, Shear NH, Yau E, Sambhara S, Barber BH, Spaner D, DeBenedette MA. Cytotoxic T lymphocyte reactiv- inhibit melanocyte proliferation. The generation of proinflam- ity to gp100, MelanA/MART-1, and tyrosinase, in HLA-A2-positive matory cytokines including IL-1b, IL-6, IL-8, and TNF-α vitiligo patients. J. Invest. Dermatol., 121, 550–556 (2003). in patients with active vitiligo were remarkably enhanced 7) Ibrahim HM, El-Elaimy IA, Saad Eldien HM, Badr BM, Rabah 29) compared with the control group. Increased concentrations DM, Badr G. Blocking type I interferon signaling rescues lympho- 30) of serum IL-13 are observed in vitiligo patients. Therefore, cytes from oxidative stress, exhaustion, and apoptosis in a strepto- it is very vital for treatment of vitiligo to decrease inflamma- zotocin- induced mouse model of type I diabetes. Oxid. Med. Cell. tory cytokines. In the present study, we revealed increased Longev., 2013, 148725 (2013). levels of serum IL-6, TNF-α, IFN-γ, and IL-13 in the model 8) Groom JR, Luster AD. CXCR3 ligands: redundant, collaborative group, supporting the hypothesis that cytokines may involve and antagonistic functions. Immunol. Cell Biol., 89, 207–215 (2011). in immunologic events in the pathogenesis of vitiligo. We also 9) Grimes PE, Morris R, Avaniss-Aghajani E, Soriano T, Meraz M, found that Baicalin could dramatically inhibit IL-6, TNF-α, Metzger A. Topical tacrolimus therapy for vitiligo: therapeutic re- sponses and skin messenger RNA expression of proinflammatory IFN-γ, and IL-13. According to the results, Baicalin may be cytokines. J. Am. Acad. Dermatol., 51, 52–61 (2004). benefit for inhibiting cytokine secretion in vitiligo. 10) Gao Z, Huang K, Xu H. Protective effects of flavonoids in the roots In summary, it is demonstrated that Baicalin has a potential of Scutellaria baicalensis Georgi against hydrogen peroxide-induced therapeutic effect on depigmentation. Baicalin slowed down oxidative stress in HS-SY5Y cells. Pharmacol. Res., 43, 173–178 the appearance of depigmentation, lessened depigmentation (2001). incidence, and decreased the depigmentation area. Baicalin 11) Ikezoe T, Chen SS, Heber D, Taguchi H, Koeffler HP. Baicalin is significantly receded histopathologic changes in the mice a major component of PC-SPES which inhibits the proliferation of skins and inhibited the infiltration of CD8+ T cells and ex- human cancer cells via apoptosis and cell cycle arrest. Prostate, 49, pression of chemokines CXCL10 and CXCR3. It also showed 285–292 (2001). that Baicalin could significantly inhibit the level of inflam- 12) Shen YC, Chiou WF, Chou YC, Chen CF. Mechanisms in mediating the anti-inflammatory effects of baicalin and baicalein in human matory mediators. Our data indicated that Baicalin may be a leukocytes. Eur. J. Pharmacol., 465, 171–181 (2003). potential agent for vitiligo treatment. 13) Rajadhyaksha M, Grossman M, Esterowitz D, Webb RH, Anderson RR. In vivo confocal scanning laser microscopy of human skin: Acknowledgments This study was supported in part by melanin provides strong contrast. J. Invest. Dermatol., 104, 946– the Natural Science Foundation of Zhejiang, China (Grant No. 952 (1995). LQ16H290001), the Chinese Medicine Science and Technol- 14) Yamashita T, Kuwahara T, Gonza’lez S, Takahashi M. Non-invasive ogy Project of Zhejiang (No. 2019ZB104 2019ZA095), the Sci- visualization of melanin and melanocytes by reflectance-mode con- ence Foundation of Ministry of zhejiang Health Department focal microscopy. J. Invest. Dermatol., 124, 235–240 (2005). (Grant No. 2019RC072), the Postdoctoral Science Foundation 15) Mir-Palomo S, Nácher A, Ofelia Vila Busó MA, Caddeo C, Manca of China (No. 2018M642494), the National Natural Science ML, Manconi M, Díez-Sales O. Baicalin and berberine ultradeform- Foundation of China (Grant No. 81071294). able vesicles as potential adjuvant in vitiligo therapy. Colloids Surf. B Biointerfaces, 175, 654–662 (2019). 16) Chen YC, Shen SC, Chen LG, Lee TJ, Yang LL. Wogonin, baicalin, Conflict of Interest The authors declare no conflict of and baicalein inhibition of inducible nitric oxide synthase and interest. cyclooxygenase-2 gene expressions induced by nitric oxide syn- thase inhibitors and lipopolysaccharide. Biochem. Pharmacol., 61, REFERENCES 1417–1427 (2001). 17) Sun J, Li L, Wu J, Liu B, Gong W, Lv Y, Luo Q, Duan X, Dong J. 1) Rezaei N, Gavalas NG, Weetman AP, Kemp EH. Autoimmunity as Effects of baicalin on airway remodeling in asthnatic mice. Planta an aetiological factor in vitiligo. J. Eur. Acad. Dermatol. Venereol., Med., 79, 199–206 (2013). 21, 865–876 (2007). 18) Zhu YP, Wang SQ, Xu AE. A mouse model of vitiligo induced by 2) Palermo B, Campanelli R, Garbelli S, Mantovani S, Lantelme E, monobenzone. Exp. Dermatol., 22, 499–501 (2013). Brazzelli V, Ardigó M, Borroni G, Martinetti M, Badulli C, Necker 19) Grojean MF, Thivolet J, Perrot H. Acquired leukomelanoderma A, Giachino C. Specific cytotoxic T lymphocyte responses against caused by topical depigmenting agents. Ann. Dermatol. Venereol., Melan-A/MART1, tyrosinase and gp100 in vitiligo by the use of 109, 641–647 (1982). major histocompatibility complex/peptide tetramers: the role of cel- 20) Badri AM, Todd PM, Garioch JJ, Gudgeon JE, Stewart DG, Goudie lular immunity in the etiopathogenesis of vitiligo. J. Invest. Derma- RB. An immunohistological study of cutaneous lymphocytes in vit- tol., 117, 326–332 (2001). iligo. J. Pathol., 170, 149–155 (1993). 3) Le Gal FA, Avril MF, Bosq J, Lefebvre P, Deschemin JC, Andrieu 21) Wu J, Zhou M, Wan Y, Xu A. CD8+ T cells from vitiligo perile- M, Dore MX, Guillet JG. Direct evidence to support the role of sional margins induce autologous melanocyte apoptosis. Mol. Med. antigen-specific CD8(+) T cells in melanoma-associated vitiligo. J. Rep., 7, 237–241 (2013). Invest. Dermatol., 117, 1464–1470 (2001). 22) Lacotte S, Brun S, Muller S, Dumortier H. CXCR3, inflammation, 4) Ogg GS, Rod Dunbar P, Romero P, Chen JL, Cerundolo V. High and autoimmune diseases. Ann. N. Y. Acad. Sci., 1173, 310–317 frequency of skin-homing melanocyte-specific cytotoxic T lympho- (2009). cytes in autoimmune vitiligo. J. Exp. Med., 188, 1203–1208 (1998). 23) Rashighi M, Agarwal P, Richmond JM, Harris TH, Dresser K, Su 5) Lang KS, Caroli CC, Muhm A, Wernet D, Moris A, Schittek B, MW, Zhou Y, Deng A, Hunter CA, Luster AD, Harris JE. CXCL10 Knauss-Scherwitz E, Stevanovic S, Rammensee HG, Garbe C. is critical for the progression and maintenance of depigmentation HLA-A2 restricted, melanocyte-specific CD8(+) T lymphocytes in a mouse model of vitiligo. Sci. Transl. Med., 6, 223ra23 (2014). detected in vitiligo patients are related to disease activity and are 24) Wang XX, Wang QQ, Wu JQ, Jiang M, Chen L, Zhang CF, Xiang Vol. 42, No. 9 (2019) Biol. Pharm. Bull. 1455

LH. Increased expression of CXCR3 and its ligands in patients with disorders. Int. Rev. Immunol., 17, 217–228 (1998). vitiligo and CXCL10 as a potential clinical marker for vitiligo. Br. 28) Laddha NC, Dwivedi M, Begum R. Increased Tumor Necrosis Fac- J. Dermatol., 174, 1318–1326 (2016). tor (TNF)-α and its promoter polymorphisms correlate with disease 25) Richmond JM, Bangari DS, Essien KI, Currimbhoy SD, Groom JR, progression and higher susceptibility towards vitiligo. PLOS ONE, Pandya AG, Youd ME, Luster AD, Harris JE. Keratinocyte-derived 7, e52298 (2012). chemokines orchestrate T cell positioning in the epidermis during 29) Swope VB, Abdel-Malek Z, Kassem LM, Nordlund JJ. Interleukins1 vitiligo and may serve as biomarkers of disease. J. Invest. Derma- alpha and 6 and tumor necrosis factor-alpha are paracrine inhibitors tol., 137, 350–358 (2017). of human melanocyte proliferation and melanogenesis. J. Invest. 26) Moretti S, Spallanzani A, Amato L, Hautmann G, Gallerani I, Dermatol., 96, 180–185 (1991). Fabiani M, Fabbri P. New insights into the pathogenesis of vitiligo: 30) Zailaie MZ. Decreased proinflammatory cytokine production by imbalance of epidermal cytokines at sites of lesions. Pigment Cell peripheral blood mononuclear cells from vitiligo patients following Res., 15, 87–92 (2002). aspirin treatment. Saudi Med. J., 26, 799–805 (2005). 27) Feldmann M, Brennan FM, Maini R. Cytokines in autoimmune