Regulating the Wnt/ β -catenin Signaling Pathway Promotes Repigmentation in Vitiligo Using Fire Needle Therapy

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Introduction
Vitiligo is an autoimmune disorder characterized by the progressive loss of melanocytes in the epidermis with an estimated global prevalence of between 0.5% and 2% [1].Te typical lesion of vitiligo is well-demarcated, nonscaly milky white macules.It can occur anywhere in the body.Te disorder can signifcantly afect the patient's quality of life, potentially leading to the development of mental health conditions, such as anxiety [2] and depression [3].Te current primary treatment strategies for vitiligo involve the systemic administration of glucocorticoids, local application of calcineurin inhibitors, and vitamin D3 derivatives.In addition, localized phototherapy is widely employed, including, narrowband ultraviolet B (NBUVB) and 308-nm excimer laser therapy [4].However, these treatments often fall short of their desired efcacy.Hence, the exploration of novel treatments for vitiligo is imperative.
Clinical observations during the treatment of vitiligo have highlighted perifollicular repigmentation patterns, where repigmentation spots emerge around hair follicles and gradually expand.Tis suggests that the hair follicle bulge serves as a melanocyte reservoir [5].Melanocyte stem cells within the hair follicle may be induced to proliferate, differentiate, and migrate, thus replenishing the depigmented epidermis [6].Te Wnt/β-catenin signalling pathway, also known as canonical Wnt signalling, plays a pivotal role in cell development and proliferation [7].It is central to the proliferation and diferentiation of these melanocyte stem cells (McSCs) [8,9].Te diferentiation of McSCs in both epidermal and hair pigmentation processes necessitates the activation of the Wnt/β-catenin signalling [10,11].Decreased β-catenin expression across the epidermal layer of vitiligo lesions has been observed by immunofuorescence staining assays [12].Moreover, Wnt signalling regulates hair follicle signalling in the epidermis by coordinating dynamic intercellular communication between the epidermal and dermal layers [13].Tis indicates that the activation of the Wnt signalling pathway might play a role in vitiligo repigmentation.
Fire needle therapy (FNT) is an ancient practice in traditional Chinese medicine and is utilized as a symptomatic treatment for vitiligo.FNT employs a slender needle which can withstand high temperatures.Te needles are heated over an open fame until red-hot and are then swiftly inserted into vitiligo lesions [14].Clinically, the technique has exhibited notable therapeutic outcomes [15].In a mouse model of androgenetic alopecia treatment, fre needles stimulated hair growth by activating the Wnt/β-catenin signalling pathway, thereby promoting the proliferation and diferentiation of hair follicle melanocyte stem cells [16].Te hair follicle bulge, as a melanocyte reservoir, plays a significant role in vitiligo repigmentation.Currently, research exploring the relationship among the Wnt/β-catenin signalling pathway, hair follicle melanocyte stem cells, and the pigment deposition induced by fre needles in vitiligo is lacking.We hypothesize that FNT may promote the recolouration of vitiligo lesions through this pathway.We tested this hypothesis by comparing the gene expression associated with the Wnt/β-catenin signalling pathway in normal skin to that in the lesions of patients with vitiligo in the Gene Expression Omnibus database (GEO database).Te database is an international public repository of functional genomic datasets, including high-throughput microarray, and next-generation sequencing data, all submitted by the research community.Te data is indexed, cross-linked, and searchable [17].Furthermore, we used a vitiligo mouse model to investigate the impact of FNT on pigmentation and its infuence on the Wnt/β-catenin signalling pathway.

Bioinformatics Analysis.
We procured normalized total RNA expression levels from skin samples of vitiligo patients through the GEO database (with the accession numbers GSE75819 and GSE65127).Tis study conducted a diferential gene expression analysis focusing on the Wnt/ β-catenin signalling pathway in vitiligo lesions compared to a control group, and the results were validated using the bootstrap hypothesis test.

Establishment of the Vitiligo Model and Groups.
C57BL/6J male mice (7 weeks old) were purchased from the Animal Center at Weifang Medical College, Weifang, China.All research procedures were approved by the Weifang Medical Ethics Committee (project number 2022SDL004).
Te vitiligo model was created following established protocols [18].Briefy, mice were subcutaneously immunized in the hind footpad once a week for two weeks with TRP2-180 (50 μg; Anaspec, Fremont, USA), lipopolysaccharide (LPS) (5 μg; InvivoGen, SanDiego, USA), and CpGoligodeoxynucleotide (CpG-ODN) (5 μg; InvivoGen).Subsequently, mice were intradermally injected twice in the tail with a one-week interval.After the fnal injection, the emergence of depigmentation in the tail area was witnessed over a span of four weeks.
Te mice were randomly divided into four groups: Blank (n � 10), Iwr-1 (n � 10), Fire needle (n � 10), and Sham (n � 10).Te fre needle group underwent fre needles once weekly for four weeks.In contrast, the Iwr-1 group received Iwr-1 (TargetMOL, Wellesley Hills, MA, USA) injections into the depigmented tail skin once weekly for four weeks.As previously reported [10], a 10 mM stock solution of Iwr-1 in DMSO was diluted with phosphate-bufered saline (PBS) to 0.1 mM and subsequently administered by injection into the tail skin of mice (4 μg per cm 2 ).Te aim was to inhibit the Wnt signal pathway.Te Sham treatment group did not receive any interventions post-modelling, while the Blank group remained procedure-free.
2.3.Fire Needle Terapy.Te fre needle (1.5 inches in length, 0.25 mm in diameter) was applied to vitiligo lesions.Mice were immobilized without anaesthesia using a specialized apparatus after sterilizing the tail skin.An alcohol lamp was positioned close to the tail skin lesion.Te fre needle tip and body were heated until they turned red.Te designated point was swiftly pricked with the needle which was then promptly withdrawn.Te fre needle was used at a depth of 0.5-1 mm, with a stimulation frequency of 30 times per square centimetre.

Haematoxylin and Eosin (H&E) Staining and Fontana-Masson
Staining.Te mice were euthanized using cervical dislocation, and their tail skin was shaved.Subsequently, the lesional skin and tail skin were fxed in 4% paraformaldehyde and embedded in parafn.Tissue sections (4 μm) were stained with H&E.Fontana-Masson staining was carried out following the instructions provided for the Masson-Fontana melanin staining solution (Shangbao, Shanghai, China).After deparafnization, tissue sections were hydrated and then exposed to the working Fontana silver ammonia solution at a temperature of 56 °C for 40 minutes.Following this, the sections underwent treatment with sodium hypochlorite solution (Shangbao) for one minute, and fnally, they were exposed to neutral red solution for fve minutes.

Quantitative Real-Time PCR (qPCR).
Total RNA was extracted from vitiligo mice tail lesions using TRIzol reagent (Vazyme, Nanjing, China), according to the manufacturer's instructions to ensure efcient RNA isolation.Te HiScript III RT SuperMix (Vazyme) was used for cDNA synthesis.Quantitative real-time PCR was conducted employing the SYBR qPCR Master Mix (Vazyme), following the protocol provided by the manufacturer.Te GAPDH gene was utilized as the reference gene, and data analysis was conducted by employing the 2 −ΔΔCT method (Table 1).

Statistical Analysis.
Te data were expressed as the mean ± standard deviation (SD).Statistical analyses were conducted using SPSS Statistics version 22.0.Two-group comparisons were performed using a t-test, and for comparisons involving more than two groups, a one-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test was applied.p < 0.05 was considered statistically signifcant.

Gene Expression Analysis of the Wnt/β-Catenin Signalling
Pathway in Lesions from Patients with Vitiligo.To comprehensively examine the status of the Wnt/β-catenin signalling pathway in vitiligo lesions, we analyzed publicly available gene array data from the GEO database (Accession number: GSE75819).Tis data included mRNA expression levels of altered components within the Wnt/β-catenin signalling pathway in skin biopsies obtained from both normal skin and vitiligo lesions in 15 patients.Upon comparing the mRNA expression levels with those of normal skin samples, we discerned a marked reduction in the transcript levels of numerous pivotal components in the lesions of vitiligo.Notably, the mRNA levels of WNT10A, WNT10B, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5B, and WNT7B displayed signifcant decreases in vitiligo lesions skin (Figure 1(a)).We explored a further gene array dataset from the GEO database (Accession number: GSE65127).In this gene array, we analyzed the gene expression patterns of the Wnt/β-catenin signalling pathway in lesional, peri-lesional, and nonlesional skin samples from 10 vitiligo patients and 10 healthy controls.Interestingly, our analysis revealed that the mRNA levels of WNT10B and WNT11 were signifcantly diminished in lesional samples compared to those in healthy skin samples.Notably, a distinctive decrease was also observed in the expression of LEF1 mRNA in lesional vitiligo skin when compared to nonlesional vitiligo samples and healthy skin samples (Figure 1(b)).To further assess the stability of the results, the bootstrap hypothesis test was used for internal validation.Te bootstrap hypothesis test is known for its efcacy in estimating the distribution of a statistic based on random resampling with replacement.We conducted a bootstrap hypothesis test to perform the sampling 1000 times to assess the diferences between the main genes, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5B, WNT7B, WNT10A, WNT10B, WNT11, and LEF1.We found stable gene expression results in dataset GSE75819.However, genes WNT10B and WNT11 were excluded from dataset GSE65127 due to unstable expression.Te diference between the LEF1 expression levels in the lesion and    2).Tis analysis was performed using the R (4.3.2).Furthermore, our exploration encompassed gene ontology (GO) enrichment (Figure 1(c)) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment (Figure 1(d)) analysis of the down-regulated genes.Tis analysis underscored the signifcant involvement of the Wnt/β-catenin signalling pathway in the intricate developmental processes underlying vitiligo.

Establishment of a Vitiligo Mouse Model and Reduced Wnt/β-Catenin Pathway-Related Gene Expression in Lesions.
Mice received two immunization cycles using TRP2, LPS, and CpG-ODN sequentially delivered to the hind footpad and tail skin at one-week intervals (Figure 2 In normal skin, keratinocytes and melanocytes stimulate the Wnt pathway, promoting diferentiation and proliferation of melanocyte stem cells.Tis dynamic process ensures a continual turnover of epidermal melanocyte pools.In vitiligo lesions, oxidative stress diminishes Wnt pathway activity in both melanocytes and keratinocytes.Tis reduction may impede the diferentiation and proliferation of melanocyte stem cells, consequently contributing to active depigmentation of the skin and rendering resistance to repigmentation strategies.(b) Scatter plot showing the expression level of Wnt signal path-related down-regulated genes in vitiligo skin, compared with that of normal skin.Te expression values were obtained from the NCBI GEO (accession number: GSE75819).Fifteen biopsies were taken from normal skin and vitiligo skin.(c) Scatter plot showing the expression levels of down-regulated genes associated with the Wnt signalling pathway in vitiligo skin, compared with that of healthy skin, perilesional skin, or non-lesional skin.Te expression values were acquired from the NCBI GEO (accession number: GSE65127).Ten samples of healthy skin and ten from patients with vitiligo were collected from lesional, peri-lesional and nondiseased skin.Gene ontology enrichment (d) and kyoto encyclopedia of genes and genomes enrichment (e), the red wireframe represents the pathway associated with the Wnt signal pathway.Data are mean ± SEM. * p < 0.05, * * p < 0.01, * * * p < 0.001, Signifcant diferences are indicated.ns p > 0.05 no signifcant diference.Histological evaluations using H&E and Fontana-Masson staining conclusively showed that FNT enhances melanocyte proliferation and melanin production (Figures 3(c) and 3(d)).

Efect of Fire Treatment on the Wnt/β-Catenin Signalling
Pathway.We analyzed the expression of the Wnt/β-catenin signalling pathway after fre FNT in mice model.Dual immunofuorescence staining showed nuclear β-catenin accumulation in the melanocyte marker DCT (Figure 4(a)).We noticed a signifcant rise in DCT+β-catenin + cells in the lesions of fre needle-treated mice relative to mice in the Sham group.Tese cells were primarily located around hair follicles.Interestingly, the Iwr-1 group showed no DCT+β-Catenin + expression, while the Sham group exhibited DCT+βcatenin-cells, pointing to the presence of melanocytes but the absence of β-catenin.We conducted a detailed analysis of the expression levels of melanogenesis-associated genes (Trp1, Trp2, Tyr, Mitf) and genes related to Wnt/β-catenin signalling (Wnt3, Wnt3a, Wnt10b, Lef1, Ctnnb1) (Figure 4(b)).Te results showed elevated levels of these genes in the treatment group relative to the Sham group, while the inhibitor group   Dermatologic Terapy exhibited a notable reduction.Additionally, protein concentrations of TRP1, TRP2, MITF, Wnt3, CTNNB, and LEF1 were higher in the treatment group (Figure 4(c)).

Discussion
Vitiligo is an autoimmune skin disease that targets melanocytes, resulting in the appearance of depigmented white patches on the skin [19].Tere are multiple mechanisms believed to be responsible for the loss of melanocytes, including genetic predisposition, environmental triggers, and immune-mediated responses [20].Current therapies for vitiligo primarily focus on the promotion of the accumulation of pigment in afected areas.Clinically, the most common pattern of repigmentation in human vitiligo is a perifollicular distribution, characterized by the presence of black pigment dots surrounding hair follicles.Tis suggests that hair follicles are the main source of repigmentation [5,21].Moreover, McSCs in hair follicles can diferentiate into epidermal melanocytes in mice after injuries or UVB treatment [22].Tese studies indicate that follicular melanocyte stem cells (McSCs) function as supplementary reservoirs for melanocytes, playing a crucial role in vitiligo repigmentation.Te Wnt/β-catenin signalling pathway can regulate a variety of biological processes, including regulating stem cell pluripotency, cell migration, self-regeneration, and cell fate determination [23,24].Tis pathway is central to melanocyte biology.Te multifunctional kinase GSK3β is displaced upon activation of the Wnt receptor complex.As a result, cytosolic β-catenin undergoes stabilisation and is translocated to the nucleus.In the nucleus, β-catenin forms complexes with the lymphoid-enhancing factor-1/T-cell factor transcription factor, leading to an upregulation of MITF expression consequently promoting melanogenesis [24,25].In healthy skin, keratinocytes and melanocytes activate the Wnt pathway, promoting the diferentiation and proliferation of melanocyte stem cells and ensuring a continuous renewal of the epidermal melanocyte population [26].Trough the analysis of publicly available gene array data from patients with vitiligo, we found that the gene expression of WNT10A, WNT10B, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5B, WNT7B, and LEF1 were downregulated in the vitiligo skin lesions.Tis suggests the possibility that Wnt signalling is inhibited.Previous research using immunofuorescence staining assays supports the idea that β-catenin expression is reduced in the entire epidermal layer of vitiligo lesions [12].Vitamin D analogues enhance repigmentation in vitiligo by protecting melanocytes from oxidative damage through Wnt/β-catenin pathway activation [27].Ex vivo studies have shown that Wnt agonists can stimulate resident stem cells to diferentiate into premelanocytes in vitiligo lesion [26].Consequently, targeting the Wnt signalling pathway holds potential in relation to vitiligo treatment.NB-UVB, an efective and common approach for vitiligo management, operates by activating melanocyte stem cells, which are considered the reservoir of melanocytes, during the therapeutic process.Tis mechanism is believed to be mediated by the action of the Wnt signalling [10].During the wound healing process, melanocytes are recruited to the wound site, resulting in pigmentation.Tis phenomenon is primarily attributed to the infuence of the Wnt signalling on McSCs [28].Here, we observed a reduction in lesion size and an increase in pigmentation post-FNT in our vitiligo mouse model.Importantly, we noted an increase in melanocytes and melanin in vitiligo lesions.Tis supports the idea that bulge McSCs relocate from the hair follicle to the epidermis following a fre needle.
FNT, an external treatment modality used in traditional Chinese medicine with historical roots dating back to the time of the Yellow Emperor (475−221 BC), has shown promise in promoting repigmentation in vitiligo [29].FNT is distinguished by its simplicity and afordability.Although the treatment requires weekly hospital visits, increasing patient attendance, its integration with conventional treatments including topical medications and phototherapy signifcantly boosts outcomes and shortens therapy duration.Systematic reviews and meta-analyses have shown that treatment including fre needles had a signifcantly therapeutic efect in a shorter time than traditional methods without fre needles [30,31].Moreover, the costefectiveness of FNT coupled with its synergistic benefts when used alongside other treatments, elevates its clinical utility [32].In terms of safety, several patients experienced minor adverse reactions, such as local itching, erythema, and, less commonly, blistering at the puncture sites.Tese reactions may be due to the procedure, the individual condition of the patient, or an infection caused by contact with foreign bodies after the procedure.Systematic evaluations have confrmed that the incidence of these adverse efects is comparable to control groups including topical medications and phototherapy [32,33], indicating a favorable safety profle for FNT.In our study, the mice in the fre needle group showed redness at the sites of needle insertion after treatment.Bleeding and scab formation may occurr in few cases; however, the scabs may fall of within one week.
FNT acts as a controlled injury method, drawing parallels with contemporary treatments such as CO2 fractional laser and microneedling [34,35].In addition, Wnt ligands in epithelial cells activate Wnt signalling in epidermal melanocytes during wound healing [28].Interestingly, our study found that there is an increase in the expression levels of WNT3A, β-CATENIN, and LEF1 proteins, as well as the mRNA levels of Wnt3, Wnt3a, Wnt10b, Lef1, and Ctnnb1 after fre needle.Tis suggests that FNT may promote the proliferation and diferentiation of McSCs by activating the Wnt/β-catenin signal pathway, increasing the production of melanin, and promoting the recovery of vitiligo.Tis research contributes to the growing body of evidence supporting the efcacy of traditional Chinese medicine in the treatment of vitiligo.
Previous studies have shown the mouse model displays epidermal depigmentation while melanocytes remain within the hair follicles, and the model may be studied in the mechanisms of repigmentation [18].However, our mouse model cannot fully refect the complexity and heterogeneity of the human condition which is a limitation of our study.For example, the epidermis of human skin is thick and multilayered, with a prominent stratum corneum, whereas mouse skin has a thinner and less stratifed epidermis [36].Terefore, the response to FNT may difer.Te depth and density of FNT should be adjusted according to individual conditions for clinical use.While our vitiligo models ofer valuable insights into the mechanism of FNT, it is essential to corroborate the fndings in human-based systems to ensure their clinical relevance which need to be further studied.
In conclusion, our study provides novel evidence that FNT efectively regulates the Wnt/β-catenin signalling pathways to promote pigmentation in vitiligo.Tis research contributes to the growing body of evidence supporting the efcacy of traditional Chinese medicine in the treatment of vitiligo.

Figure 1 :
Figure 1: Expression of Wnt/β-catenin signalling pathway in skin lesions of vitiligo.(a) Mechanism diagram.In normal skin, keratinocytes and melanocytes stimulate the Wnt pathway, promoting diferentiation and proliferation of melanocyte stem cells.Tis dynamic process ensures a continual turnover of epidermal melanocyte pools.In vitiligo lesions, oxidative stress diminishes Wnt pathway activity in both melanocytes and keratinocytes.Tis reduction may impede the diferentiation and proliferation of melanocyte stem cells, consequently contributing to active depigmentation of the skin and rendering resistance to repigmentation strategies.(b) Scatter plot showing the expression level of Wnt signal path-related down-regulated genes in vitiligo skin, compared with that of normal skin.Te expression values were obtained from the NCBI GEO (accession number: GSE75819).Fifteen biopsies were taken from normal skin and vitiligo skin.(c) Scatter plot showing the expression levels of down-regulated genes associated with the Wnt signalling pathway in vitiligo skin, compared with that of healthy skin, perilesional skin, or non-lesional skin.Te expression values were acquired from the NCBI GEO (accession number: GSE65127).Ten samples of healthy skin and ten from patients with vitiligo were collected from lesional, peri-lesional and nondiseased skin.Gene ontology enrichment (d) and kyoto encyclopedia of genes and genomes enrichment (e), the red wireframe represents the pathway associated with the Wnt signal pathway.Data are mean ± SEM. * p < 0.05, * * p < 0.01, * * * p < 0.001, Signifcant diferences are indicated.ns p > 0.05 no signifcant diference.

Figure 2 :
Figure 2: Development of the vitiligo mouse model and the expression of related genes.(a) Immunization schedule.C57BL/6J mice were immunized subcutaneously twice at a one-week interval into the hind footpad with TRP2-180, LPS and CpG ODN.One week after the same adjuvants were injected twice intradermally into the tail, again at a one-week interval (Figure 2(a)).After four weeks of fnal immunization, we observed depigmented skin lesions around the tail injection site.(b) Developed depigmented skin lesions in the tail at week seven.(c) Representative pictures show haematoxylin and eosin staining in the tail sections of the control and vitiligo mouse model (Scale bar � 100 μm).(d) Te mRNA expression levels of Trp1, Trp2, Tyr, Mitf, Wnt3, Wnt3a, Wnt10b, Lef1, Ctnnb1 in the vitiligo model mouse skin and control mouse skin by qRT-PCR.Data are mean ± SEM. * p < 0.05, * * p < 0.01, * * * p < 0.001, signifcant diferences are indicated.ns p > 0.05 no signifcant diference.

Figure 3 :
Figure 3: Fire needling induces vitiligo repigmentation.(a) Representative images showing the changes before and after treatment.Te mice were divided into four groups: the blank group, the inhibitor (Iwr-1) group, the fre needle group, and the sham treatment group.(b) Comparison of the fnal percentage change in pigmentation among the blank group, Iwr-1 group, fre needle group, and sham group.(c) Haematoxylin and eosin staining was performed on the tail skins of mice in each group (bar � 100 μm).(d) Fontana-Masson staining showing the amount and distribution of melanin in each group (bar � 100 μm).

Figure 4 :
Figure 4: Efect of FNT on the Wnt/β-catenin signalling pathway.(a) Dual immunostaining reveals the accumulation of β-catenin in the migrated DCT (+) melanocytes within each vitiligo lesion skin, as indicated by arrowheads and related quantifcations.Te yellow arrow represents DCT (+) β-catenin (−) n � 5, scale bar � 50 μm.(b) qRT-PCR analysis was performed on key factors of the Wnt/β-catenin pathway and genes related to melanin synthesis in the blank group, inhibitor group, treatment group, and sham group.(c) Te protein expressions of TRP1, TRP2, MITF, WNT3, β-catenin, and LEF1 in the tail skin were detected using western blot analysis and quantifcations of the protein expressions.Te statistical signifcance was represented as * p < 0.05, * * p < 0.01, * * * p < 0.001.Signifcantly diferent as indicated.ns p > 0.05 no signifcant diference.Tere were fve samples in each group for the qRT-PCR test.

Table 1 :
Primer used for quantitative real-time PCR.

Table 2 :
Te results of bootstrap hypothesis test.