Exploring the potential of intradermal platelet‐rich plasma in treating acquired bilateral nevus of Ota‐like macule (Hori's nevus): A pilot study

Hori's nevus is a common and challenging dermatological condition, often complicated by post‐inflammatory hyperpigmentation following treatment. Platelet‐rich plasma (PRP) has demonstrated efficacy in the treatment of hyperpigmentation disorders such as melasma and periorbital darkening. Given the benefits and minimally invasive nature of PRP treatments, exploring its application in managing Hori's nevus through further investigation is worthwhile.

Acquired bilateral nevus of Ota-like macules (ABNOM, also known as Hori's nevus) is a pigmentary skin disorder that commonly affects middle-aged Asian women with Fitzpatrick skin type II to IV. 1,2 Clinically, Hori's nevus is characterized by the presence of bilateral symmetrical multiple blue-brown and/or slate-gray macules on the malar regions and, less commonly, on the forehead, upper eyelids, temple, root, and ala of the nose.In the early stages, discrete brown macules are the most common presentation, while in the late stages, confluent slate-gray macules are present. 2,3Histopathologically, Hori's nevus displays deeply pigmented melanocytes and melanophages in the superficial dermal layer, and is characterized by their elongated, slender, and irregularly shaped highly dendritic appearance. 4Differential diagnoses include nevus of Ota, nevus of Ito, congenital dermal melanocytoses, melasma, and ephelides, which may coexist with Hori's nevus. 2,5,6though the etiology of Hori's nevus remains unclear, there are three primary mechanisms believed to underlie its development.The first mechanism involves the migration of epidermal melanocytes and hair bulb melanocytes to the dermis, though the reasons for this migration remain elusive.The second mechanism entails the reactivation of preexisting dermal melanocytes, a process that can be triggered by factors such as sun exposure, dermal inflammation, the degeneration of the epidermis and dermis, and the natural aging process, collectively inducing dermal melanogenesis. 1,7Finally, the third mechanism centers on an increase in melanogenic cytokines in the dermis.These cytokines are released by dermal fibroblasts and dermal mast cells, including stem cell factor (SCF)/c-kit and hepatocyte growth factor (HGF).These mechanisms cooperatively contribute to our understanding of Hori's nevus etiology. 8While Hori's nevus is not life-threatening, it can be cosmetically noticeable and may impact an individual's self-esteem.
Currently, there are several treatment options for Hori's nevus including lasers and dermabrasion.However, these treatments have varying degrees of success and can result in complications such as irritation, erythema, prolonged recovery, and post-inflammatory hyperpigmentation (PIH). 7,9Therefore, constant efforts to find effective therapies for Hori's nevus are still ongoing.
Platelet-rich plasma (PRP) is a blood-derived product characterized by a high concentration of platelets which play a pivotal role in the area of wound healing.Notably, platelets harbor a rich reservoir of growth factors that exert a significant influence not only in hemostasis but also in the intricate process of tissue regeneration. 10wadays, PRP has gained widespread adoption across diverse medical specialties, including orthopedics, dermatology, and dentistry, owing to its capacity to facilitate the healing process and promote tissue regeneration. 10,11In dermatology, PRP has emerged as a conventional therapeutic modality, frequently employed for various clinical objectives such as skin rejuvenation, non-scarring alopecia, acne scars, periorbital hyperpigmentation, and melasma. 10P has been proven to be successful in treating several pigmentary disorders such as melasma and periorbital hyperpigmentation. 10,11Melasma is one of the most common differential diagnoses of Hori's nevus due to their shared characteristics in clinical, histopathology and pathophysiology. 1,3,6,82][13] Consequently, the objective of this pilot study was to assess the safety and possible efficacy of intradermal PRP in the treatment of Hori's nevus.

| Patients
Ten patients, aged between 25 and 60 years old, were diagnosed with bilateral symmetrical Hori's nevus on the facial area.The diagnosis was established based on typical clinical manifestations, dermoscopic examination, and confirmation by two dermatologists.
Exclusion criteria encompassed individuals who were pregnant or lactating, had a history of PRP allergy, had experienced bleeding disorders, had been diagnosed with blood-borne diseases such as hepatitis B, hepatitis C, or HIV, had been using antioxidant supplements like vitamin C and vitamin E within the 3 months preceding the study, were undergoing chemotherapy or anticoagulant treatment within the same timeframe, had undergone laser or light therapy within the previous 3 months, had co-existing hyperpigmented lesions, or were afflicted with any other active skin diseases in the area where PRP was going to be administered, such as infection, skin cancer, or dermatitis.The study protocol received approval from the institute's ethics committee, and all patients provided informed consent.

| PRP preparation
A total of 1.5 mL of anticoagulant citrate dextrose A (ACD-A) and 18.0 mL of blood were drawn into a 20-mL syringe using a butterfly cannula.Subsequently, the product was transferred to the MINOS® PRP kit (Neogenesis, South Korea) and subjected to centrifugation within a Ugaiya Cence L500 tabletop low-speed centrifuge for 5 min at 1920 g, following the manufacturer's specified protocol.As a result of this process, the final PRP concentration reached approximately 3 300 000 cells/μL, representing a 16-fold increase compared to the normal baseline.

| Procedure
Preoperatively, the area was cleaned, and make-up was removed.
Treated areas were anesthetized with topical lidocaine 2.5% and pilocaine 2.5% (Liprikaine®) for 45 min, then washed off and wiped with isopropyl alcohol.PRP was then injected intradermally using a apart, and a 0.5-1 cm diameter was considered.The treatment was performed four times, at an interval of 2 weeks (Weeks 0, 2, 4, and 6).All 10 patients were followed up at 2, 4, 8, and 12 weeks postoperatively (Weeks 8, 10, 14, and 18).The patients were instructed to use only provided sunscreen and moisturizer and to avoid sun exposure during the entire study period.Clinical photographs of the patients were taken at baseline (Week 0) and each follow-up visit (Weeks 8, 10, 14, and 18) using the Visia® Camera system (Canfield Scientific, Inc).

| Objective assessment
The Modified Dermal Pigmentation Area and Severity Index (mD-PASI) was utilized as a primary outcome to evaluate the effectiveness of PRP in the treatment of Hori's nevus.The mDPASI is a scoring system that is modified from the original Dermal Pigmentation Area and Severity Index (DPASI) to evaluate the severity of Hori's nevus, which is calculated as follows. 14

| Statistical analysis
Data were presented as mean ± SD.Paired t-test and repeated ANOVA were used to estimate the significance of progression of each follow-up period compared to baseline.The statistical analyses were performed using SPSS software.A p value <0.05 was considered statistically significant.

| RE SULTS
All the patients were female (100%).The average age was 33 ± 5 years old with an average age of onset at 25.9 ± 1.4 years old.Six (60%) patients were skin type IV on the Fitzpatrick Scale, and 4 (40%) were type III.The malar region was affected in all patients (100%) while the central face was affected in only 40%.Regarding severity, as determined by the mDPASI, 3 patients were classified as mild, 4 patients as moderate, and an additional 3 patients as severe.It is noteworthy that all these patients had not undergone any previous treatment.Demographic data are displayed in Table 1.
An average reduction of 38.86% in the mDPASI score was observed at Week 12 after treatment.The mean mDPASI between two expert dermatologists decreased substantially from 0.929 ± 0.195 at baseline to 0.568 ± 0.131 (p < 0.05) at the end of the study.The maximum reduction in mDPASI was noticed at Week 4 post-treatment; then, there was a small increase of mDPASI at Week 8 and 12 posttreatment.Summary of mean mDPASI is shown in Table 2 and Clinical changes in the skin lesions are illustrated in Figures 4 and   5.According to the patients' self-assessments at the end of the follow-up, two patients rated their lesions as showing moderate improvement (25-50%), five patients reported marked improvement (50-75%), and the remaining three patients rated their outcomes as excellent, with improvement exceeding 75% compared to baseline (Figure 6).
Most patients encountered minimal side effects, including pain, mild edema, and bruising on the first day following the procedure.
Fortunately, these effects largely resolved on their own within a 3day period.Importantly, none of the patients reported severe side effects.

| DISCUSS ION
Hori's nevus is a challenging acquired hyperpigmented cutaneous lesion characterized by multiple speckled blue-brown and/ or slate-gray macules typically over the malar region. 1,2,6Its dermoscopic features include a homogeneous speckled pattern that does not spare sweat gland apertures or follicles. 6The main therapeutic approaches typically involve laser therapy, with dermabrasion being a less common alternative.However, it is essential to recognize the considerable risk of post-procedural dyschromia associated with these treatment modalities, which presents a significant challenge in the management of Hori's nevus. 7 when assessing Hori's nevus, one of the most common differential diagnoses to consider is melasma, which clinically presents with symmetrical irregular brown patches and with a light brown, regular and/or irregular pigment network, and sparing of hair follicles on dermoscopy. 6It is noteworthy that similarities in the pathophysiology of melasma and Hori's nevus may exist.This is evident through their shared clinical features, which include a symmetrical presentation, acquired onset, high incidence in Asian populations, and a higher prevalence among females. 8Histopathologically, both Hori's nevus and melasma exhibit heightened dermal hyperpigmentation. 1,3,15Numerous studies have provided insights into the importance of dermal melanogenic paracrine networks, with a specific on the role of the stem cell factor (SCF) and its receptor c-Kit, in the development of epidermal hyperpigmentation. 8In a study done by Lee  quantitative analysis of dermal SCF expression also showed similar increases in both Hori's nevus and melasma lesions.This suggests that SCF, as a key player in melanogenesis, might have a role in the pathogenesis of Hori's nevus as well. 813]15 Considering the shared characteristics between Hori's nevus and melasma, and the efficacy of PRP in treating melasma, it is reasonable to speculate that PRP might also be a valuable therapeutic option for Hori's nevus.Our study demonstrated that PRP could effectively improve Hori's nevus as validated by changes in mDPASI, MI, brightening scores evaluated by physicians, patient self-assessment, and clinical photographs, aligning with our initial hypothesis.
Interestingly, a component of the pathogenesis of Hori's nevus features an increase in melanogenic cytokines such as SCF/ckit, HGF, and mast cells.These cytokines are triggered by factors such as dermal inflammation, the aging process, and exposure to sunlight. 8SCF and HGF play roles in upregulating melanocytic microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein-1,2 ultimately leading to an increase in melanogenesis. 16Furthermore, evidence suggests that HGF and mast cells play a role in regulating the proliferation and migration of melanocytes to the dermal layer.They found that TGF-β1 had an inhibitory effect on the production of MITF, tyrosinase, and tyrosinase-related proteins 1 and 2. 17 This inhibition resulted in decreased melanogenesis which could potentially offer a pathway for clinical improvement. 15,17This aligned with the findings of a study conducted by Hofny et al. which investigated the impact of PRP on melasma.Their observations revealed that PRP treatment increased TGF-β1 expression and improved basal cell hyperpigmentation in the lesional skin of melasma patients. 15These findings indicate that TGF-β1 in PRP may have a crucial role in decreasing melanogenesis, not only in melasma but also potentially in Hori's nevus.the potential to modify skin volume, thereby contributing to the improvement of hyperpigmented areas and enhancing overall skin radiance. 11The improvement in the brightening score in our study may be attributed to this mechanism.
Furthermore, an analysis of the constituents of PRP has identified the presence of macromolecular activators of phagocytosis from platelets (MAPPs). 19Studies have shown that the MAPPs can enhance phagocytosis activity through Fc receptors in various cell types, including macrophages, melanophages, dermal dendritic cells, and neutrophils. 19,20The result of this heightened phagocytosis is the potential reduction of dormant melanocyte remnants and melanin granules within the dermal layer. 21The inclusion of MAPPs in PRP presents an intriguing mechanism for decreasing melanin within the dermis of Hori's nevus lesions.This process parallels the mechanism by which Q-switched Nd:YAG laser treatments can improve Hori's nevus lesions.In both approaches, macrophages are activated to phagocytose fragmented melanin, ultimately contributing to the reduction of pigmentation. 22In addition, PRP is known to contain adenosine triphosphate (ATP) and adenosine diphosphate (ADP), compounds known to boost macrophage phagocytosis. 23[21] Other interesting pathophysiological mechanisms indicate that the elevated dermal melanogenesis in Hori's nevus is exacerbated by dermal inflammation. 8Along these lines, previous studies have indicated that PRP may possess anti-inflammatory properties and can improve inflammatory skin diseases such as psoriasis and atopic dermatitis. 24In a pilot study conducted by Ghoz et al., patients with rosacea underwent six PRP sessions spaced 2 weeks apart resulting in a significant reduction in the rosacea grading scale.Tissue biopsies demonstrated a marked decrease in dermal inflammation and lower expression of nuclear factor kappa beta (NF-κB), an important inflammatory regulator in affected skin. 25These findings suggest that PRP could potentially ameliorate dermal inflammation, which could contribute to a reduction in dermal melanogenesis in Hori's nevus.We hypothesize that the combination of these effects could provide a plausible explanation for the clinical response observed in this pilot study.
Of note, a rebound in mDPASI and MI was observed at the 8week and 12-week post-treatment follow-up, respectively.This observation might be attributed to a reduction in the number of associated growth factors over time or a potential disease relapse suggesting that studies with longer follow-up are needed.
In this pilot study, all patients expressed high satisfaction, not only with the improvement of their lesions but also with the enhanced skin texture and overall facial brightening.Although some minimal side effects including discomfort, mild swelling, and bruising were noted, they all spontaneously resolved within 3 days following the injection.While the occurrence of adverse effects is infrequent, it is crucial to bear in mind that the response to PRP therapy varies among individuals, requiring careful observation of each patient's progress.However, the aforementioned results indicated that PRP therapy was a safe and efficacious intervention for Hori's nevus.
Up to our knowledge, this was the first study conducted to evaluate the efficacy of PRP in Hori's nevus.Nonetheless, the study has various limitations.Notably, the small sample size and single-center focus might limit the generalizability of the findings, while the absence of a control group and the non-randomized trial design make direct comparisons difficult.Additionally, the lack of a standardized PRP treatment protocol and the relatively short follow-up period may raise questions about the long-term effects of PRP.Furthermore, histopathological assessments and immunohistochemical examinations to confirm the mechanism of PRP are also required.

| CON CLUS ION
In summary, our study indicated that following a total of four PRP treatment sessions, there was a significant clinical improvement of

ACK N OWLED G M ENTS
We acknowledge the assistance of Dr. Onjuta Chayangsu, M.D. and Dr. Sutsarun Prunglumpoo, M.D. for their invaluable expertise as dermatologists in this study.Furthermore, we extend our appreciation to Mr. Aporn Bureethan and Miss Wanchanida Komkhong for the assistance and support on PRP preparation.Lastly, thanks to Miss Piyawan Wimolchat for taking clinical photographs.

CO N FLI C T O F I NTER E S T S TATEM ENT
There is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Figure 1
Figure 1 illustrated the area of mDPASI.The severity of the disease was assessed and classified into different grades based on observations during dermoscopy.Grade 0 was assigned when no change in color or normal patterns were noted.Grade 1 denoted mild disease, which included light brown color changes and/or the presence of dotted patterns.For moderate disease, as is in grade 2, the characteristics were a bluish or violaceous color and the appearance of Chinese letter or semi-arcuate

Figure 2 .
Figure 2. At the 12-week post-procedure follow-up, the MI significantly decreased by 12.75%, dropping from 208.650 ± 26.319 to 182.052 ± 17.028 (p < 0.0001).There was a downward trend from Week 2 to Week 8 post-PRP.Subsequently, it exhibited a slight increase to 182.052 at Week 12, aligning with the changes seen in mD-PASI.A summary of the mean MI is shown in Table 3 and Figure 3.At 2, 4, 8, and 12 weeks post-treatment, the mean brightening scores, assessed by two experts, were 0.3 (indicating slight lightening), 0.8 (indicating slight lightening), 1.3 (indicating moderate lightening), and 1.4 (indicating moderate lightening), respectively.

8 F I G U R E 4
Another potential mechanism by which PRP may also induce changes in Hori's nevus likely involves the reduction of the quantity of dermal melanocytes through the downregulation of dermal melanogenesis and an increased elimination of F I G U R E 3 Mean ± SD of melanin index at baseline, 2-week, 4-week, 8-week, and 12-week post-PRP treatment (*p < 0.05).Clinical photograph of a patient at baseline (a-c) and after 4 sessions of PRP treatment at 12-week follow-up (d-f).preexisting dermal melanocytes, which has been described in several studies.First, Kim et al. conducted a study to investigate the influence of transforming growth factor (TGF)-β1 on the melanogenesis.

F I G U R E 6
Secondly, Yun et al. made a significant discovery revealing that epidermal growth factor (EGF) inhibits prostaglandin E2 (PGE2), leading to the deactivation of tyrosinase through the cAMP signaling pathway and phospholipase C.This mechanism disrupts the melanin production process, offering a potential avenue for reducing melanogenesis.Notably, PRP contains EGF among its growth factors suggesting that it may impact melanogenesis by utilizing the tyrosinase inhibitor mechanism.18Thirdly, fibroblast growth factor (FGF) aids in the elimination of photodamaged extracellular matrix (ECM) collagen and promotes the synthesis of new collagen.Platelet-derived growth factor (PDGF) also has the capacity to stimulate the production of new collagen and hyaluronic acid.In particular, both FGF and PDGF are components found in PRP.As a result, these growth factors have F I G U R E 5 Clinical photograph of a patient at baseline (a-c) and after 4 sessions of PRP treatment at 12-week follow-up (d-f).Patient self-assessment at the 2-week, 4-week, 8-week, and 12-week post-treatment follow-up.
Hori's nevus lesions, as evidenced by both objective and subjective assessments.Therefore, PRP could serve as a potential novel therapeutic alternative for the management of Hori's nevus.Nonetheless, further research is required to elucidate the full mechanism behind these effects of PRP.Additional studies with larger sample size and longer follow-up visits are required to confirm this preliminary results.Moreover, future investigations employing control interventions such as laser therapy or dermabrasion would be pivotal in advancing the understanding of prospective therapeutic approaches for Hori's nevus.AUTH O R CO NTR I B UTI O N SPD, CC, MA, and CK designed the study and wrote the first proposal.PD, CC, and CK performed PRP injections, collected data, and analyzed the results.PD, CC, MA, and CK wrote the manuscript.All authors provided critical feedback throughout the study, reviewed and edited subsequent drafts.All authors read and approved the final manuscript. Frequently, TA B L E 1 Demographic data of participating patients.

SD) % Decreased from baseline Mean difference* (SD) 95% Confidence Interval p value Lower Bound Upper Bound
et al., dermal SCF was found to be significantly increased in the lesional skin of Hori's nevus.The TA B L E 2 Summary of the mDPASI results.Mean (*Mean compared with baseline.p<0.05 was statistically significant.F I G U R E 2 Mean ± SD of mDPASI at baseline, 2-week, 4-week, 8-week, and 12-week post-PRP treatment (*p < 0.05).TA B L E 3 Mean melanin index results.Mean (