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L'amygdaline régule la signalisation de FOXO3a pour inhiber le stress oxydatif et améliorer l'alopécie areata chez la souris

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DOI :

10.3791/73759

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29 septembre 2026

 ,  ,  ,  ,  ,  , 
1Department of Dermatology, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, 2Neurosurgery, Institute of Sichuan Cancer Hospital, 3Department of Dermatology, The Affiliated Hospital, Southwest Medical University

* These authors contributed equally

Dans cet article

Résumé

Cette étude vise à explorer les mécanismes moléculaires potentiels sous-jacents aux effets antioxydants et anti-inflammatoires de l'amygdaline dans un modèle de pelade chez des souris C3H/HeJ et dans un modèle de lésion de mélanocytes épidermiques primaires humains induit par le H2O2.

Résumé

Cet article vise à explorer le mécanisme de l'effet thérapeutique de l'amygdaline (AMG) sur l'alopécie areata (AA). La coloration à l'hématoxyline-éosine a été utilisée pour évaluer la toxicité hépatonéphrétique de l'AMG. Après établissement du modèle d'AA chez des souris C3H/HeJ et H2O2-mélanocytes primaires épidermiques humains (HPEM) endommagés, et après l'intervention par AMG, la teneur en espèces réactives de l'oxygène (ERO) ainsi que les niveaux de la protéine et du gène de la boîte forkhead O3A (FOXO3a) ont été mesurés. Des kits biochimiques ou ‌dosage immuno-enzymatique‌ ont été utilisés pour détecter les concentrations de superoxyde dismutase (SOD), de catalase (CAT), de glutathion peroxydase 4 (GPX-4), de malondialdéhyde (MDA), d'interleukine-15 (IL-15) et d'interféron-gamma (IFN-γ), et l'interleukine-6‌ (IL-6). Un immunoblotting a été réalisé pour évaluer les niveaux de CMH I, d'IL-15, de FOXO3a, de SOD, de CAT, de GPX-4 et d'IFN-γ, IL-6 et IL-15. Une analyse ChIP-PCR a été réalisée afin d'évaluer l'enrichissement d'AMG au niveau des promoteurs de SOD, CAT et GPX-4 dans FOXO3a. Parallèlement, les niveaux de ROS, d'apoptose ainsi que les niveaux protéique et génique de FOXO3a, SOD, CAT et GPX-4 dans les cellules HPEM présentant une faible expression de FOXO3a ont été étudiés. AMG ne présente aucune toxicité potentielle hépatique ou rénale et peut réduire l'expression des protéines MHC I et IL-15 dans la peau, ainsi que le taux sérique d'IL-15. AMG peut augmenter l'expression de la protéine et du gène FOXO3a dans la peau, ainsi que les niveaux de SOD, de CAT et de GPX-4, tout en diminuant les concentrations de ROS et de MDA. AMG peut inhiber le MHC I et l'IFN-γ, IL-6 et IL-15, MDA, apoptose et production de ROS dans les HPEM, et augmentent les teneurs en SOD, CAT et GPX-4. Importamment, l'AMG peut upréguler l'expression des gènes et des protéines FOXO3a, SOD, CAT et GPX-4, et enrichir FOXO3a au niveau des promoteurs de SOD, CAT et GPX-4. Ces données suggèrent que l'AMG peut favoriser l'expression de FOXO3a et inhiber le stress oxydatif ainsi que les réponses inflammatoires, améliorant ainsi l'AA chez les souris C3H/HeJ.

Introduction

Trouble auto-immun de caractérisé par une perte de cheveux soudaine et non cicatricielle, l'alopécie areata (AA) a une prévalence mondiale d'environ 2 à 3 %, et compte plus de 30 millions de patients en Chine1. Les données épidémiologiques montrent que la maladie peut toucher les individus de tous âges, mais qu'elle présente une prévalence plus élevée chez les jeunes adultes, environ 60 % des patients connaissant leur premier épisode avant l'âge de 20 ans, avec un ratio homme-femme quasi égal1,2. Dans le monde, l'incidence annuelle de l'AA est d'environ 0,2 à 0,3 %, dont 40 % de cas d'AA unilatérale, 35 % d'AA multilatérale, 5 % d'alopécie totale et 2 % d'alopécie universelle3,4,5. En ce qui concerne les complications, environ 30 % des patients présentent des dépressions unguéales, des sillons longitudinaux et d'autres lésions, et le taux de comorbidité avec des maladies auto-immunes telles que les troubles thyroïdiens et le vitiligo dépasse celui observé dans la population générale6,7. Sur le plan des effets néfastes, l'AA n'affecte pas seulement l'apparence, mais est également susceptible de provoquer des troubles psychologiques tels que la dépression et l'anxiété, environ 70 % des patients nouvellement diagnostiqués ayant vécu un événement stressant majeur dans les trois mois précédant l'apparition des symptômes8,9. En l'absence de traitement prolongé, les patients peuvent évoluer vers une alopécie totale ou universelle, ce qui affecte gravement leur fonction sociale et leur qualité de vie10.

Actuellement, la médecine occidentale utilise principalement une combinaison de médicaments locaux, de traitements oraux, de thérapie physique et de greffe de cheveux pour le traitement de l’alopécie areata11,12. Le traitement local comprend essentiellement la solution de minoxidil et les corticostéroïdes (comme la crème de propionate de fluticasone), qui stimulent les follicules pileux et inhibent les réponses immunitaires locales afin de favoriser la repousse des cheveux, mais une utilisation prolongée peut entraîner des effets indésirables tels que l’atrophie cutanée13,14. Chez les patients présentant une maladie progressive, des immunosuppresseurs oraux (comme la prednisone et la cyclosporine) peuvent réguler efficacement la réponse immunitaire systémique, mais une surveillance régulière de la fonction hépatique et rénale est nécessaire12,15. En ce qui concerne la thérapie physique, l’irradiation ultraviolette moyenne à bande étroite et la thérapie par laser de basse énergie améliorent la microcirculation pour favoriser la croissance des cheveux, mais plusieurs séances sont requises et l’efficacité varie d’un individu à l’autre16,17. Pour les cas réfractaires, la chirurgie de greffe de cheveux permet de transplanter des follicules pileux sains vers les zones dégarnies, mais comporte un risque d’échec de la greffe et nécessite un entretien médicamenteux18. Les avantages du traitement par la médecine occidentale résident dans son action rapide et sa forte standardisation, en particulier pour les formes localisées précoces d’alopécie areata19. Toutefois, ses inconvénients incluent une tendance au rebond avec les traitements hormonaux, des effets secondaires importants liés aux immunosuppresseurs, ainsi qu’une efficacité limitée chez les patients atteints de calvitie totale ou d’alopécie universelle20,21. De plus, la durée du traitement est longue (généralement de 3 à 6 mois) et le coût élevé (par exemple, le coût annuel des inhibiteurs de JAK ou des agents biologiques dépasse 100 000 yuans), ce qui limite également son utilisation généralisée19,22. Pour toutes ces raisons évoquées ci-dessus, les auteurs doivent impérativement explorer de nouveaux médicaments ciblés.

Le stress oxydatif contribue largement au mécanisme pathogène de l’alopécie areata (AA). Au cours de la synthèse du pigment des mélanocytes, une grande quantité de peroxyde d’hydrogène (H2O2) est produite23,24. Dans des conditions normales, celui-ci peut être éliminé par des enzymes antioxydantes (telles que la superoxyde dismutase (SOD) et la catalase (CAT))25. Toutefois, chez les personnes atteintes d’AA, l’activité des enzymes antioxydantes est souvent réduite, entraînant l’accumulation de produits oxydatifs et provoquant des lésions de l’ADN ainsi que l’apoptose cellulaire26,27. Cet état de stress oxydatif active ensuite la réponse inflammatoire, libérant des médiateurs pro-inflammatoires (tels que le TNF-α et l’IFN-γ), créant ainsi un cercle vicieux entre stress oxydatif et inflammation28. La réponse inflammatoire attaque les follicules pileux par l’intermédiaire des lymphocytes T, provoquant l’entrée des follicules pileux en phase de repos et leur chute29. Des essais cliniques ont montré que les patients atteints d’AA présentent souvent d’autres maladies auto-immunes (telles que les affections thyroïdiennes)30, suggérant qu’un déséquilibre immunitaire systémique constitue un facteur déclenchant essentiel. En outre, le stress oxydatif et l’inflammation favorisent conjointement l’apoptose des cellules des follicules pileux, et le désordre des signaux apoptotiques nuit davantage à la capacité régénérative du follicule pileux, empêchant la repousse des cheveux31. Par conséquent, le traitement de l’AA doit prendre en compte l’action antioxydante, l’effet anti-inflammatoire et la régulation des voies apoptotiques afin de rompre ce cercle vicieux.

L'amygdaline (AMG) est un composé glycoside cyanogène naturel, présent principalement dans les graines de plantes appartenant à la famille des Rosaceae (comme les noyaux d'abricot, d'amande, de pêche et de néflier)32,33,34. Des études antérieures ont montré que l'AMG peut améliorer le psoriasis en renforçant la fonction barrière de la peau35. Par ailleurs, les effets anti-apoptotiques et anti-stress oxydatif de l'AMG rendent possible son utilisation dans le traitement de l'AA36,37. Des preuves antérieures ont également confirmé que l'AMG peut inhiber la réponse inflammatoire afin d'atténuer les symptômes de l'AA chez des souris C3H/HeJ38. Toutefois, le mécanisme moléculaire par lequel l'AMG inhibe l'inflammation et atténue le stress oxydatif pour atténuer l'AA n'a pas été largement rapporté. Cette étude a principalement mis en évidence les événements liés au stress oxydatif médiés par la voie de signalisation de la forkhead box O3A (FOXO3a) suite à une intervention par l'AMG, dans le traitement d'un modèle d'AA chez des souris C3H/HeJ et d'un modèle de lésion de mélanocytes épidermiques primaires humains (HPEMs) induit par le H2O2.

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Protocole

Ethics approval and consent to participate
The experimental protocols involving animals were authorized by the Ethics Committee of Scientist Biotechnology Co., Ltd. (Ethical Approval Number: SYST-2024-014), following the ARRIVE 2.0, NIH Guide for the Care and Use of Laboratory Animals. And the Ethics Committee of Sichuan Provincial People’s Hospital granted approval for the ethical use of human clinical samples for in vitro experiments (Ethical Approval Number: 2024103). Informed consent was obtained from all tissue donors. All commercial details of the reagents and the equipment used in this study are provided in the Table of Materials.

Construction of the AA model in C3H/HeJ mice and AMG administration
Based on the previous literature, the AA model of C3H/HeJ mice was induced and treated with AMG38,39. In brief, thirty female C3H/HeJ mice (6–8 weeks old) underwent a one-week acclimatization period before being randomly assigned to five distinct groups: Control, AA, AMG-L, AMG-M, and AMG-H. Except for the control group, the AA model in the remaining four groups was established by applying 0.05 g imiquimod cream to the murine dorsal skin. This application was performed three times weekly, accumulating to a total of nine doses. The area of the skin that was applied was 1.5 cm × 1.5 cm. Mice in both the control and AA cohorts received 0.02 mL/g of normal saline by gavage daily. While the mice in AMG groups were given intragastric injections of 60, 90, and 120 mg/kg daily. The administration period is 21 days. For the general observation and assessment of the AA model, a well-defined circular alopecia patch ≥ 1 cm in diameter, with no scarring or severe inflammation, and with broken hair shafts, is required. If mice show diffuse whole-body depilation, severe skin ulceration, or spontaneous rapid hair regrowth, they are excluded.

HPEMs
Following the previous method40, healthy skin tissues from patients who underwent circumcision in the hospital were selected and immersed in 75% alcohol for 5 min. The skin tissues were then repeatedly washed with PBS containing ampicillin (100 U/mL) and streptomycin sulfate (100mg/mL), and incubated in dispase II solution at 4°C overnight for digestion. The separated and isolated epidermal layers were minced and filtered via a sterile membrane to yield the cell suspension. After centrifugation, the sedimentary lower-layer cells were harvested and subsequently resuspended in melanocyte 254 medium without serum and reseeded at a density of approximately 5 × 105 in culture flasks and cultured in a CO2 incubator. After three passages, pure melanocytes were employed for subsequent experiments.

Establishment of H2O2-induced human melanocyte oxidative stress injury model and AMG intervention
Human melanocytes with a fusion degree of 60% to 80% were divided into a control group and groups with different concentrations of H2O2 (200, 400, 600, 800, and 1000 µM). After a 24-h incubation, cell viability was ‌assessed‌ by the CCK-8 method. Subsequently, the optimal concentration of H2O2 was used to establish the melanocyte oxidative stress model, and different concentrations of AMG (0.1, 0.25, 0.5, 1, 2.5, 5, and 10 mg/mL) were added for a 24-hour culture. Cell viability was then chosen to assess and screen the concentration of AMG.

HE examination
Mouse liver and kidney tissues were fixed in 4% paraformaldehyde for 24 h and embedded with paraffin, followed by a dehydration process in the following steps: 70% ethanol for 4 h, 80% ethanol for 2 h, 90% ethanol for 2 h, 95% ethanol for 2 h, anhydrous ethanol, twice, 1 h each time; xylene, twice, 30 min each time38,39,41. Then, 4-µm paraffin sections underwent H&E staining, were dehydrated, cleared, and mounted with neutral gum. A 3D HISTECH scanner was employed to digitize the tissue sections, and images at 20× and 40× magnifications were captured.

Enzyme-linked immunosorbent assay (ELISA) and biochemical detection
Skin tissues were homogenized in a buffer solution at a 1:9 (w/v) ratio, then centrifuged at 1006–1789 × g for 10 min. After protein quantification in serum, skin tissue extracts, and cell supernatants using BCA, the levels of MDA, IL-6, IL-15, and IFN-γ were assessed using the corresponding ELISA kits. And the enzyme activities of SOD, CAT, and GPX-4 were detected according to the instructions of the respective biochemical kits.

Flow cytometry for detecting reactive oxygen species (ROS)
Serum-free culture medium was used to dilute 10µM DCFH-DA at a 1:1000 ratio. And then, mouse skin tissues or human melanocytes were prepared into cell suspensions of 1–20×106 cells/mL using 10µM DCFH-DA. Cells were incubated at 37°C for 20 min. During this period, cells were shaken and mixed every 3–5 min. Following three washes with serum-free medium, intracellular ROS levels were quantified via flow cytometry, setting the excitation and emission wavelengths at 488nm and 525nm, respectively42,43.

Immunofluorescence measurement
FOXO3a (1:1000) primary antibody was incubated with 4-µm skin tissue sections or fixed HPEMs overnight at 4°C. And then, the specimens were treated with secondary antibodies labeled with Alexa Fluor fluorophores and counterstained with DAPI solution. Finally, the sections were observed and captured through fluorescence microscopy. For fluorescence imaging with ROS, a 4-µm frozen section of skin tissue or HPEMs slides were rewarmed at room temperature and dried. A circle was drawn around the tissue with a histochemical pen. The slides were treated with an autofluorescence quencher for 5 min and then rinsed under running water for 10 min. ROS staining solution was dropped and incubated in the dark at 37°C for 30 min. The sections underwent three 5-minute washes in PBS using a decolorization shaker. Then, DAPI staining solution was dropped and incubated ‌for a duration of 10 min under dark conditions at room temperature. The slides were sealed with an anti-fluorescence quencher medium. The specimens were then observed, and images were collected via a fluorescence microscope. The excitation/emission profiles are 330–380/420 nm for DAPI and 510–560/590 nm for Cy3.

Western blot
For mouse skin tissue or human melanocytes, 50 mg of the skin tissue or 2 × 105 cells and 1 mL of RIPA lysis buffer, enriched with protease inhibitors, were dispensed into a 1.5-mL microcentrifuge tube. After an ice bath for 30 min, a 5-min centrifugation was performed at 12,000 g to collect the supernatant fraction. After protein concentration was determined by the BCA method, the protein solution and 5× SDS loading buffer were mixed in a volume ratio of 4:1, and then boiled in a metal bath at 95 °C for 5 min. After the sample was subjected to 10% SDS-PAGE electrophoresis at 80 V or 120 V, proteins were electrotransferred onto a 0.22-µm PVDF membrane at a constant current of 200 mA for 1 h44,45. The membranes were then blocked at room temperature on a centrifuge shaker for 1 h using 5% skimmed milk (prepared with TBST buffer). The membranes were then exposed to primary antibodies specific to ‌major histocompatibility complex class I‌ (MHC I) (1:1000), IFN-γ (1:1000), IL-6 (1:1000), IL-15 (1:1000), β-Actin (1:1000), SOD (1:1000), CAT (1:1000), glutathione peroxidase 4 (GPX-4) (1:1000), and FOXO3a (1:1000) at 4 °C overnight. And the membranes were probed with the secondary antibody (1:10000) for 1 h at room temperature. ECL chemiluminescent reagent was employed to develop and capture images using ChemiScope 6100.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Firstly, the sample RNA from mouse skin tissue or human melanocytes was obtained in accordance with the manufacturer’s instructions of the total RNA extraction kit. According to the operation instructions of the reverse transcription kit, the PCR instrument parameters were set as 25°C for 10 min, 42°C for 15 min, and 85°C for 5 min to obtain cDNA samples. Similarly, the parameters for setting up the PCR instrument were as follows to obtain sufficient DNA copy numbers: Pre-denaturation: 95°C for 5 min, 1 cycle; Denaturation: 95°C for 10 s, 40 cycles; Annealing and extension: 60°C for 30 s, 40 cycles. ‌The normalized expression of the gene of interest is calculated using the following formula: 2‑ΔΔCq (ΔΔCq = Cqtarget‑Cqβ‑Actin)46,47. The information on the gene primer sequences for qRT-PCR is shown in Table 1.

ChIP-PCR analysis
ChIP-PCR analysis was carried out in accordance with the previous protocol48. First, the cells were cross-linked with 1% formaldehyde for 10 min, and glycine solution was added to terminate the cross-linking reaction. After washing with pre-cooled PBS containing PMSF, the cells were lysed with SDS lysis buffer containing PMSF. Subsequently, the chromatin DNA was sheared into fragments mainly ranging from 200 to 1,000 bp using an ultrasonic cell disruptor, and the fragment size was verified by agarose gel electrophoresis. The sheared chromatin samples were resuspended in ChIP dilution buffer containing PMSF, and 20µL of the sample was taken as the input control group. The remaining samples were pre-treated with Protein A+G Agarose/Salmon Sperm DNA. Then, 0.5µg of primary antibody was applied, followed by an overnight incubation at 4°C with slow shaking. The negative control group was treated with an equal amount of rabbit IgG. The next day, Protein A+G agarose beads were added to capture the antigen-antibody complexes, and the complexes were washed successively with low salt immune complex wash buffer, high salt immune complex wash buffer, LiCl immune complex wash buffer, and TE buffer. The immunoprecipitated complexes were eluted with elution buffer, and both the elution products and the samples in the input control group underwent a heat treatment of 65°C for 4h to break the cross-links. Subsequent to proteinase K-mediated protein digestion, DNA was isolated via phenol-chloroform extraction followed by ethanol precipitation. The purified DNA was dissolved in TE buffer and used for subsequent qRT-PCR analysis. And the gene sequence information for ChIP-PCR is shown in Table 2.

The candidate genomic sites for ChIP-PCR were selected through in silico prediction using the JASPAR database (https://jaspar.elixir.no/). Specifically, researchers retrieved the promoter sequences spanning 2000 bp upstream of the TSS of the CAT, SOD, and GPX4 genes. These sequences were scanned for FOXO3a binding motifs using the corresponding position weight matrix. To balance sensitivity and specificity, the authors set a relative profile score threshold of >80%. Among all predicted motifs meeting this criterion, the authors selected the top two highest-scoring putative sites for each gene as candidate regions for ChIP-PCR validation. For example, in the CAT gene, the selected sites were located at -733 bp and -1259 bp relative to the TSS. The anti-FOXO3a antibody used in this study is a commercially available ChIP-grade antibody. To ensure specificity, the authors applied two independent negative controls for every ChIP-PCR assay: isotype IgG and a negative genomic locus. The isotype IgG control was performed as parallel immunoprecipitations using non-immune IgG from the same species. For the negative genomic locus, the authors designed a specific primer pair targeting a non-binding region that lacks any predicted FOXO3a consensus motif. This control region allowed the authors to exclude enrichment due to random chromatin shearing or nonspecific PCR amplification. The fold enrichment was calculated using a three-step normalization strategy.

Step 1: Input normalized ΔCt, ΔCt [normalized IP] = Ct[IP]-(Ct[Input]-log₂(DF))    (1)

ΔCt [normalized IgG] = Ct[IgG] - (Ct[Input] - log₂(DF)).     (2)

DF = Input Dilution Factor; in the experiment, DF = 10, as they used 10% input.

Step 2: ΔΔCt between IP and IgG:

ΔΔCt [IP/IgG] =ΔCt [normalized IP]-ΔCt [normalized IgG]      (3)

Step 3: Fold enrichment = 2(-ΔΔCt [IP/IgG]).       (4)

Statistical analysis
Data are expressed as mean ± standard deviation based on a minimum of three independent experiments. Statistical analyses were performed using GraphPad Prism software (version 6.0). Comparison of data among multiple groups following a normal distribution and homogeneity of variance was assessed using one-way analysis of variance (ANOVA), followed by Tukey’s post-test. Statistical significance was established at p < 0.05.

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Résultats

AMG n'entraîne aucune toxicité hépatorénale chez les souris C3H/HeJ normales
Afin d'identifier clairement, d'un point de vue expérimental, la toxicité potentielle du AMG sur le foie et les reins, les chercheurs ont administré par gavage les trois doses (60, 90 et 120 mg/kg) précédemment étudiées chez des souris C3H/HeJ normales38. Comme indiqué dans Figure 1, après administration intragastrique d'AMG à trois doses pendant 21 jours, l'architecture des lobules hépatiques...

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Discussion

L'AMG lui-même n'a pas de toxicité directe. Il est hydrolysé par les enzymes microbiennes intestinales ou par l'acide gastrique, produisant et libérant du cyanure d'hydrogène hautement toxique49. Le cyanure d'hydrogène est ensuite rapidement absorbé par le tube digestif dans la circulation sanguine et se répand dans les tissus corporels, provoquant une hypoxie dans les tissus du système nerveux, du système cardiovasculaire et du système respiratoire50. Sur le plan mécanisti...

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Déclarations de divulgation

Les auteurs n'ont aucune incompatibilité d'intérêts à déclarer.

CONTRIBUTION DES AUTEURS :
Xun He : Investigation, méthodologie, rédaction – projet original. Jingsong Liu, Jiawei Wang et Zhe Dai : gestion des données. Yugang Gong, Wei Lu et Xiaowei Sha : logiciels, visualisation et validation. Xun He : conceptualisation, rédaction – relecture & ; Édition, acquisition de financement et supervision.

Remerciements

Ce travail a été soutenu par le Comité de santé des cadres de la province du Sichuan (2024-221).

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Matériaux

Liste des matériaux utilisés dans cet article
NomEntrepriseNuméro de catalogueCommentaires
5×MasterMix tout-en-un (avec kit de suppression d'ADN génomique AccuRT)Applied Biological Materials Inc., Jiangsu, ChineG492
Anticorps anti-β-actineAffinity Biosciences Co., Ltd., Wuhan, ChineAF7018
AmygdalineChengdu Herbpurify Co., Ltd., Chengdu, ChineK-001-181216
Anticorps catalaseProteintech, Wuhan, Chine66765-1-Ig
Kit catalaseElabscience, Wuhan, ChineE-BC-K031-M
Souris C3H/HeJChengdu Yaokang Biotechnology Co., Ltd., Chengdu, ChineN000190
Kit d'analyse ChIPBeyotime Biotechnology, Shanghai, ChineP2078
Milieu de culture 254Gibco, Thermo Fisher Scientific, AmériqueM-254-500
Réactif de coloration DAPIServicebio, Wuhan, ChineG1012
Dispase IIShanghai Yuanye Bio-Technology Co., Ltd., Shanghai, ChineS25046
EvaGreen Express 2×qPCR MasterMix - sans colorantApplied Biological Materials Inc., Jiangsu, ChineMasterMix-ES
Noir rapide HBSangon Biotech Co., Ltd., Shanghai, ChineA602008-0025
Sérum de veau fœtalEvery Green13011-8611
Microscope à fluorescenceSOPTOPCX40
Anticorps FOXO3aProteintech, Wuhan, Chine10849-1-AP
Microtome cryostatThermoCryotome E
Anticorps glutathion peroxydase 4Proteintech, Wuhan, Chine67763-1-Ig
Kit glutathion peroxydase 4Elabscience, Wuhan, ChineE-BC-K883-M
Image-pro plus 6.0Media Cybernetics, Inc., Rockville, MD, États-Unis——
Pommade à l'imiquimodSichuan Mingxin Pharmaceutical Co. Ltd., Chengdu, ChineH20030129
Anticorps interféron gammaProteintech, Wuhan, Chine15365-1-AP
Kit interféron gammaElabscience, Wuhan, ChineE-EL-H0108 et E-EL-M0048
Anticorps interleukine 6Proteintech, Wuhan, Chine21865-1-AP
Kit interleukine 6Elabscience, Wuhan, ChineE-EL-H6156 et E-EL-M0044
Anticorps interleukine 15Affinity Biosciences Ltd., Wuhan, ChineDF2521
Kit interleukine 15Elabscience, Wuhan, ChineE-EL-H0222 et E-EL-M0728 
Kit malondialdéhydeElabscience, Wuhan, ChineE-EL-0060
Anticorps CMHAffinity Biosciences Ltd., Wuhan, ChineDF8558
Microscope optiqueZEISSPrimo Star
Microtome à paraffineThermo scientificHM325
Logiciel de numérisation histopathologique3DHISTECH (Hongrie)Slide Viewer2.5.0
Solution pénicilline-synthroïdeBiosharp, Pékin, ChineBL505A
IgG de lapin Beyotime Biotechnology, Shanghai, ChineA7016
Kit d'analyse des EROBeyotime Biotechnology, Shanghai, ChineS0033M
Anticorps superoxyde dismutase 1Proteintech, Wuhan, Chine10269-1-AP
Embarreuse de tissus Wuhan Junjie Electronics Co., Ltd., Wuhan, ChineJB-P5
Hydroextracteur de tissusThermo scientificSTP420 ES
Kit d'isolement d'ARN totalForegene, Chengdu, ChineRE-03014
Kit superoxyde dismutase totaleElabscience, Wuhan, ChineE-BC-K020-M
Vaseline blancheShanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, ChineP434227

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