Amygdalin Regulates FOXO3a Signaling to Inhibit Oxidative Stress and Improve Alopecia Areata in Mice

Summary

This study aims to explore the potential molecular mechanisms underlying amygdalin's antioxidant and anti-inflammatory effects in the alopecia areata model in C3H/HeJ mice and in a human primary epidermal melanocyte injury model induced by H₂O₂.

Abstract

This paper aims to explore the mechanism of amygdalin's (AMG) therapeutic effect on alopecia areata (AA). Hematoxylin-eosin staining was used to probe AMG's hepatorenal toxicity. After establishing the AA model of C3H/HeJ mice and H₂O₂-damaged human primary epidermal melanocytes (HPEMs), and following AMG intervention, the reactive oxygen species (ROS) content and the levels of forkhead box O3A (FOXO3a) protein and gene were monitored. Biochemical kits or ‌enzyme-linked immunosorbent assay‌ were used to detect the contents of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase 4 (GPX-4), malondialdehyde (MDA), interleukin-15 (IL-15), interferon-gamma (IFN-γ), and interleukin-6‌ (IL-6). A Western blot was performed to assess the levels of MHC I, IL-15, FOXO3a, SOD, CAT, GPX-4, IFN-γ, IL-6, and IL-15. ChIP-PCR was performed to assess the enrichment of AMG at the promoters of SOD, CAT, and GPX-4 in FOXO3a. Meanwhile, ROS, apoptosis, and the protein and gene levels of FOXO3a, SOD, CAT, and GPX-4 in HPEMs with low FOXO3a expression were investigated. AMG has no potential liver or kidney toxicity and can reduce the expression of MHC I and IL-15 proteins in skin, as well as the serum IL-15. AMG can enhance the expression of the FOXO3a protein and gene in the skin, as well as the levels of SOD, CAT, and GPX-4, while reducing ROS and MDA levels. AMG can inhibit MHC I, IFN-γ, IL-6, and IL-15, MDA, apoptosis, and ROS production in HPEMs, and increase SOD, CAT, and GPX-4 contents. Importantly, AMG can upregulate the expression of the FOXO3a, SOD, CAT, and GPX-4 genes and proteins, and enrich FOXO3a at the promoters of SOD, CAT, and GPX-4. These data suggest that AMG can promote FOXO3a expression and inhibit oxidative stress and inflammatory responses, thereby improving AA in C3H/HeJ mice.

Introduction

As an autoimmune disorder characterized by sudden, non-scarring hair loss, alopecia areata (AA) has a global prevalence of approximately 2–3%, and there are over 30 million patients in China¹. Epidemiological data manifest that the disease can affect individuals across all age groups, but it exhibits a higher prevalence among young adults, with about 60% of patients experiencing their first onset before the age of 20, with a nearly equal male-to-female ratio^1,2. Globally, the annual incidence of AA is approximately 0.2–0.3%, with 40% being single-site AA, 35% multi-site AA, ....

Protocol

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 Tabl....

Results

AMG has no hepatorenal toxicity in normal C3H/HeJ mice
To clearly identify the potential liver and kidney toxicity of AMG from an experimental perspective, researchers administered the three doses (60, 90, and 120 mg/kg) previously studied in normal C3H/HeJ mice by gavage 38. As shown in Figure 1, after intragastric administration of AMG at three doses for 21 days, the architecture of the hepatic lobules was distinct, with hepatocytes arranged in an orderly manner. The.......

Discussion

AMG itself has no direct toxicity. It is hydrolyzed by intestinal microbial enzymes or gastric acid, generating and releasing highly toxic hydrogen cyanide⁴⁹. Hydrogen cyanide is then rapidly absorbed through the digestive tract into the bloodstream and spreads throughout the body tissues, causing hypoxia in the nervous system, cardiovascular system, and respiratory system tissues⁵⁰. In terms of mechanism, hydrogen cyanide combines with the iron ions in cytochrome oxidase, .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5×All-In-One MasterMix (with AccuRT genomic DNA removal kit)	Applied Biological Materials Inc., Jiangsu, China	G492
β-Actin antibody	Affinity Biosciences Co., Ltd., Wuhan, China	AF7018
Amygdalin	Chengdu Herbpurify Co., Ltd., Chengdu, China	K-001-181216
Catalase antibody	Proteintech, Wuhan, China	66765-1-Ig
Catalase kit	Elabscience, Wuhan, China	E-BC-K031-M
C3H/HeJ mice	Chengdu Yaokang Biotechnology Co., Ltd., Chengdu, China	N000190
ChIP assay kit	Beyotime Biotechnology, Shanghai, China	P2078
254 culture medium	Gibco, Thermo Fisher Scientific, America	M-254-500
DAPI staining reagent	Servicebio, Wuhan, China	G1012
Dispase II	Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China	S25046
EvaGreen Express 2×qPCR MasterMix-No Dye	Applied Biological Materials Inc., Jiangsu, China	MasterMix-ES
Fast black HB	Sangon Biotech Co., Ltd., Shanghai, China	A602008-0025
Fetal bovine serum	Every Green	13011-8611
Fluorescence microscope	SOPTOP	CX40
FOXO3a antibody	Proteintech, Wuhan, China	10849-1-AP
Freezing microtome	Thermo	Cryotome E
Glutathione peroxidase 4 antibody	Proteintech, Wuhan, China	67763-1-Ig
Glutathione peroxidase 4 kit	Elabscience, Wuhan, China	E-BC-K883-M
Image-pro plus 6.0	Media Cybernetics, Inc., Rockville, MD, USA	——
Imiquimod cream	Sichuan Mingxin Pharmaceutical Co. Ltd., Chengdu, China	H20030129
Interferon gamma antibody	Proteintech, Wuhan, China	15365-1-AP
Interferon gamma kit	Elabscience, Wuhan, China	E-EL-H0108 and E-EL-M0048
Interleukin 6 antibody	Proteintech, Wuhan, China	21865-1-AP
Interleukin 6 kit	Elabscience, Wuhan, China	E-EL-H6156 and E-EL-M0044
Interleukin 15 antibody	Affinity Biosciences Ltd., Wuhan, China	DF2521
Interleukin 15 kit	Elabscience, Wuhan, China	E-EL-H0222 and E-EL-M0728
Malondialdehyde kit	Elabscience, Wuhan, China	E-EL-0060
MHC antibody	Affinity Biosciences Ltd., Wuhan, China	DF8558
Optical microscope	ZEISS	Primo Star
Paraffin slicer	Thermo scientific	HM325
Pathological scanning software	3DHISTECH (Hungary)	Slide Viewer2.5.0
Penicillin-synthroid solution	Biosharp, Beijing, China	BL505A
Rabbit IgG	Beyotime Biotechnology, Shanghai, China	A7016
ROS assay kit	Beyotime Biotechnology, Shanghai, China	S0033M
Superoxide dismutase 1 antibody	Proteintech, Wuhan, China	10269-1-AP
Tissue embedder	Wuhan Junjie Electronics Co., Ltd., Wuhan China	JB-P5
Tissue hydroextractor	Thermo scientific	STP420 ES
Total RNA isolation kit	Foregene, Chengdu, China	RE-03014
Total superoxide dismutase kit	Elabscience, Wuhan, China	E-BC-K020-M
White vasoline	Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China	P434227