Dictamnine Ameliorates DNFB-Induced Atopic Dermatitis Like Skin Lesions in Mice by Inhibiting M1 Macrophage Polarization and Promoting Autophagy

Abstract

Autophagy and M1 macrophage polarization play important roles in the regulation of inflammation in atopic dermatitis (AD). Dictamnine is one of the main ingredients in Cortex Dictamni, a widely used traditional Chinese medicine for the treatment of dermatitis. In the present study, we investigated the anti-inflammatory effects of dictamnine on AD like skin lesions and M1 macrophage polarization. A 2,4-dinitrofluorobenzene (DNFB) triggered AD like skin lesions models in mice was established to identify the ameliorative effects of dictamnine on AD in vivo. In addition, an M1 macrophage polarization model was co-stimulated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) using phorbol myristate acetate (PMA) differentiated THP-1 cells, to investigate the effect of dictamnine on promoting autophagy and inhibiting inflammatory factor release. Dictamnine suppressed DNFB-induced skin inflammation by inhibiting M1 macrophage polarization, up-regulating the expression of microtubule-associated protein 1A/1B-light chain 3 (LC3) expression, and promoting macrophage autophagy at inflammatory sites. Dictamnine also could reduce the release of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and interleukin-8 (IL-8), and down-regulate the mRNA expression of these genes in LPS-IFN-γ triggered M1 polarized macrophages. Dictamnine ameliorates AD like skin lesions by inhibiting M1 macrophage polarization and promoting autophagy. Hence, dictamnine is expected to be a potential therapeutic candidate for AD.

INTRODUCTION

Atopic dermatitis (AD) is a chronic, non-infectious, inflammatory skin disease characterized by dry, itchy, and flaky skin and frequent eczema lesions.¹⁾ Commonly affecting infants and children, 20% of AD cases occur in children under two years of age.²⁾ The worldwide prevalence of AD is approximately 1–3% in adults, and the prevalence of AD has been increasing over the past few decades, becoming a global health problem³⁾ The lives of patients with AD are affected to varying degrees.⁴⁾ Additionally, as AD progresses, patients may develop other allergic manifestations, and the severity of AD is positively correlated with the prevalence of autoimmune and psychiatric disorders.⁵⁾

Macrophages are heterogeneous cells that play key roles in inflammatory and tissue repair responses.⁶⁾ Several studies have demonstrated a strong association between macrophages and the development of AD. Molecular imaging using optical reporter genes has been used to monitor macrophage infiltration in AD like skin lesions induced by 2,4-dinitrofluorobenzene (DNFB).⁷⁾ Integrating multiple layers of “Omics” data has revealed the overexpression of M1 macrophage polarization pathways in AD.⁸⁾ Additionally, in a 2,4-dinitrochlorobenzene (DNCB)-induced AD model, the transient receptor potential ankyrin 1 (TRPA1) was found to promote AD inflammation by facilitating the infiltration of dermal macrophages.⁹⁾ These studies suggest that macrophages have a potential role in the treatment of AD.

Autophagy participates in the regulation of damaged organelles, supports normal cellular development, and maintains cellular homeostasis.¹⁰⁾ Moreover, impaired autophagy may promote human epidermal differentiation diseases.¹¹⁾ For example, autophagy activators can reduce imiquimod-induced psoriasis-like dermatitis, and the inhibition of sebaceous gland autophagy is associated with the induction of an inflammatory response in acne in addition to psoriasis.¹²⁾ In AD, lysosomal histone protease levels and enzyme activity are reduced in patient skin specimens, leading to the inhibition of autophagy.¹³⁾ Moisturizers with autophagy-stimulating properties may promote skin barrier restoration and control inflammation.¹⁴⁾ Autophagy has been reported to play an important role in the pathogenesis of various skin diseases, and its activation may help treat psoriasis and AD, which suggested that modulating autophagy may serve as a new potential therapeutic approach for AD.¹⁵⁾

Topical corticosteroids and topical calcium phosphatase inhibitors are the first line of treatment for AD and may be combined with UV phototherapy when response to treatment is inadequate. Most patients with mild-to-moderate AD respond well to topical corticosteroids, but moderate-to-severe AD is mainly treated with potent corticosteroids and systemic immunosuppressants, which can produce significant adverse effects.¹⁶⁾ Treatments currently being developed for moderate disease require further investigation in terms of safety and efficacy.^17,18) Therefore, it is important to develop safer and more effective drugs to provide targeted relief from AD symptoms.

The characteristic active components of Dictamnus are the quinoline alkaloids citrulline and dictamnine (Fig. 1a). Dictamnine is one of the main quinoline alkaloid components isolated from the genus Dictamnus,¹⁹⁾ with antibacterial, anti-allergy, anti-inflammatory, and anti-cancer biological activities.²⁰⁾ Among the species of the genus Dictamnus, the root bark of Dictamnus dasycarpus Turcz. is a traditional herbal medicine, and according to ancient medical books and the clinical experience of modern doctors, D. dasycarpus is often used in the treatment of urticaria and AD.²¹⁾ In countries such as China, Japan, and Korea, Dictamnus dasycarpus Turcz. is widely used to treat skin conditions such as eczema, itching, and urticaria.²²⁾

Fig. 1. Dictamnine Alleviated DNFB-Induced Dermatitis

(a) Chemical structure of Dictamnine. (b) Schematic diagram of the establishment of DNFB-induced AD mouse model and administration of Dictamnine (5, 10, and 20 mg/kg). The mice were randomly divided into six groups of six mice each. The six groups were the blank group, control group, 2 mg/kg dexamethasone treated group, 5 mg/kg dictamnine treated group, 10 mg/kg dictamnine treated group, and 20 mg/kg dictamnine treated group, respectively. (c) Representative images of the ears. (d) Representative images of the dorsal skin. (e) Quantification of scratching bouts in 15 min of the mice. (f) Skin severity score. (g) IgE Serum levels of the mice. Data were presented as mean ± S.E.M. These experiments were repeated three times. Statistical significance was defined as p < 0.05 (^###p < 0.001 vs. blank group, *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group).

Previous studies have shown that dictamnine can ameliorate dermatitis inflammation,²³⁾ however, the mechanism by which dictamnine regulates M1 macrophage polarization and promotes autophagy has not been reported. This study aimed to investigate the effect of dictamnine on macrophages to alleviate the symptoms of AD and to identify a lead compound for the treatment of AD.

MATERIALS AND METHODS

Drugs and Reagents

Dictamnine was purchased from Baoji Chenguang Biotechnology Co., Ltd. (Baoji, China) with purity ≥98%. Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (Shanghai, China). Interferon-γ (IFN-γ) was purchased from Peprotech (Suzhou, China). 2,4-Dinitrobenzene (DNFB) was purchased from Macklin (Shanghai, China); RPMI-1640 medium was purchased from Coring (Jiangsu, China). Phorbol myristate acetate (PMA) was purchased from MedChemExpress (Shanghai, China). Cell Counting Kit-8 was purchased from Deeyee (Shanghai, China). Mk-459 Millipore Milli-Q Plus Ultrapure Water System was used to obtain ultrapure water for use in all aqueous solutions. The rabbit anti-human Akt1 recombinant antibody (immunoglobulin G (IgG), Cat: 80457-1-RR), rabbit anti-human phospho-Akt1 recombinant antibody (IgG, Ser473, Cat: 80462-1-RR), rabbit anti-human/mouse microtubule-associated protein 1A/1B-light chain 3 (LC3) polyclonal antibody (IgG, Cat: 14600-1-AP), rabbit anti-mouse CD86 polyclonal antibody (IgG, Cat: 13395-1-AP), and rabbit anti-human β-actin polyclonal antibody (IgG, Cat: 20536-1-AP) were purchased from Proteintech (Wuhan, China). The rabbit anti-human IKKα/β monoclonal antibody (IgG, EPR16628, Cat: ab178870) was obtained from Abcam (Cambridge, U.K.). The rat anti-mouse F4/80 monoclonal antibody (IgG2a, Clone BM8, Cat: 14-4801-82), mouse anti-human CD68 monoclonal antibody (IgG1, Clone KP1, Cat: 14-0688-82), rabbit anti-mouse CD86 polyclonal antibody (IgG, Cat: PA5-114995) was obtained from Invitrogen (Carlsbad, CA, U.S.A.). The rabbit anti-human phospho-IKKα/β monoclonal antibody (IgG, Ser176/180, Cat: 2697), rabbit anti-human nuclear factor-kappaB (NF-κB)-p65 monoclonal antibody (IgG, Cat: 8242), rabbit anti-human phospho-NF-κB-p65 monoclonal antibody (IgG, Ser536, Cat: 3033), rabbit anti-human extracellular signal-regulated kinase (Erk)1/2 monoclonal antibody (IgG, Cat: 4695), rabbit anti-human phospho-Erk1/2 monoclonal antibody (IgG, Thr202/Tyr204, Cat: 4370) were obtained from Cell Signaling Technology (Danvers, MA, U.S.A.). The rabbit anti-human NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) polyclonal antibody (IgG, Cat: A14223), rabbit anti-human caspase-1 polyclonal antibody (IgG, Cat: A16792), rabbit anti-human interleukin (IL)-1β polyclonal antibody (IgG, Cat: A19635) were purchased from ABclonal (Wuhan, China). Goat Anti-Rat IgG H&L/Fluorescein isothiocyanate (FITC) antibody (bs-0293G-FITC), Goat Anti-Mouse IgG H&L/FITC antibody (bs-0296G-FITC), and Goat Anti-Mouse IgG H&L/Cy3 antibody (bs-0296G-Cy3) were purchased from Beijing Bioss Biological Technology Co., Ltd. (Beijing, China). The secondary antibodies, Peroxidase AffiniPure Goat Anti-Mouse IgG (H + L) (DY60203) and Peroxidase AffiniPure Goat Anti-Rabbit IgG (H + L) (DY60202), were purchased from Deeyee (Shanghai, China). The IL-1β enzyme-linked immunosorbent assay (ELISA) kit was purchased from R&D Systems (MN, U.S.A.), the monocyte chemotactic protein (MCP)-1, IL-6, IL-8, tumor necrosis factor-α (TNF-α) ELISA kit was purchased from Sino Best biological technology Co., Ltd. (Shanghai, China), the IgE ELISA kit was purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China). SYBR^® Green Premix Pro Taq HS qPCR kit was obtained from Accurate biology (Changsha, China). Bicinchoninic acid (BCA) kit was purchased from Shaanxi Zhonghui Hecai Biopharmaceutical Technology Co., Ltd. (Xi’an, China).

Animals

Adult male BABL/c mice (6–8 weeks old) were purchased from the Experimental Animal Center of Xi’an Jiaotong University (Xi’an, China). Animals were housed individually in cages in large group rooms with free access to water and fed a standard dry diet twice daily. The breeding environment was 20–25 °C with a relative humidity of 40% and a light/dark cycle of 12 h. All experiments involving equal treatment of animals were performed by experimenters who were unaware of the experimental conditions.

Ethical Considerations

This study was conducted in strict accordance with the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Animal Ethics Committee of Xi’an Jiaotong University, China (License Number: XJTUAE2023-1623).

The Anti-effect of Dictamnine on DNFB Induced AD Like Skin Lesions

The experimental AD like skin lesions mice model were established by using 2,4-dinitrobenzene (DNFB) according to the previous study²⁴⁾ with slight modification. The mice were randomly divided into six groups of six mice each. The six groups were named blank group, control group, 2 mg/kg dexamethasone treated group, 5 mg/kg dictamnine treated group, 10 mg/kg dictamnine treated group, and 20 mg/kg dictamnine treated group, respectively. Before modeling, the back of each mouse was de-haired. On the first day of modeling, 0.5% DNFB (100 µL, dissolved in acetone/olive oil = 4 : 1, v/v) was applied to the dorsal skin of the mice for sensitization, and 20 µL to ears. For the blank group, 100 µL same ratio of acetone/olive oil was applied to the dorsal skin, and 20 µL to ears. From the second day after stimulation, the mice were administered by gavage in the treatment groups (dexamethasone or dictamnine), and the same volume of saline was administered for control group and blank group. From days 5 to 8, the mice were stimulated by 100 µL 0.2% DNFB on the dorsal skin, and 20 µL to ears, blank group was used same volume of acetone/olive oil solution. On day 8, after stimulation and administration, the mice groups were blind to the experimenter and the number of scratches was quantified within 15 min of the application of DNFB to calculate the degree of pruritus. After quantification, mice were anaesthetized by intraperitoneal injection of sodium pentobarbital (1%, 50 mg/kg), the eyeball of each mouse was removed and the blood was collected into an anticoagulant coated tube, and finally sacrificed the mice. After the mice were sacrificed, the dorsal skin severity score was ruled by symptoms (asymptomatic: 0, mild: 1, moderate: 2, severe: 3) according to the following four indications (erythema/hemorrhage, edema, epidermis/erosion and desquamation/dryness), and the sum of the scores was recorded.²⁵⁾ The dorsal skin and the ears were then collected and fixed in 4% paraformaldehyde.

Quantification of the Scratching Behavior

Mice were allowed to familiar with the environment for at least 24 h in the behavior room, which was quiet and undisturbed, and then were transferred to the observation boxes 15 min before triggering scratching behavior. The observation box was assembled using an uncapped barrel sheet, a square acrylic sheet, and a 30-degree tilted mirror. A camera, or a mobile phone with camera function, was used to record the behavior of animal during a 15 min period immediately after the stimulation. DNFB was applied to the dorsal skin and the ears of mice to induce pruritus.

The methodology for determining and quantifying scratching behavior was informed by previous study.²⁴⁾ Scratching response was defined as the action of scratching with the hind limbs towards the ears and the dorsal area of the neck, and scratching and biting the back with the mouth or the forelimbs. The quantification of the number of scratches is as follows: the beginning to the end of a scratch is recorded as a scratch. When the duration of scratching does not exceed 3s (including 3s), the beginning to the end of scratching is recorded as one scratch (e.g., a scratch lasting 2s is recorded as one scratch); when the duration of scratching exceeds 3s, the length of scratching is recorded as the number of scratches (e.g., a scratch lasting 15s is recorded as 15 scratches).

Hematoxylin–Eosin (H&E) Staining

For H&E staining of the dorsal skin and ears, the tissues were administrated with gradient ethanol and xylene step by step, and then embedded in paraffin, sections (10-µm thick) were embedded in paraffin using a paraffin slider. The slides were dehydrated and transparent by xylene and gradient ethanol step by step, and then stained with hematoxylin and eosin, respectively. Finally, sealing the stained slides with transparent resin. Images were captured using an Eclipse Ci-L type biomicroscope (Nikon, Tokyo, Japan).

Mouse Serum IgE Level Detecting Assay

The mouse serum was separated by centrifugation at 12000 rpm for 20 min at 4 °C form the whole blood. The IgE levels in the serum were measured using an IgE commercial ELISA kit according to the manufacturer’s instructions.

LC3 Immunohistochemical Staining of the Mouse Tissues

The paraffin sections were first deparaffinized and hydrated with xylene and gradient ethanol, and then were boiled in a citrate buffer solution for antigen repairing. After blocking endogenous catalase, the section was blocked with goat serum, and then incubation with anti-LC3 antibody (1 : 500) at 4 °C for 8 h. After washing with phosphate-buffered saline (PBS), the section was incubated with Peroxidase AffiniPure Goat Anti-Rabbit IgG (H + L) (DY60202, Deeyee, Shanghai, China) at 37 °C for 1 h. After washing with PBS, the section was colored by DAB kit (Fuzhou Meixin Biotechnology Development Co., DAB-1031, Fuzhou, China), and the stained with hematoxylin. Finally, sealing the stained section with transparent resin. Images were captured using an Eclipse Ci-L type biomicroscope (Nikon).

F4/80 and CD86 Immunofluorescence Staining of the Mouse Tissues

After deparaffinizing, hydrating, and antigen repairing, the section was blocked with goat serum, and then incubated with anti-F4/80 antibody (1 : 200) and anti-CD86 antibody (1 : 100) at 4 °C for 8 h. After washing with PBS, the section was incubated with Goat Anti-Rat IgG H&L/FITC antibody (1 : 200) and Goat Anti-Mouse IgG H&L/Cy3 antibody (1 : 200) at 37 °C for 1 h away from light. After washing with PBS, the section was coated with transparent resin contained 4′,6-diamidino-2- phenylindole (DAPI). Images were immediately captured using a Leica TCS SP8 STED 3X Super-resolution Confocal Microscope (Wetzlar, Germany).

Cell Culture

The THP-1 cell line was obtained from National Collection of Authenticated Cell Cultures (Shanghai, China), and the cells were cultured with RPMI-1640 medium containing 10% fetal bovine serum (FBS), 1% penicillin and streptomycin, and 0.05 mM β-mercaptoethanol. THP-1 cells were inoculated with culture medium contained 50 ng/mL PMA for 48 h in 6/96-well plates to differentiate the cells into macrophages.

M1 Polarization Model of Macrophages

THP-1 cells were inoculated into 6/96-well plates and cultured in 50 ng/mL PMA for 48 h to induce differentiation of THP-1 macrophages into macrophages. THP-1 macrophages were cultured in PMA-containing medium with dictamnine (50, 100, 200 µM) for 2 h, and then differentiated THP-1 macrophages were stimulated with both 50 ng/mL LPS and 20 ng/mL IFN-γ for 24 h to induce pro-inflammatory M1 macrophages.

Cell Cytotoxicity Assay

Cell viability was determined using the CCK-8 assay. Logarithmic growth phase cells were inoculated in 96-well plates at a density of 10000 cells/well. After treating the cells with dictamnine (3.125, 6.25, 12.5, 25, 50, 100, 200, 400 µM) for 24 h, 10 µL of CCK-8 solution was added to each well. Incubating for another 2 h at the 37 °C incubator, and then the optical density (OD) was read at 450 nm using a microplate reader (800TS, Biotek, U.S.A.).

CD68 Immunofluorescence Staining

Cell samples were prepared using 24 × 24 mm coverslips, coated with polylysine for 1 h. The prepared coverslips were placed in a six-well plate, 1 × 10⁶ THP-1 cells were inoculated on the coverslips and cultured in the six-well plate, 50 ng/mL PMA was added to the culture medium and cultured for 48 h. The cells were then pretreated with dictamnine (50, 100, 200 µM). 2 h later, 50 ng/mL LPS and 20 ng/mL IFN-γ were added to the wells. After incubation, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 10 min, then incubated with 0.5% Triton X-100 for 5 min. After cell punching, they were blocked with 5% BSA for 1 h. Then the cells were incubated with mouse anti-human CD68 monoclonal antibody (1 : 200) for 3 h, followed by incubating with goat anti-mouse IgG H&L/FITC antibody (1 : 200) for 2 h. Then, cells were sealed with transparent resin contained DAPI and anti-fluorescence quenching reagent. Images were captured immediately using a Leica TCS SP8 STED 3X Super-resolution Confocal Microscope.

ELISA

The differentiated THP-1 macrophages were treated with dictamnine (50, 100, 200 µM) for 2 h, and then co-incubated with 50 ng/mL LPS and 20 ng/mL IFN-γ for another 24 h. An equal volume of solvent was used as a negative control. Then the cell supernatant was collected, and the levels of IL-1β, TNF-α, MCP-1, IL-6, and IL-8 were measured by ELISA kits according to the manufacturer’s instructions, respectively.

Real Time PCR

After the same treatment processing as above, total cellular RNA was extracted from THP-1 cells using TRIzol reagent and then reverse transcribed into cDNA using the Hifair^® III 1st Stand cDNA Synthesis SuperMix for qPCR kit depending on the concentration. Subsequently, real-time PCR was performed using the SYBR^® Green Premix Pro Taq HS qPCR kit. The reaction was performed on a real-time fluorescence qPCR system (Bio Rad, Hercules, CA, U.S.A.). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. The primers used in this study are listed in Table 1.

Table 1. Primer Sequences for Real-Time PCR

Gene	Primer (5′–3′)
MCP-1 forward	gTCCCAAAgAAgCTgTgATCT
MCP-1 reverse	AgTCTTCggAgTTTgggTTTg
IL-8 forward	CTgCgCCAACACAgAAATTAT
IL-8 reverse	AAACTTCTCCACAACCCTCTg
IL-6 forward	CACTCACCTCTTCAgAACgAAT
IL-6 reverse	gCTgCTTTCACACATgTTACTC
TNF-α forward	CCTggTATgAgCCCATCTATCT
TNF-α reverse	CAgggCAATgATCCCAAAgT
IL-1β forward	ggTgTTCTCCATgTCCTTTgTA
IL-1β reverse	gCTgTAgAgTgggCTTATCATC
H-gapdh-175 forward	CTCCTCCACCTTTgACgCTg
H-gapdh-175 reverse	TCCTCTTgTgCTCTTgCTgg

Western Blot Analysis

After the same treatment processing as above, the total protein was lysed using radio immunoprecipitation assay (RIPA) lysate, and then measured by a BCA kit according to the manufacturer’s instructions. The various groups of cell lysates were degraded by boiling in a loading buffer for 5 min and then separated using 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). After electrophoresis, the proteins were transferred to a polyvinylidene fluoride membrane, then blocking with 5% skim milk solution for 2 h. Thereafter, the protein bands were incubated with anti-IL-1β (1 : 1000), anti-caspase-1 (1 : 1000), anti-NLRP3 (1 : 1000), anti-Akt1 (1 : 1000), anti-phospho-Akt1 (1 : 1000), anti-NF-κB-p65 (1 : 1000), anti-phospho-NF-κB-p65 (1 : 1000), anti-IKKα/β (1 : 500), anti-phospho-IKKα/β (1 : 1000), anti-Erk1/2 (1 : 1000), anti-phospho-Erk1/2 (1 : 1000), or anti-β-actin (1 : 1000) antibodies at 4 °C for 10 h. After washing, the protein bands were incubated with secondary antibody (1 : 20000) for 1 h at 37 °C. After washing, the images of the bands were performed using an enhanced chemiluminescence (ECL) kit. Lane 1DTM retractor was used to capture the imprinted images and image-Pro Plus 6.0 was used to quantify the proteins.

Statistical Analysis

Data were expressed as mean ± standard error of the mean (S.E.M.). The normality of the data was tested using the Shapiro–Wilk test. Statistical comparisons were performed by one-way ANOVA with least significant difference of variance (LSD) post hoc (assuming homogeneous variance in most cases) or Tamhane’s t post hoc (assuming heterogeneous variance in a few cases). Statistical significance was applied at p < 0.05 compared with negative control. All statistical analyses were performed using Graphpad Prism 9.5 software.

RESULTS

Dictamnine Alleviated Inflammation Symptoms in DNFB-Induced Mouse Model of AD Like Skin Lesions

DNFB was repeatedly used to coat the skin of mice to induce AD like symptoms such as swelling, erythema, erosion and dryness. This was done to evaluate the ameliorative effects of dictamnine on inflamed skin. Dictamnine or dexamethasone was administered by gavage (Fig. 1b). The macroscopic appearance of the mouse ears and dorsal skin are shown in (Figs. 1c, d). The control group showed symptoms of erythema/haemorrhage, oedema, epidermal erosion, and desquamation/dryness. On the last day of the modelling, the scratching behaviour of the mice was monitored and quantified. The number of scratches significantly increased after DFNB stimulation, dexamethasone significantly relieved itch, and dictamnine dose-dependently inhibited DNFB-induced itch (Fig. 1e). IgE expression in mouse plasma was measured using an ELISA kit. DNFB stimulation significantly increased IgE levels in mouse plasma, whereas dictamnine dose-dependently inhibited IgE expression (Fig. 1g). After treatment with dictamnine and dexamethasone, inflammatory symptoms were alleviated (Fig. 1f).

Histopathological Analysis of Dictamnine on Suppressing Inflammation, Enhancing Autophagy, and Inhibiting M1 Macrophage Polarization in the Skin of AD Like Mice

H&E staining revealed a reduction in DNFB-induced ear thickness (Fig. 2a) and epidermal thickness (Fig. 2b), further indicating that dictamnine dose-dependently suppress inflammatory symptoms. The immumohistochemical staining revealed that dictamnine dose-dependently increased the expression of LC3 in the dorsal skin of mice (Fig. 2c), suggesting that dictamnine promotes cell autophagy in AD. Macrophage-mediated inflammation plays an important role in AD.²⁶⁾

Fig. 2. The Histopathology Analysis of Dictamnine on Inflammation in the Dorsal Skin of AD Like Mice

(a) H&E staining of the ears from the mice, lower panel was the local enlarged images in the yellow boxes for the upper panel. (b) H&E staining of the dorsal skin from the mice. (c) Immunohistochemical staining of LC3 in the dorsal skin from the mice, arrows indicate representative positive cells. (d) Immunofluorescence staining of F4/80 (green), CD86 (red) and DAPI (blue) in the dorsal skin from the mice. These experiments were repeated three times. Scale bars were labeled in the images.

The immunofluorescence staining was applied for labeling the F4/80 (green) and CD86 (red) positive cells in the skin tissues (Fig. 2d). The nuclear was also stained using DAPI (blue). The presence of F4/80 and CD86 positive cells infiltration in the dorsal skin suggested the presence of M1-type polarized macrophages in AD like mice model. However, the count of both F4/80 and CD86 positive cells was decreased in the dorsal skin tissues of DNCB-induced AD like mice by administration of dictamnine, indicating that dictamnine reduced macrophage infiltration in the dorsal skin, and inhibited M1 macrophage polarization in AD like mice model.

Dictamnine Suppressed the Transcription and Secretion of Pro-inflammatory Factors in Macrophages

A PAM-differentiated THP-1 macrophage model was used to investigate the effects of dictamnine on M1 macrophage polarization. The effect of dictamnine on THP-1 cell viability was examined using the CCK-8 kit (Fig. 3a), and cells exposed to less than 200 µM of dictamnine showed no significant decrease in cell viability. Macrophages were identified using immunofluorescence staining for CD68 (Fig. 3b). Three cell types, namely THP-1 cells, PMA-induced M0 cells, and LPS-IFN-γ-induced M1 macrophages, were comparatively analyzed. It was observed that both the M0 and M1 cells transitioned from suspension cells to adherent cells and exhibited CD68-positive properties, confirming the differentiation of THP-1 cells into macrophages. An ELISA was used to assess the expression of IL-1β, TNF-α, MCP-1, IL-6 and IL-8 in LPS and IFN-γ-stimulated M1 macrophages, the results showed that dictamnine inhibited the release of IL-1β, TNF-α, MCP-1, IL-6 and IL-8 in a dose-dependent manner (Figs. 3c–g). Moreover, the mRNA expression levels of IL-1β, TNF-α, IL-6, MCP-1, and IL-8 were significantly increased after LPS and IFN-γ stimulation, whereas dictamnine reduced the increasing trend (Figs. 3h–l). Dictamnine inhibited the release of inflammatory factors from macrophages to alleviate inflammation.

Fig. 3. Dictamnine Inhibited the Levels of Inflammatory Factors

(a) The effect of dictamnine (3.125, 6.25, 12.5, 25, 50, 100, 200, 400 µM) on THP-1 cell viability. (b) Immunofluorescence staining of CD68 (green) and DAPI (blue) in THP-1, M0 (PMA differentiated THP-1 cells), and M1 cells (LPS and IFN-γ activated M0 cells), lower panel was the local enlarged images in the White dashed boxes for the upper panel. (c–g) Enzyme-linked immunosorbent assay of IL-1β (c), TNF-α (d), IL-6 (e), MCP-1 (f), and IL-8 (g). (h–l) The mRNA levels of IL-1β (h), TNF-α (i), IL-6 (j), MCP-1 (k), and IL-8 (l) by real-time PCR. Data were presented as mean ± S.E.M. These experiments were repeated three times. Statistical significance was defined as p < 0.05 (^#p < 0.05, ^###p < 0.001 vs. blank group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group).

Dictamnine Enhanced Autophagy in Macrophages

Autophagy has been shown to play an important role in the innate immune response. To further clarify the effect of dictamnine on macrophage autophagy, THP-1 cells were induced to differentiate into macrophages using PMA and then polarized into M1 macrophages by LPS and IFN-γ stimulation. After treating with dictamnine (50, 100, and 200 µM), the cells were stained with LC3 and immunofluorescence images were obtained (Fig. 4a). The results showed that LPS and IFN-γ stimulation significantly increased the expression of LC3 in M1 macrophages compared with the blank group. Moreover, dictamnine treatment further increased the expression of LC3 in the macrophages (Figs. 4a, b). In addition, the protein expression of LC3 was assessed by Western blot, LPS and IFN-γ treatment significantly increased the expression of LC3-II compared to LC3-I, and dictamnine treatment further upregulated the ratio of LC3-II with LC-I protein expression (Figs. 4c, d), suggesting an autophagy augmentation effect of dictamnine on macrophages.

Fig. 4. Dictamnine Enhanced the Autophagy of Macrophages

(a) Effect of dictamnine on immunofluorescence staining of LC3 (green) and DAPI (blue) in M1 cells, scale bar was 50 µm. (b) Quantification of fluorescence intensity of LC3 in (a). (c) Protein expression of LC3 in M1 cells by Western blotting. (d) Quantification of (c). Data were presented as mean ± S.E.M. These experiments were repeated three times. Statistical significance was defined as p < 0.05 (^##p < 0.01, ^###p < 0.001 vs. blank group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group).

Dictamnine Decreased M1 Macrophage Polarization by Down-Regulating the Protein Expression of IL-1β, Caspase-1, and NLRP3, and the Phosphorylation Levels of Akt1, Erk1/2, IKKα/β, and NF-κB-p65

NOD-like Receptor Protein 3 (NLRP3, protein 3 containing the NACHT, LRR and PYD structural domains) is an intracellular sensor whose activation leads to mature IL-1β secretion through caspase-1 activation in a dose-dependent manner.^27,28) NLRP3 mediates the pathology of AD.²⁹⁾ Stimulated by LPS and IFN-γ, the protein expression of NLRP3, caspase-1 and IL-1β in THP-1 macrophages were significantly upregulated, whereas dictamnine treatment decreased the upregulating trend (Figs. 5a, b). In addition, dictamnine inhibited the phosphorylation levels of Akt1, Erk1/2, IKKα/β, and NF-κB-p65 in THP-1 macrophages triggered by LPS and IFN-γ (Figs. 5c, d), suggesting an inhibitory effect of inflammation.

Fig. 5. Effect of Dictamnine on Protein Levels of NLRP3 Pathway and Akt/Erk-IKK-NFκB (p65) Pathway in Activated M1 Cells

(a) The representative Western blot bands of IL-1β, Caspase-1, NLRP3, and β-actin in M1 cells. (b) Quantification of (a) by densitometric analysis. (c) The representative Western blot bands of p-Akt1, Akt1, p-Erk1/2, Erk1/2, p-IKKα/β, IKKα/β, p-NF-κB-p65, NF-κB-p65, and β-actin in M1 cells. (d–g) Quantification of (c) by densitometric analysis. Data were presented as mean ± S.E.M. These experiments were repeated for three times. Statistical significance was defined as p < 0.05 (^###p < 0.001 vs. blank group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group).

DISCUSSION

The pathogenesis of AD is complex, with an impaired skin barrier and inflammation being the main pathogenic mechanisms.³⁰⁾ The lesions of AD are characterized by CD4 T-cell infiltration, keratinocyte activity, and cytokine overexpression associated with Th1 cells and Th17 cells.³¹⁾ The release of IL-10, IL-4, and IL-13 promotes macrophage polarization.³²⁾ Given the role of macrophages in inflammation and tissue repair responses, there is growing evidence that macrophages play an important role in AD.^7–9) Excessive activation of macrophages promotes the development of chronic skin diseases in AD,³³⁾ and the activation of antiviral responses in macrophages is associated with AD severity.³⁴⁾ In the present study, it was found that the construction of DNFB-induced skin lesions in AD mice resulted in a significant increase in macrophage infiltration. More importantly, dictamnine reduced the infiltration of F4/80 and CD86-positive macrophages in the dorsal skin of the mice, suggesting that dictamnine partially ameliorates AD by inhibiting M1 macrophage polarization.

Macrophage polarization is associated with various diseases, such as liver disease,³⁵⁾ rheumatoid arthritis synovitis,³⁶⁾ Impairment of hematopoietic microenvironment,³⁷⁾ and chronic/detrimental inflammation.³⁸⁾ There are two pathways of macrophage polarization, the classical activated macrophage (M1) pathway induced by IFN-γ and LPS, which produces large amounts of pro-inflammatory factors that favour pathogen/tumour elimination but are detrimental to wound healing and exacerbate disease progression. Additionally, IL-4- or IL-13-induced M2 macrophages that express IL-10 are thought to have anti-inflammatory effects.³⁸⁾ In AD, different phenotypes play different roles in AD progression. M1-type macrophages counteract inflammation, and their continued activation leads to tissue damage and exacerbates inflammatory diseases.³⁹⁾ Thus, inhibiting M1 macrophage polarization could be a candidate treatment for AD.⁴⁰⁾ Given the role of the balance between different phenotypes of macrophages in skin inflammation, the present study found that dictamnine was able to inhibit M1 macrophages induced by LPS-IFN-γ. It was also able to significantly reduce the expression levels of reduced M1 markers (IL-6, TNF-α, IL-1β) as well as M1 polarization-associated chemokine MCP-1 mRNA, similarly to published findings,⁴¹⁾ and thus dictamnine may be able to suppress inflammatory diseases caused by M1 polarization. In addition, it was reported that promoting macrophage polarization to the M2 type significantly ameliorated skin lesions in DNCB-induced AD mice.³²⁾ During the treatment of AD, regulating M1/M2 macrophage polarization and promoting M2 macrophage polarization could help ameliorate inflammation. Though the present results indicate that dictamnine can inhibit M1 macrophage polarization, the functional studies on its promotion of M2 macrophage polarization are also of great interest, which can be a direction of follow-up study.

Autophagy plays different roles in inflammatory diseases and has been associated with the pathology of several skin diseases.⁴²⁾ There is much evidence that autophagy plays an important role in the pathogenesis of AD as well as in various skin diseases.^11–14) Autophagy blockade contributes to the development of AD,⁴³⁾ and modulation of autophagic mechanisms significantly ameliorates eosinophil-mediated allergic inflammation in AD,⁴⁴⁾ whereas LC3 is associated with autophagosome development and maturation and is commonly used to monitor autophagic activity.⁴⁵⁾ Given the important role of autophagy in AD, our study found that dictamnine enhanced the abundance of LC3 in the dorsal skin of mice, suggesting that dictamnine ameliorated AD by inducing LC3 expression, which is consistent with previous studies.^46,47) The mechanism by which autophagy deficiency promotes inflammation is through the regulation of macrophage polarization, as evidenced by increased pro-inflammatory M1 and decreased anti-inflammatory M2 polarization.⁴⁸⁾ Therefore, increased autophagy may inhibit M1 macrophage polarization, a hypothesis confirmed in the present study, where dictamnine increased the expression of LC3-II in M1-type macrophage.

Autophagy-related proteins regulate proinflammatory cytokine secretion by regulating cystatin-1 activation and NLRP3-dependent inflammation by protecting mitochondrial integrity.^47,49) NLRP3 inflammatory vesicle components assemble into large cytoplasmic complexes upon stimulation, and activation of caspase-1 ultimately leads to IL-1β maturation and secretion. Studies have shown that NLRP3 inflammatory vesicles are associated with inflammatory responses.⁵⁰⁾ Our results suggest that dictamnine reduces the expression of NLRP3, caspase-1 and IL-1β proteins in LPS-IFN-γ-induced THP-1 cells. In addition, we found that dictamnine significantly inhibited the release of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-8, as well as the mRNA levels of these cytokines in the LPS-induced macrophage inflammation model. This suggests that dictamnine may inhibit pro-inflammatory cytokine expression in macrophages by attenuating NLRP3 inflammatory vesicle activation. Akt signalling pathway components control inflammatory cytokines as well as functions such as autophagy,^51,52) Erk,⁵³⁾ NF-κB-p65 and IKK^54,55) pathways, which are associated with anti-inflammatory effects in macrophages. We found that dictamnine decreased the phosphorylation levels of Akt1, Erk1/2, IKKα/β, and NF-κB-p65, which suggests that Akt1, Erk1/2, IKKα/β, and NF-κB-p65 pathways are associated with LPS-IFN-γ-induced inflammation and autophagy in THP-1 cells.

The main treatments for AD are topical corticosteroids, topical calcium phosphatase inhibitors, UV phototherapy, and monotherapy.⁵⁶⁾ Glucocorticoids has been reported to enhance osteoclast autophagy, and the enhanced autophagy under dexamethasone treatment was verified in primary cultured osteoclasts, as shown by the increased levels of Beclin 1 and LC3-II/LC3-I.⁵⁷⁾ The reported concentration of dexamethasone in vitro was 1 µM in BMDM cells.⁵⁸⁾ For the present project, 50 µM of dictamnine were selected for the minimum concentration. Although the concentrations of dexamethasone and dictamnine are both in micromoles per liter, higher concentration of dictamnine is indeed a drawback that restricts its promotion and application. Dictamnine is an alkaloid that is poorly water-soluble, and more effective pharmaceutical dosage forms of it could be developed to improve the bioavailability. For example, dictamnine delivered by poly (lactic-co-glycolic acid) nanocarriers significantly ameliorated inflammation in a mouse model of oxazolone-induced dermatitis. The nano formulations of diamine penetrated the dermal tissue, achieved sustained drug release, and reduce the concentration of drug use.^20,23) A previous study revealed the potential signaling pathway MrgprA3-TRPA1 of dictamnine to alleviate chronic itch in AD,^20,23) we similarly demonstrated that dictamnine ameliorated itch, epidermal hyperplasia, and cutaneous inflammation in AD. However, unlike the previous study, our study found that dictamnine upregulates the autophagy marker protein LC3 in mouse skin and LC3-II in M1-polarized macrophages, revealing that dictamnine exerts its therapeutic effects by modulating autophagy. Unfortunately, dictamnine has been reported to be hepatotoxic, owing to the presence of a furan ring.^59,60) Therefore, optimizing the structure of dictamnine and reducing its hepatotoxicity may be a way to identify novel drug candidates for treating AD.

CONCLUSION

Taken together, this study provides evidence that dictamnine can attenuate DNFB-induced AD like mice and LPS-IFN-γ-induced inflammatory response in THP-1 cells. Additionally, the potential mechanism by which dictamnine alleviates AD may be closely related to autophagy. Thus, autophagy may be a potential target for the regulation of AD and M1-type macrophage hyperactivation. Together, these results suggest that dictamnine could be a potential novel therapeutic agent for patients with AD. Given its previously reported hepatotoxicity, structural optimization may be another avenue to identify potential drugs. The findings of this study provide new directions for the development of novel AD treatment strategies.

Acknowledgments

We thank Yonghui Zhang (Xi’an Jiaotong University) for providing mice experiments assistance.

Funding

This work was supported by the National Natural Science Foundation of China (Grant Number: 81872837), Natural Science Basic Research Program of Shaanxi Province (Grant Number: 2023-JC-YB-737), Natural Science Basic Research Program of Shaanxi Province (Grant Number: 2019JQ-979), and Xi’an Science and Technology Planning Project (J201901005).

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Mandlik DS, Mandlik SK. Atopic dermatitis: new insight into the etiology, pathogenesis, diagnosis and novel treatment strategies. Immunopharmacol. Immunotoxicol., 43, 105–125 (2021).
• 2) Strathie Page S, Weston S, Loh R. Atopic dermatitis in children. Aust. Fam. Physician, 45, 293–296 (2016).
• 3) Torres T, Ferreira EO, Goncalo M, Mendes-Bastos P, Selores M, Filipe P. Update on Atopic Dermatitis. Acta Med. Port., 32, 606–613 (2019).
• 4) Ali F, Vyas J, Finlay AY. Counting the burden: atopic dermatitis and health-related quality of life. Acta Derm. Venereol., 100, adv00161 (2020).
• 5) Sroka-Tomaszewska J, Trzeciak M. Molecular mechanisms of atopic dermatitis pathogenesis. Int. J. Mol. Sci., 22, 4130 (2021).
• 6) Wen JH, Li DY, Liang S, Yang C, Tang JX, Liu HF. Macrophage autophagy in macrophage polarization, chronic inflammation and organ fibrosis. Front. Immunol., 13, 946832 (2022).
• 7) Lee SB, Park H, Lee JE, Kim KS, Jeon YH. In vivo optical reporter-gene-based imaging of macrophage infiltration of DNCB-induced atopic dermatitis. Int. J. Mol. Sci., 21, 6205 (2020).
• 8) Ghosh D, Bernstein JA, Khurana Hershey GK, Rothenberg ME, Mersha TB. Leveraging multilayered “omics” data for atopic dermatitis: a road map to precision medicine. Front. Immunol., 9, 2727 (2018).
• 9) Zeng D, Chen C, Zhou W, Ma X, Pu X, Zeng Y, Zhou W, Lv F. TRPA1 deficiency alleviates inflammation of atopic dermatitis by reducing macrophage infiltration. Life Sci., 266, 118906 (2021).
• 10) Choi MS, Chae YJ, Choi JW, Chang JE. Potential therapeutic approaches through modulating the autophagy process for skin barrier dysfunction. Int. J. Mol. Sci., 22, 7869 (2021).
• 11) Akinduro O, Sully K, Patel A, Robinson DJ, Chikh A, McPhail G, Braun KM, Philpott MP, Harwood CA, Byrne C, O’Shaughnessy RFL, Bergamaschi D. Constitutive autophagy and nucleophagy during epidermal differentiation. J. Invest. Dermatol., 136, 1460–1470 (2016).
• 12) Peng G, Tsukamoto S, Ikutama R, Nguyen HLT, Umehara Y, Trujillo-Paez JV, Yue H, Takahashi M, Ogawa T, Kishi R, Tominaga M, Takamori K, Kitaura J, Kageyama S, Komatsu M, Okumura K, Ogawa H, Ikeda S, Niyonsaba F. Human beta-defensin-3 attenuates atopic dermatitis-like inflammation through autophagy activation and the aryl hydrocarbon receptor signaling pathway. J. Clin. Invest., 132, e156501 (2022).
• 13) Klapan K, Frangez Z, Markov N, Yousefi S, Simon D, Simon HU. Evidence for lysosomal dysfunction within the epidermis in psoriasis and atopic dermatitis. J. Invest. Dermatol., 141, 2838–2848.e4 (2021).
• 14) Kwon SH, Lim CJ, Jung J, Kim HJ, Park K, Shin JW, Huh CH, Park KC, Na JI. The effect of autophagy-enhancing peptide in moisturizer on atopic dermatitis: a randomized controlled trial. J. Dermatolog. Treat., 30, 558–564 (2019).
• 15) Klapan K, Simon D, Karaulov A, Gomzikova M, Rizvanov A, Yousefi S, Simon HU. Autophagy and skin diseases. Front. Pharmacol., 13, 844756 (2022).
• 16) Puar N, Chovatiya R, Paller AS. New treatments in atopic dermatitis. Ann. Allergy Asthma Immunol., 126, 21–31 (2021).
• 17) Newsom M, Bashyam AM, Balogh EA, Feldman SR, Strowd LC. New and emerging systemic treatments for atopic dermatitis. Drugs, 80, 1041–1052 (2020).
• 18) Silverberg JI, Thyssen JP, Fahrbach K, Mickle K, Cappelleri JC, Romero W, Cameron MC, Myers DE, Clibborn C, DiBonaventura M. Comparative efficacy and safety of systemic therapies used in moderate-to-severe atopic dermatitis: a systematic literature review and network meta-analysis. J. Eur. Acad. Dermatol. Venereol., 35, 1797–1810 (2021).
• 19) Lv M, Xu P, Tian Y, Liang J, Gao Y, Xu F, Zhang Z, Sun J. Medicinal uses, phytochemistry and pharmacology of the genus Dictamnus (Rutaceae). J. Ethnopharmacol., 171, 247–263 (2015).
• 20) Yang N, Shao H, Deng J, Yang Y, Tang Z, Wu G, Liu Y. Dictamnine ameliorates chronic itch in DNFB-induced atopic dermatitis mice via inhibiting MrgprA3. Biochem. Pharmacol., 208, 115368 (2023).
• 21) Qin Y, Quan HF, Zhou XR, Chen SJ, Xia WX, Li H, Huang HL, Fu XY, Dong L. The traditional uses, phytochemistry, pharmacology and toxicology of Dictamnus dasycarpus: a review. J. Pharm. Pharmacol., 73, 1571–1591 (2021).
• 22) Kim H, Kim M, Kim H, Lee GS, An WG, Cho SI. Anti-inflammatory activities of Dictamnus dasycarpus Turcz., root bark on allergic contact dermatitis induced by dinitrofluorobenzene in mice. J. Ethnopharmacol., 149, 471–477 (2013).
• 23) Lin CY, Hsieh YT, Chan LY, Yang TY, Maeda T, Chang TM, Huang HC. Dictamnine delivered by PLGA nanocarriers ameliorated inflammation in an oxazolone-induced dermatitis mouse model. J. Control. Release, 329, 731–742 (2021).
• 24) Wang Y, Tan L, Jiao K, Xue C, Tang Q, Jiang S, Ren Y, Chen H, El-Aziz TMA, Abdelazeem KNM, Yu Y, Zhao F, Zhu MX, Cao Z. Scutellarein attenuates atopic dermatitis by selectively inhibiting transient receptor potential vanilloid 3 channels. Br. J. Pharmacol., 179, 4792–4808 (2022).
• 25) Tang L, Gao J, Cao X, Chen L, Wang H, Ding H. TRPV1 mediates itch-associated scratching and skin barrier dysfunction in DNFB-induced atopic dermatitis mice. Exp. Dermatol., 31, 398–405 (2022).
• 26) Wang M, Qu S, Ma J, Wang X, Yang Y. Metformin suppresses LPS-induced inflammatory responses in macrophage and ameliorates allergic contact dermatitis in mice via autophagy. Biol. Pharm. Bull., 43, 129–137 (2020).
• 27) Zhang J, Liu X, Wan C, Liu Y, Wang Y, Meng C, Zhang Y, Jiang C. NLRP3 inflammasome mediates M1 macrophage polarization and IL-1beta production in inflammatory root resorption. J. Clin. Periodontol., 47, 451–460 (2020).
• 28) Swanson KV, Deng M, Ting JP. The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat. Rev. Immunol., 19, 477–489 (2019).
• 29) Zheng J, Yao L, Zhou Y, Gu X, Wang C, Bao K, Sun Y, Hong M. A novel function of NLRP3 independent of inflammasome as a key transcription factor of IL-33 in epithelial cells of atopic dermatitis. Cell Death Dis., 12, 871 (2021).
• 30) Simon D, Wollenberg A, Renz H, Simon HU. Atopic dermatitis: collegium internationale allergologicum (CIA) update 2019. Int. Arch. Allergy Immunol., 178, 207–218 (2019).
• 31) Weidinger S, Beck LA, Bieber T, Kabashima K, Irvine AD. Atopic dermatitis. Nat. Rev. Dis. Primers, 4, 1 (2018).
• 32) Zeng H, Zhao B, Zhang D, Rui X, Hou X, Chen X, Zhang B, Yuan Y, Deng H, Ge G. Viola yedoensis Makino formula alleviates DNCB-induced atopic dermatitis by activating JAK2/STAT3 signaling pathway and promoting M2 macrophages polarization. Phytomedicine, 103, 154228 (2022).
• 33) Valledor AF, Comalada M, Santamaria-Babi LF, Lloberas J, Celada A. Macrophage proinflammatory activation and deactivation: a question of balance. Adv. Immunol., 108, 1–20 (2010).
• 34) Zhang B, Roesner LM, Traidl S, Koeken V, Xu CJ, Werfel T, Li Y. Single-cell profiles reveal distinctive immune response in atopic dermatitis in contrast to psoriasis. Allergy, 78, 439–453 (2023).
• 35) Wang C, Ma C, Gong L, Guo Y, Fu K, Zhang Y, Zhou H, Li Y. Macrophage polarization and its role in liver disease. Front. Immunol., 12, 803037 (2021).
• 36) Cutolo M, Campitiello R, Gotelli E, Soldano S. The role of M1/M2 macrophage polarization in rheumatoid arthritis synovitis. Front. Immunol., 13, 867260 (2022).
• 37) Yuda M, Aizawa S, Tsuboi I, Hirabayashi Y, Harada T, Hino H, Hirai S. Imbalanced M1 and M2 macrophage polarization in bone marrow provokes impairment of the hematopoietic microenvironment in a mouse model of hemophagocytic lymphohistiocytosis. Biol. Pharm. Bull., 45, 1602–1608 (2022).
• 38) Funes SC, Rios M, Escobar-Vera J, Kalergis AM. Implications of macrophage polarization in autoimmunity. Immunology, 154, 186–195 (2018).
• 39) Zhou X, Li W, Wang S, Zhang P, Wang Q, Xiao J, Zhang C, Zheng X, Xu X, Xue S, Hui L, Ji H, Wei B, Wang H. YAP aggravates inflammatory bowel disease by regulating M1/M2 macrophage polarization and gut microbial homeostasis. Cell Reports, 27, 1176–1189.e5 (2019).
• 40) Yang L, Fu J, Han X, Zhang C, Xia L, Zhu R, Huang S, Xiao W, Yu H, Gao Y, Liang Q, Li W, Zhou Y. Hsa_circ_0004287 inhibits macrophage-mediated inflammation in an N⁶-methyladenosine-dependent manner in atopic dermatitis and psoriasis. J. Allergy Clin. Immunol., 149, 2021–2033 (2022).
• 41) Tsai CF, Chen GW, Chen YC, Shen CK, Lu DY, Yang LY, Chen JH, Yeh WL. Regulatory effects of quercetin on M1/M2 macrophage polarization and oxidative/antioxidative balance. Nutrients, 14, 67 (2021).
• 42) Jin M, Zhang Y. Autophagy and inflammatory diseases. Adv. Exp. Med. Biol., 1207, 391–400 (2020).
• 43) Hailfinger S, Schulze-Osthoff K. Impaired autophagy in psoriasis and atopic dermatitis: a new therapeutic target? J. Invest. Dermatol., 141, 2775–2777 (2021).
• 44) Hou T, Sun X, Zhu J, Hon KL, Jiang P, Chu IM, Tsang MS, Lam CW, Zeng H, Wong CK. IL-37 ameliorating allergic inflammation in atopic dermatitis through regulating microbiota and AMPK-mTOR signaling pathway-modulated autophagy mechanism. Front. Immunol., 11, 752 (2020).
• 45) Schaaf MB, Keulers TG, Vooijs MA, Rouschop KM. LC3/GABARAP family proteins: autophagy-(un)related functions. FASEB J., 30, 3961–3978 (2016).
• 46) Du S, Li C, Lu Y, Lei X, Zhang Y, Li S, Liu F, Chen Y, Weng D, Chen J. Dioscin alleviates crystalline silica-induced pulmonary inflammation and fibrosis through promoting alveolar macrophage autophagy. Theranostics, 9, 1878–1892 (2019).
• 47) Liu T, Wang L, Liang P, Wang X, Liu Y, Cai J, She Y, Wang D, Wang Z, Guo Z, Bates S, Xia X, Huang J, Cui J. USP19 suppresses inflammation and promotes M2-like macrophage polarization by manipulating NLRP3 function via autophagy. Cell. Mol. Immunol., 18, 2431–2442 (2021).
• 48) Liu K, Zhao E, Ilyas G, Lalazar G, Lin Y, Haseeb M, Tanaka KE, Czaja MJ. Impaired macrophage autophagy increases the immune response in obese mice by promoting proinflammatory macrophage polarization. Autophagy, 11, 271–284 (2015).
• 49) Nakahira K, Haspel JA, Rathinam VA, Lee SJ, Dolinay T, Lam HC, Englert JA, Rabinovitch M, Cernadas M, Kim HP, Fitzgerald KA, Ryter SW, Choi AM. Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat. Immunol., 12, 222–230 (2011).
• 50) Feng M, Wei SQ, Zhang SD, Yang Y. Anti-inflammation and anti-pyroptosis activities of mangiferin via suppressing NF-κB/NLRP3/GSDMD signaling cascades. Int. J. Mol. Sci., 23, 10124 (2022).
• 51) Vergadi E, Ieronymaki E, Lyroni K, Vaporidi K, Tsatsanis C. Akt signaling pathway in macrophage activation and M1/M2 polarization. J. Immunol., 198, 1006–1014 (2017).
• 52) Fang S, Wan X, Zou X, Sun S, Hao X, Liang C, Zhang Z, Zhang F, Sun B, Li H, Yu B. Arsenic trioxide induces macrophage autophagy and atheroprotection by regulating ROS-dependent TFEB nuclear translocation and AKT/mTOR pathway. Cell Death Dis., 12, 88 (2021).
• 53) Jung YC, Kim ME, Yoon JH, Park PR, Youn HY, Lee HW, Lee JS. Anti-inflammatory effects of galangin on lipopolysaccharide-activated macrophages via ERK and NF-kappaB pathway regulation. Immunopharmacol. Immunotoxicol., 36, 426–432 (2014).
• 54) Lee N, Heo YJ, Choi SE, Jeon JY, Han SJ, Kim DJ, Kang Y, Lee KW, Kim HJ. Anti-inflammatory effects of empagliflozin and gemigliptin on LPS-stimulated macrophage via the IKK/NF-kappaB, MKK7/JNK, and JAK2/STAT1 signalling pathways. J. Immunol. Res., 2021, 9944880 (2021).
• 55) Chen L, Li Y, Zeng S, Duan S, Huang Z, Liang Y. The interaction of O-GlcNAc-modified NLRX1 and IKK-alpha modulates IL-1beta expression in M1 macrophages. In Vitro Cell. Dev. Biol. Anim., 58, 408–418 (2022).
• 56) Frazier W, Bhardwaj N. Atopic dermatitis: diagnosis and treatment. Am. Fam. Physician, 101, 590–598 (2020).
• 57) Fu LJ, Wu W, Sun XJ, Zhang P. Glucocorticoids enhanced osteoclast autophagy through the PI3K/Akt/mTOR signaling pathway. Calcif. Tissue Int., 107, 60–71 (2020).
• 58) Karuppagounder V, Arumugam S, Thandavarayan RA, Sreedhar R, Giridharan VV, Pitchaimani V, Afrin R, Harima M, Krishnamurthy P, Suzuki K, Nakamura M, Ueno K, Watanabe K. Naringenin ameliorates skin inflammation and accelerates phenotypic reprogramming from M1 to M2 macrophage polarization in atopic dermatitis NC/Nga mouse model. Exp. Dermatol., 25, 404–407 (2016).
• 59) Shi F, Pan H, Cui B, Li Y, Huang L, Lu Y. Dictamnine-induced hepatotoxicity in mice: the role of metabolic activation of furan. Toxicol. Appl. Pharmacol., 364, 68–76 (2019).
• 60) Tu C, Xu Z, Tian L, Yu Z, Wang T, Guo Z, Zhang J, Wang T. Multi-omics integration to reveal the mechanism of hepatotoxicity induced by dictamnine. Front. Cell Dev. Biol., 9, 700120 (2021).