Matrine regulates Th1/Th2 inflammatory responses by inhibiting the Hsp90/NF‐κB signaling axis to alleviate atopic dermatitis

Abstract

Atopic dermatitis (AD) is a common inflammatory skin disease. Matrine is the main component of the traditional Chinese medicine Sophora flavescens, and it poses good therapeutic effects on inflammatory diseases. This study aimed to explore the pharmacological effects of matrine on AD and its underlying mechanism. An AD mouse model and inflamed human epidermal keratinocyte cells (HaCaT) cells were established. Histopathological aspects were examined using hematoxylin and eosin staining, toluidine blue staining, and immunohistochemistry. The mRNA and protein expressions were assessed using quantitative real‐time polymerase chain reaction and Western blot, respectively. The secretions of cytokines and chemokines were examined by enzyme‐linked immunosorbent assay. Flow cytometry was carried out to analyze the proportions of T‐helper (Th) 1 and Th2 cells. Herein, our results displayed that matrine diminished AD symptoms and decreased heat shock protein 90 (Hsp90) expression. Matrine decreased the Th2 cytokine levels in the ear tissues and serum, and it also significantly repressed inflammatory cytokines (thymus activation regulated chemokine and interleukin‐6) secretions by repressing the Hsp90/NF‐κB signaling axis in inflamed HaCaT cells. Furthermore, matrine inhibited Th2 differentiation of CD4⁺ T cells when co‐cultured with inflamed HaCaT cells. Matrine can regulate the Th1/Th2 inflammatory response by inhibiting the Hsp90/NF‐κB signaling axis to alleviate AD. Therefore, it may be a candidate for AD treatment.

Abbreviations

AD

atopic dermatitis

ANOVA

analysis of variance

DFE

Dermatophagoides farinae extract

DNCB

2,4‐dinitrochlorobenzene

ELISA

enzyme‐linked immunosorbent assay

HE

hematoxylin–eosin

Hsp90

heat shock protein 90

IFN

Interferon

IL

interleukin

MTT

3‐(4, 5‐dimethylthiazolyl2)‐2, 5‐diphenyltetrazolium bromide

NF‐κB

nuclear factor kappa‐B

PBMCs

peripheral blood mononuclear cells

qPCR

quantitative real‐time polymerase chain reaction

SD

standard deviation

TARC

thymus activation regulated chemokine

TARC/CCL17

C‐C motif chemokine ligand 17

TB

toluidine blue

Th

T‐helper

TNF

tumor necrosis factor

1. INTRODUCTION

Atopic dermatitis (AD) is a common recurrent inflammatory skin disease. ¹ This condition affects 20% of children and 3% of adults worldwide, and the number of AD patients is rapidly increasing. ² The pathogenesis of AD is complex and linked to a variety of factors (e.g., genetic background, environmental factors, skin barrier dysfunction, and abnormal immune response). ³ , ⁴ T‐helper (Th) 2 skewing is the key point in AD pathogenesis, and the excessive infiltration of mast cells and increased Th2 cytokine (interleukin (IL)‐4 and IL‐13) secretions contributes to AD development. ⁵ Keratinocytes act as a barrier against pathogens and allergens in the skin by releasing inflammatory cytokines. ⁶ Inflammatory cytokines secreted by keratinocytes (IL‐6 and thymus activation regulated chemokine [TARC]) enrich Th2 cells and activate Th2 immune responses in AD. ⁷ Therefore, the inhibition of inflammatory cytokine secretion, Th2 response, and Th2 cytokine secretion may be an effective treatment strategy for AD.

Sophora flavescens, a traditional Chinese medicine, has good therapeutic effects on various diseases. ⁸ Matrine, as a tetracyclic quinoline alkaloid, is the main component of Sophora flavescens. ⁹ It has been proven to have good pharmacological effects on inflammatory skin diseases. For instance, matrine treatment can alleviate psoriasiform dermatitis in mice by regulating dendritic cells. ¹⁰ In addition, as previously reported, matrine treatment can elevate the level of Th1 cytokines (interferon (IFN)‐γ) to regulate the Th1/Th2 balance in eczema, thereby alleviating eczema. ¹¹ However, the biological roles and potential mechanisms of matrine in the development of AD remain unclear.

Heat shock protein 90 (Hsp90) is a highly conserved immunomodulatory molecule upregulated when cells are stimulated by inflammation. ¹² Hsp90 is involved in various autoimmune diseases and inflammatory skin diseases, including psoriasis and AD. Hsp90 was significantly upregulated in IFN‐γ‐treated human epidermal keratinocyte cells (HaCaT) cells, and luteolin treatment can significantly alleviate psoriasis in vitro and in vivo by repressing Hsp90. ¹³ In addition, Debio 0932 (Hsp90 inhibitor) administration can effectively relieve disease symptoms in a xenograft model of psoriasis. ¹⁴ More importantly, the severity of the disease in AD patients is positively correlated with serum Hsp90 levels. ¹⁵ Nevertheless, the role of Hsp90 in AD has not been fully elucidated. Moreover, whether matrine acts on Hsp90 and regulates its downstream targets, which ultimately affects the Th1/Th2 inflammatory response in AD, remains unclear. The nuclear factor kappa‐B (NF‐κB) pathway is a classic pro‐inflammatory pathway. ¹⁶ The activation of the NF‐κB signaling pathway is related to Th2 polarization and accompanied by Th2 cytokine secretion. ¹⁷ Notably, Hsp90 inhibition inactivates the NF‐κB signaling pathway, ¹⁸ , ¹⁹ but the roles of the NF‐κB signaling pathway in AD and the regulatory relationship between the Hsp90 and NF‐κB in AD have not been fully explained.

In the present research, we speculated that matrine regulated Th1/Th2 inflammatory responses by repressing the Hsp90/NF‐κB signaling axis to alleviate AD. Our work provides a candidate for AD treatment and clarifies its treatment mechanism.

2. MATERIALS AND METHODS

2.1. Isolation and culture of CD4 ⁺ T cells

Approximately 6 ml venous blood was obtained from healthy adult volunteers, with the approval of the Second Affiliated Hospital of Hunan University of Traditional Chinese Medicine Ethics Committee. Human peripheral blood mononuclear cells (PBMCs) were obtained from the diluted blood using Ficoll–Hypaque density gradient centrifugation. PBMC suspensions were incubated with Biotin‐Antibody Cocktail (Bergisch Gladbach, Germany) for 5 min followed by incubation with anti‐Biotin MicroBeads for 10 min. Naive CD4⁺ T cells were obtained by negative selection (AutoMACS; Miltenyi Biotec, Bergisch Gladbach, Germany). In brief, PBMCs were resuspended in phosphate‐buffered saline (PBS) containing 2 mM ethylenediaminetetraacetic acid (EDTA) and then combined with an antibody (Ab) mixture containing biotin‐conjugated Abs against CD8, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD45RO, CD56, CD123, TCR γ/δ, HLA‐DR, and glycophorin A (CD235a) for 10 min at 8°C, followed by mixing with colloidal superparamagnetic MACS microbeads conjugated to a monoclonal anti‐biotin Ab for 15 min. Then, the cells were washed and applied to metal matrix columns in the AutoMACS separation apparatus. Non‐Ab‐coated cells (CD4⁺CD45RA⁺CD45RO⁻ T cells) were collected and washed for additional study. The purity of isolated naive CD4⁺ T cells was >95% by flow cytometry assay with CD4‐fluorescein isothiocyanate (FITC) (Abcam, Cambridge, UK) and CD45RA‐PE (Abcam) staining. To activate CD4⁺ T cells, we cultured CD4⁺ T cells in Roswell Park Memorial Institute (RPMI) 1640 (Gibco, MD, USA) mixed with 10% fetal bovine serum (FBS) (Gibco) and 2 mM L‐glutamine. CD4⁺ T cells were cultured in 96‐well plates (2 × 10³ cells/well) precoated with anti‐CD3 Ab (3 μg/mL; BD Biosciences) for 4 days.

2.2. Induction of AD‐like lesions in the mouse ear

Female BALB/c mice (6 weeks old) were obtained from Shanghai Jiesijie Lab Animal Co., Ltd. (Shanghai, China). The experimental protocols were approved by the Animal Ethics Committee of the Second Affiliated Hospital of Hunan University of Traditional Chinese Medicine. The AD mouse model was induced by Dermatophagoides farinae extract (DFE)/2,4‐dinitrochlorobenzene (DNCB), as previously described. ⁷ In brief, DFE (Greer Laboratories, NC, USA) was dissolved in PBS with 0.5% Tween 20 (PBS‐T). Meanwhile, 1% DNCB (Sigma‐Aldrich, Merck, Germany) was dissolved in an acetone/olive oil (1:3) solution. The mice were randomly divided into three groups (n = 8/group): control, AD, and AD + matrine groups. A surgical tape (Nichiban, Tokyo, Japan) was employed to strip the surfaces of both ear lobes gently. Then, each ear was painted with 20 μl DFE and 20 μl DNCB repeated once a week for 4 weeks. Vehicle (PBS) or matrine (30 mg/kg, 98.7% purity) (the National Institute for the Control of Pharmaceutical and Biological Products of China) was orally administrated until the end of the 4‐week induction period (6 times/week). Ear thickness was detected 24 h after DFE/DNCB treatment. Then, the mice were sacrificed, and blood samples and ear tissues were collected.

2.3. Hematoxylin–eosin and toluidine blue staining

The ear skins were fixed and embedded in paraffin. The sections (4 μm in thickness) were prepared followed by dehydration and stained with HE or 0.1% TB. After washing with water for three times, the sections were imaged under a microscope.

2.4. Immunohistochemistry

The ear sections (4 μm in thickness) were prepared. After deparaffinization and antigen retrieval (Dako, Copenhagen, Denmark), the sections were blocked and incubated with the Ab against CD4 (Abcam, 1:1000, ab183685) overnight followed by incubation with an appropriate secondary Ab (Abcam, 1:500, ab7090) for 1 h. The sections were stained with DAB, counterstained with hematoxylin, dehydrated, and mounted. The images were captured using a microscope (Olympus, Tokyo, Japan).

2.5. HaCaT cell culture and treatment

HaCaT cells were purchased from the Chinese Academy of Sciences in Shanghai, China, and cultured in RPMI 1640 (Gibco) mixed with 10% FBS (Gibco) at 37°C with 5% CO₂. Moreover, they were subjected to matrine treatment (0.4, 0.2, and 0.1 mM) (Shanghai Xinyi Pharmaceutical, Shanghai, China) for 24 h followed by treatment with 10 ng/ml recombinant tumor necrosis factor (TNF)‐α and 10 ng/ml IFN‐γ (R&D systems, MN, USA) for 6 h. For co‐culture studies, HaCaT cells were seeded in 6‐well cell culture plates. Hanging cell culture inserts (1 μm; Millipore, MA, USA) containing CD4⁺ T cells (2 × 10⁵ T cells/well, 8 T cells: 1 HaCaT) were placed on top of HaCaT cells. HaCaT cells were co‐cultured with CD4⁺ T cells for 48 h. Then, the cells were collected for flow cytometry, quantitative real‐time polymerase chain reaction (qPCR), Western blot, and enzyme‐linked immunosorbent assay (ELISA).

2.6. 3‐(4, 5‐Dimethylthiazolyl2)‐2, 5‐diphenyltetrazolium bromide assay

HaCaT cells were seeded in 96‐well plates (3 × 10³ cells/well) and incubated with 10 μL MTT (5 mg/ml) (Beyotime, Shanghai, China) for 4 h. Then, dimethyl sulfoxide (DMSO) (Sigma‐Aldrich, MO, USA) was added, and the absorbance at 490 nm was analyzed with a microplate reader (Bioteke, Beijing, China).

2.7. Cell transfection

The overexpression plasmid of Hsp90 (OE‐Hsp90) and its negative control were purchased from GenePharma (Shanghai, China). HaCaT cells at the exponential stage were used for transfection. Before transfection, 1 × 10⁶ HaCaT cells were cultured in 6‐well plates with 2 ml complete medium for 24 h until 90% confluence. The plasmid and microRNAs were transfected into HaCaT cells by Lipofectamine™ 3000 (Invitrogen, CA, USA) and cultured with Opti‐MEM serum‐free medium for 24 h following the instructions. Cells were used for follow‐up experiments following the detection of transfection efficiency by qPCR.

2.8. Flow cytometry

We stained CD4⁺ T cells with an anti‐CD4‐PerCP‐Cy5.5 Ab for 30 min (BD Biosciences, NJ, USA, 566316) followed by incubation with a fixation medium (Invitrogen) to analyze the Th1 and Th2 cells. The cells were subsequently incubated with a permeabilization medium (Invitrogen) containing anti‐IFN‐γ‐FITC (BD Biosciences, 554,700) and anti‐IL‐4‐APC (BD Biosciences, 560,671) for 30 min. The fluorescence intensity was examined by flow cytometry (BD Biosciences).

2.9. Enzyme‐linked immunosorbent assay

Each ear of mice was painted with 20 μL DFE and 20 μl DNCB repeated once a week for 4 weeks. Then, the mice were sacrificed, and blood samples were collected. Serum was separated from the whole blood collected from mice by centrifugation at 3000 rpm for 10 min and stored at −80°C. The levels of IFN‐γ, TNF‐α, IL‐2, TARC, IL‐4, IL‐5, and IL‐6 were assessed using a mouse/human IFN‐γ ELISA kit (Beyotime, Shanghai, China; PI508 and PI511), mouse/human TNF‐α ELISA kit (Beyotime; PT512 and PT518), mouse/human IL‐2 ELISA kit (Beyotime; PI575 and PI580), mouse/human TARC ELISA kit (R&D Systems, MN, USA; SDN00 and MCC170), mouse/human IL‐4 ELISA kit (Beyotime; PI612 and PI618), mouse/human IL‐5 ELISA kit (Beyotime; PI620 and PI625), and mouse/human IL‐6 ELISA kit (Beyotime; PI326 and PI330), respectively. Cell supernatant and serum were used for experiments. The experimental operation was performed strictly by the manual. The data were analyzed in a microboard reader (Bioteke, Beijing, China). All ELISA results are expressed as cytokine concentrations (pg/mL). A standard curve was generated using known amounts of the respective purified recombinant cytokines.

2.10. Quantitative real‐time polymerase chain reaction

Each mouse ear was painted with 20 μl DFE and 20 μl DNCB repeated once a week for 4 weeks. Then, the mice were sacrificed, and ear tissues were collected. Total RNA was extracted from the ear tissues and HaCaT cells with TRIzol (Thermo Fisher Scientific, MA, USA). The cDNA was synthesized using the cDNA synthesis kit (Toyobo, Tokyo, Japan). Then, SYBR (Thermo Fisher Scientific) was employed for qPCR assay. β‐actin was used as the reference mRNA gene. The data were analyzed with the 2^−ΔΔCt method. Supplementary material 1 shows the primers used in the study.

2.11. Western blot

Each mouse ear was painted with 20 μl DFE and 20 μl DNCB repeated once a week for 4 weeks. Then, the mice were sacrificed, and ear tissues were collected. To prepare samples for immunoblotting, we first placed the tissues on dry ice and then added 500 μl radioimmunoprecipitation (RIPA) lysis buffer (Thermo Fisher Scientific) containing protease inhibitor and homogenization with a tissue tearor. The supernatant was collected for the next experiment by centrifugation at 10000 rpm for 20 min. Meanwhile, total proteins were extracted from HaCaT cells after different treatments using RIPA. BCA kit (Beyotime) was used to quantify the concentration. Proteins were separated using 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gel and further transferred to a polyvinylidene difluoride membrane (Millipore, MA, USA). Then, the membranes were incubated at 4°C overnight with antibodies against Hsp90 (Abcam, Cambridge, UK, 1:1000, ab13492), IkBα (Abcam, 1:1000, ab32518), p‐IkBα (Abcam, 1:1000, ab133462), p65 (Abcam, 1:1000, ab32536), p‐p65 (Cell Signaling Technology, MA, USA, 1:1000, #3031), and β‐actin Ab (Abcam, 1:5000, ab8226). After washing with PBS‐T, the membranes were incubated with the corresponding secondary Ab (Abcam; 1:5000, ab7090, ab6789) for 60 min. Protein bands were analyzed by an ECL detection kit (Beyotime). Images were captured by a gel imaging system (Bio‐Rad, CA, USA) and analyzed by ImageJ.

2.12. Statistical analysis

All our data were obtained from three independent experiments. Statistical data were analyzed by SPSS 19.0 (IBM, Armonk, NY) and expressed as means ± standard deviation (SD). The differences between individual groups were analyzed by Student's t‐test. The one‐way analysis of variance was performed to evaluate the differences among multiple groups. p values less than 0.05 were considered significant.

3. RESULTS

3.1. Matrine reduced T lymphocyte and mast cell infiltration as well as inflammatory response in an AD mouse model

The DNCB/DFE‐induced AD mouse model was treated with matrine to explore the effects of matrine on AD. The results of surface characteristics and HE staining revealed that DNCB/DFE treatment induced hyperkeratosis and inflammatory cell infiltration in the ear skins of AD mice, and these changes were significantly ameliorated by matrine treatment (Figure 1A). The weight of the AD mouse was reduced, but matrine treatment alleviated this effect (supplementary material Figure S1). Meanwhile, epidermal, dermal thickness (Figure 1B,C), mast cell and CD4 T infiltration were evidently reduced by matrine treatment (Figure 1D,E). All these results suggested that matrine had great inhibitory effects on inflammatory cell infiltration and response in the AD mouse model.

FIGURE 1.

Matrine reduced T lymphocyte and mast cell infiltration as well as inflammatory response in an AD mouse model. DFE/DNCB was employed to induce AD‐like symptoms in mice, and the mice were orally administrated with matrine. (A) Surface characteristics of the ear skin, HE, TB, and IHC staining of ear sections. (B–C) Epidermal and dermal thickness was detected. (D–E) CD4 T and mast cells were counted. Data were expressed as mean ± SD. N = 8. *p < 0.05, **p < 0.01, ***p < 0.001.

3.2. Matrine inhibited Hsp90 expression and altered the cytokine levels of Th1 and Th2 cells

As previously reported, serum Hsp90 level notably increased in AD patients. ¹⁵ Herein, the Hsp90 protein level was markedly elevated in the ear skins of AD mice, and this change was abolished by matrine treatment (Figure 2A). As reported, Th2 skewing is the key point in the pathogenesis of AD. ⁵ In the current study, Th1 and Th2 (IFN‐γ, TNF‐α, IL‐2, IL‐4, and IL‐5) and inflammatory cytokines (TARC and IL‐6) were significantly upregulated in the ear skins of AD mice, but matrine treatment can eliminate the upregulated effects of Th2 (IL‐4 and IL‐5) and inflammatory cytokines (TARC and IL‐6) on AD mice (Figure 2B). Similar trends were observed in the serum levels of cytokines in mice (Figure 2C). Altogether, matrine treatment can inhibit the DNCB/DFE‐induced immune disorder due to Th1/Th2 balance and suppress Hsp90 expression.

FIGURE 2.

Matrine inhibited Hsp90 expression and altered the cytokine levels of Th1 and Th2 cells. (A) Hsp90 level in the ear skins of mice was analyzed by Western blot. (B) qPCR was carried out to examine the mRNA levels of IFN‐γ, TNF‐α, IL‐2, TARC, IL‐4, IL‐5, and IL‐6 in ear skins of mice. (C) Serum levels of IFN‐γ, TNF‐α, IL‐2, TARC, IL‐4, IL‐5, and IL‐6 were assessed by ELISA. Data were expressed as mean ± SD. N = 8. *p < 0.05, ** p < 0.01, ***p < 0.001.

3.3. Matrine treatment suppressed inflammatory cytokines levels in inflamed HaCaT cells, as well as Hsp90 expression and NF‐κB signaling activation

As reported, keratinocytes from AD patients secrete high concentrations of inflammatory cytokines following TNF‐α/IFN‐γ stimulation. ²⁰ Therefore, TNF‐α/IFN‐γ‐treated HaCaT cells were employed to probe the role of matrine in AD in vitro. First, we examined the cytotoxicity of matrine toward HaCaT cells, and the results of the MTT assay displayed that matrine did not show cytotoxicity until 0.4 mM treatment for 48 h (Figure 3A). The results of qPCR and ELISA subsequently demonstrated that the mRNA (Figure 3B) and secretion levels (Figure 3C) of TARC and IL‐6 in inflamed HaCaT cells were significantly reduced by matrine treatment in a dose‐dependent manner and 0.4 mM treatment produced the best results, then 0.4 mM treatment for 24 h was choosen for the subsequent experiments. Matrine can also inhibit Hsp90 expression as well as IkBα and p65 phosphorylation in inflamed HaCaT cells (Figure 3D, Figure S2 and Figure S3). Collectively, our data demonstrated that matrine treatment remarkably inhibited inflammatory cytokine secretion levels in inflamed HaCaT cells, Hsp90 upregulation, and NF‐κB signaling activation.

FIGURE 3.

Matrine treatment suppressed inflammatory cytokine levels in inflamed HaCaT cells, as well as Hsp90 expression and NF‐κB signaling activation. (A) MTT assay was used to determine the viability of HaCaT cells after different concentrations of matrine treatment (0.1, 0.2, 0.4 mM) and duration for 24, 48, and 72 h. HaCaT cells were pretreated with matrine (0.1, 0.2, 0.4 mM) for 24 h before TNF‐α and IFN‐γ stimulation for 6 h. (B) mRNA levels of TARC and IL‐6 were detected by qPCR. (C) TARC and IL‐6 secretions were assessed by ELISA. (D) Western blot was conducted to analyze Hsp90, IkBα, p‐IkBα, p65, and p‐p65 levels. Data were expressed as mean ± SD. All our data were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

3.4. Matrine attenuated the HaCaT cell inflammatory reaction via the Hsp90/NF‐κB axis

To explore the role of Hsp90 in matrine‐mediated inhibition of Th2 cytokines levels, we co‐treated inflamed HaCaT cells with matrine and overexpression of Hsp90. As displayed in Figure 4A–C, Hsp90 expression in matrine and TNF‐α/IFN‐γ‐treated HaCaT cells was remarkably increased by overexpression of Hsp90 transfection. In addition, HspP90 overexpression neutralized the inhibitory effect of matrine on mRNA (Figure 4D) and secretion levels (Figure 4E) of TARC and IL‐6 in inflamed HaCaT cells. Moreover, the inhibitory effect of matrine on NF‐κB signaling activation in inflamed HaCaT cells was partially reversed by Hsp90 overexpression (Figure 4F). Therefore, matrine treatment suppressed inflammatory cytokine levels in TNF‐α/IFN‐γ‐treated HaCaT cells by inhibiting the HSP90/NF‐κB axis.

FIGURE 4.

Matrine attenuated the HaCaT cell inflammatory reaction via the Hsp90/NF‐κB axis. (A‐B) Hsp90 expression in HaCaT cells after overexpression of Hsp90 transfection was assessed by qPCR and Western blot. (C) TNF‐α/IFN‐γ‐treated HaCaT cells were co‐treated with matrine and overexpression of Hsp90 transfection and the Hsp90 expression was assessed by Western blot. (D) mRNA levels of TARC and IL‐6 were examined by qPCR. (E) Secretion levels of TARC and IL‐6 were examined by ELISA. (F) Western blot was conducted to analyze Hsp90, IkBα, p‐IkBα, p65, and p‐p65 levels. Data were expressed as mean ± SD. All our data were obtained from three independent experiments. *p < 0.05, ** p < 0.01, ***p < 0.001.

3.5. Matrine inhibited Th2 but promoted Th1 differentiation of CD4 ⁺ T cells when co‐cultured with inflamed HaCaT cells

The purity of isolated naive CD4⁺ T cells was 96.12% according to the flow cytometry assay with CD4‐FITC and CD45RA‐PE staining (Figure 5A). As demonstrated in Figure 5B, the proportion of Th1 cells was significantly decreased by co‐culture with inflamed HaCaT cells (3.81%) compared with the control group (10.82%). Meanwhile, the proportion of Th2 cells increased (9.07%) compared with the control group (5.46%), but these changes were abolished by matrine treatment. In addition, the co‐culture with inflamed HaCaT cells resulted in decreased mRNA (Figure 5C) and secretion levels (Figure 5D) of Th1 cytokines and increased levels of TARC and Th2 cytokines in CD4⁺ T cells, which were all eliminated by matrine treatment. In summary, matrine can suppress Th2 differentiation and promote Th1 differentiation of CD4⁺ T cells co‐cultured with inflamed HaCaT cells.

FIGURE 5.

Matrine inhibited Th2 but promoted Th1 differentiation of CD4⁺ T cells when co‐cultured with inflamed HaCaT cells. CD4⁺ T cells were co‐cultured with inflamed HaCaT cells followed by matrine treatment. (A) The purity of isolated native CD4⁺ T cells was analyzed by flow cytometry assay with CD4‐FITC and CD45RA‐PE staining. (B) The proportions of Th1 and Th2 cells in CD4⁺ T cells were examined by flow cytometry. (C) mRNA levels of IFN‐γ, TNF‐α, IL‐2, TARC, IL‐4, and IL‐5 were analyzed by qPCR. (D) ELISA was used to measure the levels of IFN‐γ, TNF‐α, IL‐2, TARC, IL‐4, and IL‐5. Data were expressed as mean ± SD. All our data were obtained from three independent experiments. *p < 0.05, ** p < 0.01, ***p < 0.001.

4. DISCUSSION

The current treatment drugs for AD (e.g., cortisol) and immunosuppressants have shown good therapeutic effects on AD; however, these drugs may be inappropriate for long‐term use because of their adverse effects. ²¹ Therefore, new treatment strategies for AD should be sought. As widely reported, AD is related to many factors, including keratinocytes, T cells, and cytokines. ²² , ²³ Keratinocytes are the skin's barrier against external damage. In response to various external stimuli, activated keratinocytes can produce a variety of inflammatory cytokines and chemokines. The factors secreted by keratinocytes participate in the pathogenesis and development of AD inflammatory responses. ⁶ , ²³ For instance, IL‐6, as a pro‐inflammatory cytokine, is related to keratinocyte proliferation. ⁶ In addition, inflammatory chemokines are direct players in Th2‐mediated immune responses. ²⁴ Th2 skewing is the key point in the pathogenesis of AD. ²⁵ The polarization and recruitment of Th2 cells and subsequent secretion of Th2 cytokines play key roles in promoting the onset and development of AD. Thus, their inhibition has the potential to become an effective treatment strategy for AD. ²² Matrine possesses good therapeutic effects on inflammatory diseases. ²⁶ , ²⁷ Considering the immunomodulatory properties of matrine, we hypothesized that matrine has the potential to become a therapeutic drug for AD. Herein, we focused on the regulation of matrine on the secretion of keratinocyte pro‐inflammatory cytokines and Th1/Th2 inflammatory response in AD.

As previously reported, matrine can significantly suppress the secretion of inflammatory cytokines and chemokines in HaCaT cells. ²⁸ In addition, matrine can upregulate Th1 cytokine to regulate Th1/Th2 balance in eczema. ¹¹ All the pieces of evidence presented above suggest that matrine has huge regulatory effects on inflammatory cytokine secretions in keratinocytes and the balance of Th1/Th2, but these effects of matrine in AD have not been verified. Herein, our results revealed that matrine could effectively relieve the increase in epidermal thickness and histopathological aspects of the AD mouse model. In addition, matrine can suppress inflammatory cytokines and Th2 cytokine levels in the ear tissues and serum of AD mice. We also stimulated HaCaT cells with TNF‐α and IFN‐γ to mimic skin inflammatory injury in AD, and the results showed that matrine treatment suppressed inflammatory cytokines (TARC and IL‐6) levels in TNF‐α/IFN‐γ‐treated HaCaT cells. Furthermore, when co‐cultured with TNF‐α/IFN‐γ‐treated HaCaT cells, matrine inhibited CD4⁺ T cell differentiation into Th2 but promoted Th1 differentiation. Our results verified for the first time the immune regulation effects of matrine in AD, i.e., inhibition of inflammatory cytokine secretion in keratinocytes and regulation of Th1/Th2 inflammatory response.

Hsp90, as a cell stress‐inducing molecule, is involved in various biological process, such as cell growth, differentiation, and apoptosis. ²⁹ Hsp90 inhibition remarkably ameliorated inflammatory skin diseases, such as bullous skin disease, ²⁹ dermatitis herpetiformis, ¹² and psoriasis. ¹³ Hsp90 was significantly upregulated in the serum of AD patients, and its level was positively correlated with the severity of AD. ¹⁵ More importantly, matrine can serve as an Hsp90 inhibitor for cancer therapy. ³⁰ However, whether matrine achieves its biological function in AD by targeting Hsp90 remains unknown. Herein, Hsp90 expression was markedly elevated in ear skins of AD mice and TNF‐α/IFN‐γ‐treated HaCaT cells, but matrine treatment can eliminate these changes. In addition, Hsp90 overexpression reversed the inhibitory effect of matrine on inflammatory cytokine secretions in inflamed HaCaT cells. As widely reported, the production of inflammatory cytokines and chemokines in keratinocytes is tightly regulated by the NF‐κB pathway. ²⁴ Moreover, Hsp90 inhibition suppresses NF‐κB activation in THP‐1 cells. ¹⁹ Our findings showed that NF‐κB signaling was activated in inflamed HaCaT cells, and this activation was reduced by matrine administration. Furthermore, Hsp90 overexpression eliminated the inhibitory effect of matrine on NF‐κB activation in inflamed HaCaT cells. We inferred that matrine modulated Th1/Th2 inflammatory response by inhibiting the Hsp90/NF‐κB signaling axis based on prior experimental findings.

Overall, our findings suggested that matrine regulated Th1/Th2 inflammatory responses in AD by suppressing the Hsp90/NF‐κB signaling axis. Our work provided evidence revealing that matrine has a potential therapeutic effect on AD and clarified its mechanism, which is significant for the clinical treatment of AD.

CONFLICT OF INTEREST STATEMENT

All authors declare no conflict of interest.

Supporting information

Data S1: Supporting Information

Huang P, Hu F, Yang Z‐B, Pan Y, Zhou R, Yan Y‐N, et al. Matrine regulates Th1/Th2 inflammatory responses by inhibiting the Hsp90/NF‐κB signaling axis to alleviate atopic dermatitis. Kaohsiung J Med Sci. 2023;39(5):501–510. 10.1002/kjm2.12655