Yinxieling attenuates psoriasis in mice by regulating oxidative stress and lipid mediators to correct immune cell disorder through the NF-κB/Nrf2 signaling pathways

Abstract

Background

Psoriasis is a chronic skin disease that causes inflammation over time due to immune cell-mediated inflammation, oxidative stress, and lipid mediator imbalance. This study was to investigate the impact of Yinxieling (YXL), a reliable Chinese medicine for the treatment of psoriasis vulgaris, on the redox balance and lipid mediators in mice with IMQ-induced psoriasis.

Methods

Daily application of aqueous extract of YXL on IMQ-induced psoriasis-like mice, the efficacy and mechanism of YXL were evaluated by appearance symptoms, oxidative stress indicators, immune balance, and lipid metabolism indicators.

Results

The results demonstrated that YXL significantly enhanced therapeutic efficacy in a psoriasis mouse model, markedly improving the PASl scores and cutaneous inflammation. Upon YXL treatment, lipid peroxidation levels were significantly reduced, while antioxidant levels correspondingly increased. Additionally, YXL modulated the metabolic enzymes associated with 2-AG, which led to a decrease in CB1 receptor expression and an increase in CB2 receptor expression. In terms of immune modulation, YXL treatment promoted the regulation of T cell populations by down-regulating Th1, Th17, and γδT cells, while up-regulating Th2 and Treg cells, thereby facilitating the formation of an anti-inflammatory state. Further analysis indicated that these regulatory effects were closely associated with the down-regulation of NF-kB expression and the up-regulation of Nrf2 expression.

Conclusion

In conclusion, YXL reduces mice dermatitis by inhibiting oxidative stress, elevating endocannabinoid levels and balancing T cell populations in IMQ-induced psoriasis through the NF-κB and Nrf2 signaling pathways. Our results suggest its potential as a therapeutic option for psoriasis by targeting multiple pathways involved in the disease's development.

Graphical abstract

Highlights

• •YXL alleviates IMQ-induced psoriasis in mice by modulating the NF-κB and Nrf2 signaling pathways.
• •YXL targets oxidative stress, endocannabinoid levels, and T cell responses, mitigating IMQ-induced psoriasis-like dermatitis via multiple pathways.
• •YXL has dual regulatory effects and may serve as a potential selective CB1 receptor agonist and CB2 receptor antagonist, with potential applications as a targeted drug for the endocannabinoid system.

Abbreviations

GSH

glutathione

SOD

superoxide dismutase

MDA

malondialdehyde

H₂O₂

Hydrogen Peroxide

ROS

reactive oxygen species

H&E

hematoxylin and eosin

TBST

Tris-buffered saline -Tween

IL-17

Interleukin-17

FoxP3

Forkhead box p3

RORγt

retinoid-related orphan nuclear receptor γt

T-bet

T-box expressed in T cell

IFNγ

Interferon gamma

IL-4

Interleukin-4

Th1

T helper cell 1

Th2

T helper cell 2

Th17

T helper cell 17

Treg

Regulatory T cells

Dcs

Dendritic cells

2-AG

2-arachidonoylglycerol

CB1

Cannabinoid Receptor Type 1

CB2

Cannabinoid Receptor Type 2

PLCB1

Phospholipase C Beta 1

MAGL

Monoacylglycerol lipase

DAGLα

Diacylglycerol lipase alpha

NF-κB

Nuclear Factor-kappa B

IκBα

Inhibitor of κB alpha

Nrf2

Nuclear factor erythroid 2–related factor 2

HO-1

Heme Oxygenase-1

1. Introduction

Psoriasis is an immune cell-mediated inflammatory skin disease that generally presents as the formation of erythema, thickening and scale^1,2, affecting approximately 125 million people worldwide³. It is caused by the interplay of genes and environment, associated with multiple comorbidities and substantially weaken patients’ quality of life^4,5.

The pathogenesis of psoriasis remains elusive, but research has demonstrated that inflammation and oxidative stress are major risk factors in its development^6,7. Patients with psoriasis exhibit clear signs of oxidative stress, decreased intracellular antioxidant capacity, increased ROS production and other phenomena⁸. These factors have the potential to trigger the release of pro-inflammatory signaling molecules, which can subsequently activate dendritic cells and exacerbate the inflammatory response^9,10. It also affects the abnormal differentiation and activation of T cells, which contributes to inflammation and accelerates the progression of psoriasis¹¹.

Endocannabinoids are lipid mediators found in the skin barrier, influencing cellular redox balance and inflammation via activation of cannabinoid receptors (CB1/2)¹². Previous studies have shown altered endocannabinoid levels in psoriasis, closely linked to disease development and the inflammatory process^13,14. Endocannabinoids are synthesized via enzymatic pathways, with changes in levels associated with modifications in degrading enzyme activity and membrane receptor expression. Notably, 2-AG, a potent agonist, fully activates CB1 and CB2 receptors, synthesized by PLCB and DAGLα, and degraded by MAGL. In addition, previous research has implicated endocannabinoids in the regulation of skin immune response, specifically by modulating the activity and functions of T-helper (Th) cells¹⁵. These findings shed light on the important role that endocannabinoids play in the intricate interplay in the immune system within the skin.

Cells respond to elevated ROS levels by modulating gene expression for a protective response. ROS activation can stimulate NF-κB, a key inflammation regulator^16,17. Conversely, another significant transcription factor called Nrf2 reacts to oxidative stress and boosts cellular protection against it.¹⁸ Studies suggest a correlation between increased CB1 expression and NF-κB activation, while CB2 activation can reduce NF-κB activity¹⁹. Furthermore, Nrf2 activation enhances CB2 transcription, reinforcing anti-inflammatory effects^19,20. These findings suggest a complex interplay and mutual regulation between the NF-κB/Nrf2 signaling pathway and endocannabinoids.

Psoriasis, a multifactorial disease without a definitive cure, is often addressed using Traditional Chinese Medicine (TCM) as part of complementary and alternative medicine^21,22. Yinxieling (YXL), a long-standing prescription at Guangdong Provincial Hospital of Chinese Medicine, consists of 10 Chinese herbal medicines such as Radix Rehmanniae, Radix Angelicae Sinensis, and Paeoniae Radix Rubra (detailed in Table S1). In clinical treatment, YXL has been extensively used to alleviate symptoms in patients with psoriasis²³. Previous study suggests that YXL possesses certain anti-inflammatory activity and immunomodulatory effects. It can regulate macrophage infiltration and polarization²⁴, alleviate IMQ-induced skin inflammation by upregulating the Th2-specific transcription factor GATA3²⁵, as well as upregulating CD4+FoxP3+Treg cells in vitro²⁶, implying potential pharmacological effects in treating skin inflammation.

Psoriasis is an autoimmune disease that is immune-mediated and involves multiple factors such as oxidative stress and lipid mediators in its occurrence and development. In this study, we hypothesize that YXL, through its pharmacological properties, may attenuate oxidative stress and inflammation, regulate endocannabinoid homeostasis, and subsequently rebalance the Th1/Th2 and Th17/Treg immune profiles, via NF-κB and Nrf2/HO-1 signaling pathways. Therefore, investigating the mechanisms of YXL of regulating oxidative stress and lipid mediators to correct immune cell disorder through the NF-κB and Nrf2 signaling pathways, may pave the way for new approaches to mitigate immune dysfunction and inflammation associated with psoriasis.

2. Materials and methods

2.1. Reagents

RPMI1640, fetal bovine serum (FBS), and antibiotics (penicillin-streptomycin) were purchased from Gibco (Carlsbad, USA). Cyclosporine A (CsA) was obtained from Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd. (Hangzhou, China). Imiquimod cream was obtained from Sichuan Mingxin Pharmaceutical Co., Ltd. (Sichuan, China). RIPA buffer and BCA protein quantification kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Antibodies against NF-κB (p65), Nrf2, HO-1, CNR1, PLCβ, DAGLα, MGLL and β-actin was purchased from Affinity Biosciences (Jiangsu, China). IκBα and Goat anti-Rabbit IgG (HRP)-conjugated secondary antibody was purchased from CST (Boston, USA). CNR2 was purchased from Proteintech(Wuhan, China). Liberase and DNaseI were purchased from Roche (Basel, Switzerland). All Fluorochrome-conjugated antibodies and Mouse FoxP3 Buffer Set were obtained from BD (Becton, USA).

2.2. Preparation and analysis of YXL

The YXL formula consisted of ten botanical drugs (Table S1), obtained from Guangdong Provincial Hospital of Chinese Medicine in accordance with the quality standards specified in the Chinese Pharmacopoeia (2020 edition). The herbs were prepared by cutting into small pieces, soaking in clear water at ten times the volume of the medicinal materials, and then boiled and subjected to two additional rounds of extraction. The resulting mixture was filtered through a 75 μm filter, and the filtrate was then left undisturbed at 4 °C for 24 h. Vacuum filtration, concentration, and drying were carried out using a rotary evaporator. The freeze-dried powder of 0.1g YXL extract was then analyzed using UPLC. After ultrasonic extraction with 70 % methanol, the extracts were filtered through a 0.2 μm nylon filter membrane for UPLC analysis. The samples were separated using the Waters Acquity UPLC C₁₈ column and the Waters Acquity UPLC system. For the specific methodology, please refer to the previous studies conducted by our research group²⁷.

2.3. Mice and treatments

2.3.1. Mice

All animal experiments followed the guidelines of Guangdong Provincial Hospital of Chinese Medicine. Male BALB/c mice (6–8 weeks old) were sourced from Guangdong Experimental Animal Center (Guangzhou, China). They were housed in specific pathogen-free barrier conditions with ad libitum access to food and water. All experiments adhered to approved animal protocols and guidelines established by the Animal Care and Use Ethics Committee of Guangdong Hospital of Traditional Chinese Medicine (No. 2021095).

2.3.2. Oral treatment

A total of 72 BALB/c mice were randomly allocated into the following groups (n = 4 per group, repeat 3 times): Control group (Ctrl), Imiquimod group (IMQ), cyclosporineA group (CsA), and three Yinxieling groups (YXL-L, YXL-M, YXL-H). The dosages are determined by the standard dosages used in treating psoriasis patients with YXL using a body surface area conversion formula. Ctrl group and IMQ group were orally treated with distilled water. CsA group and YXL groups were orally treated with Cyclosporine A (CsA, 25 mg/kg) and YXL (0.5 g/kg, 1.0 g/kg, 2.0 g/kg) respectively for 7 days. Except for the control group, mice were treated with 50 mg topical IMQ cream daily for five consecutive days, to create IMQ-induced psoriasis-like mouse model. On day 8, mice were euthanized and tissues were collected for further studies.

2.4. Body weight loss

The body weights of mice were measured daily before treatment. Body weight gain or reduction was calculated, compared to the first day.

2.5. Scoring severity of skin inflammation

The severity of back skin inflammation was assessed using an objective scoring system based on the Psoriasis Area and Severity Index (PASI). Erythema, scales, and skin thickness were blindly and independently scored on a 0 to 4 scale: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked. The cumulative score of these three aspects served as the assessment of inflammation severity.

2.6. Histopathology analyses

Skin lesions were collected and fixed in 4 % paraformaldehyde for 24h at room temperature, followed by dewaxing, dehydrated in ethanol, and paraffin embedding. The skin tissues were then sectioned into 4 μm slices and stained with hematoxylin and eosin (H&E) for evaluation of skin inflammation severity.

2.7. Detection of oxidative stress in skin tissues lysate

The skin tissue samples were homogenized in ice-cold PBS to make a 10 % tissue homogenate and were centrifuged at 12000 rpm for 10min at 4 °C. The supernatant was retained for detection. The level of malondialdehyde (MDA), Hydrogen peroxide (H₂O₂), superoxide dismutase (SOD), glutathione (GSH) in skin tissue were measured according to manufacturer's protocols.

2.8. Preparation of cell suspensions

2.8.1. Preparation of cell suspensions from skin lesions

Mouse skin lesions were harvested and incubated in 0.4 mg/ml Liberase at 37 °C for 75 min, followed by the addition of an equal volume of 0.1 % DNaseI and gentle disruption and digestion at 37 °C for 15 min. The tissue was then aspirated through a 70 μm cell filter to obtain a single-cell suspension. Subsequently, one million individual cells were stimulated with Leukocyte Activation Cocktail for a 5-h duration.

2.8.2. Preparation of cell suspensions from lymph nodes²⁵

Lymph nodes were ground and passed through a 70 μm cell filter to create a single cell suspension. Subsequently, one million individual cells were stimulated with Leukocyte Activation Cocktail for a 5-h duration.

2.9. Flow cytometry analysis

After harvesting, the cells were washed and stained with antibodies, including FITC Rat Anti-Mouse CD4 (Clone: GK1.5), PE Mouse Anti-CD4 (Clone: GK1.5), PerCP-cy5.5 Rat Anti-Mouse CD25 (Clone: PC61), FITC Rat Anti-Mouse γδT (Clone:GL3), eFluor 450 Rat Anti-Mouse TCR (Clone:H57-597, eBioscience) for 30 min at 4 °C in the dark. Cells were later fixed and permeabilized with Mouse FoxP3 Buffer Set before being intracellularly stained with Alexa Fluor 647 Mouse Anti-T-bet (Clone: O4-46), BV421 Rat Anti-Mouse GATA3 (Clone: L50-823), Alexa Fluor 647 Rat Anti-Mouse RORγt (Clone: Q31-378), Percp-cy5.5 Rat Anti-Mouse IFN-γ (Clone: XMG1.2), PE-CF594 Rat Anti-Mouse IL-17A (Clone: TC11-18H10), APC Rat Anti-Mouse IL-4 (Clone: 11B11) for another 30 min at 4 °C in darkness. Cell analysis was performed using FACS Aria Ш (Becton, USA) and data acquired were analyzed with FlowJo software, version 10.8.1 for Microsoft (Sam Carlos, USA).

2.10. Western blotting analyses

The mouse skin samples (50 mg per sample) were homogenized using RIPA buffer (Beyotime, China) for 30 min on ice. The concentrations of protein in supernatant were determined using the BCA Protein Assay Kit. Then, equivalent protein (60 μg) was separated by SDS-PAGE gel electrophoresis, and transferred to PVDF membranes (Millipore, BillERICA, USA). Then, the membrane was blocked with 5 % non-fat milk in 0.1 % TBST at room temperature, and incubated with mAbs at 4 °C overnight, including CNR1 Antibody #DF4918(1:1000), CNR2 Antibody-29371-1-AP (1:1000), PLCB1 Antibody #DF6726(1:1000), MGLL Antibody #DF8444(1:1000), DAGLA Antibody#DF13509(1:1000), HO-1 Antibody#AF5393(1:1000), Nrf2 Antibody#AF7006(1:1000), NF-κB p65 Antibody-#AF5006(1:1000), IκBα (44D4) Rabbit mAb #4812(1:1000, CST) overnight at 4 °C. The membranes were washed with TBST and incubate with Goat anti-Rabbit IgG (HRP)-conjugated secondary antibody (CST, USA) for 1 h. Finally, the membranes were visualized using ECL Western blotting detection reagents (Millipore, BillERICA, USA).

2.11. Statistical analysis

This study employed Graphpad Prism 8.0 software for the visualization and graphical analysis of the data. The statistical analysis of the results was conducted using SPSS 26.0 software. For multiple sets of independent measurement data that meet the criteria of normal distribution and homogeneity of variance, the results are represented as($\bar{x}\pm s$). When the data conform to a normal distribution, a one-way analysis of variance (ANOVA) is utilized. If the homogeneity of variance test indicates equality of variances, multiple comparisons between groups are performed using the Bonferroni method. In cases where variances are not equal, Dunnett's T3 method is selected for inter-group comparisons. For measurement data that do not comply with a normal distribution, they are represented as M(P25∼P75), and the Kruskal-Wallis H test, a non-parametric method, is used to compare multiple independent samples. A p-value of less than 0.05 indicates statistical significance.

3. Results

3.1. YXL effectively ameliorated dermatitis in IMQ-induced psoriasis-like mice

The IMQ-induced psoriasis mouse model had severe psoriatic phenotypes characterized by erythema, scales, and infiltration. In order to examine the potential of YXL in ameliorating clinical symptoms in BALB/c mice, we conducted an oral administration study using three different doses: low dose (0.5 g/kg), medium dose (1.0 g/kg), and high dose (2.0 g/kg) of YXL for a duration of 7 days (Fig. 1A). The results indicate that, compared to the IMQ group, both YXL and CsA groups significantly alleviated the clinical symptoms of psoriasis-like conditions in mice, including erythema, thickening, and scaling (Fig. 1B). According to the PASI score, the mice in the blank control group did not exhibit any significant changes in their back skin. However, mice in the IMQ group displayed noticeable skin lesions starting from the second day after treatment, and these lesions reached their peak on the fifth day (Fig. 1C). In comparison, the CsA and YXL treatment group exhibited lower scores for back lesions and overall total scores when compared to the IMQ group (P < 0.01). Together, YXL had protective effects on IMQ-induced psoriasis-like mice (Fig. 1D).

Fig. 1.

Yinxieling (YXL) effectively ameliorated psoriasis symptoms in Imiquimod (IMQ)-induced psoriasis-like mice.

Mice were daily administered with varying doses of YXL or vehicle control and subjected to IMQ-induced psoriasis model. The back skin was assessed for clinical symptoms at the indicated time points. A Schematic representation of the experimental design for IMQ-induced psoriasis-like mouse models and treatment with YXL or Cyclosporine A (CsA). (Created with Biorender.com). B Clinical symptoms of psoriasis, including erythema, thickening, and scaling, were alleviated by YXL treatment. C Changes of PASI scores in each group during IMQ modeling. D On day 6, each dose of YXL and CsA significantly reduced PASI scores. E The changes of body weight loss of each group of mice during IMQ modeling. F On day 6, each dose of YXL significantly reduced body weight loss. All data were displayed as mean ± SD, n = 3. ∗P < 0.05, ∗∗P < 0.01, versus group of IMQ, one representative of three independent experiments with similar results is shown.

The mice in each group were weighed daily after treatment (Fig. 1E). The results showed that the body weight of mice in the control group had no significant change before and after treatment, and the body weight of mice in the IMQ group and the YXL treatment group decreased significantly after IMQ-treatment (P < 0.01). Notably, all YXL groups exhibited an improvement in weight loss compared to the IMQ-treated group (Fig. 1F, P < 0.01). However, the CsA group did not demonstrate a significant effect on body weight rescue (Fig. 1F, P > 0.05).

3.2. YXL relieved the IMQ-induced acanthosis and normalizes the epidermal architecture

Three microscopic areas were randomly chosen to be evaluated in every H&E stained section. Epidermal thickness was lower in all YXL treatment groups than in the IMQ group (Fig. 2A and B, P < 0.01). The epidermis structure of the control group was intact and clear, consisting only of 1–2 layers of closely connected epidermal cells. Compared to the control group, skin tissue sections from the IMQ group exhibited typical psoriatic changes, such as hyperkeratosis, parakeratosis, acanthosis, and elongation of the reticular ridges. These findings were consistent with previous studies in the literature. Treatment with CsA and YXL exhibited a significant reduction in epidermal thickness and the number of cell layers in mice skin lesions, and showed a significant reduction in epidermal hyperplasia and improvement in epidermal structure (Fig. 2A, B, C, P < 0.01).

Fig. 2.

Yinxieling (YXL) alleviated Imiquimod (IMQ)-induced skin pathological structure in BALB/c mice.

Histological analysis was performed on skin sections from IMQ-induced psoriasis-like mice treated with YXL or CsA, with H&E staining to evaluate changes in epidermal structure. A H&E staining revealed that Cyclosporine A (CsA) and YXL treatments significantly reduced skin lesion epidermal thickness and cell layer count in mice, signifying improved epidermal structure. B–C Epidermal thickness and cell layers were quantified, demonstrating the normalization of epidermal architecture in response to YXL or CsA. treatment. All data were displayed as mean ± SD, n = 3. ∗P < 0.05, ∗∗P < 0.01, versus group of IMQ, one representative of three independent experiments with similar results is shown.

3.3. YXL inhibited oxidative stress in IMQ-induced psoriasis-like mice

To observe the antioxidant effects of YXL on IMQ-induced psoriasis-like mice, the H₂O₂ content, MDA content, SOD activity and GSH activity in the back skin of mice were detected. The content of MDA and H₂O₂ in the skin of IMQ group were significantly increased, while SOD activity and GSH activity decreased significantly. The results showed that IMQ could cause the imbalance of redox homeostasis in mice (P < 0.05). Compared with IMQ group, MDA content and H₂O₂ content in YXL dosage groups were significantly decreased (Fig. 3A and B, P < 0.05). Compared with the CsA group, the declining trend of YXL group was more obvious. In addition, YXL dosage groups were found to help reverse the decreased levels of SOD activity and GSH activity (Fig. 3C, D, P < 0.05).

Fig. 3.

Yinxieling(YXL) alleviated Imiquimod (IMQ)-induced oxidative events in BALB/c mice.

Oxidative stress markers were measured in skin tissue homogenates from IMQ-induced mice treated with YXL, including MDA, H₂O₂, SOD, and GSH levels.A,B,C,D The contents of A: MDA, B: H₂O₂, C: SOD, and D: GSH in the skin lesion from each group of mice. YXL and Cyclosporine A (CsA) treatment significantly decreased MDA and H₂O₂ levels and increased the activities of SOD and GSH, indicating a restoration of redox balance. All data were displayed as mean ± SD, n = 3. ∗P < 0.05, ∗∗P < 0.01, versus group of IMQ, one representative of three independent experiments with similar results is shown.

3.4. YXL exerted anti-inflammatory effects by regulating endocannabinoid

To assess the impact of YXL on endocannabinoid receptors and 2-AG metabolic enzymes in mouse skin lesions, the Western Blot assay was employed. The Western Blot assay was employed using the medium dose of YXL (1.0 g/kg), which has been demonstrated to be effective in our preliminary studies. The results demonstrated that the IMQ group exhibited significantly elevated protein expression levels of CB1, CB2, PLCB1, and DAGLα, compared to the control group (P < 0.05). Conversely, the expression of the degradation enzyme MAGL was notably reduced (P < 0.05 or 0.01). However, Cyclosporin A seems did not stimulate the synthesis of endocannabinoids (P > 0.05), but it may elevate their levels by inhibiting the production of degrading enzymes (P < 0.05). Following YXL treatment, there was a gradual decrease in the protein expressions of CB1 and MAGL, while CB2, DAGLα, and PLCB1 showed significant increases (P < 0.05 or 0.01) as depicted in Fig. 4A and B. The aforementioned experimental findings suggested that YXL could improve the pathological condition of psoriasis by regulating endocannabinoids (Fig. 4A and B).

Fig. 4.

Yinxieling (YXL) regulated cannabinoid receptors and enzymatic responses in psoriatic mouse models.

Protein expression levels of endocannabinoid-related enzymes and receptors were assessed via Western blot in skin samples from IMQ-induced mice treated with YXL. A YXL and Cyclosporine A (CsA) regulated the expression levels of PLCB1 and DAGLα and MAGL proteins in the skin of each group of mice. B YXL and CsA regulated the levels of CB1 and CB2 proteins in the skin of each group of mice. All data were displayed as mean ± SD, n = 3. ∗P < 0.05, ∗∗P < 0.01 versus group of IMQ, one representative of three independent experiments with similar results is shown.

3.5. YXL decreased the populations of dendritic cells (DCs) in the skin lesions of psoriasis-like mice

Given that abnormal activation of DCs is involved in the development of the psoriasis, we examined the effect of YXL on DCs populations in IMQ-induced mice. We collected the lymph node samples from the IMQ-treated mice, ground them and examined the profiles of the Dcs via flow cytometry. The results demonstrated that YXL treatment significantly reduced the number of MHCII + Dcs in the skin of mice treated with IMQ, when compared to the IMQ group. These findings suggested that YXL had the ability to modulate dendritic cells in vivo (Fig. 5, P < 0.05).

Fig. 5.

Yinxieling (YXL) inhibited the Dendritic cells (DCs) in skin draining lymph nodes.

Lymph node samples from IMQ-treated mice after treatment with YXL were processed to prepare cell suspensions, and the population of dendritic cells was analyzed using flow cytometry. Representative examples for YXL inhibits the MHCII + DCs. in skin draining lymph nodes. All data were displayed as mean ± SD, n = 3. ∗P < 0.05 and ∗∗P < 0.01 versus group of IMQ, one representative of three independent experiments with similar results is shown.

3.6. YXL decreased the Th1/Th2 ratio in the psoriasis-like lesions

The Th1/Th2 ratio was analyzed by flow cytometry using a single-tissue cell suspension from the skin lesion. Our study found that the administration of YXL and CsA led to a significant reduction in the percentage of pro-inflammatory TCRβ+CD4+IFNγ+Th1 populations in the skin lesions when compared to the IMQ group. Additionally, treatment with YXL resulted in an increase in the population of anti-inflammatory TCRβ+CD4+IL-4+Th2 cells (Fig. 6A, P < 0.05).

Fig. 6.

Yinxieling (YXL) inhibited skin inflammation by decreasing the ratio of IFNγ producing Th1 cells, as well as increasing the ratio of IL-4 producing Th2 cells in skin lesion. In addition, upregulating the expression of Th2 specific transcription factors GATA3, but not down regulating the expression of Th1 transcription factors T-bet.

Flow cytometry was used to analyze the ratio of Th1 to Th2 cells in single-tissue cell suspensions from skin lesions of IMQ-induced mice treated with YXL. A YXL decreased the Th1/Th2 ratio in psoriasis-like lesions by reducing the percentage of pro-inflammatory TCRβ+CD4+IFN-γ+Th1 cells and increasing the population of anti-inflammatory TCRβ+CD4+IL-4+ Th2 cells. B YXL upregulated of Th2-specific transcription factor GATA3 without significant changes in Th1 transcription factor T-bet. All data were displayed as mean ± SD, n = 3. ∗P < 0.05 and ∗∗P < 0.01 versus group of IMQ, one representative of three independent experiments with similar results is shown.

Furthermore, we assessed the levels of characteristic transcription factors in Th1/Th2 cell populations. The findings demonstrated that medium to high doses of YXL significantly elevated the frequency of TCRβ+CD4+GATA3+Th2 populations within the skin lesions (P < 0.01). However, there were no significant changes observed in the frequencies of TCRβ+CD4+T-bet+Th1 populations in all YXL treatment groups (Fig. 6B, P > 0.05).

3.7. YXL decreased the frequency of IL-17 producing Th17 cells and γδT cells, as well as the Th17/Treg ratio in the psoriasis-like lesions

To understand the effect of YXL on the IL17 producing Th17 and γδT cells, single-tissue cell suspension were made from the skin lesions for flow cytometry. Our study found that the administration of YXL and CsA led to a significant reduction in the percentage of pro-inflammatory TCRβ+CD4+IL-17A+Th17 cells, and TCRγδ+IL-17A+γδT cells in the skin lesions when compared to the IMQ group (Fig. 7A and B, P < 0.05).

Fig. 7.

Yinxieling (YXL) relieved Imiquimod (IMQ)-induced skin lesions by decreasing the ratio of IL-17 producing Th17 cells and γδ+T cells, upregulating the expression of Treg specific transcription factors FoxP3, but not down regulating the expression of Th17 transcription factors RORγt.

Flow cytometry analysis was employed to determine the frequency of IL-17 producing Th17 and γδT cells, as well as Treg cells, in skin lesion cell suspensions from IMQ-induced mice after treatment with YXL and CsA. A YXL treatment reduced the frequency of CD4+IL-17A+Th17 cells percentage. B YXL treatment reduced the frequency of TCRγδT+IL17A+γδT cells percentage. C YXL treatment elevated the frequency of TCRβ+CD4⁺CD25+FoxP3+Treg cells, but did not affect TCRβ+CD4+RORγt +Th17 cells. All data were displayed as mean ± SD, n = 3. ∗P < 0.05 and ∗∗P < 0.01 versus group of IMQ.

In addition, we conducted an assessment of the levels of characteristic transcription factors in Th17/Treg cell populations. The findings demonstrated that medium to high doses of YXL significantly elevated the frequency of TCRβ+CD4⁺CD25+FoxP3+Treg populations within the skin lesions (P < 0.05 or 0.01), but had no effect on TCRβ+CD4+RORγt + Th17 populations (Fig. 7C, P > 0.05).

3.8. YXL attenuated psoriasis-like dermatitis by inhibiting NF-κB and activating nrf2/HO-1 signaling pathways

To validate the roles of the NF-κB and Nrf2/HO-1 signaling pathways in psoriatic skin lesions and the potential mechanisms of YXL, we employed Western blotting to assess the expression levels of NF-κB, IκBα, Nrf2, and HO-1 in the skin lesions. The specific results are presented in Fig. 8A and B. The findings indicated that IMQ treatment led to an increase in NF-κB expression, concomitant with decreased expression levels of IκBα, Nrf2, and HO-1 (Fig. 8, P < 0.05). In contrast, compared to the IMQ-treated group, YXL treatment was capable of reducing NF-κB expression while increasing the expression levels of IκBα, Nrf2, and HO-1 (Fig. 8, P < 0.05).

Fig. 8.

Yinxieling (YXL) regulated the NF-κB and Nrf2/HO-1 pathways in psoriatic mouse models.

Expression levels of NF-κB, IκBα, Nrf2, and HO-1 were evaluated in skin lesions of IMQ-induced mice by Western blotting after treatment with YXL. A Western blot analysis of skin lesions shows reduced NF-κB expression and increased IκBα expression following YXL treatment. B Western blot analysis of skin lesions shows increased Nrf2, and HO-1 expression following YXL treatment. All data were displayed as mean ± SD, n = 3. ∗P < 0.05, ∗∗P < 0.01, versus group of IMQ, one representative of three independent experiments with similar results is shown.

4. Discussion

Psoriasis is a chronic autoimmune disease with a complex pathogenesis, which makes it challenging to determine the most effective treatment methods. The main treatment methods for psoriasis currently include traditional drug therapies (such as methotrexate and vitamin D3 analogues), biological agents (such as TNF-α inhibitors and IL-17 inhibitors), phototherapy, and some emerging therapies²⁸. These treatment methods have issues with recurrence and side effects^29,30. Traditional Chinese herbal medicine is a valuable resource for the treatment of diseases such as psoriasis31, 32, 33. YXL has been clinically proven to effectively improve and control the condition, and its administration is considered safe. Thus, it is viewed as a promising therapeutic agent^34,35. As demonstrated in photographs, the findings from this research indicate that YXL could potentially be used as a therapeutic treatment for IMQ-induced psoriasis in mice by modulating the NF-κB and Nrf2 signaling pathways, suppressing oxidative stress, elevating 2-AG levels, and rectifying T cell abnormalities.

The skin, being the largest organ of the body, is often exposed to both internal and external oxidative stress36, 37, 38. According to literature reports, there is a significant antioxidant imbalance in the bodies of psoriasis patients, and supplementing antioxidants with conventional therapy can significantly improve clinical symptoms of psoriasis³⁹. The skin lesions of the IMQ group showed an increase in levels of H₂O₂ and MDA, in line with the literature, while the levels of antioxidant enzymes GSH and SOD were decreased. These results further confirm the presence of oxidative stress in psoriatic skin lesions. Notably, our study revealed that YXL has a corrective effect on this abnormal oxidation state, by reducing H₂O₂ and MDA levels as well as restoring the activity of SOD and GSH. YXL has been shown to enhance antioxidant defense and protect against oxidative damage in its treatment of psoriasis.

The endocannabinoid system in the skin represents a potential direction for treating dermatological conditions, with the primary endocannabinoid 2-AG capable of directly mitigating inflammation and oxidative stress⁴⁰. This is often considered a compensatory or adaptive mechanism in the body, showing significant potential in the development of psoriasis⁴⁰ .Upon observation, we noted the activation of CB1 and CB2 receptors in skin lesions following IMQ treatment. However, following YXL treatment, we observed the suppression of CB1 expression and activation of CB2 expression in the skin lesions. At the same time, the enzymes that generate and degrade 2-AG were regulated, which resulted in an increase in 2-AG levels. The regulatory role of endocannabinoids in psoriasis is further supported by these results, which provide new evidence for the mechanism of action of YXL in treating psoriasis. It should be noted that YXL has a dual regulatory role, acting as a selective CB1 agonist and CB2 inhibitor, resulting in its potential use as a targeted drug for the endocannabinoid system. It is expected that its regulatory effects on CB1 and CB2 will become a crucial part of future therapeutic strategies.

The endocannabinoid system has been shown to modulate immune function, a topic that has been widely reviewed^15,41,42. Immune cell changes were observed in a mouse model of psoriasis after IMQ treatment. It was observed that the skin lesions in mice exhibited increased levels of DCs, Th2, and Treg cells, while the expression of γδT, Th17, and Th1 cells showed a downward trend. Interestingly, the activation of downstream T cells was affected by a reduction in dendritic cell activation levels after YXL treatment. The decrease in the proportions of γδT, Th1, and Th17 cells caused by YXL may be attributed to this effect. YXL may achieve an immune balance shift towards Th2 and Treg by weakening the function of these cells. In recent years, researchers have noted the immunomodulatory role of the cannabinoid receptor CB2 in psoriasis⁴³. Selective activation of CB2R by AJWH-133 inhibits the production of pro-inflammatory cytokines, reduces the Th17/Treg cell ratio, and alleviates psoriasis-like dermatitis⁴⁴. This suggests that the immune balance shift caused by YXL may be achieved through its interaction with the CB2 receptor. Specifically, it may promote Th2/Treg differentiation through the activation of the CB2 receptor, thereby exerting an anti-inflammatory effect in the treatment of psoriasis.

NF-κB/Nrf2 signaling pathways are mutually regulated by the endocannabinoid system. Previous studies have indicated that endocannabinoids may exert anti-inflammatory effects by potentially inhibiting the NF-κB pathway through the CB1 receptor⁴⁵. Furthermore, the endocannabinoid system interacts with the cellular redox state and the Nrf2 pathway⁴⁶. Some reports indicate abnormal states of NF-κB and Nrf2 signaling pathways in skin lesions of psoriasis patients^47,48. Our experimental study showed that IMQ application triggers activation of NF-κB, while Nrf2 activity was inhibited. Our investigation revealed that YXL reverses these effects. We hypothesize that YXL stabilizes IκBα/NF-κB complexes by inhibiting IκBα degradation and induces the Nrf2 signaling pathway. The downstream gene HO-1 of Nrf2 further enhances the activity of SOD and GSH, crucial in anti-inflammatory and antioxidative processes. Simultaneously, we noted an upregulation of CB2 expression following YXL treatment, enhancing anti-inflammatory actions, potentially mediated through CB2 mechanisms, thereby modulating immune responses. Notably, the NF-κB signaling pathway was negatively regulated by activated Nrf2 and CB2, which reduced its activity and consequently attenuated immune responses. Further investigation suggests that these two signaling pathways interact and complement each other. Activated Nrf2 and CB2 form a coordinated regulatory network with the NF-κB signaling pathway, enhancing overall anti-inflammatory and antioxidative capabilities. This mutual regulation might represent a crucial mechanism for the successful alleviation of symptoms in a murine model of psoriasis with YXL treatment.

Based on the above data, we hypothesize that YXL exerts its therapeutic effects on psoriasis through the mutual regulation of the NF-κB and Nrf2 signaling pathways, thereby modulating oxidative stress, the endocannabinoid system, and T-cell dysregulation in a coordinated manner. Simultaneously, inflammation and immune balance are influenced by the endocannabinoid system simultaneously, which creates a positive feedback loop and contributes to therapeutic efficacy of YXL. The successful application of YXL in the mouse model provides a theoretical basis and experimental evidence for its future clinical use in the treatment of human psoriasis. By inhibiting oxidative stress and modulating immune responses, YXL shows promise as a new therapeutic option for psoriasis.

5. Conclusion

Current treatment strategies for psoriasis focus on anti-inflammatory actions and the inhibition of keratinocyte proliferation⁴⁹. Our study explores the balance of the endocannabinoid system as a starting point, investigating how the Chinese medicine YXL corrects the imbalance of this system, treating psoriasis from a systemic perspective. It also interprets the modern essence of the holistic treatment concept of traditional Chinese medicine's syndrome differentiation and treatment. To sum it up, our investigation uncovers the comprehensive effects of YXL on treating psoriasis by controlling oxidative stress, endocannabinoid levels, and T-cell balance through the regulation of the NF-κB/Nrf2 pathways. This has provided us with a new understanding of the mechanism of action of YXL in the treatment of psoriasis. While our study has uncovered potential mechanisms of YXL in modulating signaling pathways and addressing T cell abnormalities, further research is needed to elucidate additional molecular details. Therefore, our future research will focus on these aspects to deepen our understanding of the molecular basis of YXL's therapeutic effects.

Funding

This work was financially supported in part by, National Natural Science Foundation of China (NO. 81703778), Guangzhou Science and Technology Bureau City/University (College) Joint Funding Project (NO.202102010336), Bajian Talents of Guangdong Provincial Hospital of Chinese Medicine (NO. BJ2022KY14), Science and technology projects in Guangdong Provincial Hospital of Traditional Chinese Medicine (NO.YN2018MJ04), The 2020 Guangdong Provincial Science and Technology Innovation Strategy Special Fund (Guangdong-Hong Kong-Macau Joint Lab, No. 2020B1212030006), Guangdong Provincial Clinical Research Center for Chinese Medicine Dermatology (NO. 2020B1111170012), 2022 TCM Innovation Team and Talent Support Program of the State Administration of Traditional Chinese Medicine (NO. ZYYCXTD-C-202204).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article: