Indigo alleviates psoriasis through the AhR/NF-κB signaling pathway: an in vitro and in vivo study

Reagents and antibodies

IDG (Pubchem CID: 10215, 99% purity, verified by high-performance liquid chromatography) was procured from Chengdu Ruifenshi Biotechnology Co., Ltd. (Chengdu, Sichuan, China), with its chemical structure depicted in Fig. 1. Additional materials included glycerol, polysorbate-80, caprylic/capric triglyceride, p-hydroxyacetophenone, 1,2-hexanediol, and sodium polyacryloyldimethyl taurate, all sourced from Shanghai Maclean’s Biochemical Science and Technology Co., Ltd. (Shanghai, China). The imiquimod 5% cream was obtained from Sichuan Mingxin Pharmaceutical Co., Ltd. (Chengdu, China) (Approval No.: State Drug Permit H20030128), and the BCA Protein Assay Kit and MOUSE CYTOKINE/CHEMOKINE MAGNETIC BEAD PANEL were purchased from Poly Research Biotechnology Co., Ltd. (Warrington, PA, USA).

Preparation of IDG NE

To prepare the IDG NE, 5% glycerol and 0.1% IDG were individually mixed and pre-dispersed. Meanwhile, 5% glycerol, 0.3% p-hydroxyacetophenone, and 0.5% 1,2-hexanediol were dissolved by stirring in a water bath heated to 80 °C. The two mixtures were then combined, followed by the addition of 0.8% polyacryloyldimethyltaurine and 83.8% purified water, with continued stirring until homogeneously dispersed. Subsequently, 3% polysorbate-80 and 1.5% caprylic/capric triglyceride were incorporated under constant heating at 60 °C with stirring to ensure thorough mixing. The mixture was then subjected to high-speed shearing using a homogenizer for 5–8 min, resulting in the final 0.1% IDG NE. The blank nanoemulsion and 1% IDG NE were prepared using a similar procedure.

Animals

SPF-grade BALB/c mice, male, aged 5–6 weeks and weighing 15–17 g, were sourced from the Guangdong Laboratory Animal Centre (Laboratory Animal Production License No.: SCXK (Guangdong) 2022-0002; Laboratory Animal Qualification Certificate No.: 44007200117320). The mice were housed in the animal facility of Guangzhou University of Traditional Chinese Medicine, maintained under a 12-h light/12-h dark cycle at a controlled room temperature of 22–25 °C with a relative humidity of 55–70%. Six mice were housed per cage with free access to standard food and water. All experimental protocols were approved by the Laboratory Animal Ethics Committee of Guangzhou University of Traditional Chinese Medicine (No. 20221209004).

Throughout the study, continuous monitoring of the mice was conducted to assess health status, behavioral changes, and any signs of distress. This included regular body weight measurements, evaluations of coat condition, checks for skin damage, and inspections for eye and nose discharge. Additionally, food and water intake were recorded, social behaviors were observed, and any instances of abnormal grunting, unnatural body posture, or excessive licking or scratching were documented.

Euthanasia was carried out when mice exhibited severe illness, irrecoverable health issues, significant behavioral abnormalities, or upon reaching the experimental endpoint. The euthanasia procedure involved administering an overdose of anesthetic to ensure a rapid and painless death.

IMQ-induced psoriasis-like mouse model

Following a 1-week acclimatization period, the mice were shaved on their backs to create a hair-free area of approximately 3 cm × 4 cm. Thirty-six male BALB/c mice were then randomly divided into six groups using a random number table: a blank control group, a model group, a dexamethasone (DXM) control group, a blank NE group, a 0.1% IDG NE group, and a 1% IDG NE group, with six mice per group. In the blank control group, no treatment was applied to the shaved backs of the mice. In the model group, after shaving, 62.5 mg of 5% IMQ cream was applied once daily to the back for psoriasis modeling. For the DXM control group, in addition to the daily application of 5% IMQ cream, compounded DXM acetate cream was applied once in the morning and once in the evening at a dose of approximately 70 mg each time. The remaining groups, including the blank NE group, 0.1% IDG NE group, and 1% IDG NE group, received 70 mg of their respective treatments (blank NE, 0.1% IDG NE, and 1% IDG NE) once in the morning and once in the evening, in conjunction with the daily IMQ application.

Scoring system

The progression of skin lesions on the backs of the mice in each group was observed daily, with photographs taken on days 1, 4, and 7 for comparative analysis. The severity of the lesions was assessed using the Psoriasis Area and Severity Index (PASI) scale, focusing on erythema, desquamation, and infiltration. Each characteristic was scored on a scale from 0 to 4, where 0 indicated no lesions, 1 mild, 2 moderate, 3 severe, and 4 extremely severe. The total score was calculated by summing the scores for erythema, desquamation, and infiltration, resulting in a possible range of 0–12 points.

Mice in each group were weighed on days 1, 4, and 7, with weights recorded accordingly. On the seventh day of treatment, after the mice were sacrificed, their spleens were weighed to calculate the spleen index, which was determined by the ratio of spleen weight to body weight (spleen index = spleen weight/body weight).

Histopathology

All mice were euthanized on the seventh day of treatment through intraperitoneal injection of 150 mg/kg sodium pentobarbital. Criteria for early euthanasia were established to prioritize animal welfare, including signs of severe distress, weight loss exceeding 20% of body weight, or other significant health concerns. During the experiment, no mice met these criteria for early euthanasia. Post-euthanasia, skin samples from the dorsal lesions were collected and fixed in formalin. The skin tissues were then dehydrated using a gradient alcohol process, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (HE). Pathological analysis was conducted by randomly selecting five fields of view from individual tissue slices under a 200X light microscope, with pathological images captured for further analysis. In cases where mice exhibited signs of impending mortality during the experiment, euthanasia was administered immediately. At the conclusion of the experiment, all surviving mice were humanely euthanized.

Luminex liquid chip detection of cell cytokines

Luminex liquid microarray technology was employed to detect cytokines in both serum and skin tissue, specifically IL-6, IL-13, IL-17A, TNF-α, and MCP-1, with each sample subjected to three experimental repetitions. Total proteins were extracted using RIPA buffer (CST, Boston, MA, USA) and then centrifuged at 12,000 rpm for 5 min at 4 °C. Protein concentrations were determined using a BCA protein assay kit (Merck, Darmstadt, Germany). For the assay, 25 μL of each sample was added to 96-well microtiter plates. Beads were prepared by extracting 60 μL from each tube into a mixing bottle, diluting to 3 mL with bead diluent, and then adding 50 μL to each well. The plates were incubated by shaking them at room temperature for 2 h, followed by two washes with a washing buffer. Subsequently, 25 μL of detection antibody was added to each well and incubated with shaking for 1 h at room temperature. This was followed by the addition of 25 μL of SAPE, with further incubation and shaking for 30 min at room temperature. The plates were washed twice, and 150 μL of sheath solution was added to each well and shaken for 5 min at room temperature in the dark. The cytokine content was measured using the MILLIPLEX® MAGPIX system, and data analysis was performed using MILLIPLEX Analyst V5.1 software.

Cell culture and drug treatment

The human immortalized keratinocyte cell line (HaCaT) was obtained from the Chinese Academy of Science Center for Excellence in Molecular Cell Science and cultured in DMEM supplemented with 10% heat-inactivated FBS. Cells were maintained at 37 °C in a sterile incubator with 5% CO₂. HaCaT cells, which are commonly used to model oxidative stress and inflammatory injury, were stimulated with 1 μg/mL lipopolysaccharide (LPS) for 4 h to establish a psoriasis-like cell model. Following this induction, the cells were treated with 48 μM of IDG for an additional 24 h.

Cell counting kit-8 (CCK-8) assay

The IC50, or the concentration of an inhibitor required to achieve 50% inhibition, was determined in this study. After culturing the cells for 24 h, they were treated with varying concentrations of IDG for an additional 24 h. Cell viability was then assessed using the CCK-8 kit (Japan Tongren Company, Dojindo, Japan). Following the addition of the CCK-8 reaction solution, the cells were incubated in the dark for 3 h. Absorbance was measured at a wavelength of 450 nm using a full-wavelength enzyme-linked immunosorbent assay reader (Thermo Fisher Scientific, Waltham, MA, USA), and cell survival rates were calculated according to the manufacturer’s formula.

Real-time polymerase chain reaction (RT-PCR)

RT-qPCR was employed to detect the expression levels of NF-κB, TNF-α, IL-1β, AhR, and CYP1A1 genes in HaCaT cells. The experiment included three groups: a blank control group (CON), a model group (LPS), and a drug treatment group (IDG). HaCaT cells in the logarithmic growth phase were seeded in six-well plates at a density of 2 × 10⁶ cells per well. After overnight incubation to achieve complete adherence, the drug group was treated with 48 μM IDG, and 24 h later, both the drug and model groups were stimulated with 1 μg/mL LPS. Following a 4-h incubation, samples were collected from the cell culture box. Total RNA was extracted on ice using TRIzol® Reagent (#15596026; Thermo Fisher, China, Hong Kong). The concentration of RNA samples was measured using an ultra-micro spectrophotometer, with RNA purity assessed by the A260/A280 ratio, which ideally should be around 2.0; deviations from this value suggest potential contamination during the experimental process. The RNA concentration was diluted to 1 μg/μl, and genomic DNA was digested and reverse-transcribed into cDNA using the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR II (#AG11711; Accurate Biotechnology, Shenzhen, China). The reaction mixture, including 1 μl RNA, gDNA Clean Reagent, 5X gDNA Clean Buffer, and other components, was incubated at 42 °C for 5 min to remove gDNA, followed by reverse transcription at 37 °C for 15 min and at 85 °C for 5 s. The resulting cDNA was stored at −80 °C. Subsequently, the qRT-PCR reaction was set up according to the instructions of the SYBR® Green Premix Pro Taq HS qPCR Kit (#AG11701; Accurate Biotechnology, Shenzhen, China). The 20 μl reaction mixture consisted of 2 × SYBR® Green Pro Taq HS Premix, 0.2 μM each of upstream and downstream primers, cDNA, and RNase-free water. The PCR amplification was performed using the LABI 7500 system (Applied Biosystems) under the following conditions: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The melt curve analysis showed a single peak, and no amplification was observed in the No Template Control (NTC), confirming the specificity of the reaction. Primers for NF-κB, TNF-α, IL-1β, AhR, and CYP1A1 were synthesized by Shanghai Jieri Bioengineering Co., Ltd. (Table 1). The relative expression levels of target genes were calculated using the 2^{(−∆∆Ct)} method, with GAPDH serving as the internal control. The PCR efficiency was validated, with slopes ranging from −3.6 to 3.1 and an R² value of at least 0.98. A limit of detection (LOD) of 2.5 was established within a 95% confidence interval. Data analysis involved the exclusion of technical replicates that deviated significantly from the other two within each triplicate set. Each group comprised eight biological replicates, with each sample subjected to three technical replicates to ensure robust and reliable results.

Western blotting analyses

After extracting and isolating cytosolic and nuclear proteins using the Total and Nuclear Protein Extraction Kit (Solebo), protein concentrations were determined using the BCA Protein Assay Kit (Beyotime). Proteins were denatured by boiling at 100 °C for 5 min. Protein samples (25 μg per sample) were then separated by electrophoresis on an SDS-PAGE gel (Beyotime). The separated proteins were subsequently transferred to PVDF membranes, which were blocked for 1 h at room temperature in Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% skimmed milk powder. The membranes were incubated overnight at 4 °C with the respective primary antibodies: β-actin (1:10,000, affinity) and AHR (1:1,000; Affinity). Following this, the membranes were washed four times with TBST (5 min each) and incubated with horseradish peroxidase (HRP)-coupled secondary antibodies: goat anti-rabbit IgG (1:2,000; Affinity) and goat anti-mouse IgG for 2 h at room temperature. The membranes were then washed four times with TBST (5 min each). Protein detection was conducted using a Bio-Rad gel imaging system after applying the ECL luminescent solution (Beyotime) to the membranes. The grey values of the protein bands were analyzed using ImageJ software.

Statistical analysis

Data are presented as the mean ± standard error of the mean (SEM) from at least three independent experiments. The Kolmogorov-Smirnov (K-S) test was used to assess the normality of the data distribution. For data that followed a normal distribution, one-way ANOVA was performed; otherwise, the non-parametric “Independent-Samples Kruskal-Wallis Test” was used. A P-value of <0.05 was considered statistically significant. Statistical analyses were conducted using SPSS 26.0 (SPSS, Inc., Chicago, IL, USA), and figures were generated using GraphPad Prism 9.5 (GraphPad Software, Inc., La Jolla, CA, USA).

LPS stimulation promotes the expression of inflammatory factors in HaCaT keratinocytes

A psoriasis-like cell model was established by stimulating HaCaT keratinocytes with LPS, aiming to replicate key features of psoriasis in vitro (Takuathung et al., 2021; Thatikonda et al., 2020; Lin et al., 2023; Hong et al., 2022). The qRT-PCR results demonstrated that 1 μg/mL LPS stimulation of HaCaT cells for 4 h significantly upregulated the mRNA levels of TNF-α, IL-6, and NF-κB (Fig. 2A). This finding confirms that LPS at a concentration of 1 μg/mL can effectively induce HaCaT keratinocytes to mimic certain characteristics of psoriatic keratinocytes. Consequently, LPS (1 μg/mL) was used to establish an in vitro psoriasis-like cell model using HaCaT keratinocytes.

IDG inhibits inflammatory responses in LPS-stimulated keratinocytes

The IC50 of IDG on HaCaT cells was determined using the CCK-8 assay. Following a 24-h treatment with varying concentrations of IDG, the IC50 was calculated to be 118.7 μmol/L (Smolinska et al., 2018; Bai et al., 2023) (Fig. 2B). For subsequent experiments, an approximation of 40% of the IC50, equivalent to 48 μmol/L, was selected as the experimental concentration. The impact of IDG on LPS-stimulated HaCaT keratinocytes was evaluated by qPCR. The results indicated that LPS stimulation markedly elevated the mRNA levels of NF-κB, IL-1β, and TNF-α, but these levels were significantly reduced in the presence of IDG (Figs. 2C–2E).

IDG activates AhR signaling in LPS-induced HaCaT keratinocytes

The activation of the AhR has been well-established as an effective treatment strategy for psoriasis. AhR is a ligand-dependent transcription factor, and upon ligand binding, its nuclear translocation site becomes exposed, facilitating the translocation of AhR into the nucleus. Once in the nucleus, AhR forms a heterodimer with the aryl hydrocarbon receptor nuclear translocator (ARNT), which subsequently regulates downstream genes such as CYP1A1, playing a critical role in controlling cellular inflammation (Di Meglio, Perera & Nestle, 2011). To explore whether IDG mitigates the inflammatory response in HaCaT keratinocytes through the activation of the AhR pathway, we measured the mRNA levels of AhR and CYP1A1 using qPCR. The results showed a significant increase in AhR and CYP1A1 mRNA levels following IDG treatment (Figs. 3A and 3B). Additionally, to further elucidate the cellular localization of AhR, Western blotting analysis was performed. The results indicated that under normal conditions, AhR, a constitutively expressed molecule, is predominantly located in the cytoplasm. However, upon treatment with IDG, increased nuclear translocation of AhR was observed compared to the untreated condition (Figs. 3C–3E).

IDG NE ameliorates skin lesions in IMQ-induced psoriasis mice

To assess the therapeutic effect of IDG NE on an IMQ-induced psoriasis-like mouse model, topical treatments with IDG NE were administered. Initial observations of dorsal skin lesions and PASI scores following IMQ administration displayed characteristic psoriasis-like manifestations. Both IDG NE and DXM treatments significantly alleviated symptoms such as erythema, scaling, and skin thickening, resulting in a substantial reduction in PASI scores (Figs. 4A and 4C). Patients with psoriasis often exhibit splenomegaly, which may be indicative of the immune system’s chronic inflammatory response (Balato et al., 2015). Therefore, the spleen index was employed as a key indicator in this study. Findings revealed a significant increase in spleen index among IMQ-treated mice compared to the control group, while IDG NE treatment significantly reduced the spleen index in these mice (Figs. 4D–4F). H&E staining further demonstrated that IMQ induction caused distinct pathological changes in psoriasis-like lesions, characterized by excessive or incomplete epidermal keratinization, thickening of the acanthosis cell layer, and pronounced downward proliferation of epidermal ridges. Remarkably, IDG NE treatment mitigated these histological abnormalities (Figs. 4B and 4G).

Additionally, the IDG NE utilized in the study demonstrated a particle size of 180.7 ± 3.5 nm and a polydispersity index of 0.28 ± 0.01 (n = 3), as determined by laser particle size analysis. Stability tests confirmed the formulation’s excellent long-term stability.

IDG NE suppresses IMQ-triggered skin inflammatory cytokines and chemokines

To assess the impact of IDG NE on cytokine levels in skin tissue and serum in IMQ-induced mice, Luminex liquid microarrays were employed to measure inflammatory cytokines. Results indicated that IDG and DXM significantly inhibited the expression of IL-6, IL-17A, MCP-1, and TNF-α in psoriasis-like lesions, with all findings reaching statistical significance, while the effect on IL-13 was not statistically significant (Table 2). In the serum of IMQ-induced mice, levels of IL-6, IL-13, IL-17A, MCP-1, and TNF-α were significantly elevated in the IMQ group but were notably reduced in both the IDG and DXM groups, with all reductions being statistically significant (Table 3).

IDG NE reduces IMQ-induced psoriasis-like skin inflammation via activating AhR signaling

Further investigation into whether IDG NE mitigates IMQ-induced psoriasis-like skin inflammation through the activation of AhR signaling was conducted via Western blotting analysis to detect CYP1A1 protein expression in skin lesion tissues. Results revealed a significant reduction in CYP1A1 expression within the model group. In contrast, both IDG NE and DXM treatments significantly enhanced CYP1A1 expression, with IDG NE showing a more pronounced effect than DXM, suggesting that IDG NE facilitates AhR activation (Figs. 5A and 5B).

IDG NE inhibits NFκB-mediated inflammatory responses

NF-κB, a family of pleiotropic transcription factors, is ubiquitously expressed across various cell types and comprises five members: NF-κB1 (p105/p50), NF-κB2 (p100/p52), Rel-A (p65), Rel-B, and c-Rel. Activation of the classical NF-κB pathway primarily involves p50 and p65. Under resting conditions, NF-κB forms a complex with its inhibitor IκB in the cytoplasm. Upon degradation of IκB, p65 is phosphorylated into p-p65, allowing its release and translocation into the nucleus, where it initiates the transcription of target genes (Giuliani et al., 2001). In this study, the expression levels of NF-κB p65 and p-NF-κB p65 were examined in the skin lesions of each group. IMQ pretreatment significantly increased the expression of p65 and p-p65, both of which were markedly reduced by IDG NE treatment. Notably, the inhibitory effect of 1% IDG NE was more substantial compared to DXM (Figs. 6A–6D).