Introduction
Psoriasis is a chronic autoimmune skin disease characterized by plaques and scales [
1]. The global prevalence is approximately 2% [
2]. Despite chronic plaque-type psoriasis, which accounts for about 90% of psoriasis cases, some patients suffer from pustular psoriasis, guttate psoriasis, and inverse psoriasis [
3]. Patients with psoriasis have excessive proliferation of keratinocytes, dilation of dermal capillaries, and infiltration of inflammatory cells. Patients afflicted with severe psoriasis experience a range of complications, such as the development of arthritis and immune dysfunction [
4]. Dendritic cells and macrophages present in the dermis affected by psoriasis are responsible for the production of interleukin 23. This production leads to the activation of T cells, as well as the release of inflammatory cytokines including IL-17A, IL-17F, IL-22, IL-6, and TNF-α [
5]. Small molecule inhibitors and biologics are commonly used in the clinical treatment of psoriasis. In certain case series, the surgical procedure of tonsillectomy has been found to result in a notable amelioration of plaque psoriasis [
6‐
8]. Methotrexate and cyclosporine are widely used in the clinical management of psoriasis. Acitretin is used to inhibit the proliferation and differentiation of keratinocytes [
9,
10]. Nevertheless, the attainment of long-term usage is challenging due to the adverse effects and the potential for relapse following cessation [
11]. T cells play a pivotal role in mediating various inflammation and immune disorders. T cell-based immunotherapy is advancing. CXC chemokine receptor 6 (CXCR6) has been listed a novel target for immunotherapy for autoimmune, including psoriasis. [
12] With the approval of the Etanercept for clinical use by the U.S. Food and Drug Administration, the advent of biologic therapies for psoriasis commenced. Subsequently, the discovery of the IL-23/Th17 axis has led to the development of a growing number of inhibitors that target IL-17 or IL-23, including guselkumab, which have entered the market [
13]. Biologic medications, while exhibiting a reduced incidence of adverse effects, impose a significant economic strain on patients due to their exorbitant cost [
14]. Hence, it holds clinical importance to identify a treatment that is both safe, efficacious and economically accessible.
As the study of psoriasis has advanced, there has been an increasing recognition of the significant involvement of endothelial cells in the progression of the disease. The characteristic epidermal hyperplasia observed in psoriasis is closely associated with the angiogenic microenvironment influenced by vascular endothelial growth factor (VEGF) [
1]. Nonetheless, in another research, psoriasis has been linked to the genetic mutation of VEGF [
15]. In addition, suppression of gene expression has been shown to effectively alleviate psoriasis-like features in a mouse model [
16]. Furthermore, for psoriatic skin, endothelial cells become activated and release endothelial adhesion molecules, resulting in the recruitment of leukocytes and subsequent initiation of an inflammatory response [
17].
As a blend of a variety of Chinese herbs, Shi-Bi-Man (SBM) comprises ingredients such as Radix Ginseng, tea polyphenols, Radix Polygoni Multiflori (the root tuber of
Polygonum multiflorum Thunb.), Radix Angelica Sinensis from the root of
Angelica sinensis (Oliv.) Diels, Aloe vera L., linseed, and green tea extract. SBM showed no toxicity in a prior mouse model [
18]. Previous research has demonstrated that TSG and EGCG, the primary active components found in SBM, can stimulate hair regrowth by activating the fibroblast growth factor (FGF) pathway in dermal papillary cells [
18].
In this study, we aimed to explain the mechanism of SBM in the treatment of psoriasis using single-cell sequencing technology, clarify the target of SBM based on specific cell populations, offering insights that could potentially introduce a new choice for the clinical treatment of psoriasis.
Materials and methods
Reagents
Imiquimod (GTH110C, 3 M Health Care Limited, UK) was purchased from Jiangsu Provincial Hospital of Traditional Chinese Medicine. Shi-Bi-Man (SBM) was purchased from Sipimo Biotechnology Co., Ltd (Shenzhen, China). TSG was purchased from Chengdu Purifa Technology Development Co., Ltd. TNF-α was purchased from MedChemExpress USA. Benvitimod was purchased from guanhaobio (Guangdong, China).
Mice
Six–Eight weeks-old female C57BL/6 mice (2023 g) were purchased from GemPharmatech Co., Ltd (Nanjing, China). The mice were kept in a controlled environment with a 12-h light/dark cycle at a temperature of 25 ± 1 ℃. They were provided with a standard laboratory diet and water ad libitum. The procedures described in this study were approved by the Experimental Animal Care and Use Committee of Nanjing University, by the guidelines outlined in the Guide for the Care and Use of Laboratory Animals (IACUC-2306011).
Imiquimod-induced model of psoriasis and SBM treatment
After anesthesia with 1% pentobarbital sodium by intraperitoneal injection, the hair on the back of the mice was shaved. A total of 30 mice were divided into six groups: (1) vehicle group; (2) IMQ group; (3) IMQ + benvitimod group; (4) IMQ + SBM group (skin administration; SBM:200 μL, daily for 5 consecutive days); (5) IMQ + 20 mg/kg SBM group (i.g. SBM, daily for 5 consecutive days); (6) IMQ + 40 mg/kg SBM group (i.g. SBM, daily for 5 consecutive days). The vehicle group was treated with glycerol. For the establishment of psoriasis mouse model, mice were topically treated with 62.5 mg 5% IMQ cream on shaved back skin daily for 5 consecutive days. Benvitimod were skin administrated 62.5 mg daily for 5 consecutive days. SBM was dissolved in ddH2O containing 5% ethanol.
Histopathologic assessment
Mouse skin tissue was cut into 5 μm thick pieces. Paraffin sections were successively treated with xylene and ethanol according to the protocol of the hematoxylin–eosin staining kit (G1005, Servicebio, China), and after treatment with hematoxylin solution, eosin staining was used. Neutral balm was used to seal the slides.
Immunofluorescence
Immunofluorescence staining was performed on tissue and cell samples. For tissue samples, slides were deparaffinized, rehydrated, and treated with sodium citrate buffer for antigen retrieval. Slides were incubated with 3% goat serum for 30 min followed by primary antibodies: CXCL16 antibody (DF13312, Affinity Biosciences, China), VCAM1 antibody (DF6082, Affinity Biosciences, China), SELE/CD62E antibody (DF6914, Affinity Biosciences, China), IL-23A antibody (DF13760, Affinity Biosciences, China), IL-17A Polyclonal antibody (26163-1-AP, PTG, China), Purified anti-human/mouse CD3ε (362701, Biolegend, USA), CD31 antibody (ab134168, Abcam, USA), Anti-E Cadherin antibody (ab231303, Abcam, USA). The secondary antibody used were Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (A-11008, Invitrogen), Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 (A-11005, Invitrogen).
Realtime-quantitative polymerase chain reaction (RT-qPCR)
TRIzol reagent (Takara, Cat. #9109) was used for total mRNA isolation. We synthesized cDNA using iScript Reverse Transcription Supermix (Bio-Rad), and BioRad CFX96 ouch™ Real-Time PCR Detection System (BioRad, CA, USA) was used for quantitative RT-PCR. HiScript II Q RT SuperMix for qPCR (R223-01) and Taq Pro Universal SYBR qPCR Master Mix (Q712-02) were purchased from Vazyme. Relative gene expression was calculated as 2^−△△Ct, where Gapdh was used as the housekeeping gene for normalization.
Detailed information about the primers for mice used is listed below:
Gapdh, 5′-AGGTCGGTGTGAACGGATTTG-3′ (forward)
5′-GGGGTCGTTGATGGCAACA-3′ (reverse);
l17f, 5′-TGCTACTGTTGATGTTGGGAC-3′ (forward)
5′-CAGAAATGCCCTGGTTTTGGT-3′ (reverse);
Il23, 5′-ATGAGTTTTTCCCTTATGGGGAC-3′ (forward)
5′-GCTGGAAGTTGGACACCTCAA-3′ (reverse);
Detailed information about the primers for HaCaT cells used is listed below:
GAPDH, 5’-GGAGCGAGATCCCTCCAAAAT (forward)
5’-GGCTGTTGTCATACTTCTCATGG (reverse);
TNF-α, 5′- CCTCTCTCTAATCAGCCCTCTG-3′ (forward)
5′- GAGGACCTGGGAGTAGATGAG-3′ (reverse);
IL23, 5′- CTCAGGGACAACAGTCAGTTC-3′ (forward)
5′- ACAGGGCTATCAGGGAGCA-3′ (reverse);
Scoring severity of skin inflammation
The clinical PASI was applied to evaluate the progression of psoriasis in mice. According to the PASI scoring standard, erythema, scaling, and thickening were taken into account, each ranging from 0 to 4. 0, none; 1, slight; 2, moderate; 3, significant; 4, extremely significant.
Single-cell RNA-seq
Skin tissues were collected after euthanasia of the mice and rinsed with phosphate-buffered saline (PBS) thrice. Subsequently, the tissues were sectioned into smaller pieces and digested into single-cell suspensions using collagenase I (Sigma), collagenase II (Sigma), and Dispase® (Sigma).
The suspension was loaded into microfluidic devices using the Singleron Matrix® Single Cell Processing System (Singleron). The scRNA-seq library was constructed according to the protocol of the GEXSCOPE® Single Cell RNA Library Kit (Singleron). The pools were sequenced on a NovaSeq 6000 system (Illumina, USA). The data were uploaded to the GEO database.
To process the raw data and convert it into a matrix suitable for analysis in R, the CeleScope package (v 1.13.0) was employed. The Seurat package (v3.2.3) in R was used for data analysis. Cells were filtered based on gene expression levels, with a threshold of more than 3000 and less than 300 genes. Additionally, cells with mitochondrial reads exceeding 8% and cells with unique molecular identifier (UMI) counts below 800 were excluded. The 'vst' method was used to integrate the data to remove the batch effect. The RunPCA function was used for dimension reduction. We used the uniform manifold approximation and projection (UMAP) function and the FindAllMarkers function. The top 30 genes scored by log2 fold-change were used for cell definition. The R package CellChat (v 1.5.0) was used for cell–cell communication analysis.
Cell culture and treatment
The HaCaT cell line was procured from the BeNa Culture Collection in Suzhou, China. The cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% streptomycin-penicillin solution. The cells were incubated in a humidified environment at 37 °C with 5% CO2. To induce cellular response, the HaCaT cells were treated with 50 ng/mL of TNF-α and 5 μM of TSG for a duration of 24 h.
Spectrum analysis
TSG and ginsenoside Rd were purchased from Chengdu Purifa Technology Development Co., Ltd. and were compared with quality scores greater than 98%.
Chromatographic conditions (qualitative) were as follows. LC-40D liquid chromatograph (SHIMADZHU, Japan); Chromatographic column: Kinetex C18 colume (100 X 4.6 mm, 2.6 μm, Phenomenex, USA).
The injection volume was 5 µL using a mobile phase system with mobile phase flow rate of 0.6 mL/min and column temperature of 40 ℃. The mobile phase were (A) 0.025% formic acid water aqueous solution, (B) methanol–acetonitrile (containing 0.025% formic acid) mixture (50:50, v/v). The gradient elution procedure was as follows: 0–2 min, A: 95%; 2–35 min, A: 95–5%; 35–45 min, A: 5%; 45–46 min, A: 5–95%; 46–50 min, A: 95%.
The mass spectrometry conditions were as follows: Acetonitrile, methanol, and formic acid were chromatographically pure (MERCK company), the water was ultrapure water (obtained by Millipore Milli-Q Synthesis System). Electrospray ion source (ESI) negative ion scanning mode was adopted. The gas temperature was maintained at 550 ℃; The curtain gas was set at 35 psi. CAD gas was 7. The spray voltage was set as 4500 V. Declustering potential was set at 80 V and collision energy at 10 V. TOF Mass range was set at m/z 50–1500 Da. All data were collected and processed using an UPLC-Q-TOF-MS/MS (SCIEX Zeno TOF 7600, USA).
Statistical analysis
GraphPad Prism 8 (GraphPad, San Diego) was used for statistical analysis. One-way ANOVA with Tukey’s multiple comparisons and paired or unpaired Student’s t-test were applied. Differences at P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01), and ns represents no significance. All data are presented as the mean ± SEM.
Discussion
Psoriasis, a chronic relapsing autoimmune skin disease, is influenced by various risk factors. These factors include obesity, dyslipidemia, hypertension, lifestyle choices, and certain medications [
23]. Recent researches have established a consensus that the IL-23/Th17 axis plays a crucial role in the development of psoriasis [
4,
24]. Despite the emergence of drugs targeting IL-17, further investigation into the underlying mechanisms of psoriasis is ongoing. The severity of psoriasis is associated with the expression of endothelial inflammatory transcripts [
25]. Tumor necrosis factor (TNF)-α and other cytokines contribute to the creation of a pro-angiogenic microenvironment in psoriasis [
26]. Consequently, VEGF has emerged as a promising therapeutic target for psoriasis [
27]. Studies have also demonstrated that alterations in fatty acid metabolism can exacerbate the pathogenesis of psoriasis-like symptoms [
28]. Additionally, lysophosphatidic acid, a simple phospholipid found in nature, has been implicated in the development of psoriasis [
29]. Furthermore, SHP2 has been shown to exacerbate psoriasis-like skin inflammation in mice through processes such as NETosis or TLR7 activation [
30,
31].
As research progresses, it has become evident that a comprehensive understanding of psoriasis pathogenesis necessitates a global perspective that considers the interplay between different cell types and even organs. With the development of research technology, single-cell sequencing technology has gradually entered into basic scientific research [
32]. With the development of research technology, single-cell sequencing technology has gradually entered into basic scientific research [
33]. Through sequencing technology, we were able to study the molecular mechanism of SBM treatment of psoriasis at the single-cell level and explore the interactions between cells. Based on the analysis results, we can further explore the underlying molecular mechanism of complex diseases and search for potential drugs.
In recent times, there has been a growing utilization of Traditional Chinese Medicine alongside biologics in the management of immune-related disorders [
32]. This trend can be attributed to the adverse effects and exorbitant costs associated with biologics, particularly in the treatment of conditions such as psoriasis and other immune diseases. Notably, ginsenoside radix has demonstrated its efficacy in preventing lung injury through its anti-inflammatory and anti-oxidative properties [
34]. Re-Du-Ning injection ameliorates radiation-induced pneumonitis and fibrosis [
35], as well as lung injury induced by LPS [
36]. What’s more, Traditional Chinese Medicine help treat insulin resistance [
37]. Paeonol ameliorates endometrial hyperplasia in mice via ferroptosis [
38]. The herbal formula also took part in the treatment of psoriasis [
39]. Cycloastragenol inhibits NLRP3 inflammasome-mediated pyroptosis in macrophages to relieve imiquimod-induced psoriasis-like skin inflammation in mice [
40]. As a mixture of a variety of Chinese herbs, SBM includes ginsenoside radix and has no toxicity using SBM in a mouse model [
18]. TSG, the main component in the skin after applying SBM, has been found to have the ability to treat non-alcoholic fatty liver diseases [
41]. Furthermore, TSG exhibits anti-aging properties in addition to its anti-inflammatory effects [
42]. Therefore, we applied SBM to the IMQ-induced psoriasis-like phenotype mouse model to find a safe and cost-effective treatment for psoriasis patients.
Depending on the scRNA-seq analysis, the potential molecular mechanism of SBM in the treatment of psoriasis was explained. IL-23 induces Th17 cells to activate and release inflammatory cytokines, leading to the typical pathological changes of psoriatic epidermal hyperplasia [
43]. The keratinocytes secreted less IL-23 after the treatment of SBM, which leads to less Infiltration of T cells that secrete IL-17 that promotes keratinocytes proliferation [
22]. After the treatment of SBM, IL-23/Th17 axis was inhibited. Since IL-17 also promotes endothelial dysfunction, we analyzed the changes in endothelial cells and found the activation of endothelial cells was suppressed. Considering patients with psoriasis are at increased risk of cardiovascular disease, SBM may be beneficial for thrombosis [
44]. Further research is needed for specific applications in vascular-related diseases.
The single-cell transcriptomics showed the T cells produce less IL-17 after SBM administration. IL-17 induces keratinocyte proliferation [
22]. As shown by single-cell transcriptomics, the expression level of inflammatory factors in endothelial cells was decreased, which reflects the inhibition of endothelial activation. Through cell–cell communication analysis, we found the CXCL signaling network changed (Fig.
7A). In the IMQ group, keratinocytes expressed higher levels of
Cxcl16, as well as the expression level of its receptor CXCR6 in endothelial cells, which reversed after SBM treatment (Fig.
7B) and confirmed by immunofluorescence (Fig.
7C). We suspected the higher expression of
Cxcl16 in keratinocytes leads to the activation of endothelial cells. And psoriasis is associated with endothelial activation [
45].
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