Total glucosides of Rhizoma Smilacis Glabrae alleviates psoriasis by stabilizing Th17/Treg balance

Psoriasis is a chronic, recurrent, inflammatory systemic disease induced by genetics and environment, typically presenting as confined or widely distributed scaly erythematous plaques. It is classified into common, pustular, erythrodermic and arthritic psoriasis ^[1]. The exact etiology and pathogenesis of psoriasis have not been fully elucidated. Both genetic background and environmental triggers lead to keratin formation, abnormal cell proliferation and inflammatory response in synovial cells and chondrocytes, resulting psoriasis ultimately ^[2].

There is no complete cure for psoriasis. Patients often required lifelong treatment, which should be both effective and safe. Mild cases of limited psoriasis can be treated with topical medications alone, while moderate to severe cases can be treated with a combination of physical and systemic therapy ^{[3, 4]}. Among the systemic therapies, methotrexate is one of the most effective systemic drugs for psoriasis. It has anti-inflammatory, antiproliferative and immunomodulatory effects, but its use is limited due to its renal metabolic properties and adverse effects ^{[5, 6]}. Thus, safer drugs that can be used to treat psoriasis need to be developed urgently.

According to traditional Chinese medicine (TCM) theory, we deems that psoriasis was an external manifestation of blood-heat syndrome resulting from the joint action of internal and external factors ^[7]. After a long period of illness, blood circulation disorder leads to dry skin and loss of nourishment ^[1]. Rhizoma Smilacis Glabrae, the dried rhizomes of Sarsaparilla, which was firstly published in the “Famous Doctor’s Record” ^[8], is a tipical TCM with the acitivity of improving blood-heat syndrome. Total glucosides of Rhizoma Smilacis Glabrae (RSG) tablet is a selective immunosuppressant for treating rheumatoid arthritis in clinical. It has the functions of antipyretic, detoxification, dehumidification and joint benefiting ^[9]. Modern pharmacological study showed that, RSG had anti-lipid peroxidation, anti-inflammatory, immunosuppressive, anti-atherosclerosis, as well as analgesic, detoxification and other effects ^[10]. Although the antipsoriasis effect of RSG is consistent with the view of TCM in improving blood-heat syndrome, its effect on psoriasis is rarely reported.

Generally, the composition of RSG is complicated and the group of potent substances is unclear. YU et al. insist that RSG contains four kinds of functional substances, mainly divided into organic acids, polysaccharides, phytosterols, and flavonoids ^[11]. Previously, we separated and extracted 49 compounds from RSG. In this study, we aimed to investigate the antipsoriasis effect of RSG, and analyze its mechanism and material basis. The results provide a theoretical basis for the development of new indications of RSG tablets.

Mouse Regulatory T Cell Staining Kit (88-8111-40), CD3e Monoclonal Antibody (145-2C11), Functional Grade (16-0031-85), IFN gamma Monoclonal Antibody (XMG1.2), IL-17A Monoclonal Antibody (eBio17B7), PE(12-7177-81), CD25 Monoclonal Antibody (PC61.5), APC (17-0251-81), FOXP3 Monoclonal Antibody (FJK-16s), CD4 Monoclonal Antibody (GK1.5), FITC (11-0041-82), IL-4 Monoclonal Antibody (11B11) and Foxp3/Transcription Factor Staining Buffer Set (00-5523-00) were purchased from Thermo Fisher (MA, USA). PMA/TPA (S1819), Ionomycin (S1672) and Brefeldin A (S1536) were purchased from Beyotime (China). Mouse TNF-α ELISA KIT (ML720852), Mouse IFN-γ ELISA KIT (ML720140) and Mouse IL-17 ELISA KIT (ML720170) were purchased from Mlbio (Shanghai, China). Non-essential amino acids (CM1008S/L) was purchased from CellCook (Guangzhou, China).

The main chemical components in the RSG were systematically separated and extracted using chromatographic techniques such as atmospheric column chromatography and preparative high performance liquid chromatography. The fingerprints of the total glucoside of RSG were analyzed and 10 common peaks were confirmed and identified by the comparison of the t_R of each peak and the chromatographic behavior (Fig. 1A). A total of 49 compounds were identified by physicochemical and spectroscopic analysis (Fig. 1B).

Figure 1.  Main chemical components of RSG were systematically separated and extracted. (A) The fingerprints of RSG were analyzed. (B) Main components separated and extracted by HPLC

Female Balb/c mice (18−20 g) were purchased from Shanghai Lingchang Biotechnology Co., Ltd. (Shanghai, China). All the experimental mice were maintained in SPF grade barrier system at a constant temperature of 21−25 °C, steady humidity of 50%−70% and a 12-h light/dark cycle. With 7 days of adaptive feeding, mice were allowed to consume the food and drink water freely. All animal experiment procedures were performed strictly according to the animal usage guidelines by the Animal Ethics Committee of China pharmaceutic University (2022-12-005).

HaCaT cells (Guangzhou Saiku Biotechnology Co., Ltd., CC4013) were placed in MEM medium and cultured in 5% CO₂, constant temperature, 37 °C incubator.

In clinical, the low and high doses used for adult are 150 and 300 mg·d⁻¹ respectively. Thus, in both mouse tail scale epidermis model and imiquimod-induced psoriasis model, we used 23.3 and 46.6 mg·kg⁻¹ for mice based on conversion. 35.0 mg·kg⁻¹ was chosed as an intermediate dose. Treats each therapeutic group once a day continuously for 14 days.

Balb/c mice acclimated for one week were divided randomly into five groups: (1) Control group, (2) RSG 23.3 mg·kg⁻¹ group, (3) RSG 35.0 mg·kg⁻¹ group, (4) RSG 46.6 mg·kg⁻¹ group and (5) Positive control group (retinoic acid 5.0 mg·kg⁻¹). After sacrifice on the 15^th day, the skin of the dorsal side of the tail was taken at a distance of about 2 cm from the root of the tail. The formation of a granular layer on the epidermis of the scales was observed under microscope.

Sixty Balb/c mice were randomly divided into the following groups: (1) Control group, (2) Model group, (3) Positive control group (methotrexate 1.0 mg·kg⁻¹), (4) RSG 23.3 mg·kg⁻¹ group, (5) RSG 35.0 mg·kg⁻¹ group and (6) RSG 46.6 mg·kg⁻¹ group. Mice back hair was removed with hair removal cream about 2 × 2 cm in size each. 62.5 mg of imiquimod ointment was applied to the back skin in (2)−(6) group daily. The application was carried out every morning for seven days ^[12]. After seven days of imiquimod ointment application, the mice were successfully modeled when they showed redness, swelling, or even skin lesions on the back skin. The drug was then administered to the group (3)−(6).

HaCaT cells were cultured in 6-well plate with 2 × 10⁵ cells per well in an incubator for 24 h. Cells were divided into blank control group, RSG group, methotrexate group, IL-17 group, IL-17 + RSG group and IL-17 + methotrexate group, which were administered separately. After 24 h of administration, cells were washed with trypsin, digested, resuspended, fixed in pre-chilled 70% ethanol, stained, and flow-detected under 488 nm, light scattering conditions for detecting the cell cycle. The cells were washed with trypsin, digested for 15 min, centrifuged at 1000 r·min⁻¹ and 4 °C, resuspended with PBS wash, and stained with Annexin V-FITC and Staining Solution before flow-through detection of apoptosis.

Helper T cells were isolated and re-suspended at the density of 10⁵−10⁷ cells·mL⁻¹ in FACS buffer. CD4-FITC and CD25-APC monoclonal antibodies were added to mark the surface targets of Th1, Th2, Th17 and Treg cells. Each sample was adjusted to ensure a final volume of 100 µL and transferred to a refrigerator for incubation (4 °C, 30 min, protected from light). Samples were washed twice to remove unbound antibodies in preparation for intracellular staining. For intracellular staining of Treg, use the Mouse Regulatory T Cell Kit to fix and permeabilize membranes and nuclei immediately after the surface staining procedure. The Foxp3 monoclonal antibody was added and incubated with permeabilization buffer for 30 min at room temperature, protected from light. the samples are ready for assay after washing twice.

For Th1, Th2, and Th17 staining, isolated Helper T cells were first activated by growing them on plates pre-coated with CD3e and CD28 functional monoclonal antibodies prior to surface staining. Phorbol-12-myristate-13-acetate, Ionomycin and Brefeldin A were performed to block the production and secretion of cytokines, which required 4 h of incubation. After incubation, samples were collected and surface staining was performed. IC fixation buffer was taken to fix the samples. IFN-γ, IL-4 and IL-17A monoclonal antibodies are added and incubated with permeabilization buffer for 30 min at room temperature. Samples were washed twice and ready for detection. All cell samples were collected and analyzed using a BD FACSCaliburTM flow cytometer ^[13].

HaCat cells were cultured in 96-well microplates and divided into blank control group, induction group (medium containing 50 µg·L⁻¹ IL-17A), and inhibitor group (total glucoside of RSG and isolated monomeric compounds with degrees of 0, 25, 50, 100, 200 and 400 mg·L⁻¹ in that order), and positive control drug with methotrexate (concentrations of 5, 10 and 20 µmol·L⁻¹) in the control group, and the cell proliferation rate was calculated by measuring absorbance values with an enzyme standard.

The samples were routinely stained with hematoxylin-eosin (HE) and then histologically observed to assess the status of skin lesions in mice according to the Baker score ^[14].

Total mRNA was extracted from skin tissue using Trizol according to the manufacturer’s instructions. The concentration of RNA was quantified to 1 μg·μL⁻¹ (Nanodrop ND-2000, Thermo Fisher Scientific, USA). The mRNA was reverse transcribed to cDNA templates using HiScript® Q RT SuperMix for qPCR (+ gDNA Wiper) reverse transcription kit. For Quantitative RT-PCR, cDNA was amplified using AceQ®SYBR Green Master Mix (High ROX Premixed) and oligonucleotide primers for specific target sequences on an Applied Biosystems StepOneTM real-time quantitative PCR. RT-PCR parameters were as follows: denaturation at 95 °C for 10 min, followed by 40 cycles of denaturing at 95 °C for 15 s, and annealing/extension at 60 °C for 60 s. Threshold cycles (Ct) were automatically calculated by the system software. The relative gene expression was calculated as 2^−ΔΔCt by quantitative PCR. Primer sequences are listed in Table 1.

All experiments were repeated at least three times in this study. Values for experiments were defined as the Mean ± SEM. The significance of difference was evaluated by One-Way ANOVA and t-test. Value of P < 0.05 was considered to be significant. All data were analyzed by GraphPad Software.

Since mouse scales lack a granular layer due to normal epidermal keratinization, their natural keratinization formation is similar to that of the human psoriatic epidermis. Thus, it is a tipical model mimicing the incomplete keratinization of psoriasis. Drugs that promote the formation of the granular layer of the mouse tail are considered to have a possible anti-psoriatic effect ^[14]. Therefore, a mouse tail scale model was used to evaluate the role of drugs in promoting the formation of the promoted granular layer in this study. HE staining results showed that, compared with the Control group, the tail granular layer of mice in the positive control group (5.0 mg·kg⁻¹ of retinoic acid) was significantly thinner. Meanwhile, tail granular layer of mice was significantly thinner in all the three dose group of RSG as a dose-dependent manner (Fig. 2A). Compare to control group, all the other groups showed a significant increase of the number of epidermal granular layer scales in the tail of mice. Obviously, 46.6 mg·kg⁻¹ RSG had a stronger effect to 5.0 mg·kg⁻¹ retinoic acid (Fig. 2B).

Figure 2.  The effect of RSG on the number of scales in the epidermal granular layer of the mouse tail by mouse tail scale epidermis model. (A) HE staining. (B) Number of granular layer scales in mice were calculated and analyzed. The data are presented as Mean ± SEM (n = 6). ^***P < 0.001 vs Control

During the seven days of animal modeling, the body weights of the control mice showed an increasing trend, but the body weight of the five groups of mocked mice showed a decreasing trend (Fig. 3A). After 7-day establishment period, the mice showed typical skin changes of psoriasis: redness of the skin on the back of the mice, patches of skin, large scales and papules (Fig. 3B). After 14 days model group mice showed apparent redness, swelling and ulceration of the skin lesions. Methotrexate group showed no apparent redness, swelling and ulceration of the skin lesions, but still had flakes accumulation. Compared with the model group, the mice after RSG administration showed significantly less redness, swelling and ulceration of the skin lesions, but still had a slight redness and swelling, where high-dose RSG group was the most pronounced (Fig. 3C).

Figure 3.  Body weight changing and epidermal damage on the back in the imiquimod-induced psodiasis mice. (A) Mouse body weight during the 7-day model building. (B) Imiquimod-induced psodiasis appearance in mice. (C) Back skin observations were performed in several groups after modeling. (D) Pathological changes in HE staining of mouse back skin. (E) Baker scores were calculated and analyzed. The data are presented as mean ± SEM (n = 6). ^&P < 0.1 vs day 0; ^***P < 0.001 vs Control; ^#P < 0.1, ^##P < 0.01, ^###P < 0.001 vs Model

The results of HE staining showed that the skin stratum corneum of mice in the control group was thin, with clear granular layer, spiny layer, basal layer and flat epidermis. In the model group, most of the mice had hyperkeratosis and partial hyperkeratosis. The the spiny layer was thickened. Compared with the model group, the symptoms of hyperkeratosis and hyperkeratosis were significantly improved in both methotrexate and RSG groups, the granular layer was visible, and the spine layer was thinner (Fig. 3D). The skin sections of the back of six groups of mice were scored according to the Baker score. Compared with the control group, the score of model group had a significant increase. After treatment, both methotrexate and RSG resisted the increase in model group and RSG displayed as a dose-dependent manner (Fig. 3E).

Transcriptome sequencing of skin tissue samples was performed from the model group and the high-dose RSG group (Fig. 4A). The results showed that 96 genes were up-regulated and 172 genes were down-regulated after the treatment of RSG (Fig. 4B). PCA analysis showed that the transcriptome profiles of the model group and RSG group were significantly different (Fig. 4C). GO analysis showed that the differential genes are mainly enriched in the development and differentiation of epithelial and epidermal cells (Fig. 4D). KEGG analysis showed the main changed pathways after the treatment of RSG were cytokine interactions, JAK-STAT, IL-17 and Wnt pathways (Fig. 4E).

Figure 4.  Related genes expression of skin tissue samples. (A) Transcriptome sequencing was performed on skin tissue samples from the model group and the high-dose administration group of total glycosides of RSG. (B) Genes expression compared with the model group. (C) PCA analysis. (D) GO analysis. (E) KEGG analysis showed the main changed pathways. (F) Spleen weight over body weight of each group mice. (G−I) Protein expression of IL-17, IFN-γ and TNF-α in skin tissue was determined by ELISA Kit. (J) Related genes expression in skin tissues were determined by RT-PCR. The data are presented as mean ± SEM (n = 6). ^*P < 0.1, ^**P < 0.01, ^***P < 0.001 vs Control; ^#P < 0.1, ^##P < 0.01, ^###P < 0.001 vs Model

The spleen coefficients of mice were measured and found to be enlarged and increased after modeling. In methotrexate group, the spleen coefficient was significantly lower than model group. While in RSG groups, the spleen coefficients showed a slight decreasing trend without significance (Fig. 4F).The ELISA kit detected the expression of IL-17 (Fig. 4G), IFN-γ (Fig. 4H) and TNF-α (Fig. 4I) in skin damage tissue. It was found that, IL-17 had a significant increase in model group, and methotrexate and RSG inhibited the elevated IL-17 obviously. However, IFN-γ and TNF-α and no strong change in skin damage tissue. The expression of related genes was detected by RT-PCR (Fig. 4J). The levels of Rig-1, Gata3, mTor, Jak, Stat3, and p65 were found to be upregulated in model group. Methotrexate down-regulated the gene levels of Rig-1, Gata3, mTor, Jak, Stat3 and Mapk in model group. Rig-1, Gata3, mTor and Jak were downregulated by RSG as a dose-dependent manner.

When the body is in immune dysfunction, the Th1/Th2 ratio will be out of balance, and the Th1 cytokines will increase under the stimulation of internal and external infectious sources. When the organism is in an immunosuppressed state, Th2 class cytokines increase. Compared with the control group, the number of Th1 and Th2 cells in the model group increased, and methotrexate significantly resisted the increase after modeling. However, RSG increase the counts of both Th1 and Th2 cells (Fig. 5A and 5B). Th1/Th2 ratio of model group increased significantly and methotrexate significantly down-regulated it, but RSG only showed a slight reduction (Fig. 5C).

Figure 5.  Mice skin tissue Th1, Th2, Th17 and Treg counts and ratio were measured after treatment. (A) Changes in the counts of Th1 cells in mice of several groups. (B) Changes in the counts of Th2 cells in mice of several groups. (C) Th1/Th2 ratio in each group was calculated and were analyzed. (D) Changes in the counts of Th17 cells in mice of several groups. (E) Changes in the counts of Treg cells in mice of several groups. (F) Th17/Treg ratio in each group was calculated and were analyzed. (G) The cell proliferation rate was detected by CCK8. (H) Cell cycle detection by flow cytometry. The data are presented as mean ± SEM (n = 6). ^*P < 0.1, ^**P < 0.01, ^***P < 0.001 vs Control; ^#P < 0.1, ^##P < 0.01, ^###P < 0.001 vs Model

In autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, multiple sclerosis and psoriasis, Th17/Treg ratio was increased showing an abnormal immune regulation. After modeling, the number of Th17 cells increased significantly without the change of Treg cells. Methotrexate significantly inhibited the increase of Th17 cells, meanwhile obviously elevated the counts of Treg cells. RSG reduced the increase of Th17 cells without influence on Treg cells (Figs. 5D and 5E). The Th17/Treg ratio had a significant increase in model group and significantly downregulated by both methotrexate and RSG (Fig. 5F).

For further study in vitro, the survival rate of HaCaT cells was detected after the treatment of RSG. The results showed that, the survival rate decreased more lower with the stimulating of IL-17 (Fig. 5G). As revealed by the flow assay, RSG significantly reduced the cell proportion in G2 phase. Under the stimulation of IL-17, this effect was stronger. However, there was no significant change in cell cycle by methotrexate (Fig. 5H).

The disease-related targets of psoriasis were obtained using GeneCards, DisGeNET, and other common disease gene databases with Psoriasis as the keyword for searching and removing duplicate targets. 22 potential active ingredients of RSG were screened. And the active targets were searched by TCMSP database ^[15], SwissTargetPrediction database and Pharm Mapper database, and converted into gene names by Uniprot database. The targets screened from each database were combined, and the duplicate targets were removed to obtain 153 targets related to the active ingredients (Fig. 6A).

Figure 6.  RSG treated psoriasis based on network pharmacology and screening of active components. (A) Potential targets of the active ingredient of RSG for psoriasis. (B) KEGG pathway enrichment analysis. (C) Component-target-pathway network. (D−F) relative mRNA expression of IL-8 and CCL-20 among components of the total glucoside of RSG. The data are presented as mean ± SEM (n = 3). ^*P < 0.1, ^**P < 0.01, ^***P < 0.001 vs Model; ^###P < 0.001 vs Control

The KEGG pathway enrichment analysis of these targets was performed using the DAVID6.8 database with P < 0.05 and FDR < 0.05 as screening criteria. A total of 56 entries were obtained, and the top 20 pathways were selected for the KEGG pathway enrichment analysis bubble mapping (Fig. 6B). The results showed that RSG had multi-component, multi-target action characteristics and can achieve the therapeutic effect through the regulation of multiple pathways, which were found to be most relevant to that, including the IL-17 signaling pathway, TNF signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, Ras signaling pathway (Fig. 6B). The active components, targets, and signaling pathways of RSG were visualized and analyzed using Cytoscape 3.7.1 software ^[16] to build a “component-target-pathway network” (Fig. 6C). The results suggested the antipsoriasis activity components from RSG had a strong connection with the immune regulation function of IL-17.

To screen the antipsoriasis activity components from RSG, IL-17 and TNFα co-treated HaCaT cell model was established. The results showed that the mRNA expressions of CCL-20 and IL-8 were significantly up-regulated in the model group. 22 compounds had significant inhibitory effect on IL-17/TNFα-induced up-regulation of CCL-20, and 19 compounds had significant inhibitory effect on IL-8 in HaCAT cells (Fig. 6D). Among them, T2 (β-sitosterol), T3 (sitostenone), T4 (stigmasterol), T13 (smiglanin), and T14 (cinchonain Ib) showed more siginicant effects (Figs. 6E and 6F).

RSG is a selective immunosuppressant developed from a single herb, positioned as an anti-rheumatic drug with less adverse reactions. Modern pharmacological research showed that RSG have anti-lipid peroxidation, anti-inflammatory, immunosuppressive, anti-atherosclerosis, as well as analgesic, detoxification, and other effects ^[10]. In this study, we found that RSG could significantly increase the number of scales in the tail of mice. At the same time, RSG showed a good therapeutic effect on the symptoms of redness, ulceration and dandruff of skin tissue of psoriasis caused by imidazolmod. The results suggested that psoriasis is expected to become a new clinical indication of RSG.

The clinical tolerable dose of RSG tablets for adults is 600 mg·d^{−1 [10]}. After conversion, the doses selected in this study are within the tolerance range of mice (90 mg·kg⁻¹). In the mouse tail scale experiment, we found that the effect of 5 mg·kg⁻¹ retinoic acid was equivalent to that of 23.3 mg·kg⁻¹ RSG. The increase of the number of mouse tail scales in the middle and high dose RSG groups was obviously campared to the retinoic acid group. In the mouse psoriasis model induced by imidazolmod, the efficacy of methotrexate was better than 46.6 mg·kg⁻¹ RSG. However, methotrexate is an antifolate antineoplastic drug, which mainly involves the synthesis of turbine cells by embedding dihydrofuse reduce. In the list of carcinogens published by the International Cancer Research Institute of the World Health Organization, methotrexate is classified as Category 3 carcinogens (substances or mixtures that have not been classified as carcinogenic to human beings). Therefore, RSG is a safer method for the treatment of psoriasis.

In psoriasis patient lesions, IL-21 induces Th17 cell differentiation, prompting Th17 cells to secrete large amounts of inflammatory cytokines (e.g. IL-17A and IL-22) ^[17]. IL-17 promoted proliferation and inhibited apoptosis of HaCaT cells ^{[18, 19]}. It has been demonstrated that many biologic agents such as suginumab, ecrelizumab, and brodalumab achieved antipsoriasis effects by inhibiting the IL-17 signaling pathway ^{[20, 21]}. Meanwhile, IL-21 downregulates the expression of Foxp3, a key transcription factor of Treg cells, which reduces the proportion and impairs the function of Treg cells, prompting the conversion of Treg cells into Th17 cells and accelerating the progression of psoriasis ^{[22, 23]}. Accordingly, RSG could selectively inhibit activated T cells without affecting normal T cells and other tissue cells and selectively inhibit activated Th1 cells without affecting Th2 cells ^[11]. In this study, flow T-cell sorting results showed that RSG significantly reduced the ratio of Th1/Th2 and Th17/Treg in spleen tissues, which was consistent with the literature. Meanwhile, total hyoscyamine in damaged skin tissues mainly exhibited an inhibitory effect on IL-17. Similiarly, WANG et al. suggested that total flavonoids of RSG can improve the skin injury of psoriatic mice, reduce skin pruritus and inhibit the inflammatory response,which may play a regulatory role by inhibiting IL-17/Notch signaling axis ^[24]. It can be seen that the treatment of psoriasis by RSG is based on an over-immune suppression approach rather than a direct repair of localized skin damage symptoms, which is consistent with its traditional efficacy in treating blood-heat syndrome in TCM theory. At the same time, this result suggested that oral administration is a more preferable way of treating psoriasis with RSG.

Consistent with the transcriptome sequencing results, mTOR, Jak, and Rig-1 signaling pathways were inhibited by RSG ^[25-27]. Jak is targeted for the therapy of autoimmune inflammatory diseases in psoriasis, and signal transducer and activator of transcription 3 (STAT3) is involved in regulating basic cell biological processes such as cell proliferation and differentiation, malignant transformation, viability and apoptosis ^{[28, 29]}. This study suggested that these signaling pathways caused by RSG may be the mechanism of its herbal action in exerting its therapeutic effect on psoriasis. Meanwhile, this study showed that RSG had a significant blocking effect on HaCaT cell cycle under IL-17 condition, which provides a theoretical basis for the therapeutic effect of RSG against psoriasis.

The result revealed that β-sitosterol, sitostenone, stigmasterol, smiglanin, and cinchonain Ib all showed powerful inhibitory effects on the inflammatory response of Hacat cells mediated by IL-17, which is generally consistent with the active compounds screened by network pharmacology. As report, YANG et al. investigated that β-sitosterol administration reduced the expression of chemokines and inflammatory cytokines in mice ^[23].WEN et al. found that soy steroid treatment restored Treg/Th17 homeostasis and altered the gut microbiota in a model of dextran sodium sulfate-induced colitis ^[30]. FENG et al. show that flavonoid-enriched extract (44.3%, mainly astilbin, engeletin, isoastilbin, cinchonain Ib, quercetin-3-O-a-L-rhamnopyranoside and chlorogenic acid) has inhibitory effects on both acute and chronic inflammation. And it was able to concentration-dependently inhibit the over-release and/or overexpression of LPS-induced inflammatory mediators TNF-α, COX-2 and iNOS, as well as the pro-inflammatory cytokines IL-1β and IL-6 in RAW264.7 macrophages ^[31]. It is suggested that these components might be the main functional substance groups in RSG to antipsoriasis. However, whether these components individually can play a role in the treatment of psoriasis requires further investigation.

Summary, RSG has certain therapeutic effects on psoriasis, and its mechanism is primarily to improve the IL-17-induced inflammatory response through regulating the Th17/Treg balance, mediating cell cycle arrest and impeding epidermal cell proliferation and differentiation. Among the 49 components isolated, β-sitosterol, sitostenone, stigmasterol, smiglanin, and cinchonain Ib showed better inhibitory effects on the IL-17 mediated inflammatory response.