Triptolide (#38748–32–2), methotrexate (MTX, #133073–73–1) was gained from Aladdin (Shanghai, China). Imiquimod was gained from Sichuan Mingxin Pharmaceutical Co. (Sichuan, China). Recombinant Human IL-17A Protein were ordered from PeproTech Co. (200–17–100, Rocky Hill, United States). Antibodies of PCNA (#10205-2-AP), CCD1 (#26939-1-AP), GAPDH (#10494-1-AP) were ordered from Proteintech co. (Wuhan, Hubei, China). Primary antibodies against Wnt5a (#ab227229), β-Catenin (#ab32572), Bax (#ab32503) and Caspase-3 (#ab32351) were ordered from Abcam Co. (Cambridge, MA, United States). Primary antibody against Bcl-2 (#sc-7382) was gained from Santa Cruz Biotechnology Inc. (Dallas, TX, United States). The secondary anti-rabbit IgG and anti-mouse IgG were gained from Santa Cruz Biotechnology Inc. (Dallas, TX, United States). Antibodies of FITC-conjugated CD4 (#11–0041–81), PE-conjugated IL-17A (#12–7177–81) were ordered from Invitrogen Life Technologies (Carlsbad, CA, United States). Elisa Kits (IL-17A, IL-22, IL-23, IL-6, and TNF-α) and SYBR Green and 2 × Taq PCR MasterMix were gained from Solarbio Science & Technology Co. (Beijing, China). Red Blood Cell Lysis Buffer was purchased from Beyotime Co. (Shanghai, China). Cell Stimulation Cocktail (#00–4970–93), TRIzolTM Reagent, hoechst 33,342 and Propidium Iodide (#V23201) were ordered from Invitrogen Life Technologies (Carlsbad, CA, United States). Propidium Iodide and FITC Annexin V Apoptosis Detection Kit I and Mouse sero block FcR (#553141) were ordered from BD Pharmingen (Franklin Lakes, NJ, United States).

The experiments were carried out with the approval of the Animal Policy and Welfare Committee of Wenzhou Medical College (No. XMSQ 2021–0021). Female BALB/c mice (6–8 weeks old) were purchased from Vital River Laboratory Animal Technology Co. (Beijing, China) and housed in the Animal Experimental Center of Wenzhou Medical University. Adaptatively reared mice were divided randomly into five groups of equal size, and the mice were anesthetized with CO₂ and sacrificed at the end of the experiment.

All mice (7–9 weeks) were randomly assigned to control group (n = 7), IMQ group (n = 7), positive control group (n = 7), low dose triptolide group (n = 7), and high dose triptolide group (n = 7). According to a previous report (Van der fits et al., 2009), mice whose backs were shaved and rested for a day, Then 62.5 mg of 5% IMQ was applied daily to their shaved back, and control mice were applied the same amount of vaseline. The first application of IMQ or vaseline was set as day 0 for seven consecutive days. Mice in positive control group received intraperitoneal injection of MTX 1 mg/kg/day, mice in low dose and high dose triptolide groups received intraperitoneal injection of TP 0.075 mg/kg/day and 0.15 mg/kg/day, respectively. The back area of each group was photographed daily to record the skin lesions, the skin thickness was recorded using vernier calipers, and the body weight was recorded. On day 7, the mice were euthanized via CO₂ inhalation. The mouse eyeballs were removed and centrifuged to obtain serum, the skin on the back was isolated, and the spleen was taken and photographed and weighed.

Based on the Clinical Psoriasis Area and Severity Index (PASI), a modified scoring system was developed to assess the severity of skin inflammation in mice (Van der fits et al., 2009). Scales, erythema, and thickening were scored on a scale from 0 to 4 (0 = none; 1 = slight; 2 = moderate; 3 = marked; 4 = very marked). Cumulative scales and erythema and thickening scores were used as measures of skin inflammation severity (0–12).

The dorsal skin of mice was collected on the seventh day and then immersed in 4% paraformaldehyde for 48 h. Following dehydration, the skin was encased in paraffin and sliced into 5 μm sections. The skin slides were stained with hematoxylin/eosin (H&E), observed with a microscope (Leica, Tokyo, Japan), and the images were collected.

Put tissue slides in the baking oven at 65°C for 4–6 h. Paraffin was removed with xylene and ethanol. Put the slides into 0.01 M sodium citrate buffer solution and heat them in a pressure cooker for 2 min. After cooling, dropped 3%H₂O₂ on the slices at room temperature for 30 min and washed them with PBS. Proliferating cell nuclear antigen (PCNA) antibody or Wnt5a antibody working solution was added and incubated at 4°C for 12 h. Anti-rabbit antibody working solution was added and incubated at room temperature for 2 h. DAB solution is used as color developing agent for 15 min and rinsing them with water. The following steps were dehydration, transparency and sealing. The skin slides were observed with a microscope, and the images were collected.

Eyeballs were extracted to obtain blood, placed at 37°C for 0.5 h, and centrifuged at 4000 rpm for 10 min at 4°C to obtain serum. IL-17A (#SEKM-0018), IL-22 (#SEKM-0023), IL-23 (#SEKM-0024), IL-6 (#SEKM-0007), and TNF-α (#SEKM-0034) in the serum were determined by ELISA kits (Solebol, Beijing, China).

In order to count single cells, spleen cells are reduced to homogeneous single cells by using nylon cell strainers of 100 m length. Red blood cells are processed three times or more by using Red Blood Cell Lysis Buffer. For staining the Th17 cells, cells were stimulated with Cell Stimulation Cocktail for 6 h. Fc receptors were blocked with mouse sero block FcR before surface staining. The cells were stained with FITC-conjugated CD4 antibody for 30 min at 4°C, fixed for 30 min, and permeabilized two times. Cells were stained using PE-conjugated IL-17A antibody for 30 min at 4°C. Flowcytometer (BD Biosciences) was used for data acquisition and analysis.

HaCaT, an immortalized line of human epidermal keratinocytes, was obtained from Cell Resources Center of the Shanghai Institutes for Biological Sciences (Chinese Academy of Sciences, Shanghai, China). HaCaT cell line was cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin in a humidified incubator with 5% CO₂ at 37°C. Recombinant Human IL-17A Protein was added to the cell supernatant at the concentration of 100 ng/mL to induce psoriasis-like model in vitro.

8,000 cells of HaCaT were planted into every well of 96-well plate. The cells treated with different concentrations of TP with or without the existence of 100 ng/mL of IL-17A were cultured at 37°C in 5% CO₂. For further experiments, 25 μL MTT solution was added to cells for 4 h before the MTT test. The crystal was then dissolved in 150 μL DMSO and read at 490 nm using a microplate reader. Graphpad Prism 7.0 software was employed to determine the semi-maximum inhibition concentration (IC₅₀).

TP (20, 40 or 80 nM) with or without the existence of 100 ng/mL of IL-17A were used to treat HaCaT cells for 24 h. Cells were collected and fixed within cold 70% ethanol in a dropwise fashion, and then incubated for 24 h at −20°C overnight. Ethanol was then removed and cells were stained with Propidium Iodide (0.05 mg/mL) and incubated in the dark for 20 min at RT. Cell cycles were analyzed by the flowcytometer.

The ratio of apoptotic cells were analyzed using the annexin V-FITC Apoptosis Detection Kit. TP (20, 40 or 80 nM) with or without the existence of 100 ng/mL of IL-17A were used to treat HaCaT cells for 24 h. The cells were harvested and washed three times with PBS. Then, the harvested cells were stained with Annexin V-FITC and PI staining solution, according to the manufacturer’s instructions. After 30 min of incubation in the dark. The ratio of apoptotic cells were analyzed by the flowcytometer.

Animal tissues or cultured cells were lysed in lysis buffer and quantified. Proteins were separated on a sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred to polyvinylidene difluoride membrane. After blocking with 5% skimmed milk for 1.5 h at room temperature, membranes were incubated overnight with specific antibodies at 4 °C. Then, the membranes were washed three times (5 min each) with tris buffered saline tween (TBST) and required 1 h incubation at room temperature with proper secondary antibodies. After washing membranes for 5 times with TBST, immunoreactivity was detected using the electrochemiluminescence detection kit.ImageJ software was used for determination of the density of the bands.

Total RNA was isolated from HaCaT cells or mouse skin tissues using TRIzol™ Reagent according to the manufacturer’s instructions. The RNA concentration and purity were then assessed using a NanoDrop microvolume spectrophotometer (Thermo). RNA was reverse transcribed to cDNA using the PrimeScriptTM RT Reagent Kit (Takara Bio, Japan). Real-time qPCR of geen expression was performed using SYBR Green and 2 × Taq PCR MasterMix and the Eppendorf Real plex 4 (Eppendorf, Hamburg, Germany). The primer sequences were as Table 1.

HaCaT cells were cultured on a six-well culture plate. TP (20, 40 or 80 nM) with or without the existence of 100 ng/mL of IL-17A were used to treat HaCaT cells for 24 h. 4% paraformaldelyde was used to fix HaCaT, and washed three times with PBS followed by stained with 1 mg/mL Hoechst 33,342 for 10 min in the dark. The characteristic of cell nucleus was observed by employing a fluorescence microscope (Leica, Tokyo, Japan).

The cell proliferation was measured using EdU Cell Proliferation kit. HaCaT cells were cultured on glass coverslips and treated with different concentrations of TP (20, 40 or 80 nM) and/or IL-17A for 24 h and stained with EdU-labeling mixture (10 mM) for 12 h. According to the manufacturer’s instruction, 4% paraformaldehyde was used for 10 min cell fixing at room temperature, and 0.5% TritonX-100 was used to permeabilized cells. HaCaT cells were subsequently incubated with 1 × Click-iT EdU reaction mixture in the dark for 30 min, followed by stained with Hoechst 33,342 for another 10 min. Finally, the EdU assay kit analyzed cell proliferation observed by employing a fluorescence microscope.

All experiments, except animal models, were conducted at least three time. GraphPad Prism 7.0 (GraphPad Software, CA, United States) was used for all statistical analyses. Data were expressed as mean ± Standard Error of Mean (SEM). Comparison of groups was subjected to one-way ANOVA (P-value <0.05 was considered statistically significant).

We investigated the therapeutic effect of TP on the psoriasis-like skin inflammation model by applying IMQ on the shaved back of mice daily to induce psoriasis. The results showed that symptoms such as scale formation, erythema, and skin thickness decreased significantly in TP-treated mice compared with those in IMQ-treated mice (Figure 1A). Furthermore, the therapeutic effect of TP was dose-dependent, as well as this effect on psoriasis-like symptoms was similar to that of MTX (Figure 1A). Additionally, TP-treated mice showed significant body weight loss compared with control mice; however, no significant difference was observed between the body weights of TP-treated and IMQ-treated mice (Figure 1B). This result was found by calculating the PASI scores of each group of mice (Figure 1C). Histopathological analysis and PCNA staining of skin tissues showed that TP treatment significantly inhibited IMQ-induced epidermal thickening (Figure 1D). Given the observation of abundant inflammatory cells exhibiting macrophage-like nuclear morphology, we performed immunofluorescence staining for the macrophage-specific marker F4/80. TP treatment induced a dose-dependent attenuation of dermal inflammatory cell infiltration, with macrophage density being significantly reduced compared to the IMQ-induced group (Supplementary Figure S3). The number of PCNA-positive proliferating keratinocytes in the skin of TP-treated mice was significantly lower than that in IMQ-treated mice (Figure 1E).

Spleen enlargement was inhibited as well as the spleen index (the ratio of spleen weight to body weight) decreased after TP treatment in psoriasis model mice, which suggested that the anti-inflammatory effects of MTX and TP were similar (Figures 2A, B). Th17 cells play an important role in psoriasis pathogenesis; thus, we performed flow cytometry to detect Th17 cells in the mouse spleen and found that the Th17 cell proportion decreased in the spleen after TP treatment (Figures 2C, D). To further explore the inflammatory cytokines regulated by TP treatment, we extracted serum from the mice and analyzed the expression of inflammatory genes, such as IL-17A, IL-22, IL-23, TNF-α, and IL-6, which were closely related to psoriasis pathogenesis. ELISA experiments showed that in vivo serum levels of inflammatory cytokines decreased significantly in TP-treated mice, and these levels were lower in the H-TP group mice than in the L-TP group mice (Figure 2E).

To elucidate the mechanism underlying the anti-psoriasis effect of TP, we analyzed and compared the transcriptome data of the skin lesion tissues of IMQ- and H-TP-treated mice by RNA sequencing. Gene Ontology (GO) molecular function analysis showed that TP treatment significantly affected skin development in the mice (Figure 3A) and downregulated the expression of Wnt5a (Figure 3B), which is highly expressed in the skin lesions of patients with psoriasis. By performing immunohistochemistry, qRT-PCR, and Western blotting of the mouse skin lesions, we showed that IMQ treatment increased Wnt5a mRNA and protein levels in the lesions, whereas TP treatment significantly inhibited IMQ-induced Wnt5a overexpression (Figures 3C–E). However, MTX treatment failed to attenuate IMQ-induced Wnt5a overexpression in the experimental model (Supplementary Figure S4).

To evaluate our conjecture that TP can regulate Wnt5a at the cellular level, we determined mRNA and protein levels in HaCaT cells treated with different concentrations of TP by performing qRT-PCR and Western blotting. The TP concentration was determined based on the IC50 value calculated by performing the MTT assay (Supplementary Figure S1). Although 20 nM TP had no significant effect on Wnt5a expression, 40 and 80 nM TP significantly suppressed Wnt5a expression in HaCaT cells (Figures 4C, D). Additionally, Western blotting was performed to examine the level of β-catenin, an important gene downstream of Wnt5a, in HaCaT cells, and the results showed that TP significantly downregulated Wnt5a/β-catenin signaling in a dose-dependent manner (Figure 4D; Supplementary Figure S5B). Fibroblast-like synoviocytes treated with IL-17A showed upregulated Wnt5A mRNA expression (Lavocat et al., 2016). When we treated HaCaT cells with 100 ng/mL IL-17A, Wnt5a mRNA levels increased significantly (Figure 4A), and they continued to increase with increasing treatment time within 24 h (Figure 4A). The Western blotting results confirmed that IL-17A treatment increased Wnt5a and β-catenin protein levels in HaCaT cells (Figure 4A; Supplementary Figure S5C). Moreover, in HaCaT cells treated with different concentrations of TP with or without 100 ng/mL IL-17A, Wnt5a expression was successfully inhibited by TP and was not upregulated by IL-17A. Similarly, β-catenin expression was regulated by IL-17A and TP (Figures 4E, F; Supplementary Figure S5C).

HaCaT cell stimulation with IL-17A induced hyperproliferation (Di Cesare et al., 2009; Kim and Krueger, 2017). When 100 ng/mL IL-17A induced HaCaT cell hyperproliferation, their treatment with TP (20 nM, 40 nM, and 80 nM) led to decreased cell survival (Figure 5A). The EdU assay showed that the proportion of HaCaT cells to be proliferated was significantly more in the IL-17A treatment group than in the TP treatment group, and the lower proportion in the latter group was independent of IL-17A treatment (Figure 5B; Supplementary Figure S2A). Additionally, TP treatment significantly promoted HaCaT cell apoptosis (Figures 5C, D; Supplementary Figures S2B, C). The cell cycle analysis showed that TP treatment alone significantly increased the number of cells in the G1 phase compared with those in the control group (Figure 5E; Supplementary Figure S2D). Compared with the IL-17A group, IL-17A and TP co-treatment significantly increased the number of cells in the G1 phase in a dose-dependent manner (Figure 5E; Supplementary Figure S2D). Subsequently, we performed Western blotting to investigate the levels of proliferation-, apoptosis-, and cell-cycle-related proteins. TP significantly increased the levels of caspase three and Bax and decreased the level of bcl-2, thereby confirming the occurrence of apoptosis (Figure 5F). IL-17A increased PCNA expression, which was inhibited by TP, thereby suggesting the involvement of TP in suppressing keratinocyte hyperproliferation in psoriasis. The effect of TP on cell cycle arrest in HaCaT cells was attributed to CCD1 expression inhibition. Thus, TP inhibited IL-17A-induced keratinocyte hyperproliferation.

IL-17A elicits an amplified inflammatory response in keratinocyte (Di Cesare et al., 2009; Kim and Krueger, 2017; Mease, 2015). The mRNA levels of several cytokines, including CXCL1, CXCL2, CXCL8, CCL20, IL-1β, IL-6, and IL-19, in HaCaT cells increased significantly after exogenous IL-17A addition (Figure 6). However, compared with IL-17A treatment, TP monotherapy significantly reduced the mRNA levels of CXCL1, CXCL2, CCL20, IL-1β, and IL-6, whereas no significant effect was observed on the levels of CXCL8 and IL-19. However, the levels of these cytokines decreased significantly after TP and IL-17A co-treatment, which suggested that TP treatment inhibited the inflammatory response triggered by IL-17A in keratinocytes.