Triptolide Inhibits Th17 Response by Upregulating microRNA-204-5p and Suppressing STAT3 Phosphorylation in Psoriasis

Background Psoriasis is an immune and inflammation-related skin disease. Triptolide with immunosuppressive and anti-inflammatory properties has been utilized for psoriasis treatment. However, the potential immunological mechanisms of triptolide have not been fully elucidated. Methods Using an imiquimod (IMQ)-induced psoriatic mouse model, we detected the effects of triptolide on psoriasis-like lesions including scales, thickening, and erythema. Methyl thiazol tetrazolium (MTT) cytotoxicity assay was performed for evaluating the influence of triptolide on cell viability. Gene expression at mRNA and protein levels were examined by reverse transcription-quantitative polymerase chain reaction and Western blot analysis, respectively. The combination between microRNA-204-5p (miR-204-5p) and signal transduction and transcription activator-3 (STAT3) was confirmed by luciferase reporter assay. Enzyme-linked immunosorbent assay was conducted to examine interleukin (IL)-17 and interferon-γ (IFN-γ) levels using corresponding kits. Hematoxylin and eosin staining was used for the visualization of epidermal thickness. Flow cytometry analysis was employed for examining T helper (Th) 17 cells. Results Triptolide ameliorated IMQ-induced psoriatic skin lesions manifested by the decreased psoriasis area and severity indexes (PASI) scores. Triptolide inhibited Th17 cell differentiation from splenocytes. Additionally, triptolide elevated miR-204-5p expression, whereas it downregulated STAT3 expression levels both in vitro and in vivo. Moreover, miR-204-5p directly targeted STAT3 in HaCaT cells. Furthermore, triptolide repressed the expression of proinflammatory cytokines in IMQ-evoked psoriasis-like mice. Conclusion Triptolide inhibits STAT3 phosphorylation via upregulating miR-204-5p and thus suppressing Th17 response in psoriasis.


Introduction
Psoriasis is one of the chronic and immune-mediated disease, characterized by hyperplasia of the epidermis, dermal angiogenesis, and infltration of infammatory cells, afecting approximately 2-3% population worldwide [1,2]. Psoriasis can develop into psoriatic arthritis, leading to a variety of complications such as cancer, cardiovascular disease, obesity, Crohn's disease, and psoriatic arthritis [3,4]. Various factors are associated with psoriasis occurrences such as immunity, metabolic disorders, and environmental and genetic factors [5]. To date, the pathogenesis of psoriasis has not been fully elucidated, but it is widely accepted that T cell-mediated immunity exerts a crucial function in the development of psoriasis. Additionally, the imbalance between anti-infammatory Treg cells and proinfammatory T helper (T) 17 cells is responsible for multiple autoimmune diseases, including psoriasis [6]. T17 cells can produce interleukin-17 (IL-17) that has been illuminated to elicit a pivotal function in psoriasis inducing the expression of antimicrobial peptides, cytokines, and proinfammatory chemokines in keratinocytes [7]. Although conventional immunosuppressants and/or glucocorticosteroids for psoriatic patients bring about temporary alleviation, the side efects for these patients are quite serious [8,9]. Terefore, it is urgently needed to identify the etiology of psoriasis and recognize novel and efective drugs and/or targets for the treatment of psoriasis patients.
MicroRNAs (miRNAs) are small noncoding RNA molecules containing about 22 nucleotides. Te critical function of miRNAs in the modulation of RNA silencing and gene expression at the posttranscriptional level has been proved [10,11]. In addition, miRNAs are involved in various biological processes, and their aberrant expression could lead to many human diseases such as immune abnormalities, infammations, and carcinomas [12,13]. Numerous miRNAs such as miR-138 [14], miR-31 [15], and miR-203 [16] have participated in psoriasis development and are dysregulated in keratinocytes and lymphocytes from patients with psoriasis. miR-204-5p is shown to be related to immune and infammation [17,18]. However, the role of miR-204-5p in psoriasis remains unclear.
Triptolide is a compound extracted from a Chinese medicinal herb Tripterygium wilfordii hook F (TWHF), possessing potential properties such as antitumor, immunosuppression, and anti-infammation [19]. TWHF has been proven to have immunoregulation efects and is considered a promising drug in the treatment of systemic lupus erythematosus and rheumatoid arthritis [20][21][22][23]. In recent years, the role of triptolide in psoriasis has been uncovered. Studies have demonstrated that triptolide signifcantly represses the proliferation of HaCaT Cells in vitro and attenuates psoriatic skin infammation induced by imiquimod (IMQ) in vivo [24,25]. Importantly, clinical observation of the triptolide efect on psoriasis vulgaris reveals that the total efective rate of triptolide is up to 75.7% among the 103 patients within the treatment period [26]. A previous literature study validates that 100 nM triptolide upregulates miR-204 expression in S2-VP10 cells [27]. However, the exact dosage of triptolide applied to psoriasis patients and the molecular mechanism of its possible efects remain unclear.
In the current study, we aimed to study the biological function of triptolide and its related mechanism by which it regulates psoriasis development. We made a hypothesis that triptolide may alleviate psoriatic symptoms of mouse models and inhibit infammatory response via regulating miR-204-5p.

Aims.
In the current study, we aimed to study the biological function of triptolide and its related mechanism by which it regulates psoriasis development.

Establishment of Psoriatic Mouse Models.
Te dorsal area (2 × 3 cm 2 ) of mice was shaved. A total of 40 mice were randomly divided into four groups (10 per group) including the sham group, model group, model + triptolide (L) group, and model + triptolide (H) group. Normal control mice (sham) were applied with appropriate vaseline on the exposed back each day and intragastric administrated with saline (0.4 ml/day) for 8 continuous days. Te mice in the model group were applied 42 mg of 5% imiquimod (IMQ) cream (GG0080, BIOSIC, Ding Zhou Biosic Biotechnology Co., Ltd., China) on the shaved dorsal area daily and intragastric administrated with saline (0.4 ml/day) for 8 continuous days. Te mice in the model + triptolide (L) and model + triptolide (H) groups were applied 42 mg of 5% IMQ on the exposed back skin daily and treated with low and high doses of triptolide (10 and 40 mg/kg, respectively) and received intragastric administration of triptolide (0.4 ml/day) for 8 continuous days.

Psoriasis Area and Severity Indexes (PASI) Scoring.
PASI scoring was used to evaluate the severity of skin infammation and lesion by measuring erythema, scaling, and thickness. PASI scores of each category were scored independently on a scale from 0 to 4: "0" indicates none, "1" indicates "slight," "2" indicates "moderate," "3" indicates "marked," and "4" indicates "severe." Te severity of the lesion was refected via the cumulative scores of erythema, scaling, and thickening. PASI scores were scored from the day when IMQ was frst administered to the last administration during the 8 consecutive days.

Methyl-Tiazol Tetrazolium (MTT) Cytotoxicity Assay.
Following isolating splenocytes from mouse models, increased concentrations of triptolide (0-500 nM) was used to cultivate with the isolated splenocytes for 3 d. After exposure to continuous drugs, MTT (M2003, Sigma Aldrich, St Louis, MI, USA) was added into cultures. Dissolving insoluble formazan complex in DMSO was performed prior to the measurement of the absorbance at 490 nm.
Te cells collected from the spleen and draining lymph node (LN) of indicated mouse models were cultured for 72 h, followed by stimulation and induction for secreting intracellular cytokines by brefeldin A (BFA; 10 µg/ml), ionomycin (1 µg/ml), and PMA (10 ng/ml). Following culturation for 6 h, the cells were subjected to incubation with an anti-mouse CD4-FITC antibody. Ten, the cells were stained with APC-labeled anti-mouse IL-17 antibody after permeabilization. Later, a fow cytometer was adopted for analyzing CD4 + IL-17 + T cell percentage.

Hematoxylin and Eosin (H&E) Staining.
On day 8, back skin samples were collected for visualizing epidermal thickness. First, 4% neutral paraformaldehyde-fxed skin samples were embedded in parafn, followed by slicing them into 5 μm-thick sections. Tese skin sections were stained by H&E, and the stained slides were observed under a microscope.

Statistical Analysis.
All quantitative data obtained from at least three independent trials are shown as the means ± standard deviation and analyzed with SPSS 15.0 software. Te data between the two groups were analyzed by the unpaired t-test with an independent sample, while the data among multiple groups were analyzed by ANOVA followed by the post hoc Bonferroni test. Te threshold for statistical signifcance was the p value less than 0.05.

Triptolide Inhibits T17 Diferentiation.
MTT was frst utilized to detect the infuence of triptolide (Figure 1(a)) on cell viability. As a result, 1-10 nM triptolide showed no notable efect on cell viability compared with the cells treated with 0 nM triptolide, while 50 nM triptolide signifcantly suppressed cell viability, suggesting that the notoxic efect of triptolide on cells was 1-50 nM (Figure 1(b)). Ten, the infuence of triptolide on T17 diferentiation was investigated. ELISA was performed to detect IL-17 concentration by assessing the culture supernatants. Results showed that the cultures supplemented with IL-6 and TGF-β led to the signifcant upregulation of IL-17 production. Te induced production of IL-17 was then dose-dependently repressed by triptolide treatment. Triptolide at the dosage of 10 nM almost completely suppressed the production of IL-17 ( Figure 1(c)). With the aim of characterizing cell subsets of IL-17-producing cells at the signal-cell level, fow cytometry analysis was employed. Compared with the anti-CD3 group, a large fraction of IL-17 + CD4 + T cells was induced by anti-CD3 together with IL-6 and TGF-β after primary culture. However, compared with the cells without triptolide treatment, a signifcant decrease of IL-17 + CD4 + T population in triptolide-induced cells of primary culture was observed (Figure 1(d)). All these data demonstrated that triptolide suppresses the diferentiation of T17 cells from splenocytes.

Triptolide Alleviates IMQ-Evoked Psoriatic Skin Lesion.
A mouse model of IMQ-induced psoriasis was constructed to detect the efects of triptolide on psoriasis. Compared with the control mice (no apparent signs of skin infammation) in the sham group, psoriatic mice treated with IMQ showed severe psoriasis-like lesions, including scales, thickening, and erythema in the model group. Severe lesions of scales, thickening, and erythema were attenuated following treatment with low doses of triptolide (10 mg/kg) and further alleviated by high doses of triptolide (40 mg/kg) (Figure 4(a)). Compared with the mice in the sham group, PASI scores were much higher in IMQ-treated psoriatic mice. However, on the 8 th day after treatment with low and high doses of triptolide, PASI scores were gradually reduced (Figure 4(b)). Furthermore, skin tissues from mouse models were subjected to H&E staining, which demonstrated that IMQ-induced higher epidermal thickness in the model group (109.82 ± 8.55 μm) was signifcantly reduced by a low dose of triptolide (82.47 ± 5.63 μm) and further downregulated by a high dose of triptolide (42.88 ± 4.24 μm) (Figures 4(c) and 4(d)). Tese results suggested that psoriatic skin lesions induced by IMQ can be ameliorated by triptolide treatment.  MTT assay conducted to detect the viability of splenocytes which were isolated from mice in the presence or absence of increasing dosages of triptolide (1-500 nM) treatment. (c) Anti-CD3 or anti-CD3 + IL-6 + TGF-β used to stimulate the isolated splenocytes from mouse models with or without 1.25-10 nM triptolide treatment. ELISA was used for measuring IL-17 concentration in the supernatants. (d) IL-17 + CD4 + T population determined by fow cytometry analysis. Each bar presents the mean ± standard deviation obtained from three to four separate experiments. * * P < 0.01, * * * P < 0.001 compared to the model group. n.s indicates no signifcance. reduced CD4 + IL-17A + percentages in the LNs and spleens to 3.74 ± 0.36% and 2.82 ± 0.50%, respectively. Tese data suggested that triptolide treatment inhibits T17 cell development in psoriasis.

Triptolide Inhibits the Expression of Proinfammatory Cytokines in IMQ-Evoked Psoriasis-Like Mice.
To recognize the immunosuppressive infuence of triptolide on IMQinduced skin lesions, the serum concentrations of proinfammatory cytokines (IL-17 and IFN-c) of mouse models in each group were frst measured through ELISA. As presented in Figures 6(a) and 6(b), serum IL-17 and IFN-c levels were higher in psoriatic mice induced by IMQ than normal mice in the sham group. After treatment with triptolide, induced upregulation of IL-17 and IFN-c levels in serum of psoriatic-like mice was downregulated by 10 mg/kg triptolide and further reduced by 40 mg/kg triptolide. On the other hand, the expression of several cytokines at mRNA levels was detected by RT-qPCR analysis. Compared with the mice in the sham group, expression levels of T17-type cytokines, including IL-6, TNF-α, IL-17F, and IL-17A, were notably elevated in the skin of IMQ-induced psoriatic mice. However, treatment of psoriatic mice with triptolide resulted in an obvious suppression of expression levels of IL-6, TNFα, IL-17F, and IL-17A in the psoriasis-like lesions in a dosedependent manner (Figures 6(c)-6(f )). Terefore, triptolide might ameliorate mouse psoriasis evoked by IMQ via inhibiting proinfammatory cytokines.

Triptolide Upregulates miR-204-5p Expression and Inhibits STAT3 Phosphorylation In Vivo.
To investigate the role of miR-204-5p and STAT3 in triptolide-mediated psoriasis in vivo, we examined the expression of miR-204-5p and STAT3 in healthy skin tissues and triptolide-treated psoriatic lesion skin tissues of mouse models. Te results from RT-qPCR analysis demonstrated that miR-204-5p expression was decreased in IMP-induced psoriatic lesion skin tissues relative to healthy skin tissues. Following triptolide treatment, miR-204-5p expression was dose-dependently increased (Figure 7(a)). On the contrary, we found an increase in STAT3 expression in the model group relative to the sham group. Induced STAT3 expression was dose-dependently suppressed by triptolide treatment (Figure 7(b)). Moreover, a negative expression correlation between miR-204-5p and STAT3 in IMP and triptolide-treated psoriatic mice was found (Figure 7(c)). Furthermore, the protein and   Each bar presents the mean ± standard deviation obtained from three to four separate experiments. Statistical signifcance was determined using two-way ANOVA followed by the post hoc Bonferroni test. * P < 0.05, * * P < 0.01, * * * P < 0.001 compared to the model group. 6 Genetics Research phosphorylation levels of STAT3 were also detected in psoriatic lesion skin tissues by RT-qPCR, which manifested that elevation of STAT3 protein and phosphorylation levels was reduced by increasing doses of triptolide (Figures 7(d)-7(f )). Conclusively, triptolide elevates miR-204-5p expression and suppresses STAT3 phosphorylation in vivo.

Discussion
Psoriasis is a multifactorial infammatory skin disorder seriously afecting the physical and psychological health of numerous patients [29]. Recently, an immunosuppressive agent such as cyclosporine or methotrexate has been applied in psoriasis treatment, but it causes severe adverse efects to the liver and kidneys. Compared to conventional drugs, biological anti-infammatory antibodies which suppress the IL-23/IL-17 axis are much more expensive. Terefore, seeking a more efcient and less expensive drug for psoriasis treatment is important. As a leading role in psoriasis pathogenesis, T17 cells have been proven to participate in the formation of IMQ-generated psoriatic lesions in mouse models [30]. Terefore, in this investigation, we focused on detecting the wellknown anti-infammatory efects of triptolide in a mouse model with the aim of further elucidating the mechanism by which triptolide exerts anti-infammatory infuences and protects against the formation of psoriasis-like lesions induced by IMQ. Our fndings illuminated that triptolide protected against topical IMQ treatment-induced psoriasislike lesions in mouse models. Moreover, our study suggested that this protection was achieved by suppressing T17 cells and triptolide inhibited T17 response through miR-204-5pmediated suppression of STAT3 phosphorylation.
Research has validated that the dosage of triptolide under 200 μg/kg shows no discernible adverse efects, while 300-1000 μg/kg of triptolide results in liver injury [31][32][33][34][35][36], which indicates that triptolide exhibited toxicological or pharmacological efects largely depend on its dosage. In our study, the concentration of triptolide at 1-500 nM was used to treat the isolated splenocytes which showed that 10-50 nM triptolide signifcantly inhibited cell viability, while triptolide at the dosage of 500 nM led to cytotoxicity. Te combination between miR-204-5p and STAT3 validated by luciferase reporter assay. Each bar presents the mean ± standard deviation obtained from three to four separate experiments. * * P < 0.01, * * * P < 0.001 compared to the model group.

Genetics Research
Additionally, the dosage of triptolide under 10 nM suppressed T17 cell diferentiation from splenocytes. Terefore, triptolide might become a promising drug for psoriasis treatment until the identifcation of the appropriate dosage for patients. Both human psoriatic plaques and IMQ-induced mouse psoriatic plaques have numerous erythema and scales in terms of histopathological features. Our study demonstrated that following continuous IMQ treatment, signifcant thickening, erythema, and scaling on the dorsal skin of mouse models at day 8 was seen. Of note, these symptoms were alleviated by triptolide treatment, which is consistent with the results of the previous report [25]. Immune cell infltration is another key factor in psoriasis development except for epidermal cell hyperplasia. It has been illuminated that the IL-23/IL-17 axis can mediate psoriasis-like skin infammation caused by IMQ induction in mouse models [37]. Cytokines such as TNF-α, IL-22, IL-17A, IL-17F, and IL-6 are produced by IL-23-activating T17 cells, which promotes the proliferative ability of keratinocytes [38,39]. In the current investigation, compared with sham mice, signifcantly upregulated levels of T17 cytokines including IL-6, TNF-a, IL-17F, and IL-17A were identifed in skin samples of IMQ-treated mice. Additionally, a greater number of CD4 + IL-17A + cells was identifed in the LNs and spleens of IMQ-induced mouse models while triptolide elicited inhibitive efects on T17 response, which is in accordance with previous literature studies [28,40].
STAT3, which is initially identifed as acute phase response factor (APRF), is activated in hepatocytes treated by IL-6 [41]. Te activation of STAT3 is associated with pathologies such as autoinfammatory or autoimmune conditions including psoriasis [42]. Not surprisingly, STAT3 has been demonstrated as a crucial player in T17 cell biology, and the diferentiation of T17 cells can be suppressed by STAT3 depletion both in vivo and in vitro [43,44]. Many documents have proved that triptolide represses STAT3 signaling in multiple human diseases. For example, triptolide contributes to autophagy through reactive oxygen species generation in ovarian cancer SKOV3/DDP cell lines resistant to cisplatin by inhibiting STAT3 signaling [45]. Another report reveals that via suppression of the Janusactivated kinase 2/STAT3 pathway, triptolide suppresses the expression of infammatory cytokines and inhibits cell proliferative abilities in fbroblast-like synoviocytes stimulated by IL-6/sIL-6R [46]. Consistently, our study manifested that the expression and phosphorylation levels of STAT3 were suppressed by triptolide both in vivo and in vitro, which indicated that psoriasis development is inhibited by triptolide through suppression of STAT3.
To further prove how triptolide regulates STAT3, we focused on miRNAs which were associated with psoriasis or triptolide. MicroRNAs are short noncoding RNAs that could posttranscriptionally modulate protein-coding gene expression, and emerging miRNAs have been uncovered to be efective in the function of immune cells and keratinocytes in patients with psoriasis [47][48][49]. MiR-204-5p is a newly identifed microRNA, which has been elucidated to inhibit synovial fbroblast infammation in osteoarthritis [50], and is correlated with CD4 + central memory and efector memory T cell infltration [51]. In addition, the promotive efect of triptolide on miR-204 expression has been verifed in S2-   VP10 cells [27]. Consistently, our study manifested that miR-204-5p expression was also elevated by triptolide in HaCaT cells and psoriatic lesion skin tissues in a dose-dependent manner. Interestingly, through bioinformatic analysis, the binding sequences between miR-204-5p and STAT3 were predicated, and the combination was proved by luciferase reporter assay. Moreover, it was found that STAT3 expression levels were reduced upon miR-204-5p overexpression and the negative expression correlation between miR-204-5p and STAT3 was recognized. All these fndings suggested that triptolide inhibited the phosphorylation of STAT3 via elevating miR-204-5p and thus repressing T17 response and psoriasis development.
Conclusively, our results manifested that triptolide inhibited T17 response by upregulating miR-204-5p and suppressing STAT3 phosphorylation in psoriasis. However, the sample size was relatively small, more samples needed to be collected, and further studies were needed to further confrm our fndings in the future. Considering the toxicity of triptolide to mouse models, its precise safe dosage to human needs further investigation. Finally, due to the complexity of the molecular mechanism, some upstream molecules or other downstream signaling pathways of the triptolide/miR-204-5p/STAT3 axis deserve further exploration.

Data Availability
Te datasets used or analyzed during the current study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest. (d-f ) Te protein levels of STAT3 and p-STAT3 in the dorsal skin of mouse models tested via Western blot analysis. Each column is representative of data obtained from a minimum of 8 animals. ## P < 0.01 compared to the sham group; * P < 0.05, * * P < 0.01 compared to the model group.