The Effects of Sishen Wan on T Cell Responses in Mice Models of Ulcerative Colitis Induced by Dextran Sodium Sulfate

Currently, it is unclear whether Sishen Wan (SSW) could modulate the balance of Th1 cells, Th17 cells, and Tregs and we evaluated the effects of SSW on T cell responses in mice models of ulcerative colitis (UC). The mice models of acute UC (4% dextran sodium sulfate (DSS), 8 days) and chronic UC (3% DSS, 16 days) with SSW were assayed. Colon tissues were collected for immunohistochemical analysis, enzyme linked immunosorbent assay (ELISA), and flow cytometry (FCM). The expressions of cytokines associated with Tregs, transcription factors of Th17 cells, the frequencies of Th1 cells, Th17 cells, and Tregs, and the functional plasticity of Th17 cells were detected. The frequency of IFN-γ+ T cells was not changed significantly with SSW treatment in acute DSS. In chronic models, the frequency of IFN-γ+ T cells was downregulated with SSW. Meanwhile, the levels of RORγt and the frequency of IL-17A+ Th17 cells showed no significant differences after SSW treatment. Despite no significant effect on the transdifferentiation of Th17 cells in chronic UC models, SSW transdifferentiated Th17 cells into IL-10+ Th17 cells and downregulated IFN-γ+ Th17 cells/IL-10+ Th17 cells in acute DSS. Moreover, there were no significant changes of cytokines secreted by Tregs in acute DSS after SSW treatment, but SSW facilitated the expressions of IL-10 and IL-35, as well as development of IL-10+ Tregs in chronic DSS. SSW showed depressive effects on the immunoreaction of Th17 cells and might promote the conversion of Th17 cells into IL-10+ Th17 cells in acute UC, while it inhibited the excessive reaction of Th1 cells, facilitated the development of Tregs, and enhanced the anti-inflammatory effects in chronic UC.


Introduction
Ulcerative colitis (UC), a common chronic nonspecific inflammatory bowel disease (IBD), is generally manifested as diarrhea, abdominal pain, weight loss, and hematochezia [1,2]. Currently, the prevalence of UC is increasing worldwide. It is commonly recognized that extensive and long-standing UC poses a substantial cancer risk and the risk for colitis-associated colorectal cancer (CAC) was reported to be around 30% at 35 years after the onset of the disease [3,4]. In China, UC prevalence increased 3 times from 2001 to 2011 and the incidence of UC was approximately 1.18/ 100,000 in 2017 [5,6]. In USA, UC prevalence rates in 2007 and 2017 were 0.25% and 0.39%, respectively [7]. Current therapeutic options used for the treatment against UC include 5-aminosalicylate, glucocorticoid, and immunosuppressive agents. However, these available treatments are accompanied with significant adverse gastrointestinal reactions, serious complications such as tuberculosis, recurrence, and a heavy economic burden [8][9][10]. It is of vital importance to explore a series of novel, safe, and efficient methods for the treatment of UC.
Although the etiology and pathogenesis of UC remain elusive, it is generally accepted that UC is associated with disordered immune response, genetic susceptibility, intestinal microbiota disorder, and environmental factors [11]. T helper 1 ( 1) cells, T helper 17 ( 17) cells, and regulatory T cells (Tregs) have been found to play important roles in the pathogenesis of UC. 1 cells, which produce and secret proinflammatory cytokines such as Interferon-gamma (IFN-c), Interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-α), are known to play a vital role in cellular immunity [12]. With the upregulation of T-box transcription factor expressed in T-cells (T-bets), 1 cells are induced excessively by signal transducer and activator of transcription 4 (STAT4) and STAT1 signal pathways, which are activated by IL-12 derived from dendritic cells and IFN-c produced from natural killer (NK) cells [13][14][15][16][17][18]. 1 cells secrete IFN-c and exacerbate inflammatory injury [19]. 17 cells are associated with the defense against extracellular infectious factors, inflammatory responses, and the development of autoimmunity [20,21]. After the increased expression of retinoic acid-related orphan receptor gamma t (RORct), the polarization of 17 cells is mediated by overexpressed IL-1β and IL-6 and activated STAT3 in UC patients [22]. 17 cells produce and secrete IL-17 and IFN-c, which induce epithelial cells to release granulocyte colony-stimulating factor (G-CSF), accelerate neutrophil recruitment, and mediate excessively inflammatory reactions [15,23]. However, IL-22, another effector cytokine derived from 17 cells, facilitates epithelial cells to secrete antimicrobial peptides, augment epithelial repair, and restore barrier function [23,24]. With remarkable functional plasticity, IL-17A producing 17 cells readily acquire the ability to secrete IFN-c in UC model, while limiting excessive innate effector functions of dendritic cells (DCs) and macrophages by expressing IL-10. Expressing anti-inflammatory cytokines such as IL-35, transforming growth factor (TGF-β) [25][26][27], and IL-10, Foxp3 + Tregs are widely distributed in mucosal tissue of colon and depress excessive inflammatory responses [28][29][30]. In UC patients, the expression of IL-10 is downregulated with the impaired Foxp3 + Tregs and the immunologic injury is aggravated [31].
As a source of pharmaceutical material, Chinese medicine plays an important role in the therapy against UC. Sishen Wan (SSW), described in Chen shi xiao er dou zhen fang lun, written by Chen, Wenzhong in Song Dynasty, is composed of Myristica fragrans Houtt.  (Table 1).
SSW is applied to treat diarrhea of deficiency of spleenyang (the heating effect of digesting food) and kidney-yang (the source power of human life) via the efficacies of warming kidney (strengthening kidney-yang) for dispelling cold, warming yang to resolve wetness (treating a series of diseases with the syndromes of heavy sensation of head, dizziness, and aches in the limbs), and relieving diarrhea with astringents in the theory of Chinese medicine. SSW has shown beneficial effects on UC in clinic treatments and several clinical studies have demonstrated that SSW can ameliorate abdominal pain, relieve diarrhea, and alleviate intestinal mucosal hyperemia and hematochezia in UC patients [33][34][35]. Sun et al. reported that the appetite of UC patients was improved by salicylazosulfapyridine (SASP) combined with SSW, and SASP combined with SSW effectively relieved abdominal pain and diarrhea [36]. e clinically effective rate of SSW on 36 UC patients was 94.4% reported by Hu and was 92.31% on 26 UC patients described by Wang [37,38]. Modern pharmacodynamic and pharmacological studies also confirmed that SSW was effective. In dextran sodium sulfate-(DSS-) induced experiment on mouse models of colitis, SSW attenuated histopathological injuries, recovered colonic mucosa gradually, restored colonic length, downregulated contents of IL-2, IL-7, IL-12, and IL-15, upregulated IL-10 expression, regulated the quantity and subpopulation of central memory T (Tcm) and effector memory T (Tem) cells, suppressed the activation of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), and T-box protein expressed in T cells (T-bet) [39]. In DSS-induced UC mice, SSW downregulated the levels of IL-1β, IL-4, IL-9, and IL-17A and CD11C + CD103 + E-cadherin + cells, as well as increasing the beneficial bacteria and reducing the pathogenic bacteria [8]. Wang et al. reported that the expressions of nuclear factor kappa B (NF-κB) p65 and NF-κB essential modulator-(NEMO-) like kinase (NLK) were depressed and the transcriptions of IFN-c, IL-12/23, TNF-α, IL-17, and IL-1β genes were inhibited in SSWtreated UC rats [40]. After the therapy of SSW, inflammatory damage and congestive and edema were alleviated, the infiltration of inflammatory cells was ameliorated, the recoveries of epithelial damage and ulceration were accelerated, and the expression of IL-10 was upregulated in dextran sodium sulfate-(DSS-) induced UC mice [41]. In our previous studies, SSW ameliorated the infiltration of inflammatory cells in colonic tissues in TNBS and DSSinduced mice models and improved disease activity index (DAI) [42]. Moreover, SSW could alleviate inflammatory damage by depressing the activity of myeloperoxidase (MPO) in acute and chronic UC, treat acute UC by reducing the contents of IFN-c and IL-17A, and relieve the damage in chronic UC by accelerating the secretion of IL-22 [43,44]. Our study revealed that SSW was effective in ameliorating UC via T cell responses, and we discussed the different therapeutic mechanisms of SSW on T cells in acute and chronic UC mice.

Experimental Design.
After adaptation for 7 days, mice were randomly divided into six experimental series, with each group consisting of 10 mice: acute control, chronic control, acute DSS, acute DSS + SSW, chronic DSS, and chronic DSS + SSW. Mice in acute control were not challenged with DSS and treated with SSW for 5 days. Mice in chronic control were not induced by DSS and treated with SSW for 26 days. Models in acute DSS were induced through free access to 4% (w/v) DSS dissolved in distilled water for 5 days. Mice in acute DSS + SSW were given SSW by gavage from day 2 to day 9 after the start of experiment, once a day, and the dose of SSW was calculated by the following equation: where D h is the clinical dose of SSW for adult humans, D m is the administered dose of SSW for mice, W is the weight of the adult human body, and F is the dose conversion factor for mice and adult humans [45]. e clinical dose of SSW is 9-18 g per day [32], and we chose the average of 9 g and 18 g (13.5 g). W was set as 60 kg. Considering the dose conversion factor of 10 between mouse and human, D m of 2.25 g/kg was calculated in our study [45]. Mice were sacrificed by cervical dislocation. Colon tissues were collected quickly and aseptically. Mice in chronic DSS were established by drinking 3% (w/v) DSS on day 1 to day 5, day 8 to day 12, day 15 to day 19, and day 22 to day 26, respectively, and drinking water without DSS on day 6 to day 7, day 13 to day 14, and day 20 to day 21 [46][47][48]. Mice in chronic DSS + SSW were intragastrically administered by SSW on day 12 for 16 days and were euthanized by cervical dislocation on day 29. Colon tissues were harvested aseptically.

Immunohistochemistry Assay.
Colon tissues (1 cm) were fixed in 10% formalin, embedded in paraffin, and cut into 4 μm thick sections. e sections were dewaxed, hydrated, and incubated with 3% H 2 O 2 for 5-10 min block endogenous peroxidase activity and washed with phosphate buffer saline (PBS) 3 times, 2 min each time.
e tissues were processed with antibody against RORct, incubated at 37°C for 1-2 h, and washed by PBS 3 times, 2 min each time. Reagent 1 was added into tissues and tissues were incubated at 37°C for 10-20 min and washed by PBS 3 times. en, tissues were incubated with Reagent 2 at 37°C for 10-20 min and washed by PBS. Sections were colored by using 3, 3′-Diaminobenzidine tetrahydrochloride hydrate (DAB) and counterstained with hematoxylin. e samples were observed under Nikon ECLIPSE TI-U inverted microscope (Nikon, Tokyo, Japan) and images were captured. Using Image Pro Plus 6.0 system, the mean absorbance of RORct + area was measured.

Enzyme Linked Immunosorbent Assay (ELISA).
ELISA was accessed to detect the expressions of IL-10, TGFβ, and IL-35 in colon tissues. Colon tissues were cultured in 24-well plates with RPMI 1640 medium. Colon tissues were incubated at 37°C in a 5% CO 2 atmosphere for 24 h and culture supernatant was collected. Immunoreactive IL-10, TGF-β, and IL-35 in colon tissues were evaluated quantitatively by ELISA as per the manufacturer's instructions.

Cell Isolation.
e colon tissues were rinsed with Hank's buffer, incised longitudinally, and cut into pieces. e pieces were incubated with 0.37 mg/ml EDTA and 0.145 mg/ml DTT in Hank's buffer in 37°C water bath 2 times, 20 min each time, and the suspension, mainly containing epithelial cells and intraepithelial lymphocytes, was removed. e remaining tissues were supplemented with 25 ml RPMI 1640 digestive solution containing 5% fetal bovine serum (FBS), 0.5 mg/ml collagenase, and 0.1 mg/ml DNase and incubated at 37°C water bath 3 times, 30 min each time. e supernatant was collected and filtered with 1 mm strainer and 100 μm and 50 μm polyamides and separated single cells were acquired. Cells were washed by RPMI-1640 containing 5% FBS and the survival rate was measured by trypan blue.
en, cells were filtered by abacterial absorbent cotton. Discontinuous gradient centrifugation was accessed by 40% and 100% Percoll to enrich the ratio of lamina propria

Statistical Analysis.
e data were presented as mean-± standard deviation. Statistical analysis was carried out using SPSS 16.0. A one-way analysis of variance (ANOVA) with LSD test and Tamhane's T2 test was performed to determine statistical differences. P < 0.05 was considered statistically significant.

SSW Depressed the Expression of 17 Cells in Acute UC Mice Models and Inhibited 1 Cells in Chronic UC Models.
Predominant pathogenic 1 cell immune responses were considered to link to the pathogenesis of UC in classical view. However, IL-17 signaling pathway derived from 17 cells has been discovered as a distinctive signal pathway associated with the pathogenesis of UC [49]. Our previous studies elaborated that SSW might alleviate immunologic injuries by downregulating the levels of IFN-c and IL-17A in acute DSS and relieve the damage in chronic DSS by promoting the secretion of IL-22. is study revealed the frequencies of 1 and 17 cells and the expression of RORct in SSW-treated UC mice.
FCM analyses of IFN-c + T cells and IL-17A + T cells are shown in Figure 1 e results showed that RORct expression was increased significantly (P < 0.01) in acute and chronic DSS compared with control mice. With the therapy of SSW, the level of RORct was decreased in acute DSS and showed no significant change in chronic DSS (Figure 1(b)). From this result, it could be seen that the effect of SSW on the level of RORct was consistent with IL-17A.

SSW Promoted Tregs to Secrete Inhibitory Cytokines in Chronic UC Models.
e level of IL-35 was increased obviously in acute DSS when compared with healthy mice; however, there were no significant differences of IL-10 and TGF-β between mice in acute control and acute DSS. Our results suggested that the levels of some inhibitory cytokines were increased in acute DSS. ere were no significant effects on the contents of IL-10, TGF-β, and IL-35 in acute DSS treated with SSW, which might be due to the fact that the efficacy of SSW had no obvious correlation with inhibitory cytokines derived from Tregs. e level of IL-10 was upregulated significantly in chronic UC models, while the levels of TGF-β and IL-35 had no obvious variations. SSW further increased the levels of IL-10 and IL-35, which implied that SSW might repress excessive inflammatory responses by upregulating the contents of IL-10 and IL-35 (Figure 3(a), P < 0.05).
Significant upregulation in the percentage of IL-10 + Tcells was found in acute DSS and chronic DSS. SSW further increased the frequency of IL-10 + T cells in chronic DSS; however, this effect was not obvious in acute DSS (Figure 3(b), P < 0.05). Our results indicated that SSW might inhibit excessive inflammatory responses and facilitate tissue reconstruction by enhancing the frequency of IL-10 + T cells in chronic UC.

Discussion
With alteration of epithelial structure, high-level neutrophils and lymphocytes infiltration, reduction of colon length, destruction of mucosa, and loss of crypts, a series of symptoms such as significant weight loss, rectal bleeding, diarrhea, and deteriorated inflammation are evident in DSSinduced animal models [50,51]. e mechanism of mucosal injury induced by DSS has not been completely explicit and may be associated with the interaction between DSS and epithelial barrier [52,53]. Intestinal epithelial bacteria and bacterial metabolites are translocated, caused by the damage to the epithelial barrier induced by DSS, leading to a large quantity of lipopolysaccharides (LPS) entering the cytoplasm, resulting in downregulation of ioredoxin-interacting protein (TXNIP), excessively upregulating the expressions of IL-1β and IL-18, and causing intemperate inflammatory responses [54][55][56]. Furthermore, DSS accelerates the differentiation to 17 cells, increasing the expressions of IL-17 and RORct, while inhibiting the polarization to Tregs, leading to inflammatory injuries and alterations of immunocytes and cytokines [57][58][59][60]. erefore, DSS-induced mice models were used to explore the effects and immunologic mechanisms of SSW against UC.  Evidence-Based Complementary and Alternative Medicine As a famous traditional Chinese herbal medicine, SSW was applied to treat diarrhea caused by deficiency of spleen-yang and kidney-yang (the source power of human life) in the theory of Chinese medicine. SSW is frequently used in clinical therapies of ulcerative colitis, irritable bowel syndrome, and allergic colitis. e clinical benefit rate in clinical trial of SSW combined with enema of herbal medicine on 62 cases of chronic UC was 90%, as reported by Yang and Rongfeng [61]. As described by Xie and Jianjun, the clinically effective rate of SSW combined with enema on 58 cases of UC was 89.7% [62]. Hu reported that the clinical benefit rate of SSW on 36 cases of UC was 94.4% [37]. Results from experimental animals revealed that SSW and major bioactive components of SSW might alleviate chronic colitis by depressing Wnt/β-catenin pathway [63]. Accomplished by decreasing the levels of MPO and malondialdehyde (MDA), increasing the contents of superoxide dismutase (SOD), IL-4, and IL-10 mRNA expressions, SSW might have a protective effect on impaired colonic mucosa rats with UC [64]. Moreover, SSW might decrease the expressions of IFN-c, IL-1β, and IL-17, upregulate the level of IL-4, and show therapeutic effects in chronic colitis [40]. Our preliminary studies revealed that SSW reduced DAI significantly, attenuated infiltration of inflammatory cells, decreased the activity of MPO in colonic tissues, and ameliorated inflammatory damages in UC mice models [43,65]. is study exhibited the immunologic regulation of SSW on T cell responses in acute and chronic UC mice models. e dysregulation of cells induced by DSS is crucial in the damage of colon tissues during the acute and chronic stages of UC. T-bet signaling pathway is induced by the activation of STAT1 in IBD process and facilitates the differentiation to 1 cells, and 1 cells produce and secrete IFN-c and IL-2, which repress the differentiation to 2 cells and restrain the expressions of IL-5 and IL-13 derived from 2 cells, leading to the disequilibrium of 1/ 2; in addition, IFN-c promotes macrophages to secrete large amounts of proinflammatory cytokines and exacerbates the tissue damage [58,[66][67][68][69]. Our previous investigation revealed that the therapeutic effects of SSW were mediated by depressing the levels of IFN-c and IL-17A in acute UC mice models and upregulating the content of IL-22 in chronic colitis [44]. In this study, FCM showed that SSW significantly restrained the frequency of IFN-c + T cells in chronic DSS; however, this effect was not obvious in acute DSS, which might be due to the fact that the efficacy of SSW was not associated with 1 cells. Under normal conditions, 17 cells, broadly distributed on intestinal epithelial tissues, secrete cytokines such as IL-17A, IL-17F, IL-22, and IL-21 and proceed with moderate immune responses in allusion to intestinal flora to maintain the stabilization of colon mucosa; in the pathological process of UC, IL-6, IL-23, and IL-21 are produced and secreted by activated macrophages, epithelial cells, and dendritic cells (DCs), followed by facilitating the expression of RORct, a critical transcription factor for 17 cells differentiation, resulting in the differentiation to 17 cells [23,[70][71][72][73]. Immunologic responses, mediated by IL-17A, make a protective effect on immune balance by eliminating pathogen; however, excessive IL-17A causes the imbalance between 17 cells/Tregs and is strongly linked to the pathogenesis of UC [74][75][76]. Our results further showed that RORct and IL-17A + T cells were upregulated in acute DSS and downregulated after being treated with SSW; however, these variations were not significant in chronic UC mice. During the acute stage of UC, the neutrophil recruitment to the colon is promoted by 17 immune responses and contributes to the exacerbation of UC activities [77]. us, SSW might suppress the neutrophil recruitment to the colon by inhibiting 17-mediated immunologic reactions in acute DSS. Mechanistically, fibrosis is a consequence of chronic stage of UC [78]. In the chronic DSS, the fibrogenesis in murine colitis might be ameliorated with the treatment of SSW by suppressing the production of 1 type cytokines and polarizing macrophages towards the alternatively activated macrophages (M2 macrophages) [79].
By possessing the ability to repolarize towards different phenotypes, 17 cells are capable of expressing IFN-c, a distinctive cytokine of 1 cells, and IL-10, a characteristic cytokine of Tregs, which is commonly referred to as plasticity [80]. Induced by the activity of proinflammatory cytokines such as IL-12 or TNF-α, 17 cells express T-bet and transdifferentiate to 1-like 17 cells, which secrete IFN-c and GM-CSF [81].
1-like 17 cells participate in the pathogenesis of autoimmune diseases actively. In juvenile idiopathic arthritis patients, 17 cells transdifferentiated to 1-like 17 cells after being exposed to TNF-α and produced IFN-c and TNF-α, which exacerbated inflammation, as described by Maggi et al. [82]. Stimulated by anti-CD3 mAb, 17 cells shift towards IL-10 + 17 cells and produce IL-10 to play a role in immunosuppression [26,83]. Our data suggested that 17 cells participated in inflammatory responses by the coexpression of IFN-c in acute UC, while SSW upregulated the frequency of IL-10 + 17 cells. In chronic UC models, the frequency of IFN-c + 17 cells was decreased compared with acute UC, which might be because upregulated IFN-c + 17 cells/IL-10 + 17 cells facilitated the secretion of IL-10 followed by depressing inflammation and accelerating the tissue repair in the disease process of chronic UC models. Moreover, we observed that the therapeutic mechanisms of SSW did not correlate with IFN-c + 17 cells/IL-10 + 17 cells in chronic UC. e possible reason for the inconspicuous effects with SSW on the variation of IFN-c + 17 cells/IL-10 + 17 cells in chronic UC might be the decline of 17 cells, and potential immunologic mechanism of the therapy against chronic UC with SSW needed to be further studied.
Widely distributed in intestinal mucosal tissues, Tregs mediate immunologic tolerance to dietary antigens and gut microbiota, secrete inhibitory cytokines to ameliorate inflammatory injuries, and accelerate the reconstruction of tissues [84]. A previous study indicated that Foxp3 + Tregs were downregulated in peripheral blood of UC patients and upregulated in the intestinal mucosa, which depressed effector T cells in mucosal tissues [85]. Secreted by Foxp3 + Tregs, IL-10, TGF-β1, and IL-35 alleviate excessive immunologic reactions and improve UC symptoms by activating Smad signal pathway, depressing 1 cells and 17 cells [86][87][88]. We detected the variations of IL-10, TGF-β1, and Evidence-Based Complementary and Alternative Medicine IL-35 in intestinal mucosa and found that IL-10, TGF-β1, and IL-35 were increased in acute UC; however, these factors had no obvious changes with the supplement of SSW. Our results implied that the preventive and therapeutic effects of SSW might not correlate with the regulation on Tregs in acute UC. In chronic UC models, IL-35 and IL-10 were further upward with SSW. Previous studies confirmed that 2 cells play an important role during the chronic stage of UC [89] and 2 cells also secrete IL-10 [90]. 2 cells depress the excessive inflammatory responses in UC; however, 2 polarized immune responses were associated with fibrosis development [91]. Treg-derived IL-35 reduces the excessive 2 immune responses [92]. us, we speculated that SSW might promote the IL-10 expression by Tregs and 2 cells to inhibit inordinate immune responses and facilitate the expression of IL-35 to suppressive excessive 2 immune responses in chronic UC rather than acute UC. However, the mechanisms of SSW on 2 cells need to be further studied.

Conclusions
Our study disclosed the immunologic modulations of SSW on responses of 1 cells, 17 cells, and Tregs in acute and chronic UC models. SSW might react on DSS-induced acute UC models by restraining the excessive immunoreaction of 17 cells and facilitating the transdifferentiation from 17 cells into IL-10 + 17 cells, leading to the downregulation of IFN-c + 17 cells/IL-10 + 17 cells. SSW depressed the excessive reaction of 1 cells and accelerated the development of Tregs, followed by enhancing the anti-inflammatory effects in chronic UC mice. is study implied that SSW can be an alternative prescription against UC via different immunologic regulations on the balance of 1 cells, 17 cells, and Tregs in acute and chronic UC, which might be correlated with the contents of cytokines, the frequencies of 1 cells, 17 cells, and Tregs, and the functional plasticity of 17 cells.

Data Availability
e data used to support the findings of the study are available from the corresponding author upon request. Disclosure e funders have no role in the design of the study and collection, analysis, and interpretation of data or in writing the manuscript.

Conflicts of Interest
e authors declare no conflicts of interest.