Sishen Wan Treats Ulcerative Colitis in Rats by Regulating Gut Microbiota and Restoring the Treg/Th17 Balance

Objective This study was aimed to explore the mechanism of Sishen Wan (SSW) in treating ulcerative colitis (UC) in a rat model of spleen-kidney yang deficiency pattern by regulating gut microbiota and the content of butyric acid in short-chain fatty acid (SCFAs) and restoring regulatory T (Treg)/T helper type 17 (Th17) balance from the perspective of the correlation between gut microbiota and immune function. Methods The UC rat model of spleen-kidney yang deficiency pattern was established by the method of combining disease and syndrome (intragastric administration of senna leaf, subcutaneous injection of hydrocortisone, and enema with 2,4-dinitrobenzenesulfonic acid (DNBS)/ethanol solution). After successful modeling, rats were randomly divided into six groups: the blank group, model group, low-, middle-, and high-dose Sishen Wan groups, and mesalazine group. Samples were taken after continuous administration for 3 weeks. The general conditions and body weight of the rats were observed and recorded, and the disease activity index (DAI) score was calculated. Colonic mucosal injury was observed, and a colonic mucosal damage index (CMDI) score was calculated. Histopathological changes in colon tissues were determined by hematoxylin and eosin (H&E) staining, and the histopathological score (HS) was calculated. The serum levels of transforming growth factor-β1 (TGF-β1), interleukin (IL)-6, IL-10, and IL-17 were determined by enzyme-linked immunosorbent assay (ELISA) assays. The expression of TGF-β1, signal transducer and activator of transcription 3 (STAT3), and peroxisome proliferator-activated receptor γ (PPARγ) was determined by Western blot analysis. The proportion of Th17 and Treg cells in colon tissue was determined by flow cytometry. The relative abundance of gut microbiota was determined by 16S rDNA sequencing, and the concentration of butyric acid of SCFAs was determined by gas chromatography-mass spectrometry (GC-MS). Results Administration of SSW significantly improved the pathological changes of colon tissue in UC rats and could attenuate the DAI and CMDI scores, and the HS. SSW significantly decreased the serum levels of IL-6 and IL-17 and increased the serum levels of TGF-β1 and IL-10. In addition, SSW increased the expression of TGF-β1 and PPARγ and decreased the expression of STAT3 in colon tissue in a dose-dependent manner. Furthermore, SSW significantly decreased the proportion of Th17 cells and increased the proportion of Treg cells in colon tissue. Additionally, SSW altered the gut microbiota, including an increase in the relative abundance of Firmicutes and a decrease in Bacteroidota at the phylum level and an increase in the relative abundance of Lactobacillus at the genus level. Moreover, SSW significantly increased the concentration of butyric acid. Conclusions Combined, these data suggested that SSW increased the relative abundance of firmicutes and the level of butyric acid and restored the balance of Treg/Th17 immune axis and gut homeostasis, thus delaying the progress of UC.


Introduction
Ulcerative colitis (UC) is a type of infammatory bowel disease (IBD) characterized by idiopathic chronic infammation of the colonic mucosa, which begins in the rectum and continues to extend to the proximal colon [1]. Typical symptoms include abdominal pain, bloody diarrhea and mucus, fecal urgency, and tenesmus [2]. Patients with UC usually relapse and experience remission, and up to 90% of patients relapse one or more times after the frst episode [3]. Te pathogenesis of UC is unclear, but it has been confrmed to be related to immune response disorder, a change in gut microbiota, genetic susceptibility, and environmental factors [4]. In recent years, based on studying the immune mechanisms underlying chronic infammatory diseases, there has been a deeper understanding of the pathogenesis of UC.
Regulatory T (Treg)/T helper type 17 (T17) immune imbalance, a common infammatory-related mechanism mediated by T cells, is considered an important mediator of tissue damage causing colonic mucosal ulceration in UC [5]. Tregs and T17 cells are derived from CD4 + T lymphocytes, and IL-6 plays a key role in CD4 + T lymphocyte diferentiation by activating the signal transducer and activator of transcription 3 (STAT3) to phosphorylate STAT3, thus promoting the occurrence and development of UC [6]. As important proinfammatory cells, T17 cells promote intestinal infammatory reactions by specifcally secreting IL-17. Tregs inhibit the activity of immune cells and control infammation by secreting transforming growth factor-β (TGF-β) and IL-10 [7][8][9]. Te gut microbiota of UC patients is related to the Treg/T17 immune axis. In previous studies, it has been shown that the Treg/T17 balance in intestinal mucosa can be improved by increasing the production of microbial short-chain fatty acids (SCFAs), so as to prevent and treat IBD [10]. SCFAs are metabolites formed by intestinal microbial glycolysis of dietary fber, mainly including acetic acid, propionic acid, and butyric acid in which butyric acid can be used as an agonist of peroxisome proliferator-activated receptor c (PPARc) [11,12]. PPARc can restore the pathophysiological imbalance associated with IBD by inhibiting the production of infammatory cytokines, thereby improving the impaired intestinal barrier function under the infammatory state [13]. Moreover, PPARc plays an important regulatory role in T17 and Treg diferentiation [14].
Drugs that are commonly used in the clinic to treat UC include 5-aminosalicylic acids, steroids, and immunosuppressants [15,16]. However, these drugs often lead to different side efects, such as nephrotoxicity, and increase the risk of infection and surgical complications, thereby reducing the quality of life of patients, especially during longterm treatment [17]. Traditional Chinese medicine (TCM) plays an auxiliary role in the treatment of UC, and its popularity is increasing worldwide [18]. However, the active components and exact underlying mechanism of action of TCM are still unclear. UC is divided into seven types of TCM, of which the spleen-kidney yang defciency pattern is one of the most important types [19]. Te main clinical manifestations of UC with a spleen-kidney yang defciency pattern are fear of cold, soreness and weakness of the waist and knees, mental fatigue, abdominal distension, and loose stool [20]. Sishen Wan (SSW) is the main prescription for treating UC of the spleen-kidney yang defciency pattern, and it is also the classic prescription in TCM for treating colitis [19,21]. Increasing data from evidence-based medicine indicate that SSW has a good therapeutic efect on IBD, and the total efective rate of 204 patients with IBD is 75.98% [22]. At present, there are relatively few studies on the single prescription of SSW, and most of them tend to be clinical studies, with few studies on the mechanism of action of SSW. Gut microbiota imbalance and immune disorders are the main etiology and pathogenesis of UC, but the mechanism of SSW in the treatment of UC by regulating gut microbiota and restoring the Treg/T17 balance is not completely clear. In this study, the UC rat model with a spleen-kidney yang defciency pattern was established by the method of combining disease and syndrome, and the mechanism of SSW on UC was verifed by animal experiment. were purchased from Abcam (UK). A rabbit antibody directed against p-STAT3 was purchased from Cell Signaling Technology (USA). Goat anti-rabbit immunoglobulin and goat anti-mouse immunoglobulin were purchased from Immunoway (USA). Anti-Mouse CD25, FOXP3, and T17 were purchased from Multi Sciences (Lianke) Biotech Co., Ltd. (China).

Animals.
A total of 120 Sprague-Dawley (SD) rats (10 weeks of age, 200 ± 20 g, males and females) were purchased from the Experimental Animal Center of Gansu University of Chinese Medicine (Gansu, China) and attached with a health and safety certifcate of conformity administered by the Chinese government (Batch number of production license is SCXK2020-0001). Rats were housed in a specifc-pathogen-free (SPF) barrier laboratory of the Experimental Animal Center of Gansu University of Chinese Medicine, with a temperature that was maintained between 21-25°C and a relative humidity within 50%-60%, 12 h light/12 h dark cycle, and noise <50 dB. Rats were freely fed with standard feed and water. [20,23]. Te UC rat model of spleen-kidney yang defciency pattern was established by the method of combining disease and syndrome, enema with DNBS and ethanol solution, subcutaneous injection of hydrocortisone, and intragastric administration of senna leaf. All rats were adaptively fed for 7 days before modeling. Rats (11 weeks of age) were given an intragastric injection of senna leaf (10 mL/kg) combined with a subcutaneous injection of hydrocortisone (15 mg/kg) once a day for 21 days. At 21 days later, rats were fasted for 24 h and had free access to water. Ten, rats were anesthetized with 2% pentobarbital sodium (0.2 mL/100 g), and polypropylene tubes were slowly passed through the anus of each rat to 2

Preparation of the Animal Model
Evidence-Based Complementary and Alternative Medicine a depth of about 8 cm. Next, a syringe was used to quickly inject DNBS and an ethanol solution (100 mg/kg DNBS+1 mL/kg 50% ethanol), and then about 0.4 mL of air was injected. Te anus was pinched tightly, and rats were held upside down for 1 min. General conditions of the rats, such as mental state, activity, fur, stool properties, bleeding, and body weight, were observed and recorded, and the disease activity index (DAI) score was calculated. Damage to the colonic mucosa was observed, and the colonic mucosal damage index (CMDI) score was calculated. Histopathological changes in colon tissues were detected using light microscopy, and the histopathological score (HS) was calculated. To sum up, the rat model of spleen-kidney yang defciency pattern was successfully established [23].

Preparation of SSW.
Te dry Chinese herbal medicines, which include Psoraleae Fructus (the dried fruits of Psoralea corylifolia), Evodiae Fructus (the dried fruits of Evodia rutaecarpa), Myristicae Semen (the ripe seeds of Myristica fragrants Houtt), Schisandrae Chinensis Fructus (the dried fruits of Schisandra chinensis (Turcz.) Baill), Zingiber Ofcinale Roscoe (the rhizomes of Zingiber ofcinale Rosc.), and Jujubae Fructus (the ripe fruits of Ziziphus jujuba Mill.) were purchased from Huirentang pharmacy (Lanzhou, China, Production dates were 2020). Te above-mentioned six herbs (in a ratio of 4 : 1 : 2 : 2 : 2 : 2 : 2) were mixed and immersed in 8 times distilled water for 30 min, decocted for 20-30 minutes, the residue was removed and fltered, and the herbs were then decocted twice with water for 15-20 min. Tree batches of fltrates were mixed. Te fltrates were concentrated to a low dose of crude drug content (0.6 g/mL), a middle dose of crude drug content (1.2 g/mL), and a high dose of crude drug content (2.4 g/mL). Samples were stored in the dark at 4°C.

Grouping and Administration of the Animal Model.
After successful modeling, rats were randomly divided into six groups (20 rats per group): the blank group, the model group, the low-dose group (SSW-L, 6 g/kg of SSW), middle-dose group (SSW-M, 12 g/kg of SSW), the high-dose group (SSW-H, 24 g/kg of SSW), and the mesalazine group (mesalazine, 0.36 g/kg) [23]. Te administered doses were converted based on the body surface areas of humans and rats. Te blank group and model group were given an equal volume of distilled water (10 mL/kg), and each group received an intragastric administration once a day for 21 consecutive days.

Collection of Specimens.
After treatment, rat fecal samples were collected by the abdominal massage method and used for detection by 16S rDNA sequencing and chromatography-mass spectrometry (GC-MS). Rats were fasted for 24 h then anesthetized with 2% pentobarbital sodium (0.2 mL/100 g). Blood samples were collected from the abdominal aorta and centrifuged to obtain serum, which was used for enzyme-linked immunosorbent assay (ELISA). Ten the rats were euthanized. Colon samples were divided into three segments: (1) One segment was fxed with 4% paraformaldehyde for hematoxylin and eosin (H&E) staining for histological observation. (2) One segment was placed in physiological saline and immediately used for fow cytometry. (3) Te last segment was stored at −80°C for Western blot analysis.

Histopathology.
Fresh colon samples were washed in physiological saline to remove the surrounding connective tissue and fat. Colonic mucosal injury was observed with the naked eye, and the CMDI score was calculated. Te CMDI score was calculated based on the following classifcations: (a) 0 � no damage; (b) 1 � mild hyperemia, oedema, smooth surface, no erosion damage; (c) 2 � moderate hyperemia, oedema, coarse granular mucosa, erosion, or intestinal adhesions; (d) 3 � highly congestive and edematous, with necrosis and ulcer formation on the mucosal surface, maximum longitudinal diameter of the ulcer <1 cm, thickening of the intestinal wall, or necrosis and infammation on the surface; (e) 4 � maximum longitudinal diameter of the ulcer >1 cm on a 3-point basis or total necrosis of the bowel wall [25].
Colon tissue was fxed with 4% paraformaldehyde, dehydrated, and embedded in parafn. Histological evaluation of colon sections was performed by H&E staining. Scoring was done according to the literature [26], (a) 0 � normal; (b) 1 � loss of 1/3 of the crypt glands; (c) 2 � loss of 2/3 of the crypt glands; (d) 3 � loss of all the crypt glands and complete mucosal epithelium with mild signs of infammatory cell infltration; (e) 4 � erosion and destruction of the mucosal epithelium with obvious infammatory cell infltration, the tissue sections were scored for HS.

Determination of Cytokines.
According to the manufacturer's instructions, serum levels of interleukin (IL)-6, IL-10, IL-17, and TGF-β1 were determined using corresponding ELISA kits. According to the instructions for the procedure, the concentration of cytokines in the serum was calculated by using a standard curve. Results were expressed as ng/L of serum per sample.
2.11. Flow Cytometry. Fresh colon tissues from rats were placed on a superclean bench to remove the surrounding connective tissues. A proper amount of sterilized PBS was added, and the mixture was fully ground and fltered to prepare a single-cell suspension. Te cells were resuspended in 100 μL of PBS containing fetal calf serum, and 2-Acetoxy-1-methoxypropane (PMA) (1 μg/mL), ionomycin (50 μg/ mL), and monamycin (0.1 mg/mL). Cells were stimulated and cultured in an incubator for 6 hours (37°C, 5% CO 2 ). After breaking the membrane and fxation, an appropriate amount of T17 antibody was added, according to the instructions, and the percentage of T17 cells was determined by fow cytometry. An appropriate amount of CD25 and Foxp3 antibodies were added, and the percentage of Treg cells was determined by fow cytometry.
2.12. 16S rDNA Gene Microbiome Analysis. Total genome DNA from samples was extracted using the CTAB/SDS method. Te 16S rRNA gene hypervariable V3-V4 region was amplifed using specifc primers with the barcode. Te prototype primers were 515F (CCTAYGGGRBGCASCAG)-806R (GGACTACNNGGGTATCTAAT). All PCR reactions were carried out in 15 μL of Phusion ® High-Fidelity PCR Master Mix (New England Biolabs); 0.2 μM of forward and reverse primers, and 10 ng of template DNA. Te same volume of 1X loading bufer (contained SYB green) was mixed with PCR products, and electrophoresis was performed on 2% agarose gels. Sequencing libraries were generated using the TruSeq ® DNA PCR-Free Sample Preparation Kit (Illumina, USA) and index codes were added. Te library's quality was assessed on the Qubit@ 2.0 Fluorometer (Termo Scientifc) and an Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an Illumina NovaSeq platform, and 250 bp paired-end reads were generated. Sequences with ≥97% similarity were assigned to the same operational taxonomic units (OTUs). Te representative sequence for each OTU was screened for further annotation.

Measurement of Short-Chain Fatty Acids.
Te level of short-chain fatty acid was measured by GC-MS analysis. A total of 50 μL 15% phosphoric acid, 100 μL isohexic acid solution, and 400 μL diethyl ether were added to each sample, grind, centrifuged, and the supernatant was tested. Te obtained SCFAs samples were measured using a TRACE 1310-ISQ LT GC-MS system (Termo, USA). Based on the detection results, target quantifcation was carried out on the detected samples, and relevant data analysis was carried out.
2.14. Statistical Analyses. SPSS 23.0 was used for statistical analysis, and one-way ANOVA was used for data analysis. Te least signifcant diference test (LSD) was used for comparison between groups with homogeneity of variance, and the Tamhane's T2 test was used for non-homogeneity of variance. All results were expressed as the mean ± SD and P < 0.05 was considered statistically signifcant.

SSW Improves the General Condition of Rats.
Rats in the blank group were in a good mental state, and the action response was very fexible. In addition, the rat fur was smooth and lustrous, the stool was normal, and the body weight increased steadily. After modeling, rats showed a reduced food intake, mental malaise, decreased activity, slow reactions, a fondness for clustering, loose stools, and damp and dirty abdominal fur. After induction of enema with DNBS and ethanol solution, rats showed obvious bloody stool and a dirty perianal region. Te body weight of rats decreased at the late modeling stage. After treatment, compared with the model group, the symptoms of rats in the SSW-L group were slightly improved; the manifestations included slightly increased food intake, a poor mental state, less activity, a fondness for clustering , still-present lack of luster in fur color, and loose and shapeless stool. Te symptoms of rats in the SSW-M, SSW-H, and mesalazine groups were signifcantly improved with increased food intake, a better mental state, increased activity, improved huddle, restored luster in fur color, formed stool, and a gradually cleaned perianal region. Te body weight of rats in the model group did not change much during modeling (Table 1). Te body weight of rats after treatment is presented in Table 2. Compared with the model group, the body weight of rats in the SSW-M, SSW-H, and mesalazine groups increased signifcantly (P < 0.05, P < 0.01).
Compared with the blank group, the DAI score of rats in the model group was signifcantly increased at the end of modeling (P < 0.01). After drug treatment, compared with the model group, the DAI score of rats in each treatment group was signifcantly decrease (P < 0.01). No signifcant diferences were observed between rats in the mesalazine and SSW-H groups (Table 3).

SSW Improves Colonic Tissue Injury.
Te appearance of the colon was evaluated as shown in Figure 1(a). Te gross morphology of colonic tissue was observed with the naked eye. In the blank group, the intestinal mucosa of rats was smooth, without congestion, ulcers, or other abnormal manifestations. Compared with the blank group, rats in the model group had thickened and hypertrophic intestines, congestion, edema, and erosion of intestinal mucosa, obvious ulcers on the surface, and severe adhesion or necrosis of some part of the intestines with surrounding organs. Compared with the model group, the intestinal wall of rats in the SSW-L group was thickened, and congestion and edema were still observed in the intestinal mucosa without a large ulcer surface, and there was occasional adhesion and necrosis between the intestines and surrounding organs. Rats in the SSW-M group, SSW-H group, and mesalazine group showed remarkable recovery of intestinal mucosal injury, and ulcers were rarely observed. A small number of rats still showed mild congestion and edema, but there was no adhesion with surrounding organs. As shown in Table 4, compared with the blank group, the CMDI score of rats in the model group was signifcantly increased. Compared with the model group, the CMDI score of rats in all treatment groups was signifcantly reduced (P < 0.01). No signifcant diferences were observed between rats in the mesalazine group and SSW-H group.
Te results of H&E staining showed that in the blank group, the colonic tissues were stained evenly, with each structure clearly visible, the intestinal mucosa was arranged neatly, and glands were distributed evenly. Compared with the blank group, the colon tissue in the model group was seriously damaged, and the intestinal mucosa disappeared. Te glandular structure was damaged, and many infammatory cells were observed. Compared with the model group, the degree of damage to colon tissue, the intestinal mucosa, and glandular structure in the SSW groups were reduced in a dose-dependent manner, and the number of infammatory cells was reduced in a dose-dependent manner. Te efects in the SSW-H and mesalazine groups were the best (Figure 1(b)). Compared with the blank group, the HS score of rats in the model group was signifcantly increased (P < 0.01). Compared with the model group, the HS scores of all treatment groups were signifcantly decreased (P < 0.01). No signifcant diferences were observed between rats in the mesalazine group and SSW-H group ( Table 5).

SSW Regulates the Level of Cytokines in the Colon of UC
Rats. By detecting the levels of related infammatory factors in colon tissue of rats with UC, compared with the blank group, the levels of IL-6 and IL-17 were signifcantly increased, and the levels of IL-10 and TGF-β1 were signifcantly decreased (P < 0.01) in the model group. Moreover, compared with the model group, the levels of IL-6 and IL-17 were signifcantly decreased (P < 0.01), and the levels of IL-10 and TGF-β1 were increased in the SSW groups in a dose-dependent manner. No signifcant diferences were observed between rats in the mesalazine group and SSW-H group (Table 6, Figure 2).

SSW Restores the Balance of T17/Treg in UC Rats.
To investigate the efect of SSW on the Treg/T17 immune axis in rats with UC, we determined the proportion of Treg and T17 cells in colon tissue by fow cytometry. Compared with the blank group, the proportion of T17 cells in colon tissue of rats in the model group was signifcantly increased (P < 0.01), and the proportion of Treg cells was signifcantly decreased (P < 0.01). In addition, compared with the model group, the proportion of Treg cells in the colon tissue of rats in each treatment group was signifcantly increased (P < 0.01, P < 0.05), and the proportion of T17 cells was signifcantly decreased (P < 0.01) in a dose-dependent manner. No signifcant diferences were observed between rats in the SSW-H group and mesalazine group (Figure 3)

SSW Regulates Protein Expression in Colon Tissue of UC
Rats. Compared with the blank group, the expression of TGF-β1 and PPARc was signifcantly decreased, and STAT3 and p-STAT3 were signifcantly increased (P < 0.01) in the model group. Furthermore, compared with the model group, the expression of TGF-β1 and PPARc in each treatment group was signifcantly increased (P < 0.05, P < 0.01), the expression of STAT3 was signifcantly decreased (P < 0.01), and the expression of p-STAT3 was signifcantly decreased (P < 0.01) in the SSW-M,

SSW Contributes to Restore the Gut Homeostasis of UC
Rats. Compared with the blank group, the abundance of OTU was decreased in the model group in UC rats. After drug treatment, the abundance of OTU in each treatment  1.25 ± 0.46 * * ## * * P < 0.01 compared with the blank group; ## P < 0.01 compared with the model group; ◆◆ P < 0.01, ◆ P < 0.05 compared with the mesalazine group (mean ± SD, n � 8). 1.13 ± 0.35 * * ## * * P < 0.01 compared with the blank group; ## P < 0.01 compared with the model group; ◆◆ P < 0.01 compared with the mesalazine group (mean ± SD, n � 8).
group was increased ( Figure 5(a)). Phylum level analysis showed that compared with the blank group, the relative abundance of Firmicutes in the model group was decreased, and the relative abundance of Bacteroidota was increased, which were statistically signifcant (P < 0.05, P < 0.01). Compared with the model group, the relative abundance of frmicutes in all treatment groups was increased, and that of the SSW-H and mesalazine group was signifcantly increased (P < 0.01) and higher than that of the blank group. Te relative abundance of Bacteroidota was decreased, and that of SSW-M, SSW-H, and mesalazine groups was decreased signifcantly (P < 0.01). Te relative abundance was lower than that of the blank group (Table 7, Figure 5(b)). Genuslevel analysis showed that, compared with the blank group, the relative abundance of Lactobacillus in the model group was signifcantly decreased (P < 0.01). Moreover, compared with the model group, the relative abundance of Lactobacillus in all treatment groups was signifcantly increased (P < 0.05, P < 0.01) (Table 8, Figure 5(c)). Taken together, these data showed that SSW increased the abundance of

SSW Promotes Butyric Acid Production by Increasing the Abundance of Lactobacillus in Firmicutes.
Compared with the blank group, the butyric acid concentration was signifcantly decreased (P < 0.01) in the model group. Moreover, compared with the model group, the butyric acid concentration increased in each treatment group, and signifcantly increased in the SSW-H and mesalazine group (P < 0.05) ( Table 9, Figure 6(a)). Te results of correlation analysis showed that, of the phylum level, the higher the relative abundance of Firmicutes, the lower the relative abundance of Bacteroidota, and the higher the butyric acid concentration. At the genus level, the higher the relative abundance of Lactobacillus, the higher the butyric acid concentration (Figures 6(b) and 6(c)). Tese fndings prove that increased frmicutes and Lactobacillus abundance promote the production of butyric acid.

Discussion
UC is a chronic type of IBD of unknown etiology in which the immune response plays an important role [1,27]. According to the long course of the disease and the tendency for repeated attacks, UC is classifed in TCM into the categories of dysentery and diarrhea in TCM. Te basic pathogenesis of UC is spleen defciency, and UC will develop from the spleen to the kidney over time, resulting in spleenkidney yang defciency. SSW is the main prescription for the treatment of UC of the spleen-kidney yang defciency, and is composed of Psoraleae Fructus, Evodiae Fructus, Myristicae Semen, Schisandrae Chinensis Fructus, Zingiber Ofcinale Roscoe, and Jujubae Fructus. Psoraleae Fructus, bitter and warm, is a sovereign drug, warming interior for dispersing cold. Evodiae Fructus is a minister drug, warmly invigorating spleen and stomach and relieving diarrhea with astringents. Myristicae Semen is also a minister drug, relieving diarrhea with astringents. Schisandrae Chinensis Fructus with astringent is an assistant drug. Zingiber Ofcinale Roscoe warming kidney for dispelling cold is envoy drug. Jujubae Fructus, which nourishes the spleen and stomach is also an envoy drug. All herbs are matched to have the efects of warming kidney and spleen and relieving diarrhea with astringents.
Te active components of the six herbs are complex, and modern pharmacological action proves that the constituent drugs of SSW have an anti-infammatory efect. Te monoterpenoid compound isolated from Psoraleae Fructus can inhibit the expression of iNOS mRNA by inactivating nuclear factor κb (NF-κB) to exert an anti-infammatory efect [28]. Te water extract of Myristicae Semen can inhibit the expression of proinfammatory factors, such as IL-1β and IL-6 in colonic mucosa and has antidiarrheal and anti-infammatory efects [29,30]. Schisandrin B can up-regulate the expression of PPARc, and inhibit the activation of NF-κB, thereby down-regulating the production of relevant infammatory factors, such as IL-6 [31,32]. Evodiae Fructus can signifcantly improve infammatory responses in the body by regulating NLRP3 and the NF-κB infammasomes [33,34]. Te active ingredient 6-Gingerol in Zingiber Ofcinale Roscoe can prevent chronic UC by down-regulatingNF-kB and inhibiting proinfammatory cytokines and can also improve acute colitis by activating adenosine monophosphate-activated protein kinase [35][36][37]. Furthermore, the Jujubae Fructus polysaccharide can signifcantly inhibit the expression of proinfammatory cytokines, such as IL-6, IL-2, and plays an anti-infammatory role [38,39]. At present, there is no efective radical treatment for UC, and most drugs that are conventionally used for the treatment of UC will     further deteriorate the intestinal microecology and promote the progression of the disease. Te pharmacological mechanism of SSW in the treatment of intestinal diseases has been confrmed to some extent but only focuses on some indicators of infammatory factors. It is not clear whether SSW can play an immune role by regulating the gut microbiota in the treatment of UC. In this study, it was confrmed that SSW can increase the content of butyric acid in the gut microbiota of UC rats, correct the imbalance of the Treg/T17 immune axis, and play a role in alleviating infammation and tissue damage. Te intestinal immune system protects the intestinal mucosal surface from infection and injury, and the T17/ Treg imbalance is an important characteristic of UC [40,41]. We proposed that SSW could treat UC by regulating intestinal microbiota and restoring regulatory Treg/T17 balance. TGF-β is a key regulator to maintain infammation and immune responses [42,43], promote mucosal healing, and protect host tissues from UC intracavitary lesions [27,44]. TGF-β can induce CD4 + T cells to diferentiate into Treg cells, and the presence of IL-6 can diferentiate CD4 + T cells into T17 cells [27]. IL-10 can inhibit the production of infammatory factors, such as IL-6 and thus inhibiting the production of T17 cells by reducing the antigen-presenting ability of monocytes [27]. T17 cells secrete infammatory factors IL-17 and IL-23 through the specifc transcription regulator RORct, thereby aggravating infammatory responses, and Treg cells can be converted into T17 cells in the presence of IL-6 and IL-23, resulting in an imbalance of the Treg/T17 immune axis [45][46][47][48]. Treg cells play an immune regulatory role by secreting infammatory inhibitors, such as IL-10 and TGF-β through the transcription regulator Foxp3 [45][46][47][48]. Terefore, we determined the proportion of T17 cells and Treg cells and the related factors. Tese results showed that after treatment, the proportion of T17 cells was signifcantly decreased, and the proportion of Treg cells was signifcantly increased, the serum levels of the infammatory cytokines IL-6 and IL-17 were signifcantly reduced, the levels of anti-infammatory cytokines IL-10 and TGF-β1 were signifcantly increased, especially in the SSW-H and mesalazine groups.
STAT3, which plays a key role in maintaining the intestinal mucosal barrier, is an important transcription factor that causes the onset of colitis. STAT3 is activated and phosphorylated by IL-6 and thus participates in the infammatory response [49,50]. STAT3, as one of the important factors afecting UC, promotes the infammation and pathogenesis of UC, and its overexpression inhibits the production of anti-infammatory factors [51]. p-STAT3 inhibits the diferentiation of CD4 + T cells into Treg cells and promotes their diferentiation into T17 cells, leading to an imbalance of the Treg/T17 immune axis [6,7,52,53]. In previous studies, it has been shown that PPARc is highly expressed in the colon, and activation of PPARc has a protective efect on colitis, while its expression is reduced in patients with UC [54][55][56][57][58]. Activation of PPARc reduces local intestinal IL-6 production and inhibits the elimination of coreceptor SMRT by RORct promoter, thereby inhibiting the RORct-induced diferentiation of T17 cells [55,[58][59][60]. Terefore, we determined the related proteins that afect the imbalance of the Treg/T17 immune axis. Tese results showed that after treatment, the expression of TGF-β1 and PPARc in colon tissue of rats in all treatment groups was signifcantly increased, and the expression of STAT3 and p-STAT3 was signifcantly decreased, especially in rats in the SSW-H and mesalazine groups.
SSW can correct the imbalance of Treg/T17 in UC rats, but whether it improves the imbalance by regulating the intestinal microbiota is still unclear. We consulted the relevant literature and found that the occurrence of UC is accompanied by intestinal microbiota dysbiosis, which changes the diversity and abundance of gut microbiota, thereby increasing Bacteroidota and decreasing Firmicutes [61][62][63]. In previous studies, it has been shown that the gut microbiota of IBD patients can afect the balance of T17 and RORct+ Treg cells in the intestinal tract of mice [64]. Te expression of PPARc in intestinal epithelial cells  0.29 ± 0.05 ## * * P < 0.01, * P < 0.05 compared with the blank group; ## P < 0.01, # P < 0.05 compared with the model group; ◆◆ P < 0.01 compared with the mesalazine group (mean ± SD, n � 6). is closely related to the gut microbiota and can activate the expression of PPARc in the intestinal tract and maintain the intestinal mucosal homeostasis [58]. SCFAs are a type of anti-infammatory bacterial metabolites that regulate the diferentiation direction of CD4 + T cells, restore the immune axis balance, and inhibit infammatory responses [65]. As one of the main components of SCFAs, butyrate is a key energy source for colonic and intestinal cells, and can reduce the intestinal permeability, enhance the intestinal mucosal barrier, down-regulate the expression of proinfammatory cytokines IL-6, and activate PPARc, thereby reducing the severity of UC [66][67][68]. Human colonic butyric acid-producing bacteria belong to Grampositive frmicutes [69][70][71][72]. Terefore, we determined the content of butyric acid and the correlation analysis between butyric acid production and various phylum and genus. Te results showed that the concentration of butyric acid increased in all treatment groups, especially in the SSW-H and mesalazine group. Te results of the correlation analysis between gut microbiota and SCFAs showed that at the phylum level, butyric acid had a positive correlation with Firmicutes and a negative correlation with Bacteroidota. Furthermore, at the genus level, there was a positive correlation between butyric acid and Lactobacillus. Te relative abundance of intestinal microbiota in rat feces was determined. Te results obtained in this study showed that the relative abundance of Firmicutes was increased and that of Bacteroidota was decreased in all treatment groups when analyzed at the phylum level. At the genus level, the relative abundance of Lactobacillus increased. In summary, SSW may regulate the relative abundance of Firmicutes and the butyric acid concentration in the gut microbiota and regulate the balance of the Treg/T17 immune axis, thereby restoring intestinal homeostasis to treat UC.

Conclusion
In this study, it was found that SSW could correct the imbalance of the Treg/T17 immune axis in colon tissue of a DNBS-induced UC rat model and played a role in reducing infammatory responses and tissue damage. SSW can increase the concentration of butyric acid in the gut microbiota, increase the expression of PPARc, inhibit the production of infammatory cytokines, and improve damaged intestinal barrier function under the infammatory state, thereby correcting the imbalance of the Treg/T17 immune axis and reducing infammatory responses and tissue damage. Terefore, our fndings showed that SSW can regulate the gut microbiota, restore immune function, and provide a potentially efective treatment for UC.

Data Availability
All the data related to this article are described as pictures and statistical analysis in the manuscript.

Ethical Approval
Te protocol of the experiment was approved by the Ethical Committee for the Experimental Animals at Gansu University of Chinese Medicine (Gansu, China) (Approval NO. 2020-260).

Conflicts of Interest
Te authors declare that there are no conficts of interest.

Authors' Contributions
Yingyun Wang was responsible for the content of this manuscript, including the data analysis and was in charge of   the main part of the experiment. Xinze Li carried out experimental assistance, worked in data analysis, and made charts. Yan Wang and Xiangdong Zhu designed the study, analyzed the result, and revised the manuscript. Yonglin Liang revised the manuscript. Jie Li carried out experimental assistance. All authors reviewed the manuscript.