Explore the underlying oral efficacy of α-, β-, γ-Cyclodextrin against the ulcerative colitis using in vitro and in vivo studies assisted by network pharmacology

Abstract The incidence of ulcerative colitis (UC) is rising worldwide. As a refractory and recurrent disease, UC could seriously affect the patients’ quality of life. However, current clinical medical treatments for UC are accompanied by various side effects, especially for long-term applications. Here, the underlying efficacy of cyclodextrins (CDs) was studied. As common excipients, CDs endow proven safety for long-term applications. Results of predictive methods derived from network pharmacology prompted the potential anti-inflammatory effects of CDs by oral administration. RAW264.7 cell experiments verified that CDs could inhibit the excessive secretion of TNF-α (β-CD > α-CD ≈ γ-CD), IL-6, and NO (α-CD > β-CD ≈ γ-CD) as predicted. In mice with DSS-induced acute UC, oral administration of CDs could effectively mitigate the pathological damage of colon tissue and reduce the level of inflammatory mediators. Moreover, 16S rRNA sequencing displayed that gut microbes disturbed by DSS were significantly regulated by CDs. Conclusively, the study showed the therapeutic application prospects of CDs in UC treatment and indicated the feasibility and advantages of developing ‘new’ therapeutic activities of ‘old’ ingredients. Communicated by Ramaswamy H. Sarma


Introduction
The incidence of ulcerative colitis (UC), one type of inflammatory bowel disease (IBD) (Kobayashi et al., 2020), is rising worldwide (Ungaro et al., 2017).UC affects the colon's normal function, commonly accompanied by frequent clinical symptoms, such as bloody stool, diarrhea, abdominal pain, and long-term recurrent intestinal inflammation (Neurath & Leppkes, 2019), which seriously affected patients' quality of life.The pathogenesis of UC is complex and related to genetic factors, immune response disorders, and environmental factors (Jostins et al., 2012;UK IBD Genetics Consortium et al., 2009;Ungaro et al., 2017).Unfortunately, current drug therapy and surgery treatment methods are accompanied by various side effects (Grucela & Steinhagen, 2009;Ham & Moss, 2012;Peyrin-Biroulet et al., 2016;Timmer et al., 2016).UC patients with oral administration of the sulfasalazine compound might suffer from anemia, abnormal liver function test, nausea, headache, fever, and rash (Tripathi & Feuerstein, 2019).Interstitial nephritis may occur after taking 5-aminosalicylic acid (5-ASA) (Ham & Moss, 2012).Long-term use of corticosteroids can lead to obesity, hypertension, diabetes, cataract, glaucoma, depression, anxiety, insomnia, and other complications.Azathioprine and 6-mercaptopurine also have a variety of side effects, among which leucopenia and acute pancreatitis are the most common ones, and some dosedependent side effects are related to the activity of thiopurine methyltransferase (TPMT) (Timmer et al., 2016).The most common side effect of antibodies is infection.Though most infections are mild, such as cold, otitis media, and sinusitis, there is a risk of further aggravation (Tripathi & Feuerstein, 2019).
Recently, natural oligosaccharides and polysaccharides have exhibited attractive effectiveness in the treatment of IBD based on their significant anti-inflammatory, immunomodulatory, and microbial regulatory capacities with obvious advantages in low toxicity (Cui et al., 2021;K-da et al., 2020;Liang et al., 2018;Yang et al., 2022).Unfortunately, some factors limited the applications of these saccharides in clinical therapies.First, for lots of natural compounds, the poor solubility and stability always demand the design of appropriate preparations tailored to specific molecules (Wang et al., 2023;Yan et al., 2022).Second, most of the natural saccharides are extracted from functional food ingredients or natural medicinal materials, which requires onerous work in separation, purification, and quality control, especially for heteropolysaccharides [e.g.turmeric polysaccharides (Yang et al., 2021), pectin from the common cranberry Vaccinium oxycoccos L. (Popov et al., 2006), and polysaccharide isolated from Phellinus baumii (Sun et al., 2021)].Furthermore, some polysaccharides with excellent efficacy are from precious natural herbs, which results in even higher application expenses (Zheng et al., 2020;Zhou et al., 2021) that might significantly increase the economic burden on UC patients.Unlike most natural oligosaccharides and polysaccharides, cyclodextrins (CDs) are both stable and cheap ensured by a mature and reliable mass production and quality control system.
CDs, originally composed of D-(þ)-glucopyranose units connected by a-(1,4)-glucoside bonds, are produced from the enzymatic degradation of starch.The most common cyclodextrins in nature are a-, b-, and c-cyclodextrin (a-, b-, and c-CD) which are composed of 6, 7, and 8 glucose residues, separately (Duchêne & Bochot, 2016).Because of their specific cyclic structures, CDs are successfully used as pharmaceutical excipients and food additives for purposes including improving stability and solubility (Funasaki et al., 2008).Notably, as one type of natural oligosaccharides, CDs might not be so 'inert' as most other excipients we usually used.The therapeutic effects of cyclodextrin in diseases including antiviral, antiparasitic, cardiovascular diseases, Niemann-Pick disease (NPD), and focal segmental glomerulosclerosis (FSGS) have been reported (Braga, 2019).Moreover, the three CDs mentioned above are considered prebiotics (Fenyvesi et al., 2016).Based on the above characteristics, it was hypothesized that CDs would display therapeutic effects upon UC.What's more, the oral safety of CDs verified by years of applications as additives also ensure their suitability for high-dose and long-term usage in UC disease control and recurrence prevention.
However, the exploitability of the pharmacological activities of 'old' ingredients excipients is a process similar to screening new drug molecules, which means that adequate investigation and well-founded prediction are necessary for avoiding the waste of time and scientific resources.Network pharmacology (Hopkins, 2007), based on the general concepts of systems biology, was used to systematically evaluate the pharmacological effects of multi-ingredient compounds on complex diseases with multi-targets (Ge et al., 2021).Here, before the experiments, methods derived from network pharmacology were applied to predict the potential pharmacological activities of oral CDs in UC treatment and better discuss the underlying mechanism.Because CDs are wellstudied excipients, both prototypes and their metabolites were considered in the predictive process in this manuscript.As a result, it promised an appropriate application of predictive analysis to the oral administration of ingredients with sufficient metabolic studies.
In this study, the effects of a-, b-and c-CD on UC treatment were explored.The results of predictive analysis derived from network pharmacology were consistent with the experimental results when both CDs prototypes and their metabolites were considered.Experiments were carried out to evaluate the anti-inflammatory effect of a-CD, b-CD, and c-CD in macrophages (RAW264.7).The effectiveness of CDs in alleviating the illness and modulating the composition of intestinal microbiota in mice with DSS-induced acute UC disease was also explored.

Identification of the common targets between cyclodextrin and UC
The 3D structure of a-CD (1BTC The mol2 structure files were uploaded to the PharmMappper database (http://lilab-ecust.cn/pharmmapper/ index.html) to predict the structure-related targets of a-CD, b-CD, c-CD and the targets of metabolites (maximum generated conformations: 300, limited to human protein targets; Normalized Fit Score (Norm Fit)>5).UniProt (The UniProt Consortium, 2021) was used to translate the target ID into the Gene name.
The predictive targets of a-CD, b-CD, and c-CD and their possible metabolites were compared with the UC-related targets obtained according to Method S1 to identify the possible targets of using the three types of CDs to treat UC.

Protein-protein interaction analysis and network construction
The protein-protein interaction (PPI) data were obtained from STRING database version 11.5 (https://string-db.org/)by uploading the candidate targets while the species was limited to 'Homo sapiens' and the confidence score > 0.400.GO and KEGG analysis was implemented according to Method S2.The Cytoscape_v3.9.1 software (https://cytoscape.org/)was utilized for analysis and network topology construction.
The toxicity of a-CD, b-CD, and c-CD to RAW264.7 cells was measured by a classic MTT (methyl thiazolyl tetrazolium) assay (Method S3) to determine the appropriate range of concentrations for further study.To evaluate the in vitro anti-inflammatory effects of cyclodextrins, the RAW264.7 cells during the logarithmic growth phase were plated in 48-well plates (4 � 10 4 cells/well) and cultured for 24 h.Then, the cells were treated with LPS (1 mg/mL) in the presence or absence of four different concentrations of three cyclodextrins (25, 50, 100, and 200 mg/mL).After 12 h, the concentrations of NO, IL-6, and TNF-a in each well's supernatant were determined using a NO kit or ELISA kit.Dexamethasone (DEX) was chosen as a positive control.Dosages and administration were designed as shown in Figure 3(A) based on previously reported (Irie & Uekama, 1997) and pre-experiment.Briefly, the mice were randomly assigned to the control group (control), dextran sulfate sodium group (DSS), and the treatment groups including the 5-ASA positive control treatment group (5-ASA, suspension in water, 200 mg/kg), a-CD treatment groups (L-a: 200 mg/kg and H-a: 400 mg/kg), b-CD treatment groups (L-b: 200 mg/kg and H-b: 400 mg/kg), c-CD treatment groups (L-c: 200 mg/kg and H-c: 400 mg/kg) (n ¼ 5).

In vivo assays in acute ulcerative colitis induced by DSS in mice
The mice of the control group were given clean water freely from the 1st to the 7th day, while the ones in the other groups were given 2.5% (w/v) DSS water solution freely to induce the formation of the UC model.Simultaneously, from Day 1st to 7th, each treatment group was treated with one of the three cyclodextrins or 5-ASA aqueous solutions by gavage, while the control group and DSS group were given an equal volume of normal saline (0.2 mL/20 g).
During the experiment, the body weights, feces, and disease activity index (DAI, score criteria were shown in Table S1) of the mice were monitored.At the end of the experiment, the animals were dissected by cervical dislocation under the premise of ethics.All animals' serum, spleen, intestinal contents, and colon tissue were collected immediately and stored at À 80 � C. Parts of colon tissues (about 1 cm) were fixed in 4% paraformaldehyde for H&E pathological analysis and images of each group were photographed under a microscope (LEICA-DM500, Heidelberg, Germany), and the histological score was evaluated by an experienced pathologist according to the scoring criteria (Table S2).The colon length from the anus to the appendix and spleen index calculated by spleen weight (mg)/body weight (g) were recorded.The content levels of cytokines (IL-1b, IL-6, and TNF-a) in the serum of mice and the levels of sIgA and myeloperoxidase (MPO) in colon tissue were tested by kits according to the manufacturer's protocols.

Fecal microbiological analysis
The cecum is the main site of fermentation in mice and has been identified as a primary site of colonic fermentation and a reservoir of anaerobic bacteria (Brown et al., 2018).As a result, the large number of bacteria in the cecum facilitates isolation and detection.Accordingly, cecum samples were widely used in the gut microbiota analysis (Taylor et al., 2022;Zhang et al., 2016).The cecum samples were sequenced for the 16S rRNA gene at Majorbio Biotechnology Co., Ltd.(Shanghai, China).Microbial community genomic DNA was extracted from the mouse cecum samples by using the E.Z.N.A. V R soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer's instructions.Amplification of the bacterial 16S rRNA V3-V4 regions was completed by using the 338F (5 0 -ACTCCTACGGG AGGCAGCAG-3 0 ) and 806 R (5 0 -GGACTACHVGGGTWTCTAAT-3 0 ) primer pairs with an ABI GeneAmp V R 9700 PCR thermocycler (ABI, CA, USA).Sequencing libraries were constructed using NEXTflex TM Rapid DNA-Seq Kit (Bioo Scientific, USA), and sequencing was performed on an Illumina MiSeq PE300 platform (Illumina, San Diego, CA, USA).The fastp (version 0.20.0)software was used for quality control and FLASH (version 1.2.7) for splicing (Method S4).To remove variances in sequencing depths among samples, sequence flattening was first performed before downstream analysis.Operational taxonomic units (OTUs) with a 97% similarity cutoff were generated using Uparse software to conduct the QIIME with the USEARCH11-uparse clustering algorithm, and chimeric sequences were identified and removed.The taxonomy of each 16S rRNA gene sequence was analyzed by the RDP Classifier algorithm against the Silva (138) 16S rRNA database using a confidence threshold of 70%.Subsequent analyses were accomplished on the cloud platform of Majorbio Bio-Pharm Technology Co., Ltd.(Shanghai, China).The alpha and beta diversity analyses were conducted on the operational taxonomic units (OTU) level.The distance algorithm of unweighted_unifrac was used in this study to execute principal coordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS) to determine the beta diversity.

Statistical analysis
Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Tukey's or Dunnett T3 test with SPSS 22 software (IBM, Chicago, IL, USA), and a value of p < 0.05, p < 0.01, p < 0.001 was considered statistically significant.

Predicted targets of a-CD, b-CD, and c-CD were related to the inflammatory response
The crude targets generated by the PharmMapper database were translated into official gene names and the duplicates were deleted.The differences in the structure of a-CD, b-CD, and c-CD lead to different properties (such as cavity size, solubility, and intermolecular hydrogen bonds).Therefore, the numbers of predicted targets (Figure S1(A)) which were in common with UC-related targets (Method S1) differed (c-CD > b-CD > a-CD, Figures S1(C-E)).Moreover, the different z-scores (e.g.hepatocyte growth factor, as shown in Tables S3-S5) of the same targets indicated different levels of significance.Since CDs might be digested by salivary and pancreatic amylases and fermented and utilized by bacteria in the colon (W€ upper et al., 2021), the prototypes of CDs and their respective metabolites were also taken into consideration.Specifically, c-CD would be degraded before reaching the colon, so only its possible metabolites were considered) (Figure S1(B)).In this case, the three cyclodextrins had similar target genes with only slight differences.As a result, it was speculated that they might have similar anti-inflammatory abilities when orally administrated (Figure 1).From the PPI network, the difference between the three kinds of CDs became more significant.As c-CD had the most targets, it might play a more effective therapeutic role than b-CD and a-CD by interacting with more key proteins.To dig out the core regulatory genes in the network, the plug-in Figure 1.The PPI network constructed for the candidate targets for UC treatment of (A) a-CD and its possible metabolites, (B) b-CD and its possible metabolites, and (C) possible metabolites of c-CD using cytoscape_v3.9.1 software (https://cytoscape.org/).the larger the value of the betweenness centrality the darker and bigger the nodes were; and the higher the combined score, which represented the stronger interaction relationship between the two linked proteins, the thicker the edge lines were.
CytoNCA of Cytoscape_v3.9.1 software was used.Hub genes that were speculated to play a significant role in CDs treated UC were extracted through comprehensive consideration of betweenness centrality, degree centrality, and closeness centrality (Table S3).Both the total number of predictive targets and the number of hub genes increased in the sequence of a-CD, b-CD, and c-CD, presumably because of more active groups and other complex structural factors.However, when the metabolites were included, the gaps in the PPI network among CDs were narrowed (Figure 1).Therefore, in this case, it was predicted that there would be no significant differences in the results of in vivo studies in DSS-induced acute ulcerative colitis mice model, which was considered more accord with the actual situation in the oral administration of a-CD, b-CD, and c-CD.Notably, among the analysis of the three kinds of cyclodextrins alone or with their possible metabolites, one of the hub genes was common, namely AKT1.AKT1 (AKT Serine/Threonine Kinase 1), one of the 3 AKT kinases, participates in the regulation of many processes including metabolism, proliferation, cell survival, growth, and angiogenesis (Hers et al., 2011).AKT1 could promote the development of inflammation (Di Lorenzo et al., 2009) and play a critical role in several pathways related to the inflammatory response (Chen et al., 2011;Xu et al., 2013).

Predicted effects of a-CD, b-CD, and c-CD on NO, TNF-a, and IL-6 secretion in LPS-treated macrophages
To further analyze the relative strength of the anti-inflammatory ability among a-CD, b-CD, and c-CD alone to predict the effects of CDs on LPS-treated macrophages in vitro, the inflammation-related secretions, NO, TNF-a, and IL-6 (the corresponding genes are NOS2, TNF, and IL6, separately), were chosen as indicators.NO is the main mediator of oxidative stress, which can aggravate the inflammatory response.TNFa and IL-6 are pro-inflammatory cytokines.As a result, NO, TNFa, and IL-6 levels are closely related to the pathogenesis of many inflammatory diseases.The PPI of the target genes of the a-CD, b-CD, or c-CD with NOS2, TNF, and IL6 was analyzed (Table S4).Then, bscore was introduced to union the affinities of CDs to the target proteins and the possibilities of the interaction between target proteins and inflammationrelated proteins.It was computed by multiplying 'zscore' and 'combined score'.Subsequently, a 'comprehensive parameter' was obtained by averaging the bscores of all inflammationrelated genes of each CD.According to the results, the strongest inhibition on NO secretion was predicted to be caused by a-CD with the highest 'comprehensive parameter', while b-CD and c-CD might show relatively weaker effects.
Similarly, both the strongest inhibiting effects on the secretions of TNF and IL-6 might be caused by b-CD.However, the contribution of the two parameters was considered together through simple mathematical methods, rationale still needed further analysis.

a-CD, b-CD, and c-CD attenuated LPS-induced inflammation of RAW264.7 cells
The RAW264.7 was a macrophage-like cell line originating from Abelson leukemia virus transformed cell line derived from BALB/c mice (Kong et al., 2019).In macrophages, LPS, one of the main components of the outer membrane of gram-negative bacteria, could promote inflammation by binding the Toll-like receptor 4 (TLR4) receptor and successively activating signal network with a critical axis formed by p38(MAPK)-pathway and signal transducer and activator of transcription (STAT)3-mediated signal-transduction (Bode et al., 2012).Research showed that RAW264.7 cells produced high levels of NO, TNF-a, IL-1b, and IL-6 after being stimulated with LPS (Facchin et al., 2022).As a result, the LPSinduced RAW264.7 cells were commonly used as inflammatory cell models (Han et al., 2019), and were also widely applied in the in vitro study of colitis (Ajayi et al., 2022;Cui et al., 2022;Du et al., 2020).
According to the results of three cyclodextrins' MTT assays on RAW264.7 cells (Figure 2(A)), four concentrations of 25, 50, 100, and 200 lg/mL were selected for the subsequent cell tests.To verify the anti-inflammatory effects of the three cyclodextrins, the concentration of NO, TNF-a, and IL-6 in LPS-induced RAW264.7 cells was determined.As shown in Figures 2(B-D), LPS treatment significantly increased the amount of NO, TNF-a, and IL-6 secretion compared with the control group (p < 0.05).Conversely, three cyclodextrins within the concentration range of 50 � 200 lg/mL significantly suppressed the overproduction of TNF-a and IL-6 in the LPS-induced RAW 264.7 cells.Consistent with PPI analysis, b-CD had the strongest inhibiting effects on the secretions of TNF-a reflected in significant effect at a low concentration of 25 lg/mL, while a-CD and c-CD showed no significant suppression.In addition, a-CD inhibited NO secretion at the concentration of 25 mg/mL, while b-CD and c-CD need to reach a concentration of 200 mg/mL to achieve the same effect, which was coordinated with the results of predictive analysis that indicated the strongest inhibition on NO secretion was caused by a-CD according to the 'comprehensive parameter'.These results indicated that a suitable dosage of a-CD, b-CD, and c-CD could attenuate the inflammation induced by LPS in RAW264.7 cells.

a-CD, b-CD, and c-CD ameliorated DSS-induced acute UC in mice
DAI is a classic clinical symptom indicator that reflects the severity degree of IBD.DSS treatment significantly increased the levels of DAI (from the third day) (Figures 3(B-D)) and reduced the body weight (from the fifth day) (Figures 3(F-H)) of mice compared with the control group.In contrast, oral administration of a-CD, b-CD, c-CD, and 5-ASA decreased the DAI levels and raised the weight of the mice compared with the DSS group to a certain extent.It is worth noting that the DAI index of the H-b group was significantly lower than that of the L-b group, but there was no difference between the L-a, H-a, L-c, and H-c groups.We speculated that it might be that the structure and in vivo degradation ability of b-CD made it exhibit stronger effects, thus displaying a significant dose dependence.However, further study was needed to explain possible reasons.The spleen index of the mice in the DSS group was significantly increased (p < 0.05) compared with the control group (Figure 3(E)) after DSS treatment as expected.Since the increase in the spleen index of DSS-treated mice was related to edema swelling caused by inflammation (Sun et al., 2019), it was indicated that the splenic edema was significantly relieved in mice of all treatment groups (except the L-b group).Histologically, DSS significantly shortened the colon length, destroyed the intestinal epithelial barrier, demolished the local crypt structure, and killed the goblet cells, as well as increased the infiltration of inflammatory cells and caused the interstitial edema in mice, while oral 5-ASA, a-CD, b-CD, and c-CD all alleviated the destruction of the intestinal structure, retained the crypts and the goblet cells, reduced the infiltration of inflammatory cells, and lengthened the colon (Figures 4(A,B)), which was consistent with the statistical analysis of histological scores (Figure 4(C)) and colon length (Figure 4(D)).Taken together, CDs ameliorated the DSSinduced acute ulcerative colitis in mice, while b-CD appeared to be better than a-CD and c-CD at 200 mg/kg.
The MPO was considered a landmark in inflammatory cells and it was significantly increased in inflammatory mice (Han et al., 2020).Besides, sIgA was considered to be an important immune indicator that reflects the immune ability of inflammatory mice, and it was used to illustrate whether the drug promotes the immune ability of experimental animals (IGAWang et al., 2019).The MPO activity in the DSS group was significantly higher than that in the control group (Figure 4

a-CD, b-CD, and c-CD predicted to affect biological processes
To gain a deeper understanding of the function of the candidate targets for using CDs to treat UC, GO enrichment analysis was implemented.The candidate targets related to biological processes, cellular components, and molecular functions with p-value < 0.05 were obtained.The top 20 significant terms of biological processes were displayed in Figures S2(A-C).It revealed that, for b-CD and c-CD, hub genes of candidate targets were strongly associated with more biological processes (including cytokine-mediated signaling pathway for b-CD; positive regulation of apoptotic process, response to xenobiotic stimulus and positive regulation of cell migration for c-CD).
When the metabolites were included, for all three types of CDs, the candidate targets of both CDs and their metabolites could act through similar biological processes including response to hypoxia, response to xenobiotic stimulus, and response to lipopolysaccharide which were also among the top 30 significantly biological processes affecting UC (Figure 5(D)).The differences between the a-CD, b-CD, and c-CD have been narrowed (Figures 5(A-C)).

a-CD, b-CD, and c-CD predicted to affect pathways related to UC
To further analyze the mechanisms at the pathway level, the candidate targets related pathways with p < 0.05 were obtained, and the top 20 significant terms of pathways were displayed in Figures 6(A-C).After neglecting some generalized and other disease terms, the major factor for CDs treating UC was the mitogen-activated protein kinase (MAPK) signaling pathway.MAPK plays an important role in the pathogenesis of UC by regulating the transcription of inflammatory cytokines, such as TNF-a, IL-1b, and IL-6, and leading to the occurrence of intestinal mucosal inflammation (Setia et al., 2014;Zhao et al., 2011).Besides, other pathways related to inflammatory response, like the toll-like receptor signaling pathway and TNF signaling pathway, might also contribute to the effects of CDs on UC as shown in Figure 6(E).
When considering the metabolism process, the three CDs displayed almost the same results of associated pathways (since the top 20 terms were the same and the P and count values of the terms were also similar, only the pathway analysis results of a-CD and its metabolites were displayed here, as shown in Figure 6(D)).The results not only shared the inflammatory response-related pathways with CDs, metabolites but also other pathways, for example, the C-type lectin receptor signaling pathway that plays an important role in regulating adaptive immune responses.Therefore, when considering metabolites, the therapeutic effects of the three CDs on colitis, especially the changes in inflammatory cytokines, were predicted to be very similar, which was verified in subsequent experiments.

Cytokines levels in serum were coordinated with the predictive analysis incorporated with possible metabolites
As shown in Figures 4(G-I), the levels of the IL-6, IL-1b, and TNF-a pro-inflammatory cytokines in the serum of DSS-induced UC mice were increased significantly compared with the control group (p < 0.01), while oral 5-ASA, a-CD, b-CD (except the L-b group), and c-CD could significantly inhibit the secretion of IL-6, IL-1b, and TNF-a in colitis mice serum.There were no significant differences between CDs, which was also coordinated with the results from predictive analysis and indicated that the possible metabolites were not negligible.

Alpha and beta diversity
The gut microbiota consisted of a complex community of a hundred trillion bacterial and archaeal cells comprising more than a thousand species, and its structure and composition could be significantly altered in patients with IBD (Alam et al., 2020); conversely, the broken balance of the gut microbiota might also lead to inflammation (Shen et al., 2018).To prove whether a-CD, b-CD, and c-CD regulate intestinal flora, we performed 16S rRNA sequencing on mouse stool samples.There were 3,024,158 sequences obtained from 45 samples.Each sample with more than 33,641 valid sequences was analyzed for subsequent microbial diversity and Beta diversity analysis is an indicator that measures the composition of microbial communities and is accustomed to assessing the differences between microbial communities.In this study, we mainly used principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS) methods calculated by the unweighted-unifrac distance algorithm to record the beta diversity of each group.The analysis of similarities (ANOSIM) of PCoA and NMDS showed that the p-value of the DSS group and the control group was <0.05.The results of PCoA (Figure 7(C)) and NMDS (Figure 7(D)) both revealed an evident difference in the microbial community structure between the DSS group and the control group.The PCoA and NMDS data of the three CD groups were close to the normal group, indicating that oral administration of the three cyclodextrins has the effect of resisting the imbalance of the flora caused by DSS.

Cyclodextrins regulated the intestinal flora of colitis mice
The Venn diagram was performed to recognize the common and unique microbiota of different groups.As shown in Figure 8(A), the common OTUs in all the treatment groups were 358.The number of total microbial OTUs was significantly lower in the DSS group compared to the control group, while it increased to varying degrees in all treatment groups.This result corresponds to the alpha diversity data.The intestinal flora in mice at the phylum level of abundance consisted of five main groups of bacteria.The abundances of Bacteroidota and Firmicutes were the largest, over 90%.As shown in Figure 8(B), the abundance of Firmicutes in mice of the DSS group was increased, and the abundance of Bacteroidota was decreased.Oral 5-ASA, CDs had a significant effect on the reversal of the abundance levels of the Firmicutes and Bacteroidota phyla.The relative abundance of bacteria at the genus level was analyzed, and the top 30 abundance genera belonging to the 5 phyla were listed in Figure 9(A).The relative abundance of genera in the colitis mice changed significantly compared with the normal mice.Specifically, as shown in Figure 9

Discussion
The pathogenesis of UC is complex and not yet fully understood.Diet, exercise, age, and other factors all play important roles in its occurrence and development.In this study, we speculated that the a-, b-, and c-CDs might have the effects of alleviating UC, which would be very meaningful because the long-term and high-dose safety of CDs makes it very suitable for treating such a recurrent disease like UC.The application of an appropriate prediction system could largely reduce the workload, save time and resource costs, as well as assist the prediction and analysis of the underlying mechanism.Therefore, inspired by network pharmacology, predictive analysis (Gu et al., 2013;Hopkins, 2007Hopkins, , 2008) ) was applied to predict the possibility of such a hypothesis before experimental verification.In this study, the predictive analysis was creatively applied to the analysis of drugs for oral administration.However, the network pharmacology is mainly suited to the cases that drugs are ensured to interact with the receptors without being metabolized.Though in treating colitis more attention was paid to the receptors on the surface and shallow layer of the intestinal mucosa, the destruction of the intestinal physiological barrier in colitis also made it possible for the active ingredients to contact with systemic receptors.Importantly, the complex changes of oral drugs in the gastrointestinal tract should be taken into consideration, especially when the components might degrade in the gastrointestinal tract, such as c-CD in this study (Kurkov & Loftsson, 2013).Moreover, in accordance with the conventional method, the possible effects of a-CD, b-CD, and c-CD on NO, TNF-a, and IL-6 secretion in LPStreated macrophages could be predicted, while the relative strength of the effects among CDs could not.Therefore, the PPI was flexibly applied to explore the interaction between the key proteins (NOS2, TNF, and IL6) and the proteins coded by the target genes of CDs.A 'comprehensive parameter' was introduced to analyze the results and the prediction was extended from qualitative to preliminary quantitative.
The combined effects of drug prototypes and their metabolites were included in the prediction in response to the special condition of oral administration.When predicting the effects of a-CD, b-CD, and c-CD on colitis, the analysis paying attention only to prototypes of CDs took c-CD as the most effective one, which was not corresponding to the in vivo study in DSS-induced acute ulcerative colitis mice.However, when the complex effects of metabolites were introduced, the results of the cell assays could be predicted and well-explained.Furthermore, the application of network pharmacology was more suitable especially when the possible metabolites were comprehensively considered, because network pharmacology is applicable to the comprehensive analysis of multi-component effects.Moreover, for commonly used excipients, there are often sound metabolic studies, which makes our combination possible.This is very suitable for the new application of traditional excipients, while for a new active substance without fully being studied, the methods weren't so applicable.It should be noted that in in vivo analysis, the relative strength of the effect couldn't be judged according to the above methods, because the composition proportion of metabolites was hard to determine.
Notably, although the predictive analysis could reduce the workload and resource costs of assays and help predict the possible mechanisms, but could not substitute for experiments.As a result, further experimental verification was still needed.Furthermore, because the prediction was based on known data, the unknown targets and pathways and the comprehensiveness and accuracy of the databases would influence the analysis results.Therefore, it is necessary to be aware of this limitation during the application and establish a set of standards (Yang et al., 2022).
The onset of UC was related to many factors, but it was found recently that the imbalance of intestinal microbes might stimulate the development and severity of the disease (Walker et al., 2011).There were more than 100 trillion species of microorganisms in the intestinal tract of mammals.These microorganisms mainly included Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria at the phylum level.Previous studies have shown that in mice with DSS-induced acute ulcerative colitis, the abundance of Firmicutes increases while the abundance of Bacteroides decreases (Li et al., 2020), which was consistent with our result.Meanwhile, a-CD, b-CD, and c-CD could reverse the imbalance of Odoribacter.Odoribacter, as a harmful bacteria, has been found to significantly increase its species abundance in colitis mice (Wei et al., 2019).Therefore, Odoribacter could be used as a key species to verify the imbalance of intestinal flora in UC mice.
After oral administration, a-CD and b-CD are mainly fermented and utilized by bacteria in the colon (Kurkov & Loftsson, 2013).Although c-CD is readily digested by salivary and pancreatic amylases (W€ upper et al., 2021), maltotriose, the digestive product of c-CD, is also a prebiotic fiber that protects the diversity of gut microbes (Jang et al., 2020).Supplemental c-CD could boost the numbers of bifidobacteria and lactobacilli (Spears et al., 2005).Although having similar chemical structures, b-CD is the only water-insoluble one in three CDs.The solubility affects where it is fermented by which gut microbes (Holscher, 2017), which might explain why three CDs regulate cecal microbiota differently as well as the result in animal experiments that the 200 mg/kg dose of a-CD and c-CD has a better therapeutic effect than the same dose of b-CD.Researchers have suggested that many unstable drugs, such as curcumin and budesonide are encapsulated in cyclodextrin for the treatment of UC (L� azaro et al., 2020;Yadav et al., 2009), however, the synergistic therapeutic effect of CDs themselves should not be ignored.To validate the impact of gut microbiota on UC and utilize it to treat UC, FMT therapy has been developed (Guo et al., 2020;Haifer et al., 2022;He et al., 2022).In this study, although the microbiota was regulated, it cannot be concluded that changes in the gut microbiota must be one of the therapeutic mechanisms, but only be inferred.Further experiments were needed to verify it.
Moreover, predictive analysis in our study showed that CDs might be effective for the treatment of not only colitis but also other diseases (e.g.other inflammation-related diseases).It's necessary to focus on more excipients to explore their possible physiological roles in the preparations because their validated safety through long-term applications and their higher dosage in formulations make their possible physiological function more useful.

Conclusion
In this study, the effects of a-, b-, and c-Cyclodextrin against ulcerative colitis were explored.The experiments on LPSinduced cells and DSS-induced mouse colitis models revealed that three cyclodextrins could inhibit LPS-induced secretion of inflammatory mediators (NO, TNF-a, and IL-6) in RAW264.7 cells, ameliorate the inflammation of colitis mice, and regulate the intestinal microflora.Meanwhile, network pharmacology was firstly applied in the oral administration to predict the outcome of CDs for UC treatment and targets, and the results showed this predictive analysis possessed accuracy and could be used to assist the analysis with experiments combined with its specific physiological and metabolic process, especially in the development of new uses of old ingredients with mature ADME research results.In summary, cyclodextrins would be a promising strategy to treat ulcerative colitis with safety in long-term application as active ingredients or as excipients in formulations.Considering their anti-inflammatory properties and their wide applications in formulations, the therapeutic effects of CDs on other inflammation-related diseases were worth further exploring.Moreover, the results prompted us to pay more attention to the possible pharmacological effects of the excipients, which should not be ignored especially when considering the usual larger amounts of them applied in formulations.

Disclosure statement
No potential conflict of interest was reported by the author(s).
(E)), while the IgA content did not change obviously (Figure 4(F)), indicating a heavy neutrophil infiltration in the intestinal tissue of the model group and the failure of secreting enough IgA to cope with the occurrence of inflammation in UC mice.After treatment with CDs and 5-ASA, the MPO activity was significantly reduced and the sIgA secretion was increased (except in the L-b group).

Figure 2 .
Figure 2. Cell proliferation and anti-inflammatory effects of a-, b-, and c-CD (CDs).(A) the effect of CDs on the viability of Raw264.7 cells.(B) The level of NO. (C) The level of TNF-a.(D) The level of IL-6.DEX: dexamethasone.All data were presented as mean ± SD (n ¼ 5).# p < 0.05, � p < 0.01, and & p < 0.001 compared to the blank group.a p < 0.05, b p < 0.01, and c p < 0.001 compared to the LPS-induced group.

Figure 3 .
Figure 3.The a-CD, b-CD, and c-CD ameliorate DSS-induced acute ulcerative colitis in mice.(A) UC mouse treatment protocol.(B) The DAI of the a-CD group.(C) The DAI of the b-CD group.(D) The DAI of the c-CD group.(E) Spleen index.(F) Weight change of a-CD group.(G) Weight change of b-CD group.(H) Weight change of c-CD group.All data were presented as mean ± SD (n ¼ 5).� p < 0.05, and �� p < 0.01, compared with the DSS group; # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with the control group.

Figure 4 .
Figure 4.The changes of colon tissue inflammation and the effects of a-CD, b-CD, c-CD on cytokines in serum.(A) Typical histological sections stained with H&E (200 � magnification).(B) Colon pictures.(C) Colon length.(D) Statistics of the histological score.(E) MPO in colon tissue.(F) sIgA in colon tissue.(G) IL-6, (H) TNFa, and (I) IL-1b in serum.All data were presented as mean ± SD (n ¼ 5).� p < 0.05 and �� p < 0.01, compared with the DSS group; ## p < 0.01 and ### p < 0.001 compared with the control group.

Figure 5 .
Figure 5. Top significant terms of biological processes.Gene ratio equals the number of differentially expressed genes against the number of genes associated with a GO term in candidate targets.Bubble diagrams of (A) a-CD, (B) b-CD, (C) c-CD with their possible metabolites, and (D) UC.
(B), oral 5-ASA protected the abundance of norank_ f__Muribaculaceae, Parabacteroides, and Helicobacter, when c-CD maintained the abundance of Helicobacter and norank_ f__Muribaculaceae and a-CD the abundance of Parabacteroides.Additionally, all treatment groups inhibited the abnormal proliferation of Odoribacter in UC mice.The Ha group specifically increased the abundance of Lactobacillus (a probiotic).To explore the relationship between intestinal inflammation and microorganisms, we correlated five clinical indicators with microorganisms at the genus level.To exclude clinical factors with strong multicollinearity, clinical

Figure 6 .
Figure 6.The top 20 significant terms of KEGG enrichment pathways of (A) a-CD, (B) b-CD, (C) c-CD, and (D) a-CD with its possible metabolites (neglecting generalized and other disease terms); (E) CDs regulated the secretion of inflammatory cytokines through the key signaling pathways.

Figure 7 .
Figure 7. Alpha and beta-diversity analysis of gut microbiota (n ¼ 5).(A) Ace index of all samples.(B) Chao index of all samples.(C) PCoA analysis.The first two principal components, PC1 and PC2, representing the two possibly principal factors affecting bacterial profiles, and their probability explain 18.5% of variance.(D) NMDS analysis.� p < 0.05, compared with the DSS group; & p < 0.001, compared with the control group.

Figure 8 .
Figure 8. Effects of a-CD, b-CD, and c-CD treatment on gut microbiota composition (n ¼ 5).(A) Venn diagram on OTU level.(B) Community column diagram at the phylum level.