International Journal of

Article

Citation: Feng, P.; Li, Q.; Liu, L.; Wang, S.; Wu, Z.; Tao, Y.; Huang, P.; Wang, P. Crocetin Prolongs Recovery Period of DSS-Induced Colitis via Altering Intestinal Microbiome and Increasing Intestinal Permeability. Int. J. Mol. Sci. 2022, 23, 3832. https:// doi.org/10.3390/ijms23073832

Academic Editors: Massimiliano Gasparrini and Luca Mazzoni

Received: 25 February 2022 Accepted: 28 March 2022 Published: 30 March 2022

Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

1. 1 College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou 310014, China; [email protected] (P.F.); [email protected] (Q.L.); [email protected] (Y.T.)
2. 2 College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China; [email protected]
3. 3 School of Medicine, Jiangsu University, Zhenjiang 212013, China; [email protected] (S.W.); [email protected] (Z.W.)

* Correspondence: [email protected] (P.H.); [email protected] (P.W.)

Abstract: Crocetin is one of the major active constituents of saffron (Crocus sativus L.) which has a reputation for facilitating blood circulation and dispersing blood stasis in traditional Chinese medicine. However, there is little evidence showing the relationship between crocetin intake and the risk of gastrointestinal diseases such as colitis. In order to investigate the effect of crocetin on the regulation of intestinal barrier function and intestinal microbiota composition, mice were treated with crocetin after 3% dextran sulfate sodium (DSS) administration for one week. We found that crocetin intake at 10 mg/kg aggravated colitis in mice, showing increased weight loss and more serious histological abnormalities compared with the DSS group. The 16s rDNA sequencing analysis of the feces samples showed that mice treated with 10 mg/kg crocetin had lower species diversity and richness than those treated with DSS. At the genus level, a higher abundance of Akkermansia and Mediterraneibacter, and a lower abundance of Muribaculaceae, Dubosiella, Paramuribaculum, Parasutterella, Allobaculum, Duncaniella, Candidatus Stoquefichus, and Coriobacteriaceae UCG-002 were observed in the crocetin group. Untargeted metabolomic analyses revealed that crocetin reduced the levels of primary and secondary bile acids such as 12-ketodeoxycholic acid, 7-ketodeoxycholic acid, 3-sulfodeoxycholic acid, 6-ethylchenodeoxycholic acid, chenodeoxycholate, glycochenodeoxycholate-7-sulfate, glycocholate, and sulfolithocholic acid in the colon. In conclusion, crocetin intake disturbed intestinal homeostasis and prolonged recovery of colitis by promoting inflammation and altering gut microbiota composition and its metabolic products in mice. Our findings suggest that patients with gastrointestinal diseases such as inflammatory bowel disease should use crocetin with caution.

Keywords: crocetin; gut microbiota; ulcerative colitis; intestinal metabolites; inflammatory bowel disease

Ulcerative colitis (UC), a major subtype of inflammatory bowel disease (IBD), is characterized by chronic and relapsing inflammation in the colonic and rectal mucosa, leading to abdominal pain, diarrhea, intestinal blood loss, and anemia [1]. UC is becoming a serious public health problem with an increasing global incidence and prevalence [2]. However, the exact pathophysiological mechanism underlying UC remains unknown; it may be caused by a combination of environmental factors, host genetics, immune disorders, intestinal microbiota, and intestinal barrier dysfunction. Recently, one hypothesis stated that the dysbiosis of intestinal microbiota and the hypersensitive immune response to non-pathogenic commensal bacteria are thought to be critical events in the pathogenesis of UC [3,4]. Thus, the switch in the gut microbiota composition may negatively affect intestinal barrier function and exacerbate colitis.

Int. J. Mol. Sci. 2022, 23, 3832. https://doi.org/10.3390/ijms23073832 https://www.mdpi.com/journal/ijms

The human gastrointestinal tract harbors trillions of microbes including bacteria, fungi, protozoa, viruses, and archaea [5]. As the largest and most complex ecosystems in the human body, the intestinal microbiota and its metabolic products play essential roles in host nutrient digestion and absorption, mucosal immunity, intestinal epithelial cell proliferation and differentiation, and resistance to the invasion of pathogenic microorganisms. Bile acids, short-chain fatty acids (SCFAs), and endotoxins link intestinal microbiota activity and composition to the host’s health [6]. Bile acids inhibit intestinal bacterial overgrowth, and intestinal microbiota converts host-derived primary bile acids to secondary bile acids and regulate enterohepatic bile acid recycling [7]. Butyrate and other SCFAs produced by bacterial fermentation of non-digestible dietary fibers in the colon have been demonstrated to play a crucial role in maintaining epithelial integrity, promoting intestinal epithelial cell proliferation, and inhibiting inflammation [8,9]. Recent studies have shown that the intestinal microbiota and its metabolic products (e.g., ATP) mediate the balance of Th17 and Treg cells and regulate inflammatory mediator secretion [10]. Dysbiosis of the intestinal microbiota can cause many diseases including IBD, obesity, metabolic syndrome, and even neurological conditions [5]. Therefore, in-depth studies on the relationship between intestinal microbiota and hosts are needed to explore the pathogenesis of UC.

Crocetin (C20H24O4, molecular weight 328.4 g/mol) is one of the major active constituents of saffron (Crocus sativus L.) [11], a traditional food and medicinal plant commonly used in Asia. Studies show that crocetin displays various pharmacological properties such as anti-inflammatory, antidepressant, antitumor, antioxidant, and hepatoprotective properties through its ability to repress proinflammatory mediators, enhance neuroprotection, induce apoptosis, improve O2 diffusivity, and increase ATP generation [12–14]. As a more in-depth understanding was gained, crocetin has been considered as a promising drug for the treatment of depression. On the other hand, patients with IBD were associated with a substantially increased risk of psychological problems such as depression and anxiety [15]. Moreover, according to traditional Chinese medicine (TCM), saffron can activate blood and resolve stasis. However, there is little evidence examining the association between crocetin intake and the risk of gastrointestinal diseases such as colitis. Thus, knowing whether crocetin intake may result in the aggravation of certain diseases is of great interest. Therefore, we aimed to investigate the effects of crocetin on colon pathology, intestinal microbiota composition, and microbial metabolites in mice with dextran sulfate sodium (DSS)-induced colitis. Our results suggest that intake of crocetin might alter intestinal homeostasis, aggravate colitis and prolong colitis recovery in mice.

1. 2.1. Crocetin Ingestion in DSS-Induced Mice Alters Weight, without Influencing Colon Length
2. 2.2. High-Dose Crocetin Increased Intestinal Permeability of Mice with DSS-Induced Colitis

To evaluate intestinal permeability, FD4 was used for fluorescence molecular imaging (FMI) to monitor the in vivo distribution (Figure 2A–D). Fluorescence was measured using a region of interest (ROI) drawn to encompass the fluorescent region of the abdomen, thus measuring FD4 delivery from the intestine to the blood. Quantification of in vivo FD4 did not show significant differences between the groups (Figure 2D). Notably, some individuals in the Crocetin-H group had the highest intestinal permeability, which likely indicates the impairment of the intestinal barrier.

1. 2.3. Effects of Crocetin on Serum Cytokine of Mice with DSS-Induced Colitis

1. Figure 1. Genome-wide identification and expression profiles of the HEX genes in B. mori. (A) Gene structure and (B) protein domains of nine GH20 HEX genes identified in B. mori. Glycohydro 20b2 and Glyco hydro 20 are abbreviations of glycoside hydrolase 20b2 and glycoside hydrolase 20 in SMART. (C) Phylogenetic analysis of HEXs of B. mori and other species. The phylogenetic tree was constructed using neighbor-joining method and bootstrap support values on 1000 replicates by MEGA7. The HEX proteins in B. mori are labeled with a red line. (D) Heatmap of the expression level of eight HEX genes in 12 tissues (from the outside to the inside: anterior silk gland, epidermis, fat body, head, hemolymph, Mal-pighian tubule, middle silk gland, midgut, ovary, posterior silk gland, testis, and trachea) on day three of the fifth instar and wandering stage. Red dots indicate epidermis. 5L3D: day three of fifth instar and W: wandering stage. (E) Heatmap of the expression level of eight HEX genes in seven developmental stages (from the outside to the inside: day three of fourth instar; molting phase in the fourth instar; the start of the fifth instar; day three of fifth instar; W, wandering stage; PP, pre-pupa stage; and P1, day one of pupa) of the epidermis, ovary, and testis in Bombyx mori. Blue indicates low expression and red indicates high expression.

1. Figure 2. Influence of crocetin in DSS-induced colitis mice on intestinal permeability. FMI of Control

1. (A), DSS (B), Crocetin-L (C), and Crocetin-H (D) mice gavaged with FD4. (E) Quantification of in vivo FD4. Data are presented as mean ± SD.

1. 2.4. Crocetin Exacerbated DSS-Induced Colon Damage and Inflammatory
2. 2.5. Effects of Crocetin on Gut Microbial Community Abundance of Mice with DSS-Induced Colitis

Given the increased body weight loss and severe intestinal inflammation, Crocetin-L group was chosen for further analysis. Alpha diversity was analyzed to investigate the microbial diversity and richness by observing operational taxonomic unit (OTU), Chao1, Shannon, and Simpson diversity indices. All indices indicated a lower alpha diversity in the Crocetin-L group than in the DSS group (Figure 5A–D), which suggests that treatment with crocetin could reduce the richness of the gut microbiota.

A principal coordinates analysis (PCoA) of the weighted UniFrac distance matrices was performed to assess the bacterial communities among these groups. The Crocetin-L group was separated from the DSS and control groups by the first principal of PCoA (Figure 5E). At the phylum level, Bacteroidetes was the most dominant phylum in the colon of mice, followed by Firmicutes and Verrucomicrobia, which accounted for 43.87%, 26.95%, and 21.68%, respectively (Figure 5F). However, as shown in Figure 5F, the different groups had different dominant phyla. Verrucomicrobia and Bacteroidetes were identified

1. as the dominant phyla in the Crocetin-L group, whereas Bacteroidetes and Firmicutes were identified as the dominant phyla in the control and DSS groups. From the UPGMA tree (Bray–Curtis), almost all mice in the Crocetin-L group clustered together and were distinctly

1. Figure 3. Effect of crocetin on serum concentration of IL-4 (A), IL-6 (B), IL-10 (C), and TNF (D). Data are presented as mean ± SD.

1. 2.6. Crocetin Treatment Altered the Representative Bacteria in Mice with DSS-Induced Colitis

We used Linear discriminant analysis effect size (LEfSe) analysis to determine the differential microbial composition among all the groups. The cladogram presenting the hierarchical taxonomic structure of the gut microbiota from phylum to genus indicated significant differences in phylogenetic distributions among all groups (Figure 6A). Using a logarithmic LDA score cut-off of 3.0, we found that the genera Akkermansia and Mediterraneibacter were enriched in the Crocetin-L group. Duncaniella, Muribaculaceae_unclassified, Paramuribaculum, Eisenbergiella, Allobaculum, and Parasutterella were significantly overrepresented in the DSS group. The heat map further demonstrated the differences in gut microbiota among the three groups (Figure 6B).

Compared with the DSS group, the relative abundance of the genera Akkermansia and Mediterraneibacter was significantly higher in the Crocetin-L group, whereas Muribaculaceae, Dubosiella, Paramuribaculum, Parasutterella, Allobaculum, Duncaniella, Candidatus Stoquefichus, and Coriobacteriaceae UCG-002 were reduced by crocetin treatment in mice with DSS-induced colitis (Figure 7A). Some of the results overlap with those of LEfSe analysis, suggesting that these microbes may be the target microbiota of crocetin.

To understand the functions of crocetin-associated intestinal microbiota, we used PICRUSt2 to predict the potential cluster of orthologous genes (COG) pathways based on 16S rRNA gene profiles. A total of 181 COGs showed significant differences in abundance between the DSS and Crocetin-L groups (p < 0.05). The relative abundance of “CubicO

1. Figure 4. Evaluation of the effect of crocetin in DSS-induced colitis mice by histological examination and immunohistochemistry. (A) H&E staining and immunohistochemistry of E-cadherin in colon of all groups. Arrow indicates crypt loss, asterisk indicates goblet cell loss, triangle indicates infiltration of inflammatory cells. (B) Histological score in DSS induced colitis. (C) Mean optical density (MOD) of E-cadherin. MOD = sum integrated optical density/area. (D) mRNA expression levels of Ecadherin are shown relative to Control. Data are presented as mean ± SD. * p < 0.05, compared to DSS group; # p < 0.05, compared to Control.

1. 2.7. Crocetin Treatment Altered the Metabolome in Mice with DSS-Induced Colitis

Using liquid chromatography-mass spectrometry (LC/MS), untargeted metabolite analysis was performed to investigate the alteration of the metabolome and examine the correlation between gut metabolites and microbiota. Crocetin significantly altered the gut metabolites (Figure 8A). The partial least squares discriminant analysis score plot demonstrated good separation between the DSS and Crocetin-L groups, suggesting that crocetin led to alterations in gut metabolites (Figure 8B). We analyzed annotated metabolites in feces. We found that 431 features in the positive mode (157 upregulated and 274 downregulated) and 778 features in the negative mode (282 upregulated and 496 downregulated) were different in the Crocetin-L group compared to the DSS group (Figure 8C). The functions of these differential metabolites were determined using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Figure 8D). According to the analysis, 44 KEGG pathways, which are involved in the metabolism of lipids, amino acids, and carbohydrates, were identified. These results demonstrate that crocetin altered the gut metabolites. In particular, bile acid metabolites including 12-ketodeoxycholic acid, 7-ketodeoxycholic acid, 3-sulfodeoxycholic acid, 6-ethylchenodeoxycholic acid, chenodeoxycholate, glycochenodeoxycholate-7-sulfate, glycocholate, and sulfolithocholic acid were decreased (Figure 9). Compared with the DSS group, the levels of arachidonic acid, prostaglandin E2 ethanolamide, and 17-phenyl-18,19,20-trinorprostaglandin E2 were significantly decreased in the Crocetin-L group, while leukotriene F4 and prostaglandin lactone-diol were significantly increased. Moreover, dicumarol was increased in the Crocetin-L group, which may interfere with coagulation [16].

1. Figure 5. Gut microbial community abundance at the phylum level. (A) Alpha diversity based on observing OTU level. (B) Alpha diversity based on Chao1 index. (C) Alpha diversity based on Simpson index. (D) Alpha diversity based on Shannon index. (E) The PCoA plots were constructed with the weighted UniFrac PCoA method. (F) The compositions and relationship of intestine microbiota in Control, DSS, and Crocetin-L group. * p < 0.05, ** p < 0.01.
2. Figure 6. Differences in composition of the gut microbiota among Control, DSS, and Crocetin-L group. (A) Differential abundance microbiota taxonomic cladogram obtained from LEfSe analysis.

1. (B) Heatmap plot depicting the normalized abundance of each microbiota genus in Control, DSS, and Crocetin-L group.

1. Figure 7. Comparison of gut microbiota between DSS and Crocetin-L group. (A) Differences in the relative abundances of intestine microbiota between DSS and Crocetin-L group at the genus level (Mann-Whitney U test). The box presented the 95% Cls, the line inside denotes the median. (B) Comparison of COG pathway abundance between DSS and Crocetin-L group using PICRUSt2.

1. Figure 8. Gut metabolomic profiling. (A) Heatmap showing the significantly changed metabolites of individuals in Crocetin-L group compared with DSS group. (B) PLS-DA scores plot revealed a clear separation of gut metabolites between the Crocetin-L and DSS groups. (C) Volcano plots of differential metabolites between Crocetin-L and DSS groups, red dots represent significantly upregulated metabolites, blue dots represent significantly down-regulated metabolites, and grey dots represent metabolites with no significant change. (D) KEGG enriched pathways of differential gut metabolites in Crocetin-L group compared with DSS group. (E) Correlation of gut metabolites and microbiota between the Crocetin-L and DSS group. (F) Correlation network of differential metabolites and microbiota between the Crocetin-L and DSS group.

1. Figure 9. Results of absolute peak area analysis. (A) Primary and secondly bile acid. (B) Arachidonic acid and its metabolites. * p < 0.05, compared to DSS group; # p < 0.05 compared to Control.

The association of gut microbiota at the genus level and gut metabolites were analyzed using Spearman correlation analysis. We found that dicoumarol was negatively correlated with the abundance of Parasutterella, Duncaniella, and Paramuribaculum and positively correlated with Akkermansia and Mediterraneibacter (p < 0.01). Moreover, arachidonic acid was positively correlated with the abundance of Candidatus Stoquefichus, Allobaculum, Paramuribaculum, Duncaniella, and Dubosiella (Figure 8E). The correlations were shown in a Cytoscape network (Figure 8F), suggesting a fundamental relationship between the gut microbiota and metabolism after crocetin treatment.

As a medicinal plant, saffron has many therapeutic effects; crocin and crocetin are the two most biologically active components of saffron, and crocin is converted to crocetin before entering the blood [17]. Some studies have shown that crocin exerts anti-ulcerogenic and colon-protective effects by decreasing inflammation-associated cytokines, enhancing

Nrf2 and HO-1 signaling, and downregulating caspase-3 activity [18,19]. TCM theory suggests that saffron promotes blood circulation and removes blood stasis. However, patients with UC typically manifest persistent bloody diarrhea, rectal bleeding, and abdominal pain. Therefore, using blood-activating and stasis-eliminating compounds is not a suitable treatment option. Consequently, we established a DSS-induced colitis mouse model to determine the effects and mechanisms of crocetin on UC. Our findings revealed that crocetin could lead to weight loss, increased intestinal permeability, and intestinal microbiota imbalance in a mouse model of colitis. Otherwise, the level of TNF-α was found to decrease with the increasing amount of crocetin. Although this result was not surprising, TNF-α was reported to have both pro-inflammatory and anti-inflammatory effects in regulating gut inflammation [20,21]. It was proven that TNF-α suppressed the pathogenesis of acute intestinal inflammation by inducing extraadrenal production of immunoregulatory glucocorticoids in the intestinal mucosa [22]. Therefore, we supposed that crocetin reduced the level of TNF-α in serum result in worsened inflammation and delayed recovery in DSS-induced colitis mice.

E-cadherin is a key cell adhesion protein that responds to epithelial cell adhesion and maintains the architecture and function of the intestinal epithelium [23]. Furthermore, reduced E-cadherin expression is associated with more severe UC [24]. Our study found that the level of E-cadherin in the mouse colon was significantly decreased after treatment with crocetin, which may be associated with the exacerbation of colitis symptoms in the Crocetin-H group. Further research is needed to determine how crocetin regulates tight junction protein expression.

Additionally, we used an approach integrating 16s rDNA gene sequencing and LC-MSbased metabolomics to explore the effect of crocetin treatment on DSS-induced colitis mice. Our analysis of 16s rDNA gene sequencing suggested that crocetin affects the intestinal microbiota composition. The altered gut microbiota included an increase in the genera Akkermansia and Mediterraneibacter and a decrease in Muribaculaceae, Dubosiella, Paramuribaculum, Parasutterella, Allobaculum, Duncaniella, Candidatus Stoquefichus, and Coriobacteriaceae UCG-002. Akkermansia is a gram-negative anaerobe that colonizes the mucus layer of the intestinal tract and degrades mucus, which is its predominant energy source [25]. Increasing evidence has proven that the abundance of Akkermansia was significantly reduced in mice with colitis [26] and oral supplementation with pasteurized Akkermansia improved DSS-induced colitis by reducing the infiltration of macrophages and CD8+ cytotoxic T lymphocytes in the colon [27]. However, Akkermansia has also been shown to degrade mucus oligosaccharides, which results in the disruption of intestinal mucosal barrier, thus enhancing infection [28,29]. It has been reported that Akkermansia activity in mucus degradation might contribute to rotavirus in entry to enterocytes [30]. In addition, glucan oligosaccharides released following mucus can provide a key source o nutrients to non-mucin degrading bacteria, including C.difficile [31]. The increase of Akkermansia by crocetin might destroy mucus layer and compromise epithelial barrier function, thereby resulting in exacerbating colitis.

Our findings also demonstrated that, in addition to increasing the abundance of Akkermansia, crocetin significantly reduced the abundance of Muribaculaceae_unclassified, Duncaniella, and Eisenbergiella, which were negatively correlated with colitis severity [32,33]. We found members of the family Muribaculaceae (known as S24-7) in the guts of homoeothermic animals, where they were thought to play an important role in modulating the host’s health by degrading mucin [34,35]. Duncaniella is abundantly present in murine intestinal tract [36], and recent advances have indicated that it plays a protective role in DSS-induced colitis [37]. Eisenbergiella is a member of the family Lachnospiraceae, and there is evidence that it is positively correlated with dietary carbohydrates, fats, and proteins [38]. In addition, a decrease in the number of Eisenbergiella has been observed in patients with UC [33]. Although Parasutterella is a member of the healthy fecal core microbiome in the human intestinal tract [39], its relationship with UC remains controversial [40,41]. We observed increased hypoxanthine after Parasutterella colonization [42]. Hypoxanthine played a crucial role in regulating the

energy balance of the intestinal epithelium and maintaining gut barrier function in a mouse DSS-induced colitis model [43]. Our results indicated that crocetin might exacerbate UC by affecting the gut microbiota composition.

The gut microbiota participates in multiple hosts metabolic pathways and generates many fermentation metabolites, playing an important role in IBD. This study showed that crocetin treatment reduced the levels of primary (chenodeoxycholate, 6-ethylchenodeoxycholic acid, glycochenodeoxycholate-7-sulfate, and glycocholate) and secondary bile acids (12ketodeoxycholic acid, 7-ketodeoxycholic acid, 3-sulfodeoxycholic acid, and sulfolithocholic acid). Primary bile acids play significant roles in cholesterol and lipid metabolism and host– microbe interactions. Studies show that deoxycholic acid exerts anti-inflammatory effects in both acute and chronic murine colitis models and reduces the expression of key cytokines and chemokines involved in inflammation [44]. We, therefore, speculate that crocetin exacerbates IBD-associated bile acid dysmetabolism and affects gut homeostasis.

Arachidonic acid, an omega-6 polyunsaturated fatty acid, acts as a second messenger involved in intracellular signal transduction [45] and also plays a role in the production of prostaglandins and leukotrienes as eicosanoid precursors [46]. Although the role of arachidonic acid in IBD is controversial, our results demonstrated that the arachidonic acid content was significantly increased in the model group, consistent with previous studies [47]. Arachidonic acid represents the starting point of the inflammatory response and generates inflammatory mediators such as prostaglandin to maintain a pro-inflammatory microenvironment. The present study results showed that crocetin treatment reduced arachidonic acid and its metabolite prostaglandin E2, which may contribute to the attenuation of inflammation. Interestingly, we also found that leukotriene F4, a pro-inflammatory mediator belonging to the leukotriene family, was significantly increased following crocetin treatment. Although leukotriene F4 has the lowest biological activity among cysteinyl leukotrienes [48], it aggregated porcine platelets and induced the release of a plateletderived vasodilatory mediator [49], resulting in increased vascular permeability, which may augment colitis. Crocetin appeared to negatively regulate arachidonic acid, prostaglandin E2 ethanolamide, and 17-phenyl-18,19,20-trinor-prostaglandin E2, and positively regulate leukotriene F4 and prostaglandin lactone-diol, suggesting that it may play a complex role in modulating the arachidonate signaling pathway. Beyond that, dicoumarol was found to antagonize the blood clotting process by disrupting the vitamin K cycle and is thus widely used as an anticoagulant [16].

1. 4.1. Preparation of Crocetin Extract
2. 4.2. Experimental Animals

Female SPF C57BL/6J (4 weeks-old) mice weighing 19.7 ± 1.3 g were purchased from Jiangsu University (Zhenjiang, China). All mice were housed in accredited animal facilities

in groups of five in ventilated cages with free access to food and water under controlled conditions of 12 h light/dark cycle, air temperature 22 ± 3 ◦C, and 60% relative humidity. All animal procedures were performed in accordance with the animal ethics committee of Jiangsu University and Zhejiang University of Technology (No. 20210607115).

1. 4.3. Treatment Protocol
2. 4.4. Serum Cytokine Measurement
3. 4.5. Morphological Analysis and Immunohistochemistry
4. 4.6. RNA Isolation and Real-Time RT-PCR (RT-qPCR)

Total RNA was extracted using RNA simple Total RNA kit (TIANGEN, Beijing, China). Real-time RT-PCR was performed using SYBR real-time PCR Premix Ex Taq TM (Tli RNaseH Plus) (TaKaRa, Kyoto, Japan) and LightCycler 96 (Roche, Basel, Switzer-

land). RNA was quantified using the ∆∆Ct method of relative quantification. Gene expression levels were reported as fold changes over those of controls. The primer sequences of E-cadherin are listed as follows: 5 -TGGCACGGGCACTCTTCT-3 and 5 AGGCTGTGGGTTCCTCTGG-3 .

1. 4.7. Gut Microbiota Analysis
2. 4.8. Metabolomic Analysis

The sample preparation, metabolite identification and quantification, and primary quality control (QC) were performed at LC-Bio Technology Co., Ltd. (Hang Zhou, Zhejiang Province, China) as previously described [58]. After UPLC-MS/MS analyses, the Progenesis QI 2.3 (Nonlinear Dynamics, Waters, Milford, MA, USA) was used for peak detection and alignment of raw data. Afterward, a data matrix was generated, including retention time (RT), mass-to-charge ratio (m/z) values, and peak intensity. Metabolic features detected at least 80% in any set of samples were retained. After filtering, quality control, normalization, and imputation, all data were log-transformed prior to statistical analysis. The metabolites were identified based on accurate mass, MS/MS fragments spectra, and isotope ratio difference in Human metabolome database (HMDB) (http://www.hmdb.ca/, accessed on 8 September 2021) and Metlin database (https://metlin.scripps.edu/, accessed on 8 September 2021).

All multivariate statistical analysis, including principal component analysis (PCA) and orthogonal partial least squares discriminate analysis (OPLS-DA), were carried out using “ropls” package for R software.

1. 4.9. Statistical Analysis

Statistical analyses were performed with SPSS 22.0. The continuous data were presented as means + SEM or medians and interquartile ranges (IQRs). Student’s t-test or one-way analysis of variance (ANOVA) was used for the normally distributed data. A p-value < 0.05 was considered to be statistically significant.

This study provided the first evidence that crocetin supplementation significantly aggravated colitis in mice. Crocetin also disrupted intestinal homeostasis by reducing the relative abundances of the genera Muribaculaceae, Dubosiella, Paramuribaculum, Parasutterella, Allobaculum, Duncaniella, Candidatus Stoquefichus, and Coriobacteriaceae UCG-002, aggravating the imbalance of intestinal microbiota in mice with DSS-induced colitis. Additionally, crocetin reduced primary and secondary bile acid levels in the colon. Aberrant metabolism of arachidonic acid exacerbates colitis. Our findings suggest that patients with gastrointestinal diseases such as IBD should use crocetin with caution.

Our study has some methodological problems and limitations. First, the number of animal experiment was limited, which could reduce the power to detect the risk of crocetin on colitis. Second, we used CMC-Na as oral delivery systems. Although CMC-Na has some properties including hydrophilicity, biocompatibility, non-toxicity, ability to gel-forming, and pH sensitivity, which is widely used in drug delivery [59]. Another study demonstrated that CMC might increase susceptibility of mice to colitis [60]. Thus, further investigation should be needed to explore the effects of CMC on crocetin aggravating colitis. Finally, the methodological limitation might also lead to biased results. The serum levels and intestinal clearance of FD4 should be measured to further assess intestinal permeability.

Author Contributions: P.F. analyzed main content of the data and performed the experiment with the help of Q.L., L.L., S.W. and Z.W. Y.T. performed the literature search. The whole work is guided by P.W. and P.H. All authors have read and agreed to the published version of the manuscript.

Funding: This study was financially supported by the National Natural Science Foundation of China (No. 81703688), the Key Research and Development Program of Zhejiang Province (No. 2022C03050), the Key projects of international scientific and technological innovation cooperation between governments (No. 2017YFE0130100), and Postdoctoral Science Foundation of China (No. 2020M681514).

Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The datasets presented in this study can be found in NCBI BioProject repository at NCBI under the accession number SUB10830062. Conflicts of Interest: The authors declare no conflict of interest.