Abstract

   Inflammatory bowel disease (IBD) is multifactorial chronic inflammatory
   disease in the gastrointestinal tract, affecting patients’ quality of
   life profoundly. The incidence of IBD has been on the rise globally for
   the last two decades. Because the molecular mechanisms underlying the
   disease remain not well understood, therapeutic development is
   significantly impeded. Metabolism is a crucial cellular process to
   generate the energy needed for an inflammatory response and tissue
   repair. Comprehensive understanding of the metabolic pathways in IBD
   would help to unravel the disease pathogenesis/progression and
   facilitate therapeutic discoveries. Here, we investigated four
   metabolic pathways altered in experimental colitis. C57BL/6J mice were
   treated with dextran sulfate sodium (DSS) in drinking water for 7 days
   to induce experimental ulcerative colitis (UC). We conducted proteomics
   analysis for the colon samples using LC/MS, to profile key metabolic
   intermediates. Our findings revealed significant alterations in four
   major metabolic pathways: antioxidative defense, β-oxidation,
   glycolysis, and TCA cycle pathways. The energy metabolism by
   β-oxidation, glycolysis, and TCA cycle pathways were downregulated
   under UC, together with reduced antioxidative defense pathways. These
   results reveal metabolic re-programming in intestinal cells under UC,
   showing dysregulation in all four major metabolic pathways. Our study
   underscores the importance of metabolic drivers in the pathogenesis of
   IBD and suggests that the modification of metabolism may serve as a
   novel diagnostic/therapeutic approach for IBD.

   Keywords: inflammatory bowel disease, ulcerative colitis, proteomics,
   metabolism, antioxidative defense, oxidative stress, β-oxidation,
   glycolysis, TCA cycle

1. Introduction

   Inflammatory bowel disease (IBD) is a digestive disorder that results
   from excessive levels of inflammation and tissue damage in the
   gastrointestinal tract. The incidence of IBD has been growing globally
   for the last two decades, especially in developed countries [[32]1].
   IBD is commonly associated with persistent diarrhea, abdominal pain,
   rectal bleeding, and weight loss [[33]2,[34]3]. IBD is classified into
   two types, ulcerative colitis (UC) and Crohn’s disease (CD) [[35]4].
   While the symptoms of both UC and CD are similar, the inflammatory
   sites differ as CD is often accompanied by systematic inflammation in
   both the small intestine and colon, while UC typically affects the
   colon [[36]3]. Prolonged inflammation of the bowels can lead to
   permanent damage and deterioration of the epithelium, resulting in
   uncontrolled inflammation as the disease progresses [[37]5]. IBD
   severely impacts patients’ social life, economic status, physical
   health, and mental health [[38]6,[39]7,[40]8]. In addition, IBD has
   been associated with colorectal cancer, small bowel adenocarcinoma,
   intestinal lymphoma, and cholangiocarcinoma [[41]9,[42]10].
   Furthermore, IBD is associated with other comorbidities, including
   cardiovascular, hepatic, and psychiatric disorders, and obesity
   [[43]11,[44]12]. IBD is a poorly understood disease; its diagnosis is
   often delayed compared to other diseases [[45]3]. In an international
   survey, 45% of respondents stated they received a diagnosis more than a
   year after symptoms appeared, and 17% of respondents stated that their
   diagnosis was received more than 5 years after the onset of symptoms
   [[46]7]. The identification of diagnostic biomarkers for timely
   detection, and the development of effective therapies are particularly
   crucial, given the challenging nature of IBD.

   Metabolism is a fundamental process that governs all biological and
   pathological activities by supplying energy and facilitating the
   signaling transduction of macromolecules. In IBD, alterations in energy
   metabolism have been observed [[47]13,[48]14,[49]15,[50]16]. Though,
   existing metabolomics studies show discrepancies in the levels of
   metabolites in blood, urine, and tissue samples in IBD
   [[51]13,[52]14,[53]17,[54]18]. Understanding the comprehensive profile
   and regulation of metabolic pathways at the molecular level is crucial
   for unraveling the complex interplay between metabolism and
   inflammation, providing valuable insights for diagnostic biomarkers and
   therapeutic interventions. In the current study, we induced
   experimental colitis in C57BL/6J male mice using a dextran sulfate
   sodium (DSS)-induced colitis model, and colon tissue was analyzed using
   mass spectrometry to assess altered levels of enzymes that regulate
   four major metabolic signaling cascades: oxidative stress, β-oxidation,
   glycolysis, and the TCA cycle.

2. Materials and Methods

2.1. Animal Model

   The C57BL/6J background of 4–6-month-old male mice were used in this
   study. All mice were housed and bred at Texas A&M University’s
   laboratory animal facility with controlled lighting and temperature (12
   h light–dark cycle, 75 ± 1 °F), with free access to a regular diet of
   Harlan-Teklad 2018X (Harlan-Teklad, Madison, WI, USA), which contains
   58% calories from carbohydrates, 24% calories from protein, and 18%
   calories from fat. Mice were randomly divided into two groups, and then
   given either ad libitum water (H[2]O) or 2% (w/v) dextran sulfate
   sodium (DSS) in water.

2.2. DSS-Induced Colitis

   Experimental colitis was induced as described before [[55]19], by
   providing 2% (w/v) DSS (MP Biomedicals; 36–50 kDa, Santa Anna, CA, USA)
   in drinking water for 7 days ad libitum. DSS was replaced every 48 h to
   maintain its potency. At termination, the mice were euthanized under
   isoflurane anesthesia.

2.3. Tissue Collection and Protein Extraction

   Colons were excised from each mouse and flushed with ice-cold PBS. They
   were opened longitudinally and then flash frozen until further
   processing. Flash frozen tissue strips were then homogenized at a ratio
   of 1 g of tissue to 10 mL of RIPA buffer (Sigma, St. Louis, MO, USA)
   with protease inhibitors, PhosSTOP™ (Sigma, St. Louis, MO, USA) and
   cOmplete™ (Sigma, St. Louis, MO, USA). Homogenates were centrifuged at
   high speed for 5 min, and the concentration of total protein was
   measured using the PierceTM BCA protein assay kit (Thermo Fisher
   Scientific, Waltham, MA, USA) following the manufacturer’s
   instructions. In brief, 25 µL of sample was mixed with 200 µL of
   Bradford reagents, then incubated at 37 °C for 30 min in the dark.
   Then, the absorbance was read at 590 nm wavelength using a CLARIOstar
   plate reader (BMG Labtech, Cary, NC, USA). Then, supernatants were
   aliquoted into volumes equivalent to 100 µg of total protein. Samples
   were stored in −80 °C until they were subjected to mass spectrometry
   analysis.

2.4. Sample Preparation for Mass Spectrometry

   The sample proteins were prepared as we have described [[56]20,[57]21].
   For each sample, 60 µg of protein was mixed with 1% SDS and 20 μL of
   BSA internal standard. Samples were mixed and heated, then proteins
   were precipitated with acetone. Dried protein pellets were
   reconstituted in Laemmli sample buffer and then run through a short
   (1.5 cm) 12.5% SDS-PAGE gel (Bio-Rad, Berkeley, CA, USA) for
   approximately 15 min at 150 V. The gels were fixed and stained with
   Coomassie blue (GelCode blue, Pierce, Appleton, WI, USA) for
   visualization. Samples were excised from the gel as entire lanes and
   then divided into smaller fragments. Gel segments were washed to remove
   the stain and then reduced with DTT and alkylated with iodoacetamide.
   The samples were digested overnight with trypsin (Promega, Madison, WI,
   USA). Sample proteins were then extracted from the gel and evaporated
   to dry in a SpeedVac. Samples were then reconstituted in 1% acetic acid
   for further analysis.

2.5. Mass Spectrometry Analysis

   Analysis was conducted using the TSQ Quantiva triple quadrupole mass
   spectrometry system (Thermo Fisher Scientific, Waltham, MA, USA) as
   previously described [[58]20,[59]21]. In brief, the HPLC was an
   Ultimate 3000 nanoflow system with 10 cm × 75 µm i.d. C18 capillary
   column (Phenomenex Jupiter, Torrance, CA, USA). A total of 5 µL
   aliquots were injected and loaded into the column. The column was
   eluted with a 60 min gradient of acetonitrile in 0.1% formic acid. The
   mass spectrometry system was operated under the selected reaction
   monitoring mode. The detection method for each protein measured for 2
   ideal peptides, and assays for multiple proteins were grouped together
   in larger panels. Determination of the integrated peak area of the
   appropriate chromatographic peaks was completed using the Skyline data
   analysis program. Responses for each protein were calculated as the
   geometric mean of the peptide areas, and values were normalized
   according to the BSA standard.

2.6. Statistical Analysis

   Statistical analysis of protein abundance was completed using
   MetaboAnalyst 5.0 “Statistical Analysis [one factor]” module
   ([60]http://www.metaboanalyst.ca/ accessed on 1 January 2023). Partial
   least squares discriminant analysis was used to analyze the clustering
   of samples and evaluate variable importance in projection. Two-sample,
   two-tailed t tests were used for the evaluation of significance, and
   then presented as mean ± SEM using GraphPad Prism 9.5.1 (GraphPad
   Software, La Jolla, CA, USA). Pathway impact analysis was completed
   using MetaboAnalyst’s Gene Set Enrichment Analysis Algorithm (GSEA).
   Fisher’s exact test was used for enrichment analysis, and topology was
   measured using betweenness centrality.

3. Results

3.1. Antioxidative Defense Pathway Is Downregulated in Colon of Colitis

   Oxidative stress is known to contribute to the pathogenesis of various
   diseases [[61]22]. Oxidative stress exacerbates epithelial barrier
   damage in IBD through reactive oxygen species (ROS). ROS lead to the
   alteration of tight junctions and result in increased gut permeability
   in the epithelium [[62]23]. To elucidate the effect of antioxidative
   defense capacity in the colon of UC, the proteins involved in the
   antioxidative stress pathways were tested using LC/MS. We detected a
   total of 30 proteins associated with the antioxidative defense pathway.

   The DSS-treated colitis group and the control group were significantly
   separated in the partial least squares discriminant analysis (PLS-DA)
   plot ([63]Figure 1A). The heatmap and variable importance in projection
   (VIP) score plot revealed that the colitis group had a significantly
   lower expression of proteins associated with antioxidative response
   pathways ([64]Figure 1B,C). Our results showed significantly reduced
   relevance abundance of superoxide dismutases (SOD1, SOD2), glutathione
   peroxidase 1 (GPX1), glutathione S-transferase (GST) family proteins
   (GSTA3, GSTM1, GSTP1), aldo-keto reductase family 1 member B (AKR1B1),
   aldehyde dehydrogenase 2 (ALDH2), and methionine sulfoxide reductase A
   (MSRA). We also conducted pathway enrichment analysis to generate a
   pathway impact plot using the Gene Set Enrichment Analysis (GSEA)
   algorithm of MetaboAnalyst 5.0. Protein names were converted to gene
   IDs to run the GSEA algorithm. The topology analysis showed that
   colitis impacted glutathione metabolism most significantly, with lesser
   impact on glycerolipid metabolism, glycolysis/gluconeogenesis, and
   selenocompound metabolism ([65]Figure 1G and [66]Table S1). The
   schematic summary of alterations of the antioxidative stress response
   is shown in [67]Figure 2. Our data collectively indicate that the
   antioxidative defense is downregulated in DSS-induced colitis,
   supporting increased ROS and engulfed inflammation in the colon under
   colitis.

Figure 1.

   [68]Figure 1
   [69]Open in a new tab

   Altered antioxidative stress pathway in DSS-induced colitis. (A)
   Partial least squares discriminant analysis (PLS-DA) biplot; (B)
   non-clustered heatmap of correlating protein concentration with
   individual samples; (C) variable importance plot (VIP) highlighting
   significantly changed proteins; (D) relative abundance of superoxide
   dismutases (SOD1, SOD2); (E) glutathione metabolism-related enzymes
   (GPX1, GSTA3, GSTM1, GSTP1); (F) relative concentrations of ROS-related
   proteins; (G) pathway enrichment impact plot, with color and size of
   circle indicating pathway impact (larger/more red = higher impact). * p
   < 0.05 and ** p < 0.01, DSS vs. Control.

Figure 2.

   [70]Figure 2
   [71]Open in a new tab

   Schematic diagram of downregulated antioxidative responses in
   DSS-induced colitis. Multiple antioxidative stress-related pathways are
   altered in colitis. (1) SOD1 and SOD2 convert O[2]^•− into H[2]O[2],
   then GPX1 oxidizes glutathione to convert H[2]O[2] into H[2]O. (2)
   Lipid-derived aldehydes like HNE form complexes with GSH through the
   GST complex (GSTA3, GSTM1, GSTP1) to form GS-HNE. The GS-HNE is further
   converted into GS-DHN by AKR1B1. Alternatively, HNE can also be
   converted to DHN or HNA by AKR1B1 or ALDH2, respectively. (3)
   ROS-mediated oxidated methionine is converted to native methionine
   through MSRA. Under colitis, enzymes regulating all three pathways are
   downregulated as indicated by the red arrows. dotted ↓ p < 0.1, ↓ p <
   0.05, ↓↓ p < 0.01, DSS vs. control. SOD: superoxide dismutase; GPX1:
   glutathione peroxidase; GSH: reduced glutathione; HNE:
   4-hydroxynonenal; GST: glutathione S-transferase; GS-HNE:
   glutathione-HNE; DHN: 1,4-dihydroxy-2-nonene; GS-DHN:
   glutathione-1,4-dihydroxy-2-nonene; HNA: 4-hydroxy-nonenoic acid;
   AKR1B1: aldo-keto reductase 1; ALDH2: aldehyde dehydrogenase; MSRA:
   methionine sulfoxide reductase.

3.2. β-Oxidation Pathway Is Downregulated in Colon of Colitis

   Fatty acid β-oxidation is one of the major pathways generating energy
   metabolizing fatty acid to acetyl-CoA. In our study, 38 proteins
   associated with the β-oxidation pathway were detected. PLS-DA of
   DSS-induced colitis mice showed distinct separation from the control
   group ([72]Figure 3A). The heatmap and VIP score plot showed
   significantly altered β-oxidation in colitis, and the relative
   abundance of the identified proteins was significantly reduced in the
   presence of colitis ([73]Figure 3B,C). Our data identified decreases in
   long-chain acyl-CoA synthetase (ACSL1), medium-chain acyl-CoA
   dehydrogenase (ACADM), enoyl CoA delta isomerase (ECI2), enoyl-CoA
   hydratase (ECHS1), 3-hydroxyacyl CoA dehydrogenase (HADH), and
   acetyl-CoA acyltransferase 2 (ACAA2) ([74]Figure 3D). We also observed
   a decreasing trend in proteins regulating the downstream ketogenesis
   pathway in colitis, including 3-hydroxy-3-methylglutaryl-CoA lyase
   (HMGCL), 3-hydroxybutyrate dehydrogenase 1 (BDH1), and carnitine
   acetyltransferase (CRAT) ([75]Figure 3E). Topology analysis using
   MetaboAnalyst’s GSEA algorithm showed that colitis significantly
   impacted fatty acid degradation and elongation; the degradation of
   valine, leucine, and isoleucine; butanoate metabolism; and the
   synthesis/degradation of ketone bodies ([76]Figure 3F and [77]Table
   S2). A schematic summary of changes in β-oxidation and ketogenesis in
   colitis is presented in [78]Figure 4. Together, our results suggest
   significant downregulation of the FA metabolism through the β-oxidation
   pathway in colitis.

Figure 3.

   [79]Figure 3
   [80]Open in a new tab

   Altered β-oxidation pathway in DSS-induced colitis. (A) PLS-DA biplot;
   (B) non-clustered heatmap of correlating protein concentration with
   individual samples; (C) VIP highlighting significantly changed
   proteins; relative abundance of (D) fatty acid (FA) metabolism and (E)
   ketogenesis; (F) pathway enrichment impact plot, with color and size of
   circle indicating pathway impact (larger/more red = higher impact). * p
   < 0.05, DSS vs. Control.

Figure 4.

   [81]Figure 4
   [82]Open in a new tab

   Schematic diagram of β-oxidation and ketogenesis in DSS-induced
   colitis. Fatty acids are metabolized to acetyl-CoA via β-oxidation, and
   subsequent ketogenesis converts acetyl-CoA to D-b-hydroxybutyrate.
   ACSL1, ACADM, HADH, ACAA2, and CRAT were significantly downregulated
   under colitis. Also, colitis showed a decreasing trend in ECI2, ECHS1,
   HMFCL, and BDH1 as indicated by the red arrows. dotted ↓ p < 0.1, ↓ p <
   0.05, DSS vs. control. ACSL1: acyl-CoA synthetase; ECI2: enoyl CoA
   delta isomerase; ACADM: medium-chain acyl-CoA dehydrogenase; ECHS1:
   enoyl CoA hydratase; HADH: hydroxy-acyl CoA dehydrogenase; ACAA2:
   acetyl-CoA acyl transferase; CRAT: carnitine acetyltransferase; HMGCL:
   hydroxy-methylglutaryl lyase; BDH1: hydroxybutyrate dehydrogenase.

3.3. Glycolysis Pathway Is Downregulated in Colon of Colitis

   Glycolysis is a central anaerobic metabolic pathway producing energy
   for cell metabolism, and glycolysis is linked to the cellular
   inflammatory response to meet rapid energy demand upon inflammation
   [[83]24,[84]25]. We detected 22 proteins regulating glycolysis. We
   observed significant separation in the PLS-DA plot between DSS-treated
   colitis and the control mice ([85]Figure 5A). The heatmap and VIP score
   plot indicated a significant decrease in proteins regulating glycolysis
   and the glycolysis-linked TCA cycle pathway ([86]Figure 5B,C). We
   observed a reduced relevant abundance for hexokinase (HK1),
   glucose-6-phosphateisomerase (GPI), phosphofructokinase (PFKL),
   fructose 1,6-biphosphate aldolase enzymes (ALDOA, ALDOB),
   triosephosphate isomerase (TPI), phosphoglycerate kinase 1 (PGK1), and
   alpha enolase (ENO1) in colitis ([87]Figure 5D). Also, the proteins
   involved in connecting glycolysis and the TCA cycle showed a decreasing
   trend, including the pyruvate carboxylase (PC) and cytosolic malate
   dehydrogenase (MDH1) ([88]Figure 5E). Pathway topology analysis using
   MetaboAnalyst’s GSEA algorithm indicated that colitis has an impact on
   glycolysis/gluconeogenesis, the pentose phosphate pathway, and
   fructose/mannose metabolism ([89]Figure 5F and [90]Table S3). A
   schematic summary of glycolysis changes in colitis is shown in
   [91]Figure 6. Overall, our data suggest significant downregulation of
   glycolysis in experimental colitis.

Figure 5.

   [92]Figure 5
   [93]Open in a new tab

   Altered glycolysis pathway in DSS-induced colitis. (A) PLS-DA biplot;
   (B) non-clustered heatmap of correlating protein concentration with
   individual samples; (C) VIP score plot highlighting significantly
   changed proteins; relative abundance of (D) glycolysis and (E)
   glycolysis adjacent pathways; (F) pathway enrichment impact plot, with
   color and size of circle indicating pathway impact (larger/more red =
   higher impact). * p < 0.05, ** p < 0.01, DSS vs. Control.

Figure 6.

   [94]Figure 6
   [95]Open in a new tab

   Schematic diagram of glycolysis changes in DSS-induced colitis. Glucose
   is metabolized to pyruvate via glycolysis. Pyruvate then can be
   carboxylated to form oxaloacetate, which enters the TCA cycle in the
   mitochondria. In colitis, the enzymes regulating glycolysis are all
   reduced, as indicated by red arrows. dotted ↓ p < 0.1, ↓ p < 0.05, ↓↓ p
   < 0.01, DSS vs. control. HK1: hexokinase; G6P: glucose-6-phosphate;
   GPI: glucose-6-phosphate isomerase; F6P: fructose-6-phosphate; PFKL:
   phosphofructokinase; F1,6BP: fructose-1,6-biphosphate; ALDO: aldolase
   fructose biphosphate; GAP: glyceraldehyde phosphate; DHAP:
   dihydroxyacetone phosphate; TPI: triosephosphate isomerase; 1,3BPG:
   1,3-biphosphoglyceric acid; PGK1: phosphoglycerate kinase; 3PG:
   3-phosphoglyceric acid; 2PG: 2-phosphoglyceric acid; ENO1: alpha
   enolase; PEP: phosphoenolpyruvic acid; PC: pyruvate carboxylase; MDH1:
   malate dehydrogenase.

3.4. Tricarboxylic Acid (TCA) Cycle Is Downregulated in Colon of Colitis

   The tricarboxylic acid (TCA) cycle, also known as the citric acid cycle
   or Krebs cycle, is a signaling hub in mitochondria generating energy by
   breaking down pyruvate and acetyl-CoA, and regulating various cellular
   responses [[96]26,[97]27]. In our study, 49 proteins associated with
   the TCA cycle were detected in the colon with/without colitis. The
   DSS-induced colitis and control groups showed significant separation in
   the PLS-DA plot ([98]Figure 7A). The heatmap and VIP score plot showed
   alterations of TCA cycle proteins in colitis ([99]Figure 7B,C). We
   observed the robust suppression of TCA cycle-associated proteins
   including the pyruvate dehydrogenase kinase complex (PDK1, PDK2),
   dihydrolipoyl transacetylase (DLAT), dihydrolipoyl dehydrogenase (DLD),
   and pyruvate dehydrogenase E1 alpha subunit (PDHA1) ([100]Figure 7D),
   which are collectively referred to as the pyruvate dehydrogenase
   complex (PDC) [[101]28]. Also, significant decreases were observed in
   citrate synthase (CS), aconitase 2 (ACO2), isocitrate dehydrogenase
   subunits (IDH3A, IDH3B, IDH3G), 2-oxoglutarate dehydrogenase subunit
   (OGDH), succinate CoA ligase alpha subunit (SUCLG1), fumarate hydratase
   (FH1), and malate dehydrogenase 2 (MDH2) ([102]Figure 7E,F).
   Additionally, ubiquinol cytochrome c reductase core protein 1 (UQCRC1),
   a component of the mitochondrial electron transport chain (ETC) that
   acts downstream of the TCA cycle, was significantly reduced
   ([103]Figure 7F). Pathway topological analysis using the GSEA
   identified significant impacts of DSS-induced colitis on the TCA cycle,
   pyruvate metabolism, glycolysis/gluconeogenesis, and cysteine and
   methionine metabolism ([104]Figure 7G and [105]Table S4). A schematic
   summary of colitis-associated changes in the TCA cycle is shown in
   [106]Figure 8. Collectively, our data suggest a significant
   downregulation of the TCA cycle in the colon of DSS-induced colitis.

Figure 7.

   [107]Figure 7
   [108]Open in a new tab

   Altered TCA cycle profile in DSS-induced colitis. (A) PLS-DA biplot;
   (B) non-clustered heatmap of correlating protein concentration with
   individual samples; (C) VIP score plot highlighting significantly
   changed proteins; relative abundance of (D) pyruvate dehydrogenase
   complex and (E,F) the TCA cycle; (G) pathway enrichment impact plot,
   with color and size of circle indicating pathway impact (larger/more
   red = higher impact). * p < 0.05, DSS vs. Control.

Figure 8.

   [109]Figure 8
   [110]Open in a new tab

   Schematic diagram of TCA cycle re-programming in DSS-induced colitis.
   Pyruvate enters the mitochondria and converts to acetyl-CoA, which is
   metabolized via the TCA cycle. Coenzymes NADH and FADH[2] produced from
   the TCA cycle then transfer electrons to the ETC to drive OXPHOS and
   produce ATP. In colitis, most enzymes regulating the TCA cycles were
   downregulated. UQCRC1, a component of complex III of the ETC, was also
   downregulated. dotted ↓ p < 0.1, ↓ p < 0.05, DSS vs. control. PDK:
   pyruvate dehydrogenase kinase; PDHA1: pyruvate dehydrogenase E1; DLAT:
   dihydrolipoyl transacetylase; DLD: dihydrolipoamide dehydrogenase; PC:
   pyruvate carboxylase; CS: citrate synthase; ACO2: aconitase; IDH3:
   isocitrate dehydrogenase; OGDH: oxoglutarate dehydrogenase; SUCLG1:
   succinate CoA ligase; FH1: fumarate hydratase; MDH2: malate
   dehydrogenase; OXPHOS: oxidative phosphorylation; UQCRC1: ubiquinol
   cytochrome c reductase core protein.

4. Discussion

   Metabolism is essential for all biological processes including
   development, differentiation, proliferation, and functionality of
   cells. Metabolism is a dynamic cellular process that generates energy
   to meet the demand of physiological/pathological conditions, to adapt
   to the given environment or stimulus [[111]29]. A previous study using
   RNA sequencing reported that key genes and pathways changed in UC
   patients; the most upregulated pathways are related to immune
   activation and cytokine production, and the most downregulated pathways
   are the mitochondrial TCA cycle and metabolic pathways [[112]30]. Given
   the significant role of metabolic pathways in immune-cell activation
   and function, we studied four major metabolic pathways in the colons of
   mice with experimental colitis using LC/MS proteomics. Our findings
   revealed that colitis is associated with wide-ranging dysregulation of
   major metabolic pathways, including oxidative stress, β-oxidation,
   glycolysis, and the TCA cycle.

   IBD is a complex and multifactorial disease. Though most studies in the
   literature of IBD evaluate only individual pathway. To obtain a more
   complete and comprehensive understanding of metabolic dysregulation in
   IBD, we have studied four major metabolic pathways in colitis. Also,
   many previous studies have used human serum and urine samples due to
   easy accessibility of bodily fluids [[113]13,[114]31,[115]32,[116]33].
   However, several metabolomics studies indicate discrepancies of
   metabolite levels between blood/urine and colon biopsy samples
   [[117]13,[118]14,[119]17,[120]18]. It has been suggested that
   exfoliated cells in feces may serve as a noninvasive method to assess
   mucosal signatures for early detection/diagnosis of intestinal health
   [[121]34]. In our study, we analyzed colonic tissue directly and
   employed a proteomics methodology to assess the alteration of metabolic
   pathways under colitis. Notably, proteomic profiling of metabolic
   pathways at the translational level provides more relevant insights
   into functional outcomes than gene expression. Furthermore, we focused
   on examining the levels of enzymatic proteins that govern metabolic
   pathways. The proteomic approach not only provided insights into
   immediate regulatory events but also offered a better understanding of
   the mechanistic insights associated with pathogenesis of colitis.

   Oxidative stress is a crucial pathological factor that plays an
   important role in the onset and progression of IBD
   [[122]23,[123]35,[124]36,[125]37]. ROS promote mucosal epithelium
   damage, contributing to mucosal barrier dysfunction [[126]38]. Our data
   showed that colitis significantly downregulated the proteins associated
   with antioxidant defense in the colon ([127]Figure 1), suggesting a
   significant accumulation of toxins derived from ROS, including O[2]^•−,
   H[2]O[2], lipid-derived aldehydes, and oxidized methionine. It is in
   line with our previous report showing significantly increased colonic
   inflammation accompanied with an increase in disease activity (reduced
   body weight, increased rectal bleeding, and diarrhea) and inflammatory
   cytokine expressions in the colon of DSS-induced colitis compared to
   water control [[128]19]. Of note, a different degree of colitis
   severity would have differential effects on metabolomic profiles. In
   our study, we induced ulcerative colitis using 2% DSS, a concentration
   generally associated with mild induction of ulcerative colitis. The 2%
   DSS for 7 days of treatment increased the disease activity index (DAI)
   to approximately half of the maximum score, consistent with the results
   of our previous report [[129]19]. This indicates that the observed
   alterations in metabolic pathways in our current study are associated
   with mild disease activity in ulcerative colitis.

   Our current data revealed that the mice with colitis showed decreased
   enzymes regulating the clearance of detrimental superoxide including
   O[2]^•−, H[2]O[2], lipid-derived aldehydes, and oxidized methionine
   ([130]Figure 2). Superoxide dismutases (SOD) catalyze the reduction of
   superoxide radicals (O[2]^•−) into hydrogen peroxide (H[2]O[2])
   [[131]39]. Our study showed that both SOD1 and SOD2 are downregulated
   in experimental colitis ([132]Figure 1D). This result is in line with
   other reports that the polymorphism of SOD1 is associated with
   increased risk of UC [[133]40], and the polymorphism of SOD2 affects UC
   onset [[134]41], underscoring the protective role of SOD in UC. On the
   other hand, one study has reported that SOD levels are elevated in IBD
   patients, whereas the levels can be restored to normal in remission
   [[135]42]. However, the low enzymatic activity of SOD is associated
   with the active stage of IBD [[136]42,[137]43], which suggests that SOD
   activity is critical in pathogenesis of UC. Our finding is further
   supported by the evidence that SOD1 deficiency exacerbates DSS-induced
   colitis and SOD treatment significantly attenuates colonic inflammation
   in experimental colitis [[138]44,[139]45].

   Glutathione peroxidases (GPX) are selenoproteins with
   antioxidant/anti-inflammatory functions. They protect cell damage from
   peroxides by catalyzing glutathione (GSH) into an oxidized form
   (glutathione disulfide (GSSH)), reducing H[2]O[2] into H[2]O. Our data
   indicated a notable decrease in the GPX1 level in colitis ([140]Figure
   1E). This finding is consistent with the other reports showing an
   association of GPX1 and IBD onset [[141]46], and a double knockout of
   Gpx1 and Gpx2 genes triggers colitis [[142]46]. Given the study showing
   the reduced enzymatic activity of GPX and GSH in SOD1 deficiency in a
   DSS-induced colitis model [[143]45], we cannot exclude the possibility
   that a lower expression of GPX1 in our study is the result of reduced
   SOD in colitis.

   GST and ALDH2 are the enzymes that reduce lipid-derived aldehydes
   (HNE). Our results revealed that the levels of these enzymes are all
   reduced in colitis ([144]Figure 1E). Glutathione transferases (GST) are
   a group of enzymes consisting of four classes (alpha, mu, pi, and
   theta), which conjugate with glutathione to regulate scavenging
   lipid-derived aldehydes (HNE). We found that colitis significantly
   decreased GST classes alpha, mu, and pi (GSTA3, GSTM1, GSTP1)
   ([145]Figure 1E). Our data are supported by previous reports showing
   low GSTM in the early age onset of UC [[146]47,[147]48,[148]49],
   whereas limited studies can be found in regard of GSTA and GSTP in IBD.
   Similar to GST, aldehyde dehydrogenases (ALDH) have a key role in
   converting aldehydes to harmless HNA. Our result of reduced ALDH2 in
   colitis could potentially be explained by the reduced presence of the
   ALDH^+ macrophage and dendritic cells in the colon of UC [[149]50],
   considering that inflammatory myeloid cells are largely recruited to
   the lamina propria in UC [[150]51]. On the other hand, an increased
   ALDH level in colon stem cells has been shown to mediate the transition
   from colitis to cancer, suggesting the differential role of ALDH in
   different cell types [[151]52].

   MSRA is a key antioxidant enzyme that catalyzes the reduction of
   methionine S-sulfoxide. Our study showed that colitis decreased MSRA in
   the colon ([152]Figure 1F). While limited studies have investigated
   whether MSRA is involved in UC pathogenesis, it has been shown that the
   deletion of MSRA induces a higher susceptibility to LPS-induced lethal
   shock [[153]53]. In the same study, bone marrow-derived macrophages
   with MSRA deficiency showed elevated ROS levels along with higher NF-kB
   nuclear translocation. This is in line with our results, suggesting
   that lower MSRA expression likely contributes to high ROS levels, which
   in turn increases vulnerability to colitis. Together, our findings
   suggest that antioxidative defense pathways are significantly
   downregulated in colitis. This failure of antioxidative defense
   contributes to increased ROS levels, which strongly indicates colonic
   inflammation.

   Ulcerative colitis is often described as an energy-deficient state
   accompanied by oxidative stress [[154]54]. β-oxidation is an important
   energy-generating pathway. The association between IBD and fatty acid
   β-oxidation has been reported [[155]54,[156]55]. Our data showed that
   the enzymes ACSL1 and ACADM, which break down long-chain fatty acids
   and medium-chain fatty acids, respectively, were significantly
   decreased in colitis. One study reported that ACSL1 was upregulated in
   the colons of UC patients, although only mRNA levels were tested in the
   study [[157]56]. It was suggested that the primary function of ACSL1 is
   to promote triacylglycerols and de-novo synthesis of phospholipids. The
   alterations of ACSL1 in β-oxidation could be a compensatory mechanism
   for disease progression [[158]57]. β-oxidation of short-chain fatty
   acids (SCFAs), especially butyrate, is a preferred energy source in
   epithelial cells that govern the maintenance of mucosal homeostasis
   [[159]58,[160]59]. Our results showed that the enzymes regulating
   β-oxidation were significantly downregulated in the colon of colitis
   ([161]Figure 3 and [162]Figure 4), suggesting a contribution of
   diminished β-oxidation of SCFAs in UC. Our findings are in line with
   previous reports indicating decreased oxidation and uptake of butyrate
   in IBD patients [[163]54,[164]55,[165]60]. Additionally, our results
   are in line with the report that the deletion of β-oxidation in the
   epithelium develops ulcerative colitis [[166]61]. ECHS1 is associated
   with butyrate metabolism. Notably, our study showed a reduction in
   ECHS1, which is consistent with the downregulation of the gene encoding
   ECHS1 observed in the inflamed mucosa of UC patients [[167]55,[168]62].
   These findings collectively suggest that the diminished capacity of the
   colon to utilize SCFAs for energy generation contributes to mucosal
   dysbiosis and hinders epithelial regeneration in UC.

   Glycolysis is one of the major metabolic pathways which is the main
   source of energy for cells. Glycolysis converts glucose to pyruvate.
   The end products of this process enter the TCA cycle to be fully
   metabolized, generating NADH and FADH[2] to promote oxidative
   phosphorylation (OXPHOS). Our study shows a reduced expression of the
   regulatory enzymes of glycolysis in the colon under colitis
   ([169]Figure 5 and [170]Figure 6). Our finding is consistent with
   another study that demonstrated reduced levels of glycolysis-derived
   metabolites in the colon biopsies of IBD patients [[171]17].
   Specifically, our results revealed decreased levels of glycolytic
   enzymes, including HK1, GPI, PFKL, ALDOA, and ALDOB in colitis. This
   aligns with the reduced levels of myoinositol and glycerol-phosphate
   identified in IBD, both of which are products governed by the
   aforementioned enzymes that were reduced in our study. Differential
   outcomes in gene enrichment analysis of glycolysis have been reported
   in IBD by others [[172]63,[173]64]. Our study specifically assessed the
   translation of key enzymes, in contrast to prior studies that examined
   glycolysis largely at the mRNA level. It is possible that the
   downregulation of glycolysis at the translational level leads to
   compensatory stimulation of mRNA transcription to meet the cellular
   metabolic demands arising from the reduced glycolytic rate.
   Collectively, our study showed a reduced expression of glycolytic
   enzymes, suggesting the downregulation of glycolysis.

   The TCA cycle is a crucial metabolic pathway in the mitochondria. It
   plays an essential role in ATP generation. Our results revealed that
   the majority of enzymes regulating the TCA cycle were significantly
   suppressed in experimental colitis ([174]Figure 7 and [175]Figure 8),
   suggesting a significant suppression of the TCA cycle in colitis. Our
   results are in line with reports showing decreased TCA cycle-related
   metabolites in IBD, such as citrate, isocitrate, a-ketoglutarate,
   succinate, fumarate, and malate in UC
   [[176]13,[177]16,[178]17,[179]18,[180]31,[181]32,[182]33,[183]65,[184]6
   6,[185]67]. The fuels for the operation of the TCA cycle are acetyl-CoA
   and pyruvate, which are the end products of β-oxidation and glycolysis.
   Given that our results show a simultaneous downregulation of
   β-oxidation and glycolysis ([186]Figure 3, [187]Figure 4, [188]Figure 5
   and [189]Figure 7), we expect that the low availability of acetyl-CoA
   and pyruvate for the TCA cycle likely contributes to the dysregulation
   of the TCA cycle in colitis. Further studies utilizing gain-of-function
   or loss-of-function in animal and/or cell models to target the specific
   pathways would provide more insights into the necessity and sufficiency
   of the regulators, further advancing the understanding of the potential
   interplay among these dysregulated metabolic pathways in IBD.

   The TCA cycle produces electron carriers, such as NADH and FADH[2],
   which deliver electrons into the electron transport chain (ETC),
   generating a proton gradient across the inner mitochondrial membrane
   [[190]27,[191]68]. This proton gradient leads the ATP synthase complex
   to generate ATP. The energy production process is known as OXPHOS. In
   our study, we observed a significant reduction in UQCRC1 in colitis,
   which is a subunit of mitochondrial complex III in ETC ([192]Figure 7
   and [193]Figure 8). Our result is similar to previous studies showing
   the reduced gene expression of Uqcrc1 in UC and colorectal cancer
   [[194]69,[195]70]. Given the intricate connection of the TCA cycle and
   ETC in mitochondria, TCA cycle dysregulation and mitochondrial
   dysfunction may exacerbate colitis [[196]30]. Indeed, our findings
   indicated dysregulation in the resolution of oxidative stress in the
   mice with colitis ([197]Figure 1 and [198]Figure 3), suggesting the
   involvement of a defective TCA cycle and a dysfunctional ETC in the
   mitochondria in the pathogenesis of colitis. Collectively, our findings
   indicate that dysregulation of the TCA cycle and mitochondrial ETC is
   involved in the pathogenesis of colitis.

   Our current study helps to better understand metabolic pathways and
   their dysregulation in the colon under colitis. However, we are aware
   there are several limitations with our current study. CD and UC are
   characterized by both shared and distinct pathogenic features. While
   DSS-induced colitis serves as a well-established model for UC, it does
   not replicate the characteristics of CD. Hence, our findings are
   specifically relevant to ulcerative colitis. Also, our study conducted
   targeted proteomics, specifically focusing on four major metabolic
   pathways. However, it is important to note that other metabolic
   pathways such as amino acid metabolism, may also be involved in the
   pathogenesis of colitis [[199]13,[200]16]. Therefore, future study
   utilizing untargeted analysis would be beneficial in providing more
   comprehensive insights. Sexual dimorphism between males and females
   extends to various aspects of metabolism. Studies have shown that the
   severity and progression of inflammatory responses differ between male
   and female subjects; there are higher incidences and remission rates of
   colitis in males compared to females after age 45
   [[201]71,[202]72,[203]73,[204]74]. Hormonal variations, particularly
   estrogen levels in females, may influence the metabolic pathways
   involved in inflammation and tissue repair in the colon during colitis
   [[205]75,[206]76,[207]77]. Our experiments were conducted with male
   mice. For further study, it is essential to also examine the metabolic
   pathways in female mice under colitis.

   Notably, the colon lamina propria serves as a primary reservoir of a
   large body of infiltrated immune cells upon intestinal inflammation,
   which contributes significantly to the pathogenesis and progression of
   the disease [[208]78,[209]79]. We and others have previously reported
   that immune cells undergo a functional change in their microenvironment
   by shifting their metabolism from OXPHOS to glycolysis to meet the high
   demand of energy [[210]80,[211]81,[212]82,[213]83,[214]84]. The current
   study used colon tissue, which includes multiple cell types from all
   layers of the colon (for example, the epithelium, lamina propria, and
   muscularis mucosa). The detected proteomics profile reflects this
   variety of cell types. In our current study, we were unable to
   differentiate the metabolic pathways in specific cell types in colitis.
   Further exploration using cell-specific deletion models would advance
   our understanding of the cell-specific regulation of the key metabolic
   pathways in IBD.

5. Conclusions

   This study delved into the alterations in four major metabolic pathways
   including oxidative stress, β-oxidation, glycolysis, and the TCA cycle,
   in experimental colitis. The findings underscore the significance of
   energy metabolism in the pathogenesis of IBD, and specifically
   highlight the pervasive dysfunction of metabolic pathways in colitis.
   Further investigation of metabolic programming in specific cells, such
   as immune or epithelial cells, will further advance the understanding
   of tissue/cell specificity and the underlying mechanisms of IBD,
   leading to novel therapeutic interventions.

Acknowledgments