Identification of cuproptosis-related molecular classification and characteristic genes in ulcerative colitis

Ulcerative colitis (UC) is a refractory inflammatory disease with imbalances in intestinal mucosal homeostasis. Cuproptosis serves as newly identified programmed cell death (PCD) form involved in UC. In the study, UC-related datasets were extracted from the Gene Expression Omnibus (GEO) database. A comparison of UC patients and healthy controls identified 11 differentially expressed cuproptosis-related genes (DE-CRGs), where FDX1, LIAS, and DLAT were differentially expressed in UC groups from the mouse models and clinical samples, with their expression correlating with disease severity. By comprehending weighted gene co-expression network analysis (WGCNA) and differential expression analysis, the key genes common to the module genes relevant to different cuproptosis-related clusters and differentially expressed genes (DEGs) both in different clusters and patients with and without UC were identified using several bioinformatic analysis. Furthermore, the mRNA levels of four characteristic genes with diagnostic potential demonstrated significant decrease in both mouse models and clinical UC samples. Our discoveries offer a theoretical foundation for cuproptosis effect in UC

Heat shock factor 2 levels are associated with the severity of ulcerative colitis.

BACKGROUND AND AIMS: The morbidity of ulcerative colitis (UC) is increasing in China every year. In addition, there is a lack of accurate diagnostic indices with which to evaluate the activity of the disease. The aim of this study was to identify UC-associated proteins as biomarkers for the diagnosis, and objective assessment of disease activity. METHODS: Differential expression of serum proteins from UC patients compared to normal controls was analyzed by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The expression of heat shock factor 2(HSF2)in colonic mucosa in Crohn's disease, Behcet's disease, ulcerative colitis, intestinal tuberculosis, infective enteritis, intestinal lymphoma, and normal controls was investigated by immunohistochemistry (IHC). The expression of the HSF2 in colonic mucosa of UC subjects with varying severity of disease was measured by real time-PCR and Western Blots. The expression of HSF2 was inhibited by HSF2 small interfering RNA (siRNA) transfection in Caco-2 cells. The concentrations of HSF2, IL-1β, and TNF-α in serum and IL-1β, and TNF-α in the supernatants of transfected Caco-2 cells were determined by ELISA. RESULTS: HSF2 was differentially expressed in UC patients compared to normal controls. HSF2 expression was significantly higher in the intestinal mucosa of UC patients compared to other six groups. The results of immunohistochemistry, real time-PCR, Western Blots, and ELISA showed that the expression of HSF2 increased in parallel with the severity of UC. The serum concentration of HSF2 also positively correlated with levels of IL-1β and TNF-α. After down-regulation expression of HSF2 in Caco-2 cells by RNA interference, the productions of IL-1β and TNF-α stimulated by lipopolysaccharide (LPS) increased dramatically. CONCLUSIONS: HSF2 appears to be a potential novel molecular marker for UC activity, and may provide a basis for studies on the pathogenesis and novel therapeutic targets for UC

Identification of Environmental Factors Associated with Inflammatory Bowel Disease in a Southwestern Highland Region of China: A Nested Case-Control Study

<div><p>Background</p><p>The aim of this study was to examine environmental factors associated with inflammatory bowel disease (IBD) in Yunnan Province, a southwestern highland region of China.</p><p>Methods</p><p>In this nested case-control study, newly diagnosed ulcerative colitis (UC) cases in 2 cities in Yunnan Province and Crohn’s disease (CD) cases in 16 cities in Yunnan Province were recruited between 2008 and 2013. Controls were matched by geography, sex and age at a ratio of 1:4. Data were collected using the designed questionnaire. Conditional logistic regression models were used to estimate adjusted odds ratios (ORs).</p><p>Results</p><p>A total of 678 UC and 102 CD cases were recruited. For UC, various factors were associated with an increased risk of developing UC: dietary habits, including frequent irregular meal times; consumption of fried foods, salty foods and frozen dinners; childhood factors, including intestinal infectious diseases and frequent use of antibiotics; and other factors, such as mental labor, high work stress, use of non-aspirin non-steroidal anti-inflammatory drugs and allergies (OR > 1, <i>p</i> < 0.05). Other factors showed a protective effect: such as consumption of fruits, current smoking, physical activity, and drinking tea (OR < 1, <i>p</i> < 0.05). For CD, appendectomy and irregular meal times increased the disease risk (OR >1, <i>p</i> < 0.05), whereas physical activity may have reduced this risk (OR < 1, <i>p</i> < 0.05).</p><p>Conclusions</p><p>This study is the first nested case-control study to analyze the association between environmental factors and IBD onset in a southwestern highland region of China. Certain dietary habits, lifestyles, allergies and childhood factors may play important roles in IBD, particularly UC.</p></div

Transferrin Is Up-Regulated by Microbes and Acts as a Negative Regulator of Immunity to Induce Intestinal Immunotolerance

Cross-talks (e.g., host-driven iron withdrawal and microbial iron uptake between host gastrointestinal tract and commensal microbes) regulate immunotolerance and intestinal homeostasis. However, underlying mechanisms that regulate the cross-talks remain poorly understood. Here, we show that bacterial products up-regulate iron-transporter transferrin and transferrin acts as an immunosuppressor by interacting with cluster of differentiation 14 (CD14) to inhibit pattern recognition receptor (PRR) signaling and induce host immunotolerance. Decreased intestinal transferrin is found in germ-free mice and human patients with ulcerative colitis, which are characterized by impaired intestinal immunotolerance. Intestinal transferrin and host immunotolerance are returned to normal when germ-free mice get normal microbial commensalism, suggesting an association between microbial commensalism, transferrin, and host immunotolerance. Mouse colitis models show that transferrin shortage impairs host’s tolerogenic responses, while its supplementation promotes immunotolerance. Designed peptide blocking transferrin–CD14 interaction inhibits immunosuppressive effects of transferrin. In monkeys with idiopathic chronic diarrhea, transferrin shows comparable or even better therapeutic effects than hydrocortisone. Our findings reveal that by up-regulating host transferrin to silence PRR signaling, commensal bacteria counteract immune activation induced by themselves to shape host immunity and contribute for intestinal tolerance

Multivariate logistic regression analysis of UC patients and controls.

<p>Multivariate logistic regression analysis of UC patients and controls.</p

Expression of HSF2 in colonic mucosa tissues of normal control and UC was examined by IHC.

<p><b>A</b>: Representative normal colonic mucosa tissue was negatively stained with anti-HSF2 antibody; <b>B</b>, <b>C</b> and <b>D</b>: Representative UC colonic mucosal tissues of mild, moderate and severe group were positively stained with anti-HSF2 antibody, respectively (magnification, ×400); <b>E</b>: Positive cell expression of HSF2 was compared. All IHC results were read automatic image analyzer, measured by HPIAS-2000 analytic software. *, p<0.05; **, p<0.01.</p

Expression of HSF2 in colonic mucosa tissues of UC was examined by real- time PCR and Western Blotting.

<p><b>A</b>: the mRNA transcriptional levels of UC groups of varying severity. The data from the real-time PCR were analyzed with the delta–delta Ct method and normalized to the amount of GAPDH cDNA as an endogenous control. B: the protein expression levels of different UC severity groups, β-actin as an endogenous control. *, p<0.05; **, p<0.01.</p

Total proteins from serum of normal control subjects and UC patients were analyzed by 2-DE.

<p>The proteins were separated by IEF (pH 3–10) and 10% SDS-PAGE and subsequently processed with silver staining. The spot marked with white arrow represented HSF2. <b>A</b>: Total proteins from serum of normal controls that were analyzed by 2-DE. <b>B</b>: Total proteins from serum of UC patients that were analyzed by 2-DE.</p

The secretion of IL-1β and TNF-α in Caco-2 cells after enhanced expression or siRNA of HSF2.

<p>A: The HSF2 expression levels after HSF2 RNA interferenc and HSF2-FLAG recombinant plasmid transfection, β-actin as an endogenous control. B: The productions of IL-1β in Caco-2 cells supernates stimulated or not by LPS (50 ng/ml). C: The productions of TNF-α in Caco-2 cells supernates stimulated or not by LPS (50 ng/ml). *, p<0.05;**, p<0.01.</p

The concentration of HSF2, IL-1β and TNF-α in serum was determined by ELISA.

<p><b>A</b>: serum concentration of HSF2 in patients with UC of varying severity. B: serum concentrations of IL-1β and TNF-α of different UC severity groups. C: Correlation analysis between the concentrations of HSF2 and IL-1β. D: Correlation analysis between the concentrations of HSF2 and TNF-α.Correlation analysis was assessed using the Pearson Test which gives a correlation coefficient (Pearson “r”) and a “<i>p</i>” value. *, p<0.05; **, p<0.01.</p