Circadian Regulator PER2 Protects Against Epithelial Necroptosis in Ulcerative Colitis via the STAT1–ZBP1 Axis

Circadian Regulator PER2 Protects Against Epithelial Necroptosis in Ulcerative Colitis via the STAT1–ZBP1 Axis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Circadian Regulator PER2 Protects Against Epithelial Necroptosis in Ulcerative Colitis via the STAT1–ZBP1 Axis Jieru Zhou, Gangping Li, Suya Pang, Yiqing Mei, Haoran Wang, Yu Jin, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7601246/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by epithelial barrier disruption, excessive cell death, and dysregulated immune signaling. While circadian rhythms are known to influence immune homeostasis, the role of the core circadian regulator Period circadian clock 2 (PER2) in intestinal inflammation remains poorly understood. Methods: We investigated the impact of circadian rhythm disruption on colitis severity using a jet lag model in DSS-treated mice, as well as clinical UC samples from patients with or without sleep disorders. Epithelial-specific Per2 knockout ( Per2 -/- ) mice and in vitro PER2 knockdown models were employed to dissect the mechanistic role of PER2 in regulating epithelial cell death and inflammation. Bulk RNA-sequencing, molecular docking, chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays were used to identify downstream targets and interacting partners. Pharmacological inhibition of STAT1 using Nifuroxazide was tested for therapeutic potential. Results: Circadian disruption aggravated DSS-induced colitis in mice and increased epithelial apoptosis in UC patients, accompanied by marked suppression of PER2 expression. Loss of PER2 in mice resulted in exacerbated intestinal inflammation, elevated DAI scores, and histological damage. Transcriptomic profiling and functional assays revealed that PER2 deficiency promoted necroptosis by upregulating ZBP1, RIPK3, and MLKL at both transcriptional and protein levels. Mechanistically, PER2 directly interacts with STAT1 via its PAS1 domain and restrains STAT1-mediated transcription of Zbp1. Inhibition of STAT1 by Nifuroxazide ameliorated colitis severity and suppressed necroptotic signaling in Per2 -/- mice, highlighting the PER2–STAT1–ZBP1 axis as a key pathway linking circadian disruption to epithelial injury. Conclusion: Our findings identify PER2 as a critical suppressor of intestinal epithelial necroptosis and inflammation, acting through inhibition of STAT1-dependent ZBP1 activation. Circadian rhythm disruption impairs this protective pathway, exacerbating colitis severity. Pharmacological targeting of STAT1 may offer a novel therapeutic strategy for UC patients with circadian rhythm dysregulation. Ulcerative colitis Circadian rhythm Necroptosis Per2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Ulcerative colitis (UC) is a chronic, debilitating form of inflammatory bowel disease (IBD) characterized by persistent inflammation and ulceration confined to the colon's mucosal layer[ 1 ]. The incidence and prevalence of UC have risen steadily over recent decades, particularly in industrialized nations, posing significant clinical and economic burdens on global healthcare systems[ 2 ]. Although considerable advances have been made in elucidating UC pathogenesis, current treatments remain inadequate for many patients, highlighting the need for deeper insights into its underlying molecular and cellular mechanisms to identify novel therapeutic targets[ 3 ]. Central to UC pathophysiology is the disruption of the intestinal epithelial barrier, composed of specialized intestinal epithelial cells (IECs) that constitute the frontline defense against pathogenic microorganisms and harmful luminal antigens[ 4 , 5 ]. Under normal physiological conditions, IECs maintain intestinal homeostasis through intricate interactions with gut microbiota, immune cells, and environmental stimuli, preserving mucosal integrity by regulating barrier permeability, nutrient absorption, antimicrobial secretion, and epithelial regeneration[ 6 ]. In UC, however, chronic inflammation induces persistent IEC damage, compromising barrier function, enhancing permeability, and perpetuating inflammatory responses via translocation of luminal pathogens and antigens[ 7 ]. These events, in turn, drive excessive immune activation, further amplifying epithelial injury and resulting in a vicious cycle of inflammation, tissue damage, and impaired mucosal repair[ 8 , 9 ]. Consequently, understanding the regulatory mechanisms governing IEC integrity and epithelial homeostasis during inflammatory stress is crucial for developing effective therapeutic strategies for UC. Emerging evidence indicates that circadian rhythms significantly influence intestinal physiology and immune homeostasis[ 10 ]. The circadian system is an evolutionarily conserved endogenous timing mechanism that orchestrates physiological, behavioral, and cellular processes with a periodicity of approximately 24 hours. This system coordinates organismal adaptation to daily environmental cycles, such as light/dark changes and feeding/fasting patterns[ 11 ]. At the molecular level, circadian rhythms are governed by a transcriptional–translational feedback loop involving several core circadian genes, including CLOCK, BMAL1, PER1/2, CRY1/2, and NR1D1, whose rhythmic expression orchestrates downstream processes such as metabolism, immunity, and cell proliferation[ 12 ]. Disruption of circadian rhythms—through shift work, chronic jet lag, sleep deprivation, or genetic mutation—has been increasingly linked to various diseases, including metabolic disorders, cardiovascular disease, autoimmune conditions, and chronic inflammatory diseases[ 13 , 14 ]. Indeed, epidemiological studies have established correlations between circadian disruption and elevated incidence or exacerbation of IBD, suggesting circadian genes may directly impact intestinal inflammation and epithelial barrier function[ 15 ]. Of particular interest among the circadian components is the Period circadian regulator 2 (PER2), a core circadian clock protein involved in generating and maintaining circadian rhythmicity through its transcriptional repression of CLOCK–BMAL1 complexes[ 16 ]. Beyond its canonical circadian functions, PER2 has been implicated in diverse biological processes, including tumor suppression, immune modulation, and oxidative stress responses[ 17 ]. Notably, PER2 dysfunction has recently been associated with increased susceptibility to inflammatory conditions and impaired epithelial barrier integrity, highlighting its potential role as an epithelial homeostasis regulator under inflammatory conditions[ 18 , 19 ]. However, the precise mechanistic relationship between PER2 and epithelial damage in UC remains incompletely understood, particularly regarding the downstream molecular pathways mediating these effects. Given its dual role in circadian regulation and cellular stress responses, PER2 is well positioned to influence the balance of programmed cell death pathways in the intestinal epithelium. Programmed cell death (PCD)—including apoptosis, pyroptosis, and necroptosis—is essential for maintaining intestinal epithelial homeostasis. In UC, chronic inflammatory stimuli disrupt the balance of these pathways, leading to excessive epithelial loss, barrier breakdown, and release of pro-inflammatory mediators that perpetuate disease[ 20 ]. While necroptosis has received particular attention for its inflammatory nature, it functions alongside other PCD modes to determine epithelial fate. Circadian rhythms are increasingly recognized as modulators of PCD[ 21 ]. The core clock protein PER2 regulates stress responses and immune signaling, and its disruption—through environmental or pathological factors—has been linked to heightened cell death in various tissues[ 22 , 23 ]. However, the role of PER2 in controlling PCD in intestinal epithelial cells (IECs) during UC remains unclear. Here, we investigated how PER2 influences PCD in IECs under inflammatory and circadian disruption. Using IEC-specific Per2 knockout mice, transcriptomic profiling, protein–protein interaction studies, molecular docking, chromatin immunoprecipitation, luciferase assays, and pharmacological inhibition, we identified a STAT1-dependent transcriptional pathway as a key effector downstream of PER2. Inhibition of STAT1 with nifuroxazide alleviated colitis and normalized PCD signaling, revealing the PER2–STAT1 axis as a potential therapeutic target, particularly for UC patients with circadian rhythm disturbances. Methods Patient Specimens Specimens were obtained from the digestive medicine biobank at the Union Hospital affiliated with Huazhong University of Science and Technology, following informed consent. Patients were included based on the Chinese UC Diagnosis and Treatment Guidelines: (1) typical clinical manifestations (recurrent diarrhea, tenesmus, mucous and bloody stools, abdominal pain); (2) characteristic endoscopic findings; (3) pathological features such as crypt abscesses and reduced goblet cells; (4) imaging results consistent with UC; (5) a Mayo endoscopic score of 1 to 3; (6) age ≥ 18 years. Exclusion criteria included: (1) other gastrointestinal disorders (e.g., intestinal tumors, Crohn's disease, infectious enteritis); (2) intestinal strictures or severe systemic diseases precluding endoscopic biopsy. Specimens from healthy individuals without UC or other gastrointestinal diseases were also collected. Sleep quality was assessed using the Pittsburgh Sleep Quality Index (PSQI), which has a maximum score of 21, based on Buysse et al[24]. Patients scoring ≤ 7 were classified as having good sleep quality (normal sleep group), while those scoring ≥ 8 were categorized as having poor sleep quality (sleep disorder group). Generation of Intestinal Epithelial Cell-Specific Per2 Knockout Mice, Circadian Disruption Model, and DSS-Induced Colitis Protocol To investigate the role of Per2 in circadian regulation and intestinal inflammation, we generated mice with intestinal epithelial cell-specific deletion of Per2 ( Per2 -/- ) by crossing Villin-Cre transgenic mice (C57BL/6 background; Simo Biotechnology, Nanjing, China) with Per2^flox/flox mice on the same background. Offspring harboring the Villin-Cre transgene and homozygous floxed Per2 alleles (Villin-Cre;Per2^flox/flox) were designated as Per2 -/- mice, while Per2^flox/flox littermates served as wild-type controls. All mice used in experiments were male and 8 weeks of age. Animals were housed under specific pathogen-free (SPF) conditions with stable temperature (22 ± 2°C), relative humidity (60–70%), and a 12-hour light/12-hour dark cycle. Mice had ad libitum access to autoclaved food and water. To model chronic circadian rhythm disruption, a jet lag paradigm was implemented. Mice were subjected to an 8-hour advance in the light phase every 3 days for a total of 8 weeks, simulating repeated eastward travel and persistent circadian misalignment. Light cycles were precisely controlled using an automated environmental lighting system. Control animals were maintained under a constant 12:12-hour light/dark cycle for the same duration. To induce experimental colitis, mice were administered 2.5% (w/v) dextran sulfate sodium (DSS; MW 36,000–50,000; MP Biomedicals) in their drinking water for 8 consecutive days. Each experimental group ( Per2 f/f and Per2 -/- ) consisted of six mice. Upon completion of DSS treatment, mice were euthanized, and the entire colon was excised. A distal colonic segment (~0.5 cm) was fixed in 4% paraformaldehyde and processed for histological evaluation, including hematoxylin and eosin (H&E) staining and immunofluorescence microscopy. All animal experiments were conducted in accordance with institutional guidelines and approved by the Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. Preparation of Purified Colonic Epithelial Cells for Downstream Molecular Analyses Colonic epithelial cells (IECs) were isolated from mouse colon using a chelation-based dissociation protocol to enable high-quality transcriptomic and proteomic profiling[25]. Briefly, colons were excised, thoroughly rinsed with ice-cold DPBS, and sectioned into ~0.5 cm fragments. Tissues were incubated at 4°C for 75 minutes in an EDTA/DTT dissociation buffer (14 mL DPBS, 0.9 mL 0.5 M EDTA, 22.5 µL 1 M DTT) under gentle agitation to promote epithelial cell detachment. Following incubation, samples underwent three successive rounds of centrifugation and resuspension in fresh buffer to enrich for epithelial cells. After each centrifugation step, the supernatant was discarded and the pellet was resuspended in 5 mL of buffer. The final cell suspension was passed through a 40-μm nylon mesh to remove debris and yield a purified single-cell epithelial fraction. Isolated IECs were immediately processed for downstream applications, including quantitative PCR, Western blotting, and bulk RNA sequencing. High-Throughput RNA-Seq Analysis of Colonic Inflammation in PER2 Knockout Mice Total RNA was extracted from colonic tissues of Per2 -/- , Per2 f/f , Per2 -/- + DSS, Per2 f/f + DSS, and WT + DSS mice (n = 3 per group) for transcriptomic analysis. RNA isolation, quality control, library preparation, and high-throughput sequencing were performed by Frasergen Co., Ltd. (Wuhan, China) using the Illumina NovaSeq 6000 platform. Raw sequencing data were processed and analyzed using the DESeq2 package in R, with differentially expressed genes (DEGs) defined as those with an absolute fold change > 2 and adjusted P < 0.05, based on the Benjamini–Hochberg correction. Data visualization, including volcano plots and heatmaps, was generated using the ggplot2 and ComplexHeatmap packages. Functional annotation and enrichment analyses, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Gene Set Enrichment Analysis (GSEA), were performed with a significance cutoff of P < 0.05. Histological Staining Distal colonic segments (~0.5 cm) were collected from euthanized mice, fixed in 10% neutral-buffered formalin overnight at room temperature, and embedded in paraffin. Tissues were sectioned at 5 μm thickness and stained with haematoxylin and eosin (H&E) following standard protocols. Haematoxylin was used to visualize nuclei, while eosin counterstained the cytoplasm and extracellular matrix. Histological scoring was performed by two independent, blinded pathologists, who evaluated inflammatory cell infiltration, mucosal architecture, and tissue damage. Discrepancies in scoring were resolved by consensus. GEO Dataset Analysis Transcriptomic data were obtained from the Gene Expression Omnibus (GEO) database (accession number: GSE38713), which includes gene expression profiles of colonic mucosal biopsies from human subjects. The dataset comprises three groups: 13 healthy controls, 15 ulcerative colitis (UC) patients in clinical remission, and 15 patients with active UC. The normalized gene expression matrix was downloaded and analyzed to assess the expression levels of core circadian clock genes. Differential expression analysis and statistical comparisons between groups were conducted using appropriate bioinformatic tools in R, with significance thresholds defined as P < 0.05. Quantitative PCR Analysis of Gene Expression in IECs Quantitative real-time PCR (qPCR) was conducted to evaluate gene expression in isolated intestinal epithelial cells (IECs) using a LightCycler system (Roche Diagnostics) and SYBR Green PCR Master Mix (Takara, Cat# RR036A). Each reaction mixture contained cDNA template, gene-specific primers obtained from PrimerBank (https://pga.mgh.harvard.edu/primerbank/), and SYBR Green reagent. Thermal cycling conditions were as follows: initial denaturation at 95°C for 3 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. Amplification specificity was confirmed by melting curve analysis. All reactions were run in triplicate, and relative gene expression was calculated using the ΔΔCt method, with GAPDH used as the endogenous normalization control. Immunoblot Analysis of Protein Expression Proteins were extracted from IECs isolated from murine colon, human colonic biopsies, and organoids using RIPA buffer (Thermo Fisher Scientific, MA, USA) with protease and phosphatase inhibitors (Roche). Protein concentrations were measured using a BCA assay (Pierce, IL, USA). Equal amounts (20–30 µg) were separated by SDS–PAGE (10–12%) and transferred to PVDF membranes (Millipore, MA, USA). Membranes were blocked with 5% milk or BSA in TBS-T, incubated overnight at 4 °C with primary antibodies, and then with HRP-conjugated secondary antibodies for 1 hour at room temperature. Bands were visualized using ECL (ECL; GE Healthcare, NJ, USA) and imaged with a ChemiDoc system (Bio-Rad). Densitometry was performed using ImageJ, with GAPDH as the loading control. Co-immunoprecipitation and Pulldown Assays NCM460 cells were transfected with FLAG–PER2 plasmids using Lipofectamine 3000 and harvested 48 h later. Cells were lysed in RIPA buffer with protease inhibitors, and lysates were centrifuged at 12,000 × g for 15 min at 4 °C. Supernatants were incubated overnight at 4 °C with anti-FLAG (DYKDDDDK) magnetic beads. After washing, bound proteins were eluted in SDS loading buffer at 95 °C and analyzed by Western blot. Gel bands (~1 cm) were excised, digested with trypsin, and subjected to LC-MS/MS using an EASY-nLC 1200 system and Q-Exactive HF mass spectrometer. Raw data were searched against the SwissProt database using Proteome Discoverer v2.4 to identify PER2-interacting proteins. HEK293T cells were transfected with His–STAT1 plasmids and lysed in NET buffer. Lysates were incubated with anti-His magnetic beads, and bound proteins were eluted stepwise with 20 mM, 50 mM, and 250 mM imidazole. For pulldown assays, HEK293T cells were transfected with FLAG–PER2 (WT or mutants). Lysates were pre-cleared with Protein A/G beads, incubated with purified His–STAT1 for 2 h, followed by anti-His bead capture. Complexes were eluted with SDS buffer and analyzed by Western blot. Protein – Protein Docking and Domain Mapping The 3D crystal structure of human STAT1 was obtained from the Protein Data Bank (PDB), and the full-length structure of PER2 was predicted using AlphaFold. Preprocessing, including removal of water molecules and addition of hydrogen atoms, was performed in AutoDockTools v1.5.7. Protein–protein docking was carried out using the GRAMM-X server, with STAT1 set as the receptor and PER2 as the ligand. Among the generated models, the top-ranked complex was selected based on docking score and subjected to energy minimization. The predicted interaction interface was visualized and analyzed using PyMOL, with key contact residues annotated. To experimentally validate the docking predictions and define the minimal region of PER2 required for STAT1 binding, truncated mutants targeting the PAS1, PAS2, and PAC domains were constructed and cloned into FLAG-tagged expression vectors. These constructs were transfected into HEK293T cells, and protein–protein interactions were assessed via co-immunoprecipitation and pulldown assays using purified His–STAT1 protein. The PAS1 domain was identified as essential for STAT1 interaction. Chromatin Immunoprecipitation (ChIP) Assays ChIP assays were performed to assess STAT1 binding to the ZBP1 promoter in colonic epithelial cells. Cells were fixed with 1% formaldehyde to cross-link protein–DNA complexes, and chromatin was extracted and sonicated to obtain DNA fragments of 200–500 bp. Immunoprecipitation was carried out using the SimpleChIP® Enzymatic Chromatin IP Kit (Cell Signaling Technology, Cat# 9003), following the manufacturer’s instructions. Samples were incubated overnight at 4 °C with a STAT1-specific antibody or normal rabbit IgG (negative control), followed by capture with magnetic protein G beads for 2 hours. After reverse cross-linking and DNA purification, the immunoprecipitated DNA was analyzed by PCR using primers targeting the predicted STAT1 binding site within the human ZBP1 promoter. Primers were designed using SnapGene software. Luciferase Reporter Assays Dual-luciferase reporter assays were performed to evaluate the transcriptional activity of the ZBP1 promoter in STAT1-deficient and control cells. The promoter region of ZBP1, either wild-type (WT) or containing a mutated STAT1-binding site (Mut), was cloned into the pGL4.11-Basic vector (Promega). Cells were co-transfected with the reporter plasmid and a Renilla luciferase control vector (pRL-TK) using Lipofectamine 3000 (Thermo Fisher Scientific, MA, USA). After 24–48 hours, luciferase activity was measured using the Dual-Luciferase® Reporter Assay System (Promega, Madison, USA) and quantified with a luminometer. Firefly luciferase activity was normalized to Renilla luciferase to control for transfection efficiency and sample variability. Immunofluorescence Staining of Colonic Tissue For tissue immunofluorescence, paraffin-embedded sections of mouse colon were deparaffinized, rehydrated through graded alcohols, and subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0). Sections were blocked with 5% BSA to reduce non-specific binding, followed by incubation with primary antibodies (1:100 dilution) overnight at 4 °C. After washing, fluorescently labeled secondary antibodies were applied for 1 hour at room temperature in the dark. Nuclei were counterstained with Hoechst 33342. Stained sections were mounted with antifade medium and imaged using a Zeiss LSM-800 confocal microscope (Carl Zeiss AG, Oberkochen, Germany). Flow Cytometric Analysis of Apoptosis Following PER2 Knockdown and LPS Stimulation Apoptotic cell death was assessed in NCM460 human intestinal epithelial cells subjected to PER2 knockdown and lipopolysaccharide (LPS) treatment, with or without pharmacological inhibition. Cells were stained using the Annexin V-FITC/PI apoptosis detection kit (Beyotime) according to the manufacturer’s protocol. Briefly, after experimental treatments, cells were harvested, washed with cold PBS, and resuspended in binding buffer. Annexin V-FITC and propidium iodide (PI) were then added to the suspension, followed by incubation for 15 minutes at room temperature in the dark. Stained cells were analyzed immediately using flow cytometry to quantify early apoptotic (Annexin V⁺/PI⁻), late apoptotic or necrotic (Annexin V⁺/PI⁺), and viable (Annexin V⁻/PI⁻) populations. Results Circadian rhythm disruption aggravates colonic inflammation and suppresses PER2 expression in both mouse and human colitis To model the impact of circadian misalignment on intestinal inflammation, we employed a jet lag mouse protocol involving repeated 8-hour light phase advances every three days, simulating chronic rhythm disruption. Colitis was induced during the final week by administering 2.5% dextran sulfate sodium (DSS) in drinking water. Mice subjected to both jet lag and DSS treatment exhibited significantly greater weight loss compared to DSS-treated controls (WT + DSS), indicating more severe disease progression (Fig. S1A). Macroscopic examination revealed notable colon shortening in the Jet Lag + DSS group (Fig. S1B), confirmed by quantitative measurement (Fig. S1C). These morphological changes were accompanied by elevated Disease Activity Index (DAI) scores (Fig. S1D) and worsened histopathological outcomes, including epithelial erosion, crypt distortion, and inflammatory cell infiltration (Fig. S1F). Histological scoring further confirmed significantly aggravated mucosal injury in rhythm-disrupted mice relative to controls (Fig. S1E). Together, these findings suggest that circadian misalignment sensitizes the colon to inflammatory injury and amplifies DSS-induced tissue damage. To explore the underlying mechanisms, we profiled the expression of core circadian genes in colonic tissue collected at four Zeitgeber time (ZT) points (ZT0, 6, 12, and 18). WT mice exhibited clear rhythmic expression of Per2, Per1, Bmal1, Clock, Cry1, and Nr1d1 over a 24-hour cycle (Fig. S1G). DSS treatment dampened the amplitude and disrupted the phase of these transcripts, most notably Per2. In the Jet Lag + DSS group, Per2 levels were consistently suppressed across all time points, with similar but less pronounced reductions observed in Per1, Bmal1, Cry1, and Nr1d1. Clock expression was modestly affected. These results indicate that colitis perturbs circadian gene oscillations in the colon, and circadian misalignment further disrupts this regulatory network, with Per2 being especially vulnerable. To assess the relevance of these findings in humans, we clinically stratified individuals based on both UC status and sleep quality. Colonic biopsies were collected from four groups: healthy controls (HC), healthy individuals with chronic sleep disturbance (HC + Jet Lag), UC patients without sleep disorders (UC), and UC patients reporting comorbid sleep disruption (UC + Jet Lag). TUNEL staining showed low levels of epithelial apoptosis in HC and HC + Jet Lag samples, while UC patients displayed increased apoptotic signals, further amplified in the UC + Jet Lag group (Fig. 1A). These data suggest that circadian disruption may contribute to epithelial injury in colitis. We next examined PER2 protein expression in these patient groups. Immunofluorescence staining revealed abundant PER2 localization in the epithelial compartment of HC and HC + Jet Lag samples. In contrast, PER2 signal intensity was reduced in UC patients and nearly absent in those with both UC and sleep disruption (Fig. 1B), suggesting a synergistic effect of inflammation and circadian dysfunction on PER2 suppression. To support these observations, we analyzed transcriptomic data from dataset GSE38713, which includes colonic biopsies from healthy controls (HC), patients with active UC (A-UC), and patients in remission (R-UC). PER2 mRNA expression was significantly reduced in A-UC compared to HC, while PER1 levels were unchanged (Fig. 1C). Notably, both CLOCK and CRY1 transcripts were upregulated in R-UC samples relative to A-UC and HC, suggesting reprogramming of circadian machinery during recovery. Western blot analysis of fresh patient biopsies confirmed downregulation of PER2 protein in UC and UC + Jet Lag samples (Fig. 1D), corroborating the immunostaining and transcriptomic data. Altogether, these findings establish a conserved relationship between circadian rhythm disruption and impaired PER2 expression in both murine and human colitis. The concomitant increase in epithelial apoptosis under conditions of rhythm misalignment underscores a mechanistic link between circadian dysfunction and the exacerbation of mucosal inflammation. Epithelial PER2 deficiency exacerbates colitis via transcriptional reprogramming and necroptosis-driven inflammation To clarify the functional role of PER2 in the intestinal epithelium, we generated conditional knockout mice with epithelium-specific deletion of Per2 ( Per2 -/- ) using the Cre-loxP system. Intestinal epithelial cells were isolated from Per2 -/- and littermate Per2 flox/flox ( Per2 f/f ) control mice for molecular analysis. Western blotting confirmed a substantial reduction in PER2 protein levels in knockout mice compared to controls (Fig. S2A–B), and this was paralleled by a significant decrease in Per2 mRNA expression as assessed by qPCR (Fig. S2C). In agreement with these findings, immunofluorescence staining of intestinal sections revealed markedly attenuated PER2 signal in the epithelial layer of Per2 -/- mice relative to Per2 f/f controls (Fig. S2D). These data collectively validate the successful generation of a tissue-specific Per2 knockout model and confirm efficient loss of gene expression at both transcript and protein levels within the intestinal epithelium. To elucidate the global transcriptional consequences of Per2 deletion in intestinal epithelial cells, we performed bulk RNA sequencing on isolated epithelial cells from Per2 -/- and Per2 f/f mice. Principal Component Analysis (PCA) revealed clear separation between the two groups, indicating a distinct transcriptomic landscape driven by Per2 deficiency (Fig. 2A). Differential gene expression analysis visualized by volcano plot further confirmed widespread transcriptional alterations following Per2 deletion (Fig. 2B). Gene Ontology (GO) enrichment analysis highlighted significant upregulation of pathways associated with hydrolase activity, cell communication, and Toll-like receptor signaling (Fig. 2C). KEGG pathway analysis revealed that differentially expressed genes were enriched in multiple inflammatory and immune-related pathways, including TNF signaling, JAK-STAT signaling, and the inflammatory bowel disease (IBD) pathway (Fig. 2D). Of particular interest, necroptosis emerged as one of the top enriched pathways, suggesting a possible mechanistic link between Per2 loss and regulated necrotic cell death. Necroptosis is a regulated form of lytic cell death mediated by receptor-interacting protein kinase 3 (RIPK3) and its downstream effector, mixed lineage kinase domain-like pseudokinase (MLKL). Upon activation by death receptors or pattern recognition receptors, MLKL undergoes phosphorylation, oligomerization, and translocation to the plasma membrane, where it disrupts membrane integrity, leading to cell rupture and the release of pro-inflammatory contents[26]. In the intestinal epithelium, excessive necroptosis compromises barrier function and amplifies mucosal inflammation, thereby contributing to ulcerative colitis pathogenesis. To further explore this potential link, we focused on necroptosis-associated genes. Heatmap analysis of the RNA-seq data revealed upregulation of Zbp1, Ripk3, Mlkl, Stat1, and Stat2 in Per2 -/- samples relative to controls (Fig. 2E), indicating transcriptional activation of necroptosis machinery in the absence of PER2. We next validated these observations at the protein level. Western blotting confirmed increased expression of ZBP1, MLKL, and RIPK3 in Per2 -/- mice, along with enhanced phosphorylation of MLKL, a key effector of necroptosis (Fig. 2F). Quantitative analysis normalized to GAPDH corroborated these findings, with significantly elevated levels of ZBP1, MLKL, and RIPK3 proteins in the knockout group (Fig. 2G). While p-MLKL and p-RIPK3 also trended upward, only p-MLKL showed a statistically significant increase. Collectively, these results demonstrate that epithelial-specific Per2 deletion activates necroptosis-related pathways at both the transcript and protein levels. This suggests that PER2 may function as a gatekeeper of epithelial cell survival by suppressing programmed necrotic cell death, thereby contributing to the maintenance of intestinal barrier integrity. To investigate the functional impact of PER2 on intestinal inflammation, we employed a dextran sulfate sodium (DSS)-induced acute colitis model using four experimental groups: Per2 f/f , Per2 -/- , Per2 f/f + DSS, and Per2 -/- + DSS. During DSS administration, Per2 -/- mice exhibited significantly greater body weight loss compared to Per2 f/f controls (Fig. 3A), suggesting enhanced susceptibility to colonic injury in the absence of PER2. Macroscopic evaluation of colonic tissue revealed pronounced shortening in Per2 -/- + DSS mice (Fig. 3B), while histological examination demonstrated severe epithelial destruction, crypt loss, and inflammatory infiltration compared to all other groups (Fig. 3C). Notably, Per2 deletion alone did not significantly affect colon length, but when combined with DSS treatment, it resulted in marked tissue damage and shortening (Fig. 3D). Disease severity was further confirmed by increased Disease Activity Index (DAI) scores (Fig. 3E) and elevated histological scores (Fig. 3F) in Per2 -/- + DSS mice relative to Per2 f/f + DSS controls. Consistent with these pathological features, transcriptional analysis of inflammatory mediators revealed that mRNA levels of Tnfα, Ifnγ, Il1β, and Il6 were significantly upregulated in the Per2 -/- + DSS group (Fig. 3G), indicating a heightened proinflammatory response. Given our prior findings linking PER2 deficiency to necroptosis pathway activation, we next assessed the expression of necroptosis-associated proteins. Western blotting showed significantly increased expression of ZBP1, MLKL, and RIPK3, along with their phosphorylated forms p-MLKL and p-RIPK3 in Per2 -/- + DSS mice compared to Per2 f/f + DSS controls (Fig. 3H–I). To determine whether these molecular changes are conserved in human disease, we examined necroptosis-related protein expression in colonic tissues from ulcerative colitis (UC) patients with or without sleep disruption. Immunoblotting revealed increased levels of ZBP1, MLKL, and RIPK3 in UC specimens, particularly in those with concurrent jet lag (Fig. S3A), further supporting a role for PER2 in suppressing necroptotic signaling during mucosal inflammation. Finally, to localize and quantify necroptotic cell death, we performed TUNEL staining combined with MLKL immunofluorescence on mouse colon sections. Confocal microscopy revealed that Per2 -/- + DSS mice displayed significantly stronger TUNEL (green) and MLKL (red) signals than the other groups, indicating a marked increase in necroptotic epithelial cell death in the absence of PER2 under inflammatory conditions (Fig. S3B). Collectively, these findings indicate that epithelial-specific deletion of Per2 exacerbates DSS-induced colitis by amplifying inflammatory responses and promoting necroptosis, a process that may be similarly activated in human UC associated with circadian disruption. To determine the transcriptional consequences of PER2 deletion in intestinal epithelial cells under inflammatory conditions, we performed bulk RNA sequencing on epithelial cells isolated from DSS-treated Per2 -/- and Per2 f/f mice. Principal Component Analysis (PCA) revealed a distinct separation between the two groups, indicating robust transcriptomic divergence induced by Per2 loss (Fig. 4A). Differential expression analysis, visualized through a volcano plot, identified numerous upregulated and downregulated genes in the Per2 -/- + DSS group relative to controls (Fig. 4B). To explore the functional relevance of these transcriptional alterations, we conducted comprehensive enrichment analyses. Gene Ontology (GO) analysis showed that differentially expressed genes (DEGs) were significantly enriched in biological processes related to immune system activation, including “immune response,” “defense response,” and “cell adhesion” (Fig. 4C). Enrichment in cellular components (CC) included extracellular matrix, supramolecular complexes, and cytoskeletal structures (Fig. 4D), while molecular function (MF) terms such as “cytokine activity” and “signaling receptor binding” suggested a shift toward proinflammatory cytokine signaling (Fig. 4E). KEGG pathway analysis further supported this notion, identifying significant enrichment in several inflammatory and immune-regulatory signaling pathways, including the JAK-STAT signaling pathway, TNF signaling, NF-κB pathway, Toll-like receptor signaling, and the Inflammatory Bowel Disease (IBD) pathway (Fig. 4F). These findings suggest that Per2 deletion potentiates inflammatory signaling cascades in the context of colonic injury. To validate these observations, Gene Set Enrichment Analysis (GSEA) was performed. The results confirmed significant upregulation of key inflammatory pathways—namely the TNF, JAK-STAT, and NF-κB signaling axes—in the Per2 -/- + DSS group compared to Per2 f/f + DSS controls (Fig. 4G). Together, these data indicate that loss of Per2 reprograms the epithelial transcriptome toward a proinflammatory state and amplifies the activation of canonical immune pathways during acute intestinal inflammation. PER2 knockdown promotes LPS-induced necroptosis in human intestinal epithelial cells via ZBP1-dependent signaling To further explore the mechanistic role of PER2 in regulating epithelial cell death, we employed an in vitro model using human colonic epithelial NCM460 cells. Four experimental groups were established: control (NC), PER2 knockdown (PER2 KD), control with LPS stimulation (NC + LPS), and PER2 knockdown with LPS (PER2 KD + LPS). Apoptosis was assessed by flow cytometry using Annexin V-FITC and PI staining. PER2 knockdown alone did not significantly increase apoptosis compared to controls. However, when combined with LPS, PER2 silencing significantly augmented apoptotic cell death relative to LPS-treated controls (Fig. 5A), indicating that PER2 deficiency sensitizes epithelial cells to LPS-induced cell death. To determine whether this increase in apoptosis involved necroptosis, we employed the RIPK3 inhibitor GSK872. The addition of GSK872 did not significantly alter apoptosis in the NC + LPS group, but markedly reduced apoptosis in PER2 KD + LPS cells (Fig. 5B), suggesting that PER2 deficiency promotes necroptotic rather than apoptotic cell death under inflammatory stress. Consistent with this interpretation, Western blot analysis revealed that PER2 knockdown in LPS-stimulated cells led to increased expression of necroptosis-associated proteins ZBP1, MLKL, and RIPK3, as well as their phosphorylated forms p-MLKL and p-RIPK3 (Fig. 5C). These data suggest activation of the canonical ZBP1-RIPK3-MLKL necroptotic axis in the absence of PER2. Given that our previous transcriptomic data implicated ZBP1 as a key upstream mediator, we next assessed the effects of ZBP1 knockdown using siRNA. Western blotting showed that inhibition of ZBP1 in PER2 KD + LPS cells markedly reduced the expression of MLKL, RIPK3, and their phosphorylated counterparts (Fig. 5D), supporting a central role for ZBP1 in this pathway. Moreover, flow cytometry demonstrated that apoptosis was significantly attenuated in PER2 KD + LPS cells following ZBP1 knockdown (Fig. 5E), confirming that ZBP1 mediates PER2-deficiency–induced necroptosis. Together, these findings establish ZBP1 as a critical effector linking PER2 loss to necroptosis activation in intestinal epithelial cells during inflammatory challenge. STAT1 mediates PER2-deficiency – induced necroptosis through transcriptional activation of ZBP1 To investigate how PER2 regulates necroptosis at the transcriptional level, we performed immunoprecipitation (IP) of PER2 in NCM460 human intestinal epithelial cells, followed by mass spectrometry analysis to identify PER2-interacting proteins. Cross-referencing these candidates with a curated list of necroptosis-associated proteins revealed STAT1 and STAT3 as potential interactors (Fig. S4A), implicating them as downstream mediators of PER2[27]. To validate this finding in vivo, we analyzed STAT1 and STAT3 expression in colonic epithelial cells isolated from Per2 -/- and Per2 f/f mice subjected to DSS-induced colitis. Western blotting showed a significant increase in STAT1 protein levels in Per2 -/- + DSS mice compared to controls, whereas STAT3 expression remained unchanged (Fig. 6A; Fig. S4B), suggesting selective regulation of STAT1 by PER2 in inflamed colonic tissue. To determine the functional contribution of STAT1 to necroptosis, we knocked down STAT1 or STAT3 in LPS-treated PER2-deficient NCM460 cells. Western blot analysis revealed that STAT1 knockdown markedly reduced expression of ZBP1, MLKL, RIPK3, and their phosphorylated forms p-MLKL and p-RIPK3 (Fig. 6B), whereas STAT3 knockdown had no significant effect, indicating a critical role for STAT1 in mediating necroptotic signaling downstream of PER2 loss. Motif prediction using the JASPAR database identified a canonical STAT1 binding motif (Fig. 6C). To experimentally verify direct transcriptional regulation of ZBP1 by STAT1, we performed chromatin immunoprecipitation (ChIP) assays in LPS-stimulated NCM460 cells. STAT1 was found to directly bind to the ZBP1 promoter in both PER2 knockdown and control conditions (Fig. 6D), indicating that PER2 deficiency does not impair binding but may enhance activation. To further test whether STAT1 binding promotes ZBP1 transcription, we constructed dual-luciferase reporter plasmids harboring wild-type or mutated STAT1-binding sites in the ZBP1 promoter (Fig. S4C). Luciferase assays showed significantly enhanced transcriptional activity in PER2-deficient cells transfected with the wild-type construct, but not with the mutant construct (Fig. 6E). This effect was abolished by STAT1 knockdown (Fig. 6F), confirming that STAT1 is essential for ZBP1 transcriptional activation in the context of PER2 deficiency. Together, these findings establish STAT1 as a key transcriptional mediator linking PER2 loss to necroptosis via direct regulation of ZBP1 expression in intestinal epithelial cells. To validate the interaction between PER2 and key transcription factors STAT1 and STAT3, we constructed a FLAG-tagged PER2 expression vector and performed co-immunoprecipitation (Co-IP) assays in NCM460 intestinal epithelial cells. Western blot analysis confirmed that PER2 physically interacts with both STAT1 and STAT3 (Fig. 7A–B). Notably, reverse Co-IP using anti-STAT1 antibodies further validated the binding between PER2 and STAT1. To delineate the structural basis of this interaction, we performed in silico molecular docking using PyMol, which predicted multiple hydrogen bonds between PER2 and STAT1. Specifically, residues within PER2—ARG325, ARG399, ALA446, TYR407, GLU322, and SER279—were found to form stable hydrogen bonds with GLN314, ASN483, ASN233, GLU480, GLN311, and GLU141 of STAT1 (Fig. 7C). These interactions clustered within or near the PAS1, PAS2, and PAC domains of PER2, suggesting potential structural hotspots critical for protein binding. To experimentally test which domains are responsible for STAT1 interaction, we generated a series of truncated PER2 constructs targeting the PAS1, PAS2, and PAC domains, as well as N-terminal deletion mutants (Fig. S5A). Co-IP assays revealed that deletion of the PAS1 domain (ΔPAS1) or expression of the N-N2 and N-N3 truncations markedly impaired PER2–STAT1 binding (Fig. 7D–E), while deletions of PAS2 or PAC (ΔPAS2, ΔPAC) did not significantly affect the interaction. These findings identify PAS1 as the essential structural domain mediating PER2’s interaction with STAT1. To assess the functional relevance of the PAS1 domain in necroptotic signaling, we analyzed the expression of necroptosis-related proteins in LPS-stimulated cells transfected with either full-length PER2 or the ΔPAS1 mutant. Western blotting showed that loss of the PAS1 domain significantly reduced the expression of ZBP1, MLKL, RIPK3, and their phosphorylated forms (p-MLKL and p-RIPK3) compared to controls (Fig. S5B). These results indicate that the PAS1-dependent interaction between PER2 and STAT1 is essential for the transcriptional activation of necroptosis machinery. Together, these findings demonstrate that PER2 physically interacts with STAT1 via its PAS1 domain, and that this interaction is necessary for the activation of necroptosis-related pathways in intestinal epithelial cells under inflammatory stress. Pharmacological inhibition of STAT1 by Nifuroxazide attenuates colitis severity and necroptosis in Per2-deficient mice To determine whether STAT1 is a viable therapeutic target in Per2 -/- mice with colitis, we administered the selective STAT1 inhibitor Nifuroxazide during DSS-induced acute intestinal inflammation[28]. To determine whether STAT1 is a viable therapeutic target in Per2 -/- mice with colitis, we first prepared sodium alginate–Nifuroxazide hydrogel microspheres and examined their morphology by scanning electron microscopy. The microspheres displayed a smooth external surface (Fig. S6A) and a porous internal architecture (Fig. S6B), a feature expected to facilitate controlled Nifuroxazide release. Mice were divided into two groups: Per2 -/- + DSS (control, receiving blank hydrogel) and Per2 -/- + DSS + Nifuroxazide (receiving nifuroxazide-loaded hydrogel). Throughout the disease course, mice receiving Nifuroxazide exhibited significantly reduced body weight loss compared to untreated controls (Fig. 8A), indicating that STAT1 inhibition mitigates colitis severity. Gross anatomical examination revealed visibly longer colons in Nifuroxazide-treated mice (Fig. 8B), which was quantitatively confirmed by colon length measurements (Fig. 8C). Disease Activity Index (DAI) scores were also significantly lower in the treatment group (Fig. 8D), and histopathological scoring of H&E-stained colon sections showed attenuated epithelial damage and reduced inflammatory cell infiltration (Fig. 8E–F). These findings suggest that Nifuroxazide alleviates DSS-induced colonic injury in Per2 -/- mice. To investigate whether the protective effects of Nifuroxazide were mediated through inhibition of necroptosis, we analyzed the expression of necroptosis-related proteins in colonic tissue. Western blot analysis revealed markedly reduced expression of STAT1, ZBP1, MLKL, and RIPK3, along with diminished phosphorylation of MLKL and RIPK3, in the Nifuroxazide-treated group compared to controls (Fig. 8G–I). Quantitative densitometric analysis confirmed the statistical significance of these reductions. Furthermore, TUNEL/MLKL co-immunofluorescence staining demonstrated a substantial decrease in both apoptotic and necroptotic signals in colonic epithelial cells following Nifuroxazide treatment (Fig. 8J), suggesting that pharmacologic blockade of STAT1 protects epithelial integrity by suppressing necroptosis. Collectively, these results indicate that STAT1 inhibition via Nifuroxazide alleviates intestinal inflammation and epithelial injury in Per2 -/- mice by dampening necroptotic signaling, highlighting STAT1 as a potential therapeutic target in PER2-deficient colitis. Discussion Our study identifies the circadian regulator PER2 as a critical suppressor of epithelial necroptosis in ulcerative colitis (UC) via modulation of the STAT1–ZBP1 axis. Using a jet lag model of circadian disruption, we showed that altered light–dark cycles significantly aggravated DSS-induced colitis, with worsened weight loss, colon shortening, histological injury, and elevated disease scores (Fig. S1A–E). Rhythm disruption further suppressed Per2 expression, already downregulated in DSS-treated mice, indicating a link between circadian misalignment and intestinal vulnerability (Fig. S1F–G). In human UC biopsies, PER2 expression was reduced, particularly in patients with concurrent sleep disorders, and PER2 loss was associated with increased epithelial apoptosis (Fig. 1 A–D). Transcriptomic analysis from GSE38713 confirmed significant downregulation of PER2 in active UC (Fig. 1 C). To define PER2’s role functionally, we generated intestinal epithelial cell-specific Per2 knockout mice ( Per2 −/− ). These mice exhibited exacerbated DSS-induced colitis, with increased inflammatory infiltration, crypt damage, and elevated DAI and histological scores (Fig. 3 A–F). RNA-seq of Per2 −/− IECs revealed enrichment of necroptosis and inflammatory pathways, notably TNF and JAK–STAT signaling (Fig. 2 C–D), and upregulation of Zbp1, Ripk3, and Mlkl at both transcript and protein levels (Fig. 2 E–G). TUNEL and MLKL co-staining further confirmed enhanced epithelial necroptosis (Fig. S3B). Mechanistically, we demonstrated that PER2 binds STAT1 via its PAS1 domain, a requirement for repressing necroptosis-related gene expression (Fig. 7 A–E, Fig. S5). Loss of this interaction led to STAT1-dependent transcriptional activation of Zbp1, validated by ChIP and luciferase assays (Fig. 6 C–F). Targeted inhibition of STAT1 with Nifuroxazide significantly alleviated colitis severity, suppressed necroptosis markers, and restored epithelial integrity in Per2 −/− mice (Fig. 8 A–J). Together, these findings uncover a previously unrecognized PER2–STAT1–ZBP1 axis that links circadian regulation to epithelial cell death. They also suggest that targeting STAT1 may offer therapeutic benefit for UC patients experiencing circadian disruption. The intestinal epithelium forms a dynamic interface between the host immune system and the luminal environment. Its integrity is essential for preventing microbial translocation and maintaining mucosal homeostasis, especially in the context of chronic inflammatory diseases such as ulcerative colitis (UC)[ 29 ]. Prior studies have primarily focused on tight junction proteins, cytokine signaling, and epithelial regeneration as determinants of barrier function, while the contribution of circadian regulators to epithelial cell death and survival has remained largely underexplored. Among core clock components, PER2 has been previously implicated in modulating inflammation, oxidative stress, and cell cycle regulation in peripheral tissues, including the liver and vasculature. In the gut, however, its function has remained ambiguous[ 30 , 31 ]. While several studies have described diurnal oscillations of clock genes in intestinal tissues, few have directly linked PER2 to epithelial injury responses in UC[ 32 ]. Our study fills this gap by demonstrating that PER2 plays a non-redundant and protective role in maintaining epithelial integrity during acute colitis. In contrast to work emphasizing the anti-inflammatory effects of BMAL1 or NR1D1 through immune cell regulation, we show that PER2 acts within epithelial cells themselves to suppress necroptotic signaling cascades[ 33 , 34 ]. Necroptosis is a programmed, pro-inflammatory form of cell death executed through the RIPK3–MLKL axis, leading to membrane rupture and the release of intracellular damage-associated molecular patterns (DAMPs) that amplify inflammation. Recent work has highlighted necroptosis as a key contributor to epithelial loss in UC, with upregulation of RIPK3 and MLKL observed in inflamed human colonic tissues[ 35 – 37 ]. ZBP1, a sensor of nucleic acids and endogenous danger signals, has also emerged as a driver of necroptotic activation in epithelial and immune cells[ 38 , 39 ]. However, the upstream regulation of this pathway in the context of circadian rhythms has not been established. Our findings reveal that PER2 constrains ZBP1-mediated necroptosis by repressing STAT1 activity, positioning it as a central checkpoint linking the circadian clock to inflammatory epithelial cell death. Notably, unlike traditional views of PER2 as a passive output of CLOCK–BMAL1 transcription[ 40 ], our data suggest that PER2 may act as a direct modulator of transcription factor activity, specifically by binding and inhibiting STAT1’s access to inflammatory gene promoters. This is distinct from prior studies in macrophages or cancer cells where PER2 was shown to regulate inflammation indirectly through systemic cues or metabolic reprogramming[ 41 , 42 ]. Our work therefore expands the functional repertoire of PER2, positioning it as a local, cell-autonomous regulator of epithelial fate under inflammatory stress. Taken together, these findings establish PER2 not only as a circadian rhythm gene but also as an epithelial stress-response factor. They challenge the classical paradigm that circadian genes operate solely through transcriptional oscillators and suggest that disruption of circadian homeostasis—whether via environmental light shifts or intrinsic gene suppression—can directly sensitize the epithelium to inflammatory injury[ 43 – 45 ]. This mechanistic link between clock gene dysfunction and necroptotic epithelial loss may have broad implications for understanding barrier failure in UC and other chronic inflammatory disorders. The identification of a PER2–STAT1–ZBP1 regulatory axis in epithelial necroptosis not only advances our understanding of UC pathogenesis but also opens new avenues for therapeutic intervention. Our data show that pharmacological inhibition of STAT1 with Nifuroxazide significantly attenuates colitis severity in Per2 −/− mice, rescuing epithelial architecture and reducing necroptosis-associated protein expression (Fig. 8 A–J). These results support the concept that targeting STAT1 activation downstream of circadian disruption may serve as a viable strategy to protect intestinal epithelial cells under inflammatory stress. Given that circadian disruption is increasingly prevalent in modern society—affecting shift workers, frequent travelers, and individuals with sleep disorders—this mechanism has particular relevance to a growing subset of UC patients whose disease severity may be aggravated by chronobiological misalignment[ 46 , 47 ]. Moreover, transcriptomic analyses from patient biopsies revealed that PER2 is suppressed in active UC and further downregulated in individuals with disordered sleep (Fig. 1 ), suggesting that PER2 deficiency may represent a biomarker of heightened disease susceptibility or treatment resistance in these populations. The responsiveness of Per2 −/− colitis to STAT1 inhibition raises the possibility that therapeutic targeting of this pathway could offer benefit even when upstream circadian regulation is impaired. Such approaches could be integrated into personalized treatment strategies, incorporating circadian phenotyping and sleep behavior assessments into UC management frameworks. Furthermore, the fact that Nifuroxazide is already an orally available agent with anti-inflammatory potential may expedite its translational application, particularly for patients experiencing flares linked to sleep disruption or jet lag[ 48 ]. Despite the mechanistic insights offered by our study, several limitations warrant consideration. First, we employed an acute DSS-induced colitis model, which recapitulates epithelial injury and inflammation but does not fully reflect the chronicity and immune complexity of human UC. Future studies using chronic colitis models or genetic models of IBD may provide a more comprehensive understanding of PER2’s long-term regulatory role. In addition, although we observed consistent PER2 suppression and necroptotic activation in human UC specimens, functional validation in primary human epithelial cells or patient-derived organoids would strengthen translational relevance. Another open question is whether restoration of PER2 expression or activity could serve as a therapeutic strategy in itself. Pharmacological clock modulators or gene delivery systems targeting circadian machinery may offer future opportunities to re-establish epithelial resilience. Moreover, given the bidirectional relationship between the circadian clock and gut microbiota, it is plausible that PER2 deficiency alters microbial composition, further amplifying inflammation. Integrating metagenomic or metabolomic analyses will be important to define how microbiota–host clock interactions contribute to necroptosis and mucosal injury. Lastly, while this study focuses on UC, the relevance of PER2-mediated necroptotic regulation in Crohn’s disease and other chronic inflammatory disorders remains to be explored. Whether this pathway also contributes to fibrosis, epithelial regeneration, or extraintestinal manifestations is unknown. Addressing these questions may broaden the applicability of circadian-targeted interventions in inflammatory diseases. Conclusion This study establishes the circadian core protein PER2 as an epithelial-intrinsic guardian that restrains necroptosis-driven mucosal injury in ulcerative colitis. By physically sequestering STAT1 via its PAS1 domain, PER2 blocks STAT1-mediated transcription of ZBP1 and consequently shuts down the ZBP1–RIPK3–MLKL necroptotic axis. Circadian disruption or epithelial-specific Per2 deletion unleashes STAT1, precipitating ZBP1-dependent necroptosis, exaggerated cytokine responses and severe DSS-colitis. Conversely, pharmacologic inhibition of STAT1 with nifuroxazide rescues Per2-deficient mice, normalizing necroptotic signaling and mucosal architecture. The axis is conserved in humans: PER2 is markedly reduced in active UC and further suppressed in patients with comorbid sleep disturbance, correlating with enhanced necroptosis markers. Thus, the PER2–STAT1–ZBP1 circuit couples circadian integrity to epithelial survival and represents a readily druggable checkpoint whose targeting may benefit UC subjects with chronobiological dysfunction. Abbreviations ANOVA Analysis of Variance A-UC Active Ulcerative Colitis BSA Bovine Serum Albumin ChIP Chromatin Immunoprecipitation CLOCK Circadian Locomotor Output Cycles Kaput CRY1 Cryptochrome 1 DAI Disease Activity Index DAMP Damage-associated Molecular Pattern DMEM Dulbecco’s Modified Eagle’s Medium DSS Dextran Sulfate Sodium DTT Dithiothreitol EDTA Ethylenediaminetetra-acetic Acid FBS Fetal Bovine Serum FITC Fluorescein Isothiocyanate GAPDH Glyceraldehyde-3-phosphate Dehydrogenase GEO Gene Expression Omnibus GO Gene Ontology GSEA Gene Set Enrichment Analysis H&E Hematoxylin and Eosin HC Healthy Control IBD Inflammatory Bowel Disease IEC Intestinal Epithelial Cell IFN-γ Interferon gamma IgG Immunoglobulin G IL-1β Interleukin-1 beta IL-6 Interleukin-6 IP Immunoprecipitation JAK Janus Kinase KEGG Kyoto Encyclopedia of Genes and Genomes KD Knockdown LC-MS/MS Liquid Chromatography-tandem Mass Spectrometry LPS Lipopolysaccharide MLKL Mixed Lineage Kinase Domain-like NF-κB Nuclear Factor Kappa-light-chain-enhancer of Activated B Cells Nr1d1 Nuclear Receptor Subfamily 1 Group D Member 1 PBS Phosphate-buffered Saline PCA Principal Component Analysis PCR Polymerase Chain Reaction Per1/2 Period 1/2 PI Propidium Iodide PPI Protein–protein Interaction PSQI Pittsburgh Sleep Quality Index PVDF Polyvinylidene Fluoride qPCR Quantitative PCR RIPK3 Receptor-interacting Protein Kinase 3 R-UC UC in Remission SDS-PAGE Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis SPF Specific-pathogen-free STAT1/3 Signal Transducer and Activator of Transcription 1/3 TNF-α Tumor Necrosis Factor alpha TUNEL Terminal Deoxynucleotidyl Transferase dUTP Nick-end Labeling UC Ulcerative Colitis WT Wild Type ZBP1 Z-DNA-binding Protein 1 ZT Zeitgeber Time Declarations Funding declaration This study was funded by grants from the National Key Research and Development Program (Grant No. 2023YFC2307001) and the National Natural Science Foundation of China (NSFC) (Grant Nos. 81800480 and 81800465). Conflict of interest The authors declare that they have no competing interests or conflicts of interest in relation to this study. Ethical approval This study was conducted in strict accordance with ethical guidelines. Ethical approval for animal experiments was obtained from the Animal Ethics Committee of Huazhong University of Science and Technology (Approval No. 2023-4300). For human subjects, approval was granted by the Independent Ethics Committee of Wuhan Union Hospital (Approval No. 2022-S147). The study adhered to the principles set forth in the Declaration of Helsinki. Data availability All data generated or analyzed during this study are available from the corresponding author upon reasonable request. Consent to Publish declaration All participants involved in this study provided written informed consent for the collection of their data and subsequent publication of the study findings. 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1","display":"","copyAsset":false,"role":"figure","size":524215,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCircadian rhythm disruption increases epithelial apoptosis and suppresses PER2 expression in the colonic mucosa of UC patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative TUNEL staining of colonic biopsy sections from four clinical groups: healthy controls (HC), healthy individuals with sleep disruption (HC + Jet Lag), ulcerative colitis patients (UC), and UC patients with comorbid sleep disruption (UC + Jet Lag). Nuclei are stained with DAPI (blue), and apoptotic cells are marked with TUNEL (green). Increased epithelial apoptosis is observed in UC and further exacerbated in UC + Jet Lag samples. (B) Immunofluorescence staining of PER2 (red) in colonic mucosa from the same four groups. PER2 is prominently expressed in epithelial cells of HC and HC + Jet Lag groups but substantially reduced in UC, with near-complete loss in UC + Jet Lag tissues. Nuclei are counterstained with DAPI (blue). (C) Box plots showing relative mRNA expression of circadian genes (PER2, PER1, CLOCK, and CRY1) in colonic tissues from healthy controls (HC), patients with active UC (A-UC), and those in remission (R-UC) based on GSE38713 dataset. PER2 is significantly downregulated in A-UC compared to HC, while CLOCK and CRY1 are upregulated in R-UC. (D) Western blot analysis of PER2 protein expression in fresh colonic biopsy tissues from HC and UC patients, with or without sleep disruption. GAPDH serves as a loading control. PER2 levels are notably decreased in UC and further reduced with concomitant circadian disruption. Data are presented as mean ± SEM, n=6. Statistical significance was determined by one-way ANOVA followed by post hoc testing; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7601246/v1/3c86cece2388e46134c8a567.jpeg"},{"id":93335785,"identity":"64c878ee-5e7d-4ec5-957a-6015257d054b","added_by":"auto","created_at":"2025-10-12 14:00:52","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":359324,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of Per2 in intestinal epithelial cells induces necroptosis-associated transcriptional and protein changes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Principal Component Analysis (PCA) of bulk RNA-seq data from intestinal epithelial cells of \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice shows clear transcriptomic separation between groups (n = 3 per group). (B) Volcano plot displaying differentially expressed genes between \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e epithelial cells. Significantly upregulated genes are shown in red, and downregulated genes in blue; dot size reflects statistical significance. (C) Gene Ontology (GO) enrichment analysis of differentially expressed genes, highlighting biological processes related to hydrolase activity, MyD88-dependent Toll-like receptor signaling, and extracellular matrix organization. (D) KEGG pathway enrichment analysis shows significant involvement of inflammation-related pathways, including TNF signaling, JAK-STAT signaling, and inflammatory bowel disease (IBD), with necroptosis identified as the most significantly enriched pathway. (E) Heatmap of selected necroptosis-associated genes reveals upregulation in \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice, including Zbp1, Ripk3, Mlkl, Stat1, and Stat2. (F) Representative Western blot showing protein expression levels of ZBP1, MLKL, phosphorylated MLKL (p-MLKL), RIPK3, and phosphorylated RIPK3 (p-RIPK3) in isolated intestinal epithelial cells from \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice. GAPDH serves as loading control. (G) Quantification of relative protein expression normalized to GAPDH. Significant upregulation of ZBP1, MLKL, and RIPK3 is observed in the \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e group. Data are shown as mean ± s.e.m. Statistical significance determined by unpaired two-tailed Student’s t-test, n=3-6; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05,; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7601246/v1/61ba2cbb7243fdb742f570f6.jpeg"},{"id":93335791,"identity":"2778e855-044c-4400-bef3-6e7ddbe7af61","added_by":"auto","created_at":"2025-10-12 14:00:52","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":864330,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePER2 deficiency exacerbates DSS-induced colitis and activates necroptosis signaling in intestinal epithelial cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Body weight change (%) over 8 days of DSS treatment in \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e + DSS, and \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + DSS mice (n = 6 per group). \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + DSS mice exhibited significantly greater weight loss. (B) Representative images of colons from each group showing pronounced shortening in \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + DSS mice.\u003c/p\u003e\n\u003cp\u003e(C) H\u0026amp;E-stained colonic sections reveal increased inflammatory cell infiltration, crypt destruction, and epithelial damage in \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + DSS mice. Scale bar, 200 μm. (D) Quantification of colon length across groups. Significant shortening was observed only in DSS-treated \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice. (E) Disease Activity Index (DAI) scores indicate increased clinical severity in \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + DSS mice. (F) Histological scoring based on H\u0026amp;E-stained sections shows exacerbated mucosal damage in \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + DSS mice. (G) Relative mRNA expression levels of inflammatory cytokines (Tnfα, Ifnγ, Il1β, Il6) in colonic epithelial cells, as measured by qPCR. Cytokine expression is significantly elevated in \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + DSS mice. (H) Western blot analysis of necroptosis-related proteins ZBP1, MLKL, p-MLKL, RIPK3, and p-RIPK3 in epithelial cells from \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e + DSS and \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + DSS mice. GAPDH serves as the loading control. (I) Quantification of protein expression levels normalized to GAPDH. Per2 deletion significantly enhances expression and activation of necroptosis markers. Data represent mean ± SEM. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 (*),\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 by unpaired two-tailed t-test or one-way ANOVA as appropriate.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7601246/v1/b437a7f951fa0bcf81d1d597.jpeg"},{"id":93335789,"identity":"bcca6631-dd45-4c34-9217-3b1e581bdbad","added_by":"auto","created_at":"2025-10-12 14:00:52","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":387430,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of PER2 alters the epithelial transcriptome and activates inflammatory signaling during DSS-induced colitis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Principal Component Analysis (PCA) of bulk RNA-seq data from colonic epithelial cells of\u003cem\u003e Per2\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e + DSS and \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + DSS mice reveals distinct clustering between groups (n = 3 per group), indicating PER2-dependent transcriptional reprogramming. (B) Volcano plot of differentially expressed genes (DEGs) between \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + DSS and \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e + DSS groups. Upregulated and downregulated genes are colored by significance and magnitude of change. (C–E) Gene Ontology (GO) enrichment analysis of DEGs, showing significantly enriched terms in the categories of biological process (BP, panel C), cellular component (CC, panel D), and molecular function (MF, panel E). Key enriched terms include “immune response,” “extracellular matrix,” and “cytokine activity.” (F) KEGG pathway enrichment analysis identifies significant enrichment of inflammation-related pathways in the \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + DSS group, including JAK-STAT signaling, TNF signaling, Toll-like receptor signaling, NF-κB signaling, and the IBD pathway. (G) Gene Set Enrichment Analysis (GSEA) shows significant activation of the TNF, JAK-STAT, and NF-κB signaling pathways in \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + DSS mice compared to controls. Normalized enrichment scores (NES), \u003cem\u003eP\u003c/em\u003e-values, and adjusted \u003cem\u003eP\u003c/em\u003e-values are shown in the legend.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7601246/v1/a8a8f5ad3c258d007cb36655.jpeg"},{"id":93337370,"identity":"45912f48-4265-4309-9bf7-3263a64dd74d","added_by":"auto","created_at":"2025-10-12 14:08:52","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":466278,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePER2 knockdown enhances LPS-induced necroptosis via ZBP1 in human intestinal epithelial cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Flow cytometric analysis of apoptosis in NCM460 cells across four groups: negative control (NC), PER2 knockdown (PER2 KD), control with LPS treatment (NC + LPS), and PER2 KD with LPS treatment (PER2 KD + LPS). PER2 knockdown significantly increased apoptosis in response to LPS stimulation. (B) Flow cytometry showing the effect of the necroptosis inhibitor GSK872. GSK872 markedly reduced LPS-induced apoptosis in PER2 KD cells (PER2 KD + LPS + GSK872), but had minimal effect in control cells (NC + LPS + GSK872), indicating a necroptosis-dependent mechanism.\u003c/p\u003e\n\u003cp\u003e(C) Western blot analysis of necroptosis markers (ZBP1, MLKL, RIPK3) and their phosphorylated forms (p-MLKL, p-RIPK3) in LPS-treated PER2 KD and control cells. PER2 knockdown led to upregulation and activation of necroptosis proteins. (D) Western blot showing the effects of ZBP1 silencing (siZBP1) in PER2 KD + LPS cells. Knockdown of ZBP1 suppressed expression and phosphorylation of MLKL and RIPK3, suggesting ZBP1 is required for PER2-deficiency–induced necroptosis. GAPDH served as the loading control. (E) Flow cytometric analysis demonstrating reduced apoptosis in PER2 KD + LPS cells following siZBP1 treatment, confirming the regulatory role of ZBP1 in mediating necroptosis under PER2-deficient conditions. N=6.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7601246/v1/cf072f9583ab56fe75779aad.jpeg"},{"id":93335799,"identity":"d3cbfd7e-d509-41d4-98af-9790e6887adb","added_by":"auto","created_at":"2025-10-12 14:00:52","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":263758,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSTAT1 directly regulates ZBP1 transcription and mediates necroptosis in PER2-deficient intestinal epithelial cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot analysis of STAT1 and STAT3 protein expression in colonic epithelial cells from \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice following DSS-induced colitis. GAPDH serves as a loading control. (B) Western blot analysis of necroptosis-related proteins (ZBP1, MLKL, p-MLKL, RIPK3, p-RIPK3) in NCM460 cells treated with LPS following knockdown of PER2 in the presence or absence of siSTAT1 or siSTAT3. STAT1 knockdown, but not STAT3 knockdown, markedly reduced expression of necroptosis markers. (C) STAT1 consensus binding motif predicted from the JASPAR database, indicating a canonical recognition site in the ZBP1 promoter. (D) Chromatin immunoprecipitation (ChIP) assays showing STAT1 binding to the ZBP1 promoter in control (NC + LPS) and PER2 knockdown (PER2 KD + LPS) NCM460 cells. Anti-STAT1 significantly enriched ZBP1 promoter sequences compared to IgG control. (E) Dual-luciferase reporter assays assessing ZBP1 promoter activity in NCM460 cells transfected with wild-type (WT) or STAT1-binding site mutant (Mut) constructs, under control or PER2 knockdown + LPS conditions. Transcriptional activity was significantly enhanced in the WT group upon PER2 knockdown and abrogated in the mutant construct. (F) Dual-luciferase assay demonstrating that STAT1 knockdown significantly reduces ZBP1 promoter activity in PER2-deficient cells. Data are shown as mean ± SEM. Statistical significance was assessed by unpaired two-tailed Student’s t-test or one-way ANOVA; \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 (*), \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001, ns: not significant.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7601246/v1/89848d963c2b9e2233c9de78.jpeg"},{"id":93337374,"identity":"54d780c2-3b5a-4bd3-9537-55595726a5c2","added_by":"auto","created_at":"2025-10-12 14:08:52","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":399709,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe PAS1 domain of PER2 mediates its interaction with STAT1 and regulates necroptosis signaling.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Co-immunoprecipitation (Co-IP) assay showing that FLAG-tagged PER2 co-precipitates endogenous STAT1 and STAT3 in NCM460 cells. (B) Reverse Co-IP using STAT1 antibody confirms the interaction between endogenous STAT1 and FLAG-PER2. (C) Molecular docking model of PER2–STAT1 interaction, visualized by PyMol. The inset highlights hydrogen bond interactions between key residues of PER2 (e.g., ARG325, TYR407, ALA446, SER279) and STAT1 (e.g., GLN314, ASN233, GLU480). Structural domains PAS1, PAS2, and PAC are spatially associated with these contact sites. (D) Co-IP results from NCM460 cells expressing full-length or truncated FLAG-PER2 mutants, showing reduced STAT1 binding in constructs lacking the PAS1 domain (ΔPAS1, N-N2, N-N3). (E) Pulldown validation confirming that deletion of PAS1 or N-terminal truncations significantly attenuate PER2–STAT1 interaction. His-STAT1 and FLAG-PER2 expression were validated by input blots.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7601246/v1/b19f080678b7c9518d6437b4.jpeg"},{"id":93335795,"identity":"64f495c9-d474-428e-bf06-007f000f117a","added_by":"auto","created_at":"2025-10-12 14:00:52","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":634717,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSTAT1 inhibition by Nifuroxazide alleviates DSS-induced colitis and suppresses necroptosis in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePer2\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Body weight changes during DSS treatment in \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice with or without Nifuroxazide administration (n = 6 per group). Nifuroxazide significantly reduced weight loss over time. (B) Representative images of colons from \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + DSS and \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e + DSS + Nifuroxazide mice at endpoint.\u003c/p\u003e\n\u003cp\u003e(C–E) Quantitative analyses of colon length (C), Disease Activity Index (DAI) (D), and histological scores (E) showing improved clinical outcomes in Nifuroxazide-treated mice. (F) Representative H\u0026amp;E-stained colon sections demonstrating reduced inflammatory infiltration and crypt damage in the Nifuroxazide group. Scale bar, 200 μm. (G) Western blot analysis of STAT1, ZBP1, MLKL, RIPK3, and their phosphorylated forms (p-MLKL, p-RIPK3) in colon tissue lysates from the two groups. GAPDH served as the loading control. (H, I) Quantification of total protein (H) and phosphorylated protein (I) levels, normalized to GAPDH. Nifuroxazide treatment significantly reduced necroptosis marker expression. (J) Immunofluorescence staining of TUNEL (green), MLKL (red), and DAPI (blue) in colon sections, showing reduced epithelial apoptosis and necroptosis in the Nifuroxazide group. Scale bar, 100 μm.Data are presented as mean ± SEM. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 (*), \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 (**), determined by unpaired two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7601246/v1/7ba8be0570c944809bf7bc58.jpeg"},{"id":94470325,"identity":"8e29168d-039f-45ce-af5c-7c374f59e2d3","added_by":"auto","created_at":"2025-10-27 15:31:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5425747,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7601246/v1/e3e35c18-b4f0-461d-a8b8-4d371cbb6a54.pdf"},{"id":93337367,"identity":"983475bc-b391-4475-b214-7626f9054088","added_by":"auto","created_at":"2025-10-12 14:08:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":148378,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-7601246/v1/44290a063c49f6433940418b.docx"},{"id":93338088,"identity":"83c4ef50-b678-4553-9aa2-2ed8e55683f3","added_by":"auto","created_at":"2025-10-12 14:16:52","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1593613,"visible":true,"origin":"","legend":"","description":"","filename":"SuppFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-7601246/v1/59f3f4f34b2bb9dd8357c1f6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Circadian Regulator PER2 Protects Against Epithelial Necroptosis in Ulcerative Colitis via the STAT1–ZBP1 Axis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUlcerative colitis (UC) is a chronic, debilitating form of inflammatory bowel disease (IBD) characterized by persistent inflammation and ulceration confined to the colon's mucosal layer[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The incidence and prevalence of UC have risen steadily over recent decades, particularly in industrialized nations, posing significant clinical and economic burdens on global healthcare systems[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although considerable advances have been made in elucidating UC pathogenesis, current treatments remain inadequate for many patients, highlighting the need for deeper insights into its underlying molecular and cellular mechanisms to identify novel therapeutic targets[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCentral to UC pathophysiology is the disruption of the intestinal epithelial barrier, composed of specialized intestinal epithelial cells (IECs) that constitute the frontline defense against pathogenic microorganisms and harmful luminal antigens[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Under normal physiological conditions, IECs maintain intestinal homeostasis through intricate interactions with gut microbiota, immune cells, and environmental stimuli, preserving mucosal integrity by regulating barrier permeability, nutrient absorption, antimicrobial secretion, and epithelial regeneration[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In UC, however, chronic inflammation induces persistent IEC damage, compromising barrier function, enhancing permeability, and perpetuating inflammatory responses via translocation of luminal pathogens and antigens[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These events, in turn, drive excessive immune activation, further amplifying epithelial injury and resulting in a vicious cycle of inflammation, tissue damage, and impaired mucosal repair[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Consequently, understanding the regulatory mechanisms governing IEC integrity and epithelial homeostasis during inflammatory stress is crucial for developing effective therapeutic strategies for UC.\u003c/p\u003e\u003cp\u003eEmerging evidence indicates that circadian rhythms significantly influence intestinal physiology and immune homeostasis[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The circadian system is an evolutionarily conserved endogenous timing mechanism that orchestrates physiological, behavioral, and cellular processes with a periodicity of approximately 24 hours. This system coordinates organismal adaptation to daily environmental cycles, such as light/dark changes and feeding/fasting patterns[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. At the molecular level, circadian rhythms are governed by a transcriptional\u0026ndash;translational feedback loop involving several core circadian genes, including CLOCK, BMAL1, PER1/2, CRY1/2, and NR1D1, whose rhythmic expression orchestrates downstream processes such as metabolism, immunity, and cell proliferation[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Disruption of circadian rhythms\u0026mdash;through shift work, chronic jet lag, sleep deprivation, or genetic mutation\u0026mdash;has been increasingly linked to various diseases, including metabolic disorders, cardiovascular disease, autoimmune conditions, and chronic inflammatory diseases[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Indeed, epidemiological studies have established correlations between circadian disruption and elevated incidence or exacerbation of IBD, suggesting circadian genes may directly impact intestinal inflammation and epithelial barrier function[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOf particular interest among the circadian components is the Period circadian regulator 2 (PER2), a core circadian clock protein involved in generating and maintaining circadian rhythmicity through its transcriptional repression of CLOCK\u0026ndash;BMAL1 complexes[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Beyond its canonical circadian functions, PER2 has been implicated in diverse biological processes, including tumor suppression, immune modulation, and oxidative stress responses[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Notably, PER2 dysfunction has recently been associated with increased susceptibility to inflammatory conditions and impaired epithelial barrier integrity, highlighting its potential role as an epithelial homeostasis regulator under inflammatory conditions[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, the precise mechanistic relationship between PER2 and epithelial damage in UC remains incompletely understood, particularly regarding the downstream molecular pathways mediating these effects. Given its dual role in circadian regulation and cellular stress responses, PER2 is well positioned to influence the balance of programmed cell death pathways in the intestinal epithelium.\u003c/p\u003e\u003cp\u003eProgrammed cell death (PCD)\u0026mdash;including apoptosis, pyroptosis, and necroptosis\u0026mdash;is essential for maintaining intestinal epithelial homeostasis. In UC, chronic inflammatory stimuli disrupt the balance of these pathways, leading to excessive epithelial loss, barrier breakdown, and release of pro-inflammatory mediators that perpetuate disease[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. While necroptosis has received particular attention for its inflammatory nature, it functions alongside other PCD modes to determine epithelial fate.\u003c/p\u003e\u003cp\u003eCircadian rhythms are increasingly recognized as modulators of PCD[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The core clock protein PER2 regulates stress responses and immune signaling, and its disruption\u0026mdash;through environmental or pathological factors\u0026mdash;has been linked to heightened cell death in various tissues[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, the role of PER2 in controlling PCD in intestinal epithelial cells (IECs) during UC remains unclear.\u003c/p\u003e\u003cp\u003eHere, we investigated how PER2 influences PCD in IECs under inflammatory and circadian disruption. Using IEC-specific Per2 knockout mice, transcriptomic profiling, protein\u0026ndash;protein interaction studies, molecular docking, chromatin immunoprecipitation, luciferase assays, and pharmacological inhibition, we identified a STAT1-dependent transcriptional pathway as a key effector downstream of PER2. Inhibition of STAT1 with nifuroxazide alleviated colitis and normalized PCD signaling, revealing the PER2\u0026ndash;STAT1 axis as a potential therapeutic target, particularly for UC patients with circadian rhythm disturbances.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePatient Specimens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecimens were obtained from the digestive medicine biobank at the Union Hospital affiliated with Huazhong University of Science and Technology, following informed consent.\u003c/p\u003e\n\u003cp\u003ePatients were included based on the Chinese UC Diagnosis and Treatment Guidelines: (1) typical clinical manifestations (recurrent diarrhea, tenesmus, mucous and bloody stools, abdominal pain); (2) characteristic endoscopic findings; (3) pathological features such as crypt abscesses and reduced goblet cells; (4) imaging results consistent with UC; (5) a Mayo endoscopic score of 1 to 3; (6) age \u0026ge; 18 years.\u003c/p\u003e\n\u003cp\u003eExclusion criteria included: (1) other gastrointestinal disorders (e.g., intestinal tumors, Crohn\u0026apos;s disease, infectious enteritis); (2) intestinal strictures or severe systemic diseases precluding endoscopic biopsy.\u003c/p\u003e\n\u003cp\u003eSpecimens from healthy individuals without UC or other gastrointestinal diseases were also collected.\u003c/p\u003e\n\u003cp\u003eSleep quality was assessed using the Pittsburgh Sleep Quality Index (PSQI), which has a maximum score of 21, based on Buysse et al[24]. Patients scoring \u0026le; 7 were classified as having good sleep quality (normal sleep group), while those scoring \u0026ge; 8 were categorized as having poor sleep quality (sleep disorder group).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of Intestinal Epithelial Cell-Specific \u003cem\u003ePer2\u003c/em\u003e Knockout Mice, Circadian Disruption Model, and DSS-Induced Colitis Protocol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of Per2 in circadian regulation and intestinal inflammation, we generated mice with intestinal epithelial cell-specific deletion of Per2 (\u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e) by crossing Villin-Cre transgenic mice (C57BL/6 background; Simo Biotechnology, Nanjing, China) with Per2^flox/flox mice on the same background. Offspring harboring the Villin-Cre transgene and homozygous floxed Per2 alleles (Villin-Cre;Per2^flox/flox) were designated as \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice, while Per2^flox/flox littermates served as wild-type controls. All mice used in experiments were male and 8 weeks of age. Animals were housed under specific pathogen-free (SPF) conditions with stable temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C), relative humidity (60\u0026ndash;70%), and a 12-hour light/12-hour dark cycle. Mice had ad libitum access to autoclaved food and water.\u003c/p\u003e\n\u003cp\u003eTo model chronic circadian rhythm disruption, a jet lag paradigm was implemented. Mice were subjected to an 8-hour advance in the light phase every 3 days for a total of 8 weeks, simulating repeated eastward travel and persistent circadian misalignment. Light cycles were precisely controlled using an automated environmental lighting system. Control animals were maintained under a constant 12:12-hour light/dark cycle for the same duration.\u003c/p\u003e\n\u003cp\u003eTo induce experimental colitis, mice were administered 2.5% (w/v) dextran sulfate sodium (DSS; MW 36,000\u0026ndash;50,000; MP Biomedicals) in their drinking water for 8 consecutive days. Each experimental group (\u003cem\u003ePer2\u003csup\u003ef/f\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e) consisted of six mice. Upon completion of DSS treatment, mice were euthanized, and the entire colon was excised. A distal colonic segment (~0.5 cm) was fixed in 4% paraformaldehyde and processed for histological evaluation, including hematoxylin and eosin (H\u0026amp;E) staining and immunofluorescence microscopy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with institutional guidelines and approved by the Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of Purified Colonic Epithelial Cells for Downstream Molecular Analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eColonic epithelial cells (IECs) were isolated from mouse colon using a chelation-based dissociation protocol to enable high-quality transcriptomic and proteomic profiling[25]. Briefly, colons were excised, thoroughly rinsed with ice-cold DPBS, and sectioned into ~0.5 cm fragments. Tissues were incubated at 4\u0026deg;C for 75 minutes in an EDTA/DTT dissociation buffer (14 mL DPBS, 0.9 mL 0.5 M EDTA, 22.5 \u0026micro;L 1 M DTT) under gentle agitation to promote epithelial cell detachment. Following incubation, samples underwent three successive rounds of centrifugation and resuspension in fresh buffer to enrich for epithelial cells. After each centrifugation step, the supernatant was discarded and the pellet was resuspended in 5 mL of buffer. The final cell suspension was passed through a 40-\u0026mu;m nylon mesh to remove debris and yield a purified single-cell epithelial fraction. Isolated IECs were immediately processed for downstream applications, including quantitative PCR, Western blotting, and bulk RNA sequencing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHigh-Throughput RNA-Seq Analysis of Colonic Inflammation in PER2 Knockout Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from colonic tissues of \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e, \u003cem\u003ePer2\u003csup\u003ef/f\u003c/sup\u003e\u003c/em\u003e, \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e + DSS, \u003cem\u003ePer2\u003csup\u003ef/f\u003c/sup\u003e\u003c/em\u003e + DSS, and WT + DSS mice (n = 3 per group) for transcriptomic analysis. RNA isolation, quality control, library preparation, and high-throughput sequencing were performed by Frasergen Co., Ltd. (Wuhan, China) using the Illumina NovaSeq 6000 platform. Raw sequencing data were processed and analyzed using the DESeq2 package in R, with differentially expressed genes (DEGs) defined as those with an absolute fold change \u0026gt; 2 and adjusted \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, based on the Benjamini\u0026ndash;Hochberg correction. Data visualization, including volcano plots and heatmaps, was generated using the ggplot2 and ComplexHeatmap packages. Functional annotation and enrichment analyses, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Gene Set Enrichment Analysis (GSEA), were performed with a significance cutoff of \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDistal colonic segments (~0.5 cm) were collected from euthanized mice, fixed in 10% neutral-buffered formalin overnight at room temperature, and embedded in paraffin. Tissues were sectioned at 5 \u0026mu;m thickness and stained with haematoxylin and eosin (H\u0026amp;E) following standard protocols. Haematoxylin was used to visualize nuclei, while eosin counterstained the cytoplasm and extracellular matrix. Histological scoring was performed by two independent, blinded pathologists, who evaluated inflammatory cell infiltration, mucosal architecture, and tissue damage. Discrepancies in scoring were resolved by consensus.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGEO Dataset Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTranscriptomic data were obtained from the Gene Expression Omnibus (GEO) database (accession number: GSE38713), which includes gene expression profiles of colonic mucosal biopsies from human subjects. The dataset comprises three groups: 13 healthy controls, 15 ulcerative colitis (UC) patients in clinical remission, and 15 patients with active UC. The normalized gene expression matrix was downloaded and analyzed to assess the expression levels of core circadian clock genes. Differential expression analysis and statistical comparisons between groups were conducted using appropriate bioinformatic tools in R, with significance thresholds defined as \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative PCR Analysis of Gene Expression in IECs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative real-time PCR (qPCR) was conducted to evaluate gene expression in isolated intestinal epithelial cells (IECs) using a LightCycler system (Roche Diagnostics) and SYBR Green PCR Master Mix (Takara, Cat# RR036A). Each reaction mixture contained cDNA template, gene-specific primers obtained from PrimerBank (https://pga.mgh.harvard.edu/primerbank/), and SYBR Green reagent. Thermal cycling conditions were as follows: initial denaturation at 95\u0026deg;C for 3 minutes, followed by 40 cycles of 95\u0026deg;C for 15 seconds, 60\u0026deg;C for 30 seconds, and 72\u0026deg;C for 30 seconds. Amplification specificity was confirmed by melting curve analysis. All reactions were run in triplicate, and relative gene expression was calculated using the \u0026Delta;\u0026Delta;Ct method, with GAPDH used as the endogenous normalization control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoblot Analysis of Protein Expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteins were extracted from IECs isolated from murine colon, human colonic biopsies, and organoids using RIPA buffer (Thermo Fisher Scientific, MA, USA) with protease and phosphatase inhibitors (Roche). Protein concentrations were measured using a BCA assay (Pierce, IL, USA). Equal amounts (20\u0026ndash;30 \u0026micro;g) were separated by SDS\u0026ndash;PAGE (10\u0026ndash;12%) and transferred to PVDF membranes (Millipore, MA, USA). Membranes were blocked with 5% milk or BSA in TBS-T, incubated overnight at 4 \u0026deg;C with primary antibodies, and then with HRP-conjugated secondary antibodies for 1 hour at room temperature. Bands were visualized using ECL (ECL; GE Healthcare, NJ, USA) and imaged with a ChemiDoc system (Bio-Rad). Densitometry was performed using ImageJ, with GAPDH as the loading control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-immunoprecipitation and Pulldown Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNCM460 cells were transfected with FLAG\u0026ndash;PER2 plasmids using Lipofectamine 3000 and harvested 48 h later. Cells were lysed in RIPA buffer with protease inhibitors, and lysates were centrifuged at 12,000 \u0026times; g for 15 min at 4 \u0026deg;C. Supernatants were incubated overnight at 4 \u0026deg;C with anti-FLAG (DYKDDDDK) magnetic beads. After washing, bound proteins were eluted in SDS loading buffer at 95 \u0026deg;C and analyzed by Western blot. Gel bands (~1 cm) were excised, digested with trypsin, and subjected to LC-MS/MS using an EASY-nLC 1200 system and Q-Exactive HF mass spectrometer. Raw data were searched against the SwissProt database using Proteome Discoverer v2.4 to identify PER2-interacting proteins.\u003c/p\u003e\n\u003cp\u003eHEK293T cells were transfected with His\u0026ndash;STAT1 plasmids and lysed in NET buffer. Lysates were incubated with anti-His magnetic beads, and bound proteins were eluted stepwise with 20 mM, 50 mM, and 250 mM imidazole.\u003c/p\u003e\n\u003cp\u003eFor pulldown assays, HEK293T cells were transfected with FLAG\u0026ndash;PER2 (WT or mutants). Lysates were pre-cleared with Protein A/G beads, incubated with purified His\u0026ndash;STAT1 for 2 h, followed by anti-His bead capture. Complexes were eluted with SDS buffer and analyzed by Western blot.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein\u003c/strong\u003e\u003cstrong\u003e\u0026ndash;\u003c/strong\u003e\u003cstrong\u003eProtein Docking and Domain Mapping\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 3D crystal structure of human STAT1 was obtained from the Protein Data Bank (PDB), and the full-length structure of PER2 was predicted using AlphaFold. Preprocessing, including removal of water molecules and addition of hydrogen atoms, was performed in AutoDockTools v1.5.7. Protein\u0026ndash;protein docking was carried out using the GRAMM-X server, with STAT1 set as the receptor and PER2 as the ligand. Among the generated models, the top-ranked complex was selected based on docking score and subjected to energy minimization. The predicted interaction interface was visualized and analyzed using PyMOL, with key contact residues annotated.\u003c/p\u003e\n\u003cp\u003eTo experimentally validate the docking predictions and define the minimal region of PER2 required for STAT1 binding, truncated mutants targeting the PAS1, PAS2, and PAC domains were constructed and cloned into FLAG-tagged expression vectors. These constructs were transfected into HEK293T cells, and protein\u0026ndash;protein interactions were assessed via co-immunoprecipitation and pulldown assays using purified His\u0026ndash;STAT1 protein. The PAS1 domain was identified as essential for STAT1 interaction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromatin Immunoprecipitation (ChIP) Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChIP assays were performed to assess STAT1 binding to the ZBP1 promoter in colonic epithelial cells. Cells were fixed with 1% formaldehyde to cross-link protein\u0026ndash;DNA complexes, and chromatin was extracted and sonicated to obtain DNA fragments of 200\u0026ndash;500 bp. Immunoprecipitation was carried out using the SimpleChIP\u0026reg; Enzymatic Chromatin IP Kit (Cell Signaling Technology, Cat# 9003), following the manufacturer\u0026rsquo;s instructions. Samples were incubated overnight at 4 \u0026deg;C with a STAT1-specific antibody or normal rabbit IgG (negative control), followed by capture with magnetic protein G beads for 2 hours. After reverse cross-linking and DNA purification, the immunoprecipitated DNA was analyzed by PCR using primers targeting the predicted STAT1 binding site within the human ZBP1 promoter. Primers were designed using SnapGene software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLuciferase Reporter Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDual-luciferase reporter assays were performed to evaluate the transcriptional activity of the ZBP1 promoter in STAT1-deficient and control cells. The promoter region of ZBP1, either wild-type (WT) or containing a mutated STAT1-binding site (Mut), was cloned into the pGL4.11-Basic vector (Promega). Cells were co-transfected with the reporter plasmid and a Renilla luciferase control vector (pRL-TK) using Lipofectamine 3000 (Thermo Fisher Scientific, MA, USA). After 24\u0026ndash;48 hours, luciferase activity was measured using the Dual-Luciferase\u0026reg; Reporter Assay System (Promega, Madison, USA) and quantified with a luminometer. Firefly luciferase activity was normalized to Renilla luciferase to control for transfection efficiency and sample variability.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence Staining of Colonic Tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor tissue immunofluorescence, paraffin-embedded sections of mouse colon were deparaffinized, rehydrated through graded alcohols, and subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0). Sections were blocked with 5% BSA to reduce non-specific binding, followed by incubation with primary antibodies (1:100 dilution) overnight at 4 \u0026deg;C. After washing, fluorescently labeled secondary antibodies were applied for 1 hour at room temperature in the dark. Nuclei were counterstained with Hoechst 33342. Stained sections were mounted with antifade medium and imaged using a Zeiss LSM-800 confocal microscope (Carl Zeiss AG, Oberkochen, Germany).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow Cytometric Analysis of Apoptosis Following PER2 Knockdown and LPS Stimulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApoptotic cell death was assessed in NCM460 human intestinal epithelial cells subjected to PER2 knockdown and lipopolysaccharide (LPS) treatment, with or without pharmacological inhibition. Cells were stained using the Annexin V-FITC/PI apoptosis detection kit (Beyotime) according to the manufacturer\u0026rsquo;s protocol. Briefly, after experimental treatments, cells were harvested, washed with cold PBS, and resuspended in binding buffer. Annexin V-FITC and propidium iodide (PI) were then added to the suspension, followed by incubation for 15 minutes at room temperature in the dark. Stained cells were analyzed immediately using flow cytometry to quantify early apoptotic (Annexin V⁺/PI⁻), late apoptotic or necrotic (Annexin V⁺/PI⁺), and viable (Annexin V⁻/PI⁻) populations.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCircadian rhythm disruption aggravates colonic inflammation and suppresses PER2 expression in both mouse and human colitis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo model the impact of circadian misalignment on intestinal inflammation, we employed a jet lag mouse protocol involving repeated 8-hour light phase advances every three days, simulating chronic rhythm disruption. Colitis was induced during the final week by administering 2.5% dextran sulfate sodium (DSS) in drinking water. Mice subjected to both jet lag and DSS treatment exhibited significantly greater weight loss compared to DSS-treated controls (WT + DSS), indicating more severe disease progression (Fig. S1A).\u003c/p\u003e\n\u003cp\u003eMacroscopic examination revealed notable colon shortening in the Jet Lag + DSS group (Fig. S1B), confirmed by quantitative measurement (Fig. S1C). These morphological changes were accompanied by elevated Disease Activity Index (DAI) scores (Fig. S1D) and worsened histopathological outcomes, including epithelial erosion, crypt distortion, and inflammatory cell infiltration (Fig. S1F). Histological scoring further confirmed significantly aggravated mucosal injury in rhythm-disrupted mice relative to controls (Fig. S1E). Together, these findings suggest that circadian misalignment sensitizes the colon to inflammatory injury and amplifies DSS-induced tissue damage.\u003c/p\u003e\n\u003cp\u003eTo explore the underlying mechanisms, we profiled the expression of core circadian genes in colonic tissue collected at four Zeitgeber time (ZT) points (ZT0, 6, 12, and 18). WT mice exhibited clear rhythmic expression of Per2, Per1, Bmal1, Clock, Cry1, and Nr1d1 over a 24-hour cycle (Fig. S1G). DSS treatment dampened the amplitude and disrupted the phase of these transcripts, most notably Per2. In the Jet Lag + DSS group, Per2 levels were consistently suppressed across all time points, with similar but less pronounced reductions observed in Per1, Bmal1, Cry1, and Nr1d1. Clock expression was modestly affected. These results indicate that colitis perturbs circadian gene oscillations in the colon, and circadian misalignment further disrupts this regulatory network, with Per2 being especially vulnerable.\u003c/p\u003e\n\u003cp\u003eTo assess the relevance of these findings in humans, we clinically stratified individuals based on both UC status and sleep quality. Colonic biopsies were collected from four groups: healthy controls (HC), healthy individuals with chronic sleep disturbance (HC + Jet Lag), UC patients without sleep disorders (UC), and UC patients reporting comorbid sleep disruption (UC + Jet Lag). TUNEL staining showed low levels of epithelial apoptosis in HC and HC + Jet Lag samples, while UC patients displayed increased apoptotic signals, further amplified in the UC + Jet Lag group (Fig. 1A). These data suggest that circadian disruption may contribute to epithelial injury in colitis.\u003c/p\u003e\n\u003cp\u003eWe next examined PER2 protein expression in these patient groups. Immunofluorescence staining revealed abundant PER2 localization in the epithelial compartment of HC and HC + Jet Lag samples. In contrast, PER2 signal intensity was reduced in UC patients and nearly absent in those with both UC and sleep disruption (Fig. 1B), suggesting a synergistic effect of inflammation and circadian dysfunction on PER2 suppression.\u003c/p\u003e\n\u003cp\u003eTo support these observations, we analyzed transcriptomic data from dataset GSE38713, which includes colonic biopsies from healthy controls (HC), patients with active UC (A-UC), and patients in remission (R-UC). PER2 mRNA expression was significantly reduced in A-UC compared to HC, while PER1 levels were unchanged (Fig. 1C). Notably, both CLOCK and CRY1 transcripts were upregulated in R-UC samples relative to A-UC and HC, suggesting reprogramming of circadian machinery during recovery. Western blot analysis of fresh patient biopsies confirmed downregulation of PER2 protein in UC and UC + Jet Lag samples (Fig. 1D), corroborating the immunostaining and transcriptomic data.\u003c/p\u003e\n\u003cp\u003eAltogether, these findings establish a conserved relationship between circadian rhythm disruption and impaired PER2 expression in both murine and human colitis. The concomitant increase in epithelial apoptosis under conditions of rhythm misalignment underscores a mechanistic link between circadian dysfunction and the exacerbation of mucosal inflammation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEpithelial PER2 deficiency exacerbates colitis via transcriptional reprogramming and necroptosis-driven inflammation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo clarify the functional role of PER2 in the intestinal epithelium, we generated conditional knockout mice with epithelium-specific deletion of \u003cem\u003ePer2\u003c/em\u003e (\u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e) using the Cre-loxP system. Intestinal epithelial cells were isolated from \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u0026nbsp;\u003c/em\u003eand littermate \u003cem\u003ePer2\u003csup\u003eflox/flox\u003c/sup\u003e\u003c/em\u003e (\u003cem\u003ePer2\u003csup\u003ef/f\u003c/sup\u003e\u003c/em\u003e) control mice for molecular analysis. Western blotting confirmed a substantial reduction in PER2 protein levels in knockout mice compared to controls (Fig. S2A\u0026ndash;B), and this was paralleled by a significant decrease in \u003cem\u003ePer2\u003c/em\u003e mRNA expression as assessed by qPCR (Fig. S2C).\u003c/p\u003e\n\u003cp\u003eIn agreement with these findings, immunofluorescence staining of intestinal sections revealed markedly attenuated PER2 signal in the epithelial layer of \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u0026nbsp;\u003c/em\u003emice relative to \u003cem\u003ePer2\u003csup\u003ef/f\u003c/sup\u003e\u003c/em\u003e controls (Fig. S2D). These data collectively validate the successful generation of a tissue-specific Per2 knockout model and confirm efficient loss of gene expression at both transcript and protein levels within the intestinal epithelium.\u003c/p\u003e\n\u003cp\u003eTo elucidate the global transcriptional consequences of Per2 deletion in intestinal epithelial cells, we performed bulk RNA sequencing on isolated epithelial cells from \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e and\u003cem\u003e\u0026nbsp;Per2\u003csup\u003ef/f\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003emice. Principal Component Analysis (PCA) revealed clear separation between the two groups, indicating a distinct transcriptomic landscape driven by Per2 deficiency (Fig. 2A). Differential gene expression analysis visualized by volcano plot further confirmed widespread transcriptional alterations following Per2 deletion (Fig. 2B).\u003c/p\u003e\n\u003cp\u003eGene Ontology (GO) enrichment analysis highlighted significant upregulation of pathways associated with hydrolase activity, cell communication, and Toll-like receptor signaling (Fig. 2C). KEGG pathway analysis revealed that differentially expressed genes were enriched in multiple inflammatory and immune-related pathways, including TNF signaling, JAK-STAT signaling, and the inflammatory bowel disease (IBD) pathway (Fig. 2D). Of particular interest, necroptosis emerged as one of the top enriched pathways, suggesting a possible mechanistic link between Per2 loss and regulated necrotic cell death.\u0026nbsp;Necroptosis is a regulated form of lytic cell death mediated by receptor-interacting protein kinase 3 (RIPK3) and its downstream effector, mixed lineage kinase domain-like pseudokinase (MLKL). Upon activation by death receptors or pattern recognition receptors, MLKL undergoes phosphorylation, oligomerization, and translocation to the plasma membrane, where it disrupts membrane integrity, leading to cell rupture and the release of pro-inflammatory contents[26]. In the intestinal epithelium, excessive necroptosis compromises barrier function and amplifies mucosal inflammation, thereby contributing to ulcerative colitis pathogenesis.\u003c/p\u003e\n\u003cp\u003eTo further explore this potential link, we focused on necroptosis-associated genes. Heatmap analysis of the RNA-seq data revealed upregulation of Zbp1, Ripk3, Mlkl, Stat1, and Stat2 in \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e samples relative to controls (Fig. 2E), indicating transcriptional activation of necroptosis machinery in the absence of PER2. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe next validated these observations at the protein level. Western blotting confirmed increased expression of ZBP1, MLKL, and RIPK3 in \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice, along with enhanced phosphorylation of MLKL, a key effector of necroptosis (Fig. 2F). Quantitative analysis normalized to GAPDH corroborated these findings, with significantly elevated levels of ZBP1, MLKL, and RIPK3 proteins in the knockout group (Fig. 2G). While p-MLKL and p-RIPK3 also trended upward, only p-MLKL showed a statistically significant increase.\u003c/p\u003e\n\u003cp\u003eCollectively, these results demonstrate that epithelial-specific Per2 deletion activates necroptosis-related pathways at both the transcript and protein levels. This suggests that PER2 may function as a gatekeeper of epithelial cell survival by suppressing programmed necrotic cell death, thereby contributing to the maintenance of intestinal barrier integrity.\u003c/p\u003e\n\u003cp\u003eTo investigate the functional impact of PER2 on intestinal inflammation, we employed a dextran sulfate sodium (DSS)-induced acute colitis model using four experimental groups: \u003cem\u003ePer2\u003csup\u003ef/f\u003c/sup\u003e\u003c/em\u003e, \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e, \u003cem\u003ePer2\u003csup\u003ef/f\u003c/sup\u003e\u003c/em\u003e + DSS, and \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e + DSS. During DSS administration, \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice exhibited significantly greater body weight loss compared to \u003cem\u003ePer2\u003csup\u003ef/f\u003c/sup\u003e\u003c/em\u003e controls (Fig. 3A), suggesting enhanced susceptibility to colonic injury in the absence of PER2.\u003c/p\u003e\n\u003cp\u003eMacroscopic evaluation of colonic tissue revealed pronounced shortening in \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e + DSS mice (Fig. 3B), while histological examination demonstrated severe epithelial destruction, crypt loss, and inflammatory infiltration compared to all other groups (Fig. 3C). Notably, Per2 deletion alone did not significantly affect colon length, but when combined with DSS treatment, it resulted in marked tissue damage and shortening (Fig. 3D). Disease severity was further confirmed by increased Disease Activity Index (DAI) scores (Fig. 3E) and elevated histological scores (Fig. 3F) in \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e + DSS mice relative to \u003cem\u003ePer2\u003csup\u003ef/f\u003c/sup\u003e\u003c/em\u003e + DSS controls.\u003c/p\u003e\n\u003cp\u003eConsistent with these pathological features, transcriptional analysis of inflammatory mediators revealed that mRNA levels of Tnf\u0026alpha;, Ifn\u0026gamma;, Il1\u0026beta;, and Il6 were significantly upregulated in the \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e + DSS group (Fig. 3G), indicating a heightened proinflammatory response. Given our prior findings linking PER2 deficiency to necroptosis pathway activation, we next assessed the expression of necroptosis-associated proteins. Western blotting showed significantly increased expression of ZBP1, MLKL, and RIPK3, along with their phosphorylated forms p-MLKL and p-RIPK3 in \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e + DSS mice compared to \u003cem\u003ePer2\u003csup\u003ef/f\u003c/sup\u003e\u003c/em\u003e + DSS controls (Fig. 3H\u0026ndash;I).\u003c/p\u003e\n\u003cp\u003eTo determine whether these molecular changes are conserved in human disease, we examined necroptosis-related protein expression in colonic tissues from ulcerative colitis (UC) patients with or without sleep disruption. Immunoblotting revealed increased levels of ZBP1, MLKL, and RIPK3 in UC specimens, particularly in those with concurrent jet lag (Fig. S3A), further supporting a role for PER2 in suppressing necroptotic signaling during mucosal inflammation.\u003c/p\u003e\n\u003cp\u003eFinally, to localize and quantify necroptotic cell death, we performed TUNEL staining combined with MLKL immunofluorescence on mouse colon sections. Confocal microscopy revealed that \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e + DSS mice displayed significantly stronger TUNEL (green) and MLKL (red) signals than the other groups, indicating a marked increase in necroptotic epithelial cell death in the absence of PER2 under inflammatory conditions (Fig. S3B).\u003c/p\u003e\n\u003cp\u003eCollectively, these findings indicate that epithelial-specific deletion of Per2 exacerbates DSS-induced colitis by amplifying inflammatory responses and promoting necroptosis, a process that may be similarly activated in human UC associated with circadian disruption.\u003c/p\u003e\n\u003cp\u003eTo determine the transcriptional consequences of PER2 deletion in intestinal epithelial cells under inflammatory conditions, we performed bulk RNA sequencing on epithelial cells isolated from DSS-treated \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003ePer2\u003csup\u003ef/f\u003c/sup\u003e\u003c/em\u003e mice. Principal Component Analysis (PCA) revealed a distinct separation between the two groups, indicating robust transcriptomic divergence induced by Per2 loss (Fig. 4A). Differential expression analysis, visualized through a volcano plot, identified numerous upregulated and downregulated genes in the \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e + DSS group relative to controls (Fig. 4B).\u003c/p\u003e\n\u003cp\u003eTo explore the functional relevance of these transcriptional alterations, we conducted comprehensive enrichment analyses. Gene Ontology (GO) analysis showed that differentially expressed genes (DEGs) were significantly enriched in biological processes related to immune system activation, including \u0026ldquo;immune response,\u0026rdquo; \u0026ldquo;defense response,\u0026rdquo; and \u0026ldquo;cell adhesion\u0026rdquo; (Fig. 4C). Enrichment in cellular components (CC) included extracellular matrix, supramolecular complexes, and cytoskeletal structures (Fig. 4D), while molecular function (MF) terms such as \u0026ldquo;cytokine activity\u0026rdquo; and \u0026ldquo;signaling receptor binding\u0026rdquo; suggested a shift toward proinflammatory cytokine signaling (Fig. 4E).\u003c/p\u003e\n\u003cp\u003eKEGG pathway analysis further supported this notion, identifying significant enrichment in several inflammatory and immune-regulatory signaling pathways, including the JAK-STAT signaling pathway, TNF signaling, NF-\u0026kappa;B pathway, Toll-like receptor signaling, and the Inflammatory Bowel Disease (IBD) pathway (Fig. 4F). These findings suggest that Per2 deletion potentiates inflammatory signaling cascades in the context of colonic injury.\u003c/p\u003e\n\u003cp\u003eTo validate these observations, Gene Set Enrichment Analysis (GSEA) was performed. The results confirmed significant upregulation of key inflammatory pathways\u0026mdash;namely the TNF, JAK-STAT, and NF-\u0026kappa;B signaling axes\u0026mdash;in the \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e + DSS group compared to \u003cem\u003ePer2\u003csup\u003ef/f\u003c/sup\u003e\u003c/em\u003e + DSS controls (Fig. 4G). Together, these data indicate that loss of Per2 reprograms the epithelial transcriptome toward a proinflammatory state and amplifies the activation of canonical immune pathways during acute intestinal inflammation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePER2 knockdown promotes LPS-induced necroptosis in human intestinal epithelial cells via ZBP1-dependent signaling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further explore the mechanistic role of PER2 in regulating epithelial cell death, we employed an in vitro model using human colonic epithelial NCM460 cells. Four experimental groups were established: control (NC), PER2 knockdown (PER2 KD), control with LPS stimulation (NC + LPS), and PER2 knockdown with LPS (PER2 KD + LPS). Apoptosis was assessed by flow cytometry using Annexin V-FITC and PI staining. PER2 knockdown alone did not significantly increase apoptosis compared to controls. However, when combined with LPS, PER2 silencing significantly augmented apoptotic cell death relative to LPS-treated controls (Fig. 5A), indicating that PER2 deficiency sensitizes epithelial cells to LPS-induced cell death.\u003c/p\u003e\n\u003cp\u003eTo determine whether this increase in apoptosis involved necroptosis, we employed the RIPK3 inhibitor GSK872. The addition of GSK872 did not significantly alter apoptosis in the NC + LPS group, but markedly reduced apoptosis in PER2 KD + LPS cells (Fig. 5B), suggesting that PER2 deficiency promotes necroptotic rather than apoptotic cell death under inflammatory stress.\u003c/p\u003e\n\u003cp\u003eConsistent with this interpretation, Western blot analysis revealed that PER2 knockdown in LPS-stimulated cells led to increased expression of necroptosis-associated proteins ZBP1, MLKL, and RIPK3, as well as their phosphorylated forms p-MLKL and p-RIPK3 (Fig. 5C). These data suggest activation of the canonical ZBP1-RIPK3-MLKL necroptotic axis in the absence of PER2.\u003c/p\u003e\n\u003cp\u003eGiven that our previous transcriptomic data implicated ZBP1 as a key upstream mediator, we next assessed the effects of ZBP1 knockdown using siRNA. Western blotting showed that inhibition of ZBP1 in PER2 KD + LPS cells markedly reduced the expression of MLKL, RIPK3, and their phosphorylated counterparts (Fig. 5D), supporting a central role for ZBP1 in this pathway.\u003c/p\u003e\n\u003cp\u003eMoreover, flow cytometry demonstrated that apoptosis was significantly attenuated in PER2 KD + LPS cells following ZBP1 knockdown (Fig. 5E), confirming that ZBP1 mediates PER2-deficiency\u0026ndash;induced necroptosis. Together, these findings establish ZBP1 as a critical effector linking PER2 loss to necroptosis activation in intestinal epithelial cells during inflammatory challenge.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSTAT1 mediates PER2-deficiency\u003c/strong\u003e\u003cstrong\u003e\u0026ndash;\u003c/strong\u003e\u003cstrong\u003einduced necroptosis through transcriptional activation of ZBP1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate how PER2 regulates necroptosis at the transcriptional level, we performed immunoprecipitation (IP) of PER2 in NCM460 human intestinal epithelial cells, followed by mass spectrometry analysis to identify PER2-interacting proteins. Cross-referencing these candidates with a curated list of necroptosis-associated proteins revealed STAT1 and STAT3 as potential interactors (Fig. S4A), implicating them as downstream mediators of PER2[27].\u003c/p\u003e\n\u003cp\u003eTo validate this finding in vivo, we analyzed STAT1 and STAT3 expression in colonic epithelial cells isolated from \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003ePer2\u003csup\u003ef/f\u003c/sup\u003e\u003c/em\u003e mice subjected to DSS-induced colitis. Western blotting showed a significant increase in STAT1 protein levels in \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e + DSS mice compared to controls, whereas STAT3 expression remained unchanged (Fig. 6A; Fig. S4B), suggesting selective regulation of STAT1 by PER2 in inflamed colonic tissue.\u003c/p\u003e\n\u003cp\u003eTo determine the functional contribution of STAT1 to necroptosis, we knocked down STAT1 or STAT3 in LPS-treated PER2-deficient NCM460 cells. Western blot analysis revealed that STAT1 knockdown markedly reduced expression of ZBP1, MLKL, RIPK3, and their phosphorylated forms p-MLKL and p-RIPK3 (Fig. 6B), whereas STAT3 knockdown had no significant effect, indicating a critical role for STAT1 in mediating necroptotic signaling downstream of PER2 loss.\u003c/p\u003e\n\u003cp\u003eMotif prediction using the JASPAR database identified a canonical STAT1 binding motif (Fig. 6C). To experimentally verify direct transcriptional regulation of ZBP1 by STAT1, we performed chromatin immunoprecipitation (ChIP) assays in LPS-stimulated NCM460 cells. STAT1 was found to directly bind to the ZBP1 promoter in both PER2 knockdown and control conditions (Fig. 6D), indicating that PER2 deficiency does not impair binding but may enhance activation.\u003c/p\u003e\n\u003cp\u003eTo further test whether STAT1 binding promotes ZBP1 transcription, we constructed dual-luciferase reporter plasmids harboring wild-type or mutated STAT1-binding sites in the ZBP1 promoter (Fig. S4C). Luciferase assays showed significantly enhanced transcriptional activity in PER2-deficient cells transfected with the wild-type construct, but not with the mutant construct (Fig. 6E). This effect was abolished by STAT1 knockdown (Fig. 6F), confirming that STAT1 is essential for ZBP1 transcriptional activation in the context of PER2 deficiency.\u003c/p\u003e\n\u003cp\u003eTogether, these findings establish STAT1 as a key transcriptional mediator linking PER2 loss to necroptosis via direct regulation of ZBP1 expression in intestinal epithelial cells.\u003c/p\u003e\n\u003cp\u003eTo validate the interaction between PER2 and key transcription factors STAT1 and STAT3, we constructed a FLAG-tagged PER2 expression vector and performed co-immunoprecipitation (Co-IP) assays in NCM460 intestinal epithelial cells. Western blot analysis confirmed that PER2 physically interacts with both STAT1 and STAT3 (Fig. 7A\u0026ndash;B). Notably, reverse Co-IP using anti-STAT1 antibodies further validated the binding between PER2 and STAT1.\u003c/p\u003e\n\u003cp\u003eTo delineate the structural basis of this interaction, we performed in silico molecular docking using PyMol, which predicted multiple hydrogen bonds between PER2 and STAT1. Specifically, residues within PER2\u0026mdash;ARG325, ARG399, ALA446, TYR407, GLU322, and SER279\u0026mdash;were found to form stable hydrogen bonds with GLN314, ASN483, ASN233, GLU480, GLN311, and GLU141 of STAT1 (Fig. 7C). These interactions clustered within or near the PAS1, PAS2, and PAC domains of PER2, suggesting potential structural hotspots critical for protein binding.\u003c/p\u003e\n\u003cp\u003eTo experimentally test which domains are responsible for STAT1 interaction, we generated a series of truncated PER2 constructs targeting the PAS1, PAS2, and PAC domains, as well as N-terminal deletion mutants (Fig. S5A). Co-IP assays revealed that deletion of the PAS1 domain (\u0026Delta;PAS1) or expression of the N-N2 and N-N3 truncations markedly impaired PER2\u0026ndash;STAT1 binding (Fig. 7D\u0026ndash;E), while deletions of PAS2 or PAC (\u0026Delta;PAS2, \u0026Delta;PAC) did not significantly affect the interaction. These findings identify PAS1 as the essential structural domain mediating PER2\u0026rsquo;s interaction with STAT1.\u003c/p\u003e\n\u003cp\u003eTo assess the functional relevance of the PAS1 domain in necroptotic signaling, we analyzed the expression of necroptosis-related proteins in LPS-stimulated cells transfected with either full-length PER2 or the \u0026Delta;PAS1 mutant. Western blotting showed that loss of the PAS1 domain significantly reduced the expression of ZBP1, MLKL, RIPK3, and their phosphorylated forms (p-MLKL and p-RIPK3) compared to controls (Fig. S5B). These results indicate that the PAS1-dependent interaction between PER2 and STAT1 is essential for the transcriptional activation of necroptosis machinery.\u003c/p\u003e\n\u003cp\u003eTogether, these findings demonstrate that PER2 physically interacts with STAT1 via its PAS1 domain, and that this interaction is necessary for the activation of necroptosis-related pathways in intestinal epithelial cells under inflammatory stress.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePharmacological inhibition of STAT1 by Nifuroxazide attenuates colitis severity and necroptosis in Per2-deficient mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether STAT1 is a viable therapeutic target in \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice with colitis, we administered the selective STAT1 inhibitor Nifuroxazide during DSS-induced acute intestinal inflammation[28]. To determine whether STAT1 is a viable therapeutic target in \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice with colitis, we first prepared sodium alginate\u0026ndash;Nifuroxazide hydrogel microspheres and examined their morphology by scanning electron microscopy. The microspheres displayed a smooth external surface (Fig. S6A) and a porous internal architecture (Fig. S6B), a feature expected to facilitate controlled Nifuroxazide release. Mice were divided into two groups: \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e + DSS (control, receiving blank hydrogel) and \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e + DSS + Nifuroxazide (receiving nifuroxazide-loaded hydrogel). Throughout the disease course, mice receiving Nifuroxazide exhibited significantly reduced body weight loss compared to untreated controls (Fig. 8A), indicating that STAT1 inhibition mitigates colitis severity.\u003c/p\u003e\n\u003cp\u003eGross anatomical examination revealed visibly longer colons in Nifuroxazide-treated mice (Fig. 8B), which was quantitatively confirmed by colon length measurements (Fig. 8C). Disease Activity Index (DAI) scores were also significantly lower in the treatment group (Fig. 8D), and histopathological scoring of H\u0026amp;E-stained colon sections showed attenuated epithelial damage and reduced inflammatory cell infiltration (Fig. 8E\u0026ndash;F). These findings suggest that Nifuroxazide alleviates DSS-induced colonic injury in \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice.\u003c/p\u003e\n\u003cp\u003eTo investigate whether the protective effects of Nifuroxazide were mediated through inhibition of necroptosis, we analyzed the expression of necroptosis-related proteins in colonic tissue. Western blot analysis revealed markedly reduced expression of STAT1, ZBP1, MLKL, and RIPK3, along with diminished phosphorylation of MLKL and RIPK3, in the Nifuroxazide-treated group compared to controls (Fig. 8G\u0026ndash;I). Quantitative densitometric analysis confirmed the statistical significance of these reductions.\u003c/p\u003e\n\u003cp\u003eFurthermore, TUNEL/MLKL co-immunofluorescence staining demonstrated a substantial decrease in both apoptotic and necroptotic signals in colonic epithelial cells following Nifuroxazide treatment (Fig. 8J), suggesting that pharmacologic blockade of STAT1 protects epithelial integrity by suppressing necroptosis.\u003c/p\u003e\n\u003cp\u003eCollectively, these results indicate that STAT1 inhibition via Nifuroxazide alleviates intestinal inflammation and epithelial injury in \u003cem\u003ePer2\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice by dampening necroptotic signaling, highlighting STAT1 as a potential therapeutic target in PER2-deficient colitis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study identifies the circadian regulator PER2 as a critical suppressor of epithelial necroptosis in ulcerative colitis (UC) via modulation of the STAT1\u0026ndash;ZBP1 axis. Using a jet lag model of circadian disruption, we showed that altered light\u0026ndash;dark cycles significantly aggravated DSS-induced colitis, with worsened weight loss, colon shortening, histological injury, and elevated disease scores (Fig. S1A\u0026ndash;E). Rhythm disruption further suppressed Per2 expression, already downregulated in DSS-treated mice, indicating a link between circadian misalignment and intestinal vulnerability (Fig. S1F\u0026ndash;G). In human UC biopsies, PER2 expression was reduced, particularly in patients with concurrent sleep disorders, and PER2 loss was associated with increased epithelial apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;D). Transcriptomic analysis from GSE38713 confirmed significant downregulation of PER2 in active UC (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eTo define PER2\u0026rsquo;s role functionally, we generated intestinal epithelial cell-specific Per2 knockout mice (\u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e). These mice exhibited exacerbated DSS-induced colitis, with increased inflammatory infiltration, crypt damage, and elevated DAI and histological scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;F). RNA-seq of \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e IECs revealed enrichment of necroptosis and inflammatory pathways, notably TNF and JAK\u0026ndash;STAT signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u0026ndash;D), and upregulation of Zbp1, Ripk3, and Mlkl at both transcript and protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e2\u003c/span\u003eE\u0026ndash;G). TUNEL and MLKL co-staining further confirmed enhanced epithelial necroptosis (Fig. S3B).\u003c/p\u003e\u003cp\u003eMechanistically, we demonstrated that PER2 binds STAT1 via its PAS1 domain, a requirement for repressing necroptosis-related gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;E, Fig. S5). Loss of this interaction led to STAT1-dependent transcriptional activation of Zbp1, validated by ChIP and luciferase assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u0026ndash;F). Targeted inhibition of STAT1 with Nifuroxazide significantly alleviated colitis severity, suppressed necroptosis markers, and restored epithelial integrity in \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u0026ndash;J).\u003c/p\u003e\u003cp\u003eTogether, these findings uncover a previously unrecognized PER2\u0026ndash;STAT1\u0026ndash;ZBP1 axis that links circadian regulation to epithelial cell death. They also suggest that targeting STAT1 may offer therapeutic benefit for UC patients experiencing circadian disruption.\u003c/p\u003e\u003cp\u003eThe intestinal epithelium forms a dynamic interface between the host immune system and the luminal environment. Its integrity is essential for preventing microbial translocation and maintaining mucosal homeostasis, especially in the context of chronic inflammatory diseases such as ulcerative colitis (UC)[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Prior studies have primarily focused on tight junction proteins, cytokine signaling, and epithelial regeneration as determinants of barrier function, while the contribution of circadian regulators to epithelial cell death and survival has remained largely underexplored.\u003c/p\u003e\u003cp\u003eAmong core clock components, PER2 has been previously implicated in modulating inflammation, oxidative stress, and cell cycle regulation in peripheral tissues, including the liver and vasculature. In the gut, however, its function has remained ambiguous[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. While several studies have described diurnal oscillations of clock genes in intestinal tissues, few have directly linked PER2 to epithelial injury responses in UC[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Our study fills this gap by demonstrating that PER2 plays a non-redundant and protective role in maintaining epithelial integrity during acute colitis. In contrast to work emphasizing the anti-inflammatory effects of BMAL1 or NR1D1 through immune cell regulation, we show that PER2 acts within epithelial cells themselves to suppress necroptotic signaling cascades[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNecroptosis is a programmed, pro-inflammatory form of cell death executed through the RIPK3\u0026ndash;MLKL axis, leading to membrane rupture and the release of intracellular damage-associated molecular patterns (DAMPs) that amplify inflammation. Recent work has highlighted necroptosis as a key contributor to epithelial loss in UC, with upregulation of RIPK3 and MLKL observed in inflamed human colonic tissues[\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. ZBP1, a sensor of nucleic acids and endogenous danger signals, has also emerged as a driver of necroptotic activation in epithelial and immune cells[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, the upstream regulation of this pathway in the context of circadian rhythms has not been established. Our findings reveal that PER2 constrains ZBP1-mediated necroptosis by repressing STAT1 activity, positioning it as a central checkpoint linking the circadian clock to inflammatory epithelial cell death.\u003c/p\u003e\u003cp\u003eNotably, unlike traditional views of PER2 as a passive output of CLOCK\u0026ndash;BMAL1 transcription[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], our data suggest that PER2 may act as a direct modulator of transcription factor activity, specifically by binding and inhibiting STAT1\u0026rsquo;s access to inflammatory gene promoters. This is distinct from prior studies in macrophages or cancer cells where PER2 was shown to regulate inflammation indirectly through systemic cues or metabolic reprogramming[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Our work therefore expands the functional repertoire of PER2, positioning it as a local, cell-autonomous regulator of epithelial fate under inflammatory stress.\u003c/p\u003e\u003cp\u003eTaken together, these findings establish PER2 not only as a circadian rhythm gene but also as an epithelial stress-response factor. They challenge the classical paradigm that circadian genes operate solely through transcriptional oscillators and suggest that disruption of circadian homeostasis\u0026mdash;whether via environmental light shifts or intrinsic gene suppression\u0026mdash;can directly sensitize the epithelium to inflammatory injury[\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. This mechanistic link between clock gene dysfunction and necroptotic epithelial loss may have broad implications for understanding barrier failure in UC and other chronic inflammatory disorders.\u003c/p\u003e\u003cp\u003eThe identification of a PER2\u0026ndash;STAT1\u0026ndash;ZBP1 regulatory axis in epithelial necroptosis not only advances our understanding of UC pathogenesis but also opens new avenues for therapeutic intervention. Our data show that pharmacological inhibition of STAT1 with Nifuroxazide significantly attenuates colitis severity in \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, rescuing epithelial architecture and reducing necroptosis-associated protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u0026ndash;J). These results support the concept that targeting STAT1 activation downstream of circadian disruption may serve as a viable strategy to protect intestinal epithelial cells under inflammatory stress.\u003c/p\u003e\u003cp\u003eGiven that circadian disruption is increasingly prevalent in modern society\u0026mdash;affecting shift workers, frequent travelers, and individuals with sleep disorders\u0026mdash;this mechanism has particular relevance to a growing subset of UC patients whose disease severity may be aggravated by chronobiological misalignment[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Moreover, transcriptomic analyses from patient biopsies revealed that PER2 is suppressed in active UC and further downregulated in individuals with disordered sleep (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e1\u003c/span\u003e), suggesting that PER2 deficiency may represent a biomarker of heightened disease susceptibility or treatment resistance in these populations.\u003c/p\u003e\u003cp\u003eThe responsiveness of \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e colitis to STAT1 inhibition raises the possibility that therapeutic targeting of this pathway could offer benefit even when upstream circadian regulation is impaired. Such approaches could be integrated into personalized treatment strategies, incorporating circadian phenotyping and sleep behavior assessments into UC management frameworks. Furthermore, the fact that Nifuroxazide is already an orally available agent with anti-inflammatory potential may expedite its translational application, particularly for patients experiencing flares linked to sleep disruption or jet lag[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite the mechanistic insights offered by our study, several limitations warrant consideration. First, we employed an acute DSS-induced colitis model, which recapitulates epithelial injury and inflammation but does not fully reflect the chronicity and immune complexity of human UC. Future studies using chronic colitis models or genetic models of IBD may provide a more comprehensive understanding of PER2\u0026rsquo;s long-term regulatory role. In addition, although we observed consistent PER2 suppression and necroptotic activation in human UC specimens, functional validation in primary human epithelial cells or patient-derived organoids would strengthen translational relevance.\u003c/p\u003e\u003cp\u003eAnother open question is whether restoration of PER2 expression or activity could serve as a therapeutic strategy in itself. Pharmacological clock modulators or gene delivery systems targeting circadian machinery may offer future opportunities to re-establish epithelial resilience. Moreover, given the bidirectional relationship between the circadian clock and gut microbiota, it is plausible that PER2 deficiency alters microbial composition, further amplifying inflammation. Integrating metagenomic or metabolomic analyses will be important to define how microbiota\u0026ndash;host clock interactions contribute to necroptosis and mucosal injury.\u003c/p\u003e\u003cp\u003eLastly, while this study focuses on UC, the relevance of PER2-mediated necroptotic regulation in Crohn\u0026rsquo;s disease and other chronic inflammatory disorders remains to be explored. Whether this pathway also contributes to fibrosis, epithelial regeneration, or extraintestinal manifestations is unknown. Addressing these questions may broaden the applicability of circadian-targeted interventions in inflammatory diseases.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study establishes the circadian core protein PER2 as an epithelial-intrinsic guardian that restrains necroptosis-driven mucosal injury in ulcerative colitis. By physically sequestering STAT1 via its PAS1 domain, PER2 blocks STAT1-mediated transcription of ZBP1 and consequently shuts down the ZBP1\u0026ndash;RIPK3\u0026ndash;MLKL necroptotic axis. Circadian disruption or epithelial-specific Per2 deletion unleashes STAT1, precipitating ZBP1-dependent necroptosis, exaggerated cytokine responses and severe DSS-colitis. Conversely, pharmacologic inhibition of STAT1 with nifuroxazide rescues Per2-deficient mice, normalizing necroptotic signaling and mucosal architecture. The axis is conserved in humans: PER2 is markedly reduced in active UC and further suppressed in patients with comorbid sleep disturbance, correlating with enhanced necroptosis markers. Thus, the PER2\u0026ndash;STAT1\u0026ndash;ZBP1 circuit couples circadian integrity to epithelial survival and represents a readily druggable checkpoint whose targeting may benefit UC subjects with chronobiological dysfunction.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eANOVA\u0026emsp;Analysis of Variance\u003cbr\u003e\u0026nbsp;A-UC\u0026emsp;Active Ulcerative Colitis\u003cbr\u003e\u0026nbsp;BSA\u0026emsp;Bovine Serum Albumin\u003cbr\u003e\u0026nbsp;ChIP\u0026emsp;Chromatin Immunoprecipitation\u003cbr\u003e\u0026nbsp;CLOCK\u0026emsp;Circadian Locomotor Output Cycles Kaput\u003cbr\u003e\u0026nbsp;CRY1\u0026emsp;Cryptochrome 1\u003cbr\u003e\u0026nbsp;DAI\u0026emsp;Disease Activity Index\u003cbr\u003e\u0026nbsp;DAMP\u0026emsp;Damage-associated Molecular Pattern\u003cbr\u003e\u0026nbsp;DMEM\u0026emsp;Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium\u003cbr\u003e\u0026nbsp;DSS\u0026emsp;Dextran Sulfate Sodium\u003cbr\u003e\u0026nbsp;DTT\u0026emsp;Dithiothreitol\u003cbr\u003e\u0026nbsp;EDTA\u0026emsp;Ethylenediaminetetra-acetic Acid\u003cbr\u003e\u0026nbsp;FBS\u0026emsp;Fetal Bovine Serum\u003c/p\u003e\n\u003cp\u003eFITC \u0026nbsp;Fluorescein Isothiocyanate\u003cbr\u003e\u0026nbsp;GAPDH\u0026emsp;Glyceraldehyde-3-phosphate Dehydrogenase\u003cbr\u003e\u0026nbsp;GEO\u0026emsp;Gene Expression Omnibus\u003cbr\u003e\u0026nbsp;GO\u0026emsp;Gene Ontology\u003cbr\u003e\u0026nbsp;GSEA\u0026emsp;Gene Set Enrichment Analysis\u003cbr\u003e\u0026nbsp;H\u0026amp;E\u0026emsp;Hematoxylin and Eosin\u003cbr\u003e\u0026nbsp;HC\u0026emsp;Healthy Control\u003cbr\u003e\u0026nbsp;IBD\u0026emsp;Inflammatory Bowel Disease\u003cbr\u003e\u0026nbsp;IEC\u0026emsp;Intestinal Epithelial Cell\u003cbr\u003e\u0026nbsp;IFN-\u0026gamma;\u0026emsp;Interferon gamma\u003cbr\u003e\u0026nbsp;IgG\u0026emsp;Immunoglobulin G\u003cbr\u003e\u0026nbsp;IL-1\u0026beta;\u0026emsp;Interleukin-1 beta\u003cbr\u003e\u0026nbsp;IL-6\u0026emsp;Interleukin-6\u003cbr\u003e\u0026nbsp;IP\u0026emsp;Immunoprecipitation\u003cbr\u003e\u0026nbsp;JAK\u0026emsp;Janus Kinase\u003cbr\u003e\u0026nbsp;KEGG\u0026emsp;Kyoto Encyclopedia of Genes and Genomes\u003cbr\u003e\u0026nbsp;KD\u0026emsp;Knockdown\u003cbr\u003e\u0026nbsp;LC-MS/MS\u0026emsp;Liquid Chromatography-tandem Mass Spectrometry\u003cbr\u003e\u0026nbsp;LPS\u0026emsp;Lipopolysaccharide\u003cbr\u003e\u0026nbsp;MLKL\u0026emsp;Mixed Lineage Kinase Domain-like\u003cbr\u003e\u0026nbsp;NF-\u0026kappa;B\u0026emsp;Nuclear Factor Kappa-light-chain-enhancer of Activated B Cells\u003cbr\u003e\u0026nbsp;Nr1d1\u0026emsp;Nuclear Receptor Subfamily 1 Group D Member 1\u003cbr\u003e\u0026nbsp;PBS\u0026emsp;Phosphate-buffered Saline\u003cbr\u003e\u0026nbsp;PCA\u0026emsp;Principal Component Analysis\u003cbr\u003e\u0026nbsp;PCR\u0026emsp;Polymerase Chain Reaction\u003cbr\u003e\u0026nbsp;Per1/2\u0026emsp;Period 1/2\u003cbr\u003e\u0026nbsp;PI\u0026emsp;Propidium Iodide\u003cbr\u003e\u0026nbsp;PPI\u0026emsp;Protein\u0026ndash;protein Interaction\u003cbr\u003e\u0026nbsp;PSQI\u0026emsp;Pittsburgh Sleep Quality Index\u003cbr\u003e\u0026nbsp;PVDF\u0026emsp;Polyvinylidene Fluoride\u003cbr\u003e\u0026nbsp;qPCR\u0026emsp;Quantitative PCR\u003cbr\u003e\u0026nbsp;RIPK3\u0026emsp;Receptor-interacting Protein Kinase 3\u003cbr\u003e\u0026nbsp;R-UC\u0026emsp;UC in Remission\u003cbr\u003e\u0026nbsp;SDS-PAGE\u0026emsp;Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis\u003cbr\u003e\u0026nbsp;SPF\u0026emsp;Specific-pathogen-free\u003cbr\u003e\u0026nbsp;STAT1/3\u0026emsp;Signal Transducer and Activator of Transcription 1/3\u003cbr\u003e\u0026nbsp;TNF-\u0026alpha;\u0026emsp;Tumor Necrosis Factor alpha\u003cbr\u003e\u0026nbsp;TUNEL\u0026emsp;Terminal Deoxynucleotidyl Transferase dUTP Nick-end Labeling\u003cbr\u003e\u0026nbsp;UC\u0026emsp;Ulcerative Colitis\u003cbr\u003e\u0026nbsp;WT\u0026emsp;Wild Type\u003cbr\u003e\u0026nbsp;ZBP1\u0026emsp;Z-DNA-binding Protein 1\u003cbr\u003e\u0026nbsp;ZT\u0026emsp;Zeitgeber Time\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by grants from the National Key Research and Development Program (Grant No. 2023YFC2307001) and the National Natural Science Foundation of China (NSFC) (Grant Nos. 81800480 and 81800465).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests or conflicts of interest in relation to this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in strict accordance with ethical guidelines. Ethical approval for animal experiments was obtained from the Animal Ethics Committee of Huazhong University of Science and Technology (Approval No. 2023-4300). For human subjects, approval was granted by the Independent Ethics Committee of Wuhan Union Hospital (Approval No. 2022-S147). The study adhered to the principles set forth in the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants involved in this study provided written informed consent for the collection of their data and subsequent publication of the study findings. The consent process adhered to the ethical guidelines of the Independent Ethics Committee of Wuhan Union Hospital and complies with the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.J.R. performed the experiments and drafted the manuscript. P.S.Y. contributed to data collection. M.Y.Q. conducted the animal experiments. W.H.R. participated in the literature review and data analysis. J.Y. and L.R. conceptualized the study and provided critical revisions of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVoelker R. What Is Ulcerative Colitis? JAMA. 2024;331:716. \u003c/li\u003e\n\u003cli\u003eLe Berre C, Honap S, Peyrin-Biroulet L. Ulcerative colitis. Lancet. 2023;402:571\u0026ndash;84. \u003c/li\u003e\n\u003cli\u003eP W, K Y, A L. Ulcerative colitis: clinical biomarkers, therapeutic targets, and emerging treatments. 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Trends Mol Med. 2025;31:588\u0026ndash;90. \u003c/li\u003e\n\u003cli\u003ePiovani D, Danese S, Peyrin-Biroulet L, Nikolopoulos GK, Lytras T, Bonovas S. Environmental Risk Factors for Inflammatory Bowel Diseases: An Umbrella Review of Meta-analyses. Gastroenterology. 2019;157:647-659.e4. \u003c/li\u003e\n\u003cli\u003eBailly C. Toward a repositioning of the antibacterial drug nifuroxazide for cancer treatment. Drug Discov Today. 2019;24:1930\u0026ndash;6. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ulcerative colitis, Circadian rhythm, Necroptosis, Per2","lastPublishedDoi":"10.21203/rs.3.rs-7601246/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7601246/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eUlcerative colitis (UC) is a chronic inflammatory bowel disease characterized by epithelial barrier disruption, excessive cell death, and dysregulated immune signaling. While circadian rhythms are known to influence immune homeostasis, the role of the core circadian regulator Period circadian clock 2 (PER2) in intestinal inflammation remains poorly understood.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eWe investigated the impact of circadian rhythm disruption on colitis severity using a jet lag model in DSS-treated mice, as well as clinical UC samples from patients with or without sleep disorders. Epithelial-specific Per2 knockout (\u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e) mice and in vitro PER2 knockdown models were employed to dissect the mechanistic role of PER2 in regulating epithelial cell death and inflammation. Bulk RNA-sequencing, molecular docking, chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays were used to identify downstream targets and interacting partners. Pharmacological inhibition of STAT1 using Nifuroxazide was tested for therapeutic potential.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eCircadian disruption aggravated DSS-induced colitis in mice and increased epithelial apoptosis in UC patients, accompanied by marked suppression of PER2 expression. Loss of PER2 in mice resulted in exacerbated intestinal inflammation, elevated DAI scores, and histological damage. Transcriptomic profiling and functional assays revealed that PER2 deficiency promoted necroptosis by upregulating ZBP1, RIPK3, and MLKL at both transcriptional and protein levels. Mechanistically, PER2 directly interacts with STAT1 via its PAS1 domain and restrains STAT1-mediated transcription of Zbp1. Inhibition of STAT1 by Nifuroxazide ameliorated colitis severity and suppressed necroptotic signaling in \u003cem\u003ePer2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice, highlighting the PER2–STAT1–ZBP1 axis as a key pathway linking circadian disruption to epithelial injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eOur findings identify PER2 as a critical suppressor of intestinal epithelial necroptosis and inflammation, acting through inhibition of STAT1-dependent ZBP1 activation. Circadian rhythm disruption impairs this protective pathway, exacerbating colitis severity. Pharmacological targeting of STAT1 may offer a novel therapeutic strategy for UC patients with circadian rhythm dysregulation.\u003c/p\u003e","manuscriptTitle":"Circadian Regulator PER2 Protects Against Epithelial Necroptosis in Ulcerative Colitis via the STAT1–ZBP1 Axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-12 14:00:47","doi":"10.21203/rs.3.rs-7601246/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4a40c594-deb6-42e9-8c56-ef1222be861f","owner":[],"postedDate":"October 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-27T13:57:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-12 14:00:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7601246","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7601246","identity":"rs-7601246","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}