Reagents

Anti-cGAS Rabbit pAb(#A8335) were obtained from ABclonal Technology (Wuhan, China). Anti-Phospho-STING (Ser365) Rabbit mAb (#72,971), Anti-Phospho-STING (Ser366) Rabbit mAb (#50,907), Anti-STING Rabbit mAb (#13,647), Anti-Phospho-TBK1/NAK (Ser172) Rabbit mAb (#5483), Anti-TBK1/NAK Rabbit mAb (#3504), Anti-Phospho-IRF- 3 (Ser396) Rabbit mAb (#29,047), Anti-IRF- 3 Rabbit mAb (#4302), Anti-Tom20 (D8T4N) Rabbit mAb (#42,406) were obtained from Cell Signaling Technology (Danvers, USA). Anti-TFAM Polyclonal antibody (#22581-1-AP) were obtained from Proteintech (Wuhan, China). HT-DNA (#D6898) was obtained from Sigma-Aldrich (St. Louis, USA).

IIR model

Male, 8–10 week old, STING knockout (STING−/−) and wild-type C57BL/6 J mice were purchased from Cyagen Biosciences and housed in a specific pathogen-free (SPF) environment. Age- and sex-matched wild-type mice served as controls. To establish the intestinal ischemia–reperfusion (IIR) model, mice were anesthetized with intraperitoneal pentobarbital (40 mg/kg). A midline laparotomy was performed, and the superior mesenteric artery was clamped for 45 min using a microvascular clip. After 45 min, the clip was removed, allowing 2 h of reperfusion before tissue collection.

Juco Ethics statement

All experiments involving animals were conducted according to the ethical policies and procedures approved by the Ethics Committee of Renmin Hospital, Wuhan University(Approval NO. WDRM 20200308).

RNA Sequencing and Differentially Expressed Genes Analysis

The libraries were sequenced on an Illumina Novaseq 6000 platform and 150 bp paired-end reads were generated. About 6500 raw reads for each sample were generated. Raw reads of fastq format were firstly processed using fastp and the low quality reads were removed to obtain the clean reads. Then about 520,000 clean reads for each sample were retained for subsequent analyses. The clean reads were mapped to the reference genome using HISAT2. FPKM of each gene was calculated and the read counts of each gene were obtained by HTSeq-count. Gene Set Enrichment Analysis (GSEA) was performed using GSEA software. The analysis was used a predefined gene set, and the genes were ranked according to the degree of differential expression in the two types of samples. Then it is tested whether the predefined gene set was enriched at the top or bottom of the ranking list.

Chiu's Scoring System

Chiu’s scoring system is used to histologically assess the severity of intestinal ischemia–reperfusion injury. The score is based on microscopic examination of tissue damage, ranging from normal to severe injury, allowing for comparative analysis across experimental groups.Score range: 0–5. 0 (Normal): Intact tissue structure, orderly epithelial arrangement, visible nuclei, complete microvilli, and normal vasculature.1 (Mild injury): Mild epithelial swelling, slightly displaced or blurred nuclei, minor microvilli breakage, intact basement membrane, no significant endothelial damage.2 (Moderate injury): Epithelial swelling, disrupted or fragmented nuclei, significant microvilli breakage, partial basement membrane damage, endothelial injury with mild thrombus formation.3 (Severe injury): Epithelial loss, absent or fragmented nuclei, extensive microvilli disruption, basement membrane damage, severe endothelial injury with thrombus formation and vascular occlusion.4 (Very severe injury): Complete epithelial loss, widespread basement membrane damage, extensive microvilli disruption, endothelial apoptosis/necrosis, severe vascular occlusion, and significant hemorrhage and inflammation.5 (Terminal injury): Complete destruction of tissue structure, absent nuclei, loss of microvilli, extensive basement membrane destruction, complete endothelial necrosis, severe vascular occlusion, widespread tissue necrosis, and fibrosis.

HR Model

HT- 29 cells (Procell Life Science&Technology, Wuhan, China) were cultured in McCoy's 5 A medium supplemented with 10% FBS and 1% penicillin/streptomycin (P/S). Mouse small intestinal organoids were cultured in IntestiCult™ Organoid Growth Medium (STEMCELL, 06005). To establish a hypoxia-reoxygenation (HR) model, both cell lines and organoids were subjected to 12 h of hypoxia (37 °C, 5% CO2 + 1% O2 + 94% N2) in a tri-gas incubator (Thermo, 160i, USA), followed by 4 h of reoxygenation (37 °C, 5% CO2) in a standard incubator.

MtDNA Intraperitoneal Injection

To facilitate mtDNA transfection, mice were intraperitoneally injected with 5 mg/kg of mtDNA complexed with Lipomaster 3000 Transfection Reagent (TL30-1, Vazyme) [22]. The mtDNA was extracted from liver tissue using the TIANamp Genomic DNA Kit (DP304, TIANGEN). Control mice received intraperitoneal injections of Lipomaster 3000 Transfection Reagent without mtDNA.

p0 cell Establishment

HT- 29 cells were cultured in DMEM/H medium supplemented with 10% FBS, 2 mM L-glutamine, 100 μg/ml sodium pyruvate (HY-W015913, MCE), and 50 μg/ml uridine (HY-B1449, MCE). Cells were passaged twice weekly, or as needed. For experiments, approximately 1 × 10^5 cells were seeded in 100 mm dishes and treated with 50 ng/ml ethidium bromide (EtBr) after 24 h. After approximately one week of EtBr treatment, dead cells began detaching from the dishes. These were removed by washing with PBS and replacing with fresh medium containing 50 ng/ml EtBr. Around one month post-EtBr treatment, visible colonies, potentially ρ0 cells, emerged. Individual colonies were isolated using penicillin and transferred to 24-well plates for expansion. The medium was changed every two days. Cells were expanded until sufficient for total DNA isolation.

Development of a Small Intestinal Organoid Model in vitro

Following euthanasia, a 15 cm segment of small intestine was excised, flushed of luminal contents, and sectioned into 2 mm fragments. The tissue underwent 20–25 washes with chilled (2–8 °C) DPBS (BI, 020231 ACS), with the supernatant discarded after each wash. Tissue fragments were then incubated in 25 mL of DPBS containing 5 mM EDTA (Invitrogen, AM9260G) for 15 min at room temperature (15–25 °C), followed by supernatant removal.

To isolate intestinal crypts, the tissue was resuspended in 10 mL of chilled DPBS containing 0.1% BSA (Vetec, V900933). After supernatant removal and filtration through a 70 μm filter, the filtrate was centrifuged at 290 × g for 5 min at 2–8 °C. The supernatant was discarded, and the pelleted crypts were resuspended in 10 mL of chilled DPBS containing 0.1% BSA. Following a second centrifugation at 200 × g for 3 min at 2–8 °C, the supernatant was removed.

The isolated crypts were resuspended in 10 mL of chilled DMEM/F- 12 (Gibco, 12634010), and crypt density was determined using a hemocytometer under an inverted microscope. Based on this count, a volume containing approximately 500 crypts was calculated. After discarding the supernatant, the crypts were resuspended in 150 μL of IntestiCult™ Organoid Growth Medium (STEMCELL, 06005) and mixed with 150 μL of undiluted Matrigel® Matrix (Corning, 354230). 50 μL of this suspension was plated in the center of each well of a pre-warmed 24-well plate and allowed to solidify for 10 min at 37 °C. 750 μL of Complete IntestiCult™ Organoid Growth Medium was then added to each well. Plates were incubated at 37 °C and 5% CO2. Crypt budding was typically observed within 3 h, with small intestinal organoids sprouting and forming complex multi-lobed structures between 5–7 days of incubation.

Transmission Electron Microscopy (TEM)

Small intestinal tissues were harvested from IIR and control mice, fixed in 2.5% glutaraldehyde, and sectioned at 70–90 nm using an ultramicrotome. Sections were stained with uranyl acetate and lead citrate and imaged using transmission electron microscopy (TEM) to obtain high-resolution micrographs [23].

Determination of MDA and LDH Release Rate

Malondialdehyde (MDA) levels were quantified using a colorimetric assay kit (Beyotime, China) and normalized to protein content. Lactate dehydrogenase (LDH) release was measured in cell culture supernatants using a colorimetric assay kit (Beyotime, China) according to the manufacturer's instructions. Absorbance values were measured at 490 nm with a reference wavelength of 600 nm.

Western Blot

Protein lysates from intestinal tissues, HT- 29 cells (Procell Life Science&Technology), and small intestinal organoids were separated by SDS-PAGE (10%) and transferred to PVDF membranes (Millipore). Membranes were blocked with 5% BSA in TBST, incubated overnight with primary antibodies, and subsequently incubated for 1 h with corresponding secondary antibodies. Protein bands were visualized using a Bio-Rad imaging system and quantified by densitometry using ImageJ software.

qPCR Analysis

DNA was extracted from plasma, serum, or cell culture supernatant using the TIANamp Genomic DNA Kit (DP304, TIANGEN) according to the manufacturer's instructions. RNA was isolated from tissues, cells, or organoids using the Cell/Tissue Total RNA Isolation Kit (Vazyme, RC101) and reverse transcribed into cDNA using a Vazyme cDNA synthesis kit. Gene expression was then quantified by real-time PCR using SYBR Green qPCR Master Mix (Vazyme, Q711 - 02/03). The expression levels of target genes were normalized to the ACTB (β-actin) gene, which served as an internal control. Relative gene expression was calculated using the ΔΔCt method, where the Ct values of the target gene were subtracted from the Ct values of ACTB, and the relative fold change in expression was determined by comparing experimental groups to controls. Primer information is shown in Table 1.

Table 1 This data is mandatory. Please provideTUNEL

Paraffin sections were mounted on slides and stained for apoptosis using a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay kit (C1098, Beyotime), following the manufacturer's instructions. Nuclei were counterstained with DAPI [22].

Quantification and Statistical Analysis

Data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism 9. A P value < 0.05 was considered statistically significant. All animal experiments included at least three mice per group. Two-group comparisons were analyzed using unpaired, two-tailed Student's t-tests. Multiple group comparisons were analyzed using one-way ANOVA followed by Tukey's post hoc test.