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Hypoxic preconditioning accelerates the healing of ischemic intestinal injury by activating HIF-1α/PPARα pathway-mediated fatty acid oxidation – Cell Death Discovery

Animals

30 SPF-grade male SD rats weighing 250 g were randomly divided into two groups: A control group and an HPC group, with 15 rats in each group. Animal managers and experimentalists do not know the order of distribution; Researchers assessing, testing, or quantifying experimental results do not know the intervention. The Control group was fed normally. The HPC group was pretreated in a small animal hypobaric chamber for 7 days (2 h/day). Intestinal ischemia (a 45 min occlusion of the superior mesenteric artery) was performed on the eighth day. The rats were sacrificed under anesthesia on days 9, 10, 11, and 15, and the intestinal tissues were removed. The procedure for animal experiments is shown in Fig. 1A.

Animals were obtained from the Laboratory Animal Center at the Air Force Medical University and were approved by the Animal Ethics Committee of the Air Force Medical University before the experiment began. All experiments were performed in accordance with relevant named guidelines and regulations. The animals were euthanized by intraperitoneal injection of 200 mg/kg of sodium pentobarbital after the experiment. All animal experiments adhere to the ARRIVE guidelines.

Cell culture and hypoxic treatment

IEC-6 cells were purchased from Shanghai Enzyme Research Biotechnology Co., LTD., and the cells were verified by STR. The cell lines were maintained at 37 °C and 5% CO2. Cell lines were cultured in 10% FBS, 1% P/S (Penicillin-Streptomycin), 0.1 u/ml insulin, DMEM, high glucose, and a medium containing pyruvate (ThermoFisher, 11995065). The cells were passaged when the cell density reached more than 80%. The HPC model was established at the hypoxic workstation with environmental parameters of 1% O2, 5% CO2, 94% N2, and a temperature of 37 °C for 24 h.

Histopathological examination

The small intestine of the rats was rinsed with normal saline, and approximately 2 cm segments of the ileum were removed, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned laterally, and stained with H&E (hematoxylin and eosin). The injury and recovery of intestinal tissue were observed under a microscope and evaluated according to Chiu’s scoring method [31]. Intestinal goblet cells were marked by AB/PAS staining kit (Solarbio Science & Technology, Beijing).

Determination of FAO in vivo

The small intestine tissue was prepared into 10% homogenate, boiled in a boiling water bath for 10 min, mixed, and extracted for 1 min. The supernatant was taken at 3500 rpm for 10 min, and the ATP content was detected according to the kit instructions (Jiancheng Bioengineering Institutes, Nanjing). In addition, rat serum samples were used to detect the content of β-hydroxybutyrate and acetoacetic acid, and the operation procedures were as shown in the instructions (Solarbio Science & Technology, Beijing).

Assessment of intestinal barrier dysfunction

Assessment of intestinal barrier dysfunctionIntestinal barrier dysfunction was assessed by measuring serum diamine oxidase (DAO) and intestinal Zonula occludens 1 protein (ZO-1) (1:100, Abcam, ab221547). Serum DAO activity detection kit (Solaibao Technology Co., Ltd., Beijing). All procedures were performed according to the manufacturer’s protocol (n = 3 per group).

Immunohistochemistry

Small intestinal tissue was fixed with 4% paraformaldehyde, then embedded in paraffin and sectioned (about 3-4 μm thick). The paraffin sections were deparaffinized with xylene, dehydrated in an ethanol gradient, followed by hydration and antigen repair in a citrate high-temperature water bath, and blocked with BSA. Primary antibodies were incubated overnight at 4 °C ((CPT1A (1:1000, Abcam, ab234111), (LGR5 (1:400, Abcam, ab219107), Ki67(1:50, Abcam, ab279653)). The secondary antibodies were incubated at room temperature for 1 h, followed by microscopic observation.

Quantitative real-time PCR assay

Total RNA was extracted from IEC-6 cells and the intestine using TransZolTM. cDNA was synthesized according to NovoScript®Plus All-in-one 1st Strand cDNA Synthesis SuperMix (gDNA Purge). Amplification was performed on the Roche LightCycler® 480 fluorescence quantitative system according to TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) instructions. The relevant primer sequences are shown in Supplementary Table 1. β-actin was used to correct the mRNA levels, and the relative mRNA expression was calculated using the 2−Ct method. Primer sequence is shown in supplementary Table 1.

Western blotting assay

Cells and intestinal tissues were added to RIPA lysate (containing protease inhibitors) to extract protein samples, and the concentration of the samples was determined by the BCA method. Protein samples were loaded on SDS-PAGE gels (8-12%) and electrophoresed until the sample reached the bottom of the gel. The proteins were then transferred to a 0.45 um PVDF membrane (Millipore, USA) at a constant current of 300 mA. Next, sample membranes containing proteins were placed in a protein-free rapid blocking solution for 0.5 h at room temperature, followed by incubation with primary antibodies (HIF-1α (1:500, Novus, #NB100-105), PPAR alpha(1:1000, Abcam, ab215270), CPT1A (1:1000, Abcam, ab234111), β-actin(1:1000, Cell Signaling Technology, #3700 S) overnight at 4 °C. The next day, the membranes were incubated with HRP-labeled secondary antibodies (HRP-conjugated Goat Anti-Rabbit IgG (H + L) (1:10000, InCellGene, SA-10011)), or HRP-conjugated Goat Anti-mouse IgG (1:10000, Zhongshan Jinqiao Biotechnology, ZB-2305) for 1.5 h at room temperature. Finally, the ChemiScope 6000 Exp chemiluminescence imaging system and enhanced chemiluminescence were used to detect protein levels. β-actin was used to relatively correct the levels of proteins.

Transfection experiment

Cells were inoculated with 5 × 103 cells per milliliter. The transfection complex was prepared with riboFECT™ CP reagent, riboFECT CP buffer, and siRNA reserve solution at 50 nM and incubated at room temperature for 15 min. After the cells grew to 50% and fused, then they were added to the complete culture medium and mixed well. After 12 h of cell culture, the cells of each group were treated with hypoxia or normoxia.

Statistical analyses

All statistical analyses were performed with Graphpad prism 8.0.2. Continuous variables were expressed as mean ± standard deviation (SD), there were three samples, and each sample repeated the experiment three times. The Shapiro-Wilk test was used to check the normality and homogeneity of variance of all data. For two-group comparisons, Student t-tests were used to determine differences between groups for normally distributed data. For multiple group comparisons, p values were analyzed using multiple t-tests. All tests were two-sided, and p <0.05 was considered statistically significant.

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