Table of Contents
Experimental animals and groups
Sixty healthy adult SPF grade male Sprague-Dawley (SD) rats, aged 10–12 weeks and weighing 300–350 g, were provided by Hunan Xilongjiang Jinda Laboratory Animal Co., Ltd. (Laboratory Animal License Number: SCXK(Xiang)2019-0004). All animal experimental procedures and handling were approved by the Experimental Animal Ethics Committee of Guilin Medical University (Number: GLMC-IACUC-2021014), and all experiments were performed in accordance with relevant regulations and reported in accordance with the ARRIVE guidelines. Using the random number table method, rats were randomly divided into five groups (yeah = 12). Six rats were randomly selected 6 and 24 hours after I/R. (1) In the sham-operated group (Sham group), the abdomen was opened, the corresponding ligaments and blood vessels were released and exposed, the abdominal cavity was washed with warm saline, and then the abdomen was closed. (2) In the autologous liver transplantation (AOLT) I/R group (I/R group), the abdomen was opened, the corresponding ligaments and blood vessels were released and exposed, the abdominal cavity was washed with warm saline, and then the portal vein (PV), suprahepatic vena cava (SHVC), inferior hepatic vena cava (IHVC), and proper hepatic artery were blocked in sequence. A perfusion device was used to deliver 4 °C sodium lactate Ringer's solution to the liver perfusion via the PV. A part of the IHVC was cut as an outflow route. After 30 ± 1 min, the blood flow of the blocked blood vessels was restored, and the liver changed from yellowish brown to bright red. The abdominal cavity was washed, and the abdomen was closed. (3) In the ferrostatin-1 group (I/R + Fer-1 group), 5 mg/kg ferrostatin-1 (catalog number: HY-100579, MCE, China) was intraperitoneally injected 1 hour before surgery. Other procedures were the same as in the I/R group. (4) In the sulfasalazine group (I/R + SAS group), 500 mg/kg SAS (catalog number: HY-14655, MCE, China) was intraperitoneally injected twice a day 7 days before surgery. Other procedures were the same as in the I/R group. (5) In the SAS + ferrostatin-1 group (I/R + SAS + Fer-1 group), 500 mg/kg SAS was intraperitoneally injected twice a day 7 days before surgery, and 5 mg/kg ferrostatin-1 was intraperitoneally injected 1 hour before surgery. Other procedures were the same as in the I/R group.
Establishing the model
This study refers to the literature on the establishment of an I/R-induced intestinal injury model after autologous orthotopic LT in SD rats.12,13,14Before modeling, rats were fasted for 8 h and allowed to drink water ad libitum. Rats were intraperitoneally injected with 3% sodium pentobarbital (0.2 ml/100 g) to maintain anesthesia and 0.005% fentanyl (0.16 ml/100 g) to relieve pain. Each rat was fixed in a supine position, hair was removed from the abdomen for skin preparation, the skin was disinfected with iodophor, and sterile surgical towels were laid down. A midline incision was made in the abdomen, followed by layer-by-layer dissection into the abdominal cavity. The hepatic falciform ligament and left deltoid ligament were dissected, the liver was turned left to open the right retroperitoneum, and the SHVC and IHVC were carefully separated with blunt force. The hepatoduodenal ligament was then cut, exposing the PV and proper hepatic artery. The proper hepatic artery, PV, and IHVC were clamped in sequence using non-injury vascular clips to completely block liver blood flow (anhepatic phase). The liver was perfused with 1 mL of heparinized saline (25 U/mL) to remove blood in the liver, prevent thrombosis, and replenish the rat's blood volume, and then the SHVC was blocked. A No. 4 infusion needle was used to inject 4°C lactated Ringer's solution into the liver from the PV, and a small hole (1-2 mm) was opened at the top of the IHVC block as an outflow tract. The perfusion rate of sodium lactate Ringer's solution was 6-8 mL/min, and the perfusion pressure was 10 kPa. The surface of the liver was covered with fine ice chips, and the cold perfusion time was 30 ± 1 min. The liver gradually changed from bright red to an earthy tan color, indicating successful modeling. After perfusion, the PV needle site and the outflow tract of the IHVC were carefully sutured with 8-0 non-traumatic sutures. The SHVC, IHVC, PV, and proper hepatic artery were opened in sequence. After hepatic blood flow was restored, the liver changed from tan to red. After rinsing the abdominal cavity with warm saline, the abdomen was closed.
Specimen collection
Six and 24 hours after the anhepatic phase, rats were anesthetized again, and 2 ml of blood was collected from the inferior vena cava and placed in a BD tube. After centrifugation, the specimens were stored in a -80°C freezer. After blood collection, the left ventricle was perfused with 50 ml of heparinized saline at 4°C. The right atrial appendage was used as the outflow tract. After sufficient perfusion to remove residual blood from the heart and body, rat ileal tissues were harvested and stored in a -80°C freezer.
Detection Indicators
Observation of the pathological changes in the intestines of rats in each group by hematoxylin and eosin (H&E) staining and optical microscope
Ileal tissues from rats were harvested and fixed in 4% paraformaldehyde solution for more than 24 hours. After the specimens were embedded in paraffin, they were sectioned (intestinal tissue section thickness was 5 μm) and stained with H&E. The intestinal sections were observed under a light microscope (EVOS M5000, Thermo Fisher Scientific, USA) (200x magnification), and the Chiu scoring method was used to describe the pathological results. The work was performed by two experts from the Department of Pathology at the Affiliated Hospital of Guilin Medical University.
Wet/dry weight (W/D) ratio of intestinal tissue
At 6 and 24 hours after AOLT hepatic reperfusion, 5 cm of ileal tissue from each group was collected, washed with 0.9% saline, and weighed as wet weight. Then, the tissue was baked in an oven at 80°C for 24 hours, and then weighed again. This is called dry weight. The intestinal W/D ratio of each experimental group was calculated in turn.
Measurement of FeAges 2+ Intestinal tissue contents
Approximately 0.1 g of intestinal tissue and 1 mL of extract were homogenized in an ice bath. After centrifugation at 4000×,G The mixture was incubated at 4°C for 10 min and the supernatant was collected. The microplate reader was preheated for 30 min, the wavelength was adjusted to 520 nm, and the OD value was adjusted to zero with distilled water. The iron content in the intestinal tissue was detected using a tissue iron content detection kit (Beijing Solar Bioscience Technology Co., Ltd., Catalog No.: BC4355).
Analysis of the decrease in GSH content in intestinal tissue and serum
(1) Frozen intestinal tissue (0.1 g) was weighed and thoroughly ground in an automatic sample rapid grinder (Shanghai Jingxin, China). The samples were then centrifuged at 8000 rpm for 10 min at 4 °C, and the supernatants were stored at 4 °C for analysis. (2) Anticoagulated blood was centrifuged at 600 × at 4 °C.G The upper layer (plasma) was transferred to another tube and centrifuged at 8000× at 4°C for 10 min.G The mixture was incubated for 10 min. The supernatant was transferred to a new tube and stored at 4 °C for analysis. GSH in the intestinal tissue and serum was detected using a reduced GSH content assay kit (catalog number: BC1175, Beijing Solar Bioscience & Technology Co., Ltd.).
Analysis of GPX4 activity
Approximately 0.1 g of rat intestinal tissue stored at -80 °C was thoroughly ground using an automatic sample rapid grinder (Shanghai Jingxin, China). After centrifugation, the supernatant was collected. Corresponding reagents were added according to the manual provided with the Intestinal GPX4 Detection Kit (Cat. No.: K003083P, Beijing Solarbio Science & Technology Co., Ltd.). Then, the corresponding OD value was obtained at a wavelength of 450 nm using a microplate reader, and the results were analyzed.
MDA content analysis
The peroxidation product MDA combines with TBA to form a red product, with a maximum absorption peak at a wavelength of 532 nm. The OD value of the product is measured using a microplate reader, and then the MDA content is calculated. The degree of lipid oxidation can be evaluated by determining the MDA concentration. Briefly, approximately 0.1 g of intestinal tissue was collected, homogenized, and centrifuged at 8000.G The mixture was stirred at 4°C for 10 min, and the supernatant was collected and placed on ice for testing. After centrifugation of the anticoagulated blood, the upper layer (serum) was collected and the MDA content in the intestinal tissue and serum was measured using an MDA content assay kit (Cat. No.: BC0025, Beijing Solar Bioscience & Technology Co., Ltd.).
Measurement of inflammatory cytokine IL-6 levels in serum of rats
A rat IL-6 ELISA kit (product number: SEKR-0005, Beijing Solebo Biotechnology Co., Ltd.) was used to detect the levels of the inflammatory cytokine IL-6 in rat serum.
Measurement of the activity of intestinal mucosal injury markers
Diamine oxidase (DAO) is widely found in animals (intestinal mucosa, lung, liver, kidney, etc.), plants and microorganisms. The activity of catalytically oxidizing polyamines to aldehydes is closely related to protein synthesis and can reflect the integrity and damage of intestinal tissue. The activity at 500 nm was calculated by measuring the absorbance at the wavelength of DAO. A DAO activity detection kit (product number: BC1280, Beijing Solebao Biotechnology Co., Ltd.) was used to detect DAO activity in intestinal tissue.
Measurement of superoxide dismutase activity
Superoxide dismutase (SOD) is the main antioxidant enzyme in the body that scavenges oxygen and protects cells from oxidative damage.–The test should be strictly according to the SOD activity test kit (product number: BC0170, Beijing Solebao Biotechnology Co., Ltd.).
Measurement of xCT and GPX4 expression in rat intestine
The expression of GPX4 and xCT was analyzed by Western blotting. Ileal tissue (0.1 g) was harvested, homogenized, and centrifuged at 13,400 rpm.GAfter incubation at 4 °C for 5 min, the supernatant was collected. Protein concentration was quantified by BCA method. After boiling and denaturing the samples, proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a membrane. The membrane was blocked with 5% nonfat dry milk for 2 h at room temperature. Primary antibodies (anti-ferritin heavy chain (dilution 1:1000, Abcam, UK), anti-glutathione peroxidase 4 (dilution 1:1000, Abcam, UK), anti-xCT (dilution 1:1000, Abcam, UK), and anti-β-actin (dilution 1:5000, Abcam, UK)) were added separately and the membrane was incubated overnight at 4 °C. Anti-rabbit secondary antibody (dilution 1:5000, Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) was added and the membrane was incubated at room temperature for 30 min. Chemiluminescence development and fixation were performed. AlphaEaseFC software processing system was used for analysis, and the ratio of the gray value of the target protein band to the gray value of the β-actin band was used to reflect the expression level of the target protein.
Statistical analysis
SPSS 26.0 was used to construct the database and perform statistical analysis. Data following normal distribution were analyzed using the mean and standard deviation (\(\overline{x}\)± sUse repeated measures ANOVA to compare differences between multiple groups. Then compare differences between two groups. If the variances are heterogeneous, use the Dunnett test. If the variances are homogeneous, use the LSD-t test.P< 0.05 was considered statistically significant.
