We purchased male C57BL/6J mice (6–8 weeks old) from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The animals were housed in the Animal Facility of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All animal experiments were conducted after approval from the Institutional Animal Care and Use Committee of the Tongji Medical College, Huazhong University of Science and Technology, and all procedures were in compliance with the relevant guidelines and regulations of the Chinese Council on Animal Care.
2.2. Partial LIRI Model
Male C57BL/6J mice (20–22 g) were randomly assigned to one of four groups: Sham; LIRI; LIRI+PMSC; and LIRI+PMSC-Lin28. The partial LIRI model was developed using methods described in an earlier study . The mice in sham group underwent the same operative procedure, without any blood vessel blocking. The LIRI+PMSC and LIRI+PMSC-Lin28 groups received injections with PMSCs and PMSCs-Lin28 into the portal vein (100 μL, 107 cells/mL) 1 hour prior to hepatic ischemia. After 1 hour of ischemia and 6 hours of reperfusion, mice were sacrificed and serum and liver tissue samples were collected.
2.3. Serum Biochemistry Assay
Blood collected from mice was first chilled on ice (30 min) and then centrifuged at 8000 RPM (15 min). We diluted the supernatant as appropriate and used a standard automatic analyzer (Mindray BS-200) to detect serum ALT and AST levels.
2.4. Cell Culture and Hepatocyte Hypoxia Model
Human PMSCs (Shanghai Tongji University) and murine hepatocyte cells (AML12 cell line; ATCC®CRL-2254TM) were cultured in Dulbecco’s Modified Eagle Medium/F-12 (Hyclone) supplemented with 10% fetal bovine serum (Hyclone), 40 ng/mL dexamethasone (Sigma-Aldrich, D4902), and ITS Liquid Media Supplement (Sigma-Aldrich, I3146). The AML12 cells and PMSCs were placed in normoxic and anoxic environments, respectively, at 37°C. A humidified incubator containing 5% CO2 was used to maintain a normoxic environment, whereas a hypoxia incubator (Whitley H35 Hypoxystation) containing 5% CO2, 1% O2, and 94% N2 was used for maintaining an anoxic environment. To induce the ischemic and hypoxic cellular model, cells were cultured to a density of approximately 70%, Subsequently, old medium was removed and serum-free medium was added; cells were incubated in anoxic conditions for 24 h. Additionally, in the inhibitortreatment group, MK2206 (Sellect, S1078) was added to cells (final concentration, 3 μM). Based on MK2206 treatment, four treatment groups were established: PMSC, PMSC-Lin28, PMSC+MK2206 2Hcl, and PMSC-Lin28+MK2206. We maintained AML12 cells under hypoxic conditions for 24 h to construct a cellular model of ischemia and hypoxia. The cells were divided into three groups: AML12 group (AML12), AML12 co-cultured with PMSCs (AML12+ PMSC group), and AML12 co-cultured with PMSCs-Lin28 (AML12+ PMSC-Lin28 group).
2.5. Overexpression of Lin28 in Cells
When cells reached a density of approximately 70%, they were cultured in OPTI-MEM Reduced Serum Medium (Gibco) with a Lin28 overexpression lentivirus (10μL/mL; with green fluorescence; obtained from Hanbio Biotechnology Co., Ltd.) for 72 hours. Subsequently, fluorescence microscopy was used to detect transfection efficiency.
2.6. Cellular Glucose Metabolism Assay
Glucose (Solarbio,BC2500), lactic acid(LA) (Solarbio,BC2230), ATP (Solarbio,BC0300), and NADPH (Solarbio,BC1100) detection kits were used to detect the intracellular glucose levels, LA levels, ATP levels, and NADPH/NADP+ ratio, respectively. ECAR was detected using the Seahorse XF Glycolysis Stress Test Kit (103020-100).
2.7.1. Hematoxylin and Eosin (H&E) Staining
After 6 hours of ischemia, liver tissue was fixed in 4% paraformaldehyde, dehydrated using an increasing ethanol concentration gradient, and embedded in paraffin. Subsequently, 4 -μm -thick sections were obtained and subjected to H&E staining. The stained samples were examined by two independent pathologists, who assessed hepatic necrosis based on Suzuki’s scores .
2.7.2. TUNEL assay
A TUNEL Kit (Servicebio, G1501) was used based on manufacturer's instructions. First, tissue sections were deparaffinized using protease K. Subsequently, membranes were disrupted broken, the reaction solution was added, and nuclei were stained with DAPI. Finally, the sections were imaged using an upright fluorescence microscope (NIKON ECLIPSE C1, Japan). The proportion of TUNEL-positive cells was calculated using five randomly selected fields.
2.7.3. Myeloperoxidase (MPO) staining
MPO immunohistochemistry was performed using mouse anti-MPO (Servicebio, GB12224) and HRP -labeled goat anti-mpO (Servicebio, G23301) antibodies, as well as the histochemical DAB chromogenic agent kit (Servicebio, GB12224) based on manufacturer's instructions. The fixed tissue was first sealed with serum, followed by the addition of primary antibody, secondary antibody and developer, and then the nucleus was stained with DAP. Finally, the tissue was examined under a microscope; images were then captured and analyzed. The hematoxylin-stained nuclei appeared blue, whereas positive mPO expression was indicated by a brownish yellow color.
2.8. Cell Viability Assay
The CCK-8 kit (Dojindo) was used to detect cell viability. Before hypoxia treatment, 10,000 cells were planted in each well of a 96-well plate, and three holes were made in each well. After 24 hours of culture, the medium was replaced and 10μL of the CCK-8 reagent was added to each well (incubation, 37°C, 2 h). Finally, a microplate reader was used to measure the absorbance at 450 nm.
2.9.Flow Cytometry Analysis of Apoptosis
An Annexin V-FITC and propidium iodide staining kit (MULTISCIENCES, Hangzhou, China) was used to examine cell apoptosis using manufacturer’s instructions. Using a Flow Cytometer (BD FACSCelesta) and FlowJo software for data analysis, we measured the percentages of apoptotic cells.
The Total RNA Rapid Extraction Kit (Fastagen, Shanghai, China) was used to isolate the total mRNA from PMSCs, which was then reverse transcribed using a high-capacity cDNA reverse transcription kit (TAKARA, Shiga, Japan) based on manufacturer’s instructions. Target gene expression was evaluated with qRT-PCR, performed using a SYBR Green kit (Takara). StepOne software (Thermo Fisher Scientific) was used to analyze the data. For examining microRNA levels, the high-capacity microRNA reverse transcription kit (TAKARA, Shiga, Japan) was used; the other steps remained the same as those used for mRNA analysis. GADPH and U6 were chosen as internal controls for mRNA and microRNA, respectively. Each experiment was performed at least in triplicate. Primer sequences are shown in the table below.
2.11. Western Blot
IP lysis buffer (Beyotime, Shanghai, China) with protease inhibitors was used to extract the total protein from cell lysates. A Microplate Reader (BioTek) and BCA Protein Assay Kit (Beyotime, Shanghai, China) were used to measure the protein concentration. We performed 10% SDS-PAGE to separate protein samples, which were then transferred onto polyvinylidene fluoride membranes (Millipore, 0.22μm). The membranes were sealed with 5% skim milk in TBST and then incubated with primary antibodies for 16 h. Antibodies against β-catenin (ab32572), LDHA(ab52488), AKT(ab179463), pAKT(ser473), Lin28(ab191881), Total OXPHOS (ab110431), HEX I (ab150423), HEX II (ab209847), PKM2(ab85555), PDK1(ab202468), PFKFB2(ab234865) purchased from Abcam were used. Subsequently, the membranes were blocked with either the goat anti-rabbit IgG H&L or goat anti-mouse IgG H&L antibody labeled with horseradish peroxidase (Abcam, ab6721 and ab6789) at room temperature (20-25℃) for 1 hour. The ECL reagent (Beyotime, Shanghai, China) was used to quantify the expression of these proteins based on chemiluminescent detection, and the Chemiluminescence Imaging System (GeneGnome, Shanghai, China) was used to detect protein bands. Image-Pro Plus software (Media Cybernetics) was used for analysis. Proteins levels were normalized based on β-catenin concentration, and all experiments were performed at least thrice.
2.12. Statistical Analysis
All statistical analyses were performed using Prism software (GraphPad). All data were expressed as means±standard error of the mean. One-way ANOVA was used when multiple groups were being compared, whereas unpaired t-tests were used when two groups were being compared. P-values <0.05 were statistically significant.