2.1. Chemicals and materials
Antibodies against Plin5 (Proteintech,26951-1-AP), GPX4 (ABclonal, A1933) and GAPDH (Servicebio, GB15004) were purchased commercially. RSL-3 (MedChemExpress, MCE, HY-100218A), Propidium iodide (Sigma, P4170) and 11-Dodecenoic acid (Yuan Ye, B7393) were purchased commercially.
2.2. Absolute quantitative lipidomics and sample preparation
The centrifuge tube was filled with 50 mg of liver tissue, 10 pre-cooled zirconia beads, and 20 µl of deionized water. To extract metabolites, grind for three minutes and then add 120 µl of an internal standard containing methanol. After that, spun at 18000g for 20 minutes. Then, transfer the clear liquid remaining after centrifugation to the 96-well plate. The following operations were carried out on a Biomek 4000 workstation (Biomek 4000, Beckman Coulter, Inc., Brea, California, USA). Each well received 40µL of freshly prepared derivative reagent. The plates were sealed and heated to 30°C for 60 minutes, then samples were evaporated for 2 hours and reconstituted with 330 µl cold 50% methanol solution.
After that, the plates were kept at -20°C for 20 minutes before being centrifuged at 4°C for 30 minutes at 4000g. 135 µl of supernatant was collected in total, and then transferring 10 µl to a new 96-well plate. Internal standards were added to each well. Add the serial diluents of the derived stock standard to the left hole. Finally, the plate seal was utilized for LC-MS analysis.
2.3. Animals and treatments
All animal experiments were carried out in accordance with methods endorsed by East China Normal University's Animal Care and Use Committee. C57BL/6 WT and Plin5 KO mice (8 weeks) were used in the experiment. Plin5 KO mice were bought and generated by Cyagen Biosciences Inc, and created using the CRISPR/ CRISPR-associated 9 (Cas9) system. We used littermate WT mice as control mice. Mice were given a high-fat, high-cholesterol diet (HFHC) (D09100310, Research Diet) diet or normal chow for 24 weeks and were randomly allocated to different experimental groups. The mice were split into two groups: control and AAV-Plin5 group by injection of the adeno-associated virus vector (AAV-vector) and adeno-associated virus Plin5 (AAV-Plin5) respectively. Then mice were fed a Methionine Choline Deficient Diet (MCD) (A02082002BR, Research Diet) or a normal diet for 3 weeks prior to being randomly allocated to different experimental groups.
2.4. Glucose tolerance test (GTT) and Insulin tolerance test (ITT) analysis
GTT and ITT experiments were conducted on mice one week before the end of HFHC feeding. After fasting for 16 hours in advance of the GTT, the mice received an injection of glucose (Sigma 47829) into the intraperitoneal space. Glucose was dissolved in saline at 1.5g/kg body weight. Insulin was administered intraperitoneally to mice in the ITT experiment at a dose of 0.75U/kg. Venous blood was obtained from the tail of each mouse 0,15, 30, 60, 90 and 120 mins after the injection of glucose or insulin and an ACCU-CHEK Active (Roche) kit used for glucose measurement.
2.5. RNA Extraction, quantitative PCR
In accordance with the instructions, RNA was first extracted using Trizol, and then cDNA was generated through reverse transcription using the PrimeScriptTM RT reagent Kit (TaKaRa, RR047A). The RT-PCR experiment was carried out using SYBR green Premix kit (04913914001, Roche) next. Normalize gene expression levels by using β-actin or GAPDH as an internal reference. Table S1 displays the qPCR primer sequence.
2.6. Western Blot analysis
Samples of liver or cells were extracted and quantified. Protein samples were separated on a 10% gel before being transferred to membranes made of polyvinylidene difluoride. Protein expression was observed after incubation overnight at 4°C with the corresponding primary and secondary antibodies. Quantitative analysis was performed using ImageJ software.
2.7. Mouse liver lipid and function measurements
Using commercial enzymatic kits (Nanjing Jiancheng) and an automatic biochemical analyzer (Roche Cobas C702), serum lipids including triglyceride (TG), total cholesterol (TC) as well as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. All were evaluated in accordance with the manufacturer's suggested methodology.
2.8. Histological analysis and staining
Mice were sacrificed under 10% chloral hydrate, and liver samples were collected and weighed for further examination. Prussian Blue Iron Staining (Enhance with DAB), Hematoxylin and eosin (H&E), Masson staining and picrosirius red staining (PSR) were applied on paraffin sections. Oil red O staining was applied on frozen liver sections. Three randomly-selected fields were photographed using a Leica DM2500 Microscope.
2.9. NAS scoring
Two experienced pathologists scored the specimens blindly. Hepatic steatosis, lobular inflammation, and hepatocyte ballooning on HE staining were scored separately and then added together to form the NAS score. By combining the scores for steatosis (0–3), lobular inflammation (0–3), and hepatocyte ballooning (0–2), the NAS score—which ranges from 0 to 8—was determined. An NAS score ≥ 5 was strongly associated with a diagnosis of ‘NASH’.
2.10. Iron measurement
The determination of iron and Fe2 + in serum and liver tissue should use an iron Assay Kit (Abcam, ab83366). During the test, 10 µl of serum or 30 mg of liver tissue should be used, and then the test should be carried out according to the instructions. Finally, the OD value was measured at 593 nm, and the results were compared with the standard curve of known concentration.
2.11. Fluorescence staining
To conduct the following tests, OCT-embedded frozen liver slices were used. First, treat the liver for 10 minutes with 4% paraformaldehyde, and then treat it for 10 minutes with 0.2% Triton X-100 to rupture the liver membrane. Utilize C11-BODIPY (Invitrogen TM D3861,5 Mol/L) for 30 minutes following PBS washing for three times. The nucleus was then stained with DAPI after another PBS wash. At last, use a confocal microscope to observe and photograph the results.
2.12. Cell culture and siRNA transfection
SK-HEP1, a human liver cancer cell line, was obtained from ATCC and cultured in Media consisting of Dulbecco's modified Eagle's (Gibco) with 10% fetal bovine serum (Gibco) and 50 g/ml penicillin/streptomycin. We constructed Plin5-knockdown hepatocytes by transducing the Plin5 gene with small interfering RNA (siRNA). The siRNA target sequences were as follows: non-targeting control siRNA: sense, 5’UUCUCCGAACGUGUCACGUTT-3; antisense, 5’ACGUGACACGUUCGGAGAATT-3’. hPlin5 siRNA: sense, 5’CUGUGGAUGUUGUACUGGATT; antisense, 5’UCCAGUACAACAUCCACAGTT-3’. All siRNAs were validated by Western blotting to demonstrate a target knockdown of > 80%. Cell transfection was carried out using GP-transfect-Mate per the manufacturer’s instructions (GenePharma).
2.13. Propidium iodide (PI) staining and cell viability assay
PI was dissolved in PBS at a concentration of 25 µg/ml, then diluted with medium at a ratio of 1:50 and placed in an incubator at 37°C for 5 min. Use a Leica DM2500 microscope to take photographs.
A Trypan blue staining Cell Viability Assay Kit (Beyotime, C0011) was used to test cell viability. Cells were seeded into 12-well plates and incubated with the indicated treatments. The cells were then collected after trypsin digestion and stained with trypan blue. Cell counting plate was used to determine the number of live cells. Finally, the cell viability was expressed as a percentage and the number of cells was compared to the negative control.
2.14. MDA and SOD content detection
We used an MDA assay kit and a SOD assay kit (A003-1-1, A001-1-1, Nanjing Jiancheng Bioengineering Institute, China) to measure the amount of MDA and SOD in liver tissue. The level of MDA was measured following the guidelines provided by the manufacturer and TBA method. SOD level was measured following the protocols and hydroxylamine method.
2.15. Quantification and statistical analysis
Utilize GraphPad Prism 8.0 to analyze and visualize the experimental data. The figure legend describes the statistical aspects of the experiment. The unpaired Student's t-test was used to make statistical comparisons between two groups. P < 0.05 was considered statistically significant, *p < 0.05, **p < 0.01.