Human liver tissues
Liver specimens were acquired from individuals experiencing orthotopic LT at ShuLan (Hangzhou) Hospital. Moreover, all patients provided their written informed consent. The First Affiliated Hospital Ethics Committee at Zhejiang University School of Medicine in Hangzhou, China, gave its approval for this research, which complied with the 1975 Helsinki Declaration's ethical principles.
Animals
The experiment involved the maintenance of male mice in a controlled environment that was free from pathogens and had a regulated temperature of 23 ± 2°C. Additionally, the mice were subjected to a 12-hour light/dark cycle. The experimental subjects were mice aged between 8 to 10 weeks and their average weight was measured to be 25 ± 2 g. The provision of nutrition in the form of food and water was available. The Zhejiang University animal care committee gave its approval to all animal procedures that were performed in accordance with the national academy of sciences protocol for the care and use of laboratory animals, which was made public by the national institutes of health (publication NO. 85 − 23, revised 1985). Insig2 KO mice were provided by the laboratory of Di Wang, Zhejiang University School of Medicine. Moreover, genomic DNA was obtained from the newborn mice's toe tissue for examination by PCR. Fig. S1 shows the result of genotyping.
Mouse liver IR injury model
The study employed a mouse model that was subjected to partial (70%) warm IR injury. Moreover, the mice were anesthetized using Pentobarbital sodium at a dose of 50 mg/kg prior to performing a midline laparotomy. Consequently, the left lateral/median lobes of the liver were subjected to vascular occlusion by means of microvascular clips applied to the portal vein, the hepatic artery, and the bile duct above the point of bifurcation to the right lateral lobe. Following a period of 90 min of ischemia and 6 h of reperfusion, the subjects were euthanized. The sham control group of mice underwent an identical treatment, with the exception that their vasculature was not clamped. Following the trial, samples of serum and liver tissue were collected for subsequent analysis.
Liver biochemical measurement
Utilizing Chemray 800 (Shenzhen, China) and the manufacturer's instructions, serum concentrations of the enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were detected to evaluate mouse liver function. Employing commercial enzyme-linked immunosorbent assay (ELISA) kits (IL-6 Mouse Uncoated ELISA Kit, 88-7064-88; IL-1 beta Mouse Uncoated ELISA Kit, 88-7013-88; and TNF alpha Mouse Uncoated ELISA Kit, 88-7324-22 from Invitrogen) in accordance with the manufacturer's protocol, the inflammatory state was determined by quantifying serum cytokines.
Histological and immunohistochemical staining
Hepatic tissue specimens were fixed in 10% formalin, dried, immersed in paraffin, and divided (5 µm thick) to determine the degree of liver necrosis. Consequently, hematoxylin and eosin (H&E) were employed to stain the sections. Immunohistochemistry (IHC) was utilized to assess Insig2 (24766-1-AP; Proteintech) and Casepase3 (19677-1-AP; Proteintech) expression. Two expert pathologists assigned IHC staining intensity grades of 0 (no staining), 1 (weakly positive), 2 (weak-moderate positive), 3 (moderately positive), 4 (moderate-strong positive), and 5 (strongly positive). Fluorescence microscopy (OLYMPUS; IX83) was utilized to take pictures.
Immunofluorescence and terminal deoxynucleotidyl transferase-mediated deoxyguanosine triphosphate nick-end labeling (TUNEL) staining
The embedded liver segments in paraffin were also employed for immunofluorescence and TUNEL staining. In this study, primary antibodies targeting mouse MPO (22225-1-AP; Proteintech) and F4/80 (28463-1-AP; Proteintech) were employed. Furthermore, a secondary antibody, specifically goat anti-rabbit/mouse IgG-HRP (HKI0005; Haoke), was utilized. The TUNEL technique (HKI0008; Haoke) was utilized following the manufacturer’s instructions to identify the apoptosis in liver segments immersed in paraffin.
Determination of glucose-6-phosphate dehydrogenase (G6PD) activity and related redox couples
Commercial kits (Beyotime, China) were employed following the manufacturer's directions to measure various parameters in hepatic tissues, including G6PD activity, the reduced nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide phosphate (NADPH/NADP+), total Superoxide Dismutase (SOD) activity, glutathione/glutathione disulfide (GSH/GSSG), and Malondialdehyde (MDA) levels. The manufacturer's instructions were followed for all assays.
Glucose and Lactate Concentration Measurement
The concentrations of glucose, glucose-6-phosphate (G6P), and lactate in hepatic tissues were identified using an assay kit of glucose (Beyotime, China) and a lactate assay kit (Jiancheng Bio, Nanjing, China) following the protocol.
Quantitative Real-time PCR
Total mRNA was extracted from liver tissue and cultured cells using the the total RNA isolation kit (Vazyme) according to the manufacturer’s directions and measured using a Nanodrop (ThermoFisher). Consequently, quantitative real-time PCR was conducted with qPCR SYBR Green Master Mix (Vazyme). The outcomes were standardized against β-actin expression. Table S1 illustrates the primer sequences of the target genes for real-time PCR.
Western Blot analysis
Western Blot analysis
In mouse hepatic tissue specimens and cells, western blot analysis was employed to identify the levels of protein expression. Table S2 illustrates all antibodies used for western blot analysis.
Isolation of primary hepatocytes and hepatocyte hypoxia/reoxygenation (H/R) model
The two-step collagenase perfusion method was utilized to separate the primary hepatocytes of liver (18), and cells with > 80% viability were employed for more trials. Before cells were changed to sugar-free, serum-free DMEM for H/R experiments, they were cultivated in complete Dulbecco’s modified Eagle’s medium (DMEM) overnight. The establishment of cell environments (5% CO2, 1% O2 and 94% N2) was carried out using a modular incubator chamber (Astec APM-30D). To mimic liver IR damage in vivo, the cells underwent 6 h of hypoxia at the indicated time point before being restored to full medium and normal air conditions (95% air, 5% CO2). For further investigations, the cells and related culture media were collected.
Adeno-associated-virus 8 (AAV8) vectors construction and injection
AAV8 vectors for hepatocyte-specific Insig2 overexpression and knockdown were constructed by the Vigene Biosciences Co., Ltd (Shandong, China). In order to allow the selective expression of Insig2 in hepatocytes, the full-length Insig2 gene was cloned into an AAV8 vector with the thyroxine-binding globulin (TBG) promoter. The utilization of AAV8 vectors that express a triple short-hairpin RNA (shRNA) based on miR30 and were regulated by the TBG promoter was implemented for the purpose of Insig2 knockdown. The control group was subjected to the utilization of an AAV8-TBG vector, which contained a null cassette. By means of the tail vein, mice were administered with a virus containing 2 × 1011 AAV8 vector genomes in a volume of 100 µL.
Transcriptome, proteomics and metabolomics analysis
Hepatic biopsy specimens were obtained from the Insig2-overexpression mice and control WT mice liver tissues subjected to IR injury. Consequently, sample preparation and extraction for transcriptome, proteomics, and metabolomics sequencing was performed by Metware Biotechnology Co., Ltd. (Wuhan, China), depending on previously published methods. Moreover, on the Illumina sequencing platform, the cDNA libraries were sequenced for transcriptomics. Furthermore, genes greater than 2.0-fold change and P value < 0.05 between two groups were regarded as differentially expressed genes (DEGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG), GeneOntology (GO), and Metascape online databases were utilized for pathway enrichment analysis of differential genes. For proteomic analysis, the Swissprot.Mouse.20200826.fasta database was used for protein identification. In particular, differentially expressed proteins (DEPs) were those whose expression levels were greater than 1.2-fold change and P value < 0.05. The enrichment study of differential proteins was performed utilizing the internet databases including, KEGG, GO, and Metascape. Additionally, the Metware database was utilized to identify metabolites for metabolomics study. Orthogonal projections to latent structure-discriminant analysis (OPLS-DA) score plots were employed to show the data's intrinsic differences. Differentially abundant metabolites (DEMs) were significantly distinguished using variable importance plots (VIP) > 1 and P values < 0.05. Metabolites with significant differences between the two groups were used for KEGG enrichment analysis using MetaboAnalyst v.5.0.
Nanoparticle delivery system
Herein, we demonstrate a mature nanoparticle for effective delivery of D-glucose 6-phosphate (G6P) disodium salt, which is a polyplex of G6P, esterase-responsive polymer (ERP) and DC-Chol/DOPC lipids (19, 20). In summary, the G6P disodium salt was prepared by dissolving it in HEPES at a concentration of 5 mg/mL to obtain a stock solution. The ERP was dissolved in a DMSO buffer at a concentration of 50 mg/mL, and used as a stock solution. The pH 7.4 HEPES buffer solution (10 mM) was diluted to the necessary concentrations using the stock solutions, based on the mass ratios. At room temperature, equal volumes of ERP and G6P were incubated for 30 min after mixing and vortexed for 10 s. Consequently, at 25°C, Zetasizer Nano-ZS (Malvern Instruments) was employed to detect the sizes and zeta potentials of polyplexes in HEPES buffer. ERP/G6P polyplexes were prepared at an optimized mass ratio of 0.1, as described above (Fig. S2). The fabrication of ERP/G6P polyplexes coated with lipids (DC-Chol / DOPC) conjugated to DSPC-PEG-Gal was synthesized according to the previous report. A thin lipid film was obtained by dissolving neutral-charged helper lipids DOPC and cationic lipids DC-Chol (1:1) in chloroform, then evaporating the chloroform. The HEPES buffer was supplemented and stirred for 8 h at room temperature, followed by 10 min of sonication. Subsequently, the ERP/G6P polyplex solution was then added dropwise to the lipid solution and stirred for a further 2 h at room temperature. Finally, DSPE-PEG-Gal was added to the above mixture and stirred for 15 min to obtain PEG-Lipids/ERP/G6P at 50°C. At 25°C, a Zetasizer Nano-ZS (Malvern Instruments) was employed to detect the sizes and zeta potentials of PEG-Lipids/ERP/G6P (Fig. S3) to optimize the final concentration (DOPC/G6P [µM/µg] = 0.01; 5%PEG). The D-glucose 6-phosphate disodium salt was purchased from Selleck (CAS: 3671-99-6). By means of the tail vein, mice were administered with G6P or G6P nanoparticle in a volume of 200µL at 2 consecutive days.
Statistical analysis
All data are presented as the mean ± SD. The Student t-test or Mann-Whitney U test was employed to analyze distributed data between two groups for non-parametric testing, while one-way analysis of variance (ANOVA) was employed to compare data between several groups. Statistics were deemed significant at P < 0.05. All statistical analyses were performed using the SPSS program (version 21.0). The P values are shown as follows: *P < 0.05; **P < 0.01.