Clinical samples
The liver tissues of ALF patients used in this study came from patients undergoing liver transplantation at the Renmin Hospital of Wuhan University (Hubei, China). Normal tissue biopsies were obtained from the donor liver in liver transplantation. The project was approved by the institutional review board and all participants provided informed consent (Approve number: 2021-K016). The patients were screened and excluded including infectious diarrhea, primary sclerosing cholangitis, recent malignant tumors, history of hormone or immunosuppressive therapy and flora transplantation in the past two years, and those who have used antibiotics in the past six months. Formalin-fixed and paraffin-embedded liver tissues were used for fluorescence in situ hybridization (FISH) analysis and immunohistochemical staining.
Liquid Chromatography–Mass Spectrometry (LC–MS) Metabolomics:
For liver tissue samples, mixed 20 mg of tissue with 200 μL of pre-chilled water and 800 μL of pre-chilled methanol/acetonitrile (1:1, v/v). Centrifuged at 16000g for 20 min at 4 °C. Added the same amount of internal standard L-Glutamate-d5 to each sample and dried it in vacuum. And then added 100 μL of acetonitrile-water solution (1:1, v/v) for reconstitution, centrifuged at 16000g at 4 °C for 15 min, and taked the supernatant for analysis. Shimadzu Nexera X2 LC-30AD high performance liquid chromatography was used for separation. The relevant liquid phase gradient were as follows: 0-2 min, B liquid was maintained at 95%, 2-9 min, B liquid changed linearly from 95% to 70%, 9-10 min, B liquid changed linearly from 70% to 30%, 10-11 minutes, B solution maintained at 30%, 11-11.5 minutes, B solution changed linearly from 30% to 95%, 11.5-15 minutes, B solution maintained at 95%. All materials were purchased from Sigma-Aldrich or Shanghai Yuanye Biology, Acetonitrile (Millipore, 1.00030.4008), methanol (Millipore, 1.06007.4008), formic acid (Fluka, 06450), ammonium acetate (Sigma, 70221).
Bacterial strains and Cell culture
The human liver cell line L02 obtained from China Center for Type Culture Collection (CCTCC) were cultured in DMEM medium (HyClone, USA) containing 10 % heat-inactivated FBS (GIBCO, USA) and 100 U penicillin/100 g streptomycin (Sigma, USA) at CO2 atmosphere with a temperature of 37 °C and a humidity of 5 %. TNF-α (100 ng/mL, Sigma, USA) combined with D-Galactosamine (D-Gal, 44 μg/mL, Sigma, USA) were used to establish the ALF model in vitro. Constructed NAMPT overexpression plasmid and enveloped it with lentiviral vectors (LV, GeneCreate, China). Besides, the cells were treated with the fresh media containing 1 mM AMPK activator 5-aminoimidazole-4-carboxamide-1-b-D-riboside (AICAR). After 24 h treatment, the cells were harvested for further experiments. F. nucleatum (ATCC10953, Beijing, China) were cultured on tryptic soy under anaerobic conditions. Escherichia coli (E.coli) strain (Beijing, China) were cultured in Luria-Bertani (LB) medium.
Mice
Male C57BL/6J wild-type mice aged 5-6 weeks purchased from Wuhan Biomedical Research Institute were raised in the specific pathogen free (SPF) animal facility of Renmin Hospital of Wuhan University under conditions of light-controlled, room temperature 25 °C, humidity 55 ± 5 % and they were free to eat and drink. The laboratory animal facility use license number was No. SYXK (Hubei) 2015–0027. The operations were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University, China.
Animal models
All mice were randomly divided into nine treatment groups: saline control group, ALF group, E.coli group, F. nucleatum group, E.coli + ALF group, F. nucleatum + ALF group, F. nucleatum + ALF +Ad-lacZ group, F. nucleatum + ALF + Ad-SIRT1 group and F. nucleatum + ALF + Nicotinamide Riboside (NR) group. All mice were given 2 mg/ml streptomycin in drinking water for 3 days. After that, PBS-resuspended F. nucleatum (109 CFU/ml) or PBS alone was administered to mice by gavage every day for 4 weeks [20]. In NR supplement group, NR was administered to mice by gavage every day for 4 weeks [21]. The adenovirus overexpressing mouse SIRT1 was prepared with RAPAd® adenovirus expression system (Cell Biolabs, Inc) and purified to 1011 PFU. Ad-LacZ (control, 50 μl) and Ad-SIRT1 (50 μl) were injected to mice once a week. Intraperitoneal injection of LPS (100 μg/kg, L2880, Sigma) and D-gal (400 mg/kg, G0050, Sigma) [22] was used to establish the ALF model after NR and F. nucleatum pretreatment for 4 weeks. Animals were quickly euthanized at 24 h time point after D-gal and LPS administration and blood samples and liver tissues were harvested. Blood samples were collected for biochemical analyses. A small part of the liver tissue was fixed with paraformaldehyde and the other part was immediately frozen in liquid nitrogen.
Fluorescence in situ hybridization (FISH)
Performed microbial FISH as described [23]. Prepared 5-µm-thick sections and hybridized by using commercial kits (FOCOFISH, Guangzhou, China). The sequence of the probe were as follows: 5’-GCT GCC TCC CGT AGG AGT-3’ for “universal bacterial” probe (EUB338, Cy3 labeled), 5’-CTT GTA GTT CCG C(C/T) TAC CTC-3’ for F. nucleatum-targeted probe (FUS664, FITC labeled). Slides were examined using a microscope (BX53F, Olympus, Tokyo, Japan). Five random 200× magnification fields per sample were evaluated by an observer who was blinded to the experimental protocol, and the average number of bacteria per field was calculated. We defined a negative or positive of F. nucleatum as an average of < 10 or > 10 visualized FUS664 probes per field, respectively. Other bacteria were noted as positive with >5 bacteria per field with EUB 338 probe.
Histopathological examination
Fresh liver tissue was fixed with paraformaldehyde for 24 h. The liver tissues were sliced completely and the thickness is 4-6 μm and uniform, no wrinkles and no knife marks and then stained with haematoxylin–eosin (HE). Hematoxylin stained the nucleus into bright blue, eosin stained the cytoplasm into different shades of pink, and the eosinophilic particles in the cytoplasm were bright red under strong light. Light microscope (Olympus, Japan) was used to observe and evaluate the pathological changes of liver tissues. The liver histology score was used to judge the degree of liver damage in the ALF model, including inflammation and necrosis scores [24].
Immunohistochemical staining and immunofluorescent staining
The liver tissues were sliced completely and the thickness was 4-6 μm and uniform. Drop the cell suspension of L02 cells onto the cover glass and place it in an incubator with a CO2 concentration of 5 % at 37 °C until the cells are fixed (about 2h). The infiltration of macrophages and inflammatory factors were assessed using immunohistochemical assays and immunofluorescent assays. Sections were dewaxed, incubated with 3 % H2O2, blocking serum and thereafter incubated with a 1:100 dilution of polyclonal antibodies against TNF-α, IL-1β, F4/80 or CD68 (Santa Cruz Biotechnologies, CA, USA) according to the instructions of manufacturer (Beyotime, Shanghai, China). Slides were imaged using light microscope (Nikon) and fluorescent microscope (Olympus, Japan).
Biochemical analyses and Detection of NAD+
Hitachi Automatic Analyzer (Japan) was used to detect the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were used to detect the levels of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px). The levels of NAD+ in liver tissues and L02 cells were detected by commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Mixed RAPI lysis buffer and protease inhibitor to obtain the extracts. Incubated the sample at 30 °C for 10 minutes to allow NAD+ to degrade, and incubated with dithiothreitol 2 mM for 10 minutes
Detection of reactive oxygen species (ROS) production
Cell suspension of L02 cells was put onto the cover glass and place in an incubator with a CO2 concentration of 5 % at 37 °C until the cells are fixed about 2 h. Add 2 ml cell culture fluid and continue to culture for about 6 h. Pour out the culture medium and wash with PBS 3 times for 5 minutes each time. Dissolve a 5 ml solution of dihydroethidium (DHE, 2 mmol · L-1, Molecular Probes, D-1168) in DMSO, dilute the solution at a ratio of 1:1000, and store in the dark for use. Add 1 ml of diluted solution to each well and incubate at 37 °C for 30 minutes in the dark. Aspirate the solution and wash with PBS 3 times for 5 minutes each time. Aspirate the PBS and add an appropriate amount of DAPI solution to the wells, and stain for 10 minutes in the dark. Aspirate PBS, add a drop of anti-fluorescence quenching mounting plate into the hole, mount the cover glass, observe and take pictures under a fluorescent microscope (Olympus, Japan).
Western blotting
Protein extraction from cells and tissues was performed using a radioimmunoprecipitation assay (RIPA) kit following as instructed by the manufacturer (Sigma-Aldrich, USA). Added 5-10 μL of the collected protein samples to the SDS-PAGE gel sample holes. The proteins were transferred to polyvinylidene fluoride membranes after electrophoresis was completed, and incubated with the primary antibodies at 4 °C overnight, then incubated the membranes with the secondary antibodies at 37 °C for 1 h the next day. Primary antibodies against the following targets were used: SIRT1 (Cat.No. 9475, CST), NAMPT (Cat.No. 236874, Abcam), IDO (Cat.No. 13268-1-AP, Sanying), AMPK (Cat.No. 32047, Abcam), p-AMPK (Cat.No. 131357, Abcam) and GAPDH (Cat.No. 8245, Abcam). The blots where developed with ECL (GE Healthcare, Pittsburgh, PA, USA) according to the instruction of manufacturer. The intensity of the bands on the western blots was evaluated by Image Lab statistical software (Bio-Rad, USA).
Statistical analyses
Each experiment was repeated at least three times and the data are expressed as the means ± SDs. Moreover, for the data sets of Western blotting and the expression of mRNA, all the data were adjusted by the values of internal standard (such as GAPDH). Firstly, we calculated the control mean and then expressed all the individual control values and all the individual test values as fold of control mean and conducted appropriate statistical analyses on these normalized values. The Y axis was labelled as fold of control mean. Statistical analysis was performed using GraphPad Prism software version 8.0. Differences among multiple groups were evaluated using conventional Student’s t test or ANOVA followed by Tukey’s multiple comparison post hoc test (normally distributed data). Statistical significance was considered at P < 0.05.