- 1 Animals
- 2 Human liver samples
- 3 Liver IRI mannequin
- 4 Histology and immunohistochemical (IHC) staining
- 5 Fecal pellet assortment and 16S rDNA Sequencing
- 6 Focused BA evaluation utilizing UHPLC–MS/MS
- 7 Isolation of hepatic nonparenchymal cells (NPCs)
- 8 Move cytometry
- 9 Cell tradition and H/R mannequin
- 10 Enzyme-linked immunosorbent assay (ELISA)
- 11 siRNA transfection
- 12 Western blotting evaluation
- 13 Quantitative real-time polymerase chain response (qRT-PCR)
- 14 TUNEL apoptosis assays
- 15 Luciferase reporter assay
- 16 Chromatin immunoprecipitation assay
- 17 Statistical evaluation
Briefly, 6–8-week-old male C57BL/6J mice had been supplied by the Animal Heart of the Affiliated Drum Tower Hospital of Nanjing College Medical Faculty. All animals had been bred in a particular pathogen-free facility. All animal-related protocols had been licensed by the Institutional Animal Care and Use Committee of Nanjing College, China, which complied with the NIH Information for the Care and Use of Laboratory Animals.
4-week-old male C57BL/6 mice had been orally gavaged with antibiotics (1 g/L ampicillin; 0.5 g/L vancomycin; 1 g/L metronidazole; and 1 g/L neomycin) at a complete dose of 20 mL/kg/day for 4 weeks as beforehand described  with some modifications. Management mice had been gavaged with the identical dose of saline for 4 weeks.
To attain liver-specific FXR knockdown within the mouse mannequin, 4-week-old male C57BL/6 mice had been transfected with 1011 pfu AAV8-delivered quick hairpin RNA (shRNA) (GeneChem), which consists of a liver-specific thyroxine-binding globulin (TBG) promoter and a GFP tag. shFXR had the next goal sequence: GGAGAGTGAATGATCACAA. On the second day after tail vein injection, all mice had been orally gavaged with antibiotics for 4 weeks.
All mice had been randomly assigned to the experiments.
Human liver samples
Liver specimens had been harvested from topics with benign focal hepatic lesions who underwent liver surgical procedure between April 2020 and April 2021 at Affiliated Drum Tower Hospital, Medical Faculty of Nanjing College. The principal prognosis of those sufferers included focal nodular hyperplasia (FNH), hemangioma, hepatolithiasis, and angiomyolipoma, with out a historical past of autoimmune problems, hepatitis, malignancy, immune deficiencies, or HIV an infection. Liver samples had been collected and preserved instantly. All sufferers concerned had been knowledgeable and signed written consent varieties. All human experiments had been licensed by the human ethics committees of the Affiliated Drum Tower Hospital, Medical Faculty of Nanjing College, following the Declaration of Helsinki.
Liver IRI mannequin
The hepatic heat IRI mannequin was described beforehand with slight modifications . Briefly, isoflurane together with chloral hydrate was used for anesthesia, and the portal vein and arterial blood provide to the left and median liver lobes had been blocked utilizing an atraumatic clip. The clip was eliminated after 90 min of interruption. Mice had been euthanized after 6 h of reperfusion with high-dose isoflurane. Serum samples had been collected and instantly analyzed by an computerized analyzer (Fuji, Tokyo, Japan) for serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH). As well as, liver samples had been sectioned and subjected to fixation or snap-frozen.
Histology and immunohistochemical (IHC) staining
Paraffin-embedded liver tissue was subjected to hematoxylin and eosin (H&E) staining. Liver harm was assessed by Suzuki’s et al. standards  by three totally different pathologists who had been blinded to teams within the Division of Pathology, Affiliated Drum Tower Hospital, Medical Faculty of Nanjing College. For IHC assays, the sections had been subjected to an antigen retrieval course of after which incubated with antibodies towards CD68 (Abcam Cambridge, MA, USA), Ly6G (Abcam), CCR2 (Abcam), CCL2 (Proteintech, Wuhan, China), and FXR (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in a single day at 4 °C. This was adopted by incubation with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive cells had been incubated with DAB (diaminobenzidine) substrate. A minimum of three photos had been taken from random fields for every stained part, and every experimental group contained at the least three mice.
Fecal pellet assortment and 16S rDNA Sequencing
24 h after the final gavage, the mouse was positioned in a clear cage lined with sterile filter paper, and the fecal pattern had been collected instantly into sterile centrifuge tube after excretion. Every pattern weighed greater than 200 mg. After the gathering of every pattern, the filter paper ought to be changed and the collected sterile centrifuge ought to be positioned on liquid nitrogen instantly. In any case samples had been collected, the samples had been shipped on dry ice to the corporate for additional testing.
Fecal 16S rDNA assays had been carried out by Shanghai Biotree Biotech Co., Ltd. (Shanghai, China). Briefly, microbial DNA was extracted utilizing a QIAamp Quick DNA Stool Mini Package (Qiagen, CA, United States) primarily based on the really helpful protocol. PCR amplification focused the hypervariable V3–V4 area of the 16S rDNA, adopted by purification and quantification. Purified amplicons had been sequenced on the Illumina NovaSeq platform, and the info had been additional analyzed.
Focused BA evaluation utilizing UHPLC–MS/MS
The content material of endogenous BAs within the liver was decided by Shanghai Biotree Biotech Co., Ltd. (Shanghai, China). Briefly, particular person liver specimens had been exactly weighed and extracted with extraction answer (acetonitrile-methanol-water, 2:2:1, 0.1% formic acid contained and combined with isotopically labeled normal), and the ensuing supernatants had been subjected to UHPLC–MS/MS evaluation after centrifugation. UHPLC separation was performed on an Agilent 1290 Infinity collection UHPLC System (Agilent Applied sciences, USA). A collection of calibration normal options had been beforehand detected.
Isolation of hepatic nonparenchymal cells (NPCs)
Hepatic NPCs had been remoted as beforehand described . Briefly, mice had been anesthetized, a 20-G catheter was inserted into the superior vena cava, the inferior vena cava was blocked, and the portal vein was reduce to perfuse liver tissues. Prewarmed Hank’s balanced salt answer (HBSS) was used to perfuse liver tissues, adopted by incubation with digestion buffer [1 X HBSS containing 0.4% collagenase (Sigma-Aldrich, St. Louis, MO, USA), 1.25 mM CaCl2, 4 mM MgSO4, and 10 mM HEPES]. The left and median liver lobes had been used to make single-cell suspensions utilizing a 70-μm cell strainer (BD Falcon, Bedford, MA, USA), and the cells had been subsequently centrifuged at 700 × g for five min at 4 °C. The cell pellet was redistributed in 15 mL of 35% Percoll (Sigma-Aldrich, St. Louis, MO, USA) containing 50 U/mL heparin, adopted by centrifugation at 500 × g for 15 min. The ensuing cell pellet was harvested and redistributed in 1.5 mL of pink blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) for five min. HBSS containing 0.5% fetal bovine serum (FBS) was used to clean the cells. Lastly, the cell pellet was redistributed in 100 µL of HBSS containing 2% FBS for cell counting and antibody staining. The variety of whole NPCs was normalized based on the liver weight for statistical evaluation.
Briefly, 106 hepatic NPCs had been incubated with anti-mouse CD16/32 antibody (clone 93, Biolegend, San Diego, CA, USA) for 10 min to attenuate nonspecific binding. Cells had been incubated with a PerCP-conjugated anti-CD45 antibody (clone 30-F11, Biolegend), an APC-labeled anti-F4/80 antibody (clone BM8, Biolegend), a FITC-conjugated anti-CD11b antibody (clone M1/70, Biolegend), a PE-Cyanine7-conjugated anti-Ly6C antibody (clone HK1.4, Biolegend), and a PE-conjugated anti-CCR2 antibody (clone SA203G11, Biolegend) or incubated with a PerCP-conjugated anti-CD45 antibody (clone 30-F11, Biolegend), an APC-conjugated anti-CD3 antibody (clone 17A2, Biolegend), a PE-Cyanine7-conjugated anti-NK1.1 (clone PK136, Biolegend), a PE-conjugated anti-CD4 (clone GK1.5, Biolegend), and a FITC-conjugated anti-CD8a (clone 53-6.7, Biolegend). The outcomes had been collected by a BD FACSAria III Move Cytometer (BD Bioscience, San Jose, CA, USA) and analyzed utilizing FlowJo 10.4 software program (BD Bioscience).
After the cells had been handled with hypoxia-reoxygenation (H/R), apoptosis was assessed by an Annexin V-FITC/PI Apoptosis Detection Package (Vazyme Biotech, Nanjing, China) primarily based on the producer’s protocols.
Cell tradition and H/R mannequin
AML12 (mouse liver cell line) cell traces had been bought from ATCC (Manassas, VA, USA). AML12 cells had been cultured in DMEM/F12 medium combined with 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, 1% insulin-transferrin-sodium selenite (ITS, Sigma-Aldrich, St. Louis, MO, USA), and dexamethasone (40 ng/mL, Sigma-Aldrich). Mycoplasma contamination was performed each 3 months for all cells and constantly examined detrimental.
Mouse major hepatocytes had been remoted primarily based on a earlier protocol . Briefly, prewarmed answer 1 (Krebs Ringer with glucose and EGTA) and answer 2 (Krebs Ringer with glucose, CaCl2 and sort IV collagenase) had been slowly perfused the liver sequentially from the portal vein. After cell counting, cells had been seeded at a density of 5 × 105 cells per effectively in a 6-well plate, which had been precoated with rat-tail collagen I (Sigma-Aldrich). Mouse major hepatocytes had been cultured in DMEM supplemented with 4.5 g/L glucose, 10% fetal bovine serum, 1 mM dexamethasone and 0.1 mM insulin.
To evaluate H/R harm in vitro, cells had been maintained in a hypoxic gasoline combination (5% CO2, 94% N2, and 1% O2) with out FBS for 12 h at 37 °C. For reoxygenation, the tradition medium was modified to medium supplemented with 10% FBS in a 5% CO2 incubator, and the cells had been incubated for 4 h as beforehand described .
The degrees of IL-1β (MultiSciences, Hangzhou, China) in mouse serum had been decided utilizing a industrial ELISA package primarily based on the producer’s protocols.
The siRNA sequence of mouse FXR was GGAGAGTGAATGATCACAA. The siRNA sequence of mouse TLR4 was as follows: CAATCTGACGAACCTAGTA. Cells had been transiently transfected with siRNA utilizing Polyplus reagent (Polyplus, Illkirch, France) primarily based on the producer’s protocols.
Western blotting evaluation
Western blotting evaluation was carried out as beforehand described . The next antibodies had been used: anti-Bcl-2 (#ab182858), anti-Bax (#ab182733), anti-Bcl-xl (#ab32370), anti-p-P65 (#ab76302), anti-P65 (#ab32536), anti-p-IκBα (#ab133462), anti-IκBα (#ab32518) (Abcam Cambridge, MA, USA), anti-FXR (#sc-25309), anti-TLR4 (#sc-293072) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p-ERK1/2 (#4370), anti-ERK1/2 (#4695), anti-p-P38 (#4511), anti-P38 (#8690), anti-p-JNK1/2 (#4668), anti-JNK1/2 (#9252), anti-CCL2 (#66272-1-Ig), and anti-β-actin (#66009-1-Ig) (Proteintech, Wuhan, China).
Quantitative real-time polymerase chain response (qRT-PCR)
Complete RNA from liver tissues, ileum tissues, and hepatocytes was extracted utilizing TRIzolTM (Life Applied sciences, Carlsbad, CA, USA) reagent primarily based on the producer’s protocols. Complementary DNA (cDNA) was synthesized with HiScript II RT SuperMix for qPCR + gDNA wiper (Vazyme Biotech, Nanjing, China). The mRNA degree of goal genes was quantified by qRT-PCR utilizing ChamQ SYBR qPCR Grasp Combine (Vazyme Biotech, Nanjing, China) on an ABI PRISM 7500 Actual-Time PCR System (Utilized Biosystems, Foster Metropolis, CA, USA). The primers used for qRT-PCR are introduced in Supplementary Desk S1.
TUNEL apoptosis assays
A TUNEL BrightGreen Apoptosis Detection Package (Vazyme Biotech, Nanjing, China) was used primarily based on the producer’s protocols. Briefly, frozen liver sections had been dried at room temperature for 15 min after which mounted in 4% paraformaldehyde (diluted in PBS) for 30 min. Sections had been incubated with 0.2% Triton X-100 (diluted in PBS) for 15 min and equilibrated with equilibration buffer (diluted in ddH2O) for 20 min, adopted by incubation with 50 μL TdT response combination at 37 °C for 60 min and shielded from mild. Subsequently, the sections had been counterstained with DAPI and instantly examined below an immunofluorescence microscope.
Luciferase reporter assay
The expected mouse TLR4 promoter sequence was cloned into the luciferase reporter vector pGL3-basic. Mouse FXR (mFXR) overexpression plasmids had been constructed primarily based on the pcDNA3.1 vector. A dual-luciferase assay was carried out by cotransfecting pcDNA3.1-mFXR, pGL3-mTLR4-prom, and pGL4.75 plasmids into AML12 cells and mouse major hepatocytes. The management vector pGL4.75 was employed to normalize firefly luciferase exercise. Luciferase assays had been carried out with a Twin-Luciferase Reporter Assay Package (Vazyme Biotech, Nanjing, China) primarily based on the producer’s protocols. Transient transfections had been carried out with Polyplus reagent (Polyplus, Illkirch, France) primarily based on the producer’s protocols.
Chromatin immunoprecipitation assay
The ChIP assay was constructed as described beforehand . Briefly, management or FXR overexpression plasmids had been transfected into AML12 cells and mouse major hepatocytes for 48 h and crosslinked with 1% paraformaldehyde. The crosslinking was stopped after 10 min with glycine. Cells had been then washed and lysed with chilly PBS supplemented with protease inhibitor. The lysates had been sonicated for 15 s and interposed with 30 s pauses, which was repeated 5 instances. The DNA fragments had been immunoprecipitated with 5 μg of anti-FXR antibodies or IgG (Santa Cruz Biotechnology). The outcome was confirmed by qRT-PCR. The next primers had been used: 5′-TTTGGACAAGGTGATCTGGTGTT-3′ (ahead) and 5′-AAATTCAAGTCATAGGACATGGCAC-3′ (reverse).
Statistical evaluation was performed utilizing GraphPad Prism 6.01 software program (San Diego, CA, USA). All knowledge are expressed because the means ± SEM. All knowledge had been first examined for normality by way of the Kolmogorov–Smirnov take a look at. Usually distributed knowledge had been in contrast by Scholar’s t take a look at. In any other case, the Wilcoxon rank-sum take a look at was utilized. One-way evaluation of variance was adopted to check variations amongst a number of teams. P values <0.05 had been thought of statistically important.