Scientific Papers

Engineered FGF19ΔKLB protects against intrahepatic cholestatic liver injury in ANIT-induced and Mdr2-/- mice model | BMC Biotechnology

Cell based experiments

HepG2 cells (5.5 × 105 cells/cm2) from the Shanghai Cell Bank were identified by short tandem repeat (STR) (Supplementary Fig. 3) and planted on 6-well plates for a period of 24 h, where they were then allowed to adhere and grow for a further 24 h in an incubator (37 °C, 5% CO2). Williams’ E medium with fetal bovine serum (2%), L-glutamine, 1% penicillin/streptomycin, and 10 nM dexamethasone made up the culture media. Cells were exposed to FGF19 and FGF19ΔKLB (1.0 or 10 nM) for 6 h after being isolated for 12 h. Cyp7a1, Cyp8b1, Cyp27a1 and Cyp7b1 mRNA levels were extracted using Trizol reagent (Invitrogen) and quantified by real-time polymerase chain reaction (RT-PCR).

Expression and purification of recombinant FGF19WT and FGF19ΔKLB

Briefly, a DNA fragment encoding FGF19WT or FGF19ΔKLB was subcloned into the bacterial expression vector pET28a. Constructs were transformed into Escherichia coli BL21 (DE3). Protein expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C for 4 h and the cells were collected by centrifugation. FGF19WT or FGF19ΔKLB was refolded in vitro from isolated bacterial inclusion bodies using published protocols [46, 47]. Then, refolded FGF19WT or FGF19ΔKLB containing an N-terminal histidine tag was purified by nickel affinity column (HisTrap HP) and size exclusion chromatography (HiLoad 16/600 Superdex 75 column) with an AKTA purifier (GE Healthcare). The purity of FGF19WT or FGF19ΔKLB was estimated to be greater than 95% based on SDS-PAGE analysis. Protein concentrations were determined by Nanodrop.

Animals and animal welfare

Zhejiang Vital River Experimental Animal Technology Co. LTD. sold us male C57BL/6J mice weighing 20–25 g. Jackson Laboratory offered Mdr2−/− mice on an FVB/N background (stock number 002539) in addition to sex- and age-matched wild-type FVB/N mice. These Mice were raised at Wenzhou Medical University, China, and housed in a specific pathogen-free animal facility in a controlled environment and given free access to food and water. All animal care practices and research were approved by the Wenzhou Medical University in China’s Animals Care and Use Committee. At the end of the experiments, the mice were anesthetized with intraperitoneal injection of 150–200 mg/kg amobarbital sodium and sacrificed by cervical dislocation. Tissues samples were harvested for subsequent analyses.

Acute effects of FGF19 on the enzymes essential for BA biosynthesis

To assess the acute effects of FGF19WT and FGF19ΔKLB on the major enzymes in BA biosynthesis, C57BL/6J mice were given an intraperitoneal injection of PBS, FGF19WT and FGF19ΔKLB (1.0 mg/kg, n = 10 animals for each treatment). After four hours of dosing, liver tissue was obtained. Using RT-PCR, hepatic mRNA levels were determined and adjusted to β-actin mRNA levels.

Blood parameters measurement

Blood was collected after mouse death using EP tubes. Alkaline phosphatase (ALP) (A059-2), aspartate transaminase (AST) (C010-2-1), and alanine transaminase (ALT) (C009-2-1) were obtained from Nanjing Jiancheng Bioengineering Institute and measured on a microplate reader (MX190). According to the instructions provided by the manufacturer, all tests were completed.

FGF19’s therapeutic impact on intrahepatic cholestasis brought on by ANIT

Based on body weight (n = 6), three groups of mice were created and intraperitoneally injected once daily for six days with PBS, FGF19WT (1.0 mg/kg), and FGF19ΔKLB (1.0 mg/kg). A single oral dose of ANIT (75 mg/kg body weight, dissolved in olive oil) was given to mice on the fourth day. On the sixth day, 4 h after the last dose, the mice were put to death. Blood samples were collected via the retro-orbital route for subsequent liver function test. Liver tissues were collected for subsequent experiments.

Therapeutic effects of FGF19 in the Mdr2
−/− mice

Mdr2−/− mice that were four weeks old were given daily intraperitoneal injections of PBS, FGF19WT or FGF19 ΔKLB (1.0 mg/kg, n = 5 per group) for eight weeks. Blood samples were collected via the retro-orbital route for subsequent liver function test. Liver tissues were collected for subsequent experiments.

Hepatic bile acid pool size and serum BA concentration measurements

Total BAs in the liver were quantified using the Mouse Total BA Assay Kit (Crystal Chem INC). Total BAs were extracted from the homogenate by agitating the tissue for two hours at 50 °C after it had been homogenized with 75% ethanol from individual livers. After being centrifuged, the extraction’s supernatants were collected and diluted in PBS for examination. The dilution parameters for each tissue extract were determined to ensure that the BA readings obtained using a mouse BA kit were within the linear range of the standard curve. The liver’s total number of BAs was added to estimate the size of the BA pool.

An AAV-mediated delivery system to evaluate the tumorigenesis of FGF19

AAV-FGF19WT and AAV-FGF19ΔKLB vectors purchased from GeneChem Co., Ltd (Shanghai, China) were injected into eight-week-old db/db mice (2*1011 vector genomes per mice) via tail vein for 24 weeks. At the end of the experiment, liver tissue was collected, and analyzed for macroscopic morphology and liver index (the ratio of liver weight to body weight). The number of tumors per liver from db/db mice was counted. H&E staining and immunohistochemical staining using glutamine synthetase (GS), Ki67 and PCNA of liver tumors in db/db mice were performed.

Histological analysis

Formalin-fixed and implanted mouse liver was used. Sections of paraffin (5 μm) were produced for Masson’s Trichrome (G1346, Solarbio), hematoxylin and eosin (HE) staining (G1120, Solarbio), and Picro-Sirius Red (DC0041, Leagene Biotechnology) staining were used to evaluate liver fibrosis. Using a light microscope, histological pictures of tissue slices were taken (Nikon eclipse Ni, Tokyo, Japan).

Immunofluorescent staining

Liver slices were deparaffinized with xylene after being roasted for 5 h at 65 °C, and then rehydrated in a series of ethanol solutions of gradually decreasing concentrations. Livers were sliced, washed in PBS, and then boiled in a 10 mM sodium citrate buffer for 2 min at 100 °C (pH 6.0). Liver slices were blocked for 1 h with 5% BSA after being treated with 3% H2O2 for 30 min and then washed twice in PBS. After that, liver slices were incubated with primary antibodies for F4/80 (ab60343, Abcam) and Ki67 (ab16667, Abcam) for an entire night at 4 °C. After a PBS wash, slices were given a second, three-time PBS wash before being incubated with the Alexa Fluor 488-conjugated secondary antibody (ab15007, Abcam) for one hour. Nuclei were stained in contrast using DAPI. Using a Nikon C2si Confocal Microscope, fluorescent pictures were acquired. Using ImageJ, data were quantified.

Real-time polymerase chain reaction (RT-PCR)

From frozen liver tissues, total RNA was extracted with a TransZol Up Kit (ET111-01, TransGen Biotech) and was quantified using a NanoDrop One spectro- photometer (Thermo Fisher). Then, a One-Step gDNA Removal Kit was chosen to reverse-transcribe 2 µg of RNA into cDNA (AT341, TransGen Biotech). While performing at a Level One Plus The specific primers listed in Supplementary Table 1 were used for quantitative RT-PCR utilizing Real-Time PCR equipment (Applied Biosystems® Quant Studio® 3) and the ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme).

Western blot analysis

After centrifugation, the homogenate containing the liver tissues was washed with phosphatase and protease inhibitors in protein extraction buffer (Sigma-Aldrich, St. Louis, MO). To determine total protein concentration, a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Waltham, MA) was used. Electrotransfer of proteins from SDS-polyacrylamide gels to PVDF membranes (Millipore-Billerica, MA, USA). Then, the PVDF membranes were cut based on the molecular weight of the target proteins, prior to hybridisation with antibodies. Tris-buffered saline (pH 7.6) containing 0.1% Tween-20 (TBST) was used to wash the PVDF membranes three times after they were blocked in 10% nonfat milk for an hour at room temperature and then blots were incubated with primary antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (CST2118, Cell Signaling Technology); Cyp7a1 (ab65596, Abcam); Cyp8b1 (ab191910, Abcam), Cyp27a1 (ab126785, Abcam), Cyp7b1 (ab138491, Abcam), TGFβ1 (ab824, Abcam); Ki67 (ab16667, Abcam); PCNA (ab29, Abcam); phosphorylated EGFR (CST2231, Cell Signaling Technology); EGFR (CST4267, Cell Signaling Technology); phosphorylated STAT3 (CST9145, Cell Signaling Technology); STAT3 (CST8768, Cell Signaling Technology) at 4 °C overnight. After being washed, membranes were incubated with a secondary antibody labeled with horseradish peroxidase (HRP) for 1 h at room temperature. An enzyme called glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was employed to make sure everything was running well. The ChemiDocTM XRS coupled with Imaging LabTM Software was used for the visualization of immunoreactive protein bands (Bio-Rad, Hercules, CA). The protein concentrations were normalized with respect to GAPDH using Image J (National Institutes of Health, Bethesda, MD, USA).

Analytical statistics

The statistical analysis was performed using GraphPad Prism 8. In studies with just two groups, the means were compared using either the Mann-Whitney U test or a two-tailed Student’s t-test. When comparing data from different groups, we utilized one- or two-way ANOVA (ordinary or repeated measure) with post-hoc test (Tukey or Sidak) as appropriate. A p-value of less than 0.05 was considered significant.

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