(+)-Clausenamide protects against drug-induced liver injury by inhibiting hepatocyte ferroptosis

Chemicals and reagents

Erastin (S7242), fer-1 (S7243), necrostatin-1(nec-1, S8037), and ZVAD-fmk (S7023) were purchased from Selleck Ltd. (Shanghai, China). APAP (1003031), GSH (PHR1359), cycloheximide (CHX, 239763), dimethyl sulfoxide (DMSO, D2650), deferoxamine (DFO, D9533) and MG-132 (474790) were purchased from Sigma-Aldrich (Shanghai, China). (+)-CLA was supplied by the China Academy of Chinese Medical Science. Aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), and lactate dehydrogenase (LDH) detection kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Nicotinamide adenine dinucleotide phosphoric acid (NADPH) assay kit was purchased from Comin Biotechnology (Suzhou, China). Hematoxylin and eosin (H&E) staining kit, 4′,6′ diamidino-2-phenylindole (DAPI), Annexin V-FITC/PI apoptosis kit, and lysis buffer were purchased from Beyotime Technology (Shanghai, China). In situ cell death detection peroxidase dismutase (POD) kit was purchased from Roche (Germany). Pierce BCA protein assay kit and NE-PER™ nuclear and cytoplasmic extraction kit were purchased from Thermo Scientific (Shanghai, China). Trizol was purchased from Tiagen Biotechnology (Beijing, China). SYBR Green kit was bought from Transgen Biotechnology (Beijing, China). Primers for detecting Ptgs 2, GSTA1, GSTM2, heme oxygenase-1 (HO-1), glutamate-cysteine ligase (GCLM), NAD(P)H: quinone oxidoreductase 1 (NQO1), Nrf2, glucose transporter 1 (GLUT1), thioredoxin reductase 1 (TXNRD1), β-actin, and 18s mRNA were synthesized by Generay Biotechnology (Shanghai, China). Anti-Nrf2 antibodies were purchased from Abcam (ab62352) and Proteintech Group (16396-1-AP). Antibodies for solute carrier family 7 member 11 (SLC7A11, ab37185), GPX4 (ab125066), 4-hydroxynonenal (4-HNE, ab46545), and TATA binding protein (TBP) (ab125009) were purchased from Abcam (MA, USA). Anti-Keap1 antibody was purchased from Cell Signal Technology (MA, USA). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (FD0063) and FDbio-Pico ECL kit were from Fude Biological Technology (Hangzhou, China). Alexa Flour 594 and antibodies for Keap1(10503-2-AP), GCLM (14241-1-AP), hemagglutinin (HA, 66006-2-Ig), IgG (B900610) and β-actin (66009-1-lg) were purchased from Proteintech Group (IL, USA). Antibodies for NQO1 (SC-32793) and HO-1 (SC-390991) were purchased from Santa Cruz Biotechnology (CA, USA). Phosphatidylcholine hydroperoxide (PCOOH) was obtained from the University of Pittsburgh. Liperfluo dye (L248) was purchased from Dojindo Molecular Technologies, Inc. (Tokyo, Japan). Boron-dipyrromethene (BODIPY) 581/591 C11 (D3861) and lipofectamine 2000 (11668019) were purchased from Life Technologies. Corp. (CA, USA).

Animals and treatments

Male C57BL/6 mice aged 8–10 weeks were purchased from Guangdong Experimental Animal Center (Guangzhou, China). The animals were maintained on a 12 h light-dark cycle in a regulated temperature and humidity environment for 1 week before drug administration. Treatment protocols are shown in Fig. 1b. (+)-CLA (50 mg/kg/day, i.g.) or fer-1 (2.5 μmol/kg/day, i.p.)21,23 were administered for 7 consecutive days. To induce liver injury, mice were injected with erastin (100 mg/kg/day, i.p., twice a day) on both the 6th and 7th day, or a single dose of APAP (600 mg/kg/day, i.p.) on the 7th day after overnight food deprivation. The serum and livers were obtained for analysis. All the animal experiments were approved by humanistic animal care standards and authorized by the Laboratory Animal Ethics Committee of Jinan University. All animal protocols followed the guidelines for the Care and Use of Laboratory Animals which published by the US National Institutes of Health (NIH Publication No. 85–23, 1996).

Determination of serum ALT and AST levels

Liver injury was analyzed by measuring the serum levels of ALT and AST. Serum samples were collected from blood after centrifugation at 1200 × g for 10 min. ALT and AST in the serum were detected using commercial assay kits under the guidance of the manufacturer’s instructions.

H&E staining and immunohistochemical (IHC) analysis

The livers were chipped from mice at the same position and were fixed in 4% paraformaldehyde (PFA). PFA fixed tissues were embedded in paraffin and sections were sliced at 4.5 μm thickness and mounted on slides. H&E staining was used for morphological studies.

After deparaffinization with xylene and rehydration with gradient alcohol, the slices were boiled in 10 mM citrate buffer for 20 min for antigen retrieval. When the slices were cooled down, 0.1% Triton X-100 was used to permeabilize the cell membrane and 3% hydrogen peroxide was applied to quench endogenous peroxidase at room temperature for 10 min in the dark. Then the slices were incubated with anti-4-HNE antibody (rabbit, 1:200, Abcam) at 4 °C overnight in a humid cassette after blocking with goat serum for 1 h. Slices were washed with phosphate buffer saline (PBS, three times, 10 min) and incubated with biotinylated goat antirabbit secondary antibody for 1 h at room temperature. Biotin-streptavidin horseradish peroxidase (HRP) detection systems were used to detect immunoreactivity, then the sections were counterstained with hematoxylin and sealed with neutral resins.

Terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining

For the detection of hepatic nuclear DNA strand breaks, an in situ cell death detection kit, POD was used to stain the paraffin-embedded sections according to the manufacturer’s instructions. The sections were counterstained with hematoxylin and sealed with neutral resins. TUNEL-positive cells which were characterized with brown nuclei were counted using image J software.

Measurement of MDA, NADPH, and GSH contents

Hepatic MDA content was measured using an MDA assay kit. NADPH content was measured using a coenzyme NADPH II content kit. The GSH separation was achieved on a C18 column using 6% acetonitrile solution and detected by a CoulArray detector. The pH of the mobile phase was adjusted with phosphoric acid to 3.0 and the GSH samples were filtered through a 0.2 μm hydrophilic polypropylene membrane filter before analysis. GSH content was quantified based on a standard curve.

Isolation of total RNA and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)

Total RNA of liver tissue was isolated using Trizol and transcribed into cDNA with DNAase treatment. The purification of RNA was performed according to the ratio of absorbance at 260 and 280 nm. qRT-PCR was performed with SYBR Green kit. The relative gene expression was normalized by the comparative Ct (2Ct) with 18S or β-actin gene expression. The specific primer sequences applied in this study are presented in supplementary table 1.

Protein extraction and western blot analysis

Whole cell extracts from tissues or cells were extracted with cell lysis buffer. Cytosolic and nuclear proteins were isolated using a NE-PERTM nuclear and cytoplasmic extraction kit according to the manufacturer’s instructions. Protein expression was analyzed by western blot. Proteins in liver tissues or cell lysates were electrophoretically separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane, and blocked with skimmed milk powder and incubated at 4 °C overnight with primary antibodies. The membranes were washed with tris buffered saline with tween 20 (TBST) and incubated with secondary antibodies conjugated with HRP. The protein bands were detected by ECL reagents using the Tanon system. Chemiluminescent signals were analyzed using the Image J analyze system.

Determination of GPX4 enzymatic activity in liver tissue by liquid chromatograph-mass spectrometer (LC-MS)

Liver GPX4 samples were prepared as described24. In the test mixture, 10 μM of PCOOH, 200 μg of GPX4 samples and 5 mM GSH were added to a glass tube with 500 μL total volume using GPX4 reaction buffer (final buffer concentration of 25 mM sodium phosphate, 125 mM NaCl, 1 mM EDTA, 0.1 mM DFO, 0.1% Triton X-100; pH 8.0). Then the mixture was vortex mixed for 1 min and reacted for 10 min at 37 °C. Thereafter, it was extracted by a chloroform:methanol (2:1 vol/vol) solution three times. The chloroform layers were collected into another glass container, dried by nitrogen gas and re-dissolved in 100% methanol before LC-MS analysis.

LC-MS was carried out on an Ultimate 3000 Rapid Separation LC (Thermo Fisher Scientific) coupled to a quadrupole Orbitrap ion trap mass spectrometer. The lipid extract was separated on a UPLC HSS T3 C18 column (2.1 × 100 mm; 1.8 μm particle size) at 40 °C column temperature. The mobile phase was 2 mM ammonium formate and 0.1% formic acid in water (A) or methanol (B). The gradient curve was 75% B at 0 min, 100% B at 6 min, 100% B at 20 min, 75% B at 20.1 min, and 75% B at 22.5 min. The flow rate was 0.3 mL/min with an injection volume of 1 μL. The mass range between m/z 886.5809 ± 2 and 870.5860 ± 2 was recorded during the acquisition, and SIM of m/z 886.5809 and 870.5860 [M+HCOO] was analyzed to determine the amount of PCOOH and PCOH, respectively. The GPX4 enzymatic activity was expressed as the amount of PCOOH converted per mg of GPX4 samples within 30 min. The conversion rate (%) = (T0 PCOOH – T30 PCOOH)/T0 PCOOH × 100%.

MTT cell viability and LDH release assays

Cells (5000 cells per well) were seeded in 96-well plates and pretreated with (+)-CLA, ZVAD-fmk, nec-1 and fer-1 for 1 h and then treated with APAP or erastin for 24 h. After treatment, cell viability was detected by MTT assay, and LDH release was determined using a LDH cytotoxicity detection kit according to the manufacturer’s instructions.

BODIPY staining and confocal imaging

Hepa RG cells were plated in confocal dish at 37 °C and treated with or without (+)-CLA for 24 h. After treatment, the cells were washed three times with PBS for 30 min and fixed with 4% PFA for 15 min and permeabilized with 0.1% Triton-100 for 10 min at room temperature. Then the cells were incubated with BODIPY 581/591 C11 at 37 °C for 1 h. After washing, nucleus was stained with DAPI in PBS for 10 min. The confocal microscope images were obtained at 484/510 nm excitation/emission using a Carl Zeiss LSM 510 laser scanning confocal microscope (Tokyo, Japan).

Liperfluo staining and flow cytometry analysis

Cells were plated in 100 mm dishes and treated with (+)-CLA for 24 h. After treatment, cells were washed and incubated with 20 μM liperfluo dye for 1 h at 37 °C. Cells were trypsinized, centrifuged, and resuspended in sheath before analysis.

Co-immunoprecipitation (Co-IP) analysis

Cells were transfected with Nrf2 and Keap1 plasmids with a ratio of 1:1 using lipofectamine 2000, and cultured for 40 h. The transfected cells were treated with or without (+)-CLA for 6 h, and then were co-treated with 10 μM MG-132 for an additional 6 h. The cell lysates were diluted four-fold with the buffer including 1% Triton X-100, 150 mM NaCl, and 10 mM Tris-HCl. Protein A beads and anti-Keap1 antibodies were added for incubation at 4 °C overnight. Immunoprecipitated proteins were subjected to western blot analysis with Nrf2 and Keap1 antibodies.

Nrf2 immunostaining (IF) and confocal imaging

Cells were seeded in dishes which had been pre-placed with glass covers and were treated with indicated doses of (+)-CLA for 18 h. Then, the glass covers were washed with PBS three times and fixed with −20 °C methanol/acetone (1:1) for 10 min. The glass covers were then incubated with anti-Nrf2 antibody at 4 °C overnight. After washing in PBS, the glass covers were incubated with Alexa Flour 594 and DAPI for the indicated time. An Olympus BX53 fluorescence microscope coupled to an Olympus DP73 digital camera (Tokyo, Japan) was employed to image the fluorescence signals.

Ubiquitination detection

Cells were transfected with expression vectors for HA-ubiquitin, Nrf2, and Keap1. Then, the transfected cells were exposed to 10 μM MG-132 along with or without (+)-CLA for 6 h. Western blot analysis was used to analyze 20 μL cell lysates to evaluate the expression of Nrf2. The remaining cell lysates were diluted four-fold with the buffer including 1% Triton X-100, 150 mM NaCl, and 10 mM Tris-HCl. Then protein A beads, and anti-Nrf2 and anti-IgG antibodies were added at 4 °C overnight. Immunoprecipitated proteins were subjected to western blot analysis with anti-HA antibodies.

Half-life measurement of Nrf2 protein

Cells were seeded in 35 mm dishes, and pre-incubated with or without (+)-CLA for 8 h. After exposure to 50 μM CHX, cells were collected at 0, 10, 20, 30, and 40 min. Western blot analysis was then used to analyze protein level of Nrf2. The intensities of Nrf2 bands were quantified using Image J analyze system and plotted against time following CHX treatment.

Cellular thermal shift assay (CETSA)

For the cell lysate CETSA experiments, cells were collected and freeze-thawed three times using liquid nitrogen. The lysates were diluted with PBS and divided into two aliquots, with one aliquot being treated with (+)-CLA (20 μM) and the other aliquot as a control (DMSO). After 1 h incubation at 37 °C, the lysates were heated individually at different temperatures (42–62 °C) for 3 min followed by cooling for 3 min at room temperature. For the intact cell experiments, the cells treated with (+)-CLA (20 μM) for 24 h were heated as above described followed by lysis using three cycles of freeze-thawing with liquid nitrogen. All the samples were centrifuged at 16,600×g for 20 min at 4 °C to separate the supernatant and pellet. The soluble fractions were isolated and analyzed by western blot analysis.

siRNA transfection

Cells were cultured in 60 mm dishes (for western blot analysis) or 96-well plates (for cell viability assay). Control siRNA (no silencing) and Nrf2 siRNA were transfected into cells using lipofectamine 2000.

Annexin-V/PI staining and flow cytometry analysis

Annexin V-FITC/PI apoptosis kit was used to evaluate apoptotic ratio according the manufacturer’s instructions. Cells were pre-treated with or without ZVAD-fmk for 1 h, and then incubated with APAP for another 24 h. After treatment, cells were washed and stained with PI followed Annexin V-FITC at room temperature for 15 min. To quantify apoptotic ratio, cell samples were analyzed by a flow cytometry.

Statistical analysis

Results were expressed as means ± SD. GraphPad Prism version 7.0 statistical software was used for statistical analysis. Multiple groups were compared by t-tests, one-way ANOVA analysis or two-way ANOVA and a probability (P) value < 0.05 was considered statistically significant.

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