2.1 Drugs and reagents
Chlorogenic acid (CGA, purify≥98%) was purchased from Baoji Herbest Bio-Tech Co., Ltd. (Baoji, China). Acetaminophen (APAP) was obtained from Shanghai Johnson Pharmaceutical Co., Ltd. (Shanghai, China). Ammonium glycyrrhizinate (AG, purify≥98%) was provided by ALFA Biological Technology Co., Ltd. (Chengdu, China). Commercial assay kits for alanine transaminase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH) were supplied by Medical System Biotechnology Co., Ltd. (Ningbo, China). Cell Total RNA Isolation Kit was acquired from Chengdu Foregene Biotechnology Co., Ltd. (Cat. RE-03113). Anti-Tom20 was provided by Santa Cruz Biotechnology, Inc. Anti-SQSTM1/p62 and anti-Parkin were offered by Cell Signaling Technology (Danvers, MA, USA). Anti-LC3, anti-Bcl2 and anti-Bax were furnished by Proteintech Biological Technology Co., Ltd. (Wuhan, China). Anti-PINK1was purchased from Abcam (Cambridge, UK). Anti-glyceraldehyde phosphate dehydrogenase (GAPDH) was produced by Servicebio (Wuhan, China). The BCA protein assay kit was acquired from Multi Sciences Biotech CO., Ltd. (Hangzhou China).
100 male 4-6 weeks Kunming mice (20 ± 2 g) were obtained from Byrness Weil biotech Co., Ltd. (Chengdu, China). All mice were acclimated for 3 days in a constant temperature and humidity room (24°C±1°C, 50% ±10% humidity) with standard diet, water and a 12-h light/dark cycle (lights on at 8:00 am and lights off at 8:00 pm).
2.3 APAP-induced acute hepatotoxicity mice model and treatment
All mice were randomly divided into five groups (n=20), the control (Ctrl) group, the APAP group (300 mg/kg), the APAP (300 mg/kg) +AG group (200 mg/kg), and the APAP (300 mg/kg) + CGA group (20 mg/kg or 40 mg/kg). All the material under study is endotoxin free. Mice in the intervention group were respectively pre-administered with CGA (20 mg/kg or 40 mg/kg) and AG (200 mg/kg) for 14 consecutive days. Simultaneously, the control and APAP groups were congruously administrated with same volume 0.9% saline. On day 15, mice were administered a single dose of APAP (300 mg/kg) to induce APAP acute hepatotoxicity except the control group was administered 0.9% saline. Then, all mice were sacrificed on day 16 and serum and liver tissues were collected.
2.4 Mortality in mice assay
After APAP (300mg/kg) treatment, the death period of mice was recorded within 24 h.
2.5 Biochemical analysis for blood
Serum was collected after centrifugation at 3500 rpm for 10 min at room temperature. Serum enzymatic activities of ALT, AST and LDH were measured with a HITACHI 7180 automatic biochemistry analyzer (Hitachi, Japan).
2.6 Histopathology of liver tissue and TUNEL assay
Fresh liver tissues were immediately fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and then cut into 4-μm-thick sections (n=5). The tissues were stained with hematoxylin and eosin (HE) for histological examination under a light microscopy. The histological scores (including inflammation, necrosis and apoptosis) indicating the degree of liver injury were determined according to Suzike’s standard . Simultaneously, these slides were subjected to terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) for analyzing hepatocytes apoptosis according to the manufacturer's instructions of a TUNEL apoptotic detection kit. Images were captured by a fluorescence microscopy (Leica Microsystems，Wetzlar，German), and calculated semi-quantitatively at magnification ×200 to count the positive cells.
2.7 qRT-PCR analysis
Total RNA was extracted from liver samples using Trizol reagent. cDNA was synthesized with the SuperScript® IV First-Strand Synthesis System (Bio-Rad, Singapore). qRT-PCR was performed in the SYBR-Green PCR kit on Applied Biosystems StepOnePlus system. β-actin was used as the invariant control. The target gene expression levels by the 2-ΔΔCt method. The sequences of primers used in this study are as follows: PINK1: Forward 5′-AGACTCCCAGTTCTCGCCT-3′, Reverse 3′- AGGGACAGCCATCTGAGTCC-5′; Parkin: Forward5′-AGCCAGAGGTCCAGCAGTT-3′, Reverse 3′-CTGGCACTCACCACTCATCC-5′; p62: Forward 5′-AGATAGCCTTGGAGTCGGTG-3′, Reverse 3′-CCGGGGATCAGCCTC TGTAG-5′; LC3-Ⅱ: Forward 5′-ACCCTAACCCCATA GGAGCC-3′, Reverse 3′-TGCAAGCG CCGTCTGATTA-5′; β-actin: Forward 5′-GCTCCGGCATGTGCAAAG-3′, Reverse 3′-TTCC CACCATCACACCCTGG-5′.
2.8 Protein preparation and western blotting analysis
The total proteins of liver were extracted by RIPA lysis buffer containing protease and phosphatase inhibitor (100:1:1). Protein concentrations were determined using the BCA protein assay kit. Equal amounts of total protein (30 μg) were separated by 8%-15%SDS-PAGE gels before transferred to the PVDF membranes. Then, the membranes were blocked with 5% fat-free milk at room temperature for 90 min and incubated with primary antibody at 4°C overnight, including anti-GAPDH (1:5000), anti-Parkin (1:1000), anti-PINK1 (1:1000), anti-p62 (1:1000), anti-LC3 (1:1000), anti-Bcl2 (1:1000) and anti-Bax (1:1000). After washing with TBST, the membranes were incubated with secondary goat anti-rabbit IgG (1:5000) or anti-mouse IgG (1:5000) at room temperature for 90 min, and were conjugated with horseradish peroxidase. Then, proteins were detected by chemiluminescence reagent. The relative protein levels were normalized to GAPDH level.
2.9 Immunohistochemistry analysis
The liver tissue was cut into 4-μm-thick sections then deparaffinization with xylene and gradient ethanol, antigen retrieval with trisodium citrate dihydrate for 30 minutes and blocked with 1% BSA at room temperature for 30 min. the sections were incubated with primary anti‐Tom20 (1:100) and anti‐LC3Ⅱ（1：300）at 4℃ overnight and then with secondary antibodies (1:500) in the dark for 1 h. Hoechst 33258 was used to counterstain the nucleus. At last, the sections were mounted with anti-fluorescence quenching sealer and observed by confocal microscopy (Olympus, Japan).
Statistical analysis was performed by one-way analysis of variance (ANOVA) and all values were expressed as mean ± standard error of the mean (SEM), with the exception of survival rate. p＜0.05 was considered to be statistically significant.