Materials and reagents
Phillyrin was provided by Nanjing Biohybrid Pharmatech Co.,Ltd (CAS 487-41-2, Nanjing, China). The antibodies for mTOR, p-mTOR, AKT and p-AKT were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibodies for LC3 and p62 were purchased from Abcam (Cambridge, UK). The recombinant human PDPK1 protein was also purchased from Abcam (Cambridge, UK). P-PDPK1(Ser241) antibody and goat anti-rabbit/mouse immunoglobulin G (IgG) horseradish peroxidase (HRP) secondary antibody were purchased from Abmart (Shanghai, China). β-actin antibody was purchased from Bioss (Beijing, China). An ALT/GPT (C009-2-1), AST/GOT (C010-2-1) assay kit, TG(A110-1-1) assay kit and T-CHO(A111-1-1) assay kit were purchased from Jiancheng Bioengineering Institution (Nanjing, China). Nile red was provided by Shanghai Yuanye Biotechnology Co.,Ltd (CAS 7385-67-3).
Animal treatment
Male C57BL/6 mice in the background that were 6–8 weeks old, weighing >20 g, were provided from the from the Laboratory Animal Center of Anhui Medical University. All animal procedures were approved by the Institutional Animal Experimentation Ethics Committee of Anhui Medical University. These mice were maintained in the vivarium facility with 12 h light/dark cycles. Gao-Binge protocol recommended by the National Institute on Alcohol Abuse and Alcoholism (NIAAA) was adopted to establish ASH model. In this protocol, mice were given a liquid diet adaption for 5 days and ethanol feeding diet for 10 days. At the last day, mice were treated with a single binge ethanol administration (5 g/kg, body weight, 20% ethanol) by gavage. Whereas the CD-fed mice were fed with control liquid diets and treated with isocaloric maltose-dextrin by gavage at the last day. All diets were prepared fresh daily. 9 h after the last gavage alcohol, mice were euthanized, the liver tissues and blood were collected for further analysis.
Liver histological analysis
4% paraformaldehyde fixative was used to immerse part of hepatic lobes cut from C57BL/6J mice for 48 hours. Then, paraffin was used to embed those. Next, 5 μm thick sections were stained hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining of PDPK1. Livers embedded in optimum cutting temperature compound were used for oil-red-o staining for the assessment of hepatic steatosis. The sections were finally scanned under SlideViewer(3DHISTECH Ltd.).
Serum level of ALT/GPT, AST/GOT1, TG and T-CHO analysis
Serum ALT/GPT, AST/GOT, TG and T-CHO levels were assayed using GPT, GOT1, TG and T-CHO activity assay kits (Nanjing Jiancheng Bioengineering). The absorbance was measured at 510 nm (GPT, GOT, TG, T-CHO) with a Cytation5 Cell Imaging Microplate Assay System (Bioteck, US).
Electron microscopy
Mice livers were fixed with a combination of paraformaldehyde and glutaraldehyde, then livers were processed and sectioned with a diamond knife on copper grids after fixation. Grids were examined with a Hitachi (Tokyo, Japan) 7100 electron microscope, and images were captured using a MegaView III digital camera (Soft Imaging System, USA).
Cell culture
AML-12 cells were obtained from the Chinese Academy of Sciences (Shanghai, China), cultured in DMEM F12(Procell, China) supplemented with 10% FBS(Gibico, US) and incubated at 37 °C at an atmosphere of 5% CO2. Then cells were cultured by adding 200 mM Ethanol for 24 h or incubating with different concentrations of Phillyrin(50, 100, 200μg/ml) while adding 200 mM Ethanol for 24 h.
MTT assay
We measured the safe dose of Philyrin by MTT assay. AML-12 cells were seeded in 96-well plates, and the edge wells were filled with sterile PBS. After adherence, cells were cultured with various concentrations of Phillyrin for 24 hours. A total of 20 μL of 5 mg/mL MTT was added to each well and incubated with cells at 37°C for 4-6 hours. After removal of the supernatant, 150 μL of DMSO was added to each well. The optical density (OD) was measured at 490 nm. The percent of viable cells was calculated according to the formula.
EGFP-LC3 transient transfection
AML-12 cells were cultured on the surface of cover slip stably first. Then those cells were transfected with the EGFP-LC3 plasmid for 24 h using the transfect mate (GenePharma, China). After transfection for 6h, the cells were treated with Phillyrin for 24 h. Subsequently, the cells were stained with DAPI (Bioss, China) and observed with a Zeiss LSM880 confocal laser microscopy system and analyzed using the Zeiss LSM Browser.
Western blotting
Proteins from liver tissue (30 or 50 mg) and AML-12 cells were extracted with RIPA lysis buffer (Beyotime, China) with 1% phenyl methyl sulfonyl fluoride (PMSF; Beyotime), and the protein concentration was measured with a BCA protein assay kit (Beyotime, China) according to the manufacturer's instructions. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (size of 0.22μm just for LC3, size of 0.45μm for another proteins, Millipore Corp, Billerica, MA, USA). The PVDF membranes were blocked in 5% skim milk for 1.5 hours at room temperature and then, washed three times in TBST. Subsequently, the PVDF membranes were incubated with primary antibodies against PDPK1 (1:1000), β-actin (1:1000), LC3 (1:1000), p62 (1:1000), p-AKT (1:2000), AKT (1:1000), P-MTOR (1:1000), mTOR (1:1000) overnight at 4°C, followed by incubation with secondary antibodies (1:5000) for 1 hour at room temperature. The protein bands were visualized with the ECL-chemiluminescent kit (Epizyme, China). All experiments were repeated three times.
RNA interference analysis
Small interfering RNA (siRNA) oligonucleotides against the PDPK1 gene and scrambled sequences were designed and synthesized by HanzBio (Shanghai, China). The siRNA sequences were as follows:
PDPK1-siRNA (sense, 5ʹ-GCAACAUAGAGCAGUACAUTT-3ʹ and antisense, 5ʹ-AUGUACUGCUCUAUGUUGCTT-3ʹ);
Scrambled-siRNA (sense, 5ʹ-UUCUCCGAACGUGUCACGUTT-3ʹ and antisense, 5ʹ-ACGUGACACGUUCGGAGAATT-3ʹ).
AML-12 cells were transfected with 1000 ng/mL PDPK1-siRNA or scrambled-siRNA and mixed with Lipo3000 transfection reagent (Hanbio, China) according to the manufacturer's instructions. After 6 hours, Opti-MEM was replaced by DMEM F12 supplemented with 10% FBS DMEM, and cells were treated with 200mM ethanol. The silencing efficiency was determined by RT-qPCR.
RNA extraction and quantitative real-time PCR analysis
Total RNA was extracted by using TRIzol total RNA isolation reagent (Invitrogen, Carlsbad, CA, USA). One microgram of total mRNA was used for reverse transcription with the Takara RT-PCR synthesis kit (Takara, Dalian, China), according to the manufacturer's instructions. cDNA synthesis was performed using SYBR Premix Ex Taq II (Takara) on the PikoReal 96 qPCR system (Thermo Fisher Scientific, Waltham, MA, USA). The primer sequences used were as follows:
GAPDH forward, 5′-AGGTCGGTGTGAACGGATTTG-3´;
GAPDH reverse, 5′-GGGGTCGTTGATGGCAACA-3´;
PDPK1 forward, 5′-CTGTATGACGCTGTGCCCATT-3´;
PDPK1 reverse, 5′-AAGGGGTTGGTGCTTGGTC-3´.
GAPDH was used to normalize the expression values of the other genes. All experiments were repeated at least three times.
Construction of a stable PDPK1 over-expression AML-12 cell line
HBLV-m-PDPK1-3xflag-mcherry-PURO was offered by HanzBio (Shanghai, China). The multiplicity of infection (MOI) was detected first (MOI=30). To generate stable PDPK1 overexpression cell populations, AML-12 cells were infected with HBLV-m-PDPK1-3xflag-mcherry-PURO or HBLV-mCherry-PURO-Control for 24h. Then fresh DMEM F12 with puromycin (0.5 μmg/ml) was added in plates for stably transfected cells for 48 hours.
PDPK1 overexpression in the stable cell line was verified by RT-qPCR.
Surface Plasmon Resonance (SPR) technology-based assay
The Surface Plasmon Resonance (SPR) method was used with a BIAcore T200 (Cytiva) to measure the binding affinities. During the experiment, PDPK1 was immobilized on a CM5 sensor chip according to the Biacore methods. Phillyrin(20mM)was diluted with 5% DMSO Runnimg Buffer [1×PBS-P+] to different concentrations as follows: 1000μM, 333.3μM, 111.1μM, 37μM, 12.3μM , 4.1μM, 1.37μM, 0.46μM, 0.15μM, 0.05μM. A random concentration was double used. Samples were injected into the channels at a flow rate of 30 μL/min and then, washed with HBS buffer. The binding RU (Response Unit) values of Phillyrin to PDPK1 were recorded directly by the Biacore T200 instrument and analyzed by Biacore T200 Evaluation Software.
Cellular thermal shift assay (CETSA)
AML-12 cells were incubated with or without Phillyrin(200μg/ml) for 24 hours, Then the incubated cells was collected and resuspended to 1×107 cells/ml with PBS. Those cell suspension was divided into 10 parts, which was heated for 3 min under different temperature(43, 46, 49, 52, 55, 58, 61, 64, 67 and 70℃). were collected and subjected to CETSA assay (Jafari et al., 2014). The heated cells were kept at −80°C for 12 hours, then at room temperature for 5 min. Such freezing and thawing process was repeated 3 times. After that, cell lysates were extracted by centrifugation at 20,000× g, 20 min. Levels of PDPK1 were assessed by western blot.
Statistical analysis
Differences in multiple groups were determined by one-way ANOVA analysis. However, difference in two groups was determined by Student’s t tests (unpaired, two-tailed). Experimental data in this study were analyzed by using Prism 8.0 GraphPad Software (USA). P <0 .05 was considered statistically significant.