Human Liver Samples
Human liver samples with NASH used in this study were all obtained from patients in the Department of Weight Loss and Metabolism and these patients were excluded from excessive drinking (> 140 g in men or > 70 g in women per week), drug abuse or viral infection ( Liver lesions caused by hepatitis B virus and hepatitis C virus. These specimens were sent to the pathology department for evaluation by experts based on the NAFLD activity score, and patients with a ≥ 5 were considered likely to have NASH[25]. Normal human liver samples were collected from patients without NASH (patients with hepatic hemangioma) and were used as normal controls. All procedures involving human sample collection and application were approved by the Ethics Committee of Jiangsu Provincial People's Hospital and adhered to the principles of the Declaration of Helsinki. Written informed consent was obtained from patients or their families before surgery.
Animals
Gsk3βfl/fl and Lyz2-Cre mice were both acquired from Gem Phar matech Co., Ltd. (Nanjing, China). For producing myeloid-specific knockout mice, Homozygous Gsk3βfl/fl mice were first crossed with homozygous Lyz2-Cre mice, and heterozygous progeny (including Gsk3β and Cre) were backcrossed with homozygous Gsk3βfl/fl mice.In this study, mice with Lyz2-specific deletion of Gsk3β were defined as KO mice, and the Gsk3βfl/fl (WT)mice were defined as the controls. The genomic DNA isolated from the mouse tails was analyzed by PCR and primers are listed in Supplementary Table 1. This study was conducted in strict accordance with the recommendations in the 《Guide for the Care and Use of Laboratory Animals》 published by the National Institutes of Health. The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University (Protocol No. NMU08-092).
Mouse Liver Nash Model And Histological Analysis
8-week-old male Gsk3βfl/fl mice (WT) and Gsk3β KO mice were fed a low-fat diet (LFD) or HFD (Biopike) for 26 weeks. In another NASH model, 8-week-old male Gsk3βfl/fl mice(WT )and Gsk3β KO mice were fed either MCD or chow diet for 6 weeks, and all mice were reared under specific pathogen-free conditions, and free access to water and chow during a 12-hour light/dark cycle.Serum liver enzyme indexes (AST,ALT) were measured by Sevier (Wuhan, China). Part of the liver tissue was fixed in a 4% formaldehyde neutral buffer and embedded in paraffin. Paraffin embedded sections (4mm) were stained with H&E, Masson, and Sirius red. Frozen slices were obtained from frozen liver tissue in OCT medium and stained with oil red. The NAS score system was used to assess the extent of NAFLD in the liver tissue. A NAS score < 3 can exclude NASH, and a NAS score ≥ 5 can diagnose non-alcoholic fatty liver disease (NASH).
Cell Isolation And Cell Culture
Bone marrow cells were isolated from mouse femur and tibia and seeded in DMEM medium containing 100ng/mL M-CSF and 10% fetal bovine serum (FBS) until maturation. To analyze the characteristics of adipose-induced macrophages in vitro, we used palmitic acid (PA; P5585, Sigma) stimulated freshly isolated macrophages. Cultured macrophages were collected for Western blot analysis, qRTPCR analysis, and immunofluorescence analysis. Liver macrophages were isolated from diet-induced NASH mice by portal vein perfusion; in brief, livers were perfused via portal vein with heated (37°C) Hanks balanced salt solution followed by injection of collagenase IV (Sigma). Perfused livers were dissected and passed through 70 mm nylon mesh cell filters (BD Biosciences, San Diego, CA, USA). The supernatant was collected after centrifugation at 50 g for 2 min at 4°C, and this procedure was repeated three times. The supernatant was centrifuged at 500 g for 8 minutes and the non-parenchymal cells in the supernatant were collected. Non-parenchymal cells are then seeded in DMEM containing 10% fetal bovine serum, 10 mM N-(2-hydroxyethyl)piperazine-Nˊ-ethanesulfonic acid, 2 mM glutamic acid, penicillin (100 U/mL), and streptomycin (100 µg/mL) and incubated at 37°C for 15 min.Non-adherent cells were removed by replacing the medium and identified as hepatic macrophages by F4/80 immunofluorescence staining. They were then cultured in vitro for 6h. Cells and supernatants were harvested for further experiments. Primary hepatocytes were centrifuged at 50 g for 2 min, separated by 50% Percoll gradient centrifugation and seeded in DMEM medium supplemented with 10% fetal bovine serum.
Plasmid Transfection
Briefly, a Gsk3β kinase active mutant plasmid (S9A) or a PINK1 knockout plasmid was introduced into the cell using the Lipo8000™ transfection kit (Beyotime, China). After 3 days of stable incubation in the cell incubator at 37℃, the expression of the target gene was detected by western blotting, and the next experiment was conducted according to the detection results.
Immunohistochemistry And Immunofluorescence (If) Staining
For immunohistochemistry, tissue samples were fixed in 10% formalin, embedded in paraffin and sectioned. After dewaxed and rehydrated, specimens were boiled in citrate buffer to a boil and then cooled before repeated procedures were performed for antigen extraction and blocked with serum for 1 h at room temperature (RT). Then, tissue sections were incubated overnight at 4°C with the primary antibodies including Anti- p-Gsk3β (Affinity, AF2016), anti-α-SMA (CST, #19245), anti-Ly6G (CST, #87048S) and anti-CD11b(CST, #49420). After 3 washes, tissue sections were incubated at RT for 20 min with biotinylated secondary antibody reagents. 3 washes with PBS were followed by the addition of DAB oligonucleotides. Then, the slides were counterstained with haematoxylin. For immunofluorescence staining, cells were fixed at room temperature with 4% paraformaldehyde for 30 min and then washed three times with PBS. The cells were first permeated with 0.1% Triton X-100, and then blocked with 10% BSA. Then, Cells were incubated overnight at 4°C with the primary antibodies including Anti-Gsk3β༈Affinity, AF5016), Anti-Parkin༈Proteintech, 66674-1-Ig) Anti-PINK1(Beyotime, AF7755), Anti-LC3B༈Abcam, ab63817), Anti-NLRP3(CST, #15101). The cells were then washed three times with PBS and incubated with Alexa Fluor 488-/594-labeled secondary antibodies. Incubate at room temperature for 1–2 hours. After washing with PBS, they were stained with DAPI for 15 minutes and examined under a microscope.
Quantitative Reverse Transcription Pcr
According to the manufacturer's instructions, TRIzol reagent (Invitrogen) was used to extract total RNA from tissue samples or cells, and then the RNA concentration was analyzed by Nano-drop system. RNA was then reversed into cDNA using a kit containing SuperMix premix (Vazyme, Nanjing, China). Real-time quantitative analysis was performed using PCR kit with SYBR-green fluorescence labeling (Vazyme, Nanjing, China). The primers used in this study are listed in Supplementary Table 2.
Western Blotting
Proteins were extracted from liver tissue or cells with ice-cold lysis buffer (50mM Tris(pH 7.4), 150mM NaCl, 1% Triton X-100 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, Leupeptin and other various inhibitors).Proteins were separated on SDS-polyacrylamide gels, followed by transfer to a PVDF membrane. Western blotting analyses were performed with anti- rabbit IgG (CST, #7074), anti-mouse IgG (CST, #7076), Anti-GAPDH(CST, #5174), Anti- p-Gsk3β (Affinity, AF2016), Anti-Gsk3β༈Affinity, AF5016), Anti-NOX2༈Affinity, DF6520), Anti-NLRP3(CST, #15101), Anti-IL-1β༈Abcam, ab254360), Anti-Pro-IL-1β༈Abcam, ab254360), Anti-Caspase-1༈Abcam, ab207802), Anti-PINK1(Beyotime, AF7755), Anti-Parkin༈Proteintech, 66674-1-Ig), Anti-P62༈Servicebio, GB11239-1), Anti-LC3B༈Abcam, ab63817), Anti-PI3K༈Affinity, AF6241), Anti-p-PI3K༈Affinity, AF3241), Anti-AKT༈Proteintech, 10176-2-AP), Anti-p-PI3K༈Proteintech, 29163-1-AP) .Image Lab software was used to quantify protein expression.
Detection Of Ros Production In The Bmdms
ROS production levels in BMDMs were detected using Reactive oxygen species detection kit (Beyotime, China) with fluorescent probe DCFH-DA. In short, after the cells were spread in a culture dish and given appropriate stimulation, the cell culture medium was removed and the appropriate volume of diluted DCFH-DA was added. Incubate in a 37ºC cell incubator for 20 minutes. The cells were washed three times with serum-free cell culture solution to fully remove the DCFH-DA that did not enter the cells, and then observed under fluorescence microscope.
Detection Of The Mitochondrial Membrane Potential
Changes in mitochondrial membrane potential were detected using a mitochondrial membrane potential detection kit with a JC-1 probe (Beyotime, China). Following the manufacturer's instructions, after the cells were propagated in a culture dish and given appropriate stimulation, the cell culture medium was removed and an appropriate volume of diluted JC-1 was added.. Incubate in a 37ºC cell incubator for 20 minutes. The cells were washed three times with serum-free cell culture solution to fully remove the JC-1 ,and then observed under fluorescence microscope.Alternatively, the treated cells were suspended in diluted JC-1 and incubated in a 37ºC cell incubator for 20 min. The cells were rinsed with serum-free cell culture solution for 3 times, then the changes of membrane potential were detected by Beckman CytoFLEX FCM and analyzed by CytExpert 2.0 software.
Co-immunoprecipitation
For IP analysis, cells were lysed in Nonidet P-40 lysis buffer, and harvested cell lysates were mixed with specific primary antibodies and incubated overnight at 4°C on a rotating platform. Then it was conjugated with protein A/G beads (Beyotime, China) for 4–6 hours, and washed with PBS 5 times for 5 minutes each time.After washing, the immune precipitates were boiled in SDS-PAGE Sample Loading Buffe for 10 min. Immunoprecipitated proteins were detected by Western blotting.
Statistical analysis
The data are displayed as mean values ± SEM. In vitro and in vivo experiments were assessed in at least three independent experiments. Differences were evaluated using GraphPad Prism. Statistical signifificance was set at a p value < 0.05