Establishment and experimental grouping of mouse models of NAFLD. 6-8-week-old female or male BALB/c mice (20 ± 4 g) were purchased from SPF ( Beijing ) Biotechnology Co., Ltd., and raised at the Animal Center of the Laboratory of Guizhou Medical University in an environment with 22 ± 2˚C, 50–60% humidity and 12-h circulating lighting and provided with unrestricted access to chow and tap water throughout the duration of the present study. All animal experiments were approved by the Experimental Animal Ethics Committee of Guizhou Medical University (approval no. 1900035). To induce the development of NAFLD, 6-weeks-old female or male BALB/c mice (18 ± 2 g) were randomly divided into normal diet (n = 6) and HFD (D12492;Roden Diet with 60 kcal% fat, n = 6) fed for 16 weeks. To investigate whether Lrg1 improves HFD-induced NAFLD by enhancing TGF-β signaling, after being fed a HFD for 16 weeks, then the mice were divided into four groups (n = 6/group), the mice were injected with Lrg1 (50 µg/kg; cat. no. 7890-LR; R&D Systems, Inc.), TGF-β1 (500 ng/kg; cat. no. 1218209; PeproTech, Inc.) or TGF-β1 (500 ng/kg) in combined with Lrg1 (50 µg/kg) through the caudal vein three times (once/5 days). For all mouse experiments, the method of euthanasia was cervical dislocation. The serum samples (500 µl) were additionally collected from the angular veins of the mice.
Cell culture and treatment. Primary hepatocytes were cultured with RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), and were used in the experiments at a confluency of 85%. Primary hepatocytes were treated with or without oleic acid (0.5 mmol/l) for 24 h, and then stained with Oil Red O for 15 min at room temperature (cat. no. G1262; Beijing Solarbio Science & Technology Co., Ltd.) to determine the deposition of lipid droplets according to the manufacturer's instructions.
Isolation and culture of primary hepatocytes. The 8-weeks-old female or male BALB/c mice (21 ± 3 g) were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (150 mg/kg), and then sacrificed by cervical dislocation for perfusion with 20 ml D-Hank's buffer (cat. no. H1045; Beijing Solarbio Science & Technology Co., Ltd.) and 15 ml 0.1% collagenase Ⅳ (cat. no. C8160; Beijing Solarbio Science & Technology Co., Ltd.) successively. When the livers acquired a gray color, they were cut up and resuspended with 10 ml RPMI-1640 medium. The cell suspensions were filtered using a 70-µm cell filter mesh and centrifuged at 55 x g for 3 min at 4℃ to collect the cell precipitates, which were rich in hepatocytes (25). After resuspending with RPMI-1640 medium containing 10% FBS, the cells were cultured in culture plates pre-coated with rat tail collagen I type (cat. no. C8062; Beijing Solarbio Science & Technology Co., Ltd.). In order to induce steatosis, primary hepatocytes were treated with 0.5 mmol/l oleic acid for 24 h, and the medium was replaced with fresh medium for a further 12 h. For subsequent experiments, the conditioned medium (CM) was collected through centrifugation at 2,000 x g for 5 min at 4℃.
Isolation and culture of mouse liver macrophages. The 8-weeks-old female or male BALB/c mice (21 ± 3 g) were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (150 mg/kg),The liver perfusion method was similar to the aforementioned description. When the livers acquired a gray color, they were cut up and suspended with 10 ml D-Hank's buffer for centrifugation at 50 x g for 5 min at 4℃. The cell precipitation was collected for suspension with 20 ml 0.05% collagenase IV for 40 min of shaking in a water bath at 37˚C. Following filtration with 70-µm cell filter mesh, the supernatant was centrifuged at 700 x g for 6 min at 4˚C, and the cell precipitates were suspended with 30% Percoll (cat. no. P8370, Beijing Solarbio Science & Technology Co., Ltd.) and > 50% Percoll of the same volume was then added. Following centrifugation for 8 min at 700 x g at 4˚C, the cells at the adjacent interface of 30% Percoll and 50% Percoll were collected and mixed with an equal volume of D-Hank's buffer. The cell suspension was centrifuged for 6 min at 700 x g at 4˚C, and the precipitate was resuspended in RPMI-1640 medium containing 10% FBS (26). Following 20 min of culture in the culture plate, the medium was replaced with fresh medium, and the cells attached to the wall were liver macrophages.
Western blot analysis. The collected primary hepatocytes or liver tissues were lysed with RIPA buffer (P0013B; Beyotime Institute of Biotechnology) containing 1% PSMF, and centrifuged at 12,000 x g at 4˚C in a centrifuge (Thermo Fisher Scientific, Inc.) for 20 min. Protein concentrations were determined using a Bicinchoninic Acid assay kit (P0010; Beyotime Institute of Biotechnology). Following denaturation with sample loading buffer and electrophoresis with 10% SDS-PAGE gel (cat. no. P0690; Beyotime Institute of Biotechnology), the proteins were transferred to PVDF (cat. no. IPVH00010; EMD Millipore) membranes. Following inoculation with 5% skimmed milk powder (cat. no. 7BF0330; Yili Group) at room temperature for 4 h, the membranes were incubated at 4˚C with anti-inducible nitric oxide synthase (iNOS) antibody (1:1,50; cat. no. D6B6S; Cell Signaling Technology, Inc.) or anti-GAPDH antibody (1:2,000; cat. no. D16H11; Cell Signaling Technology, Inc.) for 16 h, and then incubated with HRP-labeled secondary antibodies (1:3,000; cat. no. ANR02-1; NeoBioscience) at room temperature for 2 h. After washing with TBS-Tween 20, the expression of special protein was detected using an ECL reagent (cat. no. WBKLS0500; EMD Millipore) using a chemiluminescence imager (CLINX5600, Clinx Science Instruments Co., Ltd.).
Reverse transcription-quantitative PCR (RT-qPCR). Total RNA was extracted from the collected primary hepatocytes or liver tissues using TRIzol® reagent (cat. no. 252610; Thermo Fisher Scientific, Inc.), and Monscript™ RTIIIAII-in-One Mix with dsDNase (cat. no. MR05101; Monad Biotech Co., Ltd.) was used to synthesise cDNA. The PCR reaction was performed according to the MonAMP™ SYBR-Green Qpcr Mix (cat. no. MQ10101S; Monad Biotech Co., Ltd.) experimental steps. The expression of specific genes was normalized according to the expression of GAPDH. The RT-qPCR conditions were as follows: 95˚C for 30 sec, 40 cycles of amplification (95˚C for 10 sec, 60˚C for 10 sec, and 72˚C for 30 sec). The primer sequences (Beijing Qingke Biotechnology Co., Ltd.) were as follows (27): Lrg1 (human) forward, 5’-CCTCTTGGAGCAGACAGCG-3’ and reverse, 5’-CAGGTGGTTGACAGGAGATGGA-3’; Lrg1 (mouse) forward 5’-TCTTGGCAGCATCAAGGAAG-3’ and reverse, 5’-TGGACAGTGTCGGCAGGGA-3’; iNOS (mouse) forward, 5’-GAGCGAGTTGTGGATTGTC-3’ and reverse, 5’-CCAGGAAGTAGGTGAGGG-3’; chemokine (C-X-C motif) ligand 1 (CXCL-1; mouse) forward, 5’-CCAAACCGAAGTCATAGCC-3’ and reverse, 5’-AGGGAGCTTCAGGGTCAA-3’; interleukin (IL)-1β (mouse) forward, 5’-GTTCCCATTAGACAACTGC-3’ and reverse, 5’-GATTCTTTCCTTTGAGGC-3’; GAPDH (human) forward, 5’-GGAGTCCACTGGCGTCTT-3’ and reverse, 5’-GAGTCCTTCCACGATACCAA-3’; and GAPDH (mouse) forward, 5’-TGTTTCCTCGTCCCGTAG-3’ and reverse, 5’-CAATCTCCACTTTGCCACT-3’.
Immunohistochemical analysis. After being fixed with 4% paraformaldehyde solution for 24 h, the liver tissues were successively placed in 70, 80, 95 and 100% alcohol, a mixture of 50% benzene and 50% alcohol and 100% benzene for 1 h each. The liver tissues were embedded in paraffin and then sliced into 5-µm-thick sections. They were kept in xylene for 30 min, followed by 100, 95 and 70% alcohol for 3 min each to dewax the sections, which were then stained with hematoxylin and eosin at room temperature for 5 min each, successively. For detecting the expression of F4/80, the dewaxed sections were blocked with 3% hydrogen peroxide and then sealed with goat serum (cat. no. SL038; Beijing Solarbio Science & Technology Co., Ltd.) for 30 min. The sections were then incubated with F4/80 (1:200; cat. no. ab6640; Abcam) at 4˚C for 14 h, followed by incubation with HRP Polymer (cat. no. ANR02-1; NeoBioscience) at 37˚C for 30 min. The expression of F4/80 was then observed by DAB staining (cat. no. ZLI-9018; Beijing Zhongshan Jinqiao Biotechnology Co.).
Intraperitoneal glucose tolerance test. After the mice were fasted for 16 h, the blood glucose concentrations were measured, and then re-measured at 15, 30, 60 and 120 min following an intraperitoneal injection of glucose (2 mg/g; cat. no. H50021025, Chuanyu, Chong Qing He Ping Pharmaceutical Co.,Ltd.).
Liver function test. Blood collected from the mice was centrifuged at 500 x g for 10 min at room temperature to collect serum. The contents of aminotransaminase, alanine aminotransaminase, total cholesterol (TC) and triglycerides (TG) in serum were detected using an automatic biochemical analyzer (model: XC8001, Sichuan Xincheng Biological Co., Ltd. China).
Statistical analysis. Data were analyzed using GraphPad Prism v5.01 (GraphPad Software, Inc.) and all data are presented as the mean ± SEM of at least three independent experiments. Differences between two groups were analyzed using a unpaired ‘t’-test, and differences among multiple groups were determined by one-way ANOVA followed by the least significant difference test. P < 0.05 was considered to indicate a statistically significant difference.