All experimental procedures were approved by the Laboratory Animal Committees of our institution and were performed according to the Guidelines for the Care and Use of Laboratory Animals (Approval number: MD17024).
We used Sprague–Dawley rats (weighing 200–250 g). The rats were individually housed in metabolic cages with free access to standard rat chow and water and acclimatized to their environment for seven days before the experiments. The laboratory environment was maintained at a standardized temperature (23 ± 1 °C) and humidity (50 ± 10%) and a 12-h light–dark cycle.
The rats were fasted overnight before the experiment. They underwent surgical placement of a central venous catheter and 90% small bowel resection and were then randomly divided into the following groups: (1) Oral feeding with normal chow plus continuous infusion of saline, control (C) group; (2) TPN with SOLE (SOLE) group; (3) TPN with CLE (CLE) group; and (4) TPN with FOLE (FOLE) group. We used Intralipos® (Otsuka Pharmaceutical Co., Ltd. Japan) as the SOLE, SMOFlipid® (Fresenius Kabi Australia Pty Ltd.) as the CLE and Omegaven® (Fresenius Kabi Deutschland GmbH) as the FOLE.
On the 13th day after surgery, the rats were anaesthetized and euthanized by collecting a blood sample via pericardial puncture, and laparotomy was performed for liver sample harvesting. The blood samples were immediately centrifuged at 1500g for 15 min at 4 ℃, plasma and serum were extracted, and liver samples were flash-frozen in liquid nitrogen; these samples were stored in −80 °C. Additional liver samples were fixed with 10% formaldehyde for histological analyses. Paraffin sections of formalin-fixed tissue were cut at a thickness of 3 µm for staining with hematoxylin and eosin (H&E) and oil red O.
Surgical procedure and maintenance methods
The rats were anesthetized with isoflurane (1.5% inhalation by mask), and cefazolin (50 mg/kg, subcutaneously; Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) and buprenorphine (0.01 mg/kg, subcutaneously; Otsuka Pharmaceutical Co., Ltd.) were administered. The central venous catheter, a silastic catheter with an outside diameter of 1.2 mm (NIPRO Co., Ltd., Osaka, Japan), was inserted into the jugular vein.
Regarding the surgical procedure, in brief, the jejunum 5 cm distal from the ligament of Treitz and the ileum 5 cm proximal to the ileocecal valve were dissected, and end-to-end anastomosis was performed. After the operation, rats were maintained at 60 ml/day on a low-concentration TPN solution (NEOPAREN® No. 2; Otsuka Pharmaceutical Co., Ltd.) supplemented with a lipid emulsion. The composition of the low-concentration TPN solution was as follows (%): amino acids 2.5, glucose 14.5, and lipids 3.33. After 24 h, the composition of the high-concentration TPN solution was switched to (%): amino acids 3.16, glucose 20.3 and lipids 3.33. The TPN solution provided equivalent nutrients to all TPN-fed animals at 76.4 kcal/rat/day (1.9 g protein, 2.0 g fat and 12.2 g carbohydrate). The rats in the C group were fed 23 g of regular rat chow per day (79 kcal/rat/day), which was almost equal to the calories in the TPN solution.
The biochemical examination of the liver function
In serum biochemical tests, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (T-Bil), direct bilirubin (D-Bil), total cholesterol (T-CHO) and triglyceride (TG) were measured using the Japan Society of Clinical Chemistry standardized matching method. These measurements were performed at the Kagoshima Lab of SRL, Inc., Japan, using the 7180 clinical Analyzer (Hitachi High-tech GLOBAL, Japan).
Analyses of phytosterols in serum
The phytosterols were measured following Matthan’s paper . Analyses of β-sitosterol and campesterol, samples were saponified to extract sterol components and analysed by gas chromatography. The findings were then compared with the analytical results of the standard product, and the target sterol concentrations were calculated from the peak elution position and area. These analyses were carried out at Skylight Biotech Inc., Akita, Japan.
Histological analyses and liver injury grading
The severity was assessed as the grade according to the NAFLD scoring system reported by Nabeshima et al.  as follows: steatosis grading [no lipid droplets (normal N); lipid droplets in < 33% of the hepatocytes (low L); lipid droplets in 33–66% of the hepatocytes (moderate M); and lipid droplets in > 66% of the hepatocytes (severe S)], inflammation grading [no inflammation (normal N); < 10 inflammatory foci, each consisting of > 5 inflammatory cells (low L); > 10 inflammatory foci (moderate M); or uncountable diffuse or fused inflammatory foci (severe S)]; and ballooning grading [none (normal N); few balloon cells (low L) or many balloon cells/prominent ballooning (moderate M)]. The histological evaluation of the liver was performed by two blinded pathologists.
Analyses of the lipid content of liver tissue and fatty acid composition in serum
According to the FOLCH methods , each snap-frozen tissue specimen was homogenised and extracted using chloroform–methanol, and T-CHO, TG and free cholesterol (FC) were measured at Skylight Biotech Inc. The fatty acid composition of the serum samples was assessed. All measurements were performed at Nagahama Life Science Laboratory (ORIENTAL YEAST CO., LTD., Shiga, Japan).
Quantitative real-time polymerase chain reaction (qPCR) of cytokines in the liver tissue
We evaluated the interleukin-6 (IL-6) and tumor necrosis factor-α (TNF- α) mRNA expressions in liver tissue. The procedure was used superscript 77IV reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) with oligo (dT) primer, RPr IL-6 F1: 5′-GTATGAACAGCGATGATGATGCACTGTC-3′ (forward), RPr IL-6 R1: 5′-TAGAAACGGAACTCCAGAAGACCAG-3′ (reverse), RPr TNF-α F1: 5′-ACGCTCTTCTGTCTACGAACTTCG-3′ (forward) and RPr TNF-α R1: 5′-AAGATGATCTGAGTGAGGGTCTG-3′ (reverse), and the expression of each gene was analyzed using the Bio-Rad CFX96 system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). These measurements were performed by Repertoire Genesis Inc., Osaka, Japan.
Analyses of the expression of Rubicon antibody
We assessed the expression of Rubicon antibody, a protein that suppresses autophagy, using Western blotting. GTX 31,593 Rubicon antibody from GeneTex, Inc., was used as the primary antibody. Western blotting was performed by IDEA Consultants, Inc., Tokyo, Japan.
All statistical analyses were performed with Easy R (Saitama Medical Center, Jichi Medical University, Tochigi, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, version 3.3.2). The statistical analyses of the data were performed using a one-way analysis of variance (ANOVA) followed by the Tukey–Kramer and Kruskal–Wallis test and the Steel–Dwass multiple comparisons post hoc test. P values of < 0.05 were considered to indicate statistical significance.