Animals

Our experimental rats were 6-week-old male Sprague–Dawley (SD) rats weighing 180–220 g (Kyudo Co., Ltd., Saga, Japan). The rats were allowed to acclimatize themselves to the laboratory environment for 7 days before the experiments were started. During this period, the rats were housed individually in metabolic cages and all had ad libitum access to standard rat chow and water. The laboratory environment was maintained at a constant temperature of 23 ± 1 °C and humidity of 50 ± 10% with a 12-h light–dark cycle (lights on at 7:00 AM).

Following a period of environmental acclimation, the experimental rats underwent surgical procedures and then were subsequently maintained on TPN for 7 days. The rats were euthanized on the 7th postoperative day.

Study design

The rats were randomly allocated into one of the three groups as follows: massive small bowel resection and TPN (SBS/TPN: Control group, n = 8); SBS/TPN with the intravenous administration of low-dose clodronate (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) (20 mg/kg/day; SBS/TPN/low-dose Clodronate: Low group, n = 8); SBS/TPN with the intravenous administration of high-dose clodronate (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) (60 mg/kg/day; SBS/TPN/high-dose Clodronate: High group, n = 8). The dosing regimen for clodronate was determined based on previous studies [5, 6]. Clodronate was administered as a single intravenous infusion via a central venous catheter on the day of surgery and then again on the 3rd postoperative day.

Surgical procedures and maintenance methods

All rats were anesthetized with isoflurane (1.5% inhalation by mask) and underwent jugular vein catheterization using the cut-down method. The catheter was tunneled subcutaneously into the back and then connected to a swivel device. Subsequently, all rats underwent 90% small bowel resection, preserving 5 cm of the jejunum from the ligament of Treitz and 5 cm of ileum from the ileocecal valve. The proximal jejunum was anastomosed to the distal ileum end-to-end. Buprenorphine (0.01 mg/kg per dose subcutaneously) was administered for analgesia. The rats had ad libitum access to water after the surgery. TPN was delivered via a multichannel syringe pump (KDS Legato 200 Series Syringe Pump Series; KD Scientific, Inc., Holliston, MA, USA) using a regimen determined in our previous study. Cardiac puncture and exsanguination under general anesthesia were performed for euthanasia.

Histological analysis

For the histological analysis of liver tissue, we performed a histological analysis of the liver specimens based on the NAFLD activity score, which was the degree of lipid accumulation (steatosis score) and the number of positive macrophages or T lymphocytes in ten randomly selected fields (lobular inflammation score).

RNA extraction, reverse transcription and real-time polymerase chain reaction (PCR)

The PCR amplification was performed as described in our previous study [7]. The intestines were frozen and processed using TRIzol reagent (Thermo Fisher Scientific Inc.) to extract mRNA. The extracted RNA was then purified using a spin cartridge and quantified using a spectrophotometer. Complementary DNA (cDNA) was synthesized from purified RNA. Real-time PCR was performed to analyze the expression of the target genes using the QuantStudio 3 system (Thermo Fisher Scientific Inc.). The relative expression of the target genes was calculated using one of the control groups as a reference. The primers used in this study were purchased from Takara Bio Inc. All the primers used are listed in Supplementary Table S1.

Serological analysis

The collected blood was immediately centrifuged at 1500 g for 15 min at 4 °C. All serum samples were stored at−  80 °C until use. The blood triglyceride and total cholesterol levels were measured using a point-of-care testing device (Spotchem EZ SP-4430; ARKRAY, Inc., Kyoto, Japan). The Spotchem II reagent was purchased from ARKRAY Inc.

Statistical analyses

Statistical analyses were conducted using two-factor factorial ANOVA followed by Tukey’s post-hoc test. Data are presented as the mean ± standard error. Statistical significance was set at a p-value of < 0.05. All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of R commander, which is designed to add statistical functions that are frequently used in biostatistics [8].

Ethical approval

This experiment was conducted in accordance with the ARRIVE guidelines and is described in the text according to the checklist. All experimental procedures were approved by the Laboratory Animal Committees of Kagoshima University Graduate School of Medical and Dental Sciences and were performed in accordance with the “Guidelines for the Care and Use of Laboratory Animals” (approval number: MD23026).