Twenty-four adult male Sprague-Dawley rats (weighing 180–200 g) were obtained from the Center for Animal Experiments of Fujian Medical University (license number SCXK min 2012-0002). They were housed at 20–25 °C under a standard 12/12 light-dark cycle with 60% relative humidity. The rats had ad libitum access to food and autoclaved water. All animal procedures were approved by the Animal Ethics Committee of Fuzhou General Hospital (Fuzhou, China).
Cell isolation and culture
The isolation of ADSCs was performed following our previous reports [12, 14, 15]. Briefly, the subcutaneous adipose tissues in groin were collected from male Sprague-Dawley rats (n = 6), cut into small pieces, and digested with 0.1% type I collagenase (Sigma-Aldrich, St Louis, MO, USA) in α-MEM (Hyclone, Logan, UT, USA) at 37 °C for 60 min. After, collagenase activity was neutralized by α-MEM containing 20% FBS (Gibco, Mulgrave, VIC, Australia), and filtered through a 100-μm cell strainer, and subsequently the cells were resuspended with osmotic lysates (Biyuntian Biological Co., Ltd., Shanghai, China) and incubated at room temperature for 10 min to eliminate the red blood cells. The remaining cells were seeded into T-75 flasks at a density of 1 × 106/mL, and cultured in α-MEM containing 10% FBS, 100 U/mL penicillin (Life Technologies, Grand Island, NY, USA) and 100 μg/mL streptomycin (Life Technologies, Grand Island, NY USA). Cells from the passage 3 were used in the present study. Human hepatic stellate cell line (LX2) was purchased from Bogu Biotech Co., Ltd., (Shanghai, China) and cultured in the complete medium containing RPMI 1640 (Gibco, Mulgrave, VIC, Australia) and 10% FBS (Gibco, Mulgrave, VIC, Australia).
Co-culture of ADSCs and HSCs
A co-culture system was constructed using a Transwell chamber (Merck Millipore, Billerica, MA, USA), which could be inserted into the wells of six-well plates. LX2 cells were seeded on six-well culture plates at a density of 5 × 105 cells/well, whereas ADSCs were seeded on the membrane (polyethylene terephthalate, pore size, 0.4 μm) at a density of 5 × 105 cells/well in the Transwell chamber. Cells were treated with or without TGF-β receptor 1 inhibitor (LY2157299, 56 nM; Selleck Chemicals Houston, TX, USA), and incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 24 h. After that, LX2 cells were collected for further q-PCR and Western blot analysis.
Animal model and ADSC transplantation
The T2D model was induced by high-fat diet (HFD) and streptozotocin (STZ) injections as previous descripted with minor modifications [
]. As shown schematically in Fig.
, 18 Sprague-Dawley rats were fed either normal chow (control group) or a high-fat diet (HFD) containing 66.5% normal chow, 10% lard, 20% sucrose, 2% cholesterol and 1.5% cholate for 4 weeks, followed by intraperitoneal injection of 25 mg/kg dose of STZ (dissolved in the 0.05 M citrate buffer at pH 4.5, prepared immediately before use) twice per week for 2 weeks. After the development of T2D, which was diagnosed as a non-fasting blood glucose ≥11.1 mmol/L by measuring glucose levels in the blood obtained from the tail vein. Diabetic rats with hyperglycemia (n = 12) were randomly divided into two groups: the model group (n = 6), rats were intraperitoneally injected with 20% carbon tetrachloride (CCl
) solution in olive oil (3 mL/kg body weight; twice per week for 6 weeks) and 15 mg/kg dose of STZ (once per week for 6 weeks), and treated with tail vein injection of PBS (1 mL/rat) on the weeks 7 and 9; and the ADSC therapy group (n = 6) received intraperitoneal injection of 20% CCl
solution in olive oil (3 mL/kg body weight; twice per week) and 25 mg/kg dose of STZ (once per week), and treated with tail vein injection of ADSCs (2.0 × 10
/L mL/rat) on the weeks 7 and 9. After the treatment, all rats were sacrificed with 2% pentobarbital sodium (100 mg/kg; Sigma-Aldrich). The liver tissues (approximately 500 mg/rat) and sera (approximately 3 mL/rat) were collected for further investigation.
Schematic diagram of the experimental protocol. ADSCs adipose tissue-derived stem cells, HFD high-fat diet, ND normal diet, NS normal saline,,i.p. intraperitoneal injection, i.v. tail vein injection, STZ streptozotocin
Measurement of blood glucose, insulin and HOMA-IR
After a 12-hour overnight fast, blood from the tail vein was collected and measured by Accuchek Active Meter (ACCU-CHEK® Active; Roche, Berlin, Germany). The insulin levels were analyzed using a rat insulin ELISA kit (BlueGene Biotech Co., Ltd., Shanghai, China) according to the manufacturer’s instruction. Insulin resistance was assessed by the homeostasis model assessment-insulin resistance (HOMA-IR) using the following formula: HOMA-IR = fasting blood glucose (mmol/L) × insulin (mIU/L)/22.5 .
Biochemical assays of liver function
To determine whether ADSC transplantation improved liver function of diabetic rats, the sera were separated after centrifugation at 1000 × g for 10 min at 4 °C, and stored at -80 °C. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as total bilirubin (TBIL) and albumin (ALB) were respectively measured using ALT, AST, TBIL, and ALB assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacture’s protocol. The serum liver fibrosis indices, including collagen type IV and hyaluronic acid, were measured using rat collagen type IV and hyaluronic acid ELISA kits respectively, according to the manufacturer’s instructions (BlueGene Biotech Co., Ltd., Shanghai, China). The serum levels of pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and C-reactive protein (CRP) were evaluated using commercial ELISA kits (Boster Biological Technology Co., Ltd., Wuhan, China).
Hydroxyproline, triglyceride, and total cholesterol content measurement
To further investigate whether ADSC transplantation could improve liver function, the hydroxyproline, triglyceride, and total cholesterol content in liver tissues were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), in accordance with the protocol of the manufacturer’s manual.
The obtained liver tissues were fixed in 4% paraformaldehyde for 24 h, then gradually dehydrated with ethanol and embedded in paraffin, and finally tissue sections of 5 μm thickness were stained with hematoxylin and eosin (H&E) for histological analysis. To observe the hepatic fibrosis, Masson’s trichrome staining was performed using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s protocol. To further clarify the fatty infiltration in the liver, frozen liver tissues were sectioned at 10 μm and stained with an Oil Red O staining kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s protocol. Double-blind evaluation of hepatic histopathological changes was performed by two expert pathologists. The histopathological examination was performed using an inverted phase-contrast microscope (Carl Zeiss, Oberkochen, Germany), and the fibrosis surface area was analyzed using the ZEN 2012 Light Edition imaging analysis system (Carl Zeiss).
Quantitative real-time PCR analysis
The total RNA was isolated from liver tissues or LX2 cells using TRIzol reagent (TransGen Biotech Co., Ltd., Beijing, China). Afterwards, the mRNA was reversely transcribed to cDNA by using a transcriptor first-strand cDNA synthesis kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instructions, and q-PCR analysis was performed using the ABI StepOnePlus real-time PCR system (Applied Biosystems Inc., Foster City, CA, USA) with q-PCR Master Mix (DBI Bioscience, Ludwigshafen, Germany). Cycling conditions were as follows: 40 cycles of 95 °C for 15 sec, 60 °C for 30 sec, and 70 °C for 30 sec. Primer sequences are listed in Additional file 1: Table S1. The expression of target gene was normalized to that of β-actin gene. Relative gene expression was calculated with the 2–△△Ct formula.
Western blot analysis
Samples were lysed in ice-cold RIPA buffer (0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA) with protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Protein quantification was performed by BCA assay, and equal amounts of protein lysate (40 μg) were separated by 10% SDS-PAGE. Transfer to nitrocellulose membranes was performed in transfer buffer (12 mM Tris base, 96 mM glycine, pH 8.3, and 15% methanol). Afterwards, the membranes were blocked for 2 hours in the TBST buffer with 5% BSA and probed with the TGF-β1, p-SMAD3, SMAD3 (all from Cell Signaling Technologies, Danvers, MA, USA; 1:500 dilution) and β-actin antibody (Transgen, Beijing, China; 1:5000 dilution) overnight at 4 °C. The membranes were washed with TBST buffer for three times, followed by incubating with appropriate HRP-conjugated secondary antibody (1:5000 dilution; TransGen Biotech Co., Ltd., Beijing, China) for 1 hour at room temperature. Finally, the protein expression levels were detected by enhanced chemiluminescence and visualized by autoradiography.
All quantitative data were expressed as the mean ± standard deviation (SD). All the statistical analyses were performed with GraphPad Prism version 6.0. (GraphPad Software, San Diego, CA, USA), Statistical analysis among different groups was performed using Student t test. The p < 0.05 was considered as statistically significant.