Establishment and identification of mice with GDM
Five-week-old female C57BL/6J mice were purchased from Shanghai Bikaikeyi Biotechnology Co., LTD. (License No. SCXK2018-0006) and housed in an SPF animal laboratory. Prior to the experiment, tail venous blood samples were collected from the mice to determine fasting blood glucose levels, with a normal range of 4–7 mmol/L. Sixteen mice were randomly divided into two groups: one group received a high-fat diet containing 60% fat (XTHF60, Xietong pharmaceutical bio, Nanjing, China; 8 mice), while the other group received a normal diet (8 mice). After 8 weeks, female mice were placed in the same cage with male C57BL/6J mice at a ratio of 2:1. Once pregnancy was confirmed, the mice continued to be fed their respective diets for 12 days. Blood glucose levels were recorded every 3 days during this period, and GDM was considered successfully induced if random blood glucose levels exceeded 11.1 mmol/L. On day 12, plasma was collected, and the concentrations of insulin, leptin, adiponectin, and hypersensitive C-reactive protein (hs-CRP) were determined using an enzyme-linked immunosorbent assay (ELISA) kit (Mlbio, Shanghai, China) following the manufacturer's instructions. All animal experiments were approved by the Animal Ethics Committee of Shanghai University of Medicine and Health Sciences.
ADSCs isolation and cell culture
Primary adipose stem cells (ADSCs) were isolated from GDM mice and normal gestational mice, respectively. Under aseptic conditions, the abdominal cavity of the mice was opened, and AT from the abdominal and inguinal regions was obtained. The AT was then rinsed, minced, and collected in a pre-cooled Hank’s Balanced Salt Solution (Sangon BIOTECH, Shanghai, China). Next, 2 mg/ml collagenase I (Yeason, Shanghai, China) and 3 mM CaCl2 were added in double volume to the tissue, which was then digested at 37℃ for 4 hours. Digestion was stopped by adding an equal volume of DMEM/F12 medium (Gibco, GIBCO, NY, USA) containing 10% FBS (Merck KGaA, Darmstadt, Germany), followed by centrifugation at 1200 g for 10 minutes. The cell precipitates were resuspended, washed with PBS, and cultured in DMEM/F12 medium containing 10% FBS. Mouse normal liver cells, AML12, were purchased from Shanghai Fuheng Biotechnology and cultured in DMEM/F12 medium containing 10% FBS, 1% ITS media supplement (R&D Systems, MN, USA), and 40 ng/ml dexamethasone (Merck KGaA).
Plasmid and reagent
The encoding region of mouse Thbs1 mRNA (NCBI number: NM011580) was cloned onto the pcDNA3.1-3×Flag-eGFP-C2 vector. The control vector and Flag-eGFP-Thbs1 fusion plasmid were transfected into AML12 cells using liposomal transfection reagent (Yeason). Thbs1 pharmacological inhibitory peptide LSKL and transforming growth factor (Tgf) β inhibitor ITD-1 were purchased from Selleck Chemicals (Shanghai, China).
Extraction, purification and identification of sEV
When the confluency of ADSCs reached 80–90%, the medium was changed to DMEM/F12 medium containing 10% FBS without sEVs and continued to culture for 72 hours. The supernatant was then centrifuged at a low speed, and large vesicles were removed by filtration using a 0.22 µm pore size filter. The culture medium was subjected to ultracentrifugation at 120,000 ×g for 90 minutes (OptimaTM XPN-100, Beckman Coulter, USA). The resulting sEV precipitates were resuspended in pre-cooled PBS, followed by another round of centrifugation. Subsequently, the sEV precipitates were resuspended in an appropriate volume of phosphate buffered saline (PBS) and characterized using nanoparticle tracking analysis (NTA) with ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany). The sEV solution was placed on copper grids (Zhongjingkeyi Technology, Beijing, China), stained with 50 µL of uranium acetate, and visualized using transmission electron microscopy (FEI Tecnai G2 Spirit TEM, USA) for sEV visualization.
Western blotting
AML12 cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (KeygenBio, Nanjing, China) containing a protease inhibitor cocktail (P8340, Merck KGaA). Total proteins in AML12 cell lysates or sEV solutions were quantified using a bicinchoninic acid (BCA) protein assay kit (Yeason). Ten micrograms of protein were resolved on SDS polyacrylamide gel before being transferred to a PVDF membrane (Millipore, USA). The membrane was then blocked with 5% bovine serum albumin (BSA, Sangon BIOTECH) at room temperature for 1 hour, incubated with primary antibodies overnight at 4℃, and subsequently incubated with secondary antibody for 1 hour. Images were captured using a Bioanalytical imaging system (Tanon 5200 Multi system, Shanghai, China). The primary antibodies used were as follows: anti-Cd63 (25682-1-AP, Proteintech, Wuhan, China), anti-Tsg101 (72312, Cell Signaling Technology, MA, USA), anti-Thbs1 (37879, Cell Signaling Technology), anti-Cd44 (15675-1-AP, Proteintech), anti-Phospho-Jnk (AP0631, ABclonal, Wuhan, China), anti-Jnk (A4867, ABclonal), anti-Atf4 (A0201, ABclonal), anti-Atf6 (A0202, ABclonal), anti-Ire1 (A17940, ABclonal), anti-Grp78 (A4908, ABclonal), anti-Chop (A0221, ABclonal), anti-Smad2 (A7699, ABclonal), anti-Phospho-Smad2 (AP0269, ABclonal), anti-Tgfβ2 (19999-1-AP, Proteintech), and anti-CD36 (A17339, ABclonal).
Immunofluorescence
A total of 2×104 ADSC or ADSC/GDM cells were cultured in 24-well plates with preset glass plates. After cell adhesion, they were fixed for 30 minutes in 4% paraformaldehyde. Subsequently, the cells were sealed in a PBS solution containing 0.5% Triton X-100 and 10% fetal bovine serum (FBS)for 2 hours. The glass slides were then incubated with primary Cd44 antibody and Alexa Fluor 594-labeled secondary antibody (SA00006-4, Proteintech). Following the cleaning process with PBS, the slides were stained with 4',6-diamidino-2-phenylindole (DAPI)solution for 5 minutes. Finally, the slides were embedded in fluoromount-G (SouthernBiotech, USA) and photographed using a fluorescence microscope (DS-Ri2, Nikon, Japan).
Cell viability assay
A total of 104 normal mouse liver cells (AML12) were seeded in 96-well plates. Once adhered, sEVs with final concentrations of 0, 108, 5×108, and 109 particle numbers/mL were added to serum-free DMEM/F12 medium and treated for 48 or 72 hours. Each well was supplemented with 100 µL of culture medium and 10 µL of CCK8 reagent (Laisi biotech, Shanghai, China). The cell viability was measured by microplate reader, determining the OD450nm value.
Cell apoptosis assay
AML12 cells at a density of 8×105 were placed in a 6-well plate and co-cultured with sEVs at a concentration of 108 particle numbers/mL for 48 or 72 hours. Cell apoptosis was detected using propidium iodide (PI) staining and the Annexin V apoptosis detection kit (BD Biosciences, NZ, USA) via flow cytometry (Novocyte, Agilent Technologies, CA, USA). The proportion of apoptotic cells was analyzed using NovoExpress software (version 1.5.0, Agilent Technologies).
Insulin sensitivity assay
A total of 6×105 AML12 cells were seeded in a 12-well plate. After cell adhesion, the AML12 cells were incubated in serum-free medium with sEVs at a concentration of 108 particles/mL for 48 or 72 hours. In the control wells, the same volume of serum-free medium was added. Following co-culture, the AML12 cells were treated with or without 1 µg/mL insulin for 1 hour. Glucose content was determined by collecting the culture medium and using the glucose detection kit (GAGO20, Merck KGaA). Glucose uptake was measured by subtracting the glucose level in the cultured well from that in the cell-free well, which reflected the insulin sensitivity.
Real-time polymerase chain reaction (PCR)
Total RNA was isolated from AML12 cells co-cultured with sEVs using the RNeasy Mini kit (Qiagen, Hilden, Germany). Synthesis of complementary DNA (cDNA) was performed using HiScript II Q RT SuperMix (Vazyme, Nanjing, China). Real-time PCR was conducted on QuantStudio 7 (Thermo Fisher Scientific) using SYBR Premix Ex Taq (Takara, Otsu, Japan). Gene expression was quantified using delta Ct. The primer sequences for all mRNA are provided in “Supplementary Table 1”.
Proteomics of sEV
Differences in expression of the sEV proteome secreted by normal ADSCs and GDM mice-derived ADSCs were identified using label-free protein quantification. The proteins in the sEVs were extracted and analyzed by nano-high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS, Thermo Q Exactive). The data acquisition mode was data-dependent acquisition (DDA). The series of mass spectra were analyzed using PEAK Studio version X (Bioinformatics Solutions Inc., Waterloo, Canada), and protein databases were searched using PEAK DB. These detection procedures were performed by Guangzhou Gene denovo Biotechnology Co., Ltd. (Guangzhou, China).
Co-immunoprecipitation
AML12 cells treated with sEVs or inhibitors were lysed with RIPA buffer. AML12 cell extracts were immunoprecipitated with anti-Tgfβ2 or anti-CD36 antibodies for 24 hours, then co-incubated with protein A/G magnetic beads (Bimake, Shanghai, China) for 3 hours. The magnetic beads carrying interacting proteins were heated at 100℃ for 5 minutes, and the expression levels of interacting proteins were analyzed by western blotting.
Histological analysis
Liver tissue, abdominal AT, and inguinal AT from normal mice, GDM mice, and GDM mice treated with LSKL were fixed with 4% paraformaldehyde and embedded in paraffin. AT and liver tissue morphology were observed through hematoxylin-eosin (HE) staining. For immunohistochemistry (IHC), tissue sections were sequentially dewaxed, rehydrated, and treated with 3% H2O2. Sections were blocked in 5% BSA for 30 minutes, and then incubated overnight with anti-Tsg101, Thbs1, p-Jnk, Atf6, Ire1, and p-Smad2 antibodies at 4℃. After incubation with an enzyme-labeled secondary antibody at room temperature, the positive signal was observed using diaminobenzene (DAB) chromogenic agent and imaged under an optical microscope.
Statistical analysis
Data analysis was performed using GraphPad Prism 7.0 (GraphPad Software, USA) and SPSS 20.0 software (SPSS Inc., USA). Results are expressed as mean ± standard deviation (SD). Student’s t-test was used to analyze statistical differences between two groups of samples. One-way analysis of variance (ANOVA) and Bonferroni post hoc tests were used to analyze statistically significant differences among three or more groups. A P-value of < 0.05 was considered statistically significant.