2.1. Cell culture and transfected
Human umbilical vein endothelial cells (HUVECs, #GDC166, CCTCC) were acquired from China type collection center (CCTCC, Wuhan, China) and cultured in high glucose Dulbecco’s modified eagle’s medium (DMEM; supplement with 10% fetal bovine serum (FBS; GIBCO, USA). The siKeap1(siKeap1-si1, siKeap1-si2, siKeap1-si3), and the relevant negative control (siNC) were obtained from Hanheng Biotechnology company (Shanghai, China). The sequences were shown in table S2. According to the manufacturer’s instructions, HUVECs were transfected for 48 h or 72 h using riboFECT™CP Reagent.
2.2. RT-PCR
Total RNA was isolated using the Ultrapure RNA kit (CW0581M, CWBIO), according to the manufacturer’s instruction. Using prime script®RT kit (#RR037A, TaKaRa), total RNA was reversed transcription into cDNA. On the StepOnePlus ™ platform (Applied Biosystems, USA), Real-time Quantitative PCR (qPCR) was performed using TB Green® Premix Ex Taq™ II kit (#RR820A, TaKaRa). The primer sequences were shown in table S1. Using the 2−ΔΔCt method, the relative expression level of targeted genes was calculated and normalized to β-actin.
2.3. HUVECs proliferation, migration, and intracellular ROS assessment
HUVECs from different treated groups were grown in 96-well culture plates (3,000 cells per well) for 24 h. After 2h incubation with EdU, the proliferation rates of HUVECs from different treated groups were evaluated with Cell-Light EdU Apollo In Vitro Kit (Ribobio, Guangzhou, China). The scratch closure rate was measured by scratch test to evaluate the cell migration ability. When 95% confluence was reached, the HUVECs from different treated groups were scraped the cell monolayer to form a wound and taken by microscope at 0 h and 48 h. The transfected HUVECs were treated with MGO for 24 h, and intracellular ROS levels were determined using DCFH-DA according to the manufacturer’s instructions. All the above images were processed with Image J.
2.4. Western blot
Total protein was extracted with protease inhibitor and phosphatase inhibitor (Roche, Switzerland) RIPA lysis buffer. Equal amounts of total protein (20-50 μg) were separated by SDS-PAGE (Beyotime Biotechnology, Shanghai, China), and then incubated overnight with primary antibody specificity for Keap1(Proteintech, China), HO-1(Proteintech, China), NrF2(Proteintech, China), β-actin (Proteintech, China), CD9(Abcame, USA), TSG101(ABclonal, China), Calnexin (Abcame, USA). The membrane was incubated with secondary antibodies (Aspen, China) for 2h and exposed to X-ray film (UVP, USA). The blots were analyzed using Image J.
2.5. Extraction of milk-derived exosomes
Milk-derived exosomes (mEXOs) were extracted by differential centrifugation from commodity raw milk [11] using previously described protocols. Raw milk was centrifuged at 13,000×g for 30 min at 4 ° C to remove fat globules. Discard the fat layer and particles on the bottom of the tube and collect the supernatant. The supernatant was ultra-centrifuged at 100,000×g for 60 min to remove precipitated and large vesicles. The supernatant was ultra-centrifuged at 145,000×g for 90 min to extracted exosomes. The exosome pellets were washed 3 times with sterile PBS and stored at -80 ° C until used.
2.6. Characterization of mEXOs
The morphology and size distribution of exosomes in each sample was detected by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The total protein concentration was measured by a Bicinchoninic Acid kit (Beyotime Biotechnology, China).
2.7. Ultrasonic technology
The mEXOs were loaded with siRNA-Keap1 or siRNA-FAM by ultrasonic using the SONICS&MATERIALS INC (NEWTOWN, USA) ultrasonic system. The mEXOs and siKeap1 were mixed in a ratio of 1:1 (mass/mass) in PBS, and the final concentration of mEXOs in the mixture was 4 μg/ml. For the concrete sonication method, the following settings [20] were 20% amplitude, 6 on/off cycles of 30 seconds, with a cooling time of 2 min between each cycle. After sonication, the solution was incubated at 37 ° C for 60 min to restore the exosome membrane. Try to put the solution at 4° C before used.
2.8. Confocal microscope
The mEXOs were incubated with a red fluorescent dye (Dil, biotium, USA) for 10 min in sterile PBS, and then the excess dye was removed by ultra-ionization to obtain labeled-mEXOs. The FAM-labeled siRNA was obtained from Hanheng Biotechnology company (Shanghai, China), and encapsulated into Dil-mEXOs through ultrasonic. The sequences were shown in table S2. The HUVECs were seeded in 24-wells culture plates for 24 h, and then co-cultured with Dil-mEXOs-FAM-siRNA for 24 h. After incubation, HUVECs were washed with PBS twice and stained with DAPI (Solarbio, Beijing, China). The uptake of Dil-mEXOs@FAM-siRNA by HUVECs were observed respectively by using confocal microscope.
2.9. The function of mEXOs-siKeap1 complex in vitro
The diabetic cell model was constructed with methylglyoxal (MGO). Experiment groups include the control group, MGO group, MGO+siNC group, MGO+siKeap1 group, MGO+mEXOs group, MGO+mEXOs-siKeap1 group. The procedure of cell proliferation, migration, and the assessment of ROS experiments were described above. When HUVECs grew to 30%-50%, the mEXOs-siKeap1 (4 μg per well) complex was added. After incubation for 24 h, the acquisition and observation of images were the same as above.
2.10. Animals experiment
All animal experiments were approved by the animal protection committee of Tongji Medical College. Six-week-old male C57 BL/6 mice were injected intraperitoneally with streptozotocin (STZ), 50 mg/kg continuous injection for 5 days. The blood glucose was measured by a blood glucose monitor after 4 weeks. When the blood glucose was above 16.7 mM, indicated that the diabetes mice model was induced successfully. 40 mice (including 8 normal mice and 32 diabetes mice) were anesthetized with sodium pentobarbital (Sigma-Aldrich) (1%, 50 mg/kg). After completion of back depilation and sterilization, all mice were subjected to full-thickness excision of wounds with a diameter of 10mm. 32 diabetes mice were randomly divided into 4 groups: PBS (STZ control) group, mEXOs (2 μg/wound), siRNA (2 μg/wound), mEXOs-siKeap1 (2 μg /wound). 8 normal mice constituted a PBS (normal control) group. After the injury, the wounds were treated at 0, 4, 9, and 14 post-wounding. Photos were taken at day 0, 4, 9, 14 and 19 post-wounding. The wound area was measured using Image J.
2.11. Histological analysis
After an injury on the 19 days, the whole wound bed of mice in each group was taken for histological analysis. The wound tissue was fixed with 4% paraformaldehyde. After dehydration, the tissue was embedded in paraffin and cut into an 8 μM thick longitudinal section and stained. The re-epithelialization rate of wound tissue was analyzed by hematoxylin-eosin (H&E) staining, and the collagen accumulation was evaluated by Masson staining.
2.12. Immunohistochemistry and immunofluorescence analysis
To evaluate the vascular regeneration of wound beds, the skin samples on the 19 days from the mice were fixed in 4% paraformaldehyde and proceeded to obtain tissue sections. Then the sections were incubated overnight at 4° C with primary antibodies against CD34 (Abcam, USA) and α-SAM (Proteintech, Wuhan, China). On the next day, the sections were incubated with a secondary antibody (Aspen, China) for 2 h at room temperature. To detect the expression of the Keap1 /HO-1 (Proteintech, Wuhan, China) immunofluorescent proteins in diabetes wound beds, we performed corresponding immunofluorescence staining. Images were obtained using a fluorescence microscope and four different fields were randomly assessed. Finally, ImageJ software was used for analysis. Skin tissues cut from mice with diabetes and normal were stored at room temperature -80° C for Western Blot analysis
2.13. Statistics
The group exceeded two groups, therefore a one-way analysis of variance (ANOVA) was performed by Tukey post- hoc test. All statistical analyses were performed using GraphPad Prism software (version 8.0.2, La Jolla, CA, USA). Hence, the dates are expressed as the mean ± standard deviation (SD). Statistical significance was set as P <0.05 (*p < 0.05, **p < 0. 01, ***p < 0. 001,****p < 0.0001).