Animals and groups
A total of 18 Male C57BL/6 mice, weighing 180–250 g were randomly divided into three groups: I) Control group (n = 6), treatment with saline; II) cecal ligation puncture group (n = 6, CLP), underwent CLP surgery; III) Exosome group (n = 6, Exos), after CLP surgery, the mice were treated with exosome.
Cecal ligation puncture (CLP)
Sepsis animal model was established by CLP surgery. Before surgery, the C57BL/6 mice were anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg). After disinfection, a midline incision was made in the abdomen. The cecum was then sectioned externally, ligated distally, and punctured once with a size 21 needle. Finally, the mice were resuscitated with saline.
Cell culture and treatments
BMMSCs were isolated from the red blood cells. In short, the red blood cells were first processed in the order of lysis, washing, re-suspension and culture. Then, the extracted cells are then purified and identified. Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from American Type Culture Collection and mouse aortic endothelial primary cells were obtained from CLP model mice. All cells are cultured in an environment with room temperature and saturated humidity.
Isolation of exosomes
Exosomes were extracted from the supernatant of BMMSCs by ultracentrifugation. Briefly, the supernatant was filtered with SteritopTM 0.22 m sterile membrane, and then centrifuged at 100,000×g for 1.5 h. Subsequently, the precipitate was then re-suspended at PBS and centrifuged until the final volume was reduced to about 300 µL. Finally, the purified exosomes were stored − 80°C for later use.
Observation of exosomes by Transmission Electron Microscopy (TEM)
First, the purified exosomes were stained with 2% sodium phosphowolframate for 15 min, and then washed with PBS. Dried for 0.5 h at room temperature, exosomes were stained with 1% uranyl acid and observed using a TEM (Hitachi H7500 TEM, Tokyo).
Nanoparticle tracking analysis (NTA)
The size distribution and concentration of the isolated exosomes were detected by NanoSight LM10. First, exosome was dilute to 10000 times with PBS and mix upside down. Then, the diluted exosome was tested, photographed and recorded under professional guidance.
The target cell protein was obtained by RIPA method, and the protein concentration was determined by BCA kit. Then, 50 ug prepared protein was taken for gel electrophoresis separation, and the isolated protein was electrically transferred to PVDF membrane (Roche, Switzerland). After blocked with 5% nonfat dry milk, the PVDF membrane was subjected to incubation with primary antibodies: CD63 (1:1000, ab134045, Abcam), CD9 (1:1000, ab92726, Abcam), HSP70 (1:1000, ab2787, Abcam) and VEGF (1:1000, ab32152, Abcam) at 4°C overnight. On the following day, the membrane was incubated with the secondary antibody at 37°C for 45 min. After washing the membrane film with TBST, luminescent solution was added and exposed in the gel imaging system. The protein content was analyzed using Quantity-One software.
StarBase (http://starbase.sysu.edu.cn/) was applied to speculate the target regulated by KCNQ10T1. miRwalk (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/index.html), miRDB (http://www.mirdb.org/) and ENCORI (http://starbase.sysu.edu.cn/panCancer.php) were applied to predict the target genes regulated by KCNQ10T1 and miR-154-3p.
Follow the instructions of the PKH26 Red Fluorescent Cell Linker Mini Kit, the purified exosomes were labeled under dark conditions. After 24 h of culture, PKH26 were co-cultured with HUVECs. Then, the labeled exosomes were detected by immunofluorescence assay.
Cell Counting Kit-8
In brief, the cells adjusted to the appropriate concentration were inoculated on 96-well plates and treated accordingly. Next, each well was added with CCK-8 solution and incubated for 2 h in the dark. Finally, the absorption value at 450nm was measured by a microplate reader (Rayto, RT6000).
5-ethynyl-2-deoxyuridine (EdU) assay
Briefly, after different treatments, HUVECs were inoculated in 96-well plates for 48 h. The EDU solution was diluted with cell complete medium at the ratio of 1000:1 to prepare an appropriate amount of 50 µM EDU culture medium. Each well was added 300µL EDU medium and then incubate for 2 h. After cell fixation, Apollo staining, DNA staining, the proliferation activity of cells was observed and detected under fluorescence microscope.
Wound healing assay
First, HUVECs were inoculated in a 6-well plate for 24 h. When the cells were fully fused, the pipette tip was applied to create a scratch wound on the confluent cells in the center. The migration and cell movement of the entire wound area were observed with an inverted optical microscope (Oberkochen, Germany), and the images were taken at 48 h with a camera connected to the microscope (SonyCyber shot, Shanghai Suoguang Visual Products Co., Ltd., China). The cell migration ability was statistically analyzed according to the cell healing.
HUVECs were firstly adjusted to the appropriate concentration. Subsequently, the upper chamber was added with adjusted cells, whereas the lower chamber was replaced by a medium supplement 15% FBS. After 24 h, 4% paraformaldehyde was used for fixation and 0.1% crystal violet for staining, respectively. Five fields were randomly selected under an inverted microscope to represent the invasion ability of cells in each group.
Measurement of interleukin (IL)-6 AND IL-8 levels by ELISA
The concentrations of IL-6 and IL-8 in the HUVECs and CLP model mice were assessed using a commercially available ELISA kit (USCN Business Co., Ltd, China).
Survival rate analysis
After mice undergoing CLP surgery, the number of mice in each group was observed and counted for survival analysis. The death time and number of these animals were observed and recorded by a researcher who was completely had no knowledge of the study
Hematoxylin-eosin (HE) staining
The aortic tissues were fixed with formaldehyde (10%) for 24 h, and then tissues were placed in a 5% nitric acid decalcification solution for 3–5 d. After washing with water, routine dehydration, transparency, paraffin immersion, embedding and sectioning, the tissue sections were stained with hematoxylin and eosin. Finally, the pathological changes of myocardium tissues were observed under microscope.
Quantitative real-time PCR
TRIpure (Invitrogen, USA) was applied to isolate the sample RNA and PrimeScript RT kit (TaKaRa, Otsu, Japan) was used for reverse transcription. After the RNA was prepared, the expression level was detected with FAST SYBRTM Green Master Mix, and GAPDH was set as internal parameter. 2-ΔΔCt methods represented the fold changes of gene expression and the experiments were conducted three times.
sh-NC, sh-KCNQ10T1, sh-RNF19A, NC mimic and miR-154-3p mimic and miR-154-3p inhibitor were constructed by Ribobio corporation (Guangzhou, China). When the confluence rate of HUVECs reached 70–80%, the transfection was conducted by using Lipofectamine 2000 (Invitrogen, USA).
Luciferase reporter assay
First, wt-KCNQ10T1, mut-KCNQ10T1, wt-RNF19A and mut-RNF19A were conducted by Quik Change Site-Directed Mutagenesis Kit (AgilentTechnologies). Then, HUVECs were incubated onto 24-well plates and co-transfected with wt-KCNQ10T1 or mut-KCNQ10T1 and wt-RNF19A or mut-RNF19A and miR-154-3p mimic or mimics-NC via Lipofectamine 2000. Finally, the luciferase activity was measured via the Dual-Glo Luciferase Assay System (Promega, USA).
All the data were analyzed by SPSS 19.0. One-way ANOVA followed by Dunnett’s multiple comparison was applied to assess the differences between the groups. Survival analysis was evaluated by the Kaplan-Meier method. P < 0.05 indicated significant difference between groups.