Cell culture and reagents
Mouse RAW264.7 macrophages were purchased from Wuhan Magall Biotechnology Co., Ltd. (introduced by the Chinese Academy of Sciences), and mouse C2C12 myoblasts were procured from Nanjing Kebai Biotechnology Co., Ltd. Both RAW264.7 macrophages and C2C12 myoblasts were maintained in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Genom Biotech Co. Ltd., Hangzhou, China) supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Products, Fisher Scientific, Waltham, MA, USA) and 1% penicillin and streptomycin (Beyotime Biotech Co. Ltd., Suzhou, China). The cells were cultured in a humidified incubator (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C with 5% CO2.
When the growth density of RAW264.7 macrophages reached about 90%, using the medium that contain 200 ng/mL lipopolysaccharide (Wuhan Saiweier Technology Co., Ltd.),10% exosome-depleted FBS ( heat-inactivated FBS centrifuged 4℃, 100,000×g After 18 h, the lower layer of serum containing the exosomes was discarded, and 80% of the upper layer of the centrifuge tube constituted the FBS without the exosomes), and 100 U/mL penicillin-streptomycin to culture for 24 h to obtain M1 type macrophages.
Exosome isolation and characterization
RAW264.7 macrophages were cultured in an induction medium for 24 h to complete their polarization to M1 type; the induction medium was discarded, the cells were washed three times with phosphate-buffered saline (PBS), and the cells were cultured in a medium devoid of serum and antibiotics. After 24 h, the cell supernatant was collected and used to extract the exosomes.
An ultracentrifuge (Optima XE-100 Ultracentrifuge, USA) was used to perform differential ultracentrifugation to separate and purify the exosomes[29]. First, the collected cell supernatant was centrifuged at 4℃, 3000×g for 15 min to remove cells and cell debris, and then at 12,000 ×g for 50 min to remove the microscopic particles, such as organelles, save the supernatant. The sample was then passed through a 0.22-μm filter to remove particles >200 nm. Finally, the filtered liquid was centrifuged at 4°C and 120,000 ×g for 150 min to separate the exosomes. The exosomes settled at the bottom of the six centrifuge tubes were collected, washed with PBS, and then centrifuged for 150 min at 4℃ and 120,000 ×g to collect the exosomes. The small amount of the obtained precipitate contained the enriched and purified exosomes. Finally, the exosomes were resuspended in 200 µL of PBS (pH = 7.4) and stored at -80°C for later use.
ZetaView® Nanoparticle Tracking Analyzer (Particle Metrix, Meerbusch, Germany) was used to track the particle size distribution and purity of the exosomes, and HT7700 transmission electron microscope (HITACHI, Japan) was used to observe the extracted exosomes. The morphology of the exosomes was photographed, and the exosome marker protein CD9 and tumor susceptibility gene 101 (Tsg101) were identified by western blot.
Mice and animal experimental design
3-month-old female unconceived wild-type C57BL/6 mice used in this study were provided by the Experimental Animal Center of Wuhan University, and all experimental procedures were performed after obtaining approval from the Experimental Animal Welfare Ethics Committee of Wuhan University People’s Hospital (Approval No. 20210306).
Establishment of a mouse SUI model[30]: The mice were anesthetized with isoflurane (induction concentration 3–4%, maintenance concentration 1–1.5%). After disinfection and lubrication, a modified 6-Fr-Foly catheter (Dalian Couliat Medical Products Co., Ltd.), was inserted into the vagina and fixed with a 4-0 needle suture at the external mouth. This action was performed gently and did not damage the urethra or vagina of the mouse. Subsequently, 0.3 mL of normal saline was injected into the balloon (diameter approximately equal to that of the newborn mouse's skull, i.e., 8 mm) to expand the vagina. Simultaneously, a 60-g weight was hung at the end of the catheter for traction, which was removed after 1 h. After the balloon was drained, the catheter was pulled out, and the suture was removed and disinfected.
A sneeze experiment was performed 24 h after the mice were modeled, and the mice’s maximum bladder volume (MBV) and abdominal leak point pressure (ALPP) were tested to determine whether the SUI model was successful. Then, thirty-six SUI mice were randomly divided into two equal groups. One group was locally injected with M1 type macrophages-derived exosomes (M1-Exo) 1 × 1010 particles/mL, 1mL in and around the levator ani muscle on both sides. Another group was injected with 1 mL of 0.9% normal saline locally in and around the levator ani muscle on both sides. The urodynamic parameters (MBV, ALPP) of the two groups were measured 3, 7, and 14 days after the injection (n = 6 at each time point).
Hematoxylin and eosin (H&E) staining
After measuring ALPP and MBV, the mice were sacrificed under anesthesia; the bilateral levator ani muscles were removed and fixed with muscle fixative (Wuhan Saiweier Biotechnology Co., Ltd.) for 24 h. The tissue specimens were embedded in paraffin, cut into 4-μm–thick sections, and H&E staining was performed for histological observation. The BX 63 automatic microscope (Olympus, Japan) was employed to capture the morphology of the levator ani muscle.
Cyclic mechanical strain (CMS)
To calculate the CMS, 1.5 mL of suspension, with a cell density of 1 × 105 cells/mL, was taken. The C2C12 myoblast suspension in the logarithmic growth phase was spread evenly in the center of the cell strain culture plate. The plate was placed in a 100-mm cell culture dish and moved to a constant temperature incubator. The cells were allowed to adhere to the wall overnight; on the next day, the medium was changed to serum-free DMEM high glucose medium to synchronize the cell cycle to the G0 phase. When the cell density reached about 80%, the strained culture plate covered with cells was turned upside down in the 50 mL container. A four-point bending cell mechanics loading device (Chengdu Mirui Technology Co., Ltd.) was connected to a strained petri dish holder with a volume fraction of 2% FBS and 1% penicillin-streptomycin DMEM high glucose medium.
To explore the best experimental parameters of damage to C2C12 myoblasts, the strain frequency of 1 Hz was applied and divided into three groups according to the strain force and strain time: 1333-μ strain (0 h, 4 h, 8 h, and 12 h), 2666-μ strain (0 h, 4 h, 8 h, and 12 h), and 5333-μ strain (0 h, 4 h, 8 h, and 12h). This step was followed by CCK-8 cell viability testing, and the optimal cell strain was determined to be 5333 μ. Subsequently, the senescence and apoptosis of C2C12 myoblasts were detected under the frequency of 1 Hz and strain force of 5333 μ (0 h, 4h, 8 h, and 12 h). The cyclic mechanical stress parameters that can cause significant differences in C2C12 myoblast cell activity, senescence, and apoptosis were selected as the best injury experimental parameters, namely 1 Hz, 5333-μ strain, and 8 h. The following cell experiment was divided into three groups, namely the control group (no CMS applied injury), the applied CMS injury group (additional 1 ml of PBS solution cultured for 24 h after the applied CMS injury), and the exosome treatment group after the applied CMS injury (after CMS injury, supplementing with 1 × 1010 particles/mL, 1 mL of exosomal solution was cultured for 24 h).
Cell proliferation analysis
Cell Counting Kit-8
C2C12 myoblasts treated with different parameters and cells before and after the intervention of exosomes were trypsinized and counted on a hemocytometer to prepare a 2 × 104/mL cell suspension. This suspension was inoculated in a 96-orifice plate (100 μL per well, three wells in each group) and placed in an incubator at 5% CO2 and 37°C for 30 min. After the cells adhered to the wall, 10 μL of CCK-8 (Wuhan Kerui Biotech Co., Ltd. Company) and 90 μL of serum-free DMEM high-glycemic culture solution were added to each well. Incubation was continued for 1 h at 37°C, 5% CO2, and relative saturated humidity of 95%. Later, a microplate reader (Perkin Elmer, USA) was used to detect the absorbance value of each group of cells at 450 nm. The cell viability was calculated based on the average value of each group, and the experiment was repeated three times independently.
EdU incorporation assay
The C2C12 myoblasts treated with different parameters and the cells before and after the intervention of exosomes were replaced with EdU working fluid and were placed in a CO2 incubator for 2 h. The EdU medium was discarded, 4% paraformaldehyde was added, fixed for 15 min, and then washed three times with PBS, 5 min each time. Subsequently, 1 mL of 0.3% Triton X-100 was added and incubated at room temperature for 10 min. The waste solution was discarded, 500 μL of the click reaction solution was added, incubated at room temperature for 30 min in the dark, 0.3% Triton X-100 was added, and washed three times, 10 min each time. Hoechst 33342 staining solution was added to stain the nucleus. The images were captured using a BX 63 automatic microscope (Olympus, Japan), and ImageJ software was employed to count the number of EdU-positive cells.
Cell senescence analysis
The β-Galactosidase Staining Kit (Shanghai Biyuntian Institute of Biotechnology, China) was used to detect the degree of senescence of the C2C12 myoblasts. In this procedure, 1 mL of β-galactosidase staining fixative solution was added to the C2C12 myoblasts treated with different parameters and cells before and after the exosomal intervention. They were fixed for 15 min at room temperature, washed three times with PBS, 5 min each time, and 1 mL of β-galactosidase staining working solution (the ratio of β-galactosidase staining solution A, B, and C and X-Gal solution was 1:1:93:5) was added. After placing overnight at 37°C in a constant temperature incubator without CO2, the senescent cells were stained blue and were counted as positive cells. Three fields of view were randomly selected under the BX 63 automatic microscope (Olympus, Japan), and the number of positive cells was estimated. Senescence cell rate = several positive cells/the total number of cells ×100%.
Cell apoptosis analysis
Hoechst 33258 assay
In this assay, 1 mL of fixative was added to the C2C12 myoblasts treated with different parameters and cells before and after the intervention of exosomes and fixed at room temperature for 10 min. The fixative was discarded, the strained culture plate was placed in a 100-mm sterile dish, an appropriate amount of PBS was added, and rinsed twice with slow shaking, 3 min each time. After sucking up the liquid, 1 ml Hoechst 33258 (Beyotime Biotech Co. Ltd., Shanghai, China) dye solution was added dropwise and dyed for 5 min in the dark. After sucking off the dye solution, an appropriate amount of PBS was added, exposure to light was avoided, and rinsed twice with slow shaking, 3 min each time. After mounting the slides with anti-fluorescence quenching mounting solution in the dark, the images were captured on a BX 63 automatic microscope (Olympus, Japan). The nuclei of the apoptotic cells appeared whitish and bright blue, and the number of apoptotic cells was counted using ImageJ software. Apoptotic cell rate = the number of positive cells/total number of cells x 100%.
Flow cytometric analysis
C2C12 myoblasts treated with different parameters and cells before and after the intervention of exosomes were prepared into a suspension of 1×106 cells/mL; 100 μL of the cell suspension was transferred to a 5-mL centrifuge tube, 5 μL of PE Annexin V and 5 μL of 7-AAD (Becton Dickinson, Franklin Lakes, NJ, USA) were added, gently vortexed, and incubated at room temperature for 15 min in the dark. Then, 400 μL of 1× binding buffer was added to the centrifuge tube, and FACS Calibur Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA) was used to detect cell apoptosis. The experiments were performed three times. The results were analyzed using FlowJo7.6.
Western blot analysis
The total protein of the M1 macrophages was extracted with RIPA lysis solution, and the protein concentration was determined using the BCA Protein Assay Kit (Beyotime Biotech Co. Ltd., Shanghai, China). The remaining protein solution was mixed with an appropriate amount of 5× protein loading buffer. Appropriate amounts of the marker, exosomes, and M1 macrophages protein samples were placed in the sample wells, and they were transferred to a polyvinylidene fluoride membrane after 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis separation; 5% mass fraction skimmed milk dissolved with Tris-buffered saline with Tween (TBST) was used for 1 h at room temperature. and primary antibodies against CD9 (1:1000) and Tsg101 (1:1000) (Abcam, Cambridge, UK) were added and incubated in a shaker at 4°C; the samples were washed with TBST three times, 5 min each time. Subsequently, secondary antibodies diluted to 1:4000 at room temperature were added, incubated for 1 h, and washed with TBST three times, 5 min each time. The Li-Cor Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA) was used to detect protein expression and identify the exosomes.
Statistical analysis
GraphPad Prism 8.0 software (GraphPad, San Diego, CA, USA) was used to analyze the experimental results. The measured data were expressed as mean ± SEM. One-way analyses of variance (ANOVA) were applied to compare the differences between the groups. p < 0.05 was considered a statistically difference. p < 0.01 and p < 0.001 were regarded as significant differences.