Exosomes Secreted by M1-type Macrophages Improve Stress Urinary Incontinence by Promoting the Repair of the Levator ani Muscle Injury in Mice
Authors: Cheng Jianhong , Hong Shasha , Yang Lian , Liu Jianfeng , Hong Li 
Published: July 2021

Abstract
Background: Macrophages are involved in the regeneration of skeletal muscle injury and the exosomes secreted by a variety of cells promote the regeneration of tissues after injury. However, the potential effect of exosomes secreted by polarized macrophages on the repair of skeletal muscle after injury remains unclear. This study explored the effect of exosomes derived from M1 macrophages (M1-Exo) on the repair of levator ani muscle in mice after Vaginal Dilation (VD) modeling and the viability of C2C12 myoblasts after mechanical injury.Methods: Differential ultracentrifugation was used to separate M1-Exo from 200 ng/mL lipopolysaccharide-induced polarization of M1 type macrophages culture medium. Nanoparticle tracking analysis, transmission electron microscopy, and western blotting of CD9 and Tsg101 proteins were employed to identify M1-Exo. In vivo experiment involving the vaginal balloon expansion method was used to simulate the trauma to the pelvic floor of the mouse during delivery. M1-Exo was injected into the levator ani muscle and its surroundings to detect the abdominal leak point pressure (ALPP) and the maximum bladder volume (MBV) of the VD mice at 3, 7, and 14 days. Then the levator ani muscle was taken for hematoxylin and eosin (H&E) staining to observe the muscle damage and repair. To evaluate the functional and anatomical recovery of M1-Exo on stress urinary incontinence (SUI) mice caused by VD model delivery trauma. Subsequently, an in vitro C2C12 myoblasts cyclic mechanical strain injury model was constructed to determine the best mechanical injury parameters. In the next step, through a series of in vitro functional tests, the effect of M1-Exo on the proliferation, senescence, and apoptosis of C2C12 myoblasts injured by cyclic mechanical strain was assessed. The effect of M1-Exo on the prevention and treatment of SUI caused by injury to the levator ani muscle after delivery was evaluated using animal experiments and cell-level studies.Results: Powerlab software test results showed that the injection of M1-Exo into the levator ani muscle of SUI mice and its surroundings can significantly increase the mouse's ALPP, and MBV. H&E staining results revealed that M1-Exo can prevent secondary necrosis of broken muscle fibers, reduce nuclear migration of muscle fibers, maintain the shape of the muscle bundles, and promote normal muscle regeneration. CCK-8 proliferation reagent, senescence-associated β-galactosidase (SA-β-Gal) staining, and flow cytometry (PE/7-AAD staining) were used to determine the best in vitro simulation of the C212 myoblasts. The best damage parameters of the C2C12 myoblast injury model occurred at 5333 μ strain for a duration of 8 hours at 1 Hz. Subsequently, the test results of the CCK-8 proliferation reagent and EdU cell proliferation reagent suggested that M1-Exo promoted the proliferation of C2C12 myoblasts subjected to mechanically induced damage. SA-β-Gal staining results indicated that M1-Exo delayed the senescence of C2C12 myoblasts subjected to mechanically induced injury. Hoechst 33258 staining reagent and flow cytometry (PE/7-AAD staining) revealed that M1-Exo inhibited mechanically induced apoptosis of the C2C12 myoblasts.Conclusions: Our experimental results established that M1-Exo helps in the functional and anatomical recovery of SUI mice caused by labor trauma. Furthermore, the findings imply that M1-Exo has a protective effect on C2C12 myoblasts after cyclic mechanical strain damage, promotes their proliferation, delays aging, and inhibits apoptosis.