Exosomes in acupoint area involved in the effect of electroacupuncture on muscle regeneration and repair in rats with multifidus muscle injury

To observe the effect of electroacupuncture (EA) on the expressions of paired box transcription factor 7 (Pax7), myogenic differentiation antigen (MyoD), myogenin (MyoG) and myosin heavy chain (MyHC) in the multifidus muscle, and CD63, programmed cell death protein 6 interacting protein (Alix) and tumor susceptibility gene 101 (TSG101) proteins in the serum exosomes in rats with lumbar multifidus muscle injury (MFMI), so as to explore the effect of exosomes in acupoint areas on EA improvement of muscular regeneration and repair.

Forty male SD rats were randomly divided into normal control, model, EA and EA+exosome inhibitor (EA+inhibitor) groups, with 10 rats in each group. The MFMI model was established by injection of 0.5% bupivacaine (150 μL × 4) into the 4 points of the multifidus muscle along the bilateral lumbar (L)4—L5 spinous processes. EA (2 Hz/10 Hz, 1 mA) was applied to bilateral “Weizhong”(BL40) and “Shenshu”(BL23) for 20 min, once a day for 7 d. For rats of the EA+inhibitor group, exosome inhibitor GW4869 (3 mg/mL, 50 μL/acupoint) was injected into bilateral BL40 and BL23 1 h before each EA intervention. The morphological changes of the multifidus muscle were observed after H.E. staining and Masson staining. The immunoactivity of Pax7 and MyoD was observed by immunohistochemistry. The serum exosomes were extracted and identified by transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA). The expression levels of MyoG and MyHC in the multifidus muscle tissue and CD63, Alix and TSG101 proteins in the serum exosomes were detected by Western blot.

Morphological results showed that in the model group, most of the muscle fibers were degenerated and necrotic, a large number of inflammatory cells infiltrated around the muscle fibers and more blue-stained collagen fibers were observed. In the EA group, the morphology of muscle fibers was relatively complete, with more new muscle fibers and reduced inflammatory cells in the injured area, and the collagen fibers were significantly reduced. In the EA+inhibitor group, there were still more muscle fiber destruction and inflammatory cell infiltration, new muscle fibers with uneven diameter and more collagen fibers. Compared with the normal control group, the immunoactivity of Pax7 in the multifidus muscle, the expression of Alix and CD63 proteins in the serum exosomes were significantly increased in the model group (P＜0.01, P＜0.05, P＜0.001). In comparison with the model group, the immunoactivity of Pax7 and MyoD, the expression levels of Alix and TSG101 in the serum exosomes and MyHC and MyoG proteins in the multifidus muscle were considerably up-regulated in the EA group (P＜0.01, P＜0.05). After local injection of GW4869 at BL40 and BL23, the immunoactivity of Pax7 and MyoD, the protein expression levels of TSG101, CD63, MyHC and MyoG were significantly lower in the EA+inhibitor group than those of the EA group (P＜0.01, P＜0.05, P＜0.001). The results of TEM and NTA showed that the exosomes were successfully extracted. The morphology of the exosomes was typical saucer-like under electron microscope, and the particle size range was concentrated in 70—200 nm.

EA of BL40 and BL23 can significantly up-regulate the expressions of Pax7, MyoD, MyoG and MyHC in the injured multifidus muscle, and promote the regeneration and repair of lumbar multifidus muscle, which may be related to its functions in promoting the release of exosomes in the acupoint area.