Effects of electroacupuncture on the expression of serum exosomes and pro-inflammatory factors in spinal cord of rats with spinal cord injury

Abstract

To investigate the effect of electroacupuncture (EA) on the morphology and microstructure of spinal cord tissue, the expression of serum exosomes, and the pro-inflammatory factors interleukin (IL)-1β and IL-6 in spinal cord of rats with spinal cord injury (SCI), so as to explore the underlying mechanism of EA in the treatment of SCI. Twenty-four female Wistar rats were randomly divided into sham operation group, model group, EA group, EA+GW4869 group, with 6 rats in each group. The SCI model was established by impinging spinal cord at T10 with a hammer, while the vertebral lamina was only opened without impingement for rats in sham operation group. Rats in EA group received EA intervention at "Jiaji"(EX-B7) acupoints at bilateral T9 and T10 (0.4-0.6 mA, 100 Hz), 3 h after modeling, once a day, for 7 concecutive days. Besides the treatment as EA group, rats in the EA+GW4869 group received injection of exosome inhibitor GW4869(200 μL, 300 μg/mL) once every 2 days from the day before modeling. Motor function of hind limbs of rats was evaluated using BBB scores. The histopathological changes of spinal cord were observed under light mircoscope after H.E. staining. Microstructure of spinal cord was observed and extracted serum exosomes were identified by using transmission electron microscopy. The expression of exosome marker proteins in serum exosomes, the levels of IL-1β and IL-6 in spinal cord were detected by Western blot. H.E. stanining showed severe tissue looseness, inflammatory cell infiltration, cellular hydropic degeneration in spinal cord of the model group, which were relatively milder in the EA and EA+GW4869 groups. Under transmission electron microscopy, there were nerve fiber disintegration, myelin sheath structure dispersion, axonal atrophy with submembrane edema and widened space, and mitochondrial swelling in spinal cord of rats in the model group, with the lesions in EA group milder than EA+GW4869 group, which were both moderate. Typical exosomes were detected by transmission electron microscope in the extracted serum of rats in each group after ultracentrifugation. Compared with the sham operation group, the motor function scores was significantly decreased (P<0.01), the expression of IL-6 and IL-1β in the spinal cord was significantly increased (P<0.01), while the expression of serum exosome marker protein CD81 was slightly increased in rats of the model group. Compared with the model group, the motor function scores was significantly increased (P<0.01), the expression of IL-6 and IL-1β in the spinal cord was significantly decreased (P<0.01) in rats of the EA and EA+GW4869 group, while the expression of serum CD81 protein was slightly increased in rats of the EA group. Compared with the EA+GW4869 group, the expression of IL-6 and IL-1β in the spinal cord was significantly decreased (P<0.01), while the expression of serum CD81 protein was slightly increased in rats of the EA group. However, there was no significance in expression of CD81 between each group mentioned above. EA can promote the secretion of serum exosomes and inhibit the expression of pro-inflammatory cytokines IL-6 and IL-1β, so as to improve the microenvironment of injured spinal cord and SCI.