The anti-inflammatory effect of electroacupuncture in mice with spinal cord injury and molecular mechanism based on transcriptome sequencing technology

Abstract

To observe the effect of electroacupuncture(EA) on neural function and spinal cord pathological morphology in spinal cord injury(SCI) mice and investigate the anti-inflammatory molecular mechanism of EA on SCI mice from the aspects of gene by using bioinformatics. Seventy-two female C57BL/6 mice were randomized into sham operation, model and EA groups, with 24 mice in each group. The SCI model was established by clamping the spinal cord with a serrefine after laminectomy at the 1st lumbar vertebra(L1). EA(1.5 Hz/7.5 Hz, 1.0 mA) was applied to bilateral "Jiaji"(EX-B2) and "Zusanli"(ST36) for 10 min, once a day for 14 consecutive days. Basso Mouse Scale(BMS) score was used to assess the hindlimb locomotor function of mice. Histopathological changes of the injured area of the spinal cord were determined by HE staining. The spinal cord RNA was sequenced by using RNA-Seq technology. The bioinformatic analysis was then performed to detect the diffe-rential genes between groups, and the function classification and the involved pathways were enriched. The mRNA and protein expressions of differential genes were detected and verified by using qRT-PCR and Western blot. Compared with the sham operation group, BMS score of the model group was significantly decreased(P<0.05), while that of EA group was increased relevant to the model group (P<0.05). HE staining showed loose and disordered structure and arrangement, cavitation, more inflammatory infiltration, nucleus pycnosis, and neuronal necrosis in the model group, which was alleviated in the EA group. Compared with the sham operation group, 565 differential genes were detected in the model group, including 545 up-regulated and 20 down-regulated, while 41 were detected between the EA and the model group, including 2 up-regulated and 39 down-regulated in the EA group. Fifteen genes that were all up-regulated after modeling and down-regulated after EA intervention were detected by using Venn plot, which are Retn, Adipoq, Myh1, Actn2, Pck1, Klhl41, Fabp4, Hspb7, Myot, Ankrd2, Hrc, Cox6a2, Obscn, Col2a1, Mybpc1, and 3 inflammation-related genes(Fabp4, Adipoq and Pck1) were finally acquired. The 15 differential genes were annotated into main biological processes, cell composition and molecular function in the GO function classification analysis. The 15 differential genes were then enriched into different KEGG pathways, including the peroxisome proliferatorsactivated receptor (PPAR) signaling pathway, Adipocytokine signaling pathway. The mRNA and protein expressions of Fabp4, Adipoq and Pck1 in spinal cord detected by qRT-PCR and Western blot were significantly increased in the model group (P<0.001, P<0.01), while these were significantly decreased in the EA group relevant to the model group(P<0.001, P<0.01, P<0.05). EA can promote the repair of nerve function and improve inflammatory infiltration in SCI mice. The mechanism may be closely related to the down-regulation of inflammatory factors Fabp4, Adipoq and Pck1 expression, and the regulation of PPAR and Adipocytokine signaling pathways.