Abstract:

Objective To discuss the effect of rubusoside （RUB） on the spinal cord injury （SCI） and neuroinflammation in the mice， and to clarify its mechanism. Methods A total of 48 female Kunming mice were randomly divided into sham operation group， SCI group， SCI+low dose of RUB group， and SCI+high dose of RUB group， and there were 12 mice in each group. Spinal cord injury behavior （BBB） scoring method was used to assess the motor function of the hind limbs of the SCI mice； water content method was used to detect the spinal cord edema of the SCI mice； real-time fluorscence quantitative PCR （RT-qPCR） method was used to detect the expression levels of pro-inflammatory cytokines cyclooxygenase-2 （COX-2）， interleukin-1β （IL-1β）， and tumor necrosis factor-α （TNF-α） mRNA of the mice in various groups； ELISA method was used to detect the levels of inflammatory factors in serum of the mice in various groups； HE staining was used to observe the pathomorphology of spinal cord tissue of the mice in various groups；immunofluorescence was used to detect the activation of microglia in spinal cord tissue of the mice in various groups； Western blotting method was used to detect the expression levels of related proteins in spinal cord tissue of the mice in various groups. Results The BBB score analysis results showed that compared with sham operation group， the score of the mice in SCI group was decreased to 0； compared with SCI group， the BBB scores of the mice in SCI+low dose of RUB group and SCI+high dose of RUB group were gradually increased. The spinal cord tissue water content detection results showed that compared with sham operation group， the water content in spinal cord tissue of the mice in SCI group was significantly increased （P<0.01）； compared with SCI group， the water contents in spinal cord tissue of the mice in SCI+ low dose of RUB group and SCI+ high dose of RUB group were significantly decreased （P<0.01）. The RT-qPCR results showed that compared with sham operation group， the expression levels of COX-2， IL-1β， and TNF-α mRNA in spinal cord tissue of the mice in SCI group were significantly increased （P<0.001）. Compared with SCI group， the expression levels of COX-2，IL-1，and TNF-α mRNA in spinal cord tissue of the mice in SCI+ low dose of RUB group and SCI+ high dose of RUB group were significantly decreased （P<0.001）. The ELISA results showed that compared with sham operation group，the levels of IL-1β （P<0.01） and TNF-α （P<0.001） in serum of the mice in SCI group were increased； compared with SCI group， the levels of IL-1β and TNF-α in spinal cord tissue of the mice in SCI+ low dose of RUB group and SCI+ high dose of RUB group were decreased （P<0.001）.The Western blotting results showed that compared with sham operation group， the expression levels of nuclear factor kappa B （NF-κB） inhibitor protein-α （IκB-α）， phosphorylated IκB-α （p-IκB-α）， phosphorylated p65 （p-p65）， p65， phosphorylated p38 （p-p38）， phosphorylated extracellular regulated protein kinase （p-ERK）， and phosphorylated c-Jun N-terminal kinase （p-JNK） proteins in spinal cord tissue of the mice in SCI group were significantly increased （P<0.05 or P<0.001）； compared with SCI group， the expression levels of IκB-α， p-IκB-α， p-p65， p65， p-p38， p-ERK， and p-JNK proteins in spinal cord tissue of the mice in SCI+ low dose of RUB group and SCI+ high dose of RUB group were significantly decreased （P<0.001）. The HE staining results showed that compared with sham operation group， the spinal cord tissue of the mice in SCI group had loose organization with the formation of vacuoles， and a large necrotic cavity was present in the center of the spinal cord； compared with SCI group， the central necrotic cavity area of the mice in SCI+ low dose of RUB group and SCI+ high dose of RUB group was significantly decreased （P<0.05）.The immunofluorescence results showed that compared with sham operation group， the number of positive microglia cells in spinal cord tissue of the mice in SCI group was significantly increased （P<0.001）； compared with SCI group， the numbers of positive microglia cells in spinal cord tissue of the mice in SCI+ low dose of RUB group and SCI+ high dose of RUB group were significantly decreased （P<0.001）. Conclusion Rubusoside can improve the motor function impairment in the SCI mice，mitigate the neuroinflammation in spinal cord tissue，and inhibit the activation of microglia. The mechanism may be related to the downregulation of expression of proteins associated with the NF-κB and mitogen-activated protein kinase （MAPK） signaling pathways in spinal cord tissue.

Key words: Rubusoside, Spinal cord injury, Neuroinflammation, Nuclear factor kappa-B, Mitogen-activated protein kinase