MiR-21 inhibitor improves locomotor function recovery by inhibiting IL-6R/JAK-STAT pathway-mediated inflammation after spinal cord injury in model of rat.

Abstract

To investigate the function of miRNA-21 and interleukin-6 receptor/Janus Kinase-Signal transducer and activator of transcription (IL-6R/JAK-STAT) pathway in microglia on inflammatory responses after spinal cord injury (SCI). This study first detected respectively the protein level of inflammatory factor inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNF-α) by Western blotting after transfection of miR-21 or administration of miR-21 inhibitor in activated microglia cells of rat in vitro. The quantitative Real-time polymerase chain reaction (qRT-PCR) was utilized to detect the expression of IL-6R under two different interventions. Next, we established a model of spinal cord injury in rat and inspected miR-21 and IL-6R in SCI rat by qRT-PCR. In addition, the protein levels of iNOS and TNF-α in SCI rat were detected by Western blotting. MiR-21 inhibitor was injected into the injured area of SCI rat to delve into the function of miR-21 down-expression on iNOS and TNF-α expression by Western blot as well as the RNA levels of IL-6R, JAK and STAT3 by qRT-PCR. Furthermore, the SCI rat with movement and coordination of hindlimbs was observed by Basso-Beattie-Bresnahan locomotor rating scale (BBB scale) after miR-21 down-expression. Compared with the microglia transfected with miR-21, the execution of inhibitor in microglia effectively relieved the expression of IL-6R and the breakout of iNOS and TNF-α. Meanwhile, the increase of miR-21 was significantly observed in SCI rat along with significant improvement of inflammatory response-related factors including iNOS and TNF-α. After that, we injected SCI rat with miR-21 inhibitor into the spinal cord injury area and found the inhibition of miR-21 decreased the protein levels of iNOS and TNF-α. Simultaneously, down-expression of miR-21 evidently declined the RNA levels of IL-6R, JAK, and STAT3 in SCI rat. Compared with the sham-operated rat, the movement and coordination of hindlimbs of the SCI group displayed dramatic dysfunction. However, miR-21 down-expression elevated the movement and coordination of hindlimbs of the SCI rat than those of the only injury group. Inhibition of miR-21 can promote the recovery of spinal cord injury by down-regulating IL-6R/JAK-STAT signaling pathway and inhibiting inflammation.