Zhenbao pill protects against acute spinal cord injury via miR-146a-5p regulating the expression of GPR17

Abstract

The aim of the present study was to observe the effect of zhenbao pill on the motor function of acute spinal cord injury (ASCI) rats and the molecular mechanisms involving miR-146a-5p and G-protein-coupled receptor 17 (GPR17). ASCI rat model was established by modified Allen method, and then the rats were divided into three groups. SH-SY5Y cells were cultured overnight in hypoxia condition and transfected with miR-146a-5p mimic or miR-146a-5p inhibitor. The hind limb motor function of the rats was evaluated by Basso, Beattie, Bresnahan (BBB) scoring system. Quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the expression of miR-146a-5p, GPR17, inducible nitric oxide synthase (iNOS), interleukin 1β (IL-1β), and tumor necrosis factor α (TNF-α). Neuronal apoptosis was measured using flow cytometry assay. Luciferase reporter assay was performed to determine the regulation of miR-146a-5p on GPR17. Zhenbao pill could enhance hind limb motor function and attenuate the inflammatory response caused by ASCI. Moreover, zhenbao pill increased the level of miR-146a-5p and decreased GPR17 expression in vivo and in vitro. Bioinformatics software predicted that GPR17 3′-UTR had a binding site with miR-146a-5p. Luciferase reporter assay showed that miR-146a-5p had a negative regulatory effect on GPR17 expression. Knockdown of miR-146a-5p could reverse the effect of zhenbao pill on the up-regulation of GPR17 induced by hypoxia, reversed the inhibitory effect of zhenbao pill on the cell apoptosis induced by hypoxia and the recovery of zhenbao pill on hind limb motor function in ASCI rats. Zhenbao pill could inhibit neuronal apoptosis by regulating miR-146a-5p/GPR17 expression, and then promoting the recovery of spinal cord function.

Selection of Reliable Reference Genes for Analysis of Gene Expression in Spinal Cord during Rat Postnatal Development and after Injury

“…Unfortunately, as was proven many times in the past, ideal and universal control gene(s), which is/are stably expressed across all cell types or under different conditions, do not exist [5,9,26]. Therefore, it is recommended that the combination of several validated RGs is employed to minimize the inherent variation in their expression [19].…”

“…The majority of studies dealing with gene expression during postnatal development and after spinal cord injury circumvent RG validation. Thus, the authors often rely on the routinely used housekeeping genes, mostly e.g., Gapdh [9,10], β-actin [11,12], or 18S rRNA [13,14], without testing and validating its stable expression under particular experimental conditions. To our knowledge, we are not aware of a serious survey and validation of stably expressed RGs in the rat spinal cord across postnatal development or after SCI.…”

Zhenbao Pill reduces Treg cell proportion in acute spinal cord injury rats by regulating TUG1/miR-214/HSP27 axis

“…As reported, Zhenbao Pill could repair the damaged neurones, promote microcirculation, and remove oxygen free radicals [ 8 , 9 ], suggesting the potential application of Zhenbao Pill in treating SCI patients. Indeed, Zhenbao Pill has been shown to protect SCI rats from further damage [ 10 , 11 ], with improved BBB score. Consistent with this finding, in the present study, we also found that the BBB score was significantly lower in SCI group compared with sham group, which was dramatically reversed by Zhenbao Pill.…”

“…Modern pharmacological studies have shown that Zhenbao Pill had the function of repairing damaged neurones, promoting microcirculation, and removing oxygen free radicals [ 8 , 9 ]. Our previous research has found that Zhenbao Pill had a protective effect on the nerves of SCI rats [ 10 ]. We also found that Zhenbao Pill improved the performance of SCI rats in the Basso, Beattie, and Bresnahan (BBB) score and reduced the proportion of Treg cells [ 11 ].…”

LncRNA DGCR5 suppresses neuronal apoptosis to improve acute spinal cord injury through targeting PRDM5

“…Hypoxia treatment was performed in AGE1.HN and PC12 cell lines for simulating SCI in vitro, as previously reported [14,15]. Oxygenglucose deprivation (OGD) was performed upon the above cells for hypoxia treatment in accordance with a previous study [16].…”