The Role Of Long Non-coding RNA LOC100911498 In Neuropathic Pain In Rats

BackgroundNeuropathic pain(NP)is the most debilitating of all clinical pain syndromes,may be a consequence of dysfunction or primary injury of the somatosensory nervous system.Unfortunately,the pathogenesis of NP is not fully understood yet and it cannot be cured totally.Long noncoding RNA(lncRNA)is a type of RNA molecule greater than 200 nucleotides,which cannot be translated to detectable protein and is involved in the regulation of various processes in the cell.Recent studies have found that dysregulated expression of lncRNAs plays an critical role in the facilitation of neuropathic pain.Previous study have shown that in the spared nerve injury(SNI)model,microglia in the rat spinal dorsal horn proliferate and activate,phosphorylation of P2X4 receptor(P2X4R),p38(p-p38)and brain-derived neurotrophic factor(BDNF)significantly increase,in addition,the expression level of lncRNA-LOC100911498 in the spinal cords of rats also increase.Studies have shown that NP can induce an increase in the expression of P2X4 R,which further promotes phosphorylation of p38,which can increase the expression of BDNF in microglia by activating downstream specific transcription factors to cause NP.Does LOC100911498 interact with P2X4 R to participate in the procession of NP? We used a protein-based amino acid and gene base pair sequence,secondary structure propensity,hydrogen bonding,and van der Waals force to predict and evaluate the interaction between protein and RNA.The online algorithm(CatRAPID)evaluated the interaction of the LOC100911498 RNA and P2X4 R,the results demonstrated a high score for the interaction between LOC100911498 and P2X4 R.Therefore,we hypothesized that LOC100911498 regulates the development of neuropathic pain by activating microglia and the P2X4R-p-p38-BDNF signaling pathway.ObjectiveThis study aims to investigate the role of LOC100911498 in neuropathic painand and its underlying mechanisms..Methods1.The effect of LOC100911498 expression on pain behavior and expression of LOC100911498 in the spinal cord after spared nerve injury(SNI)in rats.Sprague Dawley(SD)male rats were divided into sham,SNI,SNI rats transfected with LOC100911498 siRNA group(SNI+ LOC100911498 siRNA)and SNI rats transfected with scramble siRNA group(SNI+NC siRNA).From the seventh day after surgery,LOC100911498 siRNA or vehicle was injected at a dose of 1 OD siRNA/20 mL/rat once daily for 5 consecutive days.The postoperative measurement measured the mechanical withdrawal threshold(MWT)of the posterior plantar 1 day before surgery and 1,3,5,7,9,11,and 14 days after surgery.In addition,the expression of LOC100911498 in L4-6 spinal dorsal horn on 14 th day after surgery was detected by in situ hybridization.2.The effects of LOC100911498 expression on the activation of spinal cord microglia and the P2X4R-p-p38-BDNF signaling pathway.SD male rats were divided into sham,SNI,SNI+ LOC100911498 siRNA and SNI+NC siRNA.QRT-PCR detected LOC100911498 and P2X4 R mRNA expressions in the L4-6 spinal cord;western blot detected P2X4 R,p38,p-p38 and BDNF protein expression changes in the spinal cord;immunofluorescence double staining detected the expressions of activated microglia and P2X4 R in the spinal dorsal horn.AnalysisStatistical tests were performed with SPSS 10.0(SPSS Inc.,USA)and SigmaStat(Systat,San Jose,CA).All data are expressed as mean ± standard error.For MWT,the data were analysed by two-way(time and treatment)repeated measures analysis of variance(ANOVA)followed by Newman–Keuls post hoc test and t-test between 2 groups at the same time points was carried out.One-way ANOVA was used to test for statistical differences of western blot,immunofluorescence and real-time PCR data followed by Tukey or Dunnett tests,while in situ hybridization was tested by t-test as only 2 groups were applied.P <0.05 was considered statistically significant.Results1.SNI caused ipsilateral mechanical allodynia in rats.Compared with the sham group,the MWT of SNI rats began to decrease from the first day after surgery,reached the lowest value on the 7th day,and lasted until 14 th day after surgery.There was no significant change in the contral group.After intrathecal injection of LOC100911498 siRNA on the 7th postoperative day,SNI-induced mechanical hyperalgesia in rats significantly relieved,and MWT was significantly higher than that in the SNI group,but still lower than that in the control group.There was no statistical difference between SNI and SNI+NC siRNA groups.LOC100911498 expressed in both nucleus and cytoplasm.Compared with the control group,the expression level of LOC100911498 was significantly higher in the spinal dorsal horn of SNI rats on 14 th days after surgery.2.Compared with the control group,the expression levels of P2X4 R,p-p38 and BDNF in the spinal cord of SNI and SNI+NC siRNA groups significantly increased,and there are more activated microglia in the spinal dorsal horn.There were no statistical differences between the two groups of rats.While the expressions of these proteins were reduced and the activation of microglia was inhibited by the injection of LOC100911498 siRNA after surgery.Conclusion:Our study indicated the effects of lncRNA LOC100911498 siRNA on P2X4R-pp38-BDNF signaling pathway mediated by microglia activation in the L4-6 spinal dorsal horn of rats.Furthermore,LOC100911498 can be a novel regulator of neuropathic pain in rats by regulating the expression and function of P2X4R.