| Objective Neuropathic pain(NP)is a type of chronic pain that is increasing in prevalence each year and for which there is still no effective treatment.Exercise is increasingly recommended for the management of NP as an effective treatment modality that is inexpensive and has no side effects.However,the mechanisms of exercise treatment have not yet been elucidated.Micro RNA(miRNA)is expected to be potential target for the regulation of NP.Based on this,the objectives of this study were: 1.to examine the change of miRNA expression in the spinal dorsal horn(SDH)under NP and to search for potential targets of exercise to improve NP;2.to elucidate the effects of swimming exercise on pain behavior in mice with NP,and the expression of miR-206-3p and its target gene,brain-derived neurotrophic factor(BDNF),in SDH.MethodsIn the first part of this study,9 male Sprague-Dawley rats were randomly divided into three groups: sham group,NP group,and NP+swim group.Rats in the NP group and NP+swim group were modeled with chronic constriction injury(CCI),and rats in the NP+swim group received swimming intervention after modeling.After the intervention,the total RNA of L4-L6 SDH on the modeled side was extracted,and RNA sequencing(RNA-seq)was performed to analyze the differentially expressed miRNA between the NP group and sham group,NP+swim group and NP group,and the co-differentially expressed miRNA in both comparison groups.The co-differentially expressed miRNA was then validated by quantitative real-time PCR(qRT-PCR)through the construction of spared nerve injury(SNI)mouse model.In the second part,male C57BL/6 mice were randomly divided into naive group(n=15),sham group(n=15),sham+swim group(n=15),SNI group(n=15)and SNI+swim group(n=15).The sham+swim group and SNI+swim group underwent 6weeks swimming after modeling,and the other groups were routinely feeding.Pain behavioral tests were performed on postoperative day 0,3,7,14,21,28,35 and 42,and the mechanical withdrawal threshold(MWT)and response frequency(RF)were measured.On postoperative day 42,morphological changes of sciatic nerve and L4-L6 SDH from the modeled side were observed by HE,and BDNF localization and expression in SDH were observed by immunofluorescence.Expression of miR-206-3p and BDNF mRNA in SDH was detected by qRT-PCR and the expression of BDNF protein in SDH was detected by western blot.ResultsIn the first part,RNA-seq results showed that there were 15 miRNAs differentially expressed in the NP group compared with the sham group,of which 11 miRNAs up-regulated and 4 miRNAs down-regulated(p<0.05);and 25 miRNAs differentially expressed in the NP+swim group compared with the NP group,of which 19 miRNAs up-regulated and 6 miRNAs down-regulated(p<0.05).The Venn diagram of differential miRNA showed that miR-206-3p was the only co-differentially expressed miRNA.As verified by qRT-PCR,miR-206-3p up-regulated in the NP group compared to the sham group(p=0.005),and down-regulated in the NP+swim group compared to the NP group(p=0.008).In the second part,pain behavioral results showed that mice in the SNI group had significantly lower MWT(postoperative day 0: p=0.001;7,14,21,28,35,and 42:p<0.001),and higher RF(p<0.001),and lower MWT of affected side than the healthy side(p<0.001).The SNI+swim group had significantly higher MWT and lower RF than the SNI group starting at postoperative day 28(p < 0.001),and the MWT of affected side was not significantly different from the healthy side starting at postoperative day 35(p > 0.05).In the SNI group,the space between the sciatic nerve fibers enlarged,inflammatory cell infiltration and Schwann cell proliferation were observed in the sciatic nerve,and neuronal damage and loose surrounding tissue were observed in SDH.Compared with the SNI group,the overall morphology of both the sciatic nerve and SDH was improved in the SNI+swim group.Immunofluorescence showed that BDNF was co-localized with Neu N,and the fluorescence intensity of BDNF was reduced in the SNI group and enhanced in the SNI+swim group.miR-206-3p expression was significantly increased(p<0.001),and BDNF mRNA(p<0.001)and protein(p=0.007)expression were significantly reduced in the SNI group;after swimming intervention,miR-206-3p expression was significantly reduced(p=0.012),BDNF mRNA(p=0.036)and protein(p=0.03)expression were significantly increased,and there was a significant negative correlation between miR-206-3p and BDNF mRNA(r=-0.833,p=0.003).ConclusionNeuropathic pain can cause a significant increase in miR-206-3p expression and a significant decrease in BDNF expression in the SDH.Swimming intervention significantly improved neuropathic pain and reversed miR-206-3p and BDNF expression. |
