Mechanism Of MiR-145-5P In Improving Neuropathic Pain Induced By Chronic Constriction Injury Of Sciatic Nerve In Response To Exercise

ObjectiveThe prevalence rate of neuropathic pain in the general population is about 7% to 10%,and there is no definitive etiological treatment at present.There is accumulating evidence showing that exercise therapy may play an active role in peripheral neuropathic pain,but its mechanism is still unclear.microRNAs(miRNAs)are endogenous non-coding microRNAs with a length of about 23 nucleotides.Studies have found that miRNA may play a role in neuropathic pain by regulating pain-related target genes.The dorsal root ganglion(DRG)is the first level of neurons in nociceptive afferent pathway,and its dysfunctions lead to the development of neuropathic pain.So far,information about the adaptive mechanisms after exercise of the DRG is limited.No study systematically exploring the changes of miRNA,mRNA,and biological function of DRG tissue after peripheral neuropathic pain in response to exercise with transcriptome technology is yet available.Based on this,the purpose of this study is as follows: 1.To explore the evidencebased medicine evidence of exercise improving peripheral neuropathic pain;2.To find out transcriptomics changes(miRNA and mRNA)of DRG in rats with neuropathic pain induced by chronic constriction injury(CCI)after exercise intervention,and analyze biological functions and pathways of differentially expressed genes;3.To find the key miRNA and its target genes involved in exercise to improve neuropathic pain induced by CCI.4.To explore the role and mechanism of specific miRNA/mRNA signaling pathway in pain relief of peripheral neuropathic pain by exercise.MethodsThis study consists of four parts.In the first part of the study,PubMed,EMBASE,and Web of Science were searched.Experimental studies of peripheral neuropathic pain were included in the comparison between exercise and no exercise or placebo exercise.Pain-related behavioral tests were selected as outcomes.Mean differences(MDs)with 95% confidence intervals(CIs)were used for continuous data.The extracted data were analyzed by RevMan 5.3 software.In the second part,we chose adult Sprague Dawley rats,and established the model of CCI.The rats were randomly divided into Sham-operated(n = 6),CCI group(n = 6)and CCI-exercised group(n = 6).The CCI-exercised group received 4 weeks of swimming training,and the other two groups received regular feeding.Mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL)were conducted before surgery and on the 3rd,7th,14 th,21st and 28 th postoperative day to assess hyperalgesia following sciatic nerve injury.After 4 weeks,the sciatic nerve was stained with HE to further confirm the success of the CCI model.L4-L6 DRG tissue was taken for RNA-sequencing,and the differentially expressed genes were determined through bioinformatics analysis.Then,the Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were performed on the differentially expressed genes.Finally,quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to analyze the key differentially expressed genes.In the third part,the binding sequence of mir-145-5p and Cacna2d1 was predicted by the bioinformatics software of RNAhybrid.Then double luciferase reporter gene assay experiment was conducted to verify the targeting relationship between mir-145-5p and Cacna2d1.Cacna2d1 3’-UTR sequence and mutated Cacna2d1 3’-UTR sequence were designed and synthesized,and the two target gene fragments were cloned into psiCHECK-2 double luciferase reporter gene vector.The recombinant plasmids of Cacna2d1 wild type(psiCHECK-Cacna2d1)and mutant type(psiCHECK-Cacna2d1-mut)were constructed.The recombinant plasmids were co-transfected with mir-145-5p mimics or mir-145-5p Negative control(NC)into human renal epithelial(293T)cells.Finally,firefly luciferase activity and renilla luciferase activity were detected by double luciferase reporter gene system.In the fourth part,adult Sprague Dawley rats were randomly divided into Na?ve group(n=15),Sham-operated(n=15),Sham-exercised group(n=15),CCI group(n=15)and CCI-exercised group(n=15).The exercise group received swimming training for 4 weeks,while the rest of the groups received regular feeding.Preoperative and postoperative pain-related behavioral tests of MWT and TWL were performed.HE staining was used to observe the morphological changes of ligated sciatic nerve and DRG.The expression changes and position of Cacna2d1 were evaluated by immunofluorescence double staining.In addition,the mRNA and protein expression levels of mir-145-5p and Cacna2d1 in DRG were determined by qRT-PCR and Western blot(WB).ResultsIn the first part of the study,a total of 14 studies were included.For the MWT,rats in the exercised group exhibited significantly higher thresholds than those in the control group,with a MD of 0.91(95% CI 0.11 to 1.71)(P=0.03),3.11(95% CI 1.56 to 4.66)(P <0.001),3.48(95% CI 2.70 to 4.26)(P <0.001),4.16(95% CI 2.53 to 5.79)(P <0.001),and 5.58(95% CI 3.44 to 7.73)(P <0.001)at 1,2,3,4,and 5 weeks,respectively.Additionally,TWL increased in the exercised group compared with the control group,with a MD of 2.48(95% CI 0.59 to 4.38)(P=0.01),3.57(95% CI 2.10 to 5.05)(P <0.001),3.92(95% CI 2.82 to 5.03)(P <0.001),and 2.84(95% CI 1.29 to 4.39)(P <0.001)at 1,2,3,and 4 weeks,respectively.In the second part,CCI model was successfully established.MWT of the CCI and CCI-exercised groups was significantly lower than that of the Sham-operated group on the 3rd,7th,and 14 th postoperative day(P <0.01).On the 21 st and 28 th postoperative day,MWT in the CCI-exercised group was significantly increased compared with that of the CCI group(P <0.01).TWL of the CCI and CCI-exercised groups was significantly lower than that of the Sham-operated group on the 3rd,7th,and 14 th postoperative day(P <0.01).Swimming increased TWL in the CCI-exercised group on days 14,21,and 28 compared with that of the CCI group(P <0.01).HE staining showed that sciatic nerve fibers were scattered,myelin sheath vacuolated,Schwann cells proliferated and peripheral inflammatory cells infiltrated in the CCI group,which indicated the successful of CCI model.A total of 4 overlapping differentially expressed miRNAs(P <0.05)and 186 overlapping differentially expressed mRNAs(P <0.05)were identified in the two comparisons of the Sham-operated group versus the CCI group and the CCI group versus the CCI-exercised group.According to the disease annotations in Rat Genome Database,a total of 14 mRNAs related to neuropathic pain were found.Among these differentially expressed genes,miR-145-5p,miR-341,miR-300-5p,miR-653-5p,Atf3,Cacna2d1,Gal,and Ctss were validated by qRT-PCR.Compared with CCI group,KEGG analysis revealed that the differentially expressed genes were significantly enriched in the classifications of Inflammatory、mediator regulation of TRP channels,TNF signaling pathway,Rap1 signaling pathway,NFkappa B signaling pathway,and MAPK signaling pathway(P <0.05).After 4 weeks of exercise,the HIF-1 signaling pathway,Rap1 signaling pathway,T cell receptor signaling,B cell receptor signaling pathway,and Neurotrophin signaling pathway were enriched(P <0.05).In the third part,RNAhybrid2.2 software predicted that the 3809-3836 base position of Cacna2d1 3’-UTR and mir-145-5p had complete or incomplete complementary binding sites.The results of double luciferase reporter gene assay showed that compared with the group of mir-145-5p NC and psiCHECK-Cacna2d1,the relative fluorescence of the group of mir-145-5p and psiCHECK-Cacna2d1 was significantly reduced(P <0.05).Compared with the group of mir-145-5p NC and psiCHECK-Cacna2d1-mut,there was no significant change in relative fluorescence in the group of mir-145-5p and psiCHECK-Cacna2d1-mut(P >0.05).In the fourth part,on the 21 st and 28 th postoperative day,MWT in the CCIexercised group was significantly increased compared with that of the CCI group(P <0.01).Swimming increased TWL in the CCI-exercised group on days 14,21,and 28 compared with that of the CCI group(P <0.01).HE staining showed that nerve fibers of sciatic nerve were scattered,myelin cavitation,proliferation of Schwann cells,infiltration of peripheral inflammatory cells in CCI group,and significant improvement after exercise.HE staining showed that sciatic nerve fibers were scattered,myelin sheath vacuolated,Schwann cells proliferated and peripheral inflammatory cells infiltrated in CCI group,which were significantly improved after exercise.In addition,for the HE staining of DRG,the number of DRG glial cells increased,the neurons vacuolated,the number of Nissl bodies decreased,and the morphology of nerve fibers was incomplete in CCI group,which was significantly improved after exercise.Immunofluorescence double staining showed that the expression of Cacna2d1 in CCI group was significantly increased in the cell body of DRG neurons,and significantly improved after exercise(P <0.01).The results of qRT-PCR and WB showed that the expression of miR-145-5p mRNA was significantly decreased(P <0.01),while the expression of Cacna2d1 mRNA and protein was significantly increased in CCI group(P <0.01).After 4 weeks of exercise,the expression of miR-145-5p mRNA in the CCIexercised group was significantly higher than that in the CCI group(P <0.05),while the expression levels of Cacna2d1 mRNA and protein in the CCI group were significantly lower than those in the CCI group(P <0.01).Conclusion1.The results of Meta-analysis show that exercise can relief pain of rats with peripheral neuropathic pain,which suggests that exercise plays an important role.However,the results are affected by the risk of bias,which needs to be further verified.2.Among the differentially expressed genes observed,several genes,such as miR-145-5p,miR-341,miR-300-5p,miR-653-5p,Atf3,Cacna2d1,Gal,and Ctss,are related to exercise-induced benefits in the DRG of CCI rats.3.Cell experiments confirmed that Cacna2d1 was the target gene of mir-145-5p,and miR-145-5p inhibited the expression of Cacna2d1 at the post-transcriptional level by binding to the 3’-UTR of Cacna2d1.4.In vivo experiments,it was found that miR-145-5p may be involved in the therapeutic mechanism of exercise in improving hyperalgesia in CCI rats by targeting inhibition of Cacna2d1 expression.