| Background: Neuropathic pain characterized by hyperalgesia,allodynia and spontaneous pain is caused by the inflammation and nerve injury.This chronic neuropathy pain can persist for years seriously diminishing quality of life.However,the treatment becomes problematic as litter is known about the mechanism leading to neuropathic pain with the incidence of pain has increased in these years and current treatment provides poor efficacy.Therefore,neuropathic pain has become a challenging research project.Some recent studies indicated that the generation and maintenance of neuropathic pain are closely associated with the changes on the level of pain-related molecules transcription and expression.Thus,how pain-related genes are programmed to be activated for transcription,translation and enhancing expression or reducing expression by specific inhibition in response to modulate pain has been the new direction toward neuropathic pain mechanism,which was involved in epigenetics.Epigenetics refers to changes in gene expression that do not entail a change in DNA sequence,which reflected in methylation on the DNAbases,chromatin remodeling,histone acetylation,as well as non-coding RNA regulation.Epigenetic changes are reversible.Therefore,we can use the appropriate epigenetic drugs to make the gene slience or make the silence gene re-expresse.With molecular biology technology,up-regulation or down-regulation of pain-related genes has become a new target of the treatment for neuropathic pain.DNA methylation and non-coding mi RNA regulation are the earliest and most common ways to regulate gene expression.The DNA modification could turn off the activity of certain genes,and demethylationcoula contributed to gene reactivition and expression.microRNA(miRNA)is endogenous non-coding RNA(19-25nt).The incomplete combination of complex consisted of miRNA and RISC and non-encoding area of the 3’ end of target mRNA could prevent miRNA translating,and widely participate in post-transcriptional gene regulation.Some researches have demonstrated that miRNA took part in a variety way of regulatory pathways,playing an important role in gene expression regulation.Our team in previous studies have demonstrated that epigenetic mechanism,especially DNA methylation mechanism involved in the development of neuropathic pain.In addtion,our studies suggested that DNMT3 a methylase plays a key role in the development of neuropathic pain,whereas the research about miRNA by post-transcriptional regualtion on methylase DNMT3 a to modulate pain is still very few.This paper use rat model with SNL neuropathic pain via cell culture in vitro and experiments in vivo to confirm that mi RNA-143(miR-143)can regulate DNMT3 a post-transcriptional expression that could affect methylation level of MOR promoter,then reduce MOR protein expression,and subsequently affect the development of pain.This study provides new insight into the treatment of neuropathic pain.At last,a prospective randomized clinical trial was carried out to observe the analgesic efficacy of ropivacaine for postoperative pain following thoracolumbar spinal surgery.Method: Step 1:To detect the changes of miR-143 in neuropathic pain model : male Sprague-Dawley(SD)adult rats were random assigned sham-operated group and spinal nerve ligation(SNL)group.After rats were anesthetized in SNL group,L5 spinal nerves were exposed,ligated and cut off.The rats in sham group were performed the same procedure,and L5 spinal nerves were exposed without ligation.The mechanical and thermal pain threshold were measured at 3d and 7d after operation,then rats were decapitated under anesthesia for extracting bilateral spinal ganglion to determine mi RNA-143 expression with reverse transcription quantitative polymerase chain reaction(qRT-PCR)analysis.Step 2: To detect the effect of mimics-miR-143 overexpression in vivo on neuropathic pain: the spinal ganglions were exposed under anesthesia,then microinjected mimics-miR-143 into the spinal ganglion with the technology of spinal ganglion microinjection to observe the effect of miR-143 on neuropathic pain.Rats were random assigned five group,Naive group 、 Veh+SNL group(microinject saline 2.0μl into L5 spinal ganglion)、mi R-143+SNLgroup(microinject mi R-143 2.0μl into L5 spinal ganglion),miR-143+Sham group(microinject mi R-143 2.0μl into L5 spinal ganglion)、 negative control(NC)+SNL(microinject NC miR-143 2.0μl into L5 spinal ganglion).Each group were performed SNL surgery or sham surgery(spinal ganglion only exposed without ligation)at 3d after microinjection.The mechanical pain thermal-hyperalgesia and cold-allodyniawere measured at 3、5、7d after surgery.Step 3: To detect the effect of inhibition of miR-143 on the pain behaviors in rats: the spinal ganglions were exposed and microinjected inhibitor-mi R-143 with spinal ganglion microinjection technology to observe the effect of mi R-143 down-regulation on neuropathic pain.The group was assigned vehicle(exposed L4 and L5 DRG were microinjected saline 2.0μl respectively),miR-143 inhibitor group(exposed L4 and L5 DRG were microinjectded miR-143 inhibitor 2.0μl respectively),inhibitor control group(exposed L4 and L5 DRG were microinjected miR-143 negative inhibitor 2.0μl).The mechanical,thermal and cold pain threshold were measured at 3,5 d after microinjection.Step 4: To explore the mechanism underlying miR-143 in vitro: primary cultured the spinal ganglion cells extracted from 3-week old SD rats,then transfected mimics-miR-143.After transfection,the cells were collected to extract RNA and protein,and observe changes in the mRNA and protein of downstream target genes DNMT3 a with qRT-PCR or western-blot analysis.Step 5:To explore the mechanism underlying mimics-miR-143 overexpression in vivo on neuropathic pain: spinal ganglions were exposed under anesthesia,then microinjected mimics-miR-143 with spinal ganglion microinjection technology to observe the effect of miR-143 with high expression on downstream protein DNMT3 a.SD rats were divided into five groups: Naive group,Veh+SNl group(exposed L5 spinal ganglion were microinjected saline 2.0μl),miR-143+SNL group(exposed L5 spinal ganglions were microinjected miR-143 2.0μl),miR-143+sham group(exposed L5 spinal ganglions were microinjected miR-143 2.0μl),NC+SNL(exposed L5 spinal ganglions were microinjected NC miRNA 2.0μl).Each group were preformed SNL surgey or sham surgey(only exposed spinal ganglion without ligation)at 3d after injection.L5 spinal ganglions were extracted at 5d after SNL sugery to detect the changes of mRNA expression in miR-143 down-stream gene DNMT3 a and DNMT3 a downstream gene MOR with qRT-PCR analysis.Moreover,the spinal ganglions were extracted at 7d after SNL sugery to detect the changes of protein expression in miRNA-143 down-stream gene DNMT3 a and DNMT3 a down-stream gene MOR with western-blot analysis.Step 6:To detect the effect of suppressed expression of miR-143 on the expression changes of mRNA and protein in downs-tream DNMT3 a and DNMT3 a down-stream gene MOR: spinal ganglions were exposed under anesthesia,then microinjected inhibitor miR-143 with spinal ganglion microinjection technology.The experimental group were divided into Vehicle group(exposed L4 and L5 DRG were microinjected miR-143 inhibitor 2.0μl),miR-143 inhibitor group(exposed L4 and L5 DRG were microinjected miR-143 inhibitor 2.0μl),inhibitor group(exposed L4 and L5 DRG were microinjected mi R-143 negative inhibitor 2.0μl).The L4 and L5 DRG were extracted under anesthesia at 5d after injection to detect the changes of mRNA and protein expression in down-stream DNMT3 a and DNMT3 a down-stream gene MOR.Step 7: Seventy-one patients with elective posterior thoracolumbar spinal surgery were randomly divided into two groups.Local group received 0.33% ropivacaine by pump through the wound,and intravenous group received flurbiprofen axetil,pentazocine and palonosetron via intravenous pump.We evaluated the level of pain,the incidence of adverse reactions at 2,4,6,12,24,36 and 48 h after operation,and the occurrence of chronic pain three months later.Result: 1.The result that mechanical,thermal and cold pain threshold were measured in SNL group at 3,5d comparing with sham group implicated that the foot mechanical,thermal and cold pain threshold were drastically decreased,and the difference was statistically significant(P<0.05).miR-143 expression was measured with qRT-PCR analysis at 3 and 7d after surgery,and in comparision with sham group the miR-143 in L5 DRG was dramatically decreased in SNL group,the difference was statistically significant(P<0.05).2.The effect of mimics-miR-143 overexpression in vivo on neuropathic pain: compared with Naive group,the result of qRT-PCR in miR-143+sham group and mi R-143+SNL group diplayed that mi R-143 was successfully highexpressed with a remarkable increase,and the increased miR-143 was statistically significant(P<0.05).Behavior tests shown that compared to Naive group the mechanical,therma and cold pain threshold in Veh+SNLgroup and NC group were strikingly cincreased,and these differences were statistically significant(P<0.05).3.The qRT-PCR result shown that miR-143 inhibitor could reduce the expression of mi R-143 in L4\L5 DRG.Compared with vehicle group and inhibitor NC group,the mechanical,thermal and cold pain thresold were decreased,the differences were statistically significant(P<0.05).4.The spinal ganglion cells from primary cultured in SD rats were transfected mimics mi R-143.Westerrn-blot result shown that DNMT3 a protein expression was decreased while MOR expression was increased,both differences were statistically significant(P<0.05).Compared with Western-blot,the qRT-PCR result shown that the mRNA of DNMT3 a failed to decreased,but mRNA expression of DNMT3 a down-stream gene MOR was increased,the differences were statistically significant(P<0.05).5.The mimics-miR-143 overexpression in vivo shown that compared to Naive group,the mRNA of DNMT3 a were significantly increased in Veh +SNL group,NC+SNL group and miR-143+SNL group(P<0.05)while the mRNA of MOR were significantly decreased(P<0.05)in Veh+SNL group and NC+SNL group.Besides,DNMT3 a protein expression was significantly increased in Veh+SNL group and NC+SNL group while MOR protein espression were decreased,both differences were not statistically significant(P<0.05).Compared with Veh+SNL group and NC+SNL group,DNMT3 a protein expression was significantly decreased while MOR protein expression was increased in miR-143+SNL group,both differences were statistically significant(P<0.05).6.The microinjection miR-143 inhibitor in spinal ganglion in vivo shown that compared with Vehicle group and inhibitor NC group,the changes of DNMT3 a mRNA in miR-143 inhibitor group was not statistically significant(P>0.05)but the increased in DNMT3 a protein expression was statistically significant(P<0.05).Both decreased protein and mRNA expression of MOR were statistically significant(P<0.05).7.There were no significant differences in the pain level between the two groups.However,the incidence of nausea,vomiting and chronic pain was significantly lower in the local group.Conclusion: 1.Epigenetic plays an important role in chronic neuropathic pain where the decreased miR-143 and increased DNMT3 a are involved in the development of neuropathic pain.2.miR-143 could affect DNMT3 a protein expression through DNMT3 a transcriptional regulation,and DNMT3 a could affect the development of neuropathic pain depending on the changes on the level of MOR promoter methylation.3.Exogenous mi R-143 could treat neuropathic pain by reducing the level of DNMT3 a protein,decreasing MOR methylation and enhancing MOR expression,which may become a new drug to treat neuropathic pain.4.Our results showed that local infusion of ropivacaine achieved similar analgesic effects to intravenous delivery of analgesic drugs,but significantly reduced incidence of nausea,vomiting and chronic pain. |
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