【作者】 王宏伟；

【导师】 杨建军；

【作者基本信息】 郑州大学 ， 麻醉学（专业学位）， 2025， 博士

【摘要】 研究背景神经病理性疼痛(Neuropathic Pain,NP)是发生率高且极具困扰的慢性疼痛亚型之一,对患者的生活质量造成了严重的负面影响,已成为当前亟待解决的医学难题。背根神经节(Dorsal root ganglion,DRG)作为外周神经系统的重要组成部分包含了数量巨大的感觉神经元的胞体,在NP的发生机制中至关重要的作用。在多种因素的作用下,DRG神经元的代谢紊乱、离子通道功能异常、神经递质释放失衡等会影响神经纤维的正常传导,导致异常电信号的产生与传递,从而引发疼痛。可变剪接(Alternative splicing,AS)作为基因表达调控的重要机制,通过剪接因子调控前体m RNA(pre-m RNA)的加工过程,形成不同的剪接异构体,从而对蛋白质功能产生深远影响。神经细胞黏附分子(Neuronal cell adhesion molecule,Nrcam)作为细胞黏附分子免疫球蛋白超家族中的重要成员,其表达水平及其剪接异构体的变化与多种神经系统疾病相关。已有研究发现Nrcam基因的外显子10(E10)剪接变化,参与了神经病理性疼痛的发生和维持。Rbfox蛋白家族(RNA binding proteins,RBPs),是神经和肌肉组织中特异性剪接因子的关键成员。生物信息学分析发现,Nrcam基因E10上游的内含子区域含有Rbfox1的识别序列,提示Rbfox1可能通过调控Nrcam基因的可变剪接在神经病理性疼痛中发挥作用。基于此,本研究旨在探讨DRG水平Rbfox对Nrcam基因可变剪接的调控机制及其在NP中的作用。为神经病理性疼痛的精准治疗提供新的视角和潜在的靶点。研究目的探讨在外周神经损伤后,Rbfox1是否通过可变剪接调控背根神经节神经元中Nrcam的表达,进而影响神经病理性疼痛的发生发展。研究方法(1)对C57BL/6小鼠建立脊神经结扎(Spinal Nerve Ligation,SNL)模型或慢性压迫损伤(Chronic Constriction Injury,CCI)模型。(2)通过机械痛敏测试、热痛敏测试、冷痛敏测试进行疼痛行为学分析。(3)利用RNA测序技术(RNA-Seq)分析SNL组与假手术组之间的差异剪接事件。通过r MATS(复制转录本剪接的多变量分析,replicateMultivariate Analysis of Transcript Splicing)检测跳跃外显子(SE)、保留内含子(RI)、互斥外显子(MXE)、可变5’剪接位点(A5SS)和可变3’剪接位点(A3SS)五种剪接模式;通过RT-qPCR/Western Blot技术检测Rbfox1及Nrcam变体(L-Nrcam和S-Nrcam)的m RNA/蛋白质表达水平;通过免疫组化分析,定位Rbfox1在DRG中的表达分布;通过病理切片和免疫组化检测pERK1/2和GFAP表达水平,评估神经元和星形胶质细胞的激活情况。(4)通过AAV病毒介导的Rbfox1基因过表达与沉默,探究Rbfox1与NP发生的机制。(5)通过基于Nrcam外显子10两侧剪接位点设计的特异性反义寡核苷酸(Antisense Oligonucleotides,AON),干扰剪接体识别实现外显子跳跃。将AON通过涡旋转染剂混合后注射至DRG,检测其对Nrcam的长变体(L-Nrcam,包含外显子10)增加,和短变体(S-Nrcam,不含外显子10)表达的影响,并进行相应的行为学验证。研究结果(1)RNA测序结果显示,在脊神经结扎(SNL)诱导的神经病理性疼痛模型中,Rbfox1的表达在受损侧L4 DRG显著下降。(2)通过r MATS分析发现,SNL导致了DRG中多种可变剪接事件发生显著变化,其中包括Nrcam外显子10的插入。在SNL模型鼠DRG中,L-Nrcam转录增加,而S-Nrcam转录减少。RT-qPCR和Western blot验证表明,L-Nrcam在第3天、第7天和第14天分别增加34%、72%和66%,而S-Nrcam在相应时间点分别减少21%、47%和43%。(3)RT-qPCR验证显示,Rbfox1 m RNA在SNL术后第3天下降23%,第7天下降51%,第14天下降46%。Western blot结果表明,Rbfox1蛋白水平在术后第3天、第7天和第14天分别下降19%、45%和43%(P<0.01)。相比之下,未受损侧DRG及脊髓背角未观察到明显变化,表明Rbfox1表达的下调是受损侧DRG特异性的。(4)免疫组化分析显示,Rbfox1主要定位于DRG神经元。此外,Rbfox1阳性神经元中,约51.4%为小型神经元(胞体面积<300μm~2),30.5%为中型神经元(300μm~2≤胞体面积≤600μm~2),18.1%为大型神经元(胞体面积>600μm~2)。SNL后,Rbfox1阳性神经元数量在第7天减少39.4%(P<0.01)。进一步分析显示,小型非肽能神经元(IB4阳性)和小型肽能神经元(CGRP阳性)的Rbfox1表达显著减少。(5)通过RNA免疫共沉淀实验(RIP)验证了Rbfox1直接结合于Nrcam外显子10的上游和下游剪接调控元件。功能实验表明,Rbfox1的过表达能够减少L-Nrcam的生成,同时增加S-Nrcam的比例。反之,抑制Rbfox1表达会导致L-Nrcam的显著升高和S-Nrcam的减少。(6)通过AAV5-Rbfox1病毒介导的Rbfox1过表达实验,发现恢复Rbfox1表达能够显著缓解SNL小鼠的疼痛敏感性。在机械刺激实验中,注射Rbfox1后爪抽动频率降低约40%;热刺激实验中,爪抬起潜伏期延长约35%;冷刺激实验中,爪抬起潜伏期延长约28%(P<0.01)。非手术侧未观察到显著变化。在Rbfox1过表达的小鼠中,脊髓背角神经元活化标志物pERK1/2和星形胶质细胞活化标志物GFAP的表达显著降低,进一步证实了Rbfox1恢复对神经病理性疼痛的缓解作用。(7)通过AAV5-Rbfox1 shRNA病毒介导的Rbfox1表达抑制,发现未经历SNL的小鼠也表现出显著的机械痛敏和热/冷痛敏现象。此外,CPP实验显示,小鼠明显偏好与利多卡因配对的环境,提示小鼠出现了自发性疼痛。Rbfox1下调的小鼠中,pERK1/2和GFAP的表达水平显著升高,与SNL小鼠表现相似。这表明Rbfox1下调可以引发类似于神经病理性疼痛的病理生理变化。(8)在Nrcam反义寡核苷酸(AON)干预实验中,通过阻断Nrcam外显子10的剪接,显著减少了L-Nrcam的表达,改善了SNL诱导的机械痛敏和热/冷痛敏现象。这一结果进一步证实,Rbfox1通过调控Nrcam剪接变体的平衡,在神经病理性疼痛的发生中起关键作用。研究结论本研究揭示了RNA结合蛋白Rbfox1在神经病理性疼痛中的作用及其分子机制。Rbfox1在外周神经损伤后显著下调,通过调控Nrcam的可变剪接,导致长变体(L-Nrcam)增加、短变体(S-Nrcam)减少,进而促进疼痛敏感性的发生。恢复Rbfox1表达显著缓解了疼痛行为,并抑制神经元和胶质细胞的过度活化,而敲减Rbfox1可诱发疼痛敏感性和神经活动异常。此外,靶向干预Nrcam剪接变体的生成进一步验证了其在疼痛中的关键作用。本研究为神经病理性疼痛的分子机制提供了新见解,明确了Rbfox1和Nrcam剪接变体作为潜在治疗靶点的价值,为开发针对性的疼痛治疗策略提供了理论支持。更多还原

【Abstract】 BackgroundNeuropathic pain(NP)constitutes a particularly debilitating and widespread category of chronic pain in clinical practice.It has a serious negative impact on health-related quality of life and has constitutes a therapeutically refractory condition demanding multidisciplinary intervention..Dorsal root ganglion(DRG),as an important part of the peripheral nervous system,contains a large number of sensory neuron cell bodies and plays a vital role in the pathogenesis of NP.Under the influence of various factors,metabolic disorders,ion channel dysfunction,and imbalanced neurotransmitter release of DRG neurons will affect the normal conduction of nerve fibers,leading to the generation and transmission of abnormal electrical signals,thereby causing pain.Alternative splicing(AS)serves as a pivotal post-transcriptional regulatory strategy,generating diverse m RNA isoforms that dictate protein functionality through combinatorial inclusion/exclusion of exonic elements.It regulates the processing of pre-m RNA through splicing factors to form different splicing isoforms,which has a profound effect on protein function.As a crucial member of the immunoglobulin superfamily cell adhesion molecules,neuronal cell adhesion molecule(Nrcam)plays a significant role in cellular interactions.The changes in its expression level and splicing isoforms are related to a variety of nervous system diseases.Studies have found that splicing changes in exon 10(E10)of the Nrcam gene are involved in the occurrence and maintenance of neuropathic pain.The Rbfox protein family(RNA binding proteins,RBPs)is a key member of specific splicing factors in nerve and muscle tissues.Bioinformatics analysis found that the intron region upstream of E10of the Nrcam gene contains the recognition sequence of Rbfox1,suggesting that Rbfox1 may play a role in neuropathic pain by regulating the alternative splicing of the Nrcam gene.Based on this,this study aims to explore the regulatory mechanism of Rbfox at the DRG level on the alternative splicing of the Nrcam gene and its role in NP.Provide new perspectives and potential targets for the precise treatment of neuropathic pain.ObjectiveTo investigate whether Rbfox1 regulates the expression of Nrcam in dorsal root ganglion neurons through alternative splicing after peripheral nerve injury,thereby affecting the occurrence and development of neuropathy pain.Methods(1)Spinal nerve ligation(SNL)model or chronic constriction injury(CCI)model was established in C57BL/6 mice.(2)Pain behavior analysis was performed through mechanical pain sensitivity test,thermal pain sensitivity test,and cold pain sensitivity test.(3)RNA sequencing technology(RNA-Seq)was used to analyze the differential splicing events between the SNL group and the sham operation group.r MATS(replicate Multivariate Analysis of Transcript Splicing)was used to detect five splicing patterns,including skipped exons(SE),retained introns(RI),mutually exclusive exons(MXE),alternative 3’splicing sites(A3SS)and alternative 5’splicing sites(A5SS);RT-qPCR/Western blot technology was used to detect the m RNA/protein expression levels of Rbfox1 and Nrcam variants(L-Nrcam and S-Nrcam);immunohistochemical analysis was used to locate the expression distribution of Rbfox1 in DRG;pathological sections and immunohistochemistry were used to detect the expression levels of pERK1/2 and GFAP to evaluate the activation of neurons and astrocytes.(4)The mechanism of Rbfox1 and NP occurrence was explored through AAV virus-mediated Rbfox1 gene overexpression and silencing.(5)Exon skipping was achieved by interfering with spliceosome recognition using specific antisense oligonucleotides(AONs)designed based on the splice sites on both sides of Nrcam exon 10.AONs were mixed with dye by vortexing and then injected into DRG to detect their effects on the increase of the long variant(L-Nrcam,containing exon 10)and the expression of the short variant(S-Nrcam,excluding exon10)of Nrcam,and corresponding behavioral verification was performed.Results(1)Rbfox1 m RNA levels were significantly decreased in L4 DRGs following spinal nerve ligation(SNL),confirming this gene’s involvement in neuropathic pain pathogenesis.(2)r MATS analysis revealed that SNL caused significant changes in multiple alternative splicing events in DRG,including the insertion of Nrcam exon 10.In the DRG of SNL model mice,L-Nrcam transcription increased,while S-Nrcam transcription decreased.RT-qPCR and Western blot validation showed that L-Nrcam increased by 34%,72%and 66%on the 3rd,7th and 14th days,respectively,while S-Nrcam decreased by 21%,47%and 43%at the corresponding time points,respectively.(3)RT-qPCR validation showed that Rbfox1 m RNA decreased by 23%on the 3rd day after SNL surgery,51%on the 7th day,and 46%on the 14th day.Western blot results showed that Rbfox1 protein levels decreased by 19%,45%,and 43%on the 3rd,7th,and 14th days after surgery,respectively(P<0.01).In contrast,no significant changes were observed in the uninjured DRG and spinal cord dorsal horn,indicating that the downregulation of Rbfox1 expression was specific to the injured DRG.(4)Immunohistochemical analysis showed that Rbfox1 was mainly localized in DRG neurons.In addition,among Rbfox1-positive neurons,approximately 51.4%were small neurons(cell body area<300μm~2),30.5%were medium-sized neurons(300μm~2≤cell body area≤600μm~2),and 18.1%were large neurons(cell body area>600μm~2).After SNL,the number of Rbfox1-positive neurons decreased by 39.4%on the 7th day(P<0.01).Further analysis showed that Rbfox1 expression in small non-peptidergic neurons(IB4-positive)and small peptidergic neurons(CGRP-positive)was significantly reduced.(5)RNA immunoprecipitation(RIP)experiments verified that Rbfox1 directly bound to the upstream and downstream splicing regulatory elements of Nrcam exon 10.Functional experiments showed that overexpression of Rbfox1 could reduce the production of L-Nrcam and increase the proportion of S-Nrcam.Conversely,inhibition of Rbfox1 expression led to a significant increase in L-Nrcam and a decrease in S-Nrcam.(6)Through AAV5-Rbfox1 virus-mediated Rbfox1 overexpression experiments,it was found that restoring Rbfox1 expression could significantly alleviate the pain sensitivity of SNL mice.In the mechanical stimulation experiment,the paw twitching frequency decreased by about 40%after Rbfox1 injection;in the heat stimulation experiment,the paw lifting latency was prolonged by about 35%;in the cold stimulation experiment,the paw lifting latency was prolonged by about 28%(P<0.01).No significant changes were observed on the non-operated side.In mice with Rbfox1overexpression,the expression of pERK1/2,a marker of neuronal activation in the dorsal horn of the spinal cord,and GFAP,a marker of astrocyte activation,was significantly reduced,further confirming the alleviating effect of Rbfox1 restoration on neuropathic pain.(7)Through AAV5-Rbfox1 shRNA virus-mediated inhibition of Rbfox1expression,it was found that mice that did not experience SNL also showed significant mechanical hyperalgesia and heat/cold hyperalgesia.In addition,the CPP experiment showed that mice clearly preferred the environment paired with lidocaine,suggesting that the mice had spontaneous pain.In mice with Rbfox1 downregulation,the expression levels of pERK1/2 and GFAP were significantly increased,similar to the performance of SNL mice.This finding suggests that the knockdown of Rbfox1 may induce pathophysiological changes resembling neuropathic pain.(8)In the Nrcam antisense oligonucleotide(AON)intervention experiment,by blocking the splicing of Nrcam exon 10,the expression of L-Nrcam was significantly reduced,and the mechanical hyperalgesia and heat/cold hyperalgesia induced by SNL were improved.This result further confirms that Rbfox1 plays a key role in the occurrence of neuropathic pain by regulating the balance of Nrcam splicing variants.ConclusionThis study revealed the role of RNA binding protein Rbfox1 in neuropathic pain and its molecular mechanism.Rbfox1 is significantly downregulated after peripheral nerve injury,and by regulating the alternative splicing of Nrcam,it leads to an increase in the long variant(L-Nrcam)and a decrease in the short variant(S-Nrcam),thereby promoting the occurrence of pain sensitivity.Restoring Rbfox1 expression significantly alleviated pain behavior and inhibited the overactivation of neurons and glial cells,while knocking down Rbfox1 induced pain sensitivity and abnormal neural activity.In addition,targeted intervention in the generation of Nrcam splicing variants further verified its key role in pain.This study provides new insights into the molecular mechanism of neuropathic pain,clarifies the value of Rbfox1 and Nrcam splicing variants as potential therapeutic targets,and provides theoretical support for the development of targeted pain treatment strategies.更多还原