Skip to main content
NCBI home page
Journal of Central South University Medical Sciences logoLink to Journal of Central South University Medical Sciences
. 2024 Jan 28;49(1):29–39. [Article in Chinese] doi: 10.11817/j.issn.1672-7347.2024.230273

MiR - 21 - 5p通过下调电压门控钾离子通道Kv1.1表达减轻大鼠三叉神经痛

MiR - 21 - 5p alleviates trigeminal neuralgia in rats through down-regulation of voltage-gated potassium channel Kv1.1

ZHOU Xuewen 1,2,2,#, GUO Gangwen 1,3,#, YU Shanzi 3, HU Rong 1,✉
Editor: 彭 敏宁
PMCID: PMC11017021  PMID: 38615163

Abstract

Objective

Trigeminal neuralgia (TN) is a common neuropathic pain. Voltage-gated potassium channel (Kv) has been confirmed to be involved in the occurrence and development of TN, but the specific mechanism is still unclear. MicroRNA may be involved in neuropathic pain by regulating the expression of Kv channels and neuronal excitability in trigeminal ganglion (TG). This study aims to explore the relationship between Kv1.1 and miR-21-5p in TG with a TN model, evaluate whether miR-21-5p has a regulatory effect on Kv1.1, and to provide a new target and experimental basis for the treatment of TN.

Methods

A total of 48 SD rats were randomly divided into 6 groups: 1) a sham group (n=12), the rats were only sutured at the surgical incision without nerve ligation; 2) a sham+agomir NC group (n=6), the sham rats were microinjected with agomir NC through stereotactic brain injection in the surgical side of TG; 3) a sham+miR-21-5p agomir group (n=6), the sham rats were microinjected with miR-21-5p agomir via stereotactic brain injection in the surgical side of TG; 4) a TN group (n=12), a TN rat model was constructed using the chronic constriction injury of the distal infraorbital nerve (dIoN-CCI) method with chromium intestinal thread; 5) a TN+antagonist NC group (n=6), TN rats were microinjected with antagonist NC through stereotactic brain injection method in the surgical side of TG; 6) a TN+miR-21-5p antagonist group (n=6), TN rats were microinjected with miR-21-5p antagonist through stereotactic brain injection in the surgical side of TG. The change of mechanical pain threshold in rats of each group after surgery was detected. The expressions of Kv1.1 and miR-21-5p in the operative TG of rats were detected by Western blotting and real-time reverse transcription polymerase chain reaction. Dual luciferase reporter genes were used to determine whether there was a target relationship between Kv1.1 and miR-21-5p and whether miR-21-5p directly affected the 3'-UTR terminal of KCNA1. The effect of brain stereotaxic injection was evaluated by immunofluorescence assay, and then the analogue of miR-21-5p (agomir) and agomir NC were injected into the TG of rats in the sham group by brain stereotaxic apparatus to overexpress miR-21-5p. The miR-21-5p inhibitor (antagomir) and antagomir NC were injected into TG of rats in the TN group to inhibit the expression of miR-21-5p. The behavioral changes of rats before and after administration were observed, and the expression changes of miR-21-5p and Kv1.1 in TG of rats after intervention were detected.

Results

Compared with the baseline pain threshold, the facial mechanical pain threshold of rats in the TN group was significantly decreased from the 5th to 15th day after the surgery (P<0.05), and the facial mechanical pain threshold of rats in the sham group was stable at the normal level, which proved that the dIoN-CCI model was successfully constructed. Compared with the sham group, the expression of Kv1.1 mRNA and protein in TG of the TN group was down-regulated (both P<0.05), and the expression of miR-21-5p was up-regulated (P<0.05). The results of dual luciferase report showed that the luciferase activity of rno-miR-21-5p mimics and KCNA1 WT transfected with 6 nmol/L or 20 nmol/L were significantly decreased compared with those transfected with mimic NC and wild-type KCNA1 WT, respectively (P<0.001). Compared with low dose rno-miR-21-5p mimics (6 nmol/L) co-transfection group, the relative activity of luciferase in the high dose rno-miR-21-5p mimics (20 nmol/L) cotransfection group was significantly decreased (P<0.001). The results of immunofluorescence showed that drugs were accurately injected into TG through stereotaxic brain. After the expression of miR-21-5p in the TN group, the mechanical pain threshold and the expression of Kv1.1 mRNA and protein in TG were increased. After overexpression of miR-21-5p in the sham group, the mechanical pain threshold and the expression of Kv1.1 mRNA and protein in TG were decreased.

Conclusion

Both Kv1.1 and miR-21-5p are involved in TN and miR-21-5p can regulate Kv1.1 expression by binding to the 3'-UTR of KCNA1.

Keywords: voltage-gated potassium channel, miR-21-5p, trigeminal neuralgia, trigeminal ganglion

三叉神经痛(trigeminal neuralgia，TN)是一种局限于三叉神经分布区域的剧烈疼痛，其特征是间歇反复发作和突发突止。通常可由非伤害性刺激触发，是头面部最常见的神经病理性疼痛形式[1]。深入研究TN机制并找到有效的治疗靶点具有重要的医学价值和社会意义。

电压门控性钾通道(voltage-gated potassium channel，Kv)是一种具有特殊功能的钾离子通道，它可以通过控制细胞膜上的电压维持静息膜电位水平以及通过调节细胞内外离子平衡来介导动作电位的快速复极化来调节神经元兴奋性[2]。在临床前研究中，Kv通道介导的静息电位失调会导致半月神经节(trigeminal ganglion，TG)上神经元过度兴奋[3]，外周神经损伤后，TG上的Kv1(Kv1.1、Kv1.2、Kv1.4)表达减少[4]。Kv1.1可调节与机械感知相关的纤维的机械灵敏度，其功能丧失会降低放电阈值、机械痛阈和热痛阈[5]。因此，推测Kv1.1可能是TN研究的一个关键靶点。

近年来越来越多的研究者关注微RNA(microRNA，miR)与慢性疼痛的关系。已有多项研究[6-7]表明：miR参与神经病理性疼痛的发生、发展过程，并被认为是缓解神经病理性疼痛的新靶点。MiR可以通过调控Kv调节神经病理性疼痛。Zhang等[8]研究发现：在坐骨神经损伤大鼠模型中，背根神经节的miR-137可以调控下游的Kv1.2表达从而缓解神经病理性疼痛。Sakai等[9]研究发现：miR-17-92同样可以下调钾离子通道的表达，协同调节多个Kv亚基的功能，使脊神经结扎模型大鼠的机械痛觉过敏持续存在。

本研究通过miR靶位点预测网站TargetScan(www.targetscan.org/vert_80/)预测靶向作用于大鼠Kv1.1编码基因KCNA1，发现miR-21-5p与KCNA1高度相关。已有研究[10-13]报道miR-21-5p可通过靶向抑制肿瘤坏死因子α诱导蛋白3(tumor necrosis factor alpha-inducible protein 3，TNFAIP3)、转化生长因子β1(transforming growth factor β1，TGF-β1)、组织金属蛋白酶抑制因子3(tissue metalloproteinase inhibitor factor 3，TIMP3)等基因的表达而调控细胞增殖、分化和凋亡，参与多种癌症和神经系统疾病的发病机制，但miR-21-5p是否通过介导KCNA1参与TN发病鲜见报道。因此本研究拟检测大鼠TN模型中Kv1.1和miR-21-5p的表达，并验证miR-21-5p是否可以通过调控TG中Kv1.1的表达来缓解TN。

1. 材料与方法

1.1. 材料

1.1.1. 细胞与试剂

HEK293细胞购自上海中乔新舟生物科技有限公司；miR模拟物[包括miR-21-5p agomir和对照mimics (agomir NC)、miR抑制剂(包括miR-21-5p antagomir和antagomir NC)]等均购自中国生工生物工程股份有限公司；Kv1.1抗体购自以色列Alomone公司；β-actin购自中国武汉三鹰生物技术有限公司；双荧光素酶报告系统购自长沙维世尔生物公司；蛋白质印迹法相关试剂购自中国Biosharp公司；PCR引物购自北京擎科生物科技有限公司；miR反转录试剂盒购自中国Vazyme公司；免疫荧光实验相关抗体购自英国Abcam公司。

1.1.2. 动物和分组

SPF级雄性SD大鼠48只，体重为200~220 g，由中南大学实验动物学部提供[实验动物合格证号SCXK(湘)2019-0004]。将大鼠随机分为6组：1)假手术组(sham组，n=12)，大鼠仅在术侧切口缝合，不结扎神经；2)Sham+agomir NC组(n=6)，sham大鼠通过脑立体定位注射方法于术侧TG微量注射agomir NC；3)Sham+miR-21-5p agomir组(n=6)，sham大鼠通过脑立体定位注射方法于术侧TG微量注射miR-21-5p agomir；4)TN组(n=12)，采用铬肠线慢性缩窄性眶下远端神经损伤(chronic constriction injury of the distal infraorbital nerve，dIoN-CCI)法构建TN大鼠模型；5)TN+antagomir NC组(n=6)，TN大鼠通过脑立体定位注射方法于术侧TG微量注射antagomir NC；6)TN+miR-21-5p antagomir组(n=6)，TN大鼠通过脑立体定位注射方法于术侧TG微量注射miR-21-5p antagomir。本研究获得中南大学湘雅三医院伦理委员会批准(审批号：2018-S279)。

1.2. 方法

1.2.1. 细胞培养

选用HEK293细胞，置于细胞培养皿中，加入含10%胎牛血清的DMEM培养基，置于37 ℃恒温培养箱中，在5% CO2的环境中培养。定期进行分瓶传代，直到细胞生长状态良好、背景干净无杂质、细胞融合度达到80%后，才用于后续实验。

1.2.2. 双荧光素酶报告实验

首先用PBS缓冲液将已培养好的HEK293细胞洗涤2次，随后加入胰蛋白酶-EDTA溶液，充分混匀并放置于37 ℃下1 min。小心吸取胰酶溶液并弃去，随后加入含有10% FBS的DMEM培养液，小心轻柔吹打混匀细胞，形成单细胞悬液。通过细胞计数仪对其计数，每孔以1×106/mL的密度加入0.5 mL悬液，并培养24 h。

按照试剂盒说明书，取适量的不含有FBS的DMEM培养基与各组不同的双荧光素酶报告基因载体以及各组miRNAs共同转染。实验共分为9组：mimic NC+pmirGLO，rno-miR-21-5p mimics (6 nmol/L)+pmirGLO，rno-miR-21-5p mimics(20 nmol/L)+pmirGLO；mimic NC+KCNA1 WT，rno-miR-21-5p mimics (6 nmol/L)+KCNA1 WT，rno-miR-21-5p mimics (20 nmol/L)+KCNA1 WT；mimic NC+KCNA1 MUT，rno-miR-21-5p mimics (6 nmol/L)+KCNA1 MUT，rno-miR-21-5p mimics (20 nmol/L)+KCNA1 MUT。

按照不同组别的设定，将各组转染后的混合物滴入接种HEK293细胞的孔板内，培养5 h。随后在各孔中加入适量含有10% FBS的DMEM培养液，转染48 h后，采用双荧光素酶试剂盒测定各组荧光素酶的相对活性。

1.2.3. 动物模型制备

在正式造模前，需要将大鼠放置于安静环境下对其进行3 d的术前适应性训练。其间用VonFrey纤维丝对大鼠双侧触须垫进行测试，3 d后筛选触须完整且双侧面部机械痛阈稳定的SD大鼠用于dIoN-CCI造模。使用3%戊巴比妥钠溶液(0.2 mL/100 g)对大鼠进行腹腔注射。待大鼠麻醉后，剔除手术区域毛发，表面皮肤用络合碘消毒后，在左侧触须垫区后方和眶下颧弓下缘与第2、3排触须之间的交界处，用手术剪作一切口，长约5 mm，切口与眼裂平行。钝性分离切口下方的肌肉和筋膜，完整暴露出眶下神经后，用眼科弯镊轻轻分离。TN组大鼠用2根5-0可吸收铬肠线结扎眶下神经，2根结扎线之间的间距约 2 mm。结扎完成后4-0丝线缝合皮肤创口。Sham组大鼠在造模过程中，除了不对眶下神经进行结扎外，其余手术步骤均同TN组大鼠。

1.2.4. TG脑立体定位注射及验证

大鼠麻醉后，待四肢瘫软且对捏尾刺激无明显体动反应时，对手术区域进行剃毛，随后将大鼠俯卧位摆放并固定头部。消毒术区，使用手术剪在大鼠双眼连线至双耳连线区域纵向剪开1 cm，使大鼠的前囱、后囱位置充分暴露于视野下，并且调整两者位于一条直线上，同时观察左右是否对称并且在同一平面。

将大鼠前囱水平中点标记为原点并调零，然后定位大鼠左侧TG坐标位置。根据文献[14]，当SD大鼠体重为260~270 g时，双侧TG对称，以前囱为原点，其横向距离为(3.5±0.1) mm，纵向距离为(3.6±0.2) mm，确定左侧TG坐标约为(3.5, 3.6)。使用电动牙钻对坐标点进行缓慢、垂直打孔。用微量注射器避光抽取20 μL试剂，对准坐标位置，缓慢垂直进针，进针深度距颅面约为12 mm。到达目的深度后，以4 μL/min的速度缓慢注入试剂。当液体注射完毕后，微量注射器的针尖需要在原处停留10 min，随后将针向上移动1 mm后继续停留2 min，撤针后缝合皮肤创口。

验证实验：通过上述方法，将经10%高渗盐水稀释配制成的1%阿霉素的混合溶液注射至麻醉的正常大鼠TG内。1 d后，对麻醉大鼠进行快速灌注固定，断头后取出TG，泡入4%多聚甲醛中固定24 h，再浸入不同浓度的蔗糖溶液进行梯度脱水，用最佳切削温度化合物(optimum cutting temperature compound，OCT)胶包埋后置于液氮中迅速冷冻，采用冰冻切片机将TG切成10 μm左右的切片备用。

将TG冰冻切片用PBS冲洗3遍，每次5 min；然后用0.3%的TritonX-100打孔30 min，用PBS洗3次，每次5 min；加5%正常山羊血清封闭60 min；滴加一抗兔抗NeuN抗体(1꞉200)，然后将其放入湿盒中，在4 ℃下孵育过夜；用PBS冲洗3遍后，滴加二抗山羊抗兔IgG(1꞉200)，于37 ℃孵育1 h，PBS冲洗3次；滴加含有DAPI防淬灭用的封片剂进行封片，最后将成片放在荧光显微镜下观察。

由于阿霉素具有自体荧光的特性，在荧光显微镜蓝色激光滤镜下可显示绿色[16-17]，同时还可经轴浆逆行运输至神经元胞体，因而本研究采用阿霉素作为荧光染料来确定给药位置。

1.2.5. 行为学测试

所有实验大鼠提前适应环境3 d，于术前1 d及术后相应天数进行疼痛行为学测定。采用Von Frey纤维丝对各组大鼠术侧的触须垫区域进行测试。当大鼠完全适应周围环境，即不出现攻击性行为和探索行为时开始正式测试。用不同刺激强度的Von Frey纤维丝，缓慢接近大鼠的待测面部区域，使纤维丝弯曲。每根纤维丝在同一大鼠的同一部位重复刺激3次，每次间隔30 s以上。待大鼠重新回到适应状态后再次刺激，直到某一强度的纤维丝能刺激大鼠出现以下阳性反应3次：1)大鼠出现快速缩头的躲避刺激物的行为；2)大鼠出现攻击Von Frey纤维丝的行为(包括抓咬、撕扯等)；3)大鼠出现频繁的洗脸行为(即快速、多次的抓挠刺激区域的触须垫)。Von Frey纤维丝刺激强度的下限和上限分别为0.4 g和15.0 g。根据文献[15]所述的方法计算大鼠的50%的机械痛阈值。

1.2.6. 实时反转录聚合酶链反应

造模术后第15天，在低温无菌条件下迅速取各组大鼠术侧TG，放入无酶离心管中，用RNA提取试剂盒提取各组TG的总RNA，并检测其浓度和纯度。使用U6反向引物与miR-21-5p反转引物分别进行反转录，获得第1链cDNA；使用Evo M-MLV 反转录试剂盒进行反转录，获得cDNA。根据试剂盒说明书配置不同PCR体系，在Light Cycler 480高通量实时荧光定量PCR系统上完成PCR反应。β-actin和KCNA1的PCR反应参数为95 ℃ 30 s，95 ℃ 5 s，60 ℃ 30 s；40个循环。U6和miR-21-5p的PCR反应参数为95 ℃ 5 min，95 ℃ 10 s，60 ℃ 30 s；40个循环。融解曲线设置为95 ℃ 15 s，60 ℃ 60 s，95 ℃ 15 s。用2-ΔΔCt法计算出KCNA1及miR-21-5p的相对表达量。引物序列信息见表1。

表1.

引物序列

Table 1 Sequence of primers

名称 序列(5'-3') 长度/bp
β-actin 正向 TCAGGTCATCACTATCGGCAAT 22
反向 AAAGAAAGGGTGTAAAACGCA 21
KCNA1 正向 GGTTATTGCCATTGTATCCGTCAT 24
反向 GGTCTGTGAAGATGTTAGAAGTGT 24
U6 正向 AACGCTTCACGAATTTGCGT 20
反向 CTCGCTTCGGCAGCACA 17
miR-21-5p 正向 CGATGGGCTGTCTGACATTTT 21
反向 AGTGCAGGGTCCGAGGTATT 20
反转 GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGATACC 51
Open in a new tab

1.2.7. 蛋白质印迹法

造模术后第15天，在低温无菌条件下迅速取各组大鼠TG，放入离心管内，加入含蛋白酶抑制剂的裂解液，将组织裂解匀浆后于4 ℃，以12 000 r/min离心15 min，取上清液，应用BCA定量法测定样品蛋白质含量。加入上样缓冲液，并于100 ℃恒温水浴 5 min使蛋白质变性。根据目的蛋白KCNA1的分子量(56 kD)和内参β-actin分子量(42 kD)，选用SDS-PAGE凝胶试剂盒并按照产品说明配置10%的分离胶。设置电泳参数：80 V，40 min；110 V，60 min。电泳后将蛋白质转移至PVDF膜上，恒流转膜，290 mA转膜1 h，转膜后用5%脱脂奶粉封闭1 h。相应膜上分别加入兔抗大鼠KCNA1抗体(1꞉500)和小鼠抗大鼠β-actin抗体(1꞉1 000)于4 ℃摇床过夜。洗膜后分别加入山羊抗小鼠IgG H&L和山羊抗兔IgG H&L(1꞉10 000)二抗，于室温摇床上孵育1 h后进行荧光显影成像。以β-actin为内参进行半定量分析。

1.3. 统计学处理

采用SPSS 26.0统计学软件进行数据分析。计量资料以均数±标准差( x¯ ±s)表示。双荧光素酶报告基因检测结果及机械痛阈测定结果采用双因素方差分析。TN组和sham组实时反转录聚合酶链反应及蛋白质印迹法实验数据比较采用独立样本t检验。Sham组、sham+miR-21-5p agomir组及sham+agomir NC组，TN组、TN+miR-21-5p antagomir组及TN+antagomir NC组3组间实时反转录聚合酶链反应及蛋白质印迹法实验数据采用单因素方差分析，P<0.05为差异有统计学意义。

2. 结 果

2.1. 大鼠TN模型构建成功

机械痛阈测定结果显示：TN组大鼠在术后第3至15天的面部机械痛阈持续低于sham组。在术后第3天，TN组大鼠的机械痛阈开始降低，但与sham组相比差异无统计学意义(P>0.05)；从术后第5至15天，TN组大鼠的面部机械痛阈保持稳定，且显著低于sham组的机械痛阈(均P<0.001，图1)。由行为学结果可以得出，经dIoN-CCI造模后大鼠TN模型构建成功。

图1.

图1

Open in a new tab

TN组和sham 组在造模术后机械痛阈的变化(n=6， x¯ ±s)

Figure 1 Changes of the mechanical pain threshold in TN and sham groups (n=6, x¯ ±s) ***P<0.001 vs the sham group. TN: Trigeminal neuralgia.

2.2. 脑立体定位的注射部位确定

肉眼观察，可见TG注射部位上有阿霉素染料的红色标记(图2)。显微镜下观察，经阿霉素注射标记后，TG周围呈现出强阳性的荧光表达，神经元显示出特殊的绿色；用兔抗NeuN抗体将神经元染色，镜下可见神经元被染成红色；用含有DAPI防淬灭用的封片剂封片，镜下可见细胞核被染成蓝色(图3)。免疫荧光结果显示阿霉素已经进入三叉神经节神经元核内，这些结果证明脑立体定位注射给药法可以使药物准确到达TG。

图2.

图2

Open in a new tab

经脑立体定位对TG注射部位的大体观察

Figure 2 General view of the injection site of the TG by brain stereotaxis A: After stereotaxic injection, adriamycin (red) marked the left TG; B: TG tissue in vitro. TG: Trigeminal ganglion.

图3.

图3

Open in a new tab

经脑立体定位对TG注射部位的免疫荧光观察

Figure 3 Immunofluorescence observation of TG injection site by brain stereotaxis A and B: After adriamycin labeling, strong fluorescence labeling was observed at the TG injection site under fluorescence microscope (A, ×100; B, ×400). C: Staining with DAPI, blue represents the nucleus (×400); D: Staining with rabbit anti-NeuN antibody, red represents neurons (×400); E: Merged of C and D. Overlapping area indicates that adriamycin has entered the nucleus of TG neurons (×400). TG: Trigeminal ganglion; DAPI: 4’, 6-diamidino-2-phenylindole; NeuN: Neuronal nuclei.

2.3. MiR-21-5p 直接靶向作用于 KCNA1

选用Target Scan Human、miRDB和mirDIP等生物信息学软件，对与KCNA1相关的miRNA进行预测，发现在人类及大鼠基因中，miR-21-5p与Kv1.1的编码基因KCNA1紧密相关。预测发现：在人类基因中，miR-21-5p与KCNA1 3'-UTR第929~935位碱基序列存在互补的片段基因；在大鼠基因中，miR-21-5p与KCNA1 3'-UTR第960~966位碱基序列存在互补的片段基因(图4A)，表明两者之间存在靶标关系。

图4.

图4

Open in a new tab

MiR-21-5p 与 KCNA1 的靶向调控关系验证

Figure 4 Verification of the targeting relationship between miR-21-5p and KCNA1

A: Schematic diagram of the binding site of miR-21-5p and KCNA1 mRNA; B: Histogram of targeted regulative effect of miR-21-5p on KCNA1 by dual luciferase reporter assay. ***P<0.001 vs the mimic NC group; †††P<0.001 vs the rno-miR-21-5p (6 nmol/L) group.

双荧光素酶实验结果发现：与转染mimic NC和野生型KCNA1(KCNA1 WT)组相比，共转染6 nmol/L或20 nmol/L rno-miR-21-5p mimics的KCNA1 WT组的荧光素酶活性显著降低(P<0.001)，表明miR-21-5p与KCNA1存在靶向结合；同时对KCNA1 WT组进行不同剂量的rno-miR-21-5p mimics干预，与6 nmol/L rno-miR-21-5p mimics共转染组相比，20 nmol/L rno-miR-21-5p mimics共转染组的荧光素酶相对活动显著降低(P<0.001)。与mimic NC共转染突变型KCNA1(KCNA1 MUT)相比，KCNA1 MUT和rno-miR-21-5p mimics(6 nmol/L或20 nmol/L)共转染后荧光素酶活性差异无统计学意义(P>0.05)，提示miR-21-5p与KCNA1 3'-UTR特异性结合，具有序列特异性。与pmirGLO空载体和mimic NC共转染组相比，pmirGLO空载体和rno-miR-21-5p mimics(6 nmol/L或20 nmol/L)共转染后，荧光素酶活性表达差异无统计学意义(P>0.05，图4B)，排除了质粒上序列对实验结果的影响，表明miR-21-5p能够靶向结合并直接调控KCNA1 3'-UTR。

2.4. TN模型的术侧TG中 KCNA1 和 miR-21-5p 表达的变化

与sham组相比，TN组大鼠术侧TG中KCNA1 mRNA的相对表达量降低(P<0.05，图5A)，miR-21-5p相对表达量增多(P<0.05，图5B)，KCNA1蛋白质的相对表达量也降低(P<0.05，图5C)。

图5.

图5

Open in a new tab

KCNA1 及 miR-21-5p 在2组大鼠术侧TG中的表达( x¯ ±s)

Figure 5 Expression of KCNA1 and miR-21-5p in the TG on the operated side of the 2 groups of rats ( x¯ ±s) A: Expression of KCNA1 mRNA; B: Expression of miR-21-5p; C: Expression of KCNA1 protein. *P<0.05 vs the sham group. TG: Trigeminal ganglion; TN: Trigeminal neuralgia.

2.5. MiR-21-5pagomir和 miR-21-5p antagomir对大鼠机械痛阈值的影响

各组大鼠的基础痛阈在干预前无明显差异。造模术后第10天开始对sham组注射miR-21-5p agomir，在注射后第1(即术后第11天)至第5天，与sham组及sham+agomir NC组相比，sham+miR-21-5p agomir组的机械痛阈明显降低(均P<0.05，图6A)；对TN组注射miR-21-5p antagomir后，在注射后第1(即术后第11天)至第5天，与TN组及TN+antagomir NC组相比，TN+miR-21-5p antagomir组机械痛阈均显著升高(均 P<0.05，图6B)。

图6.

图6

Open in a new tab

各组大鼠机械痛阈的变化(n=5， x¯ ±s)

Figure 6 Changes in mechanical pain threshold of rats in different groups (n=5, x¯ ±s)

A: Mechanical pain threshold on the operated side of rats in the sham group, the sham+miR-21-5p agomir group, and the sham+miR-21-5p agomir group. ***P<0.001 vs the sham group, ††P<0.01, †††P<0.001 vs the sham+agomir NC group. B: Mechanical pain threshold on the operated side of rats in the TN group, the TN+miR-21-5p agomir group, and the TN+miR-21-5p agomir group. *P<0.05, **P<0.01, ***P<0.001 vs the TN group; ††P<0.01, †††P<0.001 vs the TN+antagomir NC group. ↑represents injection at that time point. TN: Trigeminal neuralgia.

2.6. MiR-21-5pagomir和 miR-21-5p antagomir对大鼠TG内KCNA1表达的影响

实时反转录聚合酶链反应结果显示：与sham组和sham+agomir NC组相比，sham+miR-21-5p agomir组大鼠TG中KCNA1 mRNA表达显著降低(P<0.05，图7A)；蛋白质印迹法结果显示sham+miR-21-5p agomir组大鼠TG中KCNA1蛋白质表达降低(P<0.05，图7B)。Sham组与sham+agomir NC组大鼠TG内KCNA1 mRNA及蛋白质的表达差异均无统计学意义(均P>0.05)。

图7.

图7

Open in a new tab

各组大鼠术侧TG中 KCNA1 表达的变化

Figure 7 Changes in the expression of KCNA1 in the TG of rats in different groups

A and B: Expression of KCNA1 mRNA (A, n=4) and protein (B, n=3) in the TG of rats in the sham group, the sham+agomir NC group, and the sham+miR-21-5p agomir group. *P<0.05, **P<0.01 vs the sham group; †P<0.05 vs the sham+agomir NC group; C and D: Expression of KCNA1 mRNA (C, n=4) and protein (D, n=3) in the TN group, the TN+antagomir NC group, and the TN+miR-21-5p antagomir group. **P<0.01, ***P<0.001 vs the TN group; †P<0.05, †††P<0.001 vs the TN+miR-21-5p antagomir group. TN: Trigeminal neuralgia; TG: Trigeminal ganglion.

实时反转录聚合酶链反应结果显示TN+miR-21-5p antagomir组大鼠TG中KCNA1 mRNA表达水平升高(P<0.05，图7C)；蛋白质印迹法结果显示TN+miR-21-5p antagomir组大鼠TG中KCNA1蛋白质表达升高(P<0.05，图7D)。TN组及TN+antagomir NC组大鼠TG内KCNA1 mRNA及蛋白质的表达差异均无统计学意义(均P>0.05)。

3. 讨 论

TN是人类可能经历的最痛苦的神经病理性疼痛类型之一，其不仅严重损害患者的身心健康，也会造成社会、医学资源的消耗。TN年发病率为0.03‰~0.05‰，女性发病率较高，并且随着年龄的增长而升高。40岁以上人群占到患病人数的70%~80%[18]。因而进一步阐明TN的发病机制，有望为今后该病检测、诊断、治疗新方法和新药物的研发打下基础。

近年来，有较多研究者[19-20]关注神经病理性疼痛中初级感觉神经元上存在的电压门控离子通道异常表达，这与疼痛的发生、发展密切相关。Kv1.1与神经病理性疼痛有紧密联系，当外周神经损伤后，髓鞘破坏增加了轴突上Kv1.1的暴露，这反过来可能导致动作电位幅度和持续时间降低，因为静息膜电位接近K+平衡电位[21]。多种Kv亚型参与TN的发生、发展。本研究结果显示：与sham组相比，TN组大鼠术侧TG中KCNA1 mRNA和蛋白质水平显著降低。这一结论也与Yang等[4]的研究结果一致，他们的研究发现在小鼠下牙槽神经切断模型术侧TG内Kv1.1的表达水平降低，这进一步验证了Kv1.1参与了TN的过程，但涉及的上游调节机制目前尚未完全明确。

已证实miR-21-5p的促炎作用参与神经病理性疼痛的发生和发展[22]，miR-21的上调和释放有助于感知神经元-巨噬细胞信号，从而损伤周围神经[23]。临床研究[24]发现：因患有腰椎间盘突出症而出现坐骨神经痛患者的髓核、纤维环和神经根周围软组织中的miR-21表达增加，局部组织miR-21的增加与患者病情严重程度呈正相关。慢性疼痛患者体内miR-21表达增加[25-26]，miR-21可作为预测疼痛发生和严重程度的重要标志物。

目前鲜少miR-21-5p在TN中作用的研究报道。本研究通过双荧光素酶报告基因检测发现miR-21-5p与KCNA1存在靶标关系并且具有序列特异性，随着rno-miR-21-5p mimics剂量的增加，转染KCNA1 WT组的荧光素酶相对活性降低更甚，表明两者可能存在负性调控关系。将miR-21-5p agomir通过脑立体定位仪精准注射于sham组大鼠TG后发现，从注射后第1天开始，大鼠术侧机械痛阈显著下降；对TN组大鼠TG注射miR-21-5p antagomir后，大鼠术侧机械痛阈显著回升，这与Reinhold等[27]和Karl-Schöller等[28]的研究结果一致。他们将miR-21-5p类似物应用于完整神经时会引起强烈的痛觉超敏反应和血神经屏障的破坏，观察到类似于神经病理性疼痛模型中的情况。相反，在损伤神经周围局部抑制miR-21-5p可以延缓和减弱神经病理性疼痛小鼠模型的痛觉超敏反应。在Zhang等[12]的研究中，将miR-21 antagomir单次注射到脊神经结扎小鼠模型的L5脊神经，低剂量注射组(0.3 μg)不影响小鼠的机械缩足阈值，但是高剂量注射组(0.5 μg)会使小鼠的机械缩足阈值逐渐增加并在第4天显著减轻机械性痛觉过敏反应。这与本实验结果一致。本实验对TN大鼠TG注射高剂量的miR-21 antagomir后，大鼠面部机械疼痛阈值升高。此外，Zhang等[12]的研究还证实miR-21可作为内源性配体作用于内体和溶酶体中的Toll样受体8(Toll-like reception 8，TLR8)，诱导细胞外调节蛋白激酶(extracellular regulated protein kinase，ERK)激活和炎症介质产生，进一步增加神经元兴奋性，并有助于维持神经性疼痛。这也许是注射miR-21 antagomir出现长时间镇痛效果的原因之一。

本研究实时反转录聚合酶链反应和蛋白质印迹法结果显示：与sham组及sham+agomir NC组相比，sham+miR-21-5p agomir组大鼠TG中KCNA1的表达降低。与TN组及TN+antagomir NC组相比，TN+miR-21-5p antagomir组大鼠TG中KCNA1的表达水平升高。这表明过表达miR-21-5p后Kv1.1的表达量降低，使sham组大鼠出现机械痛阈下降的疼痛反应，促使TN的发生、发展；抑制miR-21-5p表达后Kv1.1的表达量增加，并逆转了由dIoN-CCI引起的TN，使疼痛缓解。提示miR-21-5p对Kv1.1具有确切的负性调控作用，miR-21-5p antagomir可作为有效的镇痛目标参与TN的治疗。

综上，TN模型中TG高表达的miR-21-5p通过抑制Kv1.1编码基因KCNA1的表达，导致KV1.1表达降低，进而解除了对神经节髓鞘感觉轴突过度兴奋的保护性抑制，从而使TG神经元的动作电位增多，兴奋性增加。研究miR-21-5p调控KCNA1在TN中的作用机制，有利于进一步揭示miR-21-5p促炎的分子机制，有助于阐明TN的发生和演进机制，也可为TN的早期诊断和治疗干预提供新的理论基础。

基金资助

湖南省自然科学基金(2019JJ40457)。This work was supported by the Natural Science Foundation of Hunan Province, China (2019JJ40457).开放获取(Open access)：本文遵循知识共享许可协议，允许第三方用户按照署名-非商业性使用-禁止演绎4.0(CC BY-NC-ND 4.0)的方式，在任何媒介以任何形式复制、传播本作品(<ext-link>https://creativecommons.org/licenses/by-nc-nd/4.0/</ext-link>)。

利益冲突声明

作者声称无任何利益冲突。

作者贡献

周雪雯、郭刚文 实验构思和设计，数据采集和分析，论文撰写；余珊子 实验构思和设计，统计分析，论文修改；胡蓉 实验构思和设计，数据分析，对文章的知识性内容作批判性审阅。所有作者阅读并同意最终的文本。

Footnotes

http://dx.chinadoi.cn/10.11817/j.issn.1672-7347.2024.230273

原文网址

http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/20240129.pdf

参考文献

  • 1. Cruccu G, Di Stefano G, Truini A. Trigeminal neuralgia[J]. N Engl J Med, 2020, 383(8): 754-762. 10.1056/nejmra1914484. [DOI] [PubMed] [Google Scholar]
  • 2. Takeda M, Tsuboi Y, Kitagawa J, et al. Potassium channels as a potential therapeutic target for trigeminal neuropathic and inflammatory pain[J]. Mol Pain, 2011, 7: 5. 10.1186/1744-8069-7-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Abd-Elsayed AA, Ikeda R, Jia ZF, et al. KCNQ channels in nociceptive cold-sensing trigeminal ganglion neurons as therapeutic targets for treating orofacial cold hyperalgesia[J]. Mol Pain, 2015, 11: 45. 10.1186/s12990-015-0048-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Yang J, Liu F, Zhang YY, et al. C-X-C motif chemokine ligand 1 and its receptor C-X-C motif chemokine receptor 2 in trigeminal ganglion contribute to nerve injury-induced orofacial mechanical allodynia[J]. J Oral Rehabil, 2022, 49(2): 195-206. 10.1111/joor.13273. [DOI] [PubMed] [Google Scholar]
  • 5. Chi XX, Nicol GD. Manipulation of the potassium channel Kv1.1 and its effect on neuronal excitability in rat sensory neurons[J]. J Neurophysiol, 2007, 98(5): 2683-2692. 10.1152/jn.00437.2007. [DOI] [PubMed] [Google Scholar]
  • 6. López-González MJ, Landry M, Favereaux A. MicroRNA and chronic pain: from mechanisms to therapeutic potential[J]. Pharmacol Ther, 2017, 180: 1-15. 10.1016/j.pharmthera.2017.06.001. [DOI] [PubMed] [Google Scholar]
  • 7. Andersen HH, Duroux M, Gazerani P. MicroRNAs as modulators and biomarkers of inflammatory and neuropathic pain conditions[J]. Neurobiol Dis, 2014, 71: 159-168. 10.1016/j.nbd.2014.08.003. [DOI] [PubMed] [Google Scholar]
  • 8. Zhang J, Rong L, Shao J, et al. Epigenetic restoration of voltage-gated potassium channel Kv1.2 alleviates nerve injury-induced neuropathic pain[J]. J Neurochem, 2021, 156(3): 367-378.10.1111/jnc. 15117. [DOI] [PubMed] [Google Scholar]
  • 9. Sakai A, Saitow F, Maruyama M, et al. MicroRNA cluster miR-17-92 regulates multiple functionally related voltage-gated potassium channels in chronic neuropathic pain[J]. Nat Commun, 2017, 8: 16079. 10.1038/ncomms16079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Liu YP, Yang YD, Mou FF, et al. Exosome-mediated miR-21 was involved in the promotion of structural and functional recovery effect produced by electroacupuncture in sciatic nerve injury[J]. Oxid Med Cell Longev, 2022, 2022: 7530102. 10.1155/2022/7530102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Ning XJ, Lu XH, Luo JC, et al. Molecular mechanism of microRNA-21 promoting Schwann cell proliferation and axon regeneration during injured nerve repair[J]. RNA Biol, 2020, 17(10): 1508-1519. 10.1080/15476286.2020.1777767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Zhang ZJ, Guo JS, Li SS, et al. TLR8 and its endogenous ligand miR-21 contribute to neuropathic pain in murine DRG[J]. J Exp Med, 2018, 215(12): 3019-3037. 10.1084/jem.20180800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Juźwik CA, Drake SS, Zhang Y, et al. MicroRNA dysregulation in neurodegenerative diseases: a systematic review[J]. Prog Neurobiol, 2019, 182: 101664. 10.1016/j.pneurobio.2019.101664. [DOI] [PubMed] [Google Scholar]
  • 14. Tang XF, Bi RY, Meng Z, et al. Stereotaxis of mandibular nerve initial point of trigeminal ganglion in rats[J]. Chin J Dent Res, 2014, 17(2): 99-104. [PubMed] [Google Scholar]
  • 15. Dixon WJ. Efficient analysis of experimental observations[J]. Annu Rev Pharmacol Toxicol, 1980, 20: 441-462. 10.1146/annurev.pa.20.040180.002301 [DOI] [PubMed] [Google Scholar]
  • 16. 刘蔡钺, 李娜, 赵云富, 等. BKCa通道激动剂NS1619和Kv通道拮抗剂4-AP对眶下神经慢性缩窄环术大鼠面部机械痛阈的影响[J]. 生理学报, 2010, 62(5): 441-449. 10.13294/j.aps.2010.05.010. [DOI] [PubMed] [Google Scholar]; LIU Caiyue, LI Na, ZHAO Yunfu, et al. BKCa channel agonist NS1619 and Kv channel antagonist 4-AP on the facial mechanical pain threshold in a rat model of chronic constriction injury of the infraorbital nerve[J]. Acta Physiologica Sinica, 2010, 62(5): 441-449. 10.13294/j.aps.2010.05.010. [DOI] [PubMed] [Google Scholar]
  • 17. Neubert JK, Mannes AJ, Keller J, et al. Peripheral targeting of the trigeminal ganglion via the infraorbital foramen as a therapeutic strategy[J]. Brain Res Brain Res Protoc, 2005, 15(3): 119-126. 10.1016/j.brainresprot.2005.05.003. [DOI] [PubMed] [Google Scholar]
  • 18. Tai AX, Nayar VV. Update on trigeminal neuralgia[J]. Curr Treat Options Neurol, 2019, 21(9): 42. 10.1007/s11940-019-0583-0. [DOI] [PubMed] [Google Scholar]
  • 19. Alles SRA, Smith PA. Peripheral voltage-gated cation channels in neuropathic pain and their potential as therapeutic targets[J]. Front Pain Res, 2021, 2: 750583. 10.3389/fpain.2021.750583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Hartung J, Moy J, Loeza-Alcocer E, et al. Voltage gated calcium channels in human dorsal root ganglion neurons[J/OL]. Pain, 2021, 163(6): e774-e785[2022-06-30]. 10.1097/j.pain.0000000000002465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Liu CH, Chang HM, Wu TH, et al. Rearrangement of potassium ions and Kv1.1/Kv1.2 potassium channels in regenerating axons following end-to-end neurorrhaphy: ionic images from TOF-SIMS[J]. Histochem Cell Biol, 2017, 148(4): 407-416. 10.1007/s00418-017-1570-8. [DOI] [PubMed] [Google Scholar]
  • 22. Zhang XY, Zhu L, Wang XZ, et al. Advances in the role and mechanism of miRNA in inflammatory pain[J]. Biomed Pharmacother, 2023, 161: 114463. 10.1016/j.biopha.2023.114463. [DOI] [PubMed] [Google Scholar]
  • 23. Olivieri F, Prattichizzo F, Giuliani A, et al. MiR-21 and miR-146a: the microRNAs of inflammaging and age-related diseases[J]. Ageing Res Rev, 2021, 70: 101374. 10.1016/j.arr.2021.101374. [DOI] [PubMed] [Google Scholar]
  • 24. Zou ZF, He JP, Chen YL, et al. Increased local miR-21 expressions are linked with clinical severity in lumbar disc herniation patients with sciatic pain[J]. Adv Clin Exp Med, 2022, 31(7): 723-730. 10.17219/acem/146968. [DOI] [PubMed] [Google Scholar]
  • 25. Leinders M, Üçeyler N, Thomann A, et al. Aberrant microRNA expression in patients with painful peripheral neuropathies[J]. J Neurol Sci, 2017, 380: 242-249. 10.1016/j.jns.2017.07.041. [DOI] [PubMed] [Google Scholar]
  • 26. Vucetic M, Roganovic J, Freilich M, et al. Bone microRNA-21 as surgical stress parameter is associated with third molar postoperative discomfort[J]. Clin Oral Investig, 2021, 25(1): 319-328. 10.1007/s00784-020-03366-6. [DOI] [PubMed] [Google Scholar]
  • 27. Reinhold AK, Krug SM, Salvador E, et al. MicroRNA-21-5p functions via RECK/MMP9 as a proalgesic regulator of the blood nerve barrier in nerve injury[J]. Ann N Y Acad Sci, 2022, 1515(1): 184-195. 10.1111/nyas.14816. [DOI] [PubMed] [Google Scholar]
  • 28. Karl-Schöller F, Kunz M, Kreß L, et al. A translational study: involvement of miR-21-5p in development and maintenance of neuropathic pain via immune-related targets CCL5 and YWHAE[J]. Exp Neurol, 2022, 347: 113915. 10.1016/j.expneurol.2021.113915. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Central South University Medical Sciences are provided here courtesy of Central South University

RESOURCES