神经肽Y和降钙素基因相关肽在带状疱疹后遗神经痛中的表达及其相互作用机制研究

Expression and Interaction Mechanism of Neuropeptide Y and Calcitonin Gene-Related Peptide in Postherpetic Neuralgia

摘要:

目的：探讨神经肽Y（neuropeptide Y, NPY）和降钙素基因相关肽（calcitonin gene-related peptide, CGRP）在带状疱疹后遗神经痛（postherpetic neuralgia, PHN）中的功能及相互作用机制。 方法：将88只成年雄性大鼠，随机分为8组，每组n=11：正常对照组（NC组）：不进行任何处理，常规饲养；假手术组（sham组）：背部皮肤切开缝合但不接种病毒；PHN模型组（PHN组）：背部皮内注射水痘-带状疱疹病毒（VZV-Oka株，1×10? PFU/50 μl）建立神经痛模型；PHN+NPY溶剂对照组（PHN+NPY-Sol组）：造模后鞘内注射人工脑脊液（aCSF，10 μl）；PHN+NPY组（PHN+NPY组）：造模后鞘内注射NPY（5μg/10 μl aCSF）；PHN+CGRP溶剂对照组（PHN+CGRP-Sol组）：造模后鞘内注射10 μl生理盐水；PHN+CGRP组（PHN+CGRP组）；造模后鞘内注射CGRP（1 nmol/10 μl生理盐水）；PHN+NPY+CGRP组：造模后鞘内注射NPY（5μg）+CGRP（1 nmol）混合液，评估机械痛阈和热痛潜伏期指标。通过酶联免疫吸附测试（ELISA）、免疫荧光和Western blot分析NPY与CGRP在血清以及背根神经节（DRG）中的表达。ELISA检测血清白细胞介素-6（IL-6）及肿瘤坏死因子-α（TNF-α）的表达。在细胞实验中，使用大鼠神经元系H19-7，转染NPY或CGRP的特异性shRNA重组载体，对NPY和CGRP进行敲低，分为Control组、shNPY-NC组、shNPY组、shCGRP-NC组、shCGRP组以及shNPY+shCGRP组。Western blot检测NPY或CGRP表达以及细胞外信号调节激酶（extracellular signal-regulated kinase, ERK）/核因子-κB（NF-κB）信号通路蛋白的表达。CCK-8法检测细胞增殖。流式细胞术检测细胞凋亡。 结果：与PHN组相比，PHN+NPY组大鼠的机械痛阈值和热痛潜伏期显著提高（均P＜0.05）；而PHN+CGRP组的机械痛阈值和热痛潜伏期显著降低（均P＜0.05）。PHN组DRG中NPY和CGRP的表达显著下降（均P＜0.05）。在PHN+NPY组和PHN+NPY+CGRP组中，NPY的表达量显著上升，而CGRP在PHN+CGRP组的表达明显升高（均P＜0.05）。血清中，PHN+NPY组和PHN+NPY+CGRP组的炎性因子IL-6和TNF-α水平显著低于PHN组（均P＜0.05）。与shNPY-NC组比，shNPY组的NPY蛋白水平和细胞增殖率下调，而CGRP、p-ERK/ERK、p-P65/NF-κB P65的蛋白水平和细胞凋亡率上调（均P＜0.05）。与shCGRP-NC组比，shCGRP组的CGRP、p-ERK/ERK、p-P65/NF-κB P65的蛋白水平下调，而细胞凋亡率下调（均P＜0.05）。与shCGRP组比，shNPY+shCGRP组可显著降低细胞增殖率，增加CGRP、p-ERK/ERK、p-P65/NF-κB P65的蛋白水平以及细胞的凋亡率（均P＜0.05）。 结论：NPY与CGRP在带状疱疹后遗神经痛中通过相互作用共同调控疼痛的发生和维持。NPY通过抑制CGRP的表达及ERK/NF-κB信号通路的激活在疼痛调控中起到抑制作用，而CGRP则在痛觉传递和炎症反应中发挥加剧作用。

Abstract:

Objective: To explore the function and interaction mechanism of neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP) in postherpetic neuralgia (PHN). Methods: Eighty-eight adult male rats were randomly divided into eight groups, with each group n=11: Normal control group (NC group): no treatment, routine feeding; sham operation group (sham group): back skin incision and suture without virus inoculation; PHN model group (PHN group): intradermal injection of varicella-zoster virus (VZV-Oka strain, 1×10? PFU/50 μl) on the back to establish a neuralgia model; PHN+NPY solvent control group (PHN+NPY-Sol group): intrathecal injection of artificial cerebrospinal fluid (aCSF, 10 μl) after modeling; PHN+NPY group (PHN+NPY group): intrathecal injection of NPY (5μg/10 μl aCSF) after modeling; PHN+CGRP solvent control group (PHN+CGRP-Sol group): intrathecal injection of 10 μl saline after modeling; PHN+CGRP group (PHN+CGRP group); intrathecal injection of CGRP (1 nmol/10 μl saline) after modeling; PHN+NPY+CGRP group: intrathecal injection of a mixture of NPY (5μg) + CGRP (1 nmol) after modeling, evaluating mechanical pain threshold and thermal pain latency indicators. The expression of NPY and CGRP in serum and dorsal root ganglia (DRG) was analyzed by enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and Western blot. ELISA was used to detect the expression of serum interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). In cell experiments, rat neuron line H19-7 was used, transfected with specific shRNA recombinant vectors for NPY or CGRP to knock down NPY and CGRP, divided into Control group, shNPY-NC group, shNPY group, shCGRP-NC group, shCGRP group, and shNPY+shCGRP group. Western blot was used to detect the expression of NPY or CGRP and extracellular signal-regulated kinase (ERK)/nuclear factor-κB (NF-κB) signaling pathway proteins. CCK-8 assay was used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis. Results: Compared with the PHN group, the mechanical pain threshold and thermal pain latency of rats in the PHN+NPY group were significantly increased (both P<0.05); while the mechanical pain threshold and thermal pain latency in the PHN+CGRP group were significantly decreased (both P<0.05). The expression of NPY and CGRP in DRG of the PHN group was significantly decreased (both P<0.05). In the PHN+NPY group and PHN+NPY+CGRP group, the expression of NPY was significantly increased, while the expression of CGRP was significantly increased in the PHN+CGRP group (both P<0.05). In serum, the levels of inflammatory factors IL-6 and TNF-α in the PHN+NPY group and PHN+NPY+CGRP group were significantly lower than those in the PHN group (both P<0.05). Compared with the shNPY-NC group, the NPY protein level and cell proliferation rate in the shNPY group were downregulated, while the protein levels of CGRP, p-ERK/ERK, p-P65/NF-κB P65, and cell apoptosis rate were upregulated (all P<0.05). Compared with the shCGRP-NC group, the protein levels of CGRP, p-ERK/ERK, p-P65/NF-κB P65 in the shCGRP group were downregulated, while the cell apoptosis rate was downregulated (all P<0.05). Compared with the shCGRP group, the shNPY+shCGRP group significantly reduced the cell proliferation rate, increased the protein levels of CGRP, p-ERK/ERK, p-P65/NF-κB P65, and the cell apoptosis rate (all P<0.05). Conclusion: NPY and CGRP co-regulate the occurrence and maintenance of pain in postherpetic neuralgia through interaction. NPY plays an inhibitory role in pain regulation by inhibiting the expression of CGRP and the activation of the ERK/NF-κB signaling pathway, while CGRP plays an exacerbating role in nociceptive transmission and inflammatory response.