垂体腺苷酸环化酶激活肽对紫杉醇诱发的小鼠外周神经病理性疼痛的影响及机制研究

Effect and mechanism of pituitary adenylate cyclase-activating peptide on peripheral neuropathic pain induced by paclitaxel in mice

摘要:

目的：探讨垂体腺苷酸环化酶激活肽 (pituitary adenylate cyclase-activating polypeptide, PACAP) 对紫杉醇 (paclitaxel, PTX) 诱发的小鼠外周神经病理性疼痛 ( paclitaxel-induced peripheral neuropathy,PIPN) 的作用及机制。方法：细胞实验：将C57BL/6J小鼠背根神经节 (dorsal root ganglion, DRG) 组织进行原代培养,根据给药方式不同,分为对照组(Vehicle组)、紫杉醇组(PTX组)、给药组(PACAP组), 试剂盒检测各组原代背根神经节 (dorsal root ganglion, DRG) 细胞内活性氧 (reactive oxygen species, ROS)、线粒体膜电位 (mitochondrial membrane potential, MMP)。动物实验：采用随机数表法将小鼠分为四组：对照组(Vehicle组)、紫杉醇组(PTX组)、低剂量PACAP治疗组 [PTX+PACAP (30μg/kg)] 组和高剂量PACAP治疗组 [PTX+PACAP (100μg/kg)] 组,每组15只。PTX组和PACAP给药组分别注射累计剂量为32mg/kg的紫杉醇进行造模,Vehicle组在相应时间点注射对应溶剂。于紫杉醇注射前1天、第7、14天测量小鼠体重、机械刺激缩足反射阈值 (mechanical withdrawal threshold, MWT)、热缩足反射潜伏期 (thermal withdrawal latency, TWL) 以及冷板反应时间、试剂盒检测DRG组织内丙二醛 (malondialdehyde, MDA)、超氧化物歧化酶 (superoxide dismutase, SOD)、电镜观察DRG组织线粒体形态、Western blot 检测DRG组织PGC-1α、HO-1蛋白表达。结果：细胞实验：与Vehicle组相比,PTX组DRG细胞内ROS明显增多,MMP明显下降 (P<0.01)。PACAP可显著降低DRG细胞ROS含量,恢复MMP (P<0.01)。动物实验：PACAP、PTX均不能明显影响各组小鼠体重,差异无统计学意义；与Vehicle组相比,PTX组小鼠出现机械、热、冷痛觉过敏 (P<0.01),DRG组织 MDA含量增多、SOD活性降低、异常形态线粒体明显增多、PGC-1α表达量减少、HO-1表达量明显增加 (P<0.01)。与PTX组相比,PACAP (30μg/kg) 无明显效果,差异无统计学意义。而PACAP (100μg/kg) 能显著改善机械、热、冷痛觉过敏 (P<0.05),减少MDA,增强SOD活性,改善线粒体形态,并提高PGC-1α和HO-1蛋白表达水平 (P<0.01)。结论：PACAP能有效缓解紫杉醇诱发的小鼠外周神经病理性疼痛,其机制可能与抑制氧化应激、增加PGC-1α和HO-1的表达有关。

Abstract:

Objective: To investigate the effect and mechanism of pituitary adenylate cyclase-activating polypeptide (PACAP) on paclitaxel-induced peripheral neuropathy (PIPN) induced by PTX in mice. Methods: Cell experiment: The dorsal root ganglion (DRG) tissue of C57BL/6J mice was cultured in primary culture, according to the way of administration, They were divided into Vehicle group, PTX group and PACAP group. The intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) of the DRG in each group were detected by the test kit. Animal experiments: Mice were divided into four groups by random number table method: Control group (Vehicle group), paclitaxel group (PTX group), low-dose PACAP treatment group [PTX+PACAP (30μg/kg)] and high-dose PACAP treatment group [PTX+PACAP (100μg/kg)], with 15 mice in each group. PTX group and PACAP group were injected with a cumulative dose of 32mg/kg of paclitaxel for modeling, and Vehicle group was injected with the corresponding solvent at the corresponding time point. Body weight, mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL) and Cold plate reaction time were measured -1 day, 7 days and 14 days before paclitaxel injection. malondialdehyde (MDA) and superoxide dismutase (SOD) in DRG tissues were also determined by test kit. The morphology of mitochondria in DRG tissue was observed by electron microscopy. The expression of PGC-1α and HO-1 protein in DRG tissue was detected by Western blot. Results: Cell experiment: Compared with Vehicle group, ROS in DRG cells of PTX group increased significantly and MMP decreased significantly (P<0.01). PACAP could significantly reduce ROS content in DRG cells and restore MMP (P<0.01). Animal experiments: PACAP and PTX could not significantly change the body weight of mice in each group, and the difference was not statistically significant. Compared with Vehicle group, the mice in PTX group showed mechanical, hot and cold pain hypersensitivity (P<0.01), MDA content increased, SOD activity decreased, abnormal mitochondria increased significantly, PGC-1α expression decreased, and HO-1 expression increased significantly (P<0.01) in DRG tissues. Compared with PTX group, PACAP (30μg/kg) had no significant effect and the difference was not statistically significant. PACAP (100μg/kg) significantly improved mechanical, thermal and cold hyperalgesia (P<0.05), decreased MDA, increased SOD activity, improved mitochondrial morphology, and increased PGC-1α and HO-1 protein expression levels (P<0.01). Conclusion: PACAP can effectively relieve peripheral neuropathic pain induced by paclitaxel in mice, and its mechanism may be related to inhibiting oxidative stress and increasing the expression of PGC-1α and HO-1.